EP3555115A1 - Nouveaux composés en tant qu'agents anti-mycobactériens - Google Patents

Nouveaux composés en tant qu'agents anti-mycobactériens

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Publication number
EP3555115A1
EP3555115A1 EP17881453.9A EP17881453A EP3555115A1 EP 3555115 A1 EP3555115 A1 EP 3555115A1 EP 17881453 A EP17881453 A EP 17881453A EP 3555115 A1 EP3555115 A1 EP 3555115A1
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EP
European Patent Office
Prior art keywords
alkyl
bacteria
compound
salt
compound according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17881453.9A
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German (de)
English (en)
Other versions
EP3555115A4 (fr
Inventor
Richard J. Payne
Warwick John BRITTON
Anh Tran
Wendy TRAN
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University of Sydney
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University of Sydney
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Priority claimed from AU2016905229A external-priority patent/AU2016905229A0/en
Application filed by University of Sydney filed Critical University of Sydney
Publication of EP3555115A1 publication Critical patent/EP3555115A1/fr
Publication of EP3555115A4 publication Critical patent/EP3555115A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/08—Antibacterial agents for leprosy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0217—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -C(=O)-C-N-C(=O)-N-C-C(=O)-
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to antibacterial compounds.
  • the compounds are for inhibiting the growth of bacteria, particularly Mycobacterium tuberculosis (Mtb), and/or targeting bacteria having phospho-MurNAc-pentapeptide translocase.
  • Mtb Mycobacterium tuberculosis
  • the present disclosure also relates to compositions containing these compounds and methods of the use of these compounds and compositions.
  • Tuberculosis is caused by infection with the bacterium Mycobacterium tuberculosis (Mtb). According to the World Health Organization, TB was responsible for 1 .5 million deaths and the emergence of 9.6 million new cases of the disease in 2014.
  • Current treatment for TB includes a six month quadruple therapy comprising rifampicin, isoniazid, ethambutol and pyrazinamide.
  • This therapy provides an exceptional cure rate of >95% for drug-sensitive TB, however, it is not effective against multi-drug resistant (MDR) and extensively drug resistant (XDR) TB. This is of growing concern globally.
  • Second-line antibiotics administered for extended periods can be effective in treating MDR infections, but there are virtually no treatments available for XDR infections.
  • the present application provides compounds, particularly antibacterial compounds. These compounds inhibit bacterial cell wall biosynthesis and/or bacterial growth and have been shown to be particularly active against Mycobacterium tuberculosis (Mtb).
  • Mcb Mycobacterium tuberculosis
  • the invention provides a compound according to Formula I:
  • Ri is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; Ci- C6 alkoxy; C1-C6 alkylthio; C1-C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyi, C1-C6 alkylcarboxy, Ci-C 6 alkylcarboxyamide, C 3 -C 7 cycloalkyl; (CrC 4 alkyl)C 3 -C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-i- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C
  • R 2 is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; Ci- C6 alkoxy; C1-C6 alkylthio; C1-C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4
  • R 4 is selected from the group consisting of hydroxyl or optionally substituted: Ci- C-I 5 alkyl, C1-C15 alkylamino; C1-C15 alkoxy; C1-C15 alkylthio; C1-C15 haloalkyi, C1-C15 haloalkoxy, C1-C15 hydroxyalkyl, C1-C15 alkylcarboxy, C1-C15 alkylcarboxyamide, C3-C 7 cycloalkyl; (Ci-C 4 alkyl)C 3 -C 7 cycloalkyl, C 3 -C 7 heterocyclyl; (Ci-C 4 alkyl)C 3 -C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci-C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 aralkylamino, heteroaryl, (Ci-C 4 alkyl)
  • R 3 when is a double bond, R 3 is not -COOH, and when is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C 3 -C 4 alkylamino, -CH(CH 3 )OH, -CH 2 COOH, - CH 2 C(0)C(CH 3 ) 3 , -CH(OH)CH 3 or -CH 2 CH 2 SCH 3 ; and R 2 is methyl, isopropyl, isobutyl, -(CH 2 ) 4 NH 2 , -CH 2 CH 2 SCH 3 , -CH 2 CH 2 S(0)CH 3 , C C 2 aralkyl, fluoro-substituted benzyl, -CF 3 -substituted benzyl, aryl-substituted benzyl, naphthyl or -CH 2 -cyclo
  • the invention provides a compound according to Formula II:
  • R-i is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: CrC 6 alkyl, Ci-C 6 alkylamino; Ci- Ce alkoxy; C1-C6 alkylthio; C1-C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 aral
  • R 2 is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; Ci- Ce alkoxy; C1-C6 alkylthio; C1-C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C 7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C 3 -C 7 heterocyclyl; (Ci-C 4 alkyl)C 3 -C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, d- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C
  • R 3 is -C(0)R 4 or -CH 2 R 4; ;
  • R 4 is selected from the group consisting of hydroxyl or optionally substituted: Ci- C-I 5 alkyl, C1-C15 alkylamino; C1-C15 alkoxy; C1-C15 alkylthio; C1-C15 haloalkyl, C1-C15 haloalkoxy, C1-C15 hydroxyalkyl, C1-C15 alkylcarboxy, C1-C15 alkylcarboxyamide, C3-C7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci-C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 aralkylamino, heteroaryl, (CrC 4 alkyl)heteroary
  • R 3 when is a double bond, R 3 is not -COOH, and when is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C 3 -C 4 alkylamino, -CH(CH 3 )OH, -CH 2 COOH, - CH 2 C(0)C(CH 3 ) 3 , -CH(OH)CH 3 or -CH 2 CH 2 SCH 3 ; and R 2 is methyl, isopropyl, isobutyl, -(CH 2 ) 4 NH 2 , -CH 2 CH 2 SCH 3 , -CH 2 CH 2 S(0)CH 3 , C1-C2 aralkyl, fluoro-substituted benzyl, -CF 3 -substituted benzyl, aryl-substituted benzyl, naphthyl or -CH 2 -
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to Formula I and/or Formula II and a pharmaceutically acceptable excipient.
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient.
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb), comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient.
  • Mtb Mycobacterium tuberculosis
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc- pentapeptide transiocase, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient.
  • the invention provides a method of prevention and/or treatment of tuberculosis comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient.
  • the invention provides use of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by gram positive bacteria.
  • the invention provides use of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb).
  • Mtb Mycobacterium tuberculosis
  • the invention provides use of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc-pentapeptide translocase.
  • the invention provides use of a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of tuberculosis.
  • the invention provides a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by bacteria, such as gram positive or gram negative bacteria.
  • the invention provides a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb).
  • Mtb Mycobacterium tuberculosis
  • the invention provides a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc- pentapeptide translocase.
  • the invention provides a compound according to Formula I and/or Formula II or a composition comprising a compound of Formula I and/or Formula II and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of tuberculosis.
  • a pharmaceutically acceptable excipient for use in the prevention and/or treatment of tuberculosis.
  • FIG 18. Inhibition of Mtb H37Rv by analogue 34.
  • Figure 19. Inhibition of Mtb H37Rv by analogue 35.
  • Figure 20. Inhibition of Mtb H37Rv by analogue 36.
  • Figure 21. Inhibition of Mtb H37Rv by analogue 37.
  • FIG. 22 Inhibition of Mtb H37Ra in THP-1 cells by analogue 25.
  • IC 5 o represents average of 2 independent experiments, each performed in triplicate.
  • FIG. 23 Inhibition of Mtb H37Ra in THP-1 cells by analogue 36.
  • IC50 represents average of 2 independent experiments, each performed in triplicate.
  • FIG. 24 Inhibition of Mtb H37Ra in THP-1 cells by analogue 37.
  • IC 5 o represents average of 2 independent experiments, each performed in triplicate.
  • Figure 25 TLC assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 25.
  • C Log-transformed dose-response curve for inhibition of MurX by 25.
  • FIG. 26 TLC assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 36.
  • A TLC
  • B Raw dose-response curve for inhibition of MurX by 36
  • C Log-transformed dose-response curve for inhibition of MurX by 36.
  • FIG. 27 TLC assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 37.
  • A TLC
  • B Raw dose-response curve for inhibition of MurX by 37
  • C Log-transformed dose-response curve for inhibition of MurX by 37.
  • FIG. 28 TLC assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue tunicamycin.
  • A TLC
  • B Raw dose-response curve for inhibition of MurX by tunicamycin
  • C Log-transformed dose-response curve for inhibition of MurX by tunicamycin.
  • FIG. 29 Evaluation of kinetic parameters of MurX.
  • A Michaelis-Menten plot;
  • B Raw enzyme kinetics data. Data represents average of two independent experiments.
  • FIG. 30 Fluorescence assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 25.
  • FIG. 31 Fluorescence assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 36.
  • Figure 32 Fluorescence assay from Mtb mc 2 6230 membranes for the inhibition of MurX by analogue 37.
  • FIG 33 Fluorescence assay from Mtb mc 2 6230 membranes for the inhibition of MurX by tunicamycin (positive control).
  • the present invention describes compounds for the prevention and/or treatment of a disease or condition modulated by bacteria.
  • the disease or condition may be associated with, caused by, or arise from the relevant bacteria infection.
  • the bacteria are gram positive bacteria.
  • the bacteria are Mycobacterium tuberculosis (Mtb).
  • the compounds provided herein are useful in the prevention and/or treatment of conditions modulated by bacteria. Without wishing to be bound to any theory, it is postulated that the activity of these compounds is through the inhibition of bacterial growth and/or the inhibition of bacterial cell wall biosynthesis.
  • the compounds are particularly selective for Mycobacterium tuberculosis (Mtb) and may therefore be useful in the treatment of diseases such as tuberculosis.
  • the compounds may also be useful in the prevention and/or treatment of other conditions resulting from bacteria.
  • the invention provides a compound according to Formula I:
  • R-i is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1 -C6 alkylamino; Ci - C 6 alkoxy; CrC 6 alkylthio; Ci -C 6 haloalkyl, CrC 6 haloalkoxy, CrC 6 hydroxyalkyl, Ci -C 6 alkylcarboxy, C1 -C6 alkylcarboxyamide, C3-C7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C3-C7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 a
  • R 2 is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; Ci- Ce alkoxy; C1-C6 alkylthio; C1-C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C 7 cycloalkyl; (Ci-C 4 alkyl)C3-C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci-C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci- C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 a
  • R 3 is -C(0)R 4 or -CH 2 R 4; ;
  • R 4 is selected from the group consisting of hydroxyl or optionally substituted: Ci- Ci5 alkyl, C1-C15 alkylamino; C1-C15 alkoxy; C1-C15 alkylthio; C1-C15 haloalkyi, C1-C15 haloalkoxy, C1-C15 hydroxyalkyl, C1-C15 alkylcarboxy, C1-C15 alkylcarboxyamide, C 3 -C 7 cycloalkyl; (C1-C4 alkyl)C 3 -C 7 cycloalkyl, C 3 -C 7 heterocyclyl; (C1-C4 alkyl)C 3 -C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci-C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 aralkylamino, heteroaryl, (Ci-C 4 alkyl)he
  • R 3 when is a double bond, R 3 is not -COOH, and when is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C 3 -C 4 alkylamino, -CH(CH 3 )OH, -CH 2 COOH, - CH 2 C(0)C(CH 3 ) 3 , -CH(OH)CH 3 or -CH 2 CH 2 SCH 3 ; and R 2 is methyl, isopropyl, isobutyl, -(CH 2 ) 4 NH 2 , -CH 2 CH 2 SCH 3 , -CH 2 CH 2 S(0)CH 3 , C1-C2 aralkyl, fluoro-substituted benzyl, -CF 3 -substituted benzyl, aryl-substituted benzyl, naphthyl or -CH 2 -
  • Ri is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1 -C6 alkylamino; Ci - C6 alkoxy; C1-C6 alkylthio; C1 -C6 haloalkyi, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1 -C6 alkylcarboxy, Ci -C 6 alkylcarboxyamide, C3-C7 cycloalkyl; (CrC 4 alkyl)C 3 -C 7 cycloalkyl, C3-C 7 heterocyclyl; (Ci -C 4 alkyl)C3-C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci- C 4 aralkyl, Ci -C 4 aralkoxy
  • R 3 is -C(0)R 4 or -CH 2 R 4; ;
  • R 4 is selected from the group consisting of hydroxyl or optionally substituted: Ci- C-I 5 alkyl, C1-C15 alkylamino; C1-C15 alkoxy; C1-C15 alkylthio; C1-C15 haloalkyi, C1-C15 haloalkoxy, C1-C15 hydroxyalkyl, C1-C-1 5 alkylcarboxy, C1-C15 alkylcarboxyamide, C 3 -C 7 cycloalkyl; (C1-C4 alkyl)C 3 -C 7 cycloalkyl, C 3 -C 7 heterocyclyl; (C1-C4 alkyl)C 3 -C 7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, Ci-C 4 aralkyl, Ci-C 4 aralkoxy, Ci-C 4 aralkylamino, heteroaryl, (Ci-C 4 alky
  • R 3 when is a double bond, R 3 is not -COOH, and when is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C 3 -C 4 alkylamino, -CH(CH 3 )OH, -CH 2 COOH, - CH 2 C(0)C(CH 3 ) 3 , -CH(OH)CH 3 or -CH 2 CH 2 SCH 3 ; and R 2 is methyl, isopropyl, isobutyl, -(CH 2 ) 4 NH 2 , -CH 2 CH 2 SCH 3 , -CH 2 CH 2 S(0)CH 3 , C1-C2 aralkyl, fluoro-substituted benzyl, -CF 3 -substituted benzyl, aryl-substituted benzyl, naphthyl or -CH 2 -
  • the compound is a compound according to Formula III:
  • R-i , R 2 and R 4 are defined above as for Formula II, with the proviso that: when is a double bond, R 4 is not -OH, and when is a single bond and R-i is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C 3 -C 4 alkylamino, -CH(CH 3 )OH, -CH 2 COOH, - CH 2 C(O)C(CH 3 ) 3 , -CH(OH)CH 3 or -CH 2 CH 2 SCH 3 ; and R 2 is methyl, isopropyl, isobutyl, -(CH 2 ) 4 NH 2 , -CH 2 CH 2 SCH 3 , -CH 2 CH 2 S(O)CH 3 , C1 -C2 aralkyl, fluoro-substituted benz
  • Formula IV or a salt, solvate, polymorph or prodrug thereof; wherein R-i , R 2 and R 4 are defined as above for Formula II.
  • Ri is selected from the group consisting of hydrogen or optionally substituted: C1-C6 alkyl, C1-C2 aralkyl, optionally substituted (C1-C2 alkyl)heteroaryl, Ci- C 6 alkylamino; Ci-C 6 alkyloxy, Ci-C 6 alkylcarboxy, Ci-C 6 hydroxyalkyl. Even more preferably, Ri is selected from the group consisting of hydrogen, methyl, hydroxyl- substituted benzyl, naphthyl, C2-C 4 alkylamino, cyclohexyl, -CH 2 -cyclohexyl or - CH(OH)CH 3 .
  • R 2 is selected from the group consisting of optionally substituted: Ci- C6 alkyl, (C1-C2 alkyl)C3-C 7 cycloalkyl, Ci-C 4 aralkyl and C1-C6 alkylthio. Even more preferably, R 2 is selected from the group consisting of: isopropyl, -CH 2 -naphthyl, -CH 2 - cyclohexyl, -CH 2 CH 2 SCH 3 ; -CH 2 CH 2 S(0)CH 3 and
  • R 4 is selected from the group consisting of optionally substituted: Ci- Ci5 alkyloxy, Ci-C 4 aralkyl; C1-C15 alkylamino; Ci-C 4 aralkoxy, Ci-C 4 aralkylamino and C1-C15 alkylamino. More preferably, R 4 is selected from the group consisting of methoxy, hexoxy, dodecanyloxy, hydroxy, -CH 2 C(CH 3 ) 3 , -O-benzyl, -NH-benzyl, -NH- benzyl, hexylamino. Even more preferably, R 3 is selected from the group consisting of - CH 2 C(CH 3 ) 3 , -O-benzyl, -NH-benzyl and hexylamino.
  • the compound is selected from the group consisting of:
  • the compound of Formula I and/or Formula II is selected from the groups consisting of:
  • the compound of Formula I and/or Formula II is selected from the group consisting of:
  • the invention provides a compound selected from the following:
  • alkyl refers to a saturated or unsaturated, straight or branched chain hydrocarbon radical having from one to fifteen carbon atoms, or any range between, i.e. it contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 carbon atoms. The term therefore encompasses saturated alkyl groups as well as alkenyl and alkynyl groups. The alkyl group is optionally substituted with substituents, multiple degrees of substitution being allowed.
  • alkyl examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, propenyl, propynyl n-butyl, butenyl, isobutyl, t-butyl, n-pentyl, pentenyl, isopentyl, and the like.
  • Saturated alkyl groups may be mono-, di- or poly-unsaturated, and may contain double (alkyenyl) or triple (alkynyl) bonds.
  • C1 -C3 alkyl refers to an alkyl group, as defined above, containing at least 1 , and at most 3, 4, 6 or 15 carbon atoms respectively, or any range in between (e.g. alkyl groups containing 2-5 carbon atoms are also within the range of C-i-Ce).
  • halogen refers to fluorine (F), chlorine (CI), bromine (Br), or iodine (I) and the term “halo” refers to the halogen radicals fluoro (-F), chloro (- CI), bromo (-Br), and iodo (-I).
  • 'halo' is fluoro or chloro.
  • cycloalkyl refers to a non-aromatic cyclic hydrocarbon ring.
  • C3-C7 cycloalkyl refers to a non-aromatic cyclic hydrocarbon ring having from three to seven carbon atoms, or any range in between.
  • the C3-C7 cycloalkyl group would also include cycloalkyl groups containing 4 to 6 carbon atoms.
  • the alkyl group is as defined above, and may be substituted.
  • C3-C7 cycloalkyl groups useful in the present invention include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Such a ring may be optionally fused to one or more other optionally substituted cycloalkyl ring(s), "heterocyclic” ring(s), aryl ring(s) or heteroaryl ring(s).
  • heterocyclic or “heterocyclyl” refer to a nonaromatic heterocyclic ring, being saturated or having one or more degrees of unsaturation, containing one or more heteroatoms, substitution selected from S, S(O), S(O)2, O, or N.
  • C3-C7 heterocyclyl refers to a non-aromatic cyclic hydrocarbon ring having from three to seven carbon atoms containing one or more heteroatom substitutions as referred to herein. The heterocyclic moiety may be substituted, multiple degrees of substitution being allowed.
  • C3-C7 heterocyclyl also includes heterocyclyl groups containing C 4 -Cs, C5-C7, C6-C7, C 4 -C7, C 4 -C6 and C5-C6 carbon atoms.
  • the heterocyclic ring contains four to six carbon atoms and one or two heteroatoms. More preferably, the heterocyclic ring contains five carbon atoms and one heteroatom, or four carbon atoms and two heteroatom substitutions, or five carbon atoms and one heteroatom.
  • Such a ring may be optionally fused to one or more other optionally substituted "heterocyclic" ring(s), cycloalkyl ring(s), aryl ring(s) or heteroaryl ring(s).
  • heterocyclic moieties include, but are not limited to, tetrahydrofuran, pyran, oxetane, 1 ,4-dioxane, 1 ,3-dioxane, piperidine, piperazine, N- methylpiperazinyl, 2,4-piperazinedione, pyrrolidine, imidazolidine, pyrazolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene and the like.
  • One substituted heterocyclyl system of this invention is a substituted coumarin ring, such as 6,7-dimethoxy coumarin.
  • (Ci-C 2 alkyl)C 3 -C7 heterocyclyl includes heterocyclyl groups containing an alkyl group containing 1 or 2 carbon atoms as a linker between the compound and the heterocycle (i.e. heterocycle, -CH 2 -heterocycle or -CH 2 CH 2 -heterocycle). These heterocycles may be further substituted.
  • Substituted cycloalkyl and heterocyclyl groups may be substituted with any suitable substituent as described below.
  • aryl refers to an optionally substituted benzene ring or to an optionally substituted benzene ring system fused to one or more optionally substituted benzene rings to form, for example, anthracene, phenanthrene, or naphthalene ring systems.
  • aryl groups include, but are not limited to, phenyl, 2-naphthyl, 1 -naphthyl, biphenyl, as well as substituted derivatives thereof.
  • heteroaryl refers to a monocyclic five, six or seven membered aromatic ring, or to a fused bicyclic or tricyclic aromatic ring system comprising at least one monocyclic five, six or seven membered aromatic rings.
  • These heteroaryl rings contain one or more nitrogen, sulfur, and/or oxygen heteroatoms, where N-oxides and sulfur oxides and dioxides are permissible heteroatom substitutions and may be optionally substituted with up to three members.
  • heteroaryl groups used herein include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxo-pyridyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazyl, pyrazinyl, pyrimidyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, and substituted versions thereof.
  • alkylthio refers to an alkyl group as defined above that is either bonded through one or more sulfur (— S— ) atom or sulfur groups, or contains one or more sulfur atoms or groups within or at the end of the alkyl chain. It includes, for example, the following groups: thiols, thioesters, thioethers, alkylsulfides, alkylsulfanyls, alkylsulfenyls, alkylsulfonyls, alkylsulfanoic acids, alkylsulfenic acids and alkylsulfonic acids.
  • alkylthio groups include -CH 2 CH 2 S0 2 CH 3 , - CH 2 S(0)OH, -SO2CH2CH3, -CH2S-OH, -CH2SH, -CH3CH2SCH3, CH 2 CH 2 S(0)CH3, CH2CH2SO3H. These groups may be further substituted, as discussed below, to provide groups such as alkylsulfonylamino.
  • alkoxy refers to an alkyl group as defined above that is either bonded through one or more oxygen (— 0— ) atoms, or contains one or more oxygen atoms within the alkyl chain. Such groups therefore encompass esters, ethers, ketones and aldehydes. Some non-limiting examples of alkoxy groups include - CH2CH2OCH3, -OCH2CH3, -CO2CH2CH3, -CH2OH, -CH 3 CH 2 C(0)CH 3 , - CH2CH2COOCH3, -OCH2CH2OCH3, or the like. These groups may be further substituted, as discussed below.
  • alkylamino refers to an alkyl chain, as defined above, which includes an amine group within the alkyl chain, or an amine at the start of the chain adjoining the substituent to the rest of the compound, or at the end of the chain.
  • an alkylamino group may be groups such as -CH2CH2NHCH3, - NHCH2CH3, -CH 2 N(CH 3 )2, -CH2CH2NH2 or the like. Depending where the substituent is bound, this may, for example, result in an amide (for example, where the substituent is bound through the amine at R 3 ). Alkylamino groups may be further substituted, as discussed below.
  • haloalkyl refers to an alkyl group as defined above that is bonded to a halo group.
  • Some examples of an haloalkyl group include -CH 2 F, -CF 3 and -CH2CH2CH2CHCI2. Where possible, these groups may be further substituted, as discussed below. For example, -CH 2 C(0)CH 2 CHCl2.
  • hydroxyalkyl refers to an alkyl group as defined above that is bonded to a hydroxy group. These groups may be further substituted, as discussed below.
  • alkylcarboxy refers to an alkyl group as defined above that is bonded to a carboxy group. These groups may be further substituted, as discussed below.
  • haloalkoxy refers to an alkoxy group as defined above that is bonded to a halo group. These groups may be further substituted, as discussed below.
  • alkylcarboxyamide refers to an alkyl group as defined above that is bonded to a carboxyamide group. These groups may be further substituted, as discussed below.
  • aryloxy refers to an aryl group as defined above that is bonded through an oxygen (— 0— ) atom, amino group or thio group respectively.
  • the amino and thio groups may be further substituted, as discussed below.
  • aralkyl refers to an aryl group that is bonded through an alkyl group as defined above. These groups may be further substituted, as discussed below. Where the group is stated with a specific carbon atom range, such as "Ci-C 4 aralkyl” the Ci-C 4 refers to the number of carbon atoms in the alkyl component of the group.
  • aralkoxy refers to an aryl group that is bonded through an alkyloxy group as defined above. These groups may be further substituted, as discussed below. Where the group is stated with a specific carbon atom range, such as “Ci-C 4 aralkoxy” the Ci-C 4 refers to the number of carbon atoms in the alkyl component of the group.
  • aralkylamino refers to an aryl group that is bonded through an alkylamino group as defined above. These groups may be further substituted, as discussed below. Where the group is stated with a specific carbon atom range, such as "Ci-C 4 aralkylamino" the Ci-C 4 refers to the number of carbon atoms in the alkyl component of the group.
  • a “ring substituent” may be a moiety such as a halogen, alkyl group, or other substituent described herein that is covalently bonded to an atom, preferably a carbon or nitrogen atom, that is a ring member.
  • substituted means that any one or more hydrogens on the designated atom is replaced with a selection from the indicated substituents, provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound, i.e., a compound that can be isolated, characterized and tested for biological activity.
  • substituents include but are not limited to:
  • any of these groups may be further substituted by any of the above-mentioned groups, where appropriate.
  • salts of the compound are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts.
  • pharmaceutically acceptable derivative may include any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound which upon administration to a subject, is capable of providing (directly or indirectly) a compound of Formula I, Formula II, Formula III and/or Formula IV or an active metabolite or residue thereof.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, n
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium such as salts formed from triethylamine, alkoxyammonium such as those formed with ethanolamine and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine.
  • pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium such as salts formed from triethylamine, alkoxyammonium such as those formed with ethanolamine and salts formed from ethylenediamine, choline or amino acids such as arginine, lysine or histidine.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (eg, two, three or four) amino acid residues which are covalently joined to free amino, and amido groups of compounds of Formula I, Formula II, Formula III and/or Formula IV.
  • the amino acid residues include the 20 naturally occurring amino acids commonly designated by three letter symbols and also include, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone.
  • Prodrugs also include compounds wherein carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above substituents of Formula I, Formula II, Formula III and/or Formula IV through the carbonyl carbon prodrug sidechain.
  • polymorph includes any crystalline form of compounds of Formula I, Formula II, Formula III and/or Formula IV, such as anhydrous forms, hydrous forms, solvate forms and mixed solvate forms.
  • a "solvate” is formed by the interaction of a compound of the present invention with a solvent.
  • the compounds also may have use in treating other diesases or conditions arising from other bacteria, such as A. baumannii, E. coli, P. aeruginosa, S. aureus, methicillin-resistant Staphylococcus aureus, V. cholera, E. aerogenes, 0. anthropi, P. alcalifaciens, B. subtilis, E. faecium, L. ivanovii, S. epidermidis, S. typhimurium and Y. pseudotuberculosis.
  • A. baumannii E. coli, P. aeruginosa
  • S. aureus methicillin-resistant Staphylococcus aureus
  • V. cholera V. cholera
  • E. aerogenes 0. anthropi
  • P. alcalifaciens B. subtilis
  • E. faecium L. ivanovii
  • S. epidermidis S. typhimurium
  • these other bacteria are gram positive.
  • these bacteria are Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus, Mycobacterium bovis, Bacillus subtilis, Staphylococcus epidermis, Enterococcus faecium, Listeria ivanovii, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus.
  • these bacteria are Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium bovis. The compounds may therefore be useful in treating opportunistic infections in
  • HIV/AIDS and cystic fibrosis, leprosy and bronchiectasis and other diseases resulting from these or similar bacteria are associated with cystic fibrosis, leprosy and bronchiectasis and other diseases resulting from these or similar bacteria.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient.
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient.
  • the bacteria is A. baumannii.
  • the bacteria is E. coli.
  • the bacteria is P. aeruginosa.
  • the bacteria is S. aureus.
  • the bacteria is
  • the bacteria is V. cholerae. In a further embodiment, the bacteria is E. aerogenes. In another
  • the bacteria is 0. anthropi. In a further embodiment, the bacteria is P. alcalifaciens. In another embodiment, the bacteria is B. subtilis. In yet another embodiment, the bacteria is E. faecium. In a further embodiment, the bacteria is L.
  • the bacteria is S. epidermidisAn still a further embodiment, the bacteria is S. typhimurium. In another embodiment, the bacteria is V. pseudotuberculosis.
  • the bacteria are gram positive.
  • the bacteria are Mycobacterium tuberculosis (Mtb), Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus, Mycobacterium bovis, Bacillus subtilis, Staphylococcus epidermis, Enterococcus faecium, Listeria ivanovii, Staphylococcus aureus, methicillin- resistant Staphylococcus aureus.
  • the bacteria are Mycobacterium tuberculosis (Mtb), Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium bovis.
  • the bacteria are Mycobacterium tuberculosis (Mtb).
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb), comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient.
  • Mtb Mycobacterium tuberculosis
  • the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc- pentapeptide transiocase, comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient.
  • the invention provides a method of prevention and/or treatment of tuberculosis comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient.
  • the invention provides use of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by gram positive bacteria.
  • the invention provides use of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb).
  • Mtb Mycobacterium tuberculosis
  • the invention provides use of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc-pentapeptide translocase.
  • the invention provides use of a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNA
  • Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient in the preparation of a medicament for the prevention and/or treatment of tuberculosis.
  • the invention provides a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by bacteria, preferably gram positive bacteria.
  • the invention provides a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb).
  • Mtb Mycobacterium tuberculosis
  • the invention provides a compound according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof, or a composition comprising a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof and a pharmaceutically acceptable excipient for use in the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc-pentapeptide translocase.
  • the invention provides a compound according to Formula I,
  • the phospho-MurNAc-pentapeptide translocase may be MurX or MraY.
  • the phospho-MurNAc-pentapeptide translocase is MurX.
  • the tuberculosis (TB) being prevented or treated in the above methods and uses is Multi-Drug Resistant tuberculosis (MDR TB).
  • MDR TB Multi-Drug Resistant tuberculosis
  • XDR TB extensive drug resistant tuberculosis
  • the TB is resistant to treatment with rifampicin, isoniazid, ethambutol and/or pyrazinamide.
  • the Mtb is the H37Rv strain of Mtb.
  • compositions may be formulated from compounds according to Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof for any appropriate route of administration including, for example, topical (for example, transdermal or ocular), oral, buccal, nasal, vaginal, rectal or parenteral administration.
  • parenteral as used herein includes subcutaneous, intradermal, intravascular (for example, intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique.
  • compositions in a form suitable for oral use or parenteral use are preferred.
  • Suitable oral forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • a sterile aqueous solution which is preferably isotonic with the blood of the recipient.
  • Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride or glycine, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile.
  • the formulations may be present in unit or multi-dose containers such as sealed ampoules or vials. Examples of components are described in Martindale - The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.
  • administering includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
  • the dose of the biologically active compound according to the invention may vary within wide limits and may be adjusted to individual requirements.
  • Active compounds according to the present invention are generally administered in a therapeutically effective amount. Preferred doses range from about 0.1 mg to about 140 mg per kilogram of body weight per day (e.g. about 0.5 mg to about 7 g per patient per day). The daily dose may be administered as a single dose or in a plurality of doses.
  • Dosage unit forms will generally contain between about 1 mg to about 500 mg of an active ingredient. It will be understood, however, that the specific dose level for any particular subject and will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the subject), and the severity of the particular disorder undergoing therapy. The dosage will generally be lower if the compounds are administered locally rather than systemically, and for prevention rather than for treatment.
  • Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician.
  • the pharmaceutical compositions may contain the active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg.
  • a daily dose of about 0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50 mg/kg body weight, may be appropriate.
  • the daily dose can be administered in one to four doses per day. Preferably, the daily dose is administered once per day.
  • terapéuticaally effective amount refers to an amount of a compound of Formula I, Formula II, Formula III and/or Formula IV or a salt, solvate, polymorph or prodrug thereof that results in an improvement or remediation of the symptoms of a condition modulated by gram positive bacteria, such as tuberculosis.
  • treating encompasses curing, ameliorating or tempering the severity of a condition modulated by gram positive bacteria, such as tuberculosis.
  • Preventing means preventing the occurrence of a condition modulated by gram positive bacteria, such as tuberculosis, or tempering the severity of the condition if it develops subsequent to the administration of the compounds or pharmaceutical compositions of the present invention.
  • the compounds of the present invention may be administered along with a pharmaceutical carrier, diluent or excipient as described above.
  • Biological testing means preventing the occurrence of a condition modulated by gram positive bacteria, such as tuberculosis, or tempering the severity of the condition if it develops subsequent to the administration of the compounds or pharmaceutical compositions of the present invention.
  • the compounds of the present invention may be administered along with a pharmaceutical carrier, diluent or excipient as described above.
  • the compounds tested were not cytotoxic to HEK293 cells up to a concentration of 200 ⁇ . Furthermore, most of the compounds tested were selective for Mtb.
  • An assay to assess the inhibitory activity of the compounds against Mtb MurX was also performed as an initial screen. This involved generating Mtb mc 2 6230 membrane protein preparations that contained MurX, along with other membrane proteins. The inhibition of MurX was assessed by the addition of a 200 nM concentration of a given compound together with UDP-[ 14 C]GlcNAc and UDP-MurNAc pentapeptide (Park's nucleotide). Following quenching of the enzymatic reactions and an extraction-based work up, thin layer chromatography (TLC) and phosphorimaging were used to measure the degree of inhibition of the enzyme. Further details of these procedures are discussed below in the Examples section.
  • TLC thin layer chromatography
  • a TLC-based assay was employed using a range of concentrations for several compounds to determine IC50 values. The results show good activity and suggest a significant correlation between the IC50 against Mtb MurX and the activity against Mtb H37Rv in vitro.
  • the compounds tested were shown to potently inhibit the enzyme MurX, responsible for lipid I synthesis, a key intermediate en route to peptidoglycan in Mtb.
  • the compounds possess selective activity against Mtb. This selectivity provides a practical advantage for the potential use of these compounds as TB drugs as well as MurX as a TB drug target.
  • 1 H NMR spectra were recorded at 300 K unless otherwise specified using a Bruker Avance DPX 300, DPX 400, DPX 500 and DPX 600 NMR spectrometer at a frequency of 300.2, 400.2, 500.2 and 600.2 MHz respectively.
  • 1 H NMR chemical shifts are reported in parts per million (ppm) and are referenced to solvent residual signals: CDCIs ⁇ 7.26, MeOD ⁇ 3.31 , acetone-cf 6 ⁇ 2.05, DMSO-cf 6 ⁇ 2.50 and D 2 0 ⁇ 4.79.
  • Preparative reverse phase HPLC was performed using a Waters 600 Multisolvent Delivery System and Waters 500 pump with a Waters 2996 photodiode array detector or Waters 490E programmable wavelength detector operating at 254 and 280 nm using a Sunfire Prep C18 OBD, 19 x 50 mm column, operating at a flow rate of 7 ml_ min "1 .
  • Compounds were eluted with 0.1 % TFA or formic acid in water (solvent A), and 0.1 % TFA or formic acid in CH 3 CN (solvent B) using a linear gradient of 0-50% B over 40 min or 0-50% B over 45 min or 50-100% B over 40 min.
  • LC-MS was performed on a Shimadzu LC-MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV/Vis detector coupled to a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode. Separations were performed on a Waters Sunfire 5 pm, 2.1 x 150 mm column (C18), operating at a flow rate of 0.2 mL min "1 . Separations were performed using a mobile phase of 0.1 % formic acid in water (Solvent A) and 0.1 % formic acid in CH 3 CN (Solvent B) and a linear gradient of 0- 50% B over 30 min or 50-100% B over 30 min.
  • FIG. 1 Synthesis of uridylamine 18. Reagents and conditions: a) TIPSOTf, /Pr 2 NEt, DMF, rt, 2.5 h, 82%; b) 10% Pd/C, H 2 (1 atm), MeOH, rt, 30 min, 80%; c) (i) TFA: CH 2 CI 2 (9:1 v/v), rt, 15 min; (ii) Isobutylchloroformate, /Pr 2 NEt, THF, 0 °C to rt, 1 .5 h; (iii) NaBH 4 , H 2 O (dropwise), 0 °C, 1 h, 69%; d) Tosyl chloride, pyridine, rt, 18 h, 82%; e) NaN 3 , DMF, 75 °C, 4 h, 82%; f) (i) TBAF (1 M in THF), THF, rt, 1
  • ester S1 (3.64 g, 12.3 mmol) and ⁇ /,/V-diisopropylethylamine (2.1 mL, 12.3 mmol) in DMF (37 mL) was added triisopropylsilyl tnfluoromethanesulfonate (4.9 mL, 17.6 mmol) and the reaction was stirred at rt for 2.5 h. The reaction was quenched with saturated aqueous Na 2 CO3 (40 mL) and partitioned between Et 2 O (200 mL) and H 2 O (40 mL).
  • Ester S3 (456 mg, 1 .0 mmol) was dissolved in 9: 1 v/v TFA: CH 2 CI 2 (2.2 mL) and the reaction was stirred at rt for 15 min. The solvent was removed in vacuo to give a residue that was subsequently dissolved in THF (13 mL) and cooled to 0 °C prior to the addition of isobutyl chloroformate (390 ⁇ , 3 mmol) and ⁇ /,/V-diisopropylethylamine (260 ⁇ , 1 .5 mmol). The reaction mixture was allowed to warm up to rt and was stirred for a further 1.5 h before being cooled to 0 °C.
  • Tosylate S5 (1.65 g, 3.07 mmol) was dissolved in DMF (34 ml_). Sodium azide (2.0 g, 30.7 mmol) was added and the reaction was heated to 75 °C for 4 h. The reaction was diluted with EtOAc (200 ml_), washed with water (5x 40 ml_), brine (40 ml_) and dried over anhydrous MgSO 4 . The solvent was removed in vacuo to give a crude residue which was purified by column chromatography (1:1 v/v EtOAc: Hexane) to afford azide S6 as a colourless oil (1.09 g, 87%).
  • Nanoparticle zinc (2.15 g, 33 mmol) was added to a vigorously stirring solution of acid S10 (3.04 g, 6.6 mmol) in MeOH (34 mL). A solution of 2 M HCI (34 mL) was added dropwise and the reaction was allowed to stir at rt for 2 h. A further portion of nanoparticle zinc (0.31 g, 4.8 mmol) and 2 M HCI (1 1 mL) were then added and the reaction was allowed to stir at rt for 16 h. The reaction was concentrated in vacuo to half of its volume, subsequently diluted with H 2 O (400 mL) and washed with CH2CI2 (80 mL).
  • Acid S8 (0.60 g, 1 .7 mmols, 1 eq.) was dissolved in 10% aqueous sodium carbonate (14.3 mL) and cooled to 0 °C.
  • the reaction was subsequently diluted with H 2 0 (120 mL), washed with diethyl ether (2 x 40 mL) and acidified to pH 2 with 1 M HCI.
  • Oxalic acid (0.25 g, 0.5 mmol, 0.5 eq.) was then added before concentrating the reaction mixture in vacuo. The resulting residue was redissolved in 10% v/v MeOH in CH 2 CI 2 and filtered through a silica plug. The solvent was removed in vacuo to yield a yellow oil which was subsequently redissolved in MeOH (2.8 mL). To this was added benzhydrylamine (0.19 mL, 1 .1 mmol, 1.1 eq.), NaCNBH 3 (94 mg, 1 .5 mmol, 1 .5 eq.) followed by AcOH (40 ⁇ _, 0.7 mmol, 0.7 eq.) and allowed to stir for 15 h.
  • linker 88 was carried out following the procedure published by Torres-Garcia et a/. [3] 4-((3,4-dihydro-2H-pyran-2-yl)methoxy)benzoic acid (88)
  • reaction mixture was allowed to cool to room temperature before being washed with CH2CI2 (x 3).
  • aqueous phase was then acidified to pH 2 using 1 M HCI (aq) and the resulting white precipitate was filtered and dried in vacuo to yield 88 as a white powder (3.7 g, 16 mmol, quantitative).
  • Figure S6 Solid phase synthesis of sansanmycin analogues.
  • 2-Chlorotrityl chloride resin (100-200 mesh) with 1 % DVB (1 .22-1 .42 mmol/g, 50- 730 ⁇ , 1 eq.) was allowed to swell in anhydrous CH 2 CI 2 (3-8 ml_) for 30 min.
  • Isopeptides S15-S16 (100-320 ⁇ , 2 eq.) or amino acid 20 (1 .1 -2.5 mmol, 1 .2 eq.) were dissolved in anhydrous CH 2 CI 2 (2-2.6 mL/100 ⁇ for isopeptides S15-S16 and 0.6 mL/100 ⁇ for amino acid 20).
  • ⁇ /,/V-diisopropylethylamine 200-2920 ⁇ , 2-8 eq. for isopeptides S15-S16 and 2.4-3 eq. for amino acid 20
  • the resin was shaken for 16 h at rt.
  • the resin was subsequently washed with DMF (5x 5 mL), CH 2 CI 2 (5x 5 mL) and DMF (5x 5 mL).
  • the resin was capped by treatment with 17:2: 1 v/v/v CH 2 CI 2 : MeOH: /Pr 2 NEt (3-6 mL) for 40 min.
  • the resin was then washed with DMF (5x 5 mL), CH 2 CI 2 (5x 5 mL) and DMF (5x 5 mL).
  • Rink amide resin was treated with 20% v/v piperidine in DMF (x 3) with shaking for 3 min followed by washing with DMF (x 5), CH 2 CI 2 (x 5) and DMF (x 5).
  • Linker 88 (0.062 - 0.21 mmol, 4 eq.), PyBOP (0.062 - 0.21 mmol, 4 eq.) and NMM (0.062 - 0.21 mmol, 4 eq.) in DMF (0.10 M) was added to the resin and shaken for 1 h.
  • the resin was subsequently washed with DMF (x 5), CH 2 CI 2 (x 5) and DMF (x 5) before being capped by treatment with 10% v/v acetic anhydride in pyridine for 3 min with shaking followed by washing with DMF (x 5) and CH 2 CI 2 (x 10).
  • the resin was then refluxed at 80 °C in 1 ,2-dichloroethane (0.1 M) with 87 (0.038 - 0.13 mmol, 2.5 eq.) and PPTS (0.012 - 0.058 mmol, 1 .1 eq.) for 16 h.
  • Condition B A solution of Fmoc-protected amino acid (72-96 pmol, 1 .2 eq.), HOAt (72-96 Mmol, 1 .2 eq.) and DIC (72-96 Mmol, 1 .2 eq.) in DMF (0.1 M) was added to the resin (1 eq.) and shaken for 16 h at rt. The resin was then washed with DMF (5x 3 mL), CH 2 CI 2 (5x 3 mL) and DMF (5x 3 mL).
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S24 as a fluffy white solid (12.4 mg, 39%).
  • Fmoc-L-Leu-OH (1 .12 g, 3.16 mmol) was coupled to resin-bound S11 (0.79 mmol) using standard Fmoc-SPPS procedure with PyBOP (1 .64 g, 3.16 mmol) and NMM (650 ⁇ _, 6.3 mmol) in DMF (7.9 mL) (general procedure 3, condition A).
  • carbamate S12 (840 mg, 1 .58 mmol) was subsequently coupled using ⁇ /,/V-diisopropylethylamine (550 ⁇ _, 3.16 mmol) according to general procedure 4.
  • Resin-bound S25 60 ⁇ was coupled to Boc-3-Pal-OH (32 mg, 120 ⁇ ) using HATU (45 mg, 120 pmol) and ⁇ /,/V-diisopropylethylamine (33 ⁇ _, 180 pmol) in DMF (0.6 mL) according to general procedure 6.
  • HATU 45 mg, 120 pmol
  • ⁇ /,/V-diisopropylethylamine 33 ⁇ _, 180 pmol
  • DMF 0.6 mL
  • the peptide was purified by reverse phase HPLC (20-100% MeCN over 40 min, 10 min at 20% MeCN) to afford intermediate S26 as a fluffy white solid (33 mg, 63%).
  • Resin-bound S25 60 ⁇ was coupled to Boc-2-Nal-OH (38 mg, 120 ⁇ ) using HATU (45 mg, 120 pmol) and ⁇ /,/V-diisopropylethylamine (33 ⁇ _, 180 pmol) in DMF (0.6 ml_) according to general procedure 6.
  • HATU 45 mg, 120 pmol
  • ⁇ /,/V-diisopropylethylamine 33 ⁇ _, 180 pmol
  • DMF 0.6 ml_
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford intermediate S27 as a fluffy white solid (31.4 mg, 56%).
  • Resin-bound S25 60 ⁇ was coupled to Boc-Cha-OH dicyclohexylamine salt (54 mg, 120 ⁇ ) using HATU (45 mg, 120 pmol) and /V,/V-diisopropylethylamine (23 ⁇ _, 126 pmol) in DMF (0.6 ml_) for 16 h. Following cleavage from the resin (general procedure 7), the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford intermediate S28 as a fluffy white solid (22 mg, 41 %).
  • Resin-bound S25 60 ⁇ was coupled to Boc-Chg-OH (122 mg, 480 ⁇ ) using HATU (124 mg, 480 ⁇ ), HOAt (324 mg, 2.4 mmol) and /V,/V-diisopropylethylamine (132 ⁇ _, 720 pmol) in DMF (0.6 ml_) was added to resin- bound S25 and the reaction vessel was shaken at rt for 4.5 h. Following cleavage from the resin (general procedure 7), the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford intermediate S29 as a fluffy white solid (19 mg, 36%).
  • Amino acid 20 (219 mg, 805 pmol) was loaded onto 2-chlorotrityl chloride resin (514 mg, 730 pmol) in CH 2 CI 2 (2 mL) using ⁇ /,/V-diisopropylethylamine (500 pL, 400 pmol) according to general procedure 2. Allyl carbamate was subsequently removed using fefra/ /s(triphenylphosphine) palladium(O) (186 mg, 161 pmol) and phenylsilane (2.0 mL, 16.1 mmol) according to general procedure 5.
  • Boc-Gly-OH (256 mg, 1.46 mmol) was coupled using solid-phase isopeptide formation (General procedure 6) with HATU (554 mg, 160 pmol) and ⁇ /,/V-diisopropylethylamine (381 pL, 2.1 mmol) in DMF (1 .4 mL) to afford the resin-bound isopeptide S35.
  • Resin-bound isopeptide S35 (60 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-lle-OH (85 mg, 240 ⁇ ) using PyBOP (125 mg, 240 mol) and NMM (50 ⁇ _, 480 mol) in DMF (0.6 mL) according to general procedure 3.
  • Carbamate S12 64 mg, 120 ⁇ was subsequently coupled according to general procedure 4.
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S36 as a fluffy white solid (17 mg, 36%).
  • Resin-bound isopeptide S35 (45 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Val-OH (81 .5 mg, 240 pmol) using PyBOP (125 mg, 240 ⁇ ) and NMM (53 ⁇ _, 480 mol) in DMF (0.45 ml_) according to general procedure 6.
  • Carbamate S12 (63 mg, 120 pmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was purified by reverse-phase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S37 as a fluffy white solid (19.7 mg, 53%).
  • Resin-bound isopeptide S35 (70 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Aha-OH (103 mg, 280 ⁇ ) using PyBOP (144 mg, 280 mol) and NMM (58 ⁇ _, 560 mol) in DMF (0.7 mL) according to general procedure 3.
  • Carbamate S12 (75 mg, 140 ⁇ ) was subsequently coupled according to general procedure 4.
  • Resin-bound isopeptide S35 (60 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Chg-OH (91 mg, 240 ⁇ ) using PyBOP (124 mg, 240 pmol) and NMM (50 ⁇ _, 480 pmol) in DMF (0.6 mL) according to general procedure 3.
  • Carbamate S12 64 mg, 120 ⁇ was subsequently coupled according to general procedure 4 in the presence of ⁇ /,/V-diisopropylethylamine (21 ⁇ , 120 ⁇ ) in DMF (1 mL).
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S39 as a fluffy white solid (20 mg, 40%).
  • Resin-bound isopeptide S35 60 ⁇ was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Cha-OH (47 mg, 120 ⁇ ) using HATU (45 mg, 120 pmol) and ⁇ /,/V-diisopropylethylamine (33 ⁇ _, 180 pmol) in DMF (0.6 ml_) according to general procedure 6.
  • Carbamate S12 64 mg, 120 ⁇ was subsequently coupled according to general procedure 4.
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 60 min, 10 min at 50% MeCN) to afford depsipeptide S40 as a fluffy white solid (17 mg, 34%).
  • Resin-bound isopeptide S35 (40 pmol) was Fmoc-deprotected (general procedure 2) and double coupled to Fmoc-Thr-OH (13.7 mg, 80 pmol) using HATU (30.4 mg, 80 pmol) and /V,/V-diisopropylethylamine (20 ⁇ _, 120 pmol) in DMF (0.4 ml_) according to general procedure 6.
  • Carbamate S12 46 mg, 90 pmol
  • the peptide was purified by reverse-phase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S41 as a fluffy white solid (18.2 mg, 49%).
  • Resin-bound isopeptide S35 60 pmol was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Asp(O f Bu)-OH (99 mg, 240 pmol) using PyBOP (125 mg,
  • Resin-bound isopeptide S35 (45 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Lys(Boc)-OH (1 12 mg, 240 pmol) using PyBOP (125 mg,
  • Resin-bound isopeptide S35 (88 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Phe(4-CF 3 )-OH (160 mg, 352 ⁇ ) using PyBOP (183 mg, 352 pmol) and NMM (73 ⁇ _, 704 pmol) in DMF (0.9 ml_) according to general procedure 3.
  • Carbamate S12 (94 mg, 176 ⁇ ) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was purified by reverse phase HPLC (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S45 as a fluffy white solid (29 mg, 36%).
  • Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Tyr(O f Bu)-OH (1 10 mg, 240 pmol) using PyBOP
  • Resin-bound isopeptide S35 (60 ⁇ ) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-3-Pal-OH (92 mg, 240 ⁇ ) using PyBOP (125 mg, 240 mol) and NMM (50 ⁇ _, 480 mol) in DMF (0.6 mL) according to general procedure 3.
  • Carbamate S12 64 mg, 120 ⁇ was subsequently coupled according to general procedure 4.
  • the peptide was purified by reverse phase HPLC (50-100% MeCN over 60 min, 10 min at 50% MeCN) to afford depsipeptide S47 as a fluffy white solid (14 mg, 29%).
  • depsipeptide S48 was purified by column chromatography (eluent: 95:5 v/v CH2CI2: MeOH, 0.1 vol.% acetic acid on deactivated silica) to afford depsipeptide S48 as a yellow oil (30.2 mg, 52%).
  • Resin-bound isopeptide S35 (70 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Phg-OH (31 .5 mg, 84 pmol) using HOAt (1 1 .2 mg, 84 ⁇ ,) and DIC (13.3 ⁇ _, 84 pmol) in DMF (0.7 mL) according to general procedure 6.
  • Carbamate S12 (73.6 mg, 140 pmol) was subsequently coupled according to general procedure 4.
  • the peptide was provided as a 9: 1 mixture of diastereomers, which were readily separable by reverse- phase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S49 as a single diastereomer as a fluffy white solid (14.5 mg, 25%).
  • Resin-bound isopeptide S35 (70 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-HPhe-OH (33.7 mg, 84 pmol) using HOAt (11.2 mg, 84 pmol) and DIC (13.3 ⁇ _, 84 pmol) in DMF (0.7 mL) according to general procedure 6.
  • Carbamate S12 (73.6 mg, 140 pmol) was subsequently coupled according to general procedure 4.
  • the peptide was provided as a 9:1 mixture of diastereomers which were readily separable by reverse- phase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S50 as a single diastereomer as a fluffy white solid (18.8 mg, 27%).
  • Resin-bound isopeptide S35 60 ⁇ was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-2-Nal-OH (104 mg, 240 ⁇ ) using PyBOP (124 mg,
  • Resin-bound amino acid S25 (87 ⁇ ) was coupled to Fmoc-Cha-OH (68 mg, 174 ⁇ ) using HATU (65 mg, 174 pmol) and ⁇ /,/V-diisopropylethylamine (48 pL, 260 pmol) in DMF (0.9 mL) according to general procedure 6.
  • Carbamate S12 (93 mg, 174 ⁇ ) was subsequently coupled according to general procedure 4.
  • Depsipeptide S26 (24 mg, 27 ⁇ ) was reacted with amine 18 (7.2 mg, 32 ⁇ ) in CH 2 CI 2 : DMF (1 : 1 v/v, 260 ⁇ _) in the presence of HOAt (18 mg, 134 ⁇ ) and DIC (4.3 ⁇ _, 27 ⁇ ) for 1 .5 h at rt according to general procedure 8 condition A to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1 : 1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 2.7 ml_) according to general procedure 8 to afford 8 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (1 1 mg, 46% over 2 steps).
  • Depsipeptide S27 (30 mg, 33 ⁇ ) was reacted with amine 18 (9.8 mg, 40 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 320 ⁇ _) in the presence of HOAt (23 mg, 165 ⁇ ) and DIC (5.3 ⁇ _, 33 ⁇ ) for 2.5 h at rt according to general procedure 8 condition A to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 3.3 ml_) according to general procedure 8 to afford 9 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (10 mg, 32% over 2 steps).
  • Depsipeptide S28 (22 mg, 25 ⁇ ) was reacted with amine 18 (6.8 mg, 30 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 240 ⁇ _) in the presence of HOAt (17 mg, 123 ⁇ ) and EDC.HCI (5.2 mg, 25 ⁇ ) and NMM (3 ⁇ _, 25 ⁇ ) for 3 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 2.5 mL) according to general procedure 8 to afford 10 (as a TFA salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 O) as an amorphous white solid (10.4 mg, 44% over 2 steps).
  • Depsipeptide S29 (19 mg, 22 ⁇ ) was reacted with amine 18 (5.8 mg, 26 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 200 ⁇ _) in the presence of HOAt (14 mg, 107 ⁇ ) and DIC (5.2 ⁇ _, 33 ⁇ ) for 18 h at rt according to general procedure 8 condition A to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 2.2 ml_) according to general procedure 8 to afford 11 (as a TFA salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (5.4 mg, 26% over 2 steps).
  • Depsipeptide S36 (17 mg, 22 ⁇ ) was reacted with amine 18 (7.4 mg, 32 ⁇ ) in CH 2 CI 2 : DMF (1 : 1 v/v, 210 ⁇ _) in the presence of HOAt (15 mg, 108 ⁇ ) and EDC.HCI (5.4 mg, 28 ⁇ ) and NMM (3.0 ⁇ _, 28 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1 : 1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 2.2 mL) for 4 h according to general procedure 8 to afford 21 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 45 min, 10 min at 100% H 2 O, flow rate 9 mL/min) as an amorphous white solid (7.4 mg, 44% over 2 steps).
  • Depsipeptide S37 (17.8 mg, 23.4 pmol.) was reacted with amine 18 (10.5 mg, 46 pmol) in CH 2 CI 2 : DMF (1:1 v/v, 240 ⁇ _) in the presence of HOAt (15.7 mg, 115 Mmol), EDC.HCI (5.8 mg, 30 pmol) and NMM (3.3 ⁇ _, 30 pmol) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • Depsipeptide S38 (30 mg, 37 ⁇ ) was reacted with amine 18 (20 mg, 92 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 370 ⁇ _) in the presence of HOAt (25 mg, 185 ⁇ ) and EDC.HCI (9.2 mg, 48 ⁇ ) and NMM (5.0 ⁇ _, 48 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 3.7 mL) for 4 h according to general procedure 8 to afford 23 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (8.1 mg, 27% over 2 steps).
  • Depsipeptide S39 (24 mg, 30 ⁇ ) was reacted with amine 18 (14 mg, 60 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 300 ⁇ _) in the presence of HOAt (20 mg, 150 ⁇ ) and EDC.HCI (7.4 mg, 39 ⁇ ) and NMM (4.0 ⁇ _, 39 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 3.0 mL) for 4 h according to general procedure 8 to afford 24 (as a TFA salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 O) as an amorphous white solid (13.7 mg, 60% over 2 steps).
  • Depsipeptide S40 (17 mg, 20 ⁇ ) was reacted with amine 18 (9.5 mg, 42 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 200 ⁇ _) in the presence of HOAt (14 mg, 103 ⁇ ) and EDC.HCI (5.1 mg, 27 ⁇ ) and NMM (3.0 ⁇ _, 27 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 4 mL) for 4 h according to general procedure 8 to afford 25 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 45 min, 10 min at 100% H 2 0) as an amorphous white solid (6.0 mg, 35% over 2 steps).
  • Depsipeptide S41 (18.0 mg, 23.2 mol.) was reacted with amine 18 (10.5 mg, 46.4 pmol) in CH 2 CI 2 : DMF (1 : 1 v/v, 240 ⁇ _) in the presence of HOAt (15.8 mg, 1 16.0 Mmol), EDC.HCI (5.8 mg, 30.1 pmol) and NMM (3.3 ⁇ _, 30.1 ⁇ ) for 4 h at rt according to general procedure 8 to obtain the fully protected analogue.
  • This compound was then treated with a mixture of TFA and / ' -Pr 3 SiH in CH 2 CI 2 (1 : 1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /-Pr 3 SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 26 (as a formate salt) after reverse phase HPLC purification (0 to 30% MeCN over 40 min) as a fluffy white solid (8.3 mg, 46% over two steps).
  • Depsipeptide S42 (19.2 mg, 21.6 pmol) was reacted with amine 18 (12.2 mg, 53.9 pmol) in CH 2 CI 2 : DMF (1:1 v/v, 220 ⁇ _) in the presence of HOAt (14.7 mg, 107.8 ⁇ ), EDC-HCI (5.4 mg, 28.0 ⁇ ) and NMM (3.1 ⁇ _, 28.0 ⁇ ) for 4 h according to general procedure 8 condition B to obtain the fully protected analogue.
  • Depsipeptide S43 (13.4 mg, 14.8 pmol.) was reacted with amine 18 (11 mg, 48.7 pmol) in CH 2 CI 2 : DMF (1:1 v/v, 180 ⁇ ) in the presence of HOAt (10 mg, 74.0 Mmol), EDC.HCI (3.7 mg, 19.2 ⁇ ) and NMM (2.1 ⁇ _, 19.2 ⁇ ) for 4 h according to general procedure 8 condition B to obtain the corresponding protected analogue.
  • This compound was then treated with a mixture of TFA and /-Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /-Pr 3 SiH, 1.2 ml_) for 16 h according to general procedure 8 to afford 28 (as a TFA salt) after reverse phase HPLC (0 to 30% MeCN over 40 min) as a fluffy white solid (7.2 mg, 62% over two steps).
  • Depsipeptide S45 (28 mg, 32 ⁇ ) was reacted with amine 18 (15 mg, 64 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 320 ⁇ _) in the presence of HOAt (22 mg, 161 ⁇ ) and EDC.HCI (8.0 mg, 42 ⁇ ) and NMM (4.4 ⁇ _, 42 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 3.2 mL) for 4 h according to general procedure 8 to afford 30 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 45 min, 10 min at 100% H 2 0) as an amorphous white solid (5.6 mg, 20% over 2 steps).
  • Depsipeptide S47 (14 mg, 17 ⁇ ) was reacted with amine 18 (10 mg, 44 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 170 ⁇ _) in the presence of HOAt (12 mg, 86 ⁇ mol) and EDC.HCI (4.1 mg, 22 ⁇ ) and NMM (2.3 ⁇ _, 22 ⁇ ) for 3.5 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1:1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 1.7 mL) for 4 h according to general procedure 8 to afford 32 (as a TFA salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (8.2 mg, 54% over 2 steps).
  • Depsipeptide S48 (36.0 mg, 37.4 pmol.) was reacted with amine 18 (21.3 mg, 93.5 ⁇ ) in CH 2 CI 2 : DMF (1:1 v/v, 380 ⁇ _) in the presence of HOAt (25.5 mg, 187.0 ⁇ ), EDC.HCI (9.3 mg, 48.6 pmol) and NMM (5.4 ⁇ _, 48.6 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue.
  • Depsipeptide S49 (25.3 mg, 31.3 pmol.) was reacted with amine 18 (17.8 mg, 78.2 pmol) in CH 2 CI 2 : DMF (1:1 v/v, 320 ⁇ _) in the presence of HOAt (21.3 mg, 156.5 pmol), EDC.HCI (7.8 mg, 40.7 pmol) and NMM (4.5 ⁇ _, 40.7 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue.
  • Depsipeptide S50 (29.3 mg, 35.1 pmol.) was reacted with amine 18 (19.9 mg, 87.7 pmol) in CH 2 CI 2 : DMF (1:1 v/v, 360 ⁇ _) in the presence of HOAt (23.9 mg, 175.5 ⁇ ), EDC.HCI (8.7 mg, 45.6 ⁇ ) and NMM (5.0 ⁇ _, 45.6 ⁇ ) for 4 h according to general procedure 8 to obtain the fully protected analogue.
  • Depsipeptide S51 (21 mg, 25 ⁇ ) was reacted with amine 18 (14 mg, 63 ⁇ ) in CH 2 CI 2 : DMF (1 : 1 v/v, 250 ⁇ _) in the presence of HOAt (17 mg, 123 ⁇ ) and EDC.HCI (6.1 mg, 32 ⁇ ) and NMM (3.5 ⁇ _, 32 ⁇ ) for 4 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in CH 2 CI 2 (1 : 1 v/v TFA: CH 2 CI 2 , 2.5 vol.% /Pr 3 SiH, 2.5 mL) for 4 h according to general procedure 8 to afford 36 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 O, flow rate 9 mL/min) as an amorphous white solid (6.0 mg, 28% over 2 steps).
  • Depsipeptide S52 (18 mg, 17 ⁇ ) was reacted with amine 18 (12 mg, 52 ⁇ ) in CH 2 CI 2 : DMF (1 : 1 v/v, 180 ⁇ _) in the presence of HOAt (1 1 mg, 150 ⁇ ) and EDC.HCI (4.4 mg, 22 ⁇ ) and NMM (2.6 ⁇ _, 22 ⁇ ) for 2.5 h at rt according to general procedure 8 condition B to obtain the fully protected analogue.
  • This compound was treated with a mixture of TFA and /Pr 3 SiH in H 2 0 (9: 1 v/v TFA: CH 2 CI 2 , 2 vol.% /Pr 3 SiH, 1 .7 ml_) for 45 min at 0 °C and 2 h at rt to afford 37 (as a TFA salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H 2 0) as an amorphous white solid (8.0 mg, 53% over 2 steps).
  • the flask was cooled to 0 °C prior to dissolving the residue in a saturated aqueous solution of Na 2 CO 3 (5 ml_).
  • the reaction was partitioned between CHCI3 (10 ml_) and H 2 O (5 ml_).
  • the aqueous phase was extracted once with CHCI3 (10 ml_).
  • the combined organic extracts were dried (MgSO 4 ) and concentrated in vacuo.
  • the resulting foam was redissolved in CH2CI2 and concentrated in vacuo once more to yield an off white foam that was subsequently purified by column chromatography (eluent: 4: 1 v/v hexane: EtOAc) to afford 41 as a white foam (0.1 1 mg, 0.20 mmol, 52%).
  • the product was precipitated out in diethyl ether and centrifuged at 3600 g for 20 min.
  • the precipitate was redissoived in H 2 0 and lyophilised to yield a 46 as a blue oil (3.2 g, 8.1 mmol, quantitative).
  • aqueous phase was then acidified to pH 2 with 1 M HCI (aq) , extracted with EtOAc (3 x 100 mL), dried (MgSO 4 ) and concentrated in vacuo to yield 25 as a green powder (2.0 g, 3.8 mmol, 50%).
  • Resin-bound pseudo peptide was treated with a solution of tetrakis(triphenylphosphine)palladium(0) (0.028 - 0.052 mmol, 0.20 eq.) and phenylsilane (2.8 - 5.2 mmol, 20 eq.) in CH 2 CI 2 (0.04 M) with shaking for 15 min.
  • the resin was then washed with DMF (5 x 5 mL), CH 2 CI 2 (5 x 5 mL) and DMF (5 x 5 mL). Complete removal of the Alloc-protecting group was determined through LCMS of the minicleavage solution.
  • Resin-bound pseudo peptide 55 - 57, 63 - 65 was activated with /V,/V'-diispropylcarbodiimide (DIC) (0.54 - 0.98 mmol, 5 eq.) in anhydrous CH2CI2 (0.5 M) for 5 min with shaking prior to the addition of amine (0.21 - 0.39 mmol, 2 eq.) in anhydrous CH2CI2 (0.5 M).
  • the resin was shaken for 2 h, followed by washing with DMF (5 x 5 mL), CH2CI2 (5 x 5 mL) and DMF (5 x 5 mL). Successful coupling was determined by LCMS of the minicleavage solution.
  • DIC /V,/V'-diispropylcarbodiimide
  • the pseudo peptide 51 - 65 was cleaved from the solid support through treatment with 30% v/v HFIP in CH2CI2 (5 mL) for 40 min with shaking at rt. The resin was then washed with CH 2 CI 2 (6 x 10 mL), combined with the cleavage solution and concentrated in vacuo. The crude residue was subsequently used in the solution phase coupling without further purification.
  • EDC HCI 46 - 65 pmol, 1 .3 eq.
  • NMM 46 - 65 ⁇ , 1.3 eq.
  • the reaction was allowed to warm to room temperature and stirred for 3 - 4 h before being diluted with EtOAc (44 - 60 mL), washed with aqueous 0.5 M HCI (1 1 - 16 mL), saturated aqueous NaHC0 3 solution (1 1 - 16 mL), H 2 0 (1 1 - 16 mL), brine (1 1 - 16 mL), and dried (MgS0 4 ).
  • the organic phase was concentrated in vacuo before being redissolved in 1 : 1 v/v CH2CI2: TFA (5.2 - 6.2 mL) and 2.5 vol.% /Pr 3 SiH (0.1 1 - 0.16 mL).
  • Amino acid 20 (240 mg, 0.55 mmol, 1 .1 eq.) in CH 2 CI 2 (0.1 M, 5.5 mL) with / ' Pr 2 EtN (350 ⁇ _, 2 mmol, 4 eq.) was loaded on to 2-chlorotrityl (2-CTC) resin (420 - 560 mg, 0.50 mmol, 1 eq.) as described in general procedure 2, followed by Alloc deprotection and coupling of Boc-Gly-OH (190 mg, 1.1 mmol, 2 eq.) in DMF (0.1 M, 1 1 mL) with HATU (420 mg, 1 .1 mmol, 2 eq.) and / ' Pr 2 EtN (290 ⁇ _, 1 .7 mmol, 3 eq.) as described in general procedure 5 and 6.
  • the resin was washed with DMF (5 x 5 mL), CH 2 CI 2 (5 x 5 mL) and DMF (5 x 5 mL), followed by capping with 10% v/v acetic anhydride in pyridine (5 mL) with shaking for 3 min followed by washing with DMF (5 x 5 mL), CH 2 CI 2 (5 x 5 mL) and DMF (5 x 5 mL).
  • the resin-bound pseudo peptide was then preactivated with DIC (150 ⁇ _, 0.96 mmol, 5 eq.) in CH 2 CI 2 (1 .9 mL) prior to the addition of benzyl alcohol (99 ⁇ _, 0.96 mmol, 5 eq.) and DMAP (2.3 mg, 19 ⁇ , 0.1 eq.) in CH 2 CI 2 (1 .9 mL) according to general procedure 7.2 condition A to yield the resin-bound pseudo peptide 50
  • Resin-bound pseudo peptide 48 was preactivated with DIC (150 ⁇ , 0.97 mmol, 5 eq.) in CH 2 CI 2 (1 .9 mL) prior to the addition of methanol (39 ⁇ , 0.97 mmol, 5 eq.) and DMAP (2.4 mg, 19 ⁇ , 0.1 eq.) in CH 2 CI 2 (1 .9 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 51.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (140 ⁇ _, 0.88 mmol, 5 eq.) in CH 2 CI 2 (1 .8 mL) prior to the addition of hexanol (1 10 ⁇ , 0.88 mmol, 5 eq.) and DMAP (2.14 mg, 17.5 ⁇ , 0.1 eq.) in CH 2 CI 2 (1.74 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 52.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (1 10 ⁇ , 0.69 mmol, 5 eq.) in CH 2 CI 2 (1 .4 mL) prior to the addition of dodecanol (150 ⁇ , 0.69 mmol, 5 eq.) and DMAP (1 .7 mg, 14 ⁇ , 0.1 eq.) in CH 2 CI 2 (1.4 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 53.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (96 ⁇ _, 0.62 mmol, 5 eq.) in CH 2 CI 2 (1 .2 mL) prior to the addition of benzyl alcohol (64 ⁇ , 0.62 mmol, 5 eq.) and DMAP (1 .5 mg, 12 ⁇ , 0.1 eq.) in CH 2 CI 2 (1.2 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 54.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (150 ⁇ , 0.98 mmol, 5 eq.) in CH 2 CI 2 (2 mL) prior to the addition of hexyl amine (52 ⁇ , 0.39 mmol, 2 eq.) in CH 2 CI 2 (2 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 55.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (83 ⁇ , 0.54 mmol, 5 eq.) in CH 2 CI 2 (1 .1 mL) prior to the addition of neopentyl amine (25 ⁇ , 0.21 mmol, 2 eq.) in CH 2 CI 2 (1 .1 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 56.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (90 ⁇ _, 0.58 mmol, 5 eq.) in CH 2 CI 2 ( 2 mL) prior to the addition of benzyl amine (25 ⁇ _, 0.23 mmol, 2 eq.) in CH2CI2 ( .2 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 57.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (190 ⁇ _, 1 .3 mmol, 5 eq.) in CH 2 CI 2 (2.5 mL) prior to the addition of hexanol (160 ⁇ , 1 .3 mmol, 5 eq.) and DMAP (3.1 mg, 25 ⁇ , 0.1 eq.) in CH 2 CI 2 (2.5 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 59.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (200 ⁇ , 1 .3 mmol, 5 eq.) in CH 2 CI 2 (2.6 mL) prior to the addition of dodecanol (290 ⁇ , 1 .3 mmol, 5 eq.) and DMAP (3.2 mg, 26 ⁇ , 0.1 eq.) in CH 2 CI 2 (2.6 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 60.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (130 ⁇ _, 0.81 mmol, 5 eq.) in CH 2 CI 2 (1 .6 mL) prior to the addition of neopentyl alcohol (88 ⁇ , 0.81 mmol, 5 eq.) and DMAP (2.0 mg, 16 ⁇ , 0.1 eq.) in CH 2 CI 2 (1 .6 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 61.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (160 ⁇ , 1 .1 mmol, 5 eq.) in CH 2 CI 2 (2.1 mL) prior to the addition of benzyl alcohol (1 10 ⁇ , 1 .1 mmol, 5 eq.) and DMAP (2.6 mg, 21 ⁇ , 0.1 eq.) in CH 2 CI 2 (2.1 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 62.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (150 ⁇ , 0.98 mmol, 5 eq.) in CH 2 CI 2 (2 mL) prior to the addition of hexyl amine (52 ⁇ , 0.39 mmol, 2 eq.) in CH 2 CI 2 (2 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 63.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (85 ⁇ _, 0.55 mmol, 5 eq.) in CH2CI2 (1 .1 mL) prior to the addition of neopentyl amine (26 ⁇ _, 0.22 mmol, 2 eq.) in CH2CI2 (1 .1 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 64.
  • Resin-bound pseudo peptide 49 was preactivated with DIC (130 ⁇ , 0.8 mmol, 5 eq.) in CH2CI2 (1 .6 mL) prior to the addition of benzyl amine (40 ⁇ , 0.32 mmol, 2 eq.) in CH2CI2 (1 .6 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 65.
  • the pseudo peptide 51 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 51 (32 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 66.
  • the pseudo peptide 52 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (8.8 mg, 46 pmol, 1.3 eq.) and NMM (5.1 ⁇ _, 46 ⁇ , 1.3 eq.) in 2:1 v/v CH 2 CI 2 : DMF (180 ⁇ _) was added dropwise to the crude residue 52 (30 mg, 35 pmol, 1 eq.), uridyl amine 18 (24 mg, 110 ⁇ , 3 eq.) and HOAt (24 mg, 180 pmol, 5 eq.) in 1:2 v/v CH 2 CI 2 : DMF (180 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 67.
  • the pseudo peptide 53 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (9.3 mg, 48 pmol, 1 .3 eq.) and NMM (5.3 ⁇ _, 48 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (190 ⁇ _) was added dropwise to the crude residue 53 (35 mg, 37 pmol, 1 eq.), uridyl amine 18 (25 mg, 1 10 ⁇ , 3 eq.) and HOAt (25 mg, 190 pmol, 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (190 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 68.
  • the pseudo peptide 54 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1.3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1.3 eq.) in 2:1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 54 (35 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1:2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 69.
  • the pseudo peptide 55 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 55 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 70.
  • the pseudo peptide 56 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 56 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 71.
  • the pseudo peptide 57 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 57 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 72.
  • the pseudo peptide 58 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC-HCI (13 mg, 65 pmol, 1.3 eq.) and NMM (7.3 ⁇ _, 65 ⁇ , 1.3 eq.) in 2:1 v/v CH 2 CI 2 : DMF (160 ⁇ _) was added dropwise to the crude residue 58 (42 mg, 50 pmol, 1 eq.), uridyl amine 18 (34 mg, 0.15 mmol, 3 eq.) and HOAt (34 mg, 0.25 mmol, 5 eq.) in 1:2 v/v CH 2 CI 2 : DMF (161 ⁇ _) according to general procedure 8 condition B, yielding the protected analogue 73.
  • the pseudo peptide 59 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1.3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1.3 eq.) in 2:1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 59 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1:2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 74.
  • the pseudo peptide 60 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 60 (39 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 75.
  • 1 1 mixture of rotamers) ⁇ 173.5, 172.0, 170.3, 170.0, 166.7, 166.3, 163.9, 163.8, 158.8, 158.5, 158.3, 157.5, 151.2, 141 .7, 141 .5, 136.7, 135.7, 135.6, 133.5, 132.4, 128.6, 128.5, 128.3, 128.0, 128.0, 127.9, 127.8, 126.4, 125.9, 124.3, 121.5, 1 18.9, 1 18.7, 1 12.0, 109.7, 102.4, 102.0, 92.6, 92.5, 79.2, 74.6, 74.5, 64.8, 55.4, 55.0, 54.8, 54.7, 54.3, 53.5, 44.5, 44.3, 39.0, 38.8, 36.2, 36.1 , 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 28.5, 27.9, 25.8, 22.7, 15.2, 14.6; LRMS [M+H] + 995.1 ; HRMS [M+H]
  • Neopentyl (((S)-1 -(((2S,3S)-3-(2-amino-A -methylacetamido)-1 -((((2/?,4/?,5/?)- 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2- yl)methyl)amino)-1 -oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1 -oxopropan-2- yl)carbamoyl)-L-tryptophanate (76)
  • the pseudo peptide 61 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 61 (35 mg, 40 ⁇ , 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 76.
  • the pseudo peptide 62 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 62 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 77.
  • the pseudo peptide 63 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 63 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 78.
  • the pseudo peptide 64 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 64 (35 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 79.
  • the pseudo peptide 65 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1.3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1.3 eq.) in 2:1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 65 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1:2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 80.
  • the pseudo peptide 50 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 50 (39 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 81.
  • Resin-bound pseudo peptide 48 was preactivated with DIC (150 ⁇ _, 0.94 mmol, 5 eq.) in CH2CI2 (1.9 mL) prior to the addition of neopentyl alcohol (83 ⁇ _, 0.94 mmol, 5 eq.) and DMAP (2.3 mg, 19 ⁇ , 0.1 eq.) in CH 2 CI 2 (1.9 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 82.
  • Neopentyl (((S)-1 -(((2S,3S)-3-(2-amino-N-methylacetamido)-1-((((2R,4R,5R)- 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2- yl)methyl)amino)-1 -oxobutan-2-yl)amino)-3-cyclohexyl-1 -oxopropan-2- yl)carbamoyl)-L-tryptophanate (83)
  • the pseudo peptide 82 was cleaved from resin as described in general procedure 8 condition C.
  • a preactivated solution of EDC HCI (10 mg, 52 pmol, 1 .3 eq.) and NMM (5.7 ⁇ _, 52 ⁇ , 1 .3 eq.) in 2: 1 v/v CH 2 CI 2 : DMF (210 ⁇ _) was added dropwise to the crude residue 82 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 Mmol, 3 eq.) and HOAt (27 mg, 200 ⁇ , 5 eq.) in 1 :2 v/v CH 2 CI 2 : DMF (210 ⁇ _) according to general procedure 8 condition C, yielding the protected analogue 83.
  • the resin bound peptide was treated with 95:2.5:2.5 v/v/v TFA:H 2 0:TIS for 1 h with shaking. The resin was subsequently washed with TFA:H 2 0:TIS (x 10), combined with the cleavage solution and concentrated. The resulting crude residue was purified via reverse-phase HPLC.
  • Linker 88 (50 mg, 0.21 mmol, 4 eq.) was attached onto rink amide resin (112 mg, 0.053 mmol, 1 eq.) using PyBOP (110 mg, 0.21 mmol, 4 eq.) and NMM (23 ⁇ _, 0.21 mmol, 4 eq.) followed by loading of 87 (60 mg, 0.13 mmol, 2.5 eq.) with PPTS (15 mg, 0.058 mmol, 1.1 eq.) in 1 ,2-dichloroethane (0.60 mL) as described in general procedure 2 condition B.
  • the resin was then treated with a solution of Pd(PPh 3 ) 4 (4.6 mg, 4.0 ⁇ , 0.2 eq.) in CH 2 CI 2 (0.50 mL) with PhSiH 3 (49 ⁇ , 0.40 mmol, 20 eq.) as described in general procedure 5 followed by coupling of Boc-Gly-OH (7.0 mg, 0.040 mmol, 2 eq.) using HATU (15 mg, 0.040 mmol, 2 eq.) and /Pr 2 EtN (1 1 ⁇ _, 0.060 mmol, 8 eq.) in DMF (0.4 mL) according to general procedure 6 condition A.
  • the resin was Fmoc deprotected and Fmoc-Cha-OH (32 mg, 0.080 mmol, 4 eq.) was coupled using PyBOP (42 mg, 0.08 mmol, 4 eq.) and NMM (18 ⁇ , 0.16 mmol, 8 eq.) in DMF (0.8 mL) as described in general procedure 6 condition B.
  • PyBOP 42 mg, 0.08 mmol, 4 eq.
  • NMM 18 ⁇ , 0.16 mmol, 8 eq.
  • DMF 0.8 mL
  • coupling of carbamate S12 21 mg, 0.040 mmol, 2 eq.
  • Linker 88 (15 mg, 0.062 mmol, 4 eq.) was attached onto rink amide resin (30 mg, 0.016 mmol, 1 eq.) using PyBOP (32 mg, 0.062 mmol, 4 eq.) and NMM (7 ⁇ , 0.062 mmol, 4 eq.) followed by loading of 87 (17 mg, 0.038 mmol, 2.5 eq.) with PPTS (3 mg, 0.012 mmol, 1 .1 eq.) in 1 ,2-dichloroethane (0.12 mL) as described in general procedure 2 condition B.
  • the resin was then treated with a solution of Pd(PPh 3 ) 4 (2.4 mg, 2.1 pmol, 0.2 eq.) in CH 2 CI 2 (0.26 mL) with PhSiH 3 (26 ⁇ , 0.21 mmol, 20 eq.) as described in general procedure 5 followed by coupling of Boc-Gly-OH (3.7 mg, 0.021 mmol, 2 eq.) using HATU (8 mg, 0.021 mmol, 2 eq.) and /Pr 2 EtN (5.5 ⁇ , 0.032 mmol, 3 eq.) in DMF (0.20 mL) according to general procedure 6 condition A.
  • the resin was Fmoc deprotected and Fmoc-2-Nal-OH (19 mg, 0.042 mmol, 4 eq.) was coupled using PyBOP (22 mg, 0.042 mmol, 4 eq.) and NMM (9 ⁇ , 0.084 mmol, 8 eq.) in DMF (0.40 mL) as described in general procedure 6 condition B.
  • Fmoc deprotection coupling of carbamate S12 (1 1 mg, 0.021 mmol, 2 eq.) was achieved using / ' Pr 2 EtN (7 ⁇ _, 0.042 mmol, 4 eq.) in DMF (0.20 ml_) according to general procedure 4.
  • Mtb H37Rv ATCC 27294
  • Mtb H37Ra ATCC 25177 strains were grown in Middlebrook 7H9 broth medium supplemented with OADC (Difco Laboratories, Detroit, Ml, USA), 0.05% glycerol and 0.05% Tween-80. Freshly seeded cultures were grown at 37°C, for approximately 14 days, to mid-exponential phase (OD 6 oo 0.4-0.8) for use in the inhibition assays.
  • Mtb 100 ⁇ _, representing ⁇ 2 x10 4 CFU ml_ "1 ) was added to each well. Plates were incubated for 5 days at 37 °C in a humidified incubator prior to the addition of a 0.02% resazurin solution (30 ⁇ _) and 20% Tween-80 (12.5 ⁇ _) to each well. Sample fluorescence was measured after 24 h on a BMG Labtech Polarstar Omega instrument with an excitation wavelength of 530 nm and emission at 590 nm. Changes in fluorescence relative to positive control wells (Mtb H37Rv with no inhibitor) minus negative control wells (no Mtb H37Rv) were plotted for determination of M IC50 values.
  • Rifampicin was purchased from Sigma-Aldrich (R3501 ). All positive controls were dissolved in 100% dimethyl sulfoxide (DMSO) (0231 -500ml_; Amresco) and diluted in 7H9 broth (27131 ; Difco Becton Dickinson) with 10% ADC (BSAL; Moregate Biotech), 0.05% glycerol (GA010-P; Chem-Supply) and 0.05% Tween 80 (P6224-500ml; Sigma- Aldrich).
  • DMSO dimethyl sulfoxide
  • Amresco Amresco
  • 7H9 broth 27131 ; Difco Becton Dickinson
  • ADC BSAL; Moregate Biotech
  • glycerol G010-P
  • Chem-Supply Chem-Supply
  • Tween 80 P6224-500ml; Sigma- Aldrich
  • THP-1 human macrophage-like cell line
  • FBS FBS-500; Scientifix-life
  • M7522 Sigma-Aldrich
  • THP-1 cells were plated in 96 well tissue culture plates (Costar 3903; Corning) at a density of 1x10 5 cells/well with phorbol myristic acetate (PMA; Sigma-Aldrich, 100 nM) added. THP-1 cells were left to differentiate for 48 h at 37 °C at 5% C0 2 . A cell suspension of sonicated Mtb H37Ra in RPMI-1640 cell culture medium was used to infect differentiated THP-1 cells at a multiplicity of infection (MOI) of 5 for 4 h at 37 °C at 5% C0 2.
  • MOI multiplicity of infection
  • THP-1 cells were washed with 200 ⁇ _ phosphate buffered saline (PBS) (98-317-LB; Cellgro) three times and were subsequently replenished with fresh RPMI-1640 cell culture medium and incubated for a further 24 h at 37 °C and 5% C0 2 .
  • PBS phosphate buffered saline
  • the analogues were diluted in fresh RPMI-1640 cell culture medium and added to corresponding wells. After 72 h of incubation at 37 °C at 5% CO 2 , tissue culture medium containing the test compound was removed from the wells; the cells were washed with 200 ⁇ _ PBS, and then lysed with sterile water containing 0.1 % Triton X (T7253; Sigma-Aldrich). Cell lysates were serially diluted, 1 : 10, and plated on Middlebrook 7H1 1/OADC (283010; Difco) agar through to 1/10000 dilution. Agar plates were then incubated at 37 °C for 3-4 weeks, after which the bacteria colonies were counted and CFU/mL of cell lysates were determined. Intracellular anti-mycobacterial activity of analogues 25, 36 and 37 are shown in
  • Counter screen for selectivity The organisms in the screen included: Bacillus subtilis 168, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Listeria ivanovii, Enterococcus faecium, Escherichia coli, Vibrio cholerae, Salmonella typhimurium, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, Providencia alcalifaciens, Ochrobactrum anthropi, Enterobacter aerogenes, Acinetobacter baumannii
  • BSL1 Bacillus subtilis 168, Staphylococcus epidermis [ATCC 14990], Enterococcus faecium [ATCC 6569], Listeria ivanovii [BAA-139]; BSL2: S. aureus [ATCC 29213], methicillin- resistant S.
  • BSL1 Escherichia coli K12 [BW 251 13], Acinetobacter baumanii [NCIMB 12457], Enterobacter aerogenes [ATCC 35029], Ochrobactrum anthropi [ATCC 49687], Providencia alcalifaciens [ATCC 9886]; BSL2: Yersinia pseudotuberculosis [IP2666 pIBI], Pseudomonas aeruginosa [ATCC 27835], Salmonella typhimurium LT2, Vibrio cholerae 01 [biotype El Tor A1552].
  • pseudotuberculosis cultures were grown in Luria Broth (10 g tryptone, 5 g yeast extract and 10 g NaCI in 1 L distilled water; pH 7.5). All three media were autoclaved at 121 °C for 30 min. Inoculated cultures were grown overnight in a shaker (200 rpm; 30 °C).
  • UDP-/V-acetyl-D-glucosamine [glucosamine- 14 C(U)] ([ 14 C] UDP-GlcNAc, Specific activity 300 mCi/mmol) was obtained from American Radio Chemicals, UDP-MurNAc- pentapeptide was purchased from BacWAN, University of Warwick, Coventry, UK (mvw.warwick.ac.uk bacwan). Dansyl-labelled UDP-MurNAc-pentapeptide was synthesized from UDP-MurNAc-pentapeptide as described in section 9.4.3. TLC Silica gel 60 F 254 plates were procured from Merck (Germany). All other chemicals used were at least analytical grade and were obtained from Sigma-Aldrich. Mtb mc 2 6230 was a generous gift from Dr. William Jacobs, Albert Einstein College of Medicine, New York.
  • Mtb mc 2 6230 was grown in 7H9 medium (supplemented with 0.5% (v/v) oleic acid, 0.5% (w/v) albumin, 0.2% (w/v) dextrose, 24 pg/mL D-pantothenate and 0.2% casamino acids). Washed cells were resuspended in Buffer A (50 mM MOPS pH 7.9, 5 mM MgCI 2 , 5 mM DTT, 10% glycerol (v/v)), at 2 mL/g of cells, and disrupted by probe sonication on ice with a Sanyo Soniprep 150 (10 cycles of 60 sec on and 90 sec off).
  • Buffer A 50 mM MOPS pH 7.9, 5 mM MgCI 2 , 5 mM DTT, 10% glycerol (v/v)
  • the whole cell lysates were centrifuged at 5,000 X g for 20 min at 4 °C. The supernatant was further centrifuged at 100,000 X g (for 1 h at 4 °C) in an Optima TLX Ultracentrifuge (Beckman). The membrane-enriched pellets were washed in Buffer A followed by ultracentrifugation at 100, 000 X g. The washed pellets were resuspended in Buffer A, divided into aliquots and frozen at -80 °C. The protein concentration of the membrane-enriched fraction was estimated using a BCA protein assay kit (Pierce).
  • UDP-MurNAc-pentapeptide was achieved as described 4 by chemienzymatic recapitulation of the cytoplasmic synthetic pathway in vitro.
  • Desalted UDP-MurNAc-pentapeptide in sterile water was mixed with an equal volume of acetone and allowed to reacted overnight with a 42 fold molar ratio of dansyl chloride with stirring. The reaction was quenched with a 10 fold molar excess of Tris.CI pH 9 to dansyl chloride before rotary evaporation to remove solvents.
  • Assay mixtures (200 ⁇ _) contained 50 mM MOPS pH 7.9, 5 mM MgCI 2 , 5 mM DTT, 10% glycerol (v/v), 0.1 % CHAPS, 100 ⁇ ATP, 25 ⁇ UDP-MurNAc- pentapeptide, 0.5 ⁇ [ 14 C] UDP-GlcNAc, and varying concentrations of inhibitor (initial screening was carried out at a single concentration of 200 nM and the most potent compounds were screened at a range of concentrations to determine IC50 values). Reactions were initiated by the addition of 400-500 ⁇ g of Mtb mc 2 6230 membrane protein and incubated at 37 °C for 1 h.
  • Fluorescence-based MurX inhibition assay inhibition of MurX activity by analogues 25, 36 and 37

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Abstract

La présente invention concerne des composés antibactériens. En particulier, les composés sont destinés à inhiber la croissance de bactéries, en particulier de Mycobacterium tuberculosis (Mtb), et/ou à cibler des bactéries ayant phospho-MurNAc-pentapeptide translocase. La présente invention concerne également des compositions contenant ces composés et des procédés d'utilisation de ces composés et compositions.
EP17881453.9A 2016-12-16 2017-12-15 Nouveaux composés en tant qu'agents anti-mycobactériens Withdrawn EP3555115A4 (fr)

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