Patent Application
20220162262
The invention relates to novel compounds with the ability to link an immune response to a pathogen, to the use of said compounds in a disease or disorder mediated and/or caused by an infective agent, to compositions containing said compounds, processes for their preparation and to novel intermediates used in said process.
There is a need to find novel ways to recruit an individual's immune system to fight disease. The human immune system continually surveys the body seeking foreign signals to identify potentially harmful pathogens or mutated human cells (that could become a cause of cancerous growth) and target them for elimination. Natural antibodies exist that can be recruited to said pathogens or mutated human cells to drive the immune system to eliminate the threat. The invention details the use of a novel set of linker molecules that are designed to attract these natural antibodies in such a way as to be able to maximise the efficacy of immune recruitment while minimising potential side effects.
There is an urgent need to identify novel ways of treating bacterial, viral and fungal infections. Drug resistance is becoming a major global health threat. For example, more than 2 million people in the US were infected with bacteria resistant to at least one class of antibiotics (Centers for Disease Control and Prevention, 2013). Overall, the identification of new antibiotics targeting resistant strains of gram-negative organisms has been particularly difficult, in part due to the complex and evolving strategy these bacteria use to prevent antibiotic action (e.g., production of antibiotic inactivating enzymes, ability to transfer of resistance between strains, efflux pumps to prevent intracellular action) coupled with their naturally impermeable cell membranes that make it hard to identify drugs that penetrate into the cell and inhibit key targets. Further, many strains utilize multiple resistance mechanisms making it difficult for a single antibiotic to overcome.
An innovative approach to the treatment of infectious disease was disclosed in WO 01/45734 which describes a set of novel immunity linkers. Examples of said linker moieties include compounds or agents which are recognised by the immune system of said individual as foreign and which would therefore trigger an immune response. One such example is a carbohydrate molecule capable of binding to a human anti-alpha-galactosyl antibody (i.e. galactosyl-alpha-1,3-galactosyl-beta-1,4-N-acetylglucosamine) which results in redirection of the natural human serum antibody anti-alpha-galactosyl. The resultant effect of said immunity linker molecule is that the immune response of the individual is diverted from the pre-existing immune response of said individual towards the target, i.e. the pathogen.
WO 2018/051085 describes immunity linker molecules for the treatment of a disease or disorder mediated and/or caused by an infective agent, however, there is a need for alternative immunity linker molecules.
According to a first aspect of the invention, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof:
wherein:
L represents a cationic anti-microbial peptide selected from a moiety of formula (A):
wherein “—X1” represents the point of attachment of L to X1;
Z1 represents C1-10 alkyl;
Z2 represents —CH2—CH2—NH2, —CH2—NH2 or —CH2—OH;
S1 represents a bond or a spacer selected from a —(CH2)a— or —(CH2)b—(CH2—CH2—O)c—(CH2)d— group, wherein one to five of said —CH2— groups may optionally be substituted by a —C(O)NH— or —NHC(O)— group;
a represents an integer selected from 1 to 40;
b represents an integer selected from 0 to 25;
c represents an integer selected from 1 to 20;
d represents an integer selected from 1 to 15;
S2 represents a spacer selected from a —(CH2)e— or —(CH2)f—(CH2—CH2—O)g—(CH2)h— group,
wherein one to three of said —CH2— groups may optionally be substituted by a —C(O)NH— or —NHC(O)— group;
e represents an integer selected from 1 to 20;
f represents an integer selected from 1 to 10;
g represents an integer selected from 1 to 15;
h represents an integer selected from 1 to 5;
X1 represents a bond or —C(O)—;
Y1 and Y2 independently represent a bond, —O—, —S—, —NH—, —C(O)—, —NHC(O)— or —C(O)NH— group;
F represents a carbohydrate molecule capable of binding to a human anti-alpha-galactosyl antibody; and
R represents hydrogen, C1-6 alkyl, halogen or methoxy.
The invention comprises a conjugate of a cationic peptide (that specifically binds to bacteria) and the one or more units of the carbohydrate molecule capable of binding to a human anti-alpha-galactosyl antibody (i.e. alpha-Gal trisaccharide) connected via a linker. An example of a cationic peptide is polymyxin B (or polymyxin nonapeptide, colistin or a derivative thereof). This family of cationic peptides bind to lipid A on the bacterial cell surface and, when conjugated to alpha-gal linkers, will present alpha-Gal, resulting in anti-Gal antibody recruitment and cell killing. Resistance rates are likely to be low as lipid A is important in the survival of gram-negative bacteria. In fact, even polymyxin-resistant strains retain binding sites for cationic peptides and as such the peptide-alpha-Gal conjugate. Thus, the invention may retain efficacy even against these strains.
Clearly, new innovative therapies that work through novel mechanisms, and are not impacted by antibiotic resistance mechanisms, are particularly attractive. The solution provided by the invention, i.e. the combination of the broad-spectrum bacterial binding capability of a cationic peptide with the unique ability to specifically recruit naturally occurring anti-Gal antibodies to the bacterial surface, and re-direct these antibodies to promote complement activation, phagocytosis and killing is very attractive. The invention has the potential to provide a novel therapy for bacterial infections with broad-spectrum activity. Efficacy that is independent of antibiotic resistance mechanisms has the potential to be effective against multi-drug resistant strains. The invention may work as a single agent as well as with standard-of-care treatment to reduce the dose and duration of therapy.
WO 2018/051085 describes conjugates comprising a cationic peptide (that specifically binds to bacteria) and one or more units of a carbohydrate molecule capable of binding to a human anti-alpha-galactosyl antibody (i.e. alpha-Gal trisaccharide) connected via a linker which comprises a central biphenyl moiety. The inventors of the present invention have surprisingly identified that having a modified central biphenyl moiety with one of the phenyl rings bearing no Y1 or Y2 substituents provides a number of advantages over the conjugates described in WO 2018/051085. For example, the present invention provides a novel therapy with enhanced capability to recruit naturally occurring anti-Gal antibodies for re-direction to bacterial surfaces. Enhanced anti-Gal antibody recruitment to a bacterial surface has the potential to provide superior cell killing activity to those already known in the art. Such conjugates of the present invention comprise a central biphenyl moiety wherein both Y1 and Y2 are substituents of the same phenyl ring, which, in addition bears a pendant phenyl ring bearing no Y1 or Y2 substituents. Such pendant biphenyl conjugates demonstrate significantly improved anti-Gal antibody recruitment to multiple bacterial strains such as E. coli, P. aeruginosa and K. pneumoniae when compared to the exemplified conjugates described in WO 2018/051085. Examples of such improved conjugates will potentially trigger an increased immune response for enhanced bacterial killing.
In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is a compound of formula (I)a:
wherein F, S2, Y2, Y1, S1, X1, L and R are as defined herein.
In one embodiment, S1 represents a bond or a spacer selected from:
In a further embodiment, S1 represents a spacer selected from:
In a yet further embodiment, S1 represents a spacer which is:
It will be appreciated that a, b, c, d, e, f, g and h are selected to maintain a suitable linker length between groups F and L. Examples of suitable linker lengths between F and L range from about 5 Å to about 50 Å or more in length, about 6 Å to about 45 Å, about 7 Å to about 40 Å, about 8 Å to about 35 Å, about 9 Å to about 30 Å, about 10 Å to about 25 Å, about 11 Å to about 20 Å, about 12 Å to about 15 Å. Thus, in one embodiment, a, b, c, d, e, f, g and h represent a total integer of no more than 30, such as between 5 and 30, such as between 7 and 29.
In a further embodiment, a represents an integer selected from 1 to 35. In a further embodiment, a represents an integer selected from 1 to 10. In a further embodiment, a represents an integer selected from 2 to 13. In a yet further embodiment, a represents an integer selected from 2, 4, 6, 9 or 11. In a yet further embodiment, a represents an integer selected from 10 to 35. In a yet further embodiment, a represents an integer selected from 11 or 35. In a still yet further embodiment, a represents an integer selected from 11.
In one embodiment, b represents an integer selected from 0 to 24. In a further embodiment, b represents an integer selected from 0 to 3. In a further embodiment, b represents an integer selected from 0, 2 or 3. In a yet further embodiment, b represents an integer selected from 0 or 24. In a yet further embodiment, b represents an integer selected from 0.
In one embodiment, c represents an integer selected from 1 to 15. In a further embodiment, c represents an integer selected from 1 to 12. In a yet further embodiment, c represents an integer selected from 1 to 10. In a yet further embodiment, c represents an integer selected from 8.
In one embodiment, d represents an integer selected from 1 to 3. In a further embodiment, d represents an integer selected from 1 or 2. In a yet further embodiment, d represents an integer selected from 2.
In one embodiment, Y1 represents —C(O)NH— or —C(O)—. In a further embodiment, Y1 represents —C(O)NH—.
In one embodiment, S2 represents a spacer selected from:
In a further embodiment, S2 represents a spacer selected from —(CH2)e—, wherein one of said CH2— groups is optionally substituted by a —NHC(O)— group (such as —(CH2)3—NHCO—CH2—).
In one embodiment, e represents an integer selected from 1 to 17. In a further embodiment, e represents an integer selected from 1 to 10. In a further embodiment, e represents an integer selected from 4 to 10. In a yet further embodiment, e represents an integer selected from 4, 5 or 10. In a still yet further embodiment, e represents an integer selected from 5 or 17. In a still yet further embodiment, e represents an integer selected from 5.
In one embodiment, f represents an integer selected from 1 to 8. In a further embodiment, f represents an integer selected from 2 to 6. In a yet further embodiment, f represents an integer selected from 6. In a yet further embodiment, f represents an integer selected from 4.
In one embodiment, g represents an integer selected from 1 to 5. In a further embodiment, g represents an integer selected from 1 to 4. In a yet further embodiment, g represents an integer selected from 4.
In one embodiment, h represents an integer selected from 1 to 4. In a further embodiment, h represents an integer selected from 1 to 3. In a further embodiment, h represents an integer selected from 1 or 2. In a yet further embodiment, h represents an integer selected from 2. In a yet further embodiment, h represents an integer selected from 4.
In one embodiment, X1 represents —C(O)—.
In one embodiment, Y2 represents —O—.
In one embodiment, R represents hydrogen, methyl, t-butyl or chlorine. In a further embodiment, R represents hydrogen.
References herein to the term “carbohydrate molecule capable of binding to a human anti-alpha-galactosyl antibody” include sugar (i.e. carbohydrate) moieties capable of binding to an immune response component (i.e. an anti-alpha-galactosyl antibody) of said human and consequently eliciting an immune response in a human. Examples of such carbohydrate molecules include alpha-galactosyl compounds and modified derivatives thereof. Further examples of suitable carbohydrate molecules include the alpha-gal epitopes listed in US 2012/0003251 as being suitable for use in the selective targeting and killing of tumour cells, the epitopes of which are herein incorporated by reference. In one embodiment, F is selected from galactosyl-alpha-1,3-galactosyl-beta-1,4-N-acetylglucosamine, alpha1-3 galactobiose, alpha1-3-beta1-4-galactotriose or galilipentasaccharide.
In one particular embodiment, F has a structure as shown in one of the following formulae:
wherein S2 refers to the point of attachment to the S2 group.
In one particular embodiment, F has a structure as shown in the following formula:
wherein S2 refers to the point of attachment to the S2 group.
References herein to the term “binding moiety” refer to any suitable moiety which is capable of binding to a further component. The invention requires the binding moiety to be a cationic anti-microbial peptide linked to X1 by an amine.
In one embodiment, L represents a lipopeptide. In a further embodiment, the lipopeptide comprises polymyxin or a derivative thereof. Examples of suitable polymyxin and derivatives thereof are described in Velkov et al (2016) Future Med Chem 8(10), 1017-1025, the polymyxins and derivatives thereof are herein incorporated by reference. In one embodiment, the polymyxin or a derivative thereof is selected from Polymyxin B, Polymyxin B2, Polymyxin Nonapeptide, Colistin A, Colistin B, CB-182,204 (Cubist Pharmaceuticals), 5a (Pfizer), 5x (Pfizer), CA 14 (Cantab Anti-Infectives) CA824 (Cantab Anti-Infectives), NAB739 (Northern Antibiotics), NAB741 (Northern Antibiotics), NAB7061 (Northern Antibiotics), 38 (University of Queensland), FADDI-002 (Monash University), FADDI-100 (Monash University), or derivatives thereof. In a further embodiment, the polymyxin is Polymyxin B or derivative which has the following structure:
In one embodiment, Z1 represents octyl.
In one embodiment, Z2 represents —CH2—CH2—NH2 or —CH2—OH.
In a further embodiment, the Polymyxin B derivative is selected from one of the following structures (i) to (iii):
wherein X1 refers to the point of attachment to the X1 group.
In a yet further embodiment, the Polymyxin B derivative is selected from structures (i) and (iii).
It will be appreciated that the cationic anti-microbial peptides of the present invention will be configured to bind to a specific pathogen or infective agent.
In one embodiment, the invention provides a compound of formula (I) which comprises a compound of Examples 1-4 or a pharmaceutically acceptable salt thereof. In a further embodiment, the invention provides a compound of formula (I) which is the free base or trifluoroacetate salt of a compound of Examples 1-4. In a further embodiment, the invention provides a compound of formula (I) which comprises a compound of Examples 2 and 3 or a pharmaceutically acceptable salt thereof. In a further embodiment, the invention provides a compound of formula (I) which is the free base or trifluoroacetate salt of a compound of Examples 2 and 3.
A reference to a compound of formula (I) and sub-groups thereof also includes ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, isotopes and protected forms thereof, for example, as discussed below; preferably, the salts or tautomers or isomers or N-oxides or solvates thereof; and more preferably, the salts or tautomers or N-oxides or solvates thereof, even more preferably the salts or tautomers or solvates thereof. Hereinafter, compounds and their ionic forms, salts, solvates, isomers (including geometric and stereochemical isomers), tautomers, N-oxides, esters, isotopes and protected forms thereof as defined in any aspect of the invention (except intermediate compounds in chemical processes) are referred to as “compounds of the invention”.
Compounds of formula (I) can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulfonate and phosphate salts. All such salts are within the scope of this invention, and references to compounds of formula (I) include the salt forms of the compounds.
The salts of the present invention can be synthesized from the parent compound that contains a basic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the hydrogensulfate salt, also known as a hemisulfate salt.
Where the compounds of formula (I) contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of formula (I).
The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.
The salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.
Those skilled in the art of organic 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”. Pharmaceutically acceptable solvates of the compound of the invention are within the scope of the invention.
Compounds of formula (I) containing an amine function may also form N-oxides. A reference herein to a compound of formula (I) that contains an amine function also includes the N-oxide.
Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as dichloromethane.
It will be appreciated by those skilled in the art that certain protected derivatives of compounds of formula (I), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds of the invention which are pharmacologically active. Such derivatives may therefore be described as “prodrugs”. All such prodrugs of compounds of the invention are included within the scope of the invention. Examples of pro-drug functionality suitable for the compounds of the present invention are described in Drugs of Today, Volume 19, Number 9, 1983, pp 499-538 and in Topics in Chemistry, Chapter 31, pp 306-316 and in “Design of Prodrugs” by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as “pro-moieties”, for example as described by H. Bundgaard in “Design of Prodrugs” (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within compounds of the invention.
Also included within the scope of the compound and various salts of the invention are polymorphs thereof.
Compounds of formula (I) may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of formula (I) include all such forms. For the avoidance of doubt, where a compound can exist in one of several geometric isomeric or tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced by formula (I).
The present invention includes all pharmaceutically acceptable isotopically-labeled compounds of the invention, i.e. compounds of formula (I), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
Examples of isotopes suitable for inclusion in the compounds of the invention comprise isotopes of hydrogen, such as 2H (D) and 3H (T), carbon, such as 11C, 13C and 14C, fluorine, such as 18F, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O.
Certain isotopically-labelled compounds of formula (I), for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The compounds of formula (I) can also have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. 3H (T), and carbon-14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
Substitution with heavier isotopes such as deuterium, i.e. 2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.
Substitution with positron emitting isotopes, such as 11C, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.
Isotopically-labeled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.
Methods for the Preparation of Compounds of Formula (I)
In this section, as in all other sections of this application unless the context indicates otherwise, references to formula (I) also include all other sub-groups and examples thereof as defined herein.
The compounds pertaining to the invention described herein may be prepared in a stepwise synthetic sequence as illustrated in the Schemes below. Compounds of formula (I) can be prepared in accordance with synthetic methods well known to the skilled person. For example, one skilled in the art will appreciate that the chemical steps and choice of protecting groups may be managed in any order to enable synthetic success.
According to a further aspect of the invention there is provided a process for preparing a compound of formula (I) as hereinbefore defined which comprises:
(a) preparing a compound of formula (I) wherein Y1 represents —CONH— (i.e. a compound of formula (IA)) by reacting a compound of formula (II) with a compound of formula (III) followed by a suitable deprotection step:
wherein F, R, S2, Y2, S1, X1 and L are as defined hereinbefore and PG1 is a suitable peptide protecting group such as Dde, Cbz or Boc; or
(b) preparing a compound of formula (I) wherein Y1 represents —CONH—, S1 is terminated with —C(O)— and X1 is NH (i.e. a compound of formula (IB)) by reacting a compound of formula (V) with a compound of formula (IV) followed by a suitable deprotection step:
wherein F, R, S2, Y2, S1 and L are as defined hereinbefore and PG1 is a suitable peptide protecting group such as Dde, Cbz or Boc; or
(c) interconversion of a compound of formula (I) or protected derivative thereof to a further compound of formula (I) or protected derivative thereof.
Step (i) in processes (a) and (b) typically comprises an amide bond formation reaction, which typically comprises activation of the carboxylic acid with either phosphate containing reagents, triazine based reagents or carbodiimide containing reagents in the presence of an organic base in an organic solvent. Preferred conditions comprise HATU ((1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidehexafluorophosphate) with diispropylethylamine or triethylamine in DMF.
Step (ii) in processes (a) and (b) typically comprises any suitable deprotection reaction, the conditions of which will depend upon the nature of the protecting group. When the protecting group comprises Dde, such a deprotection will typically comprise the use of hydrazine in DMF. When the protecting group comprises Cbz such a deprotection will typically comprise hydrogenation over a suitable catalyst such as palladium on carbon. When the protecting group comprises tertbutoxycarbonyl, such a deprotection will be acid mediated and will typically comprise TFA in DCM.
Process (c) typically comprises interconversion procedures known by one skilled in the art. For example, in compounds of formula (I), a first substituent may be converted by methods known by one skilled in the art into a second, alternative substituent. A wide range of well known functional group interconversions are known by a person skilled in the art for converting a precursor compound to a compound of formula (I) and are described in Advanced Organic Chemistry by Jerry March, 4th Edition, John Wiley & Sons, 1992. For example possible metal catalysed functionalisations such as using organo-tin reagents (the Stille reaction), Grignard reagents and reactions with nitrogen nucleophiles are described in ‘Palladium Reagents and Catalysts’ [Jiro Tsuji, Wiley, ISBN 0-470-85032-9] and Handbook of OrganoPalladium Chemistry for Organic Synthesis [Volume 1, Edited by Ei-ichi Negishi, Wiley, ISBN 0-471-31506-0].
If appropriate, the reactions previously described in processes (a) and (b) are followed or preceded by one or more reactions known to the skilled of the art and are performed in an appropriate order to achieve the requisite substitutions on R, S2, Y2, S1, X1, Y1, L and F defined above to afford other compounds of formula (I). Non-limiting examples of such reactions whose conditions can be found in the literature include:
Compounds of formula (V) may be prepared according to the methods described in Scheme 1 from compounds of formula (II) and (VI), according to process steps (i) and (ii) as described hereinbefore.
wherein F, R, S2, Y2, and S1 are as defined hereinbefore and PG2 is a protecting group comprising methyl.
Wherein step ii) comprises deprotection of a methyl ester, typical phase-transfer conditions are employed. Preferred conditions comprise triethylamine in water at room temperature for three hours.
Compounds of formula (II) may be prepared according to the methods described in Scheme 2 from compounds of formula (VII) and (VIII), according to process steps (i) and (ii) as described hereinbefore.
wherein F, R, S2, Y2, are as defined hereinbefore and PG2 is a protecting group comprising benzyl.
Wherein step ii) comprises deprotection of a benzyl ester, typical palladium-mediated conditions are employed. Preferred conditions comprise 10% Pd/C in MeOH and DMF under a balloon of hydrogen at room temperature for overnight.
Compounds of formula (VIII) may be prepared according to the methods described in Scheme 3 from compounds of formula (IX) and (X), according to process step (iii), an alkylation reaction followed by deprotection step (ii) as described hereinbefore.
wherein F, R, S2, Y2, are as defined hereinbefore, PG3 is a protecting group comprising tert-butyl and Hal is a halogen comprising I, Br or Cl;
Step (iii) typically comprises alkylation conditions with compounds of formula (IX) in an inorganic base in a polar organic solvent at room temperature. Preferred conditions comprise potassium carbonate in DMF.
Wherein step ii) comprises deprotection of a tert-butyl ester, typical acid-mediated conditions are employed. Preferred conditions comprise TFA in DCM at room temperature for 4 hours.
Compounds of formula (X) may be prepared by employment of a Suzuki reaction to construct the biphenyl unit. Preferred conditions comprise tetrakistriphenyl phosphine palladium (0) with sodium carbonate in dioxane and water at 100° C. If suitable required protecting groups are employed, such as TBS, such protecting groups may be deprotected using a fluoride mediated deprotection. Preferred conditions comprise TBAF in THF at room temperature.
Compounds of formula (III), (IV), (VI), (VII) and (IX) are either commercially available, prepared according to the methods described herein or prepared according to the literature.
Pharmaceutical Compositions
While it is possible for the compound of formula (I) to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation).
Thus, according to a further aspect, the invention provides a pharmaceutical composition, and methods of making a pharmaceutical composition comprising (e.g admixing) at least one compound of the invention where L represents a cationic anti-microbial peptide, together with one or more pharmaceutically acceptable excipients and optionally other therapeutic or prophylactic agents, as described herein.
The pharmaceutically acceptable excipient(s) can be selected from, for example, carriers (e.g. a solid, liquid or semi-solid carrier), adjuvants, diluents, fillers or bulking agents, granulating agents, coating agents, release-controlling agents, binding agents, disintegrants, lubricating agents, preservatives, antioxidants, buffering agents, suspending agents, thickening agents, flavouring agents, sweeteners, taste masking agents, stabilisers or any other excipients conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical compositions are set out in more detail below.
The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity (i.e. generally recognised as safe (GRAS)), irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Pharmaceutical compositions containing compounds of the invention can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA.
The pharmaceutical compositions can be in any form suitable for parenteral, intranasal, intrabronchial, sublingual, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump or syringe driver.
Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, surface active agents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents (R. G. Strickly, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21(2) 2004, p 201-230).
The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules, vials and prefilled syringes, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
The pharmaceutical formulation can be prepared by lyophilising a compound of the invention. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
Pharmaceutical compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as sunflower oil, safflower oil, corn oil or olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of thickening or coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various anti-bacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents to adjust tonicity such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and gelatin.
In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion. For intravenous or subcutaneous administration, the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.
In another preferred embodiment, the pharmaceutical composition is in a form suitable for subcutaneous (s.c.) administration.
The compound of the invention may be formulated with a carrier and administered in the form of nanoparticles, the increased surface area of the nanoparticles assisting their absorption. In addition, nanoparticles offer the possibility of direct penetration into the cell. Nanoparticle drug delivery systems are described in “Nanoparticle Technology for Drug Delivery”, edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published 13 Mar. 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and in Sinha et al., Mol. Cancer Ther. August 1, (2006) 5, 1909.
The pharmaceutical compositions typically comprise from approximately 1% (w/w) to approximately 95% (w/w) active ingredient and from 99% (w/w) to 5% (w/w) of a pharmaceutically acceptable excipient or combination of excipients. Preferably, the compositions comprise from approximately 20% (w/w) to approximately 90% (w/w) active ingredient and from 80% (w/w) to 10% of a pharmaceutically acceptable excipient or combination of excipients. The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, pre-filled syringes, dragées, tablets or capsules.
The pharmaceutically acceptable excipient(s) can be selected according to the desired physical form of the formulation and can, for example, be selected from diluents (e.g solid diluents such as fillers or bulking agents; and liquid diluents such as solvents and co-solvents), disintegrants, buffering agents, lubricants, flow aids, release controlling (e.g. release retarding or delaying polymers or waxes) agents, binders, granulating agents, pigments, plasticizers, antioxidants, preservatives, flavouring agents, taste masking agents, tonicity adjusting agents and coating agents.
The skilled person will have the expertise to select the appropriate amounts of ingredients for use in the formulations. For example tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% flow aids and/or 0-99% (w/w) fillers/or bulking agents (depending on drug dose). They may also contain 0-10% (w/w) polymer binders, 0-5% (w/w) antioxidants, 0-5% (w/w) pigments. Slow release tablets would in addition contain 0-99% (w/w) release-controlling (e.g. delaying) polymers (depending on dose). The film coats of the tablet or capsule typically contain 0-10% (w/w) polymers, 0-3% (w/w) pigments, and/or 0-2% (w/w) plasticizers.
Parenteral or subcutaneous formulations typically contain 0-20% (w/w) buffers, 0-50% (w/w) cosolvents, and/or 0-99% (w/w) Water for Injection (WFI) (depending on dose and if freeze dried). Formulations for intramuscular depots may also contain 0-99% (w/w) oils.
The compounds of the invention can also be formulated as solid dispersions. Solid dispersions are homogeneous extremely fine disperse phases of two or more solids. Solid solutions (molecularly disperse systems), one type of solid dispersion, are well known for use in pharmaceutical technology (see (Chiou and Riegelman, J. Pharm. Sci., 60, 1281-1300 (1971)) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.
The pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions. One example of a patient pack includes a prefilled syringe. Such pre-filled syringes already contain the drug substance. The front end portion of a pre-filled syringe to which a needle is to be attached is sealed with a nozzle cap. Prior to injection, the nozzle cap is removed from the front end portion and a needle is attached thereto. A gasket is then slid by pushing a plunger rod toward the front end portion so that the drug is expelled.
Compositions for nasal delivery include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
Examples of formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound. Solutions of the active compound may also be used for rectal administration.
Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.
The compound of the invention will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within these ranges, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).
The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.
Therapeutic Uses
According to a further aspect of the invention, there is provided a compound of formula (I) as defined herein for use in therapy.
According to a further aspect of the invention, there is provided a compound of formula (I) as defined herein for use in the treatment of a disease or disorder mediated and/or caused by an infective agent.
According to a further aspect of the invention, there is provided the use of a compound of formula (I) as defined herein in the manufacture of a medicament for use in the treatment of a disease or disorder mediated and/or caused by an infective agent.
According to a further aspect of the invention, there is provided a method of treating a disease or disorder mediated and/or caused by an infective agent which comprises administering to an individual in need thereof a compound of formula (I) as defined herein.
Examples of infective agents include any pathogen such as a bacteria, fungus, parasite or virus. Thus, in one embodiment, the disease or disorder mediated by and/or caused by an infective agent is bacterial infection.
Examples of such as bacterial infection include infection by the following bacteria: Staphylococcus sp. such as Staphylococcus aureus (including methicillin resistant Staphylococcus aureus (MRSA)), Clostridia sp (e.g. Clostridium difficile, Clostridium tetani and Clostridium botulinum), Enterobacter species, Mycobacterium tuberculosis, Shigella sp. such as Shigella dysenteriae, Campylobacter sp. such as Campylobacter jejuni, Enterococcus sp. such as Enterococcus faecalis, Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Streptococcal species, Salmonella thyphimurim, Salmonella enterica, Chlamydia species, Treponema pallidum, Neisseria gonorrhoeae, Borrelia burgdorferi, Vibrio cholerae, Corynebacterium diphtheriae, Helicobacter pylori, Gram-negative pathogens, such as Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli (and including strains that are resistant to one or more classes of anti-biotics, especially multi-drug resistant (MDR) strains).
The compound of the invention is generally administered to a subject in need of such administration, for example a human or animal patient, preferably a human.
The compound of the invention will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic. However, in certain situations (for example in the case of life threatening diseases), the benefits of administering a compound of the invention may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer a compound of the invention in amounts that are associated with a degree of toxicity.
The compound of the invention may be administered over a prolonged term (i.e. chronic administration) to maintain beneficial therapeutic effects or may be administered for a short period only (i.e. acute administration). Alternatively they may be administered in a continuous manner or in a manner that provides intermittent dosing (e.g. a pulsatile manner).
A typical daily dose of the compound of the invention can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) per kilogram of bodyweight although higher or lower doses may be administered where required. The compound of the invention can either be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example. Alternatively, the compound of the invention can be administered by infusion, multiple times per day.
The compound of the invention may be administered in a range of doses, for example 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, e.g. 2 to 200 mg or 10 to 1000 mg, particular examples of doses including 10, 20, 50 and 80 mg. The compound of the invention may be administered once or more than once each day. The compound of the invention can be administered continuously (i.e. taken every day without a break for the duration of the treatment regimen). Alternatively, the compound of the invention can be administered intermittently (i.e. taken continuously for a given period such as a week, then discontinued for a period such as a week and then taken continuously for another period such as a week and so on throughout the duration of the treatment regimen). Examples of treatment regimens involving intermittent administration include regimens wherein administration is in cycles of one week on, one week off; or two weeks on, one week off; or three weeks on, one week off; or two weeks on, two weeks off; or four weeks on two weeks off; or one week on three weeks off—for one or more cycles, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles.
In one particular dosing schedule, a patient will be given an infusion of a compound of the invention for periods of one hour daily for up to ten days in particular up to five days for one week, and the treatment repeated at a desired interval such as two to four weeks, in particular every three weeks.
More particularly, a patient may be given an infusion of a compound of the invention for periods of one hour daily for 5 days and the treatment repeated every three weeks.
In another particular dosing schedule, a patient is given an infusion over 30 minutes to 1 hour followed by maintenance infusions of variable duration, for example 1 to 5 hours, e.g. 3 hours.
In a further particular dosing schedule, a patient is given a continuous infusion for a period of 12 hours to 5 days, and in particular a continuous infusion of 24 hours to 72 hours.
Ultimately, however, the quantity of compound of the invention administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.
It will be appreciated that the compound of the invention can be used as a single agent or in combination with other therapeutic agents. Combination experiments can be performed, for example, as described in Chou T C, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regulat 1984; 22: 27-55.
Where the compound of the invention is administered in combination therapy with one, two, three, four or more other therapeutic agents (preferably one or two, more preferably one), the agents can be administered simultaneously or sequentially. In the latter case, the two or more agents will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. When administered sequentially, they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s). These dosages may be administered for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days.
It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular other medicinal agent and compound of the invention being administered, their route of administration, the particular tumour being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
The weight ratio of the compound of the invention and the one or more other therapeutic agent(s) when given as a combination may be determined by the person skilled in the art. Said ratio and the exact dosage and frequency of administration depends on the particular compound of the invention and the other therapeutic agent(s) used, the particular condition being treated, the severity of the condition being treated, the age, weight, gender, diet, time of administration and general physical condition of the particular patient, the mode of administration as well as other medication the individual may be taking, as is well known to those skilled in the art. Furthermore, it is evident that the effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compound of present invention. A particular weight ratio for the compound of the invention and another therapeutic agent may range from 1/10 to 10/1, more in particular from 1/5 to 5/1, even more in particular from 1/3 to 3/1.
The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples. Compounds are named using an automated naming package (ChemDraw) or are as named by the chemical supplier.
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 synthesised according to the step in the description given.
HPLC (Method A):
Instrumentation: Agilent 1290 Infinity (Binary pump, PDA)
Column: Waters XBridge C18 5 μm 3.0×30 mm (Part no. 186003111)
Conditions:
Solvent: A=0.1% TFA in water; B=0.1% TFA in acetonitrile
Column temperature: 40° C., injection volume: 1-20 μL
LCMS (Method B)
Instrumentation: LCMS Agilent 1100 (quaternary pump)
Mass spectrometer: Waters Micromass ZQ
Column: Waters XBridge C18 4.6×50 mm, 5 μm.
Conditions:
Solvent: A=acetonitrile, B=10 mm ammonium formate in water
Column temperature: 25° C., injection volume: 5 μL
UPLC (Method C):
Instrumentation: Waters Acquity UPLC H-Class (Quaternary pump, PDA, ELSD)
Mass spectrometer: Waters Acquity QDA
Column: CSH analytical column C18, 1.7 μm, 2.1×50 mm (cat. 186005296),
Conditions:
Wherein A=0.1% formic acid in water, B=0.1% formic acid in MeCN
Column temperature: 40° C., Injection volume: 0.5 μL
NMR
NMR details were recorded on an Oxford Instruments AS400.
MS
Wherein MS data is reported for large molecular weight compounds, a mass-to-charge ratio (m/z) is typically observed.
Abbreviations
Wherein the following abbreviations have been used, the following meanings apply:
Abu is aminobutyric acid;
Alloc is allyloxycarbonyl;
Boc is tert-butyloxycarbonyl;
CDCl3 is deuterochloroform;
CTC resin is chlorotrityl chloride resin;
Dab is 2,4-diaminobutyric acid;
Dap is 2,3-diaminopropionic acid;
DCM is dichloromethane;
Dde is (1,(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-ethyl);
DIPEA/DIEA is diisopropylethylamine;
DMF is dimethylformamide;
DMSO is dimethylsulfoxide;
ES is electrospray ionisation technique;
Eq is equivalents;
EtOAc is ethyl acetate;
Fmoc is 9-fluorenylmethoxycarbonyl;
g is gram;
Gly is glycine;
HATU is O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate;
HCl is hydrochloric acid;
HOBt is hydroxybenzotriazole;
HPLC is high performance liquid chromatography;
K2CO3 is potassium carbonate;
L is litre;
LCMS is liquid chromatography mass spectrometry;
m is multiplet;
M is molar;
MeCN is acetonitrile;
MeOH is methanol;
mg is milligram;
MgSO4 is magnesium sulfate;
MHz is megaHertz,
mins is minutes;
mL is millilitre;
mm is millimetre;
mmol is millimole;
MS is mass spectrometry;
m/z is mass to charge ratio;
nm is nanometre;
NMM is N-methylmorpholine;
NMR is nuclear magnetic resonance;
Pd(PPh3)2Cl2 is palladium(II)bis(triphenylphosphine) dichloride;
Phe is phenylalanine;
PhSiH3 is phenylsilane;
ppm is parts per million;
Rt is retention time;
s is singlet;
t is triplet;
tBu is tert-butyl;
TBME is tert-butylmethyl ether;
TBTU is O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate;
TEA is triethylamine;
Thr is threonine;
TIS is triisopropylsilane;
TFA is trifluoroacetic acid;
μL is microliter;
μm is micrometre;
UPLC is ultra performance liquid chromatography; and
v is volume.
Wherein alpha-Gal is referred to, the following intermediate applies:
3-(((2R,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4S,5S,6R)-3,5-dihydroxy-6-(hydroxymethyl)-4-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)amine
This intermediate may be prepared according to the methods described by Bovin et al (Mendeleev Communications (2002), (4), 143-145).
Synthesis of Peptide Intermediates
Peptide scaffolds were constructed according to standard Solid Phase Peptide Synthesis (SPPS) using appropriately protected amino acids and CTC resin. The scaffolds were cyclised at an appropriate place in the synthesis. All protected amino acids and linker starting materials are commercially available or prepared according to the references cited herein.
Purification Conditions for Preparative HPLC of Scaffolds:
Dissolution solvent: water/MeCN
Instrument: Gilson GX-215
Mobile phase A: 0.075% TFA in water
Mobile phase B: MeCN
Gradient: see individual experimental
Column: Luna 25×200 mm, C18 10 μm, 110 Å+Gemini 150×30 mm, C18 5 μm, 110 Å
Flow rate: 20 mL/min
Wavelength: 220/254 nm
Room temperature
Peptide Analytical HPLC Method A
Instrumentation: Agilent 1200
Column: Gemini-NX C18 5 um, 110 Å, 150×4.6 mm
Conditions:
Solvent: A=0.1% TFA in water; B=0.1% TFA in acetonitrile
Column temperature: 50° C.
H2N-Ahx-Ahx-[L-octylgly]-Dab(Boc)-Thr(OH)-Dab(Boc)-Dab*-Dab(Boc)-[D-Phe]-Leu-Dab(Boc)-Dab(Boc)-Thr)OH*
The peptide chain was elongated on CTC resin commencing with Fmoc-Dab(Boc)-O-CTC-Resin.
20% piperidine in DMF (for de-blocking) was added with mixing for 30 minutes. The reaction was drained and washed with DMF (×5). Construction of the desired peptide sequence was continued using HATU (1.9-2.85 eq) and DIPEA (4-6.0 eq) in DMF (10 mL) followed by 20% piperidine in DMF for each amino acid to afford H2N-Ahx-Ahx-[L-octylGly]-Dab(Boc)-Thr(OH)-Dab(Boc)-Dab(Dde)-Dab(Boc)-[D-Phe]-Leu-Dab(Boc)-O-CTC-resin. The peptide was then treated with a mixture of DCM and DIPEA (4 eq) with Cbz-Cl (2 eq).
At this point the resin was treated with 3% hydrazine hydrate in DMF to effect Dde deprotection. The resin was washed with DMF (×5) and the peptide was further elongated as above with the required remaining amino acids. The peptide was treated with 1% TFA/DCM (2×50 mL) for 2 minutes and adjusted to pH=7 with DIPEA and diluted with DCM. TBTU (2 eq) and HOBt (2 eq) were added followed by DIPEA (2 eq), and the mixture was stirred for 1 hour to effect cyclisation. The reaction was washed with 5% aqueous HCl and concentrated in vacuo to afford Cbz-Abx-Abx-[L-octylGly]-Dab(Boc)-Thr(OH)-Dab(Boc)-Dab*-Dab(Boc)-[D-Phe]-Leu-Dab(Boc)-Dab(Boc)-Thr(OH)*.
The crude peptide was treated with 4:1 DMF:MeOH (50 mL) and Pd(OH)2/C (50%, 2 g) was added under nitrogen. The suspension was purged with hydrogen several times and stirred under 15 psi hydrogen for 4 hours at 35° C. The crude peptide was purified using preparative HPLC as described above using a gradient of between 30-70% MeCN in water (with 0.075% TFA) over 60 minutes to afford the title compound.
HPLC (Method A): Rt=9.54 minutes, ES+ MS m/z 980 [M+2]/2 and 930 [M−Boc+2]/2; theoretical mass: 1958
The peptide was synthesized using standard Fmoc chemistry commencing with Fmoc-Dab(Boc)-CTC Resin.
DMF was added to a vessel containing Fmoc-Dab(Boc)-CTC Resin (5.0 mmol, 8.33 g, 0.6 mmol/g) with N2 bubbling for 2 hours.
3% NH2NH2H2O/DMF was used for removal of Dde and the resin was washed with DMF 5 times. The final two amino acids (below) were added and 20% piperidine in DMF was used for the final Fmoc deprotection.
The resin was washed with DMF 5 times followed by MeOH 3 times, then dried under N2.
Peptide Cleavage and Purification:
HPLC (Method A): Rt=12.758 minutes, ES+ MS m/z 816.3 [M−Boc+2]/2; theoretical mass: 1733.1
The peptide was synthesized using standard Fmoc chemistry commencing with Fmoc-Dab(Boc)-CTC Resin.
DMF was added to a vessel containing Fmoc-Dab(Boc)-CTC Resin (3.0 mmol, 5.0 g, 0.6 mmol/g) with N2 bubbling for 2 hours.
3% NH2NH2H2O/DMF was used for the removal of Dde and the resin was washed with DMF 5 times. The final two amino acids (below) were added and 20% piperidine in DMF was used for the final Fmoc deprotection.
The resin was washed with DMF 5 times followed by MeOH 3 times, then dried under N2.
Peptide Cleavage and Purification:
HPLC (Method A): Rt=8.64 minutes, ES+ MS m/z 760.4 [M−Boc+2]/2; theoretical mass: 1620.0
The title compound was prepared according to the method described for Preparation 2, substituting in Fmoc-Dap(Boc)-OH at amino acid #5 in the table. The crude peptide was purified using preparative HPLC as described above using a gradient of between 45-75% MeCN in water (with 0.075% TFA) over 60 minutes.
HPLC (Method A): Rt=9.067 minutes, ES+ MS m/z 810.4 [M−Boc+2]/2; theoretical mass: 1719.1
To methyl 1-{[(tert-butoxy)carbonyl]amino}-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate (Preparation 6, 223 mg, 0.40 mmol) in dioxane (1.0 mL) was added 4M HCl in Dioxane (1.0 mL) and the reaction mixture stirred for 2 hours. The solvent was concentrated in vacuo to afford an orange oil that was used directly in the next step.
UPLC (Method C): Rt=1.84 minutes, ES+ MS m/z 456.4 [M+H]+
To 1-{[(tert-butoxy)carbonyl]amino}-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid (1.01 g, 1.86 mmol) dissolved in THF (20 mL) was added K2CO3 (1.03 g, 7.46 mmol), iodomethane (1.59 g, 11.19 mmol) and the reaction heated at 50° C. for 18 hours. The reaction mixture was filtered through a syringe filter washing through with EtOAc (5 mL). The solution was concentrated in vacuo to afford the title compound as a brown oil (1.10 g>100%).
UPLC (Method C): Rt=2.75 minutes, ES+ MS m/z 456.4 [M+H−Boc]+
1HNMR (396 MHz, CDCl3): δ ppm 3.74 (2H, t), 3.67 (3H, s), 3.66-3.63 (20H, m), 3.63-3.61 (5H, m), 3.53 (2H, t), 3.30 (2H, q), 2.60 (2H, t), 1.77 (2H, s), 1.42 (9H, s)
Preparation 7
1-[(5-{[(3-{[(2R,5S)-5-{[(2S,5S)-3,5-Dihydroxy-6-(hydroxymethyl)-4-{[(2R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3-acetamido-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}propyl)carbamoyl]methoxy}-[1,1′-biphenyl]-3-yl)formamido]-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid
Methyl 1-[(5-{[(3-{[(2R,5R)-5-{[(2S,5S)-3,5-dihydroxy-6-(hydroxymethyl)-4-{[(2S,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3-acetamido-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}propyl)carbamoyl]methoxy}-[1,1′-biphenyl]-3-yl)formamido]-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate (Preparation 8, 150 mg, 0.116 mmol) was dissolved in water:TEA (10 mL, 10:1) and stirred at room temperature for 3 hours. The reaction mixture was concentrated in vacuo and freeze-dried to afford the title compound as a white solid (146 mg, 92%).
LCMS (Method B): Rt=1.78 minutes, ES+ MS m/z 1278.8 [M−H]−
To 5-{[(3-{[(2R,5S)-5-{[(2S,5S)-3,5-dihydroxy-6-(hydroxymethyl)-4-{[(2R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3-acetamido-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}propyl)carbamoyl]methoxy}-[1,1′-biphenyl]-3-carboxylic acid (Preparation 9, 117 mg, 0.137 mmol) dissolved in DMF (4.0 mL) was added TEA (77 μL, 0.552 mmol) and methyl 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate hydrochloride (101 mg, 0.205 mmol) dissolved in DMF (2.0 mL). HATU (78 mg, 0.205 mmol) was added and the reaction stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo and purified using reverse phase chromatography eluting with 5-50% MeCN in water to afford the title compound as a white solid (150 mg, 85%).
LCMS (Method B): Rt=2.06 minutes, ES+ MS m/z 1292.9 [M−H]−
To benzyl 5-{[(3-{[(2R,5S)-5-{[(2S,5S)-3,5-dihydroxy-6-(hydroxymethyl)-4-{[(2R,5R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}oxan-2-yl]oxy}-3-acetamido-4-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}propyl)carbamoyl]methoxy}-[1,1′-biphenyl]-carboxylate (Preparation 10, 35 mg, 0.037 mmol) dissolved in MeOH:DMF (3 mL, 5:1) was added 10% palladium on carbon (3.5 mg). The reaction was degassed and stirred under an atmosphere of hydrogen (balloon) overnight. The reaction was filtered through Hyflo and the solution concentrated in vacuo to afford the title compound as a white solid (42 mg>100%).
LCMS (Method B): Rt=1.80 minutes, ES+ MS m/z 857.6 [M+H]+
To 2-({5-[(benzyloxy)carbonyl]-[1,1′-biphenyl]-3-yl}oxy)acetic acid (Preparation 11, 20 mg, 0.055 mmol) in DMF (1.0 mL) and TEA (5 drops), was added alpha-Gal (37 mg, 0.061 mmol) and a few drops of DMSO. HATU (25 mg, 0.066 mmol) was added and the reaction mixture was stirred at room temperature for 3 hours before concentrating in vacuo. The residue was purified using reverse phase column chromatography eluting with 5-50% MeCN in water to afford title compound as a white solid (35 mg, 67%).
LCMS (Method B): Rt=2.46 minutes, ES+ MS m/z 947.7 [M+H]+
Benzyl 5-hydroxy-[1,1′-biphenyl]-3-carboxylate (Preparation 12, 18 mg, 2.19 mmol) was dissolved in TFA:DCM (4 mL, 3:8) and stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and azeotroped with dioxane. The residue was dissolved in EtOAc (5 mL) and concentrated in vacuo to afford the title compound as an orange oil (828 mg, >100%).
LCMS (Method B): Rt=2.36 minutes, ES− MS m/z 361.1 [M−H]−
1HNMR (400 MHz, CDCl3): δ ppm 7.98-7.97 (1H, t), 7.60-7.35 (12H, m), 5.39 (2H, s), 4.78 (2H, s).
To benzyl-3-bromo-5-hydroxybenzoate (Preparation 13, 687 mg, 2.26 mmol) in DMF (13 mL) was added K2CO3 (343 mg, 2.48 mmol). After stirring for five minutes, t-butyl bromoacetate (0.37 mL, 2.48 mmol) was added dropwise over 5 minutes. The reaction was stirred at room temperature for 60 hours. The reaction mixture was concentrated in vacuo, diluted with water (75 mL) and extracted with EtOAc (2×50 mL). The combined organic extracts were washed with water (75 mL) and brine (75 mL), and dried (MgSO4) before concentrating in vacuo to afford the title compound as a yellow oil (918 mg, 97%).
LCMS (Method B): Rt=3.85 minutes, ES+ MS m/z 436.2 [M+NH4]+
1HNMR (400 MHz, CDCl3): δ ppm 7.96-7.95 (1H, t), 7.60-7.35 (12H, m), 5.38 (2H, s), 4.60 (2H, s), 1.48 (9H, s).
Benzyl-3-bromo-5-hydroxybenzoate (1.5 g, 4.88 mmol), phenylboronic acid (0.71 g, 5.86 mmol) and sodium carbonate (1.81 g, 17.1 mmol) were suspended in dioxane (30 mL) and water (10 mL). This was degassed before adding Pd(PPh3)4 (0.57 g, 0.49 mmol). The mixture was heated at 100° C. for 5 hours. The reaction was cooled and diluted with 2M HCl (aq) (60 mL) and EtOAc (60 mL). The EtOAc layer was separated and the aqueous layer extracted with EtOAc (2×60 mL). The combined organic extracts were washed with brine (2×150 mL), dried (MgSO4) and concentrated in vacuo. The residue was purified using silica gel column chromatography eluting with 0-50% EtOAc in heptane to afford the title compound as a yellow oil (687 mg, 46%).
LCMS (Method B): Rt=3.18 minutes, ES+ MS m/z 305.1
1HNMR (400 MHz, CDCl3): δ ppm 7.89 (1H, s), 7.59-7.28 (12H, m), 5.38 (2H, s).
The following Examples 1-4 were isolated as TFA salts.
To a solution of Preparation 9 (10 mg, 0.012 mmol) in DMF (0.5 mL) was added triethylamine (3 drops) followed by a solution of Preparation 1 (Boc-Scaffold 1, 29 mg, 0.014 mmol) in DMF (0.5 mL) and HATU (6.2 mg, 0.016 mmol). The reaction was stirred at room temperature for 18 hours. Toluene (5 mL) was added and the reaction concentrated in vacuo. The residue was purified using reverse phase chromatography eluting with 5-90% MeCN in water. The product (13 mg, 0.005 mmol) was dissolved in DCM (1.2 mL) and treated with TFA (0.8 mL) for 5 minutes at room temperature. The reaction was quenched by addition to ice-cold TBME and concentrated in vacuo. The residue was purified using reverse phase chromatography eluting with 3-40% MeCN in 0.1% TFA in water to afford the title compound, 7.6 mg.
HPLC (Method A): Rt=2.32 minutes
UP-LCMS (Method C): Rt=2.06 minutes; ES+ MS m/z 1149.9 [M+2]/2; theoretical mass: 2297.7
The title compound was prepared according to the method described for Example 1 using Preparation 2 (Boc-Scaffold 2) and Preparation 7 and wherein the intermediate was purified with 10-90% MeCN in water.
HPLC (Method A): Rt=2.32 minutes
UP-LCMS (Method C): Rt=2.10 minutes; ES+ MS m/z 832.8 [M+3]/3; theoretical mass:
2494.9
The title compound was prepared according to the method described for Example 1 using Preparation 3 (Boc-Scaffold 3) and Preparation 7 and wherein the intermediate was purified with 10-80% MeCN in water and the title compound was purified with 1-40% MeCN in 0.1% TFA in water.
HPLC (Method A): Rt=4.86 minutes
UP-LCMS (Method C): Rt=2.10 minutes; ES+ MS m/z 828.3 [M+3]/3; theoretical mass: 2481.8
The title compound was prepared according to the method described for Example 1 using Preparation 4 (Boc-Scaffold 4) and Preparation 7 and wherein the intermediate was purified with 10-90% MeCN in water.
HPLC (Method A): Rt=2.85 minutes
UP-LCMS (Method C): Rt=2.21 minutes; ES+ MS m/z 828.1 [M+3]/3; theoretical mass: 2480.8
Reference Compounds
Reference Example A
Reference Compound A may be prepared according to the method described for Example 1 and is exemplified in WO 2018/051085 (Example 6).
Reference Example B
Reference Compound B may be prepared according to the method described for Example 1 and is exemplified in WO 2018/051085 (Example 17).
Reference Example C
Reference Compound C may be prepared according to the method described for Example 1.
Reference Example D
Reference Compound C may be prepared according to the method described for Example 1.
Biological Assays
Flow Cytometric Antibody Recruitment Assay Using an Anti-Galα1-3Galβ1-4GlcNAc (Anti-Gal) Antibody
Flow cytometry was used to demonstrate binding of L (as a cationic anti-microbial peptide selected from a moiety of formula (A)) to Gram negative bacteria and the interaction of F (the alpha-Gal carbohydrate moiety of the anti-microbial peptides) with an anti-Gal IgM antibody. A secondary fluorescently labelled anti-IgM antibody was used to detect the anti-Gal antibody binding.
Method
The assays were carried out in polystyrene 96-well U bottom plates (Costar). Frozen stocks of bacteria in mid-exponential phase were thawed, centrifuged and resuspended in LB broth, Miller (Fisher BP1426-500) at OD595=0.2. The bacteria was grown to mid-exponential phase (OD595=0.4), washed once with Hank's Balanced Salt Solution with calcium and magnesium (HBSS+/+) and then resuspended in HBSS+/+ at a bacterial D595=0.6. Bacteria was diluted ⅙ and placed in the 96-well U bottom plate. The bacteria were then incubated for 45 minutes with the compounds of Example 1-4 and reference compounds A-D (see Table 1) at 20 μM concentrations, Polymyxin B at 20 μM concentrations, or buffer alone (vehicle control), at room temperature and shaking at 450 rpm. The bacteria were then washed three times with 200 μL HBSS+/+, prior to adding 50 μL of the mouse/human chimeric anti-Gal IgM antibody M86 (Absolute Antibody Ab00532) at a final concentration of 25 μg/mL in HBSS+/+. The samples were incubated for 1 hour at room temperature shaking at 450 rpm. The bacteria were washed three times as above, prior to adding 100 μL of a FITC-labeled anti-human IgM secondary antibody (Biolegend 314506) at 10 μg/mL in HBSS+/+ and incubating at room temperature for 1 hour at 450 rpm. After three final washes with 200 μL HBSS+/+, the bacteria were resuspended in 200 μL HBSS+/+ and evaluated for anti-Gal antibody binding on a Cytoflex flow cytometer (Beckman Coulter). 50,000 counts of bacterial particles were sampled and the median fluorescent shift was recorded in the FITC-A channel. Data from all samples were analysed using Kaluza software (Beckman Coulter). All samples were run in technical triplicates and biological experiments repeated as indicated in the table.
Table 1 and
Table 1 demonstrates significantly improved anti-Gal antibody recruitment to multiple bacterial strains such as E. coli, P. aeruginosa and K. pneumoniae for Examples 1-4 (that contain a pendant biphenyl linker as described by compounds of formula (I)) when compared to similar previously exemplified conjugates (that contain a linear biphenyl linker) as described in WO 2018/051085 or to reference compounds described herein (comparing Example 1 vs Reference Example B, Example 2 vs Reference Example A, Example 3 vs Reference Example C and Example 4 vs Reference Example D).
E. coli
K1
P. aeruginosa
K. pneumoniae
| Number | Date | Country | Kind |
|---|---|---|---|
| 1904534.3 | Apr 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2020/050861 | 4/1/2020 | WO | 00 |
