SE543054C2 - Cyclic peptides - Google Patents
Cyclic peptidesInfo
- Publication number
- SE543054C2 SE543054C2 SE1851022A SE1851022A SE543054C2 SE 543054 C2 SE543054 C2 SE 543054C2 SE 1851022 A SE1851022 A SE 1851022A SE 1851022 A SE1851022 A SE 1851022A SE 543054 C2 SE543054 C2 SE 543054C2
- Authority
- SE
- Sweden
- Prior art keywords
- dapa
- gram
- alkyl
- nr11r12
- daba
- Prior art date
Links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The present invention relates to novel compounds, which presents a peptide structure and an unexpected antibacterial effect even against certain multiresistant bacteria. The compounds are cyclic peptides, sometimes depsipeptides, and are defined by Formula (I). Further, the invention relates to the medical use of the herein claimed compounds, methods for the production of the compounds as well as methods of treatment including the compounds.In addition, the invention relates to a pharmaceutical preparation comprising one or more of the herein described and claimed compounds combined with suitable carrier(s) and/or adjuvant(s).Finally, the invention is the use of one or more of the claimed compounds in a method for decolonization of a surface of Gram-positive and/or Gram-negative bacteria.
Description
CYCLIC PEPTIDES TECHNICAL FIELD This invention relates to the area of peptides, and especially to peptides useful asantibacterial compounds. More specifically, the invention relates to a compound includingat least one cyclic peptide as well as methods of treating, eliminating and/or preventing a Gram-negative or Gram-positive bacterial infection in a subject using such compound(s).BACKGROUND Antibiotic resistant bacteria and fungi pose a serious threat to global health and there isan urgent need to develop novel antibiotics against priority human pathogens. The WorldHealth Organization (WHO) recently published the Priority Pathogens List, whichsuggests “Drug discovery and development strategies should focus urgently on newantibiotics specifically active against Mycobacterium tuberculosis (including multi- andextensively drug-resistant strains) and against multi- and extensively drug-resistant Gram-negative bacteria that cause acute clinical infections in both hospital and communitysettings worldwide." Among the Gram-negative bacteria, the highest priority was given tocarbapenem resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae.
Rational drug design and synthesis against selected “druggable” targets as well as high-throughput screening of large synthetic combinatorial libraries have not been assuccessful at producing novel classes of antibiotics as anticipated. Harvey, A.L. et al.(2015) Nat Rev Drug Discov 14: 111-129 present theoretical analysis that shows thatnatural products, which have evolved to penetrate into their target bacterial cells and toavoid subsequent expulsion, cover more chemical space compared to syntheticcombinatorial libraries in terms of compound lipophilicity, size, rigidity and aromaticity.Recent efforts at antibiotics discovery have thus returned to mining the wealth of antimicrobial compounds produced by bacteria and fungi.
There is a wealth of natural secondary metabolites of many different types that is, and hasbeen, used as medicinal drugs. The first and probably most prominent example is theantibiotic drug penicillin G, produced by fungi of the genus Penici/lium, and was brought to the market more than 70 years ago. The penicillins belong to a group of compounds called non-ribosomal peptides (NRPs), and in addition to the penicillins, there are manyNRPs that have been developed into important antibiotic drugs, for example the related cephalosporins, the vancomycins and daptomycin.
US 4,537,717 (Eli Lilly Company) describes daptomycin, which is an NRP based on 13amino acids with a fatty acid linked to the N-terminal amino acid, and a |actone bet\Neenthe C-terminal amino acid and a threonine residue, forming a 31-membered ring. Thisgroup of compounds are cyclic lipodepsipeptides. Daptomycin was approved by the FDAin 2003 for use against Gram-positive bacterial infections, and it has been described to disrupt several functions of bacterial cell membranes.
There are numerous microbial cyclic lipodepsipeptides with structures similar todaptomycin, i.e. 31-membered lactones formed as esters between the C-terminal aminoacid and a threonine or serine residue, and with an N-terminal fatty acyl residue. Analternative mode of |actone formation to obtain a cyclic 31-membered lipodepsipeptide, isbetween the C-terminal amino acid and an N-terminal 3-hydroxyfatty acid residue. Thereare twelve examples of such structures in the literature: JP 05 200 324 describes thepeptides B12489A-C, Kozuma, S. et al describes pedopeptin A-C (in Kozuma, S. et al.,J. Antibiot., 2014, 67, 237- 242; Hirota-Takahata, Y. et al., J. Antibiot., 2014, 67,243-251 ), several publications describe pelgipeptins A-D (see e.g. Takahara, Y. et al., J.Antibiot., 1979, 32, 115- 120, 121 - 129; Sugawara, K. et al., J. Antibiot., 1984, 37, 1257-1259; Murai, A. et al., J. Antibiot., 1985, 38, 1610- 1613; Wu, X.-C. et al., FEMSMicrobiol. Lett., 2010, 310, 32- 38; Ding, R. et al., J. Microbiol. (Seoul, Repub. Korea),2011, 49, 942- 949; Cochrane, S.A. and Vederas, J.C. Medicinal Research Reviews, 36,No. 1, 4-31, 2016), and polypeptin A and B (McLeod, C., J. Bacteriol., 1948, 56, 749-754, Howell, S.F., J. Biol. Chem., 1950, 186, 863- 877; Sogn, J.A., J. Med. Chem., 1976,19, 1228- 1231; CN102030819-A; CN102030819-B).
The peptides B12489A-C and the three pedopeptins were all isolated from Pedobactersp., and the structures are very similar or identical, with differences in aa8, Val or 3-hydroxyvaline (OHVal), and in the 3-hydroxyfatty acid residue (3-hydroxyoctanoyl or 3-hydroxy-7-methyloctanoyl), and possibly also a difference in geometry of the 2-amino-2-butenoyl (ABA) residue and the configuration of some amino acid residues (aa1, 3, 5-7).The pedopeptins and B12489A-C have been described to have anti-inflammatory action by interfering the binding of bacterial lipopolysaccharides (LPS) to receptors on cell surfaces, as well as antibacterial activities, with minimal inhibitory concentration (MIC) against Escherichia coli: 2-4 pg/mL and against Staphy/ococcus aureus: 4-64 pg/mL.
Pelgipeptin A-D and polypeptin A-B are similar to the pedopeptins and B12489-A-C, andthese peptides have aa3 and aa5 in common, and similar amino acid residues on aa1,aa6, aa7 and aa8. Pelgipeptin A-D and polypeptin A-B differ from each other by exchangeof a C-terminal serine (aa9) for threonine and isoleucine for valine (aa2), and also by theposition of the methyl branching position on the N-terminal fatty acyl group (Table 1). Thepelgipeptins and the polypeptins have been described to have activity against Gram- positive and negative bacteria, as well as against fungi.
Despite the above-described technologies, there is still an ongoing and increasing need of novel antibacterial preparations.
SUMMARY The present invention relates to novel compounds, which have been identified andcharacterized from nature; as well as use of said compounds preferably as antibacterial compounds or as part of pharmaceutical preparations.
A first aspect of the invention is an isolated or synthesised compound comprising theformula (I) as presented below: aa2Rz R N10 R3 aa3RmNaa1 R o1 O NR10 OR10N O OR4aa4oH-FA \RJY NRmX R5 aa5aa9 RQO O NR10R10N O Oo RÖ aa6NR1oRs N888 R10R?aa7 (I)whereiny: 0-5; X: O, NR10, or (CH2),, with n=0 to 10; R: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkyl branches; wherein double bonds may have E or Z configuration; R1: -(CH2),,-NR11R12, n=0 to 5, or -(CH2),,-CONR11R12, n=0 to 5, or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches; R2: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkyl branches, wherein double bonds may have E or Z configuration; Rs: -(CH2),,-NR11R12, n=0 to 5, or -(CH2),,-CONR11R12, n=0 to 5, or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches; R4: -(CH2),,-CH=C[(CH2)m-H](CH2)0-H, with n=0 to 10, m=0 to 10, o=0 to 5, and withE or Z double bond, or =C[(CH2)m-H](CH2)0-H, with m=0 to 10, o=0 to 5, with E or Zdouble bond; R5: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkyl branches; wherein double bonds may have E or Z configuration, and 0 to 2 Hmay be exchanged to any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-Hwith o=1 to 5; RB: -(CH2),,-NR11R12, n=0 to 5, or -(CH2),,-CONR11R12, n=0 to 5, or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches; R7: -(CH2),,-Ph(Y)m, with Y= any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-H, and with n=0 to 5, m=0 to 5, and o=1 to 20; Ra: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or CO-Czo n-alkenyl with 0 to 5C1-C5 n-alkyl branches, wherein double bonds may have E or Z configuration and 0to 2 H may be exchanged to any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-H with o=1 to 5; RQ: -(CH2),,-CONR11R12 or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5; R10: -H, -OR13, -(CH2)0-H with o=1 to 5, -F, -Cl, -Br, or -l, independently; R11 and R12: any combination of -H, C1-C5 n-alkyl, and C1-C5 n-alkenyl, or anycommon N-protecting group; and R13: -(CH2),,-H, with n=0 to 5; or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt thereof, wherein each stereocenter may be either R or S.
A second aspect of the invention is a method of producing a compound according to theinvention.This aspect includes methods and tools for use therein, such as intermediate peptides, microorganisms as well as expression systems.
A third aspect of the invention is the medical use of a compound according to theinvention. Thus, a compound according to the invention may be used as a medicament,such as in the treatment or prevention of infections caused by Gram-positive and/or Gram-negative bacteria and/or fungi.
A fourth aspect of the invention is a method of treatment or prevention of infectionscaused by one or more Gram-positive and/or Gram-negative bacteria, wherein the herein described and claimed compound is administered to an individual in need thereof.
A fifth aspect of the invention is a pharmaceutical preparation comprising one or more ofthe herein described and claimed compounds combined with suitable carrier(s) and/or adjuvant(s).
A sixth aspect of the invention is the use of one or more of the herein described andclaimed compounds in a method for decolonization of a surface of Gram-positive and/or Gram-negative bacteria.BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is scheme 1, detailing Part I of the solid-phase synthesis of illustrative compoundU747 according to the invention as described in Example 2 below.Figure 2 is scheme 2, detailing Part ll of the solid-phase synthesis of illustrative compound U747 as described in Example 2 below.DEFINITIONS The term "peptide" is used herein in its broadest aspect i.e. for any short chain of aminoacid monomers linked by peptide (amide) bonds and includes e.g. depsipeptides and lipodepsipeptides.
A “depsipeptide” is a peptide in which one or more of its amide, -C(O)NHR-, groups arereplaced by the corresponding ester, -C(O)OR, or more generally, is a molecule that hasboth peptide and ester linkages in proximity in the same amino acid-containing small molecule or chain.
The term “stereocenter” is used herein in its conventional meaning of a chirality center.
The term “DABA” refers herein to The term “ABA” refers herein to 2-amino-2-butenoic acid.The term “DAPA” refers herein to 2,3-diaminopropanoic acid.The term “Leu” refers herein to leucine.
The term ”Thr” refers herein to threonine.
The term ”Phe” refers herein to phenylalanine.
The term ”Asp” refers herein to aspartic acid.
The term “Asn” refers herein to asparagine.
The term ”Val” refers herein to valine.
The term “OHVa|” refers herein to 3-hydroxyvaline.
The abbreviation aa is used herein for amino acid, and aa followed by a number indicatesthe number position of the aa starting from the N-terminal amino end of a peptide or protein.
The term “HCTU” refers herein to O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate.
The term “D|PEA” refers to N,N-diisopropylethylamine The term “H FIP” refers to hexafluoroisopropanol.
The term “PyC|ocK” refers to 6-chlorobenzotriazole-1-yloxy-tris-pyrrolidino-phosphoniumhexafluorophosphate.
The term ”TIPS” refers to triisopropylsilane.
DETAILED DESCRIPTION OF THE INVENTION As appears from the above, the present invention relates to novel cyclic peptides. Morespecifically, the invention relates to novel cyclic peptides, which may be depsipeptides, as such or as part of a composition or preparation.
Thus, a first aspect of the invention is a compound of Formula I: isolated or synthesised compound comprising the formula (I) as presented below: aa2Rz RN10 R3 aa3R10Naa1 R o1 O O NR10R10N O OOH-FA RJ), NRmR5 aa5aa9 Rgw/g oaa R4 aa4 NR1o XOR10N O Oo RÖ aa6NR1oRs N8 R1oR?aa7 (I)whereiny: 0-5;X: O, NR10, or (CH2)n with n=0 to 10;R: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkyl branches; wherein double bonds may have E or Z configuration;R1É -(CH2)n-NR11R12, n=0 tO 5, Of -(CH2)n-CONR11R12, n=0 tO 5, Or -(CH2)n-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches;R2: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n- alkyl branches, wherein double bonds may have E or Z configuration; Rs: -(CH2),,-NR11R12, n=0 to 5, or -(CH2),,-CONR11R12, n=0 to 5, or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches; R4: -(CH2),,-CH=C[(CH2)m-H](CH2)0-H, with n=0 to 10, m=0 to 10, o=0 to 5, and withE or Z double bond, or =C[(CH2)m-H](CH2)0-H, with m=0 to 10, o=0 to 5, with E or Zdouble bond; R5: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkyl branches; wherein double bonds may have E or Z configuration, and 0 to 2 Hmay be exchanged to any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-Hwith o=1 to 5; RB: -(CH2),,-NR11R12, n=0 to 5, or -(CH2),,-CONR11R12, n=0 to 5, or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkylbranches; R7: -(CH2),,-Ph(Y)m, with Y= any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-H, and with n=0 to 5, m=0 to 5, and o=1 to 20; Ra: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or CO-Czo n-alkenyl with 0 to 5C1-C5 n-alkyl branches, wherein double bonds may have E or Z configuration and 0to 2 H may be exchanged to any combination of -OR13, -NR11R12, -F, -Cl, -Br, -l, -(CH2)0-H with o=1 to 5; RQ: -(CH2),,-CONR11R12 or -(CH2),,-COO-(CH2)m-H, n=0 to 5 and m=0 to 5; R10: -H, -OR13, -(CH2)0-H with o=1 to 5, -F, -Cl, -Br, or -l, independently; R11 and R12: any combination of -H, C1-C5 n-alkyl, and C1-C5 n-alkenyl, or anycommon N-protecting group; and R13: -(CH2),,-H, with n=0 to 5.
As will be discussed in relation to the various aspects of the invention, the compoundaccording to the invention may be represented by Formula I above, or, alternatively, itmay be a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a prodrug thereof.
Further, as will be discussed below in relation to the method of preparation, the compoundaccording to the invention may be obtained by peptide synthesis; by recombinant DNA technology; or by isolation from one or more native microorganisms.
The compound(s) of the invention, where X: O and y: 0, 1, or 2, are cyclic depsipeptides with an ester between a C-terminal amino acid either to a 2-hydroxyfatty acid, forming a -membered cyclic structure; to a 3-hydroxyfatty acid, forming a 31-membered cyclicstructure; or to a 4-hydroxyfatty acid, forming a 32-membered cyclic structure.
Some compounds of the invention, where X: NH, are cyclic peptides which are ring-closed from the C-terminal amino acid to an amino-substituted fatty acid residue. ln further compounds of the invention, cyclic depsipeptides or cyclic peptides havesingly/multiply exchanged R10: -H to R10: -OH and/or -CH3. ln one compound of the invention, y, X, and R-R13 are chosen such that the compoundcomprises one 3-hydroxyfatty acid, and the amino acid residues 2,3-diaminopropanoicacid (DAPA, aa1 and aa6), |eucine (Leu, aa2), 2,4-diaminobutanoic acid (DABA, aa3), 2-amino-2-butenoic acid (ABA, aa4), threonine (Thr, aa5), phenylalanine (Phe, aa7), va|ine(Val) or 3-hydroxyvaline (OHVaI, aa8), aspartic acid (Asp, aa9), forming a 31-memberedcyclic lipodepsipeptide. Such a compound has the general structure cyc/o(3-hydroxyalkanoyl - DAPA - Leu - DABA - ABA - Thr - DAPA - Phe - Val/OHVal - Asp). ln further compounds comprising the general structure cyc/o(3-hydroxya|kanoy| - DAPA -Leu - DABA - ABA - Thr - DAPA - Phe - Val/OHVal - Asp), there are single or multipleside-chain N- and/or O-substitutions with alkyl groups, or common N- or O-protectinggroups, and/or -NH2 or -OH substitutions on the Phe residue.
One illustrative compound of the invention, which will be denoted U747 herein, has y: 1,X: O; R: n-heptyl; R1: -CH2-NH2; R2: -CH2-CH(CH3)-CH3; Rs: -CH2-CH2-NH2; R4: (E)=CH-CH3; R5: CHOH-CH3; RB: -CH2-NH2; R7: -CHz-Ph; Ra: -COH(CH3)-CH3; RQ: -CHz-COOH;and R10, R11, R12 and R13: H. This compound is a cyclic lipodepsipeptide (31-memberedlactone), with ring-closure from the C-terminal L-Asp residue to a 3-hydroxydecanoylresidue and with the sequence cyc/o-((R)-3-hydroxydecanoyl - D-DAPA - L-Leu - L-DABA - (E)-ABA - L-Thr - L-DAPA - D-Phe - L-OHVal - L-Asp).
As the skilled person will appreciate, a large number of minor variations to the basicstructure described herein may be performed without deviating from the invention. Thus,the present invention will embrace any compound that fulfill the criteria of Formula (l) and presents the herein discussed advantageous properties when exposed to pathogens.
Another illustrative compound of the invention, which will be denoted U773 herein, has y:1; X: O; R: 8-methylnonyl; R1: -CH2-NH2; R2: -CH2-CH(CH3)-CH3; Rs: -CH2-CH2-NH2; R4:(E)=CH-CH3; R5: CHOH-CHS; RB: -CH2-NH2; R7: -CHz-Ph; Ra: -COH(CH3)-CH3; Rg: -CH2- 11 COOH; and R10, R11, R12 and R1s: H. This compound is a cyclic lipodepsipeptide (31-membered lactone), with ring-closure to a 3-hydroxy-11-methyldodecanoyl residue andwith the sequence cyclo-((R)-3-hydroxy-11-methyldodecanoyl - D-DAPA - L-Leu - L-DABA - ABA - L-Thr - L-DAPA - D-Phe - L-OHVaI - L-Asp).
A further illustrative compound of the invention, which will be denoted U793 herein, has y:1; X: O; R: 8-methylnonyl; R1: -CHs-NHs; Rs: -CH2-CH(CHs)-CHs; Rs: -CH2-CH2-NH2; R4:(E)=CH-CHs; Rs: CHOH-CHs; Rs: -CHs-NHs; R7: -CHs-Ph; Rs: -CH(CHs)-CHs; Rs: -CHs-COOH; and R10, R11, R12 and R1s: H. This compound is a cyclic lipodepsipeptide (31-membered lactone), with ring-closure to a 3-hydroxy-11-methyldodecanoyl residue andwith the sequence cyclo-((R)-3-hydroxy-11-methyldodecanoyl - D-DAPA - L-Leu - L-DABA - ABA - L-Thr - L-DAPA - D-Phe - L-Val - L-Asp).
Yet one illustrative compound of the invention, which will be denoted U824 herein, has y:1; X: O; R: 8-methylnonyl; R1: -CHs-CO-NHZ; Rs: -CH2-CH(CHs)-CHs; Rs: -CHs-CHs-NHZ;R4: (E)=CH-CHs; Rs: CHOH-CHs; Rs: -CHs-NHs; R7: -CHs-Ph; Rs: -CH(CHs)-CHs; Rs: -CHs-COOH; and R10, R11, R12 and R1s: H. This compound is a cyclic lipodepsipeptide (31-membered lactone), with ring-closure to a 3-hydroxy-11-methyldodecanoyl residue andwith the sequence cyclo-((R)-3-hydroxy-11-methyldodecanoyl - L-Asn - L-Leu - L-DABA- ABA - L-Thr - L-DAPA - D-Phe - L-Val - L-Asp).
One additional compound of the invention, which will be denoted U756 herein, has y: 1; X: O; R: 2-nonenyl; R1: -CHs-NHs; Rs: -CH2-CH(CHs)-CHs; Rs: -CHs-CHs-NHZ; R4: (E)=CH-CHs; Rs: CHOH-CHs; Rs: -CHs-NHs; R7: -CHs-Ph; Rs: -COH(CHs)-CHs; Rs: -CHs-COOH,and R10, R11, R12 and R1s: H. This compound is a cyclic lipodepsipeptide (31-memberedlactone), with ring-closure to a dodec-5-enoy| residue and with the sequence cyclo-((R)-3-hydroxydodec-5-enoyl - D-DAPA - L-Leu - L-DABA - ABA - L-Thr - L-DAPA - D-Phe -L-OHVaI - L-Asp). ln further compounds of the invention, similar to the U747, U773, U793, U824, and U756prototypes, the configuration of single/multiple amino acid residues are inversed, as wellas the configuration of the 3-hydroxy fatty acids. lt has been noted, that in compounds similar to U747, with identical R1-R1s, but differentR, the cytotoxicity (lCso) of the compounds may increase with the length of the alkylchains, e.g. R: C7H1s to C10H21. 12 lt has also been noted, that in compounds similar to U747, with R1 or RB exchanged from-CH2-NH2 to -CHz-CO-NHZ, the antibacterial activity may be lower under certainconditions, i.e. higher MIC values. Further, it has also been noted, that in compounds similar to U747, but with R < C7H15, the antibacterial activity may be lower under certainconditions, i.e. higher MIC values.
As the skilled person will appreciate, when developing products based on peptides suchas the ones described herein, routine optimization may be used to identify which specific structure is most advantageous for certain applications, such as in terms of effect againstcertain bacteria, and/or against certain medical conditions.
There are t\Nelve cyclic lipodepsipeptides in the literature possessing a 31-memberedmacrocyclic lactone, with the ring-closure to a 3-hydroxyfatty acid, as discussed in the“Background of the invention”, and with structures described in Table 1.
Table 1 Comparison of composition of the twelve known cyclic lipodepsipeptides With a 31-membered lactone to a 3-hydroxyfatty acid (OH FA), along with the composition of thenovel cyclic lipodepsipeptide U747. Me=methyl, Oct=octanoyl, Hex=hexanoyl, Dec=decanoyl, DAPA=2,3-diaminopropanoyl, DABA=2,4-diaminobutanoyl, ABA=2-amino-2-butenoyl, OHVal=3-hydroxyvaline få ä ä” f; ä få 2 i “åä ä ä ä ä ä ä ä ä ä ä ää ä ä g ä ä ä ä ä ä ä ä äE E E ä ä ä ä ä ä ä ä ä 5OHFA 7-MeOct Oct 7-MeOct 7-MeOct 7-MeOct Oct 5-MeHex 4-MeHex 4-MeHex 5-MeHex 4-MeHex 5-MeHex Decaa1 DAPA DAPA DAPA DAPA DAPA DAPA L-DABA L-DABA L-DABA L-DABA L-DABA L-DABA D-DAPAaa2 L-Phe L-Phe L-Phe L-Phe L-Phe L-Phe L-Val L-lle L-Val L-lle L-lle L-lle L-Leuaa3 DABA DABA DABA L-DABA DABA DABA L-DABA L-DABA L-DABA L-DABA L-DABA L-DABA L-DABAaa4 Z-ABA Z-ABA Z-ABA E-ABA E-ABA E-ABA D-Phe D-Phe D-Phe D-Phe D-Phe D-Phe E-ABAaa5 L-Leu L-Leu L-Leu Leu Leu Leu L-Leu L-Leu L-Leu L-Leu L-Leu L-Leu L-Thraa6 DAPA DAPA DAPA DAPA DAPA DAPA L-DABA L-DABA L-DABA L-DABA L-DABA L-DABA L-DAPAaa? L-Leu L-Leu L-Leu Leu Leu Leu D-Val D-Val D-Val D-Val D-Val D-Val D-Pheaa8 L-Val L-OHVal L-OHVal L-OHVal L-Val L-OHVal L-Leu L-Leu L-Leu L-Leu L-Leu L-Leu L-OHValaa9 L-Asp L-Asp L-Asp L-Asp L-Asp L-Asp L-Ser L-Ser L-Ser L-Ser L-Thr L-Thr L-Asp The peptides B12489A-C and pedopeptin A-C are very similar to each other, whereaspelgipeptin A-D and polypeptin A-B are very similar to each other. U747 is more similar toB12489A-C and pedopeptin A-C than to pelgipeptin A-D and polypeptin A-B, as U747 13 shares six of nine amino acids (same amino acid in the same position) with B12489A-Cand pedopeptin A-C (aa1, 3, 4, 6, 8, 9), and only one amino acid, aa3, with pelgipeptin A-D and polypeptin A-B. However, U747 still differs substantially from B12489A-C andpedopeptin A-C, with aa2 and aa7 having switched positions and with a polar amino acid (Thr) as aa5 instead of a non-polar (Leu).
Pedopeptins A-C have been described to interact with the LPS of Gram-negative bacteriato exert its anti-inflammatory activity. Possibly, also the antibacterial properties of thepedopeptins depend on the interaction with LPS, and given the similarity of thecompounds, it may be speculated that also U747 and similar compounds of this invention,interact with the LPS of Gram-negative bacteria. However, in its broad sense, the present invention is not limited by any definition of its mode of action.
Pedopeptin A-C were described to have MIC values of 2-4 ug/mL against tvvo E. colistrains, which is similar to the MIC values for U747, and similar compounds of theinvention, against E. coli, but also against many other Gram-negative bacteria (Example3). The compound U747 has also been found to have potent activity against carbapenemresistant strains of E. coli, P. aeruginosa and A. baumannii (MlCs between 0.5-1 ug/mL and 2-4 ug/mL), which are all human pathogens ranked by the WHO as critical.
Along with the very promising antibacterial properties of U747, this compound of theinvention has also shown low toxicity (IC5O) against human cell lines, and acceptable haemolysis rates, and also a very low rate of formation of resistant mutants. ln a second aspect, the invention relates to a method of producing any one of the herein defined compounds according to the invention.
Thus, the method may be a method of synthesis wherein a peptide comprising at leastnine amino acids and including at least one first amino acid followed by the sequence ofLeu; DABA; ABA; Thr; DAPA; Phe; Val; and Asp; wherein the first amino acid is DAPA or Asn is used as an intermediate.
More specifically, the intermediate may be a peptide as defined by any one of SEQ IDNOs: 1-5; or a longer peptide including such sequence. Thus, an intermediate useful inthe synthesis of a compound according to the invention may be the above-mentionedpeptide linked to a first amino acid which is DAPA or Asn. Further, the Val may have beenmodified with OH into OHVal. 14 Peptide synthesis may be performed using standard equipment, such as solid phasepeptide synthesis, and instruments as well as suitable protocols are widely available fromcommercial sources. The skilled person may utilise conventional methods for ring-closingof the peptide, such as esterification. An illustrative method of producing a compound according to the invention will be provided below, as Example 2.
Alternatively, the method of producing any one of the compounds according to theinvention may include cultivation of a microorganism or recombinant DNA technologycomprising the expression of parts or all of a compound as defined by any one of claims 1-6; or an intermediate peptide as discussed above.
Thus, the invention relates to a microorganism which is capable of expressing acompound according to the invention. More specifically, such a microorganism maybelong to the genus Pedobacter. A particular microorganism according to the inventionwas deposited with BCCM-LMG on July 3, 2018 in accordance with the Budapest Treaty,where it obtained patent deposit ref LMG P-30868.
Further, the invention also relates to a recombinant expression system arranged for theproduction of a compound according to the invention. The expression system maycomprise one or more microorganisms, such as bacteria or fungi, which has beenmodified by recombinant DNA technology To increase its production of compoundaccording to the invention; to grow under certain circumstances; or any other conventionalmodification that improves or otherwise changes the native microorganism's function as an expression system More specifically, an expression system according to the invention derived from the genus Pedobacter.
Furthermore, a compound according to the invention may be produced by isolation frommicroorganisms such as bacteria which natively produces such cyclic peptide(s). Onemethod of isolating bacteria for production of a compound according to the inventionincludes the steps of isolating it from a natural sample, such as soil. The actual isolationmay be performed following standard protocols, such as Thaker, M. et al. (2013) NatBiotechnol 31 (10):922-929 or the like. A specific example of isolating a compound according to the invention from soil bacteria will be presented below, as Example 1. ln a third aspect, the present invention relates to one or more of the compounds accordingto the invention for use as a medicament, such as in the treatment or prevention ofinfections caused by one or more Gram-positive and/or Gram-negative bacteria.
The infections may be caused by one or more Gram-positive bacteria, such as Gram-positive bacteria selected from the group consisting of Actinomyces; Bacillus; C/ostridium;Corynebacterium; Enterococcus; Gardnerella; Lactobacillus; Listeria; Micrococcus; Mycobacteria; Nocardia; Staphylococcus; and Streptococcus.
Alternatively, or additionally, the infections may be caused by one or more Gram-negativebacteria, such as Gram-negative bacteria selected from the group consisting ofAcinetobacter; Batronella; Bordetella; Borrelia; Bruce/la; Burkho/deria; Campylobacter;Chlamydia; Chlamydophilia; Citrobacter; Coxiella; Ehrlichia; Enterobacter; Escherichia;Francisella; Fusobacterium; Haemophilus; Helicobacter; Klebsiel/a; Legionella;Leptospira; Moraxella; Mycoplasma; Neisseria; Proteus; Pseudomonas; Rickettsia; Salmonella; Serratia: Shigella; Treponema; Vibrio; and Yersinia. ln a fourth aspect, the invention relates to a method of treatment or prevention ofinfections caused by one or more Gram-positive and/or Gram-negative bacteria, in whichmethod one or more of the compounds according to the present invention are used. Thebacteria may be as discussed above in relation to the third aspect of the invention, orelsewhere in the present application. Thus, medical conditions that may be preventedand/or treated in accordance with the present invention may be selected from the groupconsisting of Actinomyces; Bacillus; C/ostridium; Corynebacterium; Enterococcus;Gardnerel/a; Lactobacillus; Listeria; Micrococcus; Mycobacteria; Nocardia;Staphylococcus; Streptococcus; Acinetobacter; Batronella; Bordetella; Borrelia; Bruce/la;Burkho/deria; Campylobacter; Chlamydia; Chlamydophilia; Citrobacter; Coxiella;Ehrlichia; Enterobacter; Escherichia; Francisella; Fusobacterium; Haemophilus;Helicobacter; K/ebsiella; Legionella; Leptospira; Moraxella; Mycoplasma; Neisseria;Proteus; Pseudomonas; Rickettsia; Salmonella; Serratia: Shigella; Treponema; Vibrio; and Yersinia. ln a fifth aspect, the invention relates to a pharmaceutical preparation comprising one ormore of the herein described and claimed compounds combined with one or morepharmaceutically acceptable carrier(s) and/or adjuvant(s). Tha Carrier rliay be any solid orliqulfl Carrier material trilhicii is compatible iltfitil the corrlpounlïlls) according to the invention, and with any a-:lditionai coinpaiwent lncluda-tl. *vtfhl-all is suitable for ttiarapeutic 16 administration Le. pharrnaoeuti-catiy aooegstabie. Thue, the carrier may be ari crrgartic orinorganšo carrier material irrhirgti ia suitabie for ehterai (eg, oral) or pareriteraiadministration. išxarnpiee of such Carrier ritaterisiis are vrater, geiatin, iaotose, starehes,magnesium etearate, taic, vegetabie oiie, gurn arabia, poiysiikyieneghfooia, petroleum jeiiyetr. The pharmareiitioai preparations can be made :ip in a soiid form (eg. as tebiets,drageos. supoc-eitorios, or oapsuies) or in a iiouid form (eg. ae soiuticrie, stiegaohsicirie or ernuisions), "the ptiarmaoouticai preparatic-ns according to the intfenticiri may be oropared according tomethods kriovvn in the art, may be eiibjected sto converitionei pharmaceiiticai operationssircit steriiiaatiort and rnay contain one or rnore adjuvants etioh aa preserving agents,stabiiising agents, tftfetting agents, eniuisifying agents, eaite for varg/ing the osinoticpressure or bufters. inlheri a butter is used, the pii of the pharrnaceuti-cat preparation tviit, of course, varv trrithin e range whioii ie weitknrgwn in pharmaceiiticat rarargtice.
The pharmaceutical preparation according to the invention may be used as ivformulations, oral formulations, topical formulations and other formulations giving anoptimal effect against the disease causing bacteria, while at the same time giving minimal inhibition of harmless and beneficial bacteria.
The disease-causing bacteria against which the present preparation is medically effectiveare as discussed above, and may be selected from the group consisting ofAcinetobacter;Bacil/us; Campylobacter; Ch/amydia: C/ostridium; Enterobacter; Enterococcus;Escherichia; Haemophilus; Helicobacter; Klebsiel/a; Legionella; Mycobacteria; Neisseria;Pseudomonas; Salmonella; Shigel/a; Staphy/ococcus; Streptococcus; Vibrio; and Yersinia.
Advantageously, the preparation according to the invention is effective against one ormore disease-causing bacteria which are resistant, and/or multiresistant, to antibioticsselected from the group consisting of aminoglycosides; ansamycins; carbapenems;cephalosporins; fluoroquinolons; glycopeptides; lincosamides; lipopeptides; macrolides;monobactams; nitrofurans; oxazolidinones; penicillin ; polypeptides; quinolones; sulphonamides; and tetracyclines. ln a sixth aspect, the invention relates to the use of one or more of the herein described and claimed compounds in a method for decolonization of a surface of Gram-positive 17 and/or Gram-negative bacteria. The surface may a body surface, or a part of clinical equipment, such as the surface of a metal or plastic article.DETAILED DESCRIPTION OF THE DRAWINGS Figure 1 is scheme 1, detailing Part I of the solid-phase synthesis of illustrative compoundU747 according to the invention. More specifically, scheme 1 includes i) l\/lartin's sulfuranedehydrating reagent, ii) formic acid, iii) 2-chlorotrityl resin/collidine/DMF, iv) 20%piperidine/DMF, v) Fmoc-L-Leu/HCTU/DIPEA, vi) Fmoc-D-DAPA(I\IV-Boc)/HCTU/DIPEA,vii) Fmoc-L-Asp(O-tBu)-O-CH[(CH2)6-CH3]-CH2-COOH /HCTU/DIPEA, viii) Fmoc-L-OHVal(O-tBu)/HCTU/DIPEA, ix) Fmoc-D-Phe/HCTU/DIPEA. After each coupling step, the Fmoc group is removed with 20% piperidine in DMF.
Figure 2 is scheme 2, detailing Part ll of the solid-phase synthesis of illustrativecompound U747 according to the invention. More specifically, scheme 2 includes x)Fmoc-L-DAPA(I\IV-Boc)/HCTU/DIPEA, xi) Fmoc-L-Thr(O-tBu)/HCTU/DIPEA, xii) 20%HFIP in DMF, xiii) PyclocK/DIPEA, xiv) TFA/TIPS. After each coupling step, the Fmocgroup is removed with 20% piperidine in DMF.
EXPERIMENTAL PART The present examples are provided for illustrative purposes only, and are not intended tobe limiting the invention as defined by the appended claims. All references provided below and elsewhere in the present application are hereby included herein via reference.
Example 1: lsolation and identification of Pedobacter sp. UP508 The bacterial isolate, herein denoted UP508 (deposited with the Belgian CoordinatedCollections of Micro-organisms -Laboratorium voor Microbiologie (BCCM-LMG) on July 3,2018, as LMG P-30868) was isolated from a soil sample collected at Ultuna, Uppsala,Sweden, based on a standard protocol (see e.g. Thaker, M. et al. (2013) NatBiotechnol31 (10):922-929). The isolate was identified as closest related to the strain A37 ofPedobacter cryoconitis or to the strain WB 3.3-22 of Pedobacter westerhofensis by sequencing 16 S rRNA.Production of U747 The isolate Pedobacter sp. UP508 was cultivated in 32 >< 1000-mL E-flasks, each with300 mL of half strength Vegetable Peptone Broth (VPB; 15 g VPB (Oxoid Ltd) in 1000 mL 18 deionized H20), on a rotary shaker (130 rpm) at 15 °C. ln order to collect U747 and otherextracellular metabolites, 16 to 24 hours after inoculation, sterile cotton/paper bags with apolymeric resin Sepabeads® SP850 (Mitsubishi Chemical Corporation; approximately 10 gper bag) were submerged in actively growing cultures. After 6-7 days, the bags wereremoved, Washed With deionized water and finally extracted with approximately 2 X 20 mLMeOH and 2 >< 20 mL MeCN per bag. The pooled extract was dried under reducedpressure. The dried extract was dissolved in 40 mL aqueous 50% MeCN (0.2% formicacid), centrifuged (5 min at 13000 rpm), and injected (40 >< 1 mL) onto a semi-preparativeHPLC column (Phenomenex Luna Omega 5 um PS C18, 100Å; 100 >< 21.2 mm), elutedwith a two-step gradient of MeCN in Water (15-50% in 15 min and 50-95% in 2 min,followed by 95% for 4 min, “HPLC1”), with 0.2% formic acid, at 10 mL/min. Fractions (2mL) were collected in 2.2-mL deep-well plates and the chromatography was monitored bya UV-detector at 210 nm. The HPLC-fractions were analyzed by UHPLC-MS (0.5 uL;Accucore Vanquish C18+, 50 >< 2.1 mm, 1.5 um; 10-95% MeCN in 3 min and 1.2 min at95% MeCN, 0.2% formic acid, 0.9 mL/min; Agilent 1290 lnfinity ll UHPLC and BrukermaXis lmpact QTOF) in positive mode. Fractions 57-59, which contained the peptideU747 with tR 1.4 min and m/z 373.2143 (3+) (UHPLC-MS), were pooled and dried in avacuum centrifuge. The dry residue was fractionated on the same semi-preparativecolumn eluted with a linear gradient of MeCN in water (20-37.5% in 26 min, and 37.5-60%in 9 min, 0.2% formic acid, 10 mL/min, “HPLC2”). Fractions 54-61 which contained U747(UHPLC-MS as above) were pooled and dried in a vacuum centrifuge, were furtherseparated on the same column as above and using a gradient of MeCN in water (0.2%formic acid; 17.5-35% MeCN in 26 min and 35-57.5% in 9 min, “HPLC3”). Fractions 39-54which contained U747 were pooled and dried in a vacuum centrifuge, and were subjectedto one final round of HPLC on the same column (17.5-35% MeCN in 28 min and 35-57.5% in 7 min, 0.2% formic acid, 10 mL/min, “HPLC4”). Fractions 48-53, which containedU747, were pooled and dried in a vacuum centrifuge. Fractions with less pure U747 fromHPLC3 were subjected to HPLC4 and additionally HPLC5 (same conditions as inHPLC4), and the fractions containing U747 were pooled and dried in a vacuum centrifuge.The total yield of U747 Was 33 mg.
Other compounds of the invention were isolated using the same method, but with appropriate adaptations of the HPLC conditions used.
Structure determination of U747 and similar compounds 19 NMR U747 was dissolved in 0.6 mL DlVlSO-dß and was analyzed on a Bruker Avance-lll NMRspectrometer, equipped with a 5-mm cryo-probe. The NMR data of U747 from 1D 1H and”C NMR experiments, along with the 2D experiments 1H-1H COSY, 1H-1H TOCSY,HSQC-TOCSY, and lH-”C HSQC, suggested the presence of the following commonamino acids: 1 X Leu, 1 X Thr, 1 X Phe, and 1 X Asp, and several non-standard aminoacids. Subsequently, analysis of the NMR data suggested the presence of 2 X 2,3-diaminopropanoic acid (DAPA), 1 X 2,4-diaminobutanoic acid (DABA), 1 X 2-amino-2-butenoic acid (ABA), and 1 X 3-hydroxyvaline (OHVal). ROESY experiments proved theABA to be present as (E)-ABA.
The NMR data also indicated the presence of a 3-hydroxydecanoic acid, and judging fromthe chemical shift of the ß-CH, the 3-hydroxy function should be esterified, suggesting thata lactone is formed by an ester to this function. Finally, combination of ROESY and HMBCdata suggested the structure of U747 to be cyclo(3-hydroxydecanoyl - DAPA -Leu -DABA - ABA - Thr - DAPA - Phe - OHVaI - Asp).
MSMS.
To open the putative lactone of the peptide U747, a small sample (ca 50 pg) was treatedwith 1% NaOMe in 200 uL MeOH for 20 min at room temperature, and the solution wasneutralized by 10 uL 2 M HCl (aq). The resulting sample Was analyzed by UHPLC-MSMS(same instruments and column as above) using a gradient of MeCN in water (0.2% formicacid; 30-60% MeCN in 3 min, 0.9 mL/min). The ion at m/z 575.328? (2+) corresponding tothe linear ring-opened peptide U747 (tR 33 s) was selected for MSMS (21 .5 eV). Thefragment ion B1 (with the N-linked 3-hydroxydecanoyl group), along With Bz-Bg, allowed a full sequence coverage, and confirmed the sequence proposed by NMR data.Confiquration of amino acids U747 (0.5 mg) was dissolved in 200 uL 6 M HCl, and was treated in an evacuated glassvial over night at 120°C. The vial was opened and the solution dried under N2. Theconfigurations of the amino acids were subsequently determined using the advancedMarfey method (Fujii et al. 1997. Anal. Chem. 69, 5146-5151.). Derivatized samples wereanalyzed by UHPLC-MS (column as above, 20-50% MeCN in 6 min, 0.9 mL/min, 0.2% formic acid) and by comparison with authentic reference samples, the amino acids were determined to be L-Thr, L-OHVal, L-Asp, L-Leu, D-Phe, L-DAPA, D-DAPA and L-DABA.The D-DAPA was determined to be aa1 and L-DAPA aa6, by comparing results fromdifferent duration of the acidic hydrolysis, which showed the intensity of D-DAPA to increase with time and a residual DAPA-Leu dipeptide to decrease with time.
Confiquration of the 3-hvdroxv fatty acid The configuration of the 3-hydroxy fatty acid part of the peptides of the invention wasdetermined on prototype U752, using the modified l\/losher's method (Ohtani et al, J. Am.Chem. Soc. 1991, 113, 4092-4096). U752 (6 mg) was dissolved in 200 uL 6 M HCI, andwas treated at 120°C in an evacuated glass vial for 6 h. After cooling, the vial was openedand the contents were dried under N2. The residues were partitioned between EtOAc andwater and the EtOAc extract was dried under N2. The sample (ca 0.5 mg) was dissolvedin 1.2 mL pyridine-d5 and the solution was split into two equal portions (ca 1.2 umol each)in 1.5-mL glass vials. Each sample was treated with 5 uL R- or S-MTPA-Cl (3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride, ca 27 umol each, ca 22-fold excess) at roomtemperature to produce the S- and R-MTPA ester, resp., of the fatty acid. After 6 h thesamples were analysed by NMR, and the chemical shifts of H2-2, H2-4 and H2-5, weredetermined for the two diastereomeric samples. Finally, the differences between theabove chemical shifts for the S- and R-MTPA esters were calculated, and theconfiguration of the 3-hydroxy fatty acid of U752 was determined to be R, and this configuration appears to be the same in all peptides of the invention including U747.
Thus, the structure of U747 was determined to be cyc/o((R)-3-hydroxydecanoyl - D-DAPA- L-Leu - L-DABA - (E)-ABA - L-Thr - L-DAPA - D-Phe - L-OHVal - L-Asp).
The structure of other compounds of the invention were determined using a combinationof NMR, MSMS and the advanced l\/larfey's method, as needed. 21 a The structure of U747 and other compounds of the invention. DAPA: 2,3-diaminopropanoic acid, DABA: 2,4-diaminobutanoic acid, ABA: 2-amino-2-butenoic acid,OHVal: 3-hydroxyvaline. Only L-configuration Was observed for Leu, DABA, Thr, Val,OHVal, Asp and Asn, whereas both D- and L- configuration was observed for DAPA, andPhe. name R aa-1 aa-2 aa-3 aa-4 aa-5 aa-6 aa-7 aa-8 aa-9 U733 C6H13 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U736 C6H13 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U737 C6H13 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U741 C6H13 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U744 C7H15 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U747 C7H15 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U749 C7H15 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U751 C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U752 C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U753 C7H15 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U754 C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U755a C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U755b C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U756 C9H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U757 C9H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U758 C8H17 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U765 C8H17 DAPA Leu DABA ABA Thr DAPA Phe Val Asp U771 C9H17 DAPA Leu DABA ABA Thr DAPA Phe Val Asp U773 C10H21 DAPA Leu DABA ABA Thr DAPA Phe OHVal Asp U775 C9H17 DAPA Leu DABA ABA Thr DAPA Phe Val Asp U787 C10H21DAPA Leu DABA ABA Thr DAPA Phe Val Asp U793 C10H21DAPA Leu DABA ABA Thr DAPA Phe Val Asp U794 C10H21DAPA Leu DABA ABA Thr DAPA Phe Val Asp U8OO C10H21DAPA Leu DABA ABA Thr DAPA Phe Val Asp U802 C9H19 Asn Leu DABA ABA Thr DAPA Phe OHVal Asp U809 CsHw Asn Leu DABA ABA Thr DAPA Phe Val Asp U810 C10H21 Asn Leu DABA ABA Thr DAPA Phe OHVal Asp U824 C10H21 Asn Leu DABA ABA Thr DAPA Phe OHVal Asp U826 CQHW DAPA Leu DABA ABA Thr Asn Phe Val Asp U828 C10H21 Asn Leu DABA ABA Thr DAPA Phe Val Asp U829 C10H21 Asn Leu DABA ABA Thr DAPA Phe OHVal Asp U833 CQHW DAPA Leu DABA ABA Thr Asn Phe Val Asp U834 C9H19 Asn Leu DABA ABA Thr DAPA Phe Val Asp U840 C9H19 DAPA Leu DABA ABA Thr Asn Phe Val Asp U842 C10H21 Asn Leu DABA ABA Thr DAPA Phe Val Asp 22 Example 2: Synthesis of U747 The Synthesis of U747 is made in analogy with the Synthesis of the polypeptin PE2, asdescribed by Mountford et al (Org. Biomol. Chem., 2017, 15, 7173-7180). Most aminoacids are commercially available in their N“-Fmoc protected form (L-Leu, L-DABAUVV-Boc), L-Thr(O-tBu), L-DAPA(Nß-Boc), D-DAPA(Nß-Boc), D-Phe and L-Asp(O-tBu))whereas unprotected L-OHVaI is commercially available and is easily N“-Fmoc and O-tBu protected. The terf-butyl ester of L-a/lo-Thr is commercially available.
First, a dipeptide is formed by linking Fmoc-L-DABAUVV-Boc) to L-allo-Thr(OH) terf-butylester, using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/triethylamine for thecoupling. Then, (E)-ABA is obtained in this dipeptide by dehydration using lVlartin'ssulfurane [Yokokawa and Shioiri, Tetrahedron Letters 43 (2002) 8679-8682] (Figure 1; inScheme 1), followed by preparative HPLC to isolate the E-isomer from the predominant Z-isomer. The dipeptide Fmoc-L-DABAUVV-Boc) - (E)-DABA is linked to the 2-chlorotritylresin using collidine in DMF, and the linear peptide Fmoc-D-DAPA(Nß-Boc) - L-Leu - L-DABAUVV-Boc) - (E)-ABA - Resin is synthesized by solid-phase peptide synthesis using20% piperidine in DMF for Fmoc deprotection of the resin bound peptide before eachcoupling step and using HCTU (O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)/DIPEA (N,N-diisopropylethylamine) in Dl\/IF ineach coupling step (Figures 1 and 2; in Scheme 1 and 2). The required ester buildingblock (Fmoc-L-Asp(O-tBu)-O-CH[(CH2)6-CH3]-CH2-COOH) of the depsipeptide issynthesized from Fmoc-L-Asp(O-tBu) and allyl (R/S)-3-hydroxydecanoate, in analogy withthe procedure of Mountford et al (Org. Biomol. Chem., 2017, 15, 7173-7180). Followingremoval of the allyl group using palladium tetrakis triphenylphosphine, the ester buildingblock is linked to the resin bound linear peptide using HCTU/DIPEA in DMF.Subsequently, the linear depsipeptide Fmoc-L-Thr - L-DAPA(Nß-Boc) - D-Phe - L-OHVaI- L-Asp(O-tBu) - O-CH[(CH2)6-CH3]-CH2-CO - D-DAPA(Nß-Boc) - L-Leu - L-DABAUVV -Boc) - (E)-ABA - Resin is synthesized as above. After Fmoc deprotection, the N-Boc andO-tBu protected depsipeptide is released from the resin using HFIP(hexafluoroisopropanol) in dichloromethane. Subsequently, the depsipeptide is cyclizedby creating a peptide bond between the N-terminal L-Thr and the C-terminal (E)-ABAresidues, using PyClocK(6-chlorobenzotriazole-1-yloxy-tris-pyrrolidino-phosphoniumhexafluorophosphate)/DIPEA in DMF. Finally, the side chain protecting groups (N-Bocand O-tBu) are removed by treatment with TFA/TIPS (triisopropylsilane) and the peptide 23 U747 is purified from the (S)-3-hydroxydecanoic acid diastereomer by preparative HPLC(column as above, 17.5-35% MeCN in 28 min and 35-57.5% in 7 min, 0.2% formic acid,10 mL/min).
Example 3: Bioloqical Characterization of U747 Minimal inhibitory concentration (MIC) of U747 against a panel of different pathogenicbacteria was measured by means of two methods A) a broth micro-dilution method in 96-wells microtiter plates and B) according to Clinical and Laboratory Standards Institute(CLSI) guidelines (Clinical and Laboratory Standards Institute (CLSI). 2015. M7-A10.Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically,10th ed. Approved standard. CLSI, Wayne, PA).
A) ln the broth micro-dilution method, the test media were either AM3 Broth (BD DifcoLtd) mixed with phosphate buffered saline (PBS, Amresco LLC, Solon, USA) in 1:4 ratio(E. coli, A. baumannii, Enterobacter c/oacae, K/ebsie/la pneumonia and P. aeruginosa),AM3 Broth mixed with PBS in 1:1 ratio (S. aureus) or Malt Extract (ME; 15 g Malt ExtractDico Ltd. in 1000 mL deionized H20; Aspergi/lus fumigatus). Prior to MIC- tests, thebacterial cell concentration/fungal spore concentration was adjusted to betvveen 1 and 5 ><105 cells/spores per mL. Cell concentration was, always, confirmed by viable cell countingdone according to standard dilution method and plating the respective bacterial pathogenon VPA-plates (10 g VPB (0xoid Ltd.), 15 g Bacto Agar (Saveen Werner AB) in 1000 mLdeionized H20) and As. fumigatus on Malt Extract plates (15 g Malt Extract Dico Ltd., 15 gBacto Agar (Saveen Werner AB) in 1000 mL deionized H20 directly after each of MIC-tests. Plates were, subsequently, incubated at 37°C in the dark, and viable coloniescounted after 16 to 24 h. ln order to estimate MIC (concentration range bet\Neen 0.05 and64 ug/mL), the appropriate sample volumes of tested compounds out of the stocksolutions of 100, 10 and 1 and if needed 0.1 ug/mL in Me0H were dispensed into thewells of microtiter plate(s) and the solvent was evaporated in a fume-hood. AftenNards,suspensions of pathogen cells in the appropriate medium were dispensed to the wells ofmicrotiter plates and the growth of pathogens were monitored after 16 to 20 h ofincubation at 37°C in the dark. Positive control comprised respective pathogen cellssuspended in untreated sterile appropriate media without addition of compounds(s) andnegative control comprised the medium only. The MIC was defined as the lowestconcentration of each compound with no visible growth of pathogen. All MIC tests were performed in duplicate and/or triplicate and repeated at least twice. 24 B) Additionally, MIC values for U747 against a selection of pathogens were determinedaccording to Clinical and Laboratory Standards Institute (CLSI) guidelines.lC50, haemolysis rate and frequency of resistance (FoR, Table 4) was determined using standard protocols by different CROs.
Table 3 MIC values (pg/mL) for U747, determined using method A or method B. WT: wild type;ESC: Extended-spectrum cephalosporin; STX: Trimethoprim-sulfa; GEN: Gentamicin;AMK: Amikacin; CIP: Ciprofloxacin; TET: Tetracycline; CML: Chloramphenicole; CARBA:carbapenem; ESBL: extended spectrum ß-lactamase; MRSA: methicillin-resistant Staphylococcus aereus. pathogenltest strain ID Type/Resistance pattern MIC MeähA. baumannii ENOO7 WT 8 BA. baumannii EN016 BM4454 8 8 BA. baumannii EN017 BM4652 (efflux-defective) 8 BA. baumannii LMG1041T WT 1-2 AA. baumannii A219 CARBA 1-2 BA. baumannii A250 CARBA 1-2 BAs. fumigatus 16-32 AE. c/oacae LMG2783T WT 2-4 AE. coli LMG15862 ß-lactamase 2-4 AE. coli EC4163 CARBA 0.5-1 BE. coli EC4129 CARBA 1-2 BE. coli ATCC25922 control 0.5-1 BE. coli En001 WT 8 BE. coli EN002 DtolC (efflux-defective) 8 BE. coli EN0137 ESC, CIP, GEN, SXT 8 BE. coli EN003 D22 (lps mutant, hypersensitive) 4 BE. coli EN0134 ESC, SXT 4 BE. coli EN0135 ESC (I), GEN, TET 4 BE. coli EN0136 ESC, GEN, AMK, SXT 4 BE. coli EN0138 ESC, CIP, SXT, TET, CML 4 BE. coli EN0139 ESC, CIP, AMK (I), SXT, TET 4 BK. pneumoniae LMG20218 ESBL 2-4 AK. pneumoniae EN011 1161486a (efflux- defective) 8 BK. pneumoniae EN0142 ESC, ERT, CIP, GEN+TOB, SXT 8 BK. pneumoniae EN006 WT 4 BK. pneumoniae EN010 1161486 8 BK. pneumoniae EN0140 ESC, CARBA, CIP, GEN+AMK, SXT, CML 8 BK. pneumoniae EN0141 ESC, CIP, GEN, SXT, TET, CML 8 BK. pneumoniae EN0143 ESC, CIP, AMK (I), TOB, SXT 8 BK. pneumoniae EN0144 ESC, CARBA, CIP, GEN, AMK, SXT, TET 8 BP. aeruginosa LMG6395 WT 8-16 AP. aeruginosa PS992 CARBA 2-4 BP. aeruginosa PS826 CARBA 2-4 BP. aeruginosa ATCC27853 control 2-4 BP. aeruginosa EN004 PAO1 WT 32 BP. aeruginosa EN005 PAO750 (efflux-defective) 32 BS. aureus LMG15975 MRSA >32 AS. aureus EN008 ATCC 29213 WT 64 B 26 Table 4 lC50 and haemolysis rate and Frequency of Resistance (FoR) for U747, as determined bydifferent CROs.
Test Cellslstrain ID comment lC50 HepG2 Human liver cell line 50 (ug/mL)Haemolysis rate 100 ulVl 0.6% FoR E. coli EN001 @4 >< MIC and 8 >< MIC <2 ><10'9 FoR E. coli EN002 @4 >< MIC and 8 >< MIC <2 ><10'9 MIC values for other compounds (Table 5) of the invention were determined using methodA.
Table 5 MIC values (ug/mL) and lC50 (ug/mL) for selected compounds of the invention. MICvalues were determined according to method A and lC50 was determined by a CRO using a standard protocol. All MIC values were determined as concentration ranges, and only the lower limits are shown here (i.e. ”'16” means “16-32” and “1” means “1-2”. (nd = not determined).<1- |~ f f g å co |~ f oo Lo oo o o s2 1s >s2fmfëlzeägä”ia s 2 4 4 s s 2 s 2 1s 1s >32 >32 >32 >32 >s2 >s2'fmêgéäggæa 1s s s s s 4 4 s 2 s 4 s 1s >s2 >s2 1s 1sEMÉTÉS§5 >s2 >s2 1s s 1s 1s 4 1s 2 1s 1s >s2 1s 1s >s2 1s s
Claims (1)
1. An isolated or synthesised compound formula (I) as presented below: aa8R?aa? (i)whereiny: 0-5; X: O, NR10, or (CH2),, with n=0 to 10; R: C0-C20 n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkylbranches; wherein double bonds may have E or Z configuration; R1: -(CH2),.-NR11R12, n=0 to 5, or -(CH2)n-CONR11R12, n=0 to 5, or -(CH2)n-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkyl branches; R2: C0-C20 n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkylbranches, wherein double bonds may have E or Z configuration; Rs: -(CH2),.-NR11R12, n=0 to 5, or -(CH2)n-CONR11R12, n=0 to 5, or -(CH2)n-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkyl branches; R4: -(CH2),,-CH=C[(CH2)m-H](CH2)0-H, with n=0 to 10, m=0 to 10, o=0 to 5, and with E orZ double bond, or =C[(CH2)m-H](CH2)0-H, with m=0 to 10, o=0 to 5, with E or Z doublebond; R5: C0-C20 n-alkyl with 0 to 5 C1-C5 n-alkyl branches or n-alkenyl with 0 to 5 C1-C5 n-alkylbranches; wherein double bonds may have E or Z configuration, and 0 to 2 H may be exchanged to any combination of -OR1s, -NR11R12, -F, -C|, -Br, -|, -(CH2)0-H with o=1 to RB: -(CH2)n-NR11R12, n=0 to 5, or -(CH2)n-CONR11R12, n=0 to 5, or -(CH2)n-COO-(CH2)m-H, n=0 to 5 and m=0 to 5, each of which can have 0 to 5 C1-C5 n-alkyl branches; Ry: -(CH2)n-Ph(Y)m, with Y= any combination of -OR13, -NR11R12, -F, -C|, -Br, -|, -(CH2)0-H, and with n=0 to 5, m=0 to 5, and o=1 to 20; Ra: CO-Czo n-alkyl with 0 to 5 C1-C5 n-alkyl branches or C0-C20 n-alkenyl with 0 to 5 C1-C5n-alkyl branches, wherein double bonds may have E or Z configuration and 0 to 2 H maybe exchanged to any combination of -OR1s, -NR11R12, -F, -C|, -Br, -|, -(CH2)0-H with o=1to 5; Rg: -(CH2)n-CONR11R12 or -(CH2),.-COO-(CH2)m-H, n=0 to 5 and m=0 to 5; R10: -H, -OR13, -(CH2)0-H with o=1 to 5, -F, -C|, -Br, or -|, independently; R11 and R12: any combination of -H, C1-C5 n-alkyl, and C1-C5 n-alkenyl, or any commonN-protecting group; and R13: -(CH2),.-H, with n=0 to 5; or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt thereof, wherein each stereocenter may be either R or S. _ A compound according to c|aim 1, wherein .~¿.~_.\~..«\,-\ -,~_.~\ .\;.-_¿».-~«.~ _: ¿_-_..-\_ A compound <.=~..\..\.=:\..=_=_\_~ t._,<..:=== ~_~_.=i _ Acompound accordingto “=ë~~ y = 1; R: C6-C10 n-alkyl or C6-C10 n-alkenyl, with 0 or 1 methy| branch; R1 and RB: both -CH2-NR11R12 or one -CH2-NR11R12 and one -CH2-CO-NR11R12;R2: -CH2-CH(CH3)-CH3; Rs: -CH2-CH2-NR11R12; R4: E or Z =CH-CH3; R5: -CH(OR13)-CH3; Ry: -CHz-Ph; Ra: -CH(CH3)-CH3 or -COR13(CH3)-CH3; and Rg: -CHz-COOH or -CH2-CONR11R12_ f/'ï-j.) wherein R10: H; and X: O. 10. 11. 12. 13. 14. 15. 16. ' L " ' ~ “ i' =^ " '*'*'*“*** LW aa2-aa5, aa8, and aa9 are o. :xrgci :::ï~ï.:*:;:::::::ñ*of 3:: _ A compound according to any one of the preceding claims, wherein-RQ: -CHz-COOH; R11and R12: H; Rs: -CHOH-CHg, wherein the stereocenter is R; and Ra: -CH(CH3)-CH3 or -COH(CH3)-CH3. Ra: -COH(CHs)-C|-le.. . isReadi 1-aaišfi aa6, aa8, and aa9 are S; and the stereocenter of of aa7 is R. Acompound aocordingtoanyoneofthe precedingdaims,\A/heiein_;<==== . .was-z ~.«\.-\\.«\.~ _.\. , »V z .\\.~:cc\~ Qxsqsgf.. .tz _. . s; “ .-'.».~'\.-.-. .ut .ams-v .w . ss» w =f.-~,\\~'^“ 3 .:i\\:rmt'e$§.-\.\G.= må vvïkš rr-“s .i \>\\. EcSXI ::§\\.1:=.\'“\s~ vi . .C _ A. \,~\.\.~\.\.\ ._ “nu \ . _» o.. A method of producing a compound according to any one of claims by synthesis or culture of a microorganism. A method according to claim---I='“i§, which is a method of synthesis wherein a peptidecomprising at least nine amino acids and including at least one first amino acid followedby the sequence of Leu; DABA; ABA; Thr; DAPA; Phe; Val; and Asp; wherein the firstamino acid is DAPA or Asn, and wherein Val may be modified to OHVal, is used as an intermediate. A method according to wherein the intermediate is peptide is defined by any oneofSEQ ID NOs: 1-5. A method according to claimåï-šå, Which includes the culture of a microorganism whichnatively produces one or more compounds or peptides as defined by any one of claims1-6, 8 and 9. A method according to claim--fšilšiågjjšd wherein the microorganism LMG P-30868 depositedwith BCCM-LMG is used. A method according to claim--}ï"-;š§_, Which includes recombinant DNA expression of one or An expression system arranged for the production of one or more compounds or peptides as defined by any one of claims and An expression system according to claimßlšäïš t å; which comprises one or more microorganisms, such as a bacteria or fungi. An expression system according to wherein the microorganism(s) is derived from the genus Pedobacter. 17. 18. 19. 20. 21. 22. 23. 24 25. 26. .A compound An expression system according to claimlëšå;30868 deposited with BCCM-LMG. wherein the microorganism is LMG P- An expression system according to any one of claims~~1ï5~§~> which is a recombinant expression system.A compound according to any one of c|aims for use as a medicament. A compound according to any one of c|aims 1-š§~f§f__for use in the treatment or preventionof at least one infection caused by one or more Gram-positive or Gram-negative bacteria. A compound f_°_;_§_fj__g=s_§§-3.___according to wherein the infection is caused by one or more Gram-positive bacteria. A compound _according to claimä; wherein said one or more Gram-positivebacteria are selected from the group consisting of Actinomyces; Bacillus; Clostridium;Corynebacterium; Enterococcus; Gardnerella; Lactobacillus; Listeria; Micrococcus; Mycobacteria; Nocardia; Staphylococcus; and Streptococcus. A compoundiçgg1ggggg>i according to claim-~~'I:'~~$.š§§j§_, wherein the infection is caused by one or more Gram-negative bacteria. gggaccording to wherein said one or more Gram-negativebacteria are selected from the group consisting of Acinetobacter; Batronella; Bordetella; Borrelia; Bruce/la; Burkho/deria; Campylobacter; Ch/amydia; Ch/amydophi/ia; Citrobacter; Coxie/la; Ehrlichia; Enterobacter; Escherichia; Francisel/a; Fusobacterium; Haemophilus; Helicobacter; Klebsiel/a; Legionella; Leptospira; Moraxella; Mycoplasma;Neisseria; Proteus; Pseudomonas; Rickettsia; Salmonella; Serratia: Shigel/a; Treponema; Vibrio; and Yersinia. A pharmaceutical preparation comprising a compound according to any one of c|aims 1- together with pharmaceutically acceptable carrier(s) and/or adjuvant(s). A preparation according to claim~~iš~ífl , which is medically effective against bacteriaselected from the group consisting of Acinetobacter; Bacillus; Campylobacter;Chlamydia: Clostridium; Enterobacter; Enterococcus; Escherichia; Haemophilus;Helicobacter; Klebsiel/a; Legionella; Mycobacteria; Neisseria; Pseudomonas; Salmonella; Shigel/a; Staphy/ococcus; Streptococcus; Vibrio; and Yersinia. 27. A preparation according to claim<§š= 28. resistant, and/or multiresistant, to antibiotics selected from the group consisting ofaminoglycosides; ansamycins; carbapenems; cephalosporins; fluoroquinolons;glycopeptides; lincosamides; lipopeptides; macrolides; monobactams; nitrofurans; oxazolidinones; penicillin ; polypeptides; quinolones; sulphonamides; and tetracyclines. Use of a compound according to any one of claims in a method for decolonizationof a surface of Gram-positive and/or Gram-negative bacteria where the surface is a part of clinical equipment, such as the surface of a metal or plastic article.
Priority Applications (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE1851022A SE543054C2 (en) | 2018-08-28 | 2018-08-28 | Cyclic peptides |
| CN201980056367.6A CN112912383A (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
| CA3110143A CA3110143A1 (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
| PCT/SE2019/050789 WO2020046190A1 (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
| AU2019329130A AU2019329130A1 (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
| KR1020217007592A KR20210049837A (en) | 2018-08-28 | 2019-08-27 | Peptide compound |
| EP19762500.7A EP3844174A1 (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
| JP2021510977A JP7492503B2 (en) | 2018-08-28 | 2019-08-27 | Peptide Compounds |
| US17/268,694 US11981755B2 (en) | 2018-08-28 | 2019-08-27 | Peptide compounds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE1851022A SE543054C2 (en) | 2018-08-28 | 2018-08-28 | Cyclic peptides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| SE1851022A1 SE1851022A1 (en) | 2020-02-29 |
| SE543054C2 true SE543054C2 (en) | 2020-09-29 |
Family
ID=69941533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SE1851022A SE543054C2 (en) | 2018-08-28 | 2018-08-28 | Cyclic peptides |
Country Status (1)
| Country | Link |
|---|---|
| SE (1) | SE543054C2 (en) |
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2018
- 2018-08-28 SE SE1851022A patent/SE543054C2/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| SE1851022A1 (en) | 2020-02-29 |
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