AU2017377671A1

AU2017377671A1 - Novel compounds as anti-mycobacterials

Info

Publication number: AU2017377671A1
Application number: AU2017377671A
Authority: AU
Inventors: Warwick John BRITTON; Richard J. Payne; Anh Tran; Wendy TRAN
Original assignee: University of Sydney
Current assignee: University of Sydney
Priority date: 2016-12-16
Filing date: 2017-12-15
Publication date: 2019-07-11
Also published as: EP3555115A1; CN110300759A; EP3555115A4; WO2018107236A1

Abstract

The present disclosure relates to antibacterial compounds. In particular, the compounds are for inhibiting the growth of bacteria, particularly

Description

Novel compounds as anti-mycobacterials

Field of the invention

The present disclosure relates to antibacterial compounds. In particular, the compounds are for inhibiting the growth of bacteria, particularly Mycobacterium tuberculosis (Mtb), and/or targeting bacteria having phospho-MurNAc-pentapeptide translocase. The present disclosure also relates to compositions containing these compounds and methods of the use of these compounds and compositions.

Background of the invention

Tuberculosis (TB) 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.

Recently, the drugs bedaquiline and delamanid, were approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), respectively, for drug resistant TB. These drugs, however, are only to be used as a last resort due to potential toxicity issues.

To provide effective, low toxicity options for the treatment of drug-sensitive, MDR and XDR TB, new TB drugs with novel modes of action are desperately needed.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

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Summary of the invention

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 5 tuberculosis (Mtb).

In one aspect, the invention provides a compound according to Formula I:

Formula I or a salt, solvate, polymorph or prodrug thereof;

wherein

------is a single or double bond; wherein when------is a single bond, the stereochemistry at this position is of R-configuration.

Ri is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: Ο-ι-Οβ alkyl, Ο-ι-Οβ alkylamino; C-r 15 Οβ alkoxy; Ο-ι-Οβ alkylthio; Ο-ι-Οβ haloalkyl, Ο-ι-Οβ haloalkoxy, Ο-ι-Οβ hydroxyalkyl, Ο-ι-Οβ alkylcarboxy, Ci-C₆ alkylcarboxyamide, C₃-C₇ cycloalkyl; (C1-C4 alkyl)C₃-C₇ cycloalkyl, C₃-C₇ heterocyclyl; (C1-C4 alkyl)C₃-C₇ heterocyclyl, aryl, aryloxy, arylamino, arylthio, C12

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C4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: Ο-ι-Οβ alkyl, Ο-ι-Οβ alkylamino; C-r Οβ alkoxy; Ο-ι-Οβ alkylthio; Ο-ι-Οβ haloalkyl, Ο-ι-Οβ haloalkoxy, Ο-ι-Οβ hydroxyalkyl, Ο-ι-Οβ alkylcarboxy, Ο-ι-Οβ alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C? cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C? heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R3 is -C(O)R4 or-CH₂R_4;; and

R₄ is selected from the group consisting of hydroxyl or optionally substituted: C-r C15 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; (C1-C4 alkyl)C₃-C₇ cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C₃-C₇heterocyclyl, aryl, aryloxy, arylamino, arylthio, C1-C4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl.

with the proviso that:

when------is a double bond, R₃ is not -COOH, and when------is a single bond and R1 is hydrogen, methyl, isobutyl, benzyl, fluoroor hydroxy-substituted benzyl, -C3-C4 alkylamino, -CH(CH₃)OH, -CH₂COOH, CH₂C(O)C(CH₃)3, -CH(OH)CH₃ or -CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl, -(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₃ is not COOH.

In another aspect, the invention provides a compound according to Formula II:

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Formula II or a salt, solvate, polymorph or prodrug thereof;

wherein

R-ι is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C-i-C₆ alkyl, C-i-C₆ alkylamino; C-r Οβ alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 10 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C? cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC₄ aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; C-r Οβ alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C7 cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C₃-C₇ heterocyclyl, aryl, aryloxy, arylamino, arylthio, C14

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R₃ is -C(O)R₄ or-CH₂R4_;; and

R₄ is selected from the group consisting of hydroxyl or optionally substituted: C-r C15 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; (C1-C4 alkyl)C3-C? cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C1-C4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl.

with the proviso that:

when------is a double bond, R₃ is not -COOH, and when------is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoroor hydroxy-substituted benzyl, -C3-C4 alkylamino, -CH(CH₃)OH, -CH₂COOH, CH₂C(O)C(CH₃)3, -CH(OH)CH₃ or -CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl, -(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₃ is not COOH.

In another aspect, the invention provides a pharmaceutical composition comprising a compound according to Formula I and/or Formula II and a pharmaceutically acceptable excipient.

In another aspect, 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 bacteria may be gram positive or gram negative.

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In one aspect, therefore, 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.

In another aspect, the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAcpentapeptide translocase, 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.

In an aspect, 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.

In another aspect, 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.

In one aspect, 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).

In another aspect, 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

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PCT/AU2017/051394 medicament for the prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAc-pentapeptide translocase.

In an aspect, 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.

In yet another aspect, 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.

In yet another aspect, 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).

In yet another aspect, 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-MurNAcpentapeptide translocase.

In an aspect, 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.

As used herein, except where the context requires otherwise, the term comprise and variations of the term, such as comprising, comprises and comprised, are not intended to exclude further additives, components, integers or steps.

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Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Inhibition of Mtb H37Rv by analogue 7.

Figure 2. Inhibition of Mtb H37Rv by analogue 8.

Figure 3. Inhibition of Mtb H37Rv by analogue 9.

Figure 4. Inhibition of Mtb H37Rv by analogue 10.

Figure 5. Inhibition of Mtb H37Rv by analogue 11.

Figure 6. Inhibition of Mtb H37Rv by analogue 21.

Figure 7. Inhibition of Mtb H37Rv by analogue 22.

Figure 8. Inhibition of Mtb H37Rv by analogue 23.

Figure 9. Inhibition of Mtb H37Rv by analogue 24.

Figure 10. Inhibition of Mtb H37Rv by analogue 25.

Figure 11. Inhibition of Mtb H37Rv by analogue 26.

Figure 12. Inhibition of Mtb H37Rv by analogue 27.

Figure 13. Inhibition of Mtb H37Rv by analogue 28.

Figure 14. Inhibition of Mtb H37Rv by analogue 30.

Figure 15. Inhibition of Mtb H37Rv by analogue 31.

Figure 16. Inhibition of Mtb H37Rv by analogue 32.

Figure 17. Inhibition of Mtb H37Rv by analogue 33.

Figure 18. Inhibition of Mtb H37Rv by analogue 34.

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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.

Figure 22. Inhibition of Mtb H37Ra in THP-1 cells by analogue 25. IC50 represents average of 2 independent experiments, each performed in triplicate.

Figure 23. Inhibition of Mtb H37Ra in THP-1 cells by analogue 36. IC50 represents average of 2 independent experiments, each performed in triplicate.

Figure 24. Inhibition of Mtb H37Ra in THP-1 cells by analogue 37. IC50 represents average of 2 independent experiments, each performed in triplicate.

Figure 25. TLC assay from Mtb me² 6230 membranes for the inhibition of MurX by analogue 25. A: TLC; B: Raw dose-response curve for inhibition of MurX by 25; C: Log-transformed dose-response curve for inhibition of MurX by 25.

Figure 26. TLC assay from Mtb me² 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.

Figure 27. TLC assay from Mtb me² 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.

Figure 28. TLC assay from Mtb me² 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.

Figure 29. Evaluation of kinetic parameters of MurX. A: Michaelis-Menten plot; B: Raw enzyme kinetics data. Data represents average of two independent experiments.

Figure 30. Fluorescence assay from Mtb me² 6230 membranes for the inhibition of MurX by analogue 25. A: Raw dose-response curve for inhibition of MurX by 25; B:

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Log-transformed dose-response curve for inhibition of MurX by 25. Data presented is the average of two independent experiments.

Figure 31. Fluorescence assay from Mtb me² 6230 membranes for the inhibition of MurX by analogue 36. A: Raw dose-response curve for inhibition of MurX by 36; B: Log-transformed dose-response curve for inhibition of MurX by 36. Data presented is the average of two independent experiments.

Figure 32. Fluorescence assay from Mtb me² 6230 membranes for the inhibition of MurX by analogue 37. A: Raw dose-response curve for inhibition of MurX by 37; B: Log-transformed dose-response curve for inhibition of MurX by 37. Data presented is the average of two independent experiments.

Figure 33. Fluorescence assay from Mtb me² 6230 membranes for the inhibition of MurX by tunicamycin (positive control). A: Raw dose-response curve for inhibition of MurX by tunicamycin; B: Log-transformed dose-response curve for inhibition of MurX by tunicamycin. Data presented is the average of two independent experiments.

Figure 34. Inhibition of Mtb H37Rv by analogue 71.

Figure 35. Inhibition of Mtb H37Rv by analogue 79.

Figure 36. Inhibition of Mtb H37Rv in THP-1 cells by analogue 78.

Figure 37. Inhibition of Mtb H37Rv in THP-1 cells by analogue 79.

Detailed description of the embodiments

The present invention describes compounds for the prevention and/or treatment of a disease or condition modulated by bacteria. According to the invention, the disease or condition may be associated with, caused by, or arise from the relevant bacteria infection. Preferably, the bacteria are gram positive bacteria. Even more preferably, the bacteria are Mycobacterium tuberculosis (Mtb).

In one aspect, the compounds provided herein are useful in the prevention and/or treatment of conditions modulated by bacteria. Without wishing to be bound to

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In one aspect, the invention provides a compound according to Formula I:

Formula I or a salt, solvate, polymorph or prodrug thereof;

wherein

R-ι is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: Ο-ι-Οβ alkyl, Ο-ι-Οβ alkylamino; C-r C₆ alkoxy; Ci-C₆ alkylthio; Ci-C₆ haloalkyl, Ci-C₆ haloalkoxy, Ci-C₆ hydroxyalkyl, Ci-C₆alkylcarboxy, Ο-ι-Οβ alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C₃-C7 cycloalkyl,

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C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; C-r Οβ alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C7 cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC₄ aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R3 is -C(O)R4 or-CH₂R_4;; and

R₄ is selected from the group consisting of hydroxyl or optionally substituted: C-r C15 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; (C1-C4 alkyl)C3-C7 cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C1-C4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl.

with the proviso that:

when------is a double bond, R₃ is not -COOH, and when------is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoroor hydroxy-substituted benzyl, -C3-C4 alkylamino, -CH(CH₃)OH, -CH₂COOH, CH₂C(O)C(CH₃)₃, -CH(OH)CH₃ or -CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl, -(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₃ is not COOH.

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In one aspect, the invention provides a compound according to Formula II:

Formula II or a salt, solvate, polymorph or prodrug thereof;

wherein

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; C-r 10 Οβ alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C-i-C₆ alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C₃-C₇ cycloalkyl, C3-C7 heterocyclyl; (C1-C4 alkyl)C3-C? heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C-i-C₆ alkyl, C-i-C₆ alkylamino; C113

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Ce alkoxy; C-i-Ce alkylthio; C-i-Ce haloalkyl, C-i-Ce haloalkoxy, C-i-Ce hydroxyalkyl, C-i-Ce alkylcarboxy, C-i-Ce alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C? cycloalkyl,

C3-C7 heterocyclyl; (C1-C4 alkyl)C₃-C₇ heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-iC4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C1-C4 alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₃ is -C(O)R4 or-CH₂R_4;; and

with the proviso that:

when------is a double bond, R₃ is not -COOH, and when------is a single bond and R1 is hydrogen, methyl, isobutyl, benzyl, fluoroor hydroxy-substituted benzyl, -C3-C4 alkylamino, -CH(CH₃)OH, -CH₂COOH, CH₂C(O)C(CH₃)₃, -CH(OH)CH₃ or -CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl, -(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₃ is not COOH.

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In one embodiment, the compound is a compound according to Formula III:

Formula III or a salt, solvate, polymorph or prodrug thereof;

wherein Ri, R₂ and R₄ are defined above as for Formula II, with the proviso that:

when------is a double bond, R₄ is not -OH, and when------is a single bond and R-ι is hydrogen, methyl, isobutyl, benzyl, fluoroor hydroxy-substituted benzyl, -C3-C₄ alkylamino, -CH(CH₃)OH, -CH₂COOH, 10 CH₂C(O)C(CH₃)3, -CH(OH)CH₃ or-CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl,

-(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₄ is not OH.

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In another embodiment, the compound is a compound according to Formula IV:

Formula IV or a salt, solvate, polymorph or prodrug thereof;

wherein Ri, R₂ and R₄ are defined as above for Formula II.

Preferred embodiments for compounds of Formulae I, II, III and/or IV are described below.

Preferably,------is a single bond.

Preferably, Ri is selected from the group consisting of hydrogen or optionally substituted: Ο-ι-Οβ alkyl, C-i-C₂ aralkyl, optionally substituted (C-i-C₂ alkyl)heteroaryl, C-r C₆ alkylamino; Ci-C₆ alkyloxy, Ci-C₆ alkylcarboxy, Ci-C₆ hydroxyalkyl. Even more preferably, Ri is selected from the group consisting of hydrogen, methyl, hydroxylsubstituted benzyl, naphthyl, C₂-C₄ alkylamino, cyclohexyl, -CH₂-cyclohexyl or 15 CH(OH)CH₃.

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Preferably R₂ is selected from the group consisting of optionally substituted: C-r

Οβ alkyl, (C-i-C₂ alkyl)C₃-C7 cycloalkyl, C1-C4 aralkyl and Ο-ι-Οβ alkylthio. Even more preferably, R₂ is selected from the group consisting of: isopropyl, -CH₂-naphthyl, -CH₂cyclohexyl, -CH₂CH₂SCH₃; -CH₂CH₂S(O)CH₃ and

Preferably R₄ is selected from the group consisting of optionally substituted: C-r C15 alkyloxy, C-i-C₄ aralkyl; C1-C15 alkylamino; C-i-C₄ aralkoxy, C-i-C₄ aralkylamino and C1-C15 alkylamino. More preferably, R₄ is selected from the group consisting of methoxy, hexoxy, dodecanyloxy, hydroxy, -CH₂C(CH₃)₃, -O-benzyl, -NH-benzyl, -NH10 benzyl, hexylamino. Even more preferably, R₃ is selected from the group consisting of CH₂C(CH₃)₃, -O-benzyl, -NH-benzyl and hexylamino.

In some preferred embodiments, consisting of:

the compound is selected from the group

x.y·

Η ..—Ζ «Άιμ.M

... .,. K -,,../0

T h 0\

Λ y ¥ / QH

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Η Η

θ%'·

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Ο Ο

or a salt, solvate, polymorph or prodrug thereof.

In one embodiment, the compound of Formula I and/or Formula II is selected from the groups consisting of:

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or a salt, solvate, polymorph or prodrug thereof.

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In one preferred embodiment, the compound of Formula I and/or Formula II is selected from the group consisting of:

o

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In one aspect, the invention provides a compound selected from the following:

o

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Preferably, the compound is

Definitions

As used herein the term 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, 11, 12, 13, 14 or 15 carbon atoms. The term therefore encompasses saturated alkyl groups as well as alkenyl and 10 alkynyl groups. The alkyl group is optionally substituted with substituents, multiple degrees of substitution being allowed. Examples of alkyl as used herein 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) 15 bonds.

As used herein, the terms C1-C3 alkyl, C1-C4 alkyl, C1-C6 alkyl and C1-C15 alkyl refer to an alkyl group, as defined above, containing at least 1, and at most 3, 4, 6

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PCT/AU2017/051394 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).

As used herein, the term halogen refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) and the term halo refers to the halogen radicals fluoro (-F), chloro (Cl), bromo (-Br), and iodo (-I). Preferably, ‘halo’ is fluoro or chloro.

As used herein, the term cycloalkyl refers to a non-aromatic cyclic hydrocarbon ring. In a like manner the term C3-C7 cycloalkyl refers to a non-aromatic cyclic hydrocarbon ring having from three to seven carbon atoms, or any range in between. For example, 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. Exemplary 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).

As used herein, the terms 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. The term 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. The term C3-C7 heterocyclyl also includes heterocyclyl groups containing C4-C5, C5-C7, C6-C7, C4-C7, C4-C6 and C5-C6 carbon atoms. Preferably, 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). Examples of heterocyclic moieties include, but are not limited to, tetrahydrofuran, pyran, oxetane, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, Nmethylpiperazinyl, 2,4-piperazinedione, pyrrolidine, imidazolidine, pyrazolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene and the like. One

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PCT/AU2017/051394 substituted heterocyclyl system of this invention is a substituted coumarin ring, such as

6,7-dimethoxy coumarin.

The term “(Ci-C₂alkyl)C₃-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, -CH₂-heterocycle or -CH₂CH₂-heterocycle). These heterocycles may be further substituted.

Substituted cycloalkyl and heterocyclyl groups may be substituted with any suitable substituent as described below.

As used herein, the term 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. Examples of aryl groups include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl, biphenyl, as well as substituted derivatives thereof.

As used herein, the term 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. Examples of 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.

As used herein, the term “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. Some non-limiting examples of alkylthio groups include -CH₂CH₂SO₂CH₃, 28

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CH₂S(O)OH, -SO₂CH₂CH_3i -CH₂S-OH, -CH₂SH, -CH₃CH₂SCH₃, CH₂CH₂S(O)CH₃,

CH₂CH₂SO₃H. These groups may be further substituted, as discussed below, to provide groups such as alkylsulfonylamino.

As used herein, the term “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, -OCH₂CH_3i -CO₂CH₂CH_3i -CH₂OH, -CH₃CH₂C(O)CH₃, CH₂CH₂COOCH₃, -OCH₂CH₂OCH_3i or the like. These groups may be further substituted, as discussed below.

As used herein, the term “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. For example, an alkylamino group may be groups such as -CH₂CH₂NHCH₃, NHCH₂CH₃, -CH₂N(CH₃)_2i -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 R₃). Alkylamino groups may be further substituted, as discussed below.

As used herein, the term “haloalkyl” refers to an alkyl group as defined above that is bonded to a halo group. Some examples of an haloalkyl group include -CH₂F, -CF₃and -CH₂CH₂CH₂CHCI₂. Where possible, these groups may be further substituted, as discussed below. For example, -CH₂C(O)CH₂CHCI₂.

As used herein, the term “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.

As used herein, the term “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.

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As used herein, the term “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.

As used herein, the term “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.

As used herein, the terms “aryloxy”, “arylamino” or “arylthio” refer to an aryl group as defined above that is bonded through an oxygen (—O—) atom, amino group or thio group respectively. The amino and thio groups may be further substituted, as discussed below.

As used herein, the term “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 “C1-C4 aralkyl” the C1-C4 refers to the number of carbon atoms in the alkyl component of the group.

As used herein, the term “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 “C1-C4 aralkoxy” the C1-C4 refers to the number of carbon atoms in the alkyl component of the group.

As used herein, the term “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 “C1-C4 aralkylamino” the C1-C4 refers to the number of carbon atoms in the alkyl component of the group.

A substituent as used herein, refers to a molecular moiety that is covalently bonded to an atom within a molecule of interest. For example, 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. The term substituted, as used herein, means that any one or more

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PCT/AU2017/051394 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.

The terms optionally substituted or “may be substituted” and the like, as used throughout the specification, denotes that the group may or may not be further substituted or fused (so as to form a polycyclic system), with one or more non-hydrogen substituent groups. Suitable chemically viable substituents for a particular functional group will be apparent to those skilled in the art.

Examples of substituents include but are not limited to:

Ci-C₆ alkyl, Ο-ι-Οβ haloalkyl, Ο-ι-Οβ haloalkoxy, Ο-ι-Οβ hydroxyalkyl, C3-C7 heterocyclyl, C3-C7 cycloalkyl, Ο-ι-Οβ alkoxy, Ο-ι-Οβ alkylthio, Ο-ι-Οβ alkylcarboxy, alkylcarboxyamide, oxo, hydroxy, thio, amino, acyl, carboxy, carbamoyl, aryl, aryloxy, heteroaryl, aminosulfonyl, aroyl, aroylamino, heteroaroyl, acyloxy, aroyloxy, heteroaroyloxy, alkoxycarbonyl, nitro, cyano, halogen, ureido or Ο-ι-Οβ perfluoroalkyl.

Any of these groups may be further substituted by any of the above-mentioned groups, where appropriate. For example, alkylamino, or dialkylamino, Ο-ι-Οβ alkoxy, etc.

The 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.

The term “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

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PCT/AU2017/051394 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.

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. General information on types of pharmaceutically acceptable salts and their formation is known to those skilled in the art and is as described in general texts such as “Handbook of Pharmaceutical salts’’ P.H.Stahl, C.G.Wermuth, 1st edition, 2002, Wiley-VCH.

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.

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.

The term “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.

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A “solvate” is formed by the interaction of a compound of the present invention with a solvent.

Therapeutic Uses of the Compounds

Surprisingly, the inventors have discovered that compounds according to Formula I, Formula II, Formula III and/or Formula IV have exhibited potent and/or selective activity against Mtb. These compounds have been found to disrupt the activity of Mtb Phospho-MurNAc-pentapeptide translocase (MurX or translocase I), the integral membrane enzyme responsible for the biosynthesis of lipid I, a key intermediate in mycobacterial peptidoglycan synthesis.

These compounds may therefore be useful in the prevention and/or treatment of diseases or conditions modulated by Mycobacterium tuberculosis (Mtb) and/or bacteria having the enzyme Mtb phospho-MurNAc-pentapeptide translocase (MurX). The compounds may therefore be useful in the prevention and/or treatment of tuberculosis.

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.

Preferably, these other bacteria are gram positive. Preferably, 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. Even more preferably, 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.

In one aspect, therefore, the invention provides a pharmaceutical composition comprising a compound according to Formula I, Formula II, Formula III and/or Formula

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IV or a salt, solvate, polymorph or prodrug thereof, and a pharmaceutically acceptable excipient.

In another aspect, 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.

In one embodiment, the bacteria is A baumannii. In another embodiment, the bacteria is E. coli. In yet another embodiment, the bacteria is P. aeruginosa. In another embodiment, the bacteria is S. aureus. In another embodiment, the bacteria is methicillin-resistant Staphylococcus aureus. In another embodiment, the bacteria is V. cholerae. In a further embodiment, the bacteria is E. aerogenes. In another embodiment, the bacteria is O. 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. ivanovii. In another embodiment, the bacteria is S. epidermidisAn still a further embodiment, the bacteria is S. typhimurium. In another embodiment, the bacteria is Y. pseudotuberculosis.

Preferably, the bacteria are gram positive. Preferably, the bacteria are Mycobacterium tuberculosis (Mtb), Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus, Mycobacterium bovis, Bacillus subtilis, Staphylococcus epidermis, Enterococcus faecium, Listeria ivanovii, Staphylococcus aureus, methicillinresistant Staphylococcus aureus. Even more preferably, the bacteria are Mycobacterium tuberculosis (Mtb), Mycobacterium avium, Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium bovis. Most preferably, the bacteria are Mycobacterium tuberculosis (Mtb).

In another aspect, 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

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PCT/AU2017/051394 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.

In another aspect, the invention provides a method of prevention and/or treatment of a disease or condition modulated by bacteria having phospho-MurNAcpentapeptide translocase, 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.

In one aspect, 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.

In yet another aspect, 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.

In yet another aspect, 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

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PCT/AU2017/051394 prevention and/or treatment of a disease or condition modulated by Mycobacterium tuberculosis (Mtb).

In yet another aspect, 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.

In yet another aspect, 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 tuberculosis.

In another aspect, 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.

In another aspect, 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).

In another aspect, 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

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PCT/AU2017/051394 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.

In a further aspect, 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 tuberculosis.

The phospho-MurNAc-pentapeptide translocase may be MurX or MraY. Preferably, the phospho-MurNAc-pentapeptide translocase is MurX.

In one embodiment of the invention, the tuberculosis (TB) being prevented or treated in the above methods and uses is Multi-Drug Resistant tuberculosis (MDR TB). In another embodiment, the tuberculosis being prevented or treated in the above methods and uses is extensive drug resistant tuberculosis (XDR TB).

In one embodiment, the TB is resistant to treatment with rifampicin, isoniazid, ethambutol and/or pyrazinamide.

In one aspect, the Mtb is the H37Rv strain of Mtb.

Administration

Pharmaceutical 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. The term 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. In certain embodiments,

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PCT/AU2017/051394 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. For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, one or more compounds may be combined with 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.

In the context of this specification the term “administering” and variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.

For the inhibition of Mtb and/or Mtb phospho-MurNAc-pentapeptide translocase (MurX), such as H37Rv strain of Mtb, and for the prevention and/or treatment of tuberculosis and other diseases and conditions modulated by Mtb and other bacteria, 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. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. Dosage unit forms will generally contain between about 1 mg to about 500 mg of an active ingredient.

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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. A person skilled in the art will appreciate that the dosage regime or therapeutically effective amount of a compound according to Formula I, Formula II, Formula III and/or Formula IV to be administered may need to be optimized for each individual. 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.

The terms “therapeutically effective amount” or “effective amount” refer 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.

The terms “treating”, “treatment” and “therapy” are used herein to refer to curative therapy, prophylactic therapy and preventative therapy. Thus, in the context of the present disclosure the term “treating” encompasses curing, ameliorating or tempering the severity of a condition modulated by gram positive bacteria, such as tuberculosis.

“Preventing” or prevention 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.

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Biological testing

Compounds of the present invention were screened against the virulent H37Rv strain of Mtb using a resazurin assay, then counter-screened against HEK293 cells to gauge selectivity against a panel of 15 pathogenic Gram negative and Gram positive bacterial strains.

The compounds tested were not cytotoxic to HEK293 cells up to a concentration of 200 μΜ. 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 me²6230 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-[¹⁴C]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.

Inhibition of polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA) by these compounds was then investigated. The compounds of the present invention tested did not, however, inhibit WecA, suggesting these compounds are selective inhibitors of lipid I formation in peptidoglycan biosynthesis.

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.

These compounds were also assessed for their anti-mycobacterial activity in an intracellular assay utilising THP-1 macrophages, which were infected with Mtb and the inhibition of mycobacterial growth was measured in the presence of a range of

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PCT/AU2017/051394 concentrations of the compounds. The compounds tested maintained antimycobacterial activity against intracellular mycobacterial growth.

Finally, these compounds were tested for stability in mouse and human plasma and mouse and human liver microsomes. Each of the compounds showed excellent stability, with degradation half-lives of > 7 h for human and mouse plasma and >160 min for human and mouse liver microsomes.

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.

Further details of the biological testing can be seen in the Examples section.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

The methods and compounds described herein are described by the following illustrative and non-limiting examples.

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EXAMPLES

1. Materials and Methods

Analytical thin layer chromatography (TLC) was performed on commercially prepared silica plates (Merck Kieselgel 60 0.25 mm F254). Flash column chromatography was performed using 230-400 mesh Kieselgel 60 silica eluting with distilled solvents as described. Ratios of solvents used for TLC and column chromatography are expressed in v/v as specified. Compounds were visualised by UV light at 254 nm or using vanillin or cerium molybdate stain. Commercial materials were used as received unless otherwise noted. DCM and MeOH were distilled from calcium hydride, and THF and diethyl ether were distilled over sodium/benzophenone. tertbutanol was dried over activated 3 A molecular sieves at least 24 h before use. Anhydrous DMF was purchased from Sigma Aldrich.

Apparatus information ¹H 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. ¹H NMR chemical shifts are reported in parts per million (ppm) and are referenced to solvent residual signals: CDCIs δ 7.26, MeOD δ 3.31, acetone-cfe δ 2.05, DMSO-cfe δ 2.50 and D₂O δ 4.79. ¹H NMR data is reported as chemical shift (δμ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, td = triplet of doublets, tt = triplet of triplets, qd = quartet of doublets), coupling constant (J Hz) and assignment where possible. In the presence of rotamers, ¹H and ¹³C NMR data for both rotamers were reported when the ratio of the rotamers is smaller than 1.5:1. For cases in which the ratio is greater or equal to 1.5, only the major rotamer was reported.

Low resolution mass spectra were recorded on a Finnigan LCQ Deca ion trap mass spectrometer (ESI). High resolution mass spectra were recorded on a Bruker 7T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR).

Melting points were recorded using a Stanford Research Systems OptiMelt Automated Melting Point System. Infrared (IR) absorption spectra were recorded on a

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Bruker ALPHA Spectrometer with Attenuated Total Reflection (ATR) capability, using

OPUS 6.5 software. Optical rotations were measured using a Perkin Elmer Model 341 polarimeter and [αβ⁵ values are reported in 10'¹ deg cm² g’¹.

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 CH₃CN (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 UVA/is 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 CH₃CN (Solvent B) and a linear gradient of 050% B over 30 min or 50-100% B over 30 min.

Synthesis

Synthesis of uridylamine 18

The synthesis of uridylamine was carried out using modifications to the route previously published by Boojamra et al.¹

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Figure S1. Synthesis of uridylamine 18. Reagents and conditions: a) TIPSOTf, /Pr₂NEt, DMF, rt, 2.5 h, 82%; b) 10% Pd/C, H₂ (1 atm), MeOH, rt, 30 min, 80%; c) (i) TFA: CH₂CI₂ (9:1 v/v), rt, 15 min; (ii) Isobutylchloroformate, /Pr₂NEt, THF, 0 °C to rt,

1.5 h; (iii) NaBH₄, H₂O (dropwise), 0 °C, 1 h, 69%; d) Tosyl chloride, pyridine, rt, 18 h, 82%; e) NaN₃, DMF, 75 °C, 4 h, 82%; f) (i) TBAF (1 M in THF), THF, rt, 1 h, (ii) Dowex 8-400, CaCO3, MeOH, rt, 1 h, quant.; g) 1,3-propanedithiol, Et₃N, MeOH, rt, 16 h, 68%.

iert-butyl (4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4((triisopropylsilyl)oxy)-4,5-dihydrofuran-2-carboxylate (S2)

OTIPS

To a solution of ester S1 (3.64 g, 12.3 mmol) and Λ/,/V-di isopropylethylamine (2.1 mL, 12.3 mmol) in DMF (37 mL) was added triisopropylsilyl trifluoromethanesulfonate (4.9 mL, 17.6 mmol) and the reaction was stirred at rt for 2.5 h. The reaction was quenched with saturated aqueous Na₂CO3 (40 mL) and partitioned between Et₂O (200 mL) and H₂O (40 mL). The organic layer was washed with 0.2 M HCI (50 mL), H₂O (5x 50 mL), brine (50 mL) and dried over anhydrous Na₂SO4. The organic layer was concentrated in vacuo to give a crude residue that was purified by column chromatography (2:1 v/v Hexane: EtOAc) to afford ester S2 as a white foam (4.57 g, 82%).

[a]p⁵ = -182° (c = 0.27 in CH2CI2). ¹H NMR (500 MHz, CDCI3): δ 8.70 (s, 1H, NH), 7.03 (d, J = 8.1 Hz, 1H, H-6), 6.27 (d, J = 3.2 Hz, 1H, H-3’), 5.96 (d, J = 2.6 Hz, 1H, H-T), 5.77 (d, J = 8.1 Hz, 1H, H-5), 5.23 (dd, J = 3.3, 2.6 Hz, 1H, H-2’), 1.54 (s, 9H, CO₂ ^fBu), 1.12-1.07 (m, 3H, [CH(CH₃)₂]₃Si), 1.06-1.02 (m, 18H, [CHfCH₃)₂]₃Si). ¹³C NMR (126 MHz, CDCI3): δ 162.5 (C=O), 158.2, 151.3, 149.3, 140.0, 111.2, 103.7,

95.1, 83.5, 79.7, 28.0, 17.8, 17.8, 12.0. IR (ATR): 2944, 2868, 1725,

1698 cm’¹. LRMS [/W+H⁺] 453.0. HRMS (ESI m/z) [/W+Na⁺] calcd.

for C₂₂H₃₆N₂O₆SiNa, 475.2240; found, 475.2238.

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PCT/AU2017/051394 iert-butyl (2R, 4R, 5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4((triisopropylsilyl)oxy)tetrahydrofuran-2-carboxylate (S3)

OTIPS

To a solution of ester S2 (568 mg, 1.25 mmol) in MeOH (21 mL) was added 10% Pd/C (86 mg) and the reaction was degassed by passing through 3 cycles of alternating high vacuum and nitrogen. The reaction mixture was allowed to stir under an atmosphere of hydrogen for 30 min. At this point, the reaction was filtered over Celite®, and the filter cake was washed thoroughly with MeOH (100 mL). The solvent was removed in vacuo to give a crude residue that was purified by column chromatography (1:1 v/v Hexane: EtOAc) to afford ester S3 as a viscous colourless oil (480 mg, 80%).

[a]p⁵ = -14° (c = 0.41 in CH2CI2). ¹H NMR (500 MHz, CDCI3): δ 7.18 (d, J = 8.1 Hz, 1H, H-6), 5.72-5.68 (m, 2H, H-1 ’ + H-5), 4.78-4.74 (m, 2H, H-2’ + H-4’), 2.57 (ddd, J = 13.2, 8.7, 6.2 Hz, 1H, H-3’), 2.24 (ddd, J = 13.3, 5.2, 3.8 Hz, 1H, H-3’), 1.49 (s, 9H, CO/Bu), 1.15-1.05 (m, 3H, [ΟΗ(ΟΗ₃)₂]₃δί), 1.05-1.01 (m, 18H, [CH(CH₃J₂]₃Si). ¹³C NMR (126 MHz, CDCI3): δ 170.0 (C=O), 163.0 (C=O), 149.7 (C=O), 141.4, 102.2, 97.2, 82.2,

78.3, 75.1,37.8, 28.0, 17.8, 17.8, 11.9. IR (ATR): 2943, 2867, 1688 cm’¹. LRMS [/W+H⁺]

455.3. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₂₂H₃8N₂O₆SiNa, 477.2397; found, 477.2400.

-{{2R,3R, 5/?)-5-(hydroxymethyl)-3-((triisopropylsilyl)oxy )tetrahydrofuran-2yl)pyrimidine-2,4(1 H,3H)-dione (S4)

OTIPS

Ester S3 (456 mg, 1.0 mmol) was dissolved in 9:1 v/v TFA: CH₂CI₂ (2.2 mL) and the reaction was stirred at rt for 15 min. The solvent was removed in vacuo to give a

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PCT/AU2017/051394 residue that was subsequently dissolved in THF (13 mL) and cooled to 0 °C prior to the addition of isobutyl chloroformate (390 pL, 3 mmol) and /V,/V-diisopropylethylamine (260 pL, 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. Sodium borohydride (265 mg, 7 mmol) was added, followed by dropwise addition of water (2.3 mL) over 20 min to dissolve the sodium borohydride. The reaction was allowed to stir at 0 °C for 1 h before being concentrated in vacuo. The residue was partitioned between EtOAc (40 mL) and water (10 mL). The organic phase was separated, washed with 1 M HCI (10 mL), brine (10 mL) and dried over anhydrous MgSO4. The solvent was removed in vacuo to give a crude residue that was purified by column chromatography (4:1 v/v EtOAc: Hexane -a EtOAc) to afford alcohol S4 as a white foam (265 mg, 69%).

[a]p⁵ = -11° (c = 0.44 in CH2CI2). ¹H NMR (400 MHz, CDCI3): δ 8.94 (s, 1H), 7.25 (d, J = 8.1 Hz, 1H, H-6), 5.72 (d, J = 8.1 Hz, 1H, H-5), 5.69 (d, J = 2.1 Hz, 1H, H-T),

4.73 (ddd, J = 5.7, 3.5, 2.1 Hz, 1 Η, H-2’), 4.61 (app. dq, J = 9.0, 4.7 Hz, 1 Η, H-4’), 3.80-

3.72 (m, 2H, H-5’), 2.60 (s, 1H, OH), 2.27 (ddd, J = 13.9, 8.3, 5.8 Hz, 1H, H-3’), 1.96 (ddd, J = 13.6, 5.0, 3.4 Hz, 1H, H-3’), 1.20-1.08 (m, 3H, [CH(CH₃)₂]₃Si), 1.06 (d, J = 7.0 Hz, 18H, [CH(CH₃)₂]₃Si). ¹³C NMR (101 MHz, CDCI3): δ 163.2 (C=O), 150.0 (C=O), 140.2, 102.1, 95.3, 82.4, 76.1, 65.1, 34.8, 17.9, 17.8, 12.0. IR (ATR): 2943, 2866, 1686 cm’¹. LRMS [/W+Na⁺] 407.0. HRMS (ESI m/z) [/W+Na⁺] calcd. for Ci8H₃₂N₂O₅SiNa, 407.1978; found, 407.1973.

((2R, 4R, 5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4((triisopropylsilyl)oxy)tetrahydrofuran-2-yl)methyl 4-methylbenzenesulfonate (S5)

OTIPS

Alcohol S4 (1.53 g, 3.97 mmol) and p-toluenesulfonyl chloride (3.70 g, 19.3 mmol) were dissolved in pyridine (33 mL) and the reaction was allowed to stir at rt for 18 h. At this point, the reaction was concentrated in vacuo to afford a residue that was dissolved in EtOAc (200 mL), washed with water (50 mL), 1 M HCI (50 mL), brine (50

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PCT/AU2017/051394 mL) and dried over anhydrous MgSO₄. The solvent was removed in vacuo to give a crude residue that was purified by column chromatography (1:1 v/v Hexane: EtOAc) to afford tosylate S5 as a colourless oil (1.73 g, 82%).

[a]p⁵ = -7.3° (c = 0.31 in CH2CI2). ¹H NMR (500 MHz, CDCI3): δ 8.86 (s, 1H, NH),

7.80 (app. d, J = 8.0 Hz, 2H, Ar-H), 7.34 (d, J = 8.0 Hz, 2H, Ar-H), 7.13 (d, J = 8.1 Hz, 1H, H-6), 5.70 (d, J = 8.1 Hz, 1H, H-5), 5.54 (d, J = 2.0 Hz, 1H, H-T), 4.75-4.67 (m, 1H, H-4’), 4.65 (app. dt, J = 5.1,2.0 Hz, 1H, H-2’), 4.21 (dd, J= 10.4, 7.0 Hz, 1H, H-5’), 4.12 (dd, J = 10.4, 4.5 Hz, 1H, H-5’), 2.45 (s, 3H, CH₃), 2.22 (ddd, J= 13.7, 8.3, 5.4 Hz, 1H, H-3’), 1.88 (app. dt, J= 13.7, 3.6 Hz, 1H, H-3’), 1.15-1.02 (m, 3H, [CH(CH₃)₂]₃Si), 1.00 (d, J = 6.7 Hz, 18H, [CH(CH₃)₂]₃Si). ¹³C NMR (126 MHz, CDCI₃): δ 163.0, 149.8, 145.0, 140.0, 132.7, 129.9, 128.0, 102.1, 95.7, 79.0, 75.8, 71.5,

35.2, 21.7, 17.9, 17.8, 11.9. IR (ATR): 2944, 2866, 1686 cm’¹. LRMS [/W+Na⁺] 561.0. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₂₅H₃8N₂O₇SSiNa, 561.2067; found, 561.2061.

1-((2/?,3/?,5/?)-5-(azidomethyl)-3-((triisopropylsilyl)oxy)tetrahydrofuran-2yl)pyrimidine-2,4(1 H,3H)-dione (S6)

N

OTIPS

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 MgSO₄. 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%).

[a]p⁵ = -20° (c = 0.32 in CH2CI2). ¹H NMR (400 MHz, CDCI3): δ 9.01 (s, 1H, NH),

7.21 (d, J = 8.1 Hz, 1H, H-6), 5.72 (d, J = 8.1 Hz, 1H, H-5), 5.66 (d, J = 2.1 Hz, 1H, H-

T), 4.75 (ddd, J = 5.7, 3.6, 2.1 Hz, 1H, H-2’), 4.62 (tt, J = 7.4, 4.7 Hz,

1H, H-4’), 3.60 (dd, J = 12.7, 7.1 Hz, 1H, H-5’), 3.39 (dd, J = 12.7, 4.6 Hz, 1H, H-5’),

2.28 (ddd, J = 13.5, 7.9, 5.7 Hz, 1H, H-3’), 1.93 (ddd, J = 13.5, 4.9, 3.5 Hz, 1H, H-3’),

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1.19-1.08 (m, 3H, [CH(CH₃)₂]₃Si), 1.06 (d, J = 6.7 Hz, 18H, [CH(CH₃J₂]₃Si). ¹³C NMR (101 MHz, CDCIs): δ 163.2 (C=0), 149.9 (C=0), 140.4, 102.1, 96.1, 80.6, 76.1, 55.1,

36.3, 17.9, 17.9, 12.0. IR (ATR): 2944, 2867, 2102, 1686 cm’¹. LRMS [/W+H⁺] 410.0.

HRMS (APCI m/z) [/W+Na⁺] calcd. for CisHsiNsCUSiNa, 432.2043; found, 432.2038.

1-((2/7,3R, 5/?)-5-(azidomethyl)-3-hydroxytetrahydrofuran-2-yl)pyrimidine2,4(1 H,3H)-dione (S7)

OH

A 1 M solution of TBAF in THF (280 μΙ_, 0.28 mmol, 1.15 eq.) was added dropwise to a solution of azide S6 (0.10 g, 0.24 mmol, 1 eq.) in THF (1.8 mL) and allowed to stir for 1 h at rt. The reaction mixture was then diluted with MeOH (2.8 mL) and stirred with calcium carbonate (0.39 g) and Dowex8-400 (1.2 g) for a further 1 h at rt. The reaction mixture was then filtered over Celite ® and concentrated in vacuo to afford the desired azide S7 as a colourless oil (53.5 mg, 88%) which was used without further purification.

[a]_D ²⁵ = +25.8° (c = 0.21 in CH₂CI₂). ¹H NMR (300 MHz, CDCI3): δ 10.53 (brs, 1H, NH), 7.35 (d, J = 7.5 Hz, 1H, H-6), 5.81 (m, 1H, H-5), 5.72 (d, J =7.5 Hz, 1H, Η-Γ), 4.77-4.74 (m, 2H, H-2’ + H-4’), 3.64 (m, 1H, H-5’), 3.42 (m, 1H, H-5’), 2.39 (m, 1H, H3’), 1.99 (m, 1H, H-3’). IR (ATR): 3056, 2101, 1682, 1461 cm’¹. LRMS [/W+H⁺] 253.1. These data are in agreement with those reported by Boojamra et aF

1-((2/7,3/7,5/?)-5-(aminomethyl)-3-hydroxytetrahydrofuran-2-yl)pyrimidine2,4(1 H,3H)-dione (18)

OH

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To a solution of azide S7 (50.0 mg, 198 pmol, 1 eq.) in MeOH (1.0 mL) was added 1,3-propanedithiol (200 pL, 2.0 mmol, 10 eq.) and Et₃N (210 pL, 1.5 mmol, 7.6 eq.). The reaction was allowed to stir for 16 h at rt, before the solvent was removed in vacuo. The resulting residue was redissolved in H₂O (45 mL) and washed with CH₂CI₂(10x 6 mL). The aqueous layer was lyophilised to afford the desired amine 18 as a white fluffy solid (46.4 mg, 0.134 mmol, 68%), which was used without further purification.

[a]o⁵ = -1.6° (c = 0.25 in CH3OH). ¹H NMR (500 MHz, CD3OD): δ 7.56 (d, J = 8.0 Hz, 1 Η, H-6), 5.75 (d, J = 2.2 Hz, 1 Η, H-1 ’), 5.69 (d, J = 8.0 Hz, 1 Η, H-5), 4.68-4.48 (m, 1H, H-4’), 4.42 (ddd, J = 6.1, 3.6, 2.2 Hz, 1H, H-2’), 2.91 (dd, J = 13.6, 3.7 Hz, 1H, H-5’), 2.83 (dd, J = 13.5, 5.9 Hz, 1H, H-5’), 2.34 (ddd, J = 14.1, 8.1, 6.2 Hz, 1H, H-3’), 1.81 (ddd, J = 13.7, 4.8, 3.6 Hz, 1H, H-3’). IR (ATR): 3375, 1684, 1629 cm’¹. LRMS [/W+H⁺] 228.0. These data are in agreement with those previously reported by Boojamra et al.^

Synthesis of DABA fragments S8 and 20

S11

Figure S2. Synthesis of DABA fragments S8 and 20. Reagents and conditions:

a) i. TFA: CH₂CI₂ (1:1 v/v), rt, 30 min, ii. Fmoc-OSu, THF: H₂O (1.2:1 v/v), NaHCOs, 70% over two steps; b) Nanoparticle Zn, 2 M HCI, MeOH, rt, 16 h, 78%; c) i. 20 (1 eq.), 10% Na₂CO₃, 0 °C, ii. Allyl chloroformate (1.1 eq.), dioxane, 0 °C to rt, 1.5 h, 83%; d) i. 2-chlorotrityl chloride resin, /Pr₂NEt, rt, 16 h; ii. 20 vol.% piperidine in DMF, rt, 2x 4 min.

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PCT/AU2017/051394 (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3((benzyloxy)(methyl)amino)butanoic acid (S10)

Acid S9 (synthesized following procedures previously reported by Boojamra et al¹) (4.42 g, 13 mmol) in a mixture of TFA and CH2CI2 (1:1 v/v, 30 mL) was stirred at rt for 30 min. The solvent was removed in vacuo to give a residue which was dissolved in THF (70 mL). Saturated aqueous NaHCO₃ solution (70 mL) as well as solid NaHCO₃(approx. 10 g) were added (until the reaction mixture was basic), followed by the addition of Fmoc-succinimide (4.62 g, 14 mmol). The reaction was allowed to stir at rt for 16 h. At this point, H₂O (100 mL) was added and the reaction mixture was washed with diethyl ether (3x 50 mL). The aqueous layer was acidified to pH 2 with 1 M HCI and extracted with EtOAc (3x 250 mL). The combined organic layers were dried over anhydrous MgSO4 and the solvent was removed in vacuo to give a crude residue which was purified by column chromatography (3:1 v/v Hexane: EtOAc -d 1:1 v/v Hexane: EtOAc) to afford Fmoc-protected acid S10 as a colourless oil (4.19 g, 70%).

[a]o⁵ = +1.8° (c = 0.30 in CH2CI2). ¹H NMR (500 MHz, CDCI3): δ 7.96-7.69 (m, 2H, Ar-H), 7.59 (dd, J = 7.7, 3.4 Hz, 2H, Ar-H), 7.41-7.27 (m, 9H, Ar-H), 5.60 (d, J = 8.3 Hz, 1H, NH), 4.75 (s, 2H, CH₂Ph), 4.43 (m, 3H, oc-CH + Fmoc-CH₂), 4.23 (app. t, J =7.2 Hz, 1H, Fmoc-CH), 3.30-3.05 (m, 1H, β-ΟΗ), 2.68 (s, 3H, NCH₃), 1.22 (d, J = 6.9 Hz, 3H, y-CH₃). ¹³C NMR (126 MHz, CDCI₃): δ 173.5, 156.8, 143.8, 141.5, 136.0, 129.0, 128.8, 128.6, 127.3, 127.2, 125.3, 120.1, 74.7, 67.4, 64.2, 55.3, 47.3,

41.2, 10.1. IR (ATR): 2955, 1720 cm’¹. LRMS [/W+H⁺] 461.4. HRMS (ESI m/z) [/W+Na⁺] calcd. for C27H₂8N2O₅Na, 483.1896; found, 483.1890.

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PCT/AU2017/051394 (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3(methylamino)butanoic acid (S8)

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 (11 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 H₂O (400 mL) and washed with CH₂CI₂ (80 mL). The aqueous layer was extracted with EtOAc (5x 150 mL) and EtOAc/MeOH mixture (9:1 v/v EtOAc: MeOH, 2x 150 mL). The combined organic layers were dried over anhydrous MgSO₄, the solvent was removed in vacuo to give acid S8 as a white solid (1.82 g, 78%) that was used without further purification.

[a]o⁵ = +2.4° (c = 0.40 in MeOH). m.p. 133-135 °C (decomp.). ¹H NMR (500 MHz, CD₃OD): δ 7.86-7.76 (m, 2H, Ar-H), 7.70 (app. t, J = 7.4 Hz, 2H, Ar-H), 7.40 (dd, J = 8.1, 6.7 Hz, 2H, Ar-H), 7.32 (ddd, J =7.7, 6.8, 1.6 Hz, 2H, Ar-H), 4.72-4.67 (m, 1H, DABA-oc-CH), 4.55 (dd, J = 10.5, 6.7 Hz, 1H, Fmoc-CH₂), 4.41-4.32 (m, 1H, FmocCH₂), 4.27 (app. t, J = 6.7 Hz, 1H, Fmoc-CH), 3.77-3.70 (m, 1H, DABA-β-ΟΗ), 2.75 (s, 3H, NCH₃), 1.26 (d, J = 6.5 Hz, 3H, DABA-y-CH₃). ¹³C NMR (100 MHz, CDCI3): δ 174.2 (C=O), 156.7 (C=O), 144.0, 141.4, 136.2, 129.0, 128.6, 128.4, 127.8, 127.2, 125.2, 120.0, 74.5, 67.3, 64.1, 55.7, 47.2, 41.4, 14.3. IR (ATR): 3335, 3049, 1704 cm’¹. LRMS [/W+H⁺] 355.1. HRMS (ESI m/z) [/W+Na⁺] calcd. for C27H₂₈N₂O₅Na, 483.1896; found, 483.1890.

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PCT/AU2017/051394 (2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3(((allyloxy)carbonyl)amino)butanoic acid (20)

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. A solution of allyl chloroformate (196 uL, 1.9 mmol, 1.1 eq.) in 1,4-dioxane (6.2 mL) was added dropwise to the above reaction mixture and the reaction was allowed to warm to rt and stirred for 1.5 h. The reaction was subsequently diluted with H₂O (120 mL), washed with diethyl ether (2 x 40 mL) and acidified to pH 2 with 1 M HCI. The aqueous layer was then extracted with EtOAc (3 x 200 mL), with the organic phases dried over MgSO₄, combined and concentrated in vacuo to a yellow oil which contained the desired acid 20 as a white foam (0.61 g, 83%).

¹H NMR (400 MHz, CDCI₃): δ 7.75 (d, J = 7.5 Hz, 2H, Ar-H), 7.61-7.55 (m, 2H, Ar-H), 7.39 (app. t, J = 7.5 Hz, 2H, Ar-H), 7.30 (app. t, J = 7.5 Hz, 2H, Ar-H), 5.97-5.81 (m, 2H, NH + ΑΙΙοο-β-CH), 5.33-5.14 (m, 2H, ΑΙΙοο-γ-ΟΗ₂), 4.73-4.32 (m, 6H, Alloc-ocCH₂ + Fmoc-CH₂ + DABA-a-CH + DABA-β-ΟΗ), 4.20 (t, J = 6.8 Hz, 1H, Fmoc-CH),

2.85 (s, 3H, NCH₃), 1.25 (d, J = 6.7 Hz, 3H, DABA-y-CH₃). ¹³C NMR (101 MHz, CDCI3): δ 173.0 (C=O), 156.1 (C=O), 143.8, 143.6, 141.3, 132.5, 127.7, 127.1, 125.0, 120.0,

117.6, 67.2, 66.6, 57.1, 53.4, 47.2, 29.5, 14.1.

IR (ATR): 3312, 2984, 1688, 1683, 1528 cm’¹. LRMS [/W+H⁺] 439.3. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₂4H₂₆N₂O₆Na, 461.1688; found, 461.1683.

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PCT/AU2017/051394 (2S,3S)-3-(((allyloxy)carbonyl)amino)-2-aminobutanoic acid on 2-chlorotrityl chloride resin (S11)

Amino acid 20 (515 mg, 1.1 mmol) was loaded onto 2-chlorotrityl chloride resin 5 (745 mg, 1.05 mmol) in CH2CI2 (6.5 mL) at rt for 16 h using /V,/V-diisopropylethylamine (550 pL, 3.1 mmol) and Fmoc-deprotected according to general procedure 2 to afford resin-bound S11 (65% loading as determined by spectroscopic measurement of the fulvene-piperidine adduct at λ= 301 nm).

Synthesis of tryptophan 4-nitrophenylcarbamate S12

Figure S3. Synthesis of tryptophan 4-nitrophenyl carbamate S12. Reagents and conditions: a) 20 vol.% piperidine in MeCN, rt, 30 min, 75%; b) 4-nitrophenyl chloroformate, CH₂CI₂, rt, 18 h, 80%.

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PCT/AU2017/051394 iert-butyl (S)-3-(3-(iert-butoxy)-2-(((4-nitrophenoxy)carbonyl)amino)-3oxopropyl)-1 H-indole-1 -carboxylate (S12)

fe/Y-Butyl ester S13 (2.76 g, 4.74 mmol) was treated with 20 vol.% piperidine in acetonitrile (20 mL) and the reaction was allowed to stir at rt for 20 min. The solvent was removed in vacuo to give a crude residue that was purified by column chromatography (3:1 v/v Hexane: EtOAc, 0.1 vol.% Et₂NH) to afford the deprotected tryptophan S14 as a yellow oil (1.28 g, 75%). To a solution of S14 (1.28 g,

3.56 mmol) and /V,/V-diisopropylethylamine (620 pL, 3.56 mmol) in CH2CI2 (26 mL) was added p-nitrophenyl chloroformate (860 mg, 4.27 mmol) and the reaction was allowed to stir at rt for 19 h. The solvent was removed in vacuo to give a crude residue that was purified by column chromatography (7:1 v/v Hexane: EtOAc -a 5:1 v/v Hexane: EtOAc) to afford carbamate S12 as a white foam (1.49 g, 80%).

[a]o⁵ = +75° (c = 0.36 in CH2CI2). ¹H NMR (400 MHz, CDCI3): δ 8.23 (d, J = 9.1 Hz, 2H, H-3 + H-5), 7.57 (app. dt, J = 7.9, 1.0 Hz, 1H, Ar-H), 7.46 (s, 1H, Ar-H), 7.34 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H, Ar-H), 7.29-7.21 (m, 3H, Ar-H), 5.74 (d, J = 7.8 Hz, 1H, NH), 4.70-4.58 (m, 1H, oc-CH), 3.35 (dd, J = 14.8, 5.8 Hz, 1H, β-ΟΗ2), 3.25 (dd, J =

14.9, 5.5 Hz, 1H, β-ΟΗ₂), 1.66 (s, 9H, 3x CH₃), 1.46 (s, 9H, 3x CH₃). ¹³C NMR (101 MHz, CDCI₃): δ 170.0 (C=O), 155.7 (C=O), 152.5 (C=O), 144.9, 130.7, 125.5, 125.1,

124.7, 124.2, 122.7, 122.0, 121.6, 118.9, 115.4, 114.8, 83.8, 83.1, 54.9, 28.2, 28.0,

27.5. IR (ATR): 2988, 2973, 1724 cm’¹. LRMS [/W(-NO2Ph+Me)+Na⁺] 441.0. HRMS (ESI m/z) [/W(-NO2Ph+Me)+Na]⁺ calcd. for C₂2H₃oN₂06Na 441.2002; found, 441.1995.

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p-TSA,

Trimethyl orthoformate

Quantitative

1. DMSO, EDC.HCI, Pyridine, TFA, DMF, 2 h, rt.

2. Benzylhydrylamine, NaCNBH₃, AcOH, MeOH, 15 h, rt.

29% over 2 steps

(DH

TIPS-OTf, /Pr₂EtN, DMF, 1 h, rt.

84%

bTIPS

1. 10% w/w Pd/C, AcOH, MeOH, 3.5 h, rt.

2. 4 M HCI in 95% EtOH, 3 h, rt.

3. Fmoc-OSu, 1:1 v/v

THF: sat. NaHCO_3(aq), 16 h, rt.

47% over 3 steps

FmocHN

Figure S4. Synthesis of Fmoc-Uridylamine 87.

1-((3a/?,4/?,6/?,6a/?)-6-(Hydroxymethyl)-2-methoxytetrahydrofuro[3,4-i/|[1,3]dioxol-

4-yl)pyrimidine-2,4(1 H,3H)-dione (84)

To a solution of uridine (5.0 g, 21 mmol, 26 eq.) in trimethyl orthoformate (13 mL) was added p-toluenesulfonic acid (0.14 g, 1.6 mmol, 1 eq.) and allowed to stir at room temperature for 16 h. The reaction was subsequently cooled to 0 °C prior to the addition of 28% w/v NaOMe in MeOH (0.15 mL) followed by toluene (5 mL) and allowed to stir at 0 °C for a further 1 h. The reaction mixture was then filtered and washed with toluene to yield 84 as a diastereomerically pure white precipitate (5.5 g, 19 mmol, 94%).

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PCT/AU2017/051394 ¹H NMR (400 MHz, (CD3)2SO) δ 11.38 (s, 1H, NH), 7.77 (d, J= 8.0 Hz, 1H, H-6), 6.09 (s, 1H, CH), 5.80 (d, J = 2.7 Hz, 1H, H-1’), 5.63 (d, J = 8.0 Hz, 1H, H-5), 5.09 (t, J = 5.4 Hz, 1H, OH), 5.00 (dd, J = 6.5, 2.6 Hz, 1 Η, H-2’), 4.85 (dd, J = 6.4, 3.8 Hz, 1H, H-3’), 4.06 (dt, J = 4.5 Hz, 1H, H-4’), 3.59 (dd, J = 11.9, 5.3 Hz, 2H, CH2), 3.21 (s, 3H, CH3); ¹³C NMR (100 MHz, (CD3)₂SO) δ 163.7, 150.8, 142.6, 117.3, 102.2, 91.5, 86.2,

83.2, 80.5, 61.6, 50.7; LRMS [M+H]⁺ 287.0. These data are consistent with those reported by Yasuda et alV^]

1-((2/?,3/?)-5-((Benzhydrylamino)methyl)-3-hydroxy-2,3-dihydrofuran-2yl)pyrimidine-2,4(1 H,3H)-dione (85)

Ό

OH

To a solution of 84 (0.29 g, 1 mmol, 1 eq.) in DMF (6.3 mL) was added DMSO (0.43 mL,6 mmol, 6 eq.), EDC-HCI (0.58 g, 3 mmol, 3 eq.) followed by a premixture of TFA (40 pL, 2 mmol, 2 eq.) and pyridine (80 pL, 1 mmol, 1 eq.) in DMF (1.9 mL). The reaction was allowed to stir for 2 h before the addition of Et₃N (0.56 mL, 4 mmol, 4 eq.) and stirred for a further 30 min. 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 CH₂CI₂ 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.), NaCNBH₃ (94 mg, 1.5 mmol, 1.5 eq.) followed by AcOH (40 pL, 0.7 mmol, 0.7 eq.) and allowed to stir for 15 h. The solvent was removed in vacuo and the resulting residue was redissolved in CH₂CI₂ and washed with a saturated aqueous solution of NaHCO₃, brine and dried (MgSO₄). The organic phase was concentrated in vacuo and the residue was purified by column chromatography eluting with 1:4 v/v hexane:EtOAc followed by neat EtOAc, to yield 85 as a yellow foam (0.12 g, 0.29 mmol, 29% over 2 steps).

¹H NMR (400 MHz, CDCI₃) δ 7.42-7.39 (m, 4H, Ar-H), 7.33-7.30 (m, 4H, Ar-H), 7.25-

7.21 (m, 2H, Ar-H), 7.19 (d, J = 8.1 Hz, 1H, H-6), 6.27 (d, J = 1.45 Hz, 1H, H-1’) 5.64 (d, J = 8.1 Hz, 1 Η, H-5), 5.21 (d, J = 2.3 Hz, 1 Η, H-3’), 4.92 (s, 2H, CH + H-2’), 3.40 (s, 2H,

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CH₂); ¹³C NMR (100 MHz, CDCI₃) δ 163.4, 161.2, 150.6, 143.0, 138.9, 128.7, 128.6,

127.4, 127.3, 127.2, 127.1, 103.0, 100.7, 93.3, 79.7, 66.4, 44.1; HRMS Calcd for

C₂₂H₂iN₃O₄: MNa⁺, 414.14243. Found: MNa⁺, 414.14247.

1-((2/?,3/?)-5-((Benzhydrylamino)methyl)-3-((triisopropylsilyl)oxy)-2,3dihydrofuran-2-yl)pyrimidine-2,4(1 H,3H)-dione (86)

(DTI PS

To a solution of 85 (0.12 g, 0.29 mmol, 1 eq.) and /Pr₂EtN (56 μΙ_, 0.32 mmol, 1.1 eq.) in DMF (0.8 mL) was added dropwise TIPS-OTf (0.12 mL, 0.44 mmol, 1.5 eq.) and allowed to stir for 1 h. The reaction mixture was concentrated in vacuo and the resulting residue was redissolved CH₂CI₂. The organic phase was washed with a saturated aqueous solution of NaHCOs, H₂O followed by brine, dried (Na₂SO₄) and concentrated in vacuo. The resulting residue was purified by column chromatography eluting with 2:1 v/v hexane: EtOAc to yield 86 as a white foam (0.13 g, 0.24 mmol, 84%).

¹H NMR (400 MHz, CDCI3) δ 7.43-7.41 (m, 4H, Ar-H), 7.35-7.31 (m, 4H, Ar-H),

7.27-7.23 (m, 2H, Ar-H), 7.14 (d, J = 8.2 Hz, 1H, H-6), 6.35 (d, J = 2.1 Hz, 1H, H-1’),

5.72 (d, J = 8.1 Hz, 1H, H-5), 5.15 (d, J = 2.3 Hz, 1H, H-3’), 5.05 (t, J = 2.2 Hz, 1H, H-2’), 4.93 (s, 1H, CH), 3.41 (s, 2H, CH₂), 1.10-1.08 (m, 21H, [CH(CH₃)₂]3); ¹³C NMR (100 MHz, CDCI3) δ 162.9, 161.0, 149.7, 143.2, 143.1, 139.5, 128.6, 127.3, 103.4,

101.4, 93.3, 80.4, 66.1, 44.2, 17.9, 12.1; HRMS Calcd for C₃iH₄₂N₃O₄Si: MNa⁺, 570.27585. Found: MNa⁺, 570.27586.

(9H-fluoren-9-yl)methyl (((2/?,4R,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2//)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)carbamate (87)

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Ο Η

FmocHN V^N\=iO λΧ 6 ⁵

0Η

To a solution of 86 (0.13 g, 0.24 mmol, 1 eq.) in MeOH (12 mL) was added 10% Pd/C (51 mg, 0.05 mmol, 0.2 eq.) and AcOH (69 pL, 1.2 mmol, 5 eq.). The solvent was degassed for 10 min before the flask was evacuated and back filled with N_2(g) three times. The reaction was then allowed to stir under an atmosphere of H_2(g) for 3.5 h. Following the completion of the reaction as determined by LCMS, H_2(g) was removed and the reaction mixture was filtered over Celite® and concentrated in vacuo. The resulting residue was redissolved in 4 M HCI in 95% EtOH (6 mL) and stirred for 3 h before being concentrated in vacuo. The resulting residue was then redissolved in H₂O and washed with EtOAc (x 3) and lyophilised. The residue was redissolved in 1:1 v/v THF: sat. NaHCO3(_aq) (1.3 mL). To this solution was added Fmoc-OSu (48 mg, 0.14 mmol, 1.1 eq.) and allowed to stir for 16 h. The reaction mixture was then diluted with EtOAc and washed with H₂O followed by brine, dried (MgSO₄) and concentrated in vacuo. The resulting crude residue was subsequently purified by column chromatography eluting with CH₂CI₂ followed by 10% MeOH in CH₂CI₂ to yield 87 as a white foam (41 mg, 0.090 mmol, 47% over 3 steps).

¹H NMR (400 MHz, CDCI₃) δ 7.76-7.74 (m, 2H, Ar-H), 7.60-7.58 (m, 2H, Ar-H),

7.40-7.36 (m, 2H, Ar-H), 7.31-7.27 (m, 3H, 2 x Ar-H + 1 x H-6), 5.77 (m, 2H, 1 x NH + 1 x H-1 ’), 5.69 (d, J = 7.9 Hz, 1H, H-5), 5.06 (br s, 1H, OH), 4.53-4.50 (m, 2H, 1 x H-2’ + 1 x h-4’), 4.42-4.37 (m, 2H, Fmoc-CH₂), 4.21 (t, J = 6.8 Hz, 1H, Fmoc-CH), 3.52-3.39 (m, 2H, H-5’), 2.23-2.20 (m, 1H, H-3’), 1.95-1.91 (m, 1H, H-3’); ¹³C NMR (100 MHz, CDCI3) 0 163.9, 156.7, 151.3, 143.9, 143.8, 141.3, 139.1, 127.7, 127.1, 125.1, 120.0, 102.3,

94.2, 80.9, 66.8, 50.7, 47.2, 45.4, 33.7, 29.7; LRMS [M+H]⁺ 450.0. These data in agreement with those reported by Tran.^{1 [2]}

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1. 3,4-Dihydro-2H-pyran-2-methanol, PPh₃ DIAD, THF z 2. LiOH’l:1 v/v MeOH:H₂O O -----------------------Quantitative

Figure S5. Synthesis of Linker 88.

The synthesis of linker 88 was carried out following the procedure published by Torres-Garcia et al^{1 * [3]}

4-((3,4-dihydro-2H-pyran-2-yl)methoxy)benzoic acid (88) o

A solution of methyl 4-hydroxybenzoate (2.0 g, 13 mmol, 1 eq.) and PPh₃ (3.8 g, 14 mmol, 1.2 eq.) in THF (22 mL) was stirred for 30 min. This was then followed by the addition of DIAD (2.8 mL, 14 mmol, 1.2 eq.) at 0 °C. The reaction was allowed to warm to room temperature and stirred for 16 h. The solvent was removed in vacuo and redissolved in 2:1 v/v H₂O:MeOH. LiOH (1.4 g, 59 mmol, 4.5 eq.) was added and the mixture was refluxed at 120 °C for 16 h. The reaction mixture was allowed to cool to room temperature before being washed with CH₂CI₂ (x 3). The 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).

¹H NMR (400 MHz, (CD₃)₂SO) 5 12.61 (s, 1H, COOH), 7.91-7.87 (m, 2H, Ar-H),

7.05-7.01 (m, 2H, Ar-H), 6.40 (m, 1H, OCH=CH), 4.72-4.69 (m, 1H, OCH=CH), 4.13-4.12 (m, 3H, 1 x CH + 2 x OCH₂), 2.10-2.03 (m, 1H, H-3), 1.98-1.95 (m, 2H, 1 x H-3 + 1 x H-4), 1.74-1.64 (m, 1H, H-4); ¹³C NMR (100 MHz, (CD₃)₂SO) δ 167.0, 162.0,

143.2, 131.4, 123.2, 114.3, 100.5, 72.8, 69.9, 23.6, 18.7. LRMS [M+H]⁺235.0. These data are in agreement with those reported by Torres-Garcia et al^[3]

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PyBOP, NMM, 1 h, rt.

FmocHN^/

FmocHN

HATU, /Pr₂EtN, DMF, 16 h, rt.

FmocHN

1. Pd(PPh₃)₄, PhSiH₃, CH₂CI₂, 15 min, rt.

2. Boc-Gly-OH, HATU, /Pr₂EtN, DMF, 3 h, rt.

3.10% v/v acetic anhydride in pyridine, 3 min, rt.

1.20% v/v piperidine in DMF, 3x3 min, rt.

2. Fmoc-AA-OH, PyBOP, NMM, DMF, 2 h, rt.

3. 10% v/v acetic anhydride in pyridine, 3 min, rt.

1.20% v/v piperidine in DMF, 3x3 min, rt.

2. S12, /Pr₂EtN, DMF, 7 h, rt.

3. 10% v/v acetic anhydride in pyridine, 3 min, rt.

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95:2.5:2.5 v/v/v TFA:H₂O:TIS, 1 h, rt.

Figure S6. Solid phase synthesis of sansanmycin analogues.

General procedure 1: Synthesis of isopeptides

To a solution of a Boc-protected amino acid (1 eq.) in DMF (4-8 mL/mmol) was added HATU (1 eq.) and the solution was cooled to 0 °C after first stirring at rt for 10 min. S8 (1 eq.) was added, followed by NMM or /V,/\/-diisopropylethylamine (3 eq.) and the reaction was allowed to warm to rt and stirred for 3-6 h. Upon completion, the reaction was diluted with EtOAc (60-100 mL) and washed successively with 0.2 M HCI (15-20 mL), water (5x 15 mL) and brine (15 mL). The solvent was removed in vacuo to give a crude residue that was purified by column chromatography or reverse phase HPLC to afford isopeptides S15-S16.

(2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((S)-2-((ieributoxycarbonyl)amino)-3-(3-((iert-butyldimethylsilyl)oxy)phenyl)-A/methylpropanamido)butanoic acid (S15)

Boc-L-/77-Tyr(O^fBu)-OH (384 mg, 1.14 mmol) was reacted with S8 (406 mg, 1.14 mmol) in the presence of HATU (433 mg, 1.14 mmol) and NMM (376 pL, 3.42 mmol) in DMF (8.9 mL) according to general procedure 1 for 6 h to obtain isopeptide S15 after

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PCT/AU2017/051394 column chromatography (1:1 v/v EtOAc: Hexane -a EtOAc) as a white amorphous solid (384 mg, 50%).

[a]o⁵ = -3.8° (c = 0.39 in CH2CI2). ¹H NMR (400 MHz, CDCI3): δ 7.81-7.68 (m, 2H, Ar-H), 7.64-7.53 (m, 2H, Ar-H), 7.43-7.34 (m, 2H, Ar-H), 7.33-7.27 (m, 2H, Ar-H), 7.08 (d, J = 7.8 Hz, 2H, Ar-H, H-2 + H-6), 6.94-6.83 (m, 2H, Ar-H, H-3 + H-5), 5.91 (s, 1H, NH), 5.58 (d, J = 8.6 Hz, 1H, NH), 4.92-4.83 (m, 1H, Tyr2-oc-CH), 4.80-4.67 (m, 1H, DABA1-p-CH), 4.40-4.29 (m, 3H, Fmoc-CH₂ + DABA1-a-CH), 4.20 (app. t, J = 6.7 Hz, 1H, Fmoc-CH), 3.05-2.87 (m, 4H, NCH₃ + Tyr2-p-CH₂), 2.81-2.68 (m, 1H, Tyr2-p-CH₂), 1.56-1.00 (m, 21H, Boc + ^fBu + DABA1-y-CH3). IR (ATR): 3335, 3045, 1704 cm’¹. LRMS [/W+H⁺] 673.3. HRMS (ESI m/z) [/W+Na⁺] calcd. for C38H₄7N₃O₈Na, 696.3261; found, 696.3266.

(2S,3S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((S)-2-((ieributoxycarbonyl)amino)-/V-methyl-3-phenylpropanamido)butanoic acid (S16)

BocHN y

FmocHN

Boc-L-Phe-OH (70 mg, 0.26 mmol) was reacted with S8 (100 mg, 0.28 mmol) in the presence of HATU (106 mg, 0.28 mmol) and NMM (9.2 pL, 0.84 mmol) in DMF (1.1 mL) according to general procedure 1 for 4 h to obtain isopeptide S16 after column chromatography (95:5 v/v CH₂CI₂: MeOH) as a white foam (73 mg, 46%).

[a]o⁵ = -3.2° (c = 0.31 in CH₂CI₂). ¹H NMR (300 MHz, CDCI3, major rotamer) δ

7.82 (d, J = 7.5 Hz, 2H, 2 x Ar-H), 7.69 (m, 2H, 2 x Ar-H), 7.39-7.34 (m, 2H, 2 x Ar-H),

7.30-6.96 (m, 7H, 4 x Ar-H + H-2 + H-3 + H-5), 5.93 (d, J = 8.4 Hz, 1H, N-H), 5.78 (d, J = 8.2 Hz, 1H, N-H), 5.03 (m, 1H, Phe2-a-CH), 4.68 (m, 1H, DABAI-β-ΟΗ), 4.53 (m, 1H, DABAI-a-CH), 4.40-4.27 (m, 3H, Fmoc-CH₂ + Fmoc-CH), 3.00-2.77 (m, 4H, NCH₃ + Phe2-3-CH₂), 2.74 (m, 1H, Phe2-3-CH₂), 1.34-1.05 (m, 12H, Boc + DABA1-y-CH₃). IR

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PCT/AU2017/051394 (ATR): 3316, 2979, 2919, 1712, 1640 cm’¹. LRMS [M⁺H⁺] 602.4. HRMS (ESI m/z) [/W+Na⁺] calcd. for Cs^sgNsOyNa, 624.2685; found, 624.2683.

Synthesis of depsipeptides S24-S43, S45-S52

General procedure 2: Loading amino acids onto 2-chlorotrityl chloride resin

Condition A:

2-Chlorotrityl chloride resin (100-200 mesh) with 1% DVB (1.22-1.42 mmol/g, 50730 pmol, 1 eq.) was allowed to swell in anhydrous CH2CI2 (3-8 mL) for 30 min. Isopeptides S15-S16 (100-320 pmol, 2 eq.) or amino acid 20 (1.1-2.5 mmol, 1.2 eq.) were dissolved in anhydrous CH₂CI₂ (2-2.6 mL/100 pmol for isopeptides S15-S16 and 0.6 mL/100 pmol for amino acid 20). /V,/\/-diisopropylethylamine (200-2920 pmol, 2-8 eq. for isopeptides S15-S16 and 2.4-3 eq. for amino acid 20) was added and the resin was shaken for 16 h at rt. The resin was subsequently washed with DMF (5x 5 mL), CH₂CI₂ (5x 5 mL) and DMF (5x 5 mL). The resin was capped by treatment with 17:2:1 v/v/v CH₂CI₂: MeOH: /Pr₂NEt (3-6 mL) for 40 min. The resin was then washed with DMF (5x 5 mL), CH₂CI₂ (5x 5 mL) and DMF (5x 5 mL).

Condition B:

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), CH₂CI₂ (x 5) and DMF (x 5). The loading of the resin was determined through spectroscopic measurement of the fulvene piperidine adduct at λ = 301 nm (ε = 7800 cm'¹). 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), CH₂CI₂ (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 CH₂CI₂ (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 (0.0120.058 mmol, 1.1 eq.) for 16 h. The resin was then washed with CH₂CI₂ (x 5) and DMF (x 5) before being treated with 20% piperidine in DMF as previously described above from which the loading efficiency was determined.

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General procedure 3: Fmoc-strategy solid phase peptide synthesis

Fmoc deprotection: A solution of 10 vol.% piperidine/DMF (5 mL) was added to the resin and shaken for 4 min (x 2). The resin was subsequently washed with DMF (5x 3 mL), CH2CI2 (5x 3 mL) and DMF (5x 3 mL). The efficiency of the previous amino acid coupling was determined by spectroscopic measurement of the resulting fulvene piperidine adduct at λ = 301 nm.

Amino Acid Coupling:

Condition A: A solution of protected amino acid (200-640 pmol, 4 eq.), PyBOP (200-640 pmol, 4 eq.) and NMM (0.4-1.28 mmol, 8 eq.) in DMF (0.1 M) was added to the resin (1 eq.) and shaken for 1 h at rt. The resin was then washed with DMF (5x 5 mL), CH2CI2 (5x 5 mL) and DMF (5x 5 mL).

Condition B: A solution of Fmoc-protected amino acid (72-96 pmol, 1.2 eq.), HOAt (72-96 pmol, 1.2 eq.) and DIC (72-96 pmol, 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), CH2CI2 (5x 3 mL) and DMF (5x 3 mL).

Capping: A solution of 10 vol.% acetic anhydride/pyridine (5 mL) was added to the resin and shaken for 3 min. The resin was subsequently washed with DMF (5x 5 mL), CH2CI2 (5x 5 mL) and DMF (5x 5 mL).

General procedure 4: Solid-phase urea formation

A solution of carbamate S12 (90-320 pmol, 2 eq.) and Λ/,/V-di isopropylethylamine (180-640 pmol, 4 eq.) in DMF (17 pL/pmol) was added to the resin (45-160 pmol, 1 eq.). The resin was shaken at rt for 6 h. The resin was subsequently washed with DMF (5x 5 mL), CH₂CI₂ (5x 5 mL) and DMF (5x 5 mL).

General Procedure 5: Solid Phase Deprotection of Allyl Carbamate

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A solution of tefrak/s(triphenylphosphine) palladium(O) (20 pmol, 0.2 eq.) and phenylsilane (2 mmol, 20 eq.) in CH2CI2 (80 mM) was added to the resin (1 eq.) and shaken for 15 min at rt. The solvents were subsequently removed from the resin and the resin washed with CH2CI2 (5x5 mL), DMF (5x5 mL) and CH2CI2 (5x5 mL). The treatment was then repeated once.

General Procedure 6: Solid Phase Coupling to A/-methylated Amino Acid Residues

Amino acid coupling: A solution of Boc-protected amino acid (80-580 pmol, 2 eq.), HATU (80-580 pmol, 2 eq.) and DIPEA (120-870 pmol, 2-3 eq.) in DMF (0.1 M) was added to the resin (1 eq.) and shaken for 2 h at rt. The resin was then washed with DMF (5x5 mL), CH₂CI₂ (5x5 mL) and DMF (5x5 mL).

Capping: A solution of 10 vol.% acetic anhydride in pyridine (5 mL) was added to the resin and shaken for 3 min. The resin was then washed with DMF (5x3 mL), CH2CI2 (5x3 mL) and DMF (5x3 mL).

General procedure 7: Cleavage from 2-chlorotrityl chloride resin and work-up

A solution of 30 vol.% hexafluoroisopropanol (HFIP) in CH2CI2 (5-10 mL) was added to the resin and shaken for 30 min at rt. The resin was subsequently washed with CH2CI2 (6x 10 mL) and the combined cleavage and washing solutions were concentrated in vacuo. The resulting residue was dissolved in 9:1 v/v MeCN: H₂O and purified by reverse phase HPLC.

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PCT/AU2017/051394 (2S,5S,9S)-9-((1-(ieri-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-2((ieri-butoxycarbonyl)amino)-A/-methyl-3-phenylpropanamido)ethyl)-5-isobutyl12,12-dimethyl-4,7,10-trioxo-11 -oxa-3,6,8-triazatridecan-1 -oic acid (S24)

Isopeptide S16 (105 mg, 175 pmol) was loaded onto 2-chlorotrityl chloride resin (62 mg, 88 pmol) in CH2CI2 (2 mL) using /V,/V-diisopropylethylamine (61 pL, 350 pmol) and Fmoc-deprotected according to general procedure 2. Fmoc-L-Leu-OH (124 mg, 350 pmol) was subsequently coupled with PyBOP (182 mg, 350 pmol) and NMM (77 pL, 700 pmol) in DMF (1 mL) (general procedure 3). Carbamate S12 (92 mg, 175 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 (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S24 as a fluffy white solid (12.4 mg, 39%).

IR (ATR): 3340, 2976, 2931, 1730, 1635 cm’¹. ¹H NMR (400 MHz, CDCI3): δ 8.12 (1H, d, J= 8.0 Hz, Ar-H), 7.83 (1H, d, J = 8.0 Hz, Ar-H), 7.69 (1H, d, J = 4.4 Hz, Ar-H), 7.52 (1H, s, Ar-H), 7.34-7.15 (7H, m, 7x Ar-H), 6.24-6.14 (1H, m, N-H), 6.07-5.87 (1H, m, N-H), 5.05 (1H, m, ΟΑΒΑΙ-β-CH), 4.80-4.66 (3H, m, DABA1-O-CH + Phe2-aCH + Trp4-a-CH), 4.43-4.38 (1H, m, Leu3-a-CH), 3.20-3.06 (5H, m, Trp4^-CH2 + NCH3), 2.82-2.76 (2H, m, Phe2^-CH2), 1.77-1.48 (12H, m, 3x CH3 + Leu3^-CH2 + Leu3-y-CH), 1.40-1.28 (18H, m, 6x CH3), 1.23-1.18 (3H, m, DABA1-y-CH3), 0.93-0.89 (6H, m, 2 x Leu3-5-CH3). LRMS [M⁺H⁺] 879.5. HRMS (ESI m/z) [M+Na⁺] calcd. for C46H66N₆0ii 901.4687, found 901.4687.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-5-isobutyl12,12-dimethyl-2-((S)-1 -(methylamino)ethyl)-4,7,10-trioxo-11 -oxa-3,6,8triazatridecanoic acid (S25)

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 μΙ_, 6.3 mmol) in DMF (7.9 mL) (general procedure 3, condition A). Following Fmoc deprotection, carbamate S12 (840 mg, 1.58 mmol) was subsequently coupled using /V,/V-diisopropylethylamine (550 pL, 3.16 mmol) according to general procedure 4.

Allyl carbamate was subsequently removed using fefrak/s(triphenylphosphine) palladium(O) (2x 194 mg, 2x 158 pmol) and phenylsilane (2x 1.9 mL, 2x 15.8 mmol) according to general procedure 5 to afford resin bound S25.

(2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-215 ((ieri-butoxycarbonyl)amino)-A/-methyl-3-(pyridin-3-yl)propanamido)ethyl)-5isobutyl-12,12-dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S26)

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Resin-bound S25 (60 pmol) was coupled to Boc-3-Pal-OH (32 mg, 120 pmol) using HATU (45 mg, 120 pmol) and /V,/V-diisopropylethylamine (33 pl_, 180 pmol) in DMF (0.6 mL) according to general procedure 6. Following cleavage from the resin (general procedure 7), 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%).

IR (ATR): 3361, 2977, 1730, 1639 cm’¹. ¹H NMR (500 MHz, Acetone-d₆): δ 8.548.46 (1H, m), 8.46-8.33 (1H, m, Ar-H), 8.15-8.07 (1H, m, Ar-H), 7.81 (1H, app. d, J = 8.9 Hz), 7.73-7.62 (2H, m, Ar-H), 7.49 (1H, s), 7.36-7.15 (3H, m, Ar-H), 6.23 (1H, d, J = 8.0 Hz), 6.12 (1H, s), 6.03 (1H, d, J = 8.6 Hz), 4.81 (1H, app. t, J = 7.8 Hz, DABA1oc-CH), 4.73-4.62 (2H, m, Pal3-oc-CH + Trp4-oc-CH), 4.46-4.36 (2H, m, DABA1-p-CH + Leu3-oc-CH), 3.22-2.97 (6H, m, NCH₃ + Pal3-p-CH₂ + Trp4-p-CH₂), 2.82-2.74 (1H, m, Pal3-3-CH₂), 1.78-1.69 (1H, m, Leu3-y-CH), 1.65 (9H, s, 3x CH₃), 1.62-1.56 (1H, m, Leu3-p-CH₂), 1.50 (1H, m, Leu3-p-CH₂), 1.36 (9H, s, 3x CH₃), 1.28 (9H, s, 3x CH₃), 1.24-1.14 (3H, m, DABA1-y-CH₃), 0.93-0.86 (6H, m, 2x Leu3-5-CH₃). LRMS [/W+H⁺]

880.5. HRMS (ESI m/z) [/W+H⁺] calcd. for C45H66N7O11 880.4818, found 880.4824.

(2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-2((ieri-butoxycarbonyl)amino)-A/-methyl-3-(naphthalen-2-yl)propanamido)ethyl)-5isobutyl-12,12-dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S27)

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PCT/AU2017/051394

Resin-bound S25 (60 pmol) was coupled to Boc-2-Nal-OH (38 mg, 120 pmol) using HATU (45 mg, 120 pmol) and /V,/V-diisopropylethylamine (33 pL, 180 pmol) in DMF (0.6 mL) according to general procedure 6. 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 S27 as a fluffy white solid (31.4 mg, 56%).

IR (ATR): 3334, 2979, 1708, 1660 cm’¹. ¹H NMR (400 MHz, Acetone-d₆): δ 8.10 (1H, app. t, J = 7.6 Hz, Ar-H), 7.87-7.72 (4H, m, Ar-H), 7.67-7.58 (1H, m, Ar-H), 7.51 -7.38 (4H, m, Ar-H), 7.29 (1H, ddd, J = 8.4, 7.3, 1.4 Hz, Ar-H), 7.23 (1H, app. td, J= 7.5, 1.2 Hz, Ar-H), 6.18 (1H, d, J = 8.1 Hz), 6.11 (1H, d, J = 12.7 Hz), 5.98 (1H, d, J = 8.8 Hz), 5.12-5.04 (1H, m, DABA1-p-CH), 4.94-4.84 (1H, m, DABA1-oc-CH), 4.80 (1H, app. dt, J = 9.1, 4.6 Hz, Nal2-a-CH), 4.72-4.59 (1H, m, Trp4-a-CH), 4.50-4.32 (1H, m, Leu3-a-CH), 3.27 (1H, dd, J = 14.1, 4.0 Hz, Nal2-3-CH₂), 3.13 (3H, s, NCH₃), 3.113.01 (2H, m, Trp4-3-CH₂), 2.98-2.89 (1H, m, Nal2-3-CH₂), 1.81-1.69 (1H, m, Leu3-yCH), 1.68-1.58 (10H, m, 3x CH₃ + Leu3-3-CH₂), 1.57-1.45 (1H, m, Leu3-3-CH₂), 1.39-

1.31 (9H, m, 3x CH₃), 1.27-1.08 (12H, m, 3x CH₃ + DABA1-y-CH₃), 0.96-0.85 (6H, m, 2x Leu3-5-CH₃). LRMS [/W+H⁺] 929.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for CsoHesNeOnNa 951.4838, found 951.4846.

(2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-2((ieri-butoxycarbonyl)amino)-3-cyclohexyl-A/-methylpropanamido)ethyl)-5isobutyl-12,12-dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S28)

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Resin-bound S25 (60 pmol) was coupled to Boc-Cha-OH dicyclohexylamine salt (54 mg, 120 pmol) using HATU (45 mg, 120 pmol) and /V,/V-diisopropylethylamine (23 pl_, 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%).

IR (ATR): 3346, 2924, 1728, 1627 cm’¹. ¹H NMR (500 MHz, CDCI₃): δ 7.99 (1H, app. s, Ar-H), 7.86 (1H, s), 7.63-7.53 (1H, m, Ar-H), 7.41 (1H, s, Ar-H), 7.28-7.20 (2H, m, Ar-H), 6.23 (1H, s), 6.03 (1H, s), 5.64 (1H, s), 5.05-4.87 (1H, m, DABA1-p-CH), 4.79-

4.66 (1H, m, Trp4-oc-CH), 4.65-4.45 (2H, m, DABA1-a-CH + Leu3-oc-CH), 4.45-4.15 (1H, m, Cha2-oc-CH), 3.27-3.07 (2H, m, Trp4-p-CH₂), 2.98 (3H, s, NCH₃), 1.97-1.81 (1H, m, CH₂), 1.64 (12H, m, 3x CH₃ + 0.5x CH₂ + Leu-p-CH₂ + CH), 1.51-1.01 (23H, m, 6x CH₃, CH₂ + CH + DABA1-y-CH₃), 0.95-0.65 (7H, m, 0.5x CH₂ + 2x Leu3-5-CH₃). LRMS [/W+H⁺] 885.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C45H₇oN₆OiiNa 907.5151, found 907.5150.

(2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-2((ieri-butoxycarbonyl)amino)-2-cyclohexyl-A/-methylacetamido)ethyl)-5-isobutyl12,12-dimethyl-4,7,10-trioxo-11 -oxa-3,6,8-triazatridecanoic acid (S29)

OH

Resin-bound S25 (60 pmol) was coupled to Boc-Chg-OH (122 mg, 480 pmol) using HATU (124 mg, 480 pmol), HOAt (324 mg, 2.4 mmol) and /V,/V-diisopropylethylamine (132 pL, 720 pmol) in DMF (0.6 mL) was added to resinbound S25 and the reaction vessel was shaken at rt for 4.5 h. Following cleavage from

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PCT/AU2017/051394 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%).

IR (ATR): 3336, 2977, 1729, 1641, 1522 cm’¹.¹H NMR (500 MHz, Acetone-d₆): δ

8.12 (1H, app. d, J= 8.1 Hz, Ar-H), 7.72 (1H, d, J = 8.9 Hz, Ar-H), 7.71-7.65 (1H, m, ArH), 7.50 (1H, s, Ar-H), 7.31 (1H, ddd, J = 8.3, 7.3, 1.4 Hz, Ar-H), 7.26 (1H, app. td, J =

7.5, 1.2 Hz, Ar-H), 6.09 (1H, d, J = 8.1 Hz), 6.04 (1H, d, J = 9.8 Hz), 5.73 (1H, d, J = 9.3 Hz), 5.06-4.98 (1H, m, DABA1-p-CH), 4.73-4.69 (1H, m, DABA1-a-CH), 4.64 (1H, app. t, J = 6.5 Hz, Trp4-oc-CH), 4.41-4.31 (2H, m, Chg2-oc-CH + Leu3-oc-CH), 3.23-3.06 (2H, m, Trp4-3-CH₂), 3.01 (3H, s, NCH₃), 1.75-1.70 (1H, m, Leu3-y-CH), 1.67 (9H, s, 3x CH₃), 1.62-1.52 (2H, m, Leu3-3-CH₂ + CH₂), 1.52-1.44 (2H, m, Leu3-3-CH₂ + CH₂), 1.32-1.14 (7H, m, DABA1-y-CH₃ + 2x CH₂), 1.14-1.02 (3H, m), 1.02-0.94 (1H, m), 0.950.87 (6H, m, 2x Leu3-5-CH₃). LRMS [/W+H⁺] 871.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C45H₇oN₆OiiNa 893.4994, found 893.5001.

(2S,3S)-methyl 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-((iertbutoxycarbonyl)amino)-/V-methylacetamido)butanoate (S35)

BocHN ^NH

FmocHN

Amino acid 20 (219 mg, 805 pmol) was loaded onto 2-chlorotrityl chloride resin (514 mg, 730 pmol) in CH₂CI₂ (2 mL) using Λ/,/V-diisopropylethylamine (500 pL, 400 pmol) according to general procedure 2. Allyl carbamate was subsequently removed using tefrak/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.

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PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-5-((/?)-sec-butyl)-12,12dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S36)

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-lle-OH (85 mg, 240 pmol) using PyBOP (125 mg, 240 pmol) and NMM (50 pL, 480 pmol) in DMF (0.6 mL) according to general procedure

3. Carbamate S12 (64 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 (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S36 as a fluffy white solid (17 mg, 36%).

IR (ATR): 3308, 2978, 2929, 1726, 1637 cm’¹. ¹H NMR (500 MHz, Acetone-d₆, major rotamer)·. δ 8.12 (1H, d, J = 7.9 Hz), 7.80 (1H, app. d, J = 8.9 Hz, Ar-H), 7.69 (1H, d, J = 7.3 Hz), 7.59 (1H, app. d, J = 8.6 Hz, Ar-H), 7.51 (1H, s, Ar-H), 7.31 (1H, app. t, J = 7.7 Hz, Ar-H), 7.27 (1H, app. t, J = 7.5 Hz, Ar-H), 6.17-6.01 (2H, m), 5.85-5.75 (1H, m), 5.01-4.91 (1H, m, DABA1-p-CH), 4.75 (1H, app. t, J = 7.2 Hz, DABA1-a-CH), 4.64 (1H, app. t, J = 7.3 Hz, Trp4-oc-CH), 4.31-4.19 (1H, m, lle3-a-CH), 3.92-3.83 (1H, m, Gly2-a-CH₂), 3.75-3.68 (1H, m, Gly2-a-CH₂), 3.20-3.07 (2H, m, Trp4-p-CH₂), 2.85 (3H, s, NCH₃), 1.87-1.72 (1H, m, lle3-p-CH), 1.67 (9H, s, 3x CH₃), 1.60-1.49 (1H, m, Ile3-yCH₂), 1.44-1.33 (18H, s, 6x CH₃), 1.21 (3H, d,

J = 6.9 Hz, DABA1-y-CH₃), 1.17-1.07 (1H, m, lle3-y-CH₂), 0.96-0.76 (6H, m, 2x CH₃). LRMS [/W+H⁺] 789.4. HRMS (ESI m/z) [/W+Na⁺] calcd. for CsgHeoNeOnNa 811.4212, found 811.4227.

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PCT/AU2017/051394 (2S,5S,9S)-9-((1-(tert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((tertbutoxycarbonyl)amino)-/V-methylacetamido)ethyl)-5-isopropyl-12,12-dimethyl4,7,10-trioxo-11-oxa-3,6,8-triazatridecan-1-oic acid (S37)

BocHN

OH

Resin-bound isopeptide S35 (45 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Val-OH (81.5 mg, 240 pmol) using PyBOP (125 mg, 240 pmol) and NMM (53 pL, 480 pmol) 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%).

IR (ATR): 3034, 1735, 1639, 1384 cm’¹. ¹H NMR (400 MHz, (Acetone-d₆, major rotamer): δ 8.14 (1H, d, J = 8.3 Hz, Ar-H), 7.70 (1H, d, J = 7.4 Hz, Ar-H), 7.52 (1H, s, Ar-H), 7.35-7.26 (2H, m, 2x Ar-H), 6.21-6.13 (2H, m, 2x N-H), 5.86 (1H, m, N-H), 5.04-

4.97 (1H, m, ΟΑΒΑΙ-β-CH), 4.77 (1H, m, DABA1-O-CH), 4.69 (1H, m, Trp4-a-CH), 4.26 (1H, m, Val3-a-CH), 4.05-3.72 (2H, m, Gly2-a-CH₂), 3.17 (2H, d, J = 6.0 Hz, Τφ4-βCH₂), 2.88 (3H, s, NCH₃), 2.08 (1H, m, Val3^-CH), 1.68 (9H, s, 3x CH₃), 1.42 (18H, m, 6x CH₃), 1.24 (3H, d, J = 7.0 Hz, DABA1-y-CH₃), 0.95 (3H, d, J = 6.7 Hz, Val3-y-CH₃), 0.91 (3H, d, J = 6.8 Hz, Val3-y-CH₃). LRMS [M⁺H⁺] 775.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for CssHssNeOnNa 797.4061, found 797.4063.

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PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10-trioxo-5pentyl-11-oxa-3,6,8-triazatridecanoic acid (S38)

BocHN

Resin-bound isopeptide S35 (70 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Aha-OH (103 mg, 280 pmol) using PyBOP (144 mg, 280 pmol) and NMM (58 pL, 560 pmol) in DMF (0.7 mL) according to general procedure

3. Carbamate S12 (75 mg, 140 pmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was purified by column chromatography (98:2 v/v CH2CI2: MeOH -a· 9:1 v/v CH2CI2: MeOH, 0.5 vol.% AcOH) to afford intermediate S38 as a white foam (30 mg, 53%).

IR (ATR): 3356, 2977, 2932, 1731, 1638 cm’¹. ¹H NMR (500 MHz, Acetone-d₆, major rotamer): δ 8.12 (1H, d, J = 8.2 Hz, Ar-H), 7.69 (1H, d, J = 7.6 Hz, Ar-H), 7.60 (1H, d, J =8.5 Hz), 7.52 (1H, s, Ar-H), 7.33-7.24 (2H, m, Ar-H), 6.19-6.01 (1H, m), 5.86-

5.74 (1H, m), 5.04-4.91 (1H, m, DABA1-p-CH), 4.80-4.68 (1H, m, DABA1-a-CH), 4.64 (1H, app. t, J = 6.4 Hz, Trp4-oc-CH), 4.30 (1H, app. t, J = 6.8 Hz, Aha3-oc-CH), 3.90-3.80 (1H, m, Gly2-a-CH₂), 3.79-3.66 (1H, m, Gly2-oc-CH₂), 3.19-3.10 (2H, m, Trp4-3-CH₂), 2.83 (3H, s, NCH₃), 1.76 (1H, dd, J = 16.3, 8.3 Hz, Aha3-3-CH₂), 1.67 (9H, s, 3x CH₃), 1.61-1.48 (1H, m, Aha3-3-CH₂), 1.45-1.33 (20H, s, 6x CH₃ + CH₂), 1.32-1.24 (4H, m, 2x CH₂), 1.20 (3H, d, J = 7.1 Hz, DABA1-y-CH₃), 0.90-0.81 (3H, m). LRMS [/W+H⁺] 803.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₄oH62N₆OiiNa 825.4368, found 825.4364.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-5-cyclohexyl-12,12-dimethyl4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S39)

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Chg-OH (91 mg, 240 pmol) using PyBOP (124 mg, 240 pmol) and NMM (50 pl_, 480 pmol) in DMF (0.6 mL) according to general procedure

3. Carbamate S12 (64 mg, 120 pmol) was subsequently coupled according to general procedure 4 in the presence of /V,/\/-diisopropylethylamine (21 pL, 120 pmol) in DMF (1 mL). 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 S39 as a fluffy white solid (20 mg, 40%).

IR (ATR): 3308, 2979, 2929, 1726, 1637 cm’¹. ¹H NMR (400 MHz, Acetone-d₆, major rotamer): δ 8.11 (1H, app. d, J = 8.1 Hz, Ar-H), 7.74-7.60 (1H, m, Ar-H), 7.50 (1H, s, Ar-H), 7.36-7.22 (2H, m, Ar-H), 6.25-6.12 (2H, m), 5.90-5.79 (1H, m), 5.06-4.91 (1H, m, DABA1-p-CH), 4.81-4.70 (1H, m, DABA1-oc-CH), 4.70-4.58 (1H, m, Trp4-oc-CH),

4.29-4.19 (1H, m, Chg3-oc-CH), 3.90-3.81 (1H, m, Gly2-oc-CH₂), 3.77-3.66 (1H, m, Gly2oc-CH₂), 3.17-3.06 (2H, m, Trp4-p-CH₂), 1.79-1.54 (15H, 3x CH₃ + 2.5x CH₂ + Chg3-pCH), 1.43-1.32 (18H, m, 6x CH₃), 1.25-0.93 (8H, m, DABA1-y-CH₃ + 2.5x CH₂). LRMS [/W+H⁺] 815.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₄iH₆₂N₆0ii 815.4549, found 815.4548.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-5-(cyclohexylmethyl)-12,12dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S40)

BocHN

OH

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Cha-OH (47 mg, 120 pmol) using HATU (45 mg, 120 pmol) and /V,/\/-diisopropylethylamine (33 pl_, 180 pmol) in DMF (0.6 mL) according to general procedure 6. Carbamate S12 (64 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 (50-100% MeCN over 60 min, 10 min at 50% MeCN) to afford depsipeptide S40 as a fluffy white solid (17 mg, 34%).

IR (ATR): 3390, 2977, 2936, 1730, 1644 cm’¹. ¹H NMR (500 MHz, Acetone-de, rotamers in 4:1 ratio, major rotamer): δ 8.12 (1H, app. d, J = 8.2 Hz, Ar-H), 7.72-7.66 (1H, m), 7.61 (1H, app. d, J = 8.9 Hz, Ar-H), 7.52 (1H, s, Ar-H), 7.32 (1H, ddd, J = 8.3,

7.1, 1.4 Hz, Ar-H), 7.27 (1H, app. td, J = 7.5, 1.2 Hz, Ar-H), 6.16-5.97 (2H, m), 5.89-5.69 (1H, m), 5.00-4.93 (1H, m, DABA1-p-CH), 4.79-4.70 (1H, m, DABA1-a-CH), 4.68-4.60 (1H, m, Trp4-oc-CH), 4.42-4.35 (1H, m, Cha3-oc-CH), 3.96 (1H, t, J 5.5 Hz), 3.87-3.81 (1H, m, Gly2-a-CH₂), 3.75-3.69 (1H, m, Gly2-oc-CH₂), 3.19-3.11 (2H, m, Trp4-p-CH₂),

2.82 (3H, s, NCH₃), 1.84-1.75 (1H, m, CH₂), 1.67-1.56 (13H, m, 3x CH₃ + 1.5x CH₂ + Cha3-3-CH₂), 1.48-1.31 (22H, m, 6x CH₃ + Cha3-3-CH₂ + Cha3-y-CH + CH₂), 1.24-1.08 (5H, m, DABA1-y-CH₃ + CH₂), 1.00-0.77 (2H, m, CH₂). LRMS [/W+H⁺] 829.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₄2H64N₆0iiNa 851.5425, found 851.4526.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-AZ-methylacetamido)ethyl)-5-((S)-1 -hydroxyethyl)-12,12dimethyl-4,7,10-trioxo-11 -oxa-3,6,8-triazatridecan-1 -oic acid (S41)

BocHN °

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, pmol) and /V,/V-diisopropylethylamine (20 pl_, 120 pmol) in DMF (0.4 mL) according to general procedure 6. Carbamate S12 (46 mg, 90 pmol) was subsequently coupled according to general procedure. 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 S41 as a fluffy white solid (18.2 mg, 49%).

IR (ATR): 3004, 1709, 1422, 1359 cm’¹. ¹H NMR (400 MHz, Acetone-d₆, major rotamer)·. δ 8.08 (1H, d, J= 8.1 Hz, Ar-H), 7.66 (1H, d, J = 8.6 Hz, Ar-H), 7.49 (1H, s, Ar-H), 7.30-7.21 (2H, m, 2x Ar-H), 6.37 (1H, m, N-H), 6.24 (1H, m, N-H), 5.85 (1H, m, N-H), 4.97 (1H, m, ΟΑΒΑΙ-β-CH), 4.69 (1H, m, DABA1-O-CH), 4.63 (1H, m, Trp4-aCH), 4.24 (2H, m, Thr3-a-CH + Thr3-3-CH), 3.92-3.77 (2H, m, Gly2-a-CH₂), 3.14 (2H, m, Trp4-3-CH₂), 2.80 (3H, s, NCH₃), 1.63 (9H, s, 3x CH₃), 1.38-1.34 (18H, m, 6x CH₃),

1.18 (3H, d, J = 7.1 Hz, DABA1-y-CH₃), 1.08 (3H, d, J = 6.4 Hz, Thr3-y-CH₃). LRMS [/W+H⁺] 777.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for CsyHseNeO^Na 799.3853, found 799.3850.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-5-(2-(iert-butoxy)-2-oxoethyl)-9-((1-(ieri-butoxycarbonyl)-1Hindol-3-yl)methyl)-2-((S)-1-(2-((iert-butoxycarbonyl)amino)-A/methylacetamido)ethyl)-12,12-dimethyl-4,7,10-trioxo-11-oxa-3,6,8-triazatridecan-1oic acid (S42)

BocHN

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Asp(O^fBu)-OH (99 mg, 240 pmol) using PyBOP (125 mg,

240 pmol) and NMM (53 pL, 480 pmol) in DMF (0.6 mL) according to general procedure

6. Carbamate S12 (62 mg, 120 pmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was purified by column chromatography (eluent 95:5 v/v CH2CI2: MeOH, 0.1 vol.% AcOH) to afford depsipeptide S42 as a yellow oil (37.2 mg, 69%).

IR (ATR): 3359, 2978, 2933, 1726, 1640 cm’¹. ¹H NMR (300 MHz, Acetone-d₆, major rotamer): δ 8.11 (1H, d, J = 7.8 Hz, Ar-H), 7.79 (1H, d, J = 6.9 Hz, Ar-H), 7.52 (1H, s, Ar-H), 7.34-7.25 (2H, m, 2x Ar-H), 6.36-6.24 (2H, m, 2x N-H), 5.83 (1H, m, N-H),

4.93 (1H, m, ϋΑΒΑΙ-β-CH), 4.74-4.63 (3H, m, DABA1-O-CH + Asp3-a-CH + Trp4-aCH), 3.94-3.66 (2H, m, Gly2-a-CH₂), 3.17 (2H, d, J = 6.3 Hz, ΤΓρ4-β-ΟΗ₂), 2.75 (3H, s, NCH₃), 2.67-2.60 (2H, m, Asp3^-CH₂), 1.66 (9H, m, 3x CH₃),

1.41-1.38 (27H, m, 9x CH₃), 1.10 (3H, d, J = 6.9 Hz, DABA1-y-CH₃). LRMS [/W+H⁺]

847.6. HRMS (ESI m/z) [/W+Na⁺] calcd. for C₄iH62N₆0i₃Na 869.4272, found 869.4272.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-5-(4-((ieri-butoxy carbonyl) amino)butyl)-12,12-dimethyl-4,7,10-trioxo-11 -oxa-3,6,8-triazatridecan-1 -oic acid (S43)

Resin-bound isopeptide S35 (45 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Lys(Boc)-OH (112 mg, 240 pmol) using PyBOP (125 mg,

240 pmol) and NMM (53 pl_, 480 pmol) in DMF (0.6 mL) according to general procedure

6. Carbamate S12 (63 mg, 126pmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 6), the peptide was purified by column chromatography (97:3 v/v CH2CI2: /'-PrOH,

0.1 vol.% acetic acid - 9:1 v/v CH2CI2: /-PrOH, 0.1% acetic acid) to afford depsipeptide S43 as a white foam (15.4 mg, 38%).

IR (ATR): 3325, 2977, 1701, 1647 cm’¹. ¹H NMR (400 MHz, Acetone-de, major rotamer)·. δ 8.08 (1H, d, J = 10.4 Hz, Ar-H), 7.66 (1H, d, J = 9.2 Hz, Ar-H), 7.47 (1H, s, Ar-H), 7.30-7.20 (2H, m, 2x Ar-H), 6.09 (2H, m, 2x N-H), 5.88 (1H, m, N-H), 4.96 (1H, m, ϋΑΒΑΙ-β-CH), 4.72-4.61 (2H, m, DABA1-O-CH + Trp4-a-CH), 4.30 (1H, m, Lys3-aCH), 3.94-3.66 (2H, m, Gly2-a-CH2), 3.12 (2H, d, J = 8.0 Hz, ΤΓρ4-β-ΟΗ2), 2.99 (2H, d, J= 8.4 Hz, Lys3-3-CH2), 2.80 (3H, s, NCH3), 1.63 (6H, m, Lys-y-CH2 + Lys-d-CH2 + Lyse-CH2), 1.37-1.24 (36H, m, 12 x CH3), 1.16 (3H, d, J = 9.2 Hz, DABA1-y-CH3). LRMS [/W+H⁺] 904.6. HRMS (ESI m/z) [/W+Na⁺] calcd. for C44H69N₇Oi₃Na926.4851, found 926.4854.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10-trioxo-5(4-(trifluoromethyl)benzyl)-11 -oxa-3,6,8-triazatridecanoic acid (S45)

BocHN

Resin-bound isopeptide S35 (88 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Phe(4-CF₃)-OH (160 mg, 352 pmol) using PyBOP (183 mg, 352 pmol) and NMM (73 pL, 704 pmol) in DMF (0.9 mL) according to general procedure 3. Carbamate S12 (94 mg, 176 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 (50-100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S45 as a fluffy white solid (29 mg, 36%).

IR (ATR): 3374, 2979, 2932, 1731, 1666 cm’¹. ¹H NMR (400 MHz, CDCI₃, major rotamer)·. δ 8.04 (1H, app. d, J = 8.2 Hz, Ar-H), 7.76-7.60 (1H, m), 7.64 (1H, d, J = 6.8 Hz), 7.53 (1H, app. d, J = 7.5 Hz, Ar-H), 7.47-7.36 (4H, m, Ar-H), 7.30-7.14 (3H, m, Ar-H), 5.93 (1H, s), 5.82 (1H, s), 5.54 (1H, s), 4.98-4.82 (1H, m, DABA1-p-CH), 4.76-

4.32 (3H, m, DABA1-a-CH + 4-CF₃-Phe3-oc-CH + Trp4-oc-CH), 3.85-3.67 (2H, m, Gly2oc-CH₂), 3.17-3.05 (3H, m, 4-CF₃-Phe3-3-CH₂ + Trp4-3-CH₂), 3.00-2.91 (1H, m, Trp4-pCH₂), 2.73 (3H, s, NCH₃), 1.62 (9H, s, 3x CH₃), 1.42-1.19 (18H, m, 6x CH₃), 1.15 (3H, d, J = 6.9 Hz, DABA1-y-CH₃). LRMS [/W+H⁺] 891.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₄₃H₅7F₃N₆Oi 1 891.4110, found 891.4103.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-5-(4-(tert-butoxy)benzyl)-9-((1-(tert-butoxycarbonyl)-1H-indol-3yl)methyl)-2-((S)-1-(2-((iert-butoxycarbonyl)amino)-A/-methylacetamido)ethyl)12,12-dimethyl-4,7,10-trioxo-11 -oxa-3,6,8-triazatridecan-1 -oic acid (S46)

BocHN

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Tyr(O^fBu)-OH (110 mg, 240 pmol) using PyBOP (125

240 pmol) and NMM (53 pL, 480 pmol) in DMF (0.6 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 column chromatography (eluent: 95:5 v/v CH2CI2: MeOH, 0.1 vol.% acetic acid) to afford depsipeptide S46 as a yellow oil (34.8 mg, 56%).

IR (ATR): 3350, 2978, 2929, 1726, 1637 cm’¹. ¹H NMR (400 MHz, CDCI3, major rotamer): δ 8.06 (1H, d, J = 7.2 Hz, Ar-H), 7.55 (1H, d, J = 7.6 Hz, Ar-H), 7.39 (1H, s, Ar-H), 7.29-7.18 (2H, m, 2x Ar-H), 7.04 (2H, d, J = 8.4 Hz, 2x Ar-H), 6.82 (2H, d, J = 8.4 Hz, 2x Ar-H), 5.72-5.61 (2H, m, 2 x N-H), 4.84 (1H, m, ΟΑΒΑΙ-β-CH), 4.65 (1H, m, DABA1-O-CH), 4.57 (1H, m, Trp4-a-CH), 4.52 (2H, m, Tyr3-a-CH), 3.91-3.71 (2H, m, Gly2-a-CH2), 3.12 (2H, m, Trp4^-CH2), 2.98 (2H, m, Trp4^-CH2), 2.79 (3H, s, NCH3), 1.63 (9H, s, 3x CH3), 1.40-1.25 (27H, m, 9x CH3), 1.14 (3H, d, J = 6.8 Hz, DABA1-y-CH3). LRMS [/W+H⁺] 895.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C46H66N₆Oi2Na 895.4817, found 895.4810.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10-trioxo-5(pyridin-3-ylmethyl)-11 -oxa-3,6,8-triazatridecanoic acid (S47)

BocHN

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-3-Pal-OH (92 mg, 240 pmol) using PyBOP (125 mg, 240 pmol) and NMM (50 pL, 480 pmol) in DMF (0.6 mL) according to general procedure

3. Carbamate S12 (64 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 (50-100% MeCN over 60 min, 10 min at 50% MeCN) to afford depsipeptide S47 as a fluffy white solid (14 mg, 29%).

IR (ATR): 3366, 2979, 2936, 1726, 1660 cm’¹. ¹H NMR (500 MHz, Acetone-de, major rotamer)'. δ 8.70-8.61 (2H, m, NH + Ar-H), 8.21 (1H, app. d, J = 7.9 Hz, Ar-H), 8.11 (1H, app. d, J = 8.2 Hz, Ar-H), 7.98 (1H, d, J = 8.8 Hz, Ar-H), 7.83-7.81 (1H, m, ArH), 7.64 (1H, d, J = 7.0 Hz, Ar-H), 7.50 (1H, s, Ar-H), 7.33-7.28 (1H, m, Ar-H), 7.25 (1H, app. td, J = 7.5, 1.2 Hz, Ar-H), 6.37-6.21 (2H, m, 2x NH), 5.86-5.73 (1H, m), 4.97-4.89 (1H, m, DABA1-p-CH), 4.81-4.73 (1H, m, J = 7.0 Hz, 3-Pal3-a-CH), 4.71-4.62 (1H, m, DABA1-a-CH), 4.61-4.52 (1H, m, Trp4-a-CH), 3.94-3.81 (1H, m, Gly2-oc-CH₂), 3.783.69 (1H, m, Gly2-a-CH₂), 3.31 (1H, app. dt, J = 15.6, 7.6 Hz, 3-Pal3-3-CH₂), 3.20-3.06 (3H, m, 3-Pal3-p-CH₂ + Trp4-p-CH₂), 2.83 (3H, s, NCH₃), 1.65 (9H, s, 3x CH₃), 1.431.31 (18H, s, 6x CH₃), 1.18 (3H, d, J = 7.1 Hz, DABA1-y-CH₃). LRMS [/W+H⁺] 829.5. HRMS (ESI m/z) [/W+H⁺] calcd. for C41H57N7O11 824.4188, found 824.4182.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-5,9-bis((1-(ieri-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2((ieri-butoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10trioxo-11-oxa-3,6,8-triazatridecan-1-oic acid (S48)

BocHN

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-Trp(Boc)-OH (126.4 mg, 240 pmol) using PyBOP (125 mg, 240 pmol) and NMM (53 pl_, 480 pmol) in DMF (0.6 mL) according to general procedure 7. Carbamate S12 (90.4 mg, 172 pmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide 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%).

IR (ATR): 3352, 2980, 2928, 1731, 1646, 1553 cm’¹. ¹H NMR (500 MHz, CDCI3, major rotamer)·. δ 8.04 (1H, m, 2x Ar-H), 7.53 (1H, d, J = 7.6 Hz, 2x Ar-H), 7.40 (2H, m, Ar-H), 7.28-7.11 (4H, m, 4x Ar-H), 6.29 (1H, m, N-H), 6.06 (1H, m, N-H), 5.57 (1H, m, N-H), 4.84 (1H, m, ϋΑΒΑΙ-β-CH), 4.68-4.52 (2H, m, Trp3-a-CH + Trp4-a-CH), 4.47 (1H, m, DABA1-a-CH), 3.75-3.48 (2H, m, Gly2-a-CH₂), 3.16-2.91 (4H, m, ΤΓρ3-β-ΟΗ₂ + ΤΓρ4-β-ΟΗ₂), 2.88 (3H, s, NCH₃), 1.61 (18H, s, 6x CH₃), 1.42-1.22 (18H, m, 6x CH₃), 1.11 (3H, d, J = 6.6 Hz, DABA1-y-CH₃). LRMS [/W+H⁺] 962.6. HRMS (ESI m/z) [/W+H⁺] calcd. for C49H68N7O11 962.4876, found 962.4869.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10-trioxo-5phenyl-11-oxa-3,6,8-triazatridecan-1-oic acid (S49)

BocHN 'ΐγ⁰

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 (11.2 mg, 84 pmol,) and DIC (13.3 pL, 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. Following cleavage from the resin (general procedure 7), the peptide was provided as a 9:1 mixture of diastereomers, which were readily separable by reversephase 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%).

IR (ATR): 3343, 2978, 2933, 1727, 1700, 1638 cm’¹. ¹H NMR (400 MHz, Acetone-de, major rotamer)·. δ 8.13 (1H, d, J = 7.6 Hz, Ar-H), 7.72 (1H, d, J = 6.8 Hz, ArH), 7.52 (1H, s, Ar-H), 7.46 (2H, d, J 7.2 Hz, 2x Ar-H), 7.47-7.24 (5H, m, 5x Ar-H), 6.66 (1H, m, N-H), 6.27 (1H, m, N-H), 5.80 (1H, m, N-H), 5.57 (1H, m, Phg3-a-CH), 4.99 (1H, m, ϋΑΒΑΙ-β-CH), 4.79 (1H, m, DABAI-a-CH), 4.65 (1H, m, Trp4-a-CH), 3.95-3.71 (2H, m, Gly2-a-CH₂), 3.17 (2H, m, Trp4-3-CH₂), 2.88 (3H, s, NCH₃), 1.67 (9H, s, 3x CH₃),

1.40 (9H, m, 3x CH₃), 1.35 (9H, m, 3x CH₃), 1.25 (3H, d, J = 6.8 Hz, DABA1-y-CH₃). LRMS [/W+H⁺] 809.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C^HseNeOnNa 809.4085, found 809.4080.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(tert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2((tert-butoxycarbonyl)amino)-A/-methylacetamido)ethyl)-12,12-dimethyl-4,7,10trioxo-5-phenethyl-11-oxa-3,6,8-triazatridecan-1-oic acid (S50)

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 pl_, 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. Following cleavage from the resin (general procedure 7), the peptide was provided as a 9:1 mixture of diastereomers which were readily separable by reversephase 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%).

IR (ATR): 3342, 2977, 2931, 1730 cm’¹. ¹H NMR (500 MHz, CDCI3, major rotamer): δ 8.11 (1H, d, J = 8.0 Hz, Ar-H), 7.69 (1H, d, J = 7.5 Hz, Ar-H), 7.53 (1H, s, Ar-H), 7.32-7.12 (7H, m, 7x Ar-H), 6.39 (1H, m, N-H), 6.25 (1H, m, N-H), 5.95 (1H, m, N-H), 4.96 (1H, m, ΟΑΒΑΙ-β-CH), 4.71 (1H, m, DABA1-O-CH), 4.64 (1H, m, Trp4-aCH), 4.36 (1H, m, HPhe3-a-CH), 3.96-3.69 (2H, m, Gly2-a-CH2), 3.15 (2H, m, ΤΓρ4-β-ΰΗ2), 2.80 (3H, s, NCH3), 2.64 (2H, m, HPhe3^-CH2), 2.02 (2H, m, HPhe3-yCH2), 1.63 (9H, s, 3x CH3), 1.36-1.34 (18H, m, 6x CH3), 1.16 (3H, d, J = 7.1 Hz, DABA1-y-CH3). LRMS [/W+H⁺] 837.5. HRMS (ESI m/z) [/W+Na⁺] calcd. for C43H6oN₆OiiNa 859.4218, found 859.4205.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-(2-((iertbutoxycarbonyl)amino)-/V-methylacetamido)ethyl)-12,12-dimethyl-5-(naphthalen2-ylmethyl)-4,7,10-trioxo-11-oxa-3,6,8-triazatridecanoic acid (S51)

BocHN _ .hl·

Resin-bound isopeptide S35 (60 pmol) was Fmoc-deprotected (general procedure 2) and coupled to Fmoc-2-Nal-OH (104 mg, 240 pmol) using PyBOP (124 mg,

240 pmol) and NMM (50 pl_, 480 pmol) in DMF (0.6 mL) according to general procedure

3. Carbamate S12 (64 mg, 120 pmol) was subsequently coupled according to general procedure 4 in the presence of DIPEA (21 pL, 120 pmol) in DMF (1 mL). 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 S51 as a fluffy white solid (22 mg, 42%).

IR (ATR): 3390, 2978, 2936, 1730, 1644 cm’¹. ¹H NMR (500 MHz, Acetone-d₆, major rotamer): δ 8.11 (1H, d, J = 8.2 Hz, Ar-H), 7.86-7.61 (6H, m, 5x Ar-H + NH), 7.50 (1H, s, Ar-H), 7.45-7.37 (3H, m, Ar-H), 7.34-7.21 (2H, m, Ar-H), 6.22-6.12 (2H, m, NH),

5.82 (1H, s, NH), 5.01-4.92 (1H, m, DABA1-p-CH), 4.82-4.69 (2H, m, DABA1-a-CH + 2Nal3-oc-CH), 4.66-4.58 (1H, m, Trp4-oc-CH), 3.93-3.83 (1H, m, Gly2-oc-CH₂), 3.72 (1H, dd, J = 17.0, 4.2 Hz, Gly2-oc-CH₂), 3.39-3.25 (1H, m,

2-Nal3-p-CH₂), 3.19-3.05 (3H, m, 2-Nal3-p-CH₂ + Trp4-p-CH₂), 2.84 (3H, s, NCH₃), 1.65 (9H, s, 3x CH₃), 1.46-1.30 (18H, m, 6x CH₃), 1.17 (3H, d, J = 7.2 Hz, DABA1-y-CH₃). LRMS [/W+H⁺] 873.4. HRMS (ESI m/z) [/W+Na⁺] calcd. for C46H₆oN₆OiiNa 895.4212, found 895.4212.

WO 2018/107236

PCT/AU2017/051394 (2S,5S,9S)-9-((1-(iert-butoxycarbonyl)-1H-indol-3-yl)methyl)-2-((S)-1-((S)-2((ieri-butoxycarbonyl)amino)-3-(3-((ieri-butyldimethylsilyl)oxy)phenyl)-A/methylpropanamido)ethyl)-5-(cyclohexylmethyl)-12,12-dimethyl-4,7,10-trioxo-11oxa-3,6,8-triazatridecanoic acid (S52)

OTBS

Resin-bound amino acid S25 (87 pmol) was coupled to Fmoc-Cha-OH (68 mg, 174 pmol) using HATU (65 mg, 174 pmol) and /V,/V-diisopropylethylamine (48 pL, 260 pmol) in DMF (0.9 mL) according to general procedure 6. Carbamate S12 (93 mg, 174 pmol) was subsequently coupled according to general procedure 4. Allyl carbamate was subsequently removed using tefrak/s(triphenylphosphine) palladium(O) (20 mg, 17 pmol) and phenylsilane (20 pL, 170 pmol) in CH2CI2 (1 mL) according to general procedure 5. Boc-/77-Tyr(OTBS)-OH (51 mg, 130 pmol) was subsequently coupled using HATU (49 mg, 130 pmol), HOAt (25 mg, 130 pmol) and Λ/,Λ/Diisopropylethylamine (23 pL, 130 pmol) in DMF (0.87 mL). Following cleavage from the resin (general procedure 7), the peptide was purified by reverse phase HPLC (50100% MeCN over 40 min, 10 min at 50% MeCN) to afford depsipeptide S52 as a fluffy white solid (18 mg, 20%).

IR (ATR): 3314, 2928, 2856, 1730, 1630 cm’¹. ¹H NMR (500 MHz, CDCI3, major rotamer)·. δ 8.08-7.92 (1H, m, Ar-H), 7.79 (1H, s), 7.61-7.52 (1H, m, Ar-H), 7.45-7.36 (1H, m, Ar-H), 7.31-7.15 (2H, m, Ar-H), 7.01-6.87 (1H, m, Ar-H), 6.72-6.46 (3H, m, Ar87

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PCT/AU2017/051394

Η), 6.11-5.73 (1H, m, NH), 5.69-5.53 (1H, m, NH), 4.94-4.84 (1H, m, DABA1-p-CH),

4.76-4.60 (3H, m, DABA1-a-CH + m-Tyr2-oc-CH + Trp4-oc-CH), 4.46-4.19 (1H, m, Cha3oc-CH), 3.30-3.07 (2H, m, Trp4-p-CH₂), 3.02 (3H, s, NCH₃), 2.98-2.87 (1H, m, m-Tyr2-pCH₂), 2.61-2.46 (1H, m, m-Tyr2-3-CH₂), 1.78-0.74 (58H, m). LRMS [/W+H⁺] 1049.4.

HRMS (ESI m/z) [/W+H⁺] calcd. for C₅5H₈4N₆Oi₂Si 1049.5989, found 1049.5981.

Synthesis of analogues

General procedure 8: synthesis of analogues via fragment condensation strategy

Condition A:

To a solution of amine 18 (15-25 pmol, 1.2 eq.) in CH₂CI₂ and DMF (1:1 v/v CH₂CI₂: DMF, 0.1 M) was added depsipeptide S24, S26, S27, S29, and S53 (13-21 pmol, 1 eq.) and HOAt (5-5.5 eq.), immediately followed by DIC (1 eq.). The reaction was allowed to stir at rt for 1.5-2 h, at which point, the reaction mixture was diluted with H₂O (5 mL) and saturated aqueous NaHCO₃ solution (5 mL) and partitioned with EtOAc (30 mL). The organic phase was washed with a further portion of saturated aqueous NaHCO₃ solution (5 mL), dried over anhydrous MgSO₄ and concentrated in vacuo to afford a crude residue which was then resuspended in a mixture of TFA and /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 0.1 mL/pmol) and the reaction stirred at rt (1.5-16 h). The solvent was removed in vacuo to afford a crude residue which was purified via reverse phase HPLC.

Condition B:

To a solution of EDC.HCI (12-30 pmol, 1.3-3.3 eq.) in 1:1 v/v CH₂CI₂: DMF (180-240 pL) was added NMM (19-30 pmol, 1.3 eq.) and the solution was shaken for 15 min at rt. This solution was then added dropwise to a solution of depsipeptide S28, S36S43, S45-S52 (15-25 pmol, 1 eq.), amine 18 (28 -50 pmol, 1.2-3.3 eq.) and HOAt (70120 pmol, 5 eq.) and the reaction stirred at rt for 3-4 h. At this point, the reaction was diluted with EtOAc (15-25 mL), and washed with HCI (0.5 M, 4-7mL), saturated aqueous NaHCO₃ solution (5 x 4-7mL), H₂O (4-7 mL) and brine (4-7 mL) and dried over MgSO₄. The solvent was then removed in vacuo to afford a crude residue which was redissolved

WO 2018/107236

PCT/AU2017/051394 in a mixture of CH2CI2, TFA and /Pr₃SiH (1:1 CH2CI2: TFA, 2.5 vol.% /Pr₃SiH, 0.1 mL/pmol) and the reaction stirred at rt (5-16 h). The solvent was removed in vacuo to afford a crude residue which was purified via reverse phase HPLC.

(((S)-1-(((2S,3S)-3-((S)-2-amino-A/-methyl-3-(pyridin-3-yl)propanamido)-1((((2R,4R,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)amino)-1-oxobutan-2-yl)amino)-4-methyl-1oxopentan-2-yl)carbamoyl)-L-tryptophan (8)

Depsipeptide S26 (24 mg, 27 pmol) was reacted with amine 18 (7.2 mg, 32 pmol) in CH2CI2: DMF (1:1 v/v, 260 pL) in the presence of HOAt (18 mg, 134 pmol) and DIC (4.3 pL, 27 pmol) 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 /Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (11 mg, 46% over 2 steps).

IR (ATR): 2951, 2836, 1662 cm’¹. ¹H NMR (500 MHz, CD₃OD, rotamers in 1.3:1 ratio, rotamer 1\. δ 8.69-8.64 (1H, m, NH), 8.63-8.57 (1H, m, NH), 8.57-8.49 (1H, m, NH), 8.12 (1H, d, J= 7.5 Hz, Ar-H), 7.98 (1H, d, J = 8.0 Hz, Ar-H), 7.66-7.59 (2H, m, ArH + H-6), 7.58-7.51 (1H, m, Ar-H), 7.35-7.23 (1H, m, Ar-H), 7.11 (1H, s, Ar-H), 7.1089

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PCT/AU2017/051394

7.04 (1H, m, Ar-H), 7.03-6.93 (1H, m, Ar-H), 5.74 (1H, d, J = 2.7 Hz, H-1’), 5.69 (1H, d, J = 8.0 Hz, H-5), 5.00-4.91 (1H, m, Pal2-a-CH), 4.89-4.81 (1H, m, DABA1-p-CH), 4.76-4.72 (1H, m, DABA1-a-CH), 4.64-4.37 (3H, m, H-2’ + H-4’ + Trp4a-CH), 4.30-4.04 (2H, m, Leu3-a-CH), 3.52-3.41 (1H, m, H-5’), 3.30-3.24 (2H, m, H-5’ + Trp4-3-CH₂), 3.23-3.11 (3H, m, Pal2-p-CH₂ + Trp4-p-CH₂), 2.81 (3H, s, NCH₃), 2.31 (1H, dt, J = 13.8, 6.9 Hz, H-2’), 2.23 (1H, ddd, J = 13.7, 7.5, 6.2 Hz, H-2’), 1.86-1.77 (1H, m, H-2’), 1.78-1.61 (3H, m, H-2’ + Leu3-y-CH), 1.60-1.40 (2H, m, Leu3-3-CH₂),

1.28 (3H, d, J = 7.1 Hz, DABA1-y-CH₃), 1.24 (3H, d, J = 7.1 Hz, DABA1-y-CH₃), 0.980.86 (6H, m, 2x Leu3-5-CH₃). ¹³C NMR (125 MHz, CD₃OD, ratio 1.3:1, rotamer 7): δ 176.0 (C=O), 175.6 (C=O), 171.3 (C=O), 169.2 (C=O), 166.1 (C=O), 159.8 (C=O),

151.9 (C=O), 147.6, 144.4, 142.0, 141.6, 137.9, 128.9, 126.5, 124.6 (x2), 122.2, 119.6,

119.1, 111.9, 110.6, 102.2, 94.9, 80.6, 76.2, 56.4, 54.7, 54.3, 53.0, 52.3, 44.6, 41.6,

35.6, 34.8, 28.8, 28.4, 25.4, 22.4, 12.5.

¹H NMR (500 MHz, CD3OD, rotamers in 1.3:1 ratio, rotamer 2)-. δ 8.06 (1H, d, J= 8.0 Hz, Ar-H), 7.48 (1H, d, J = 8.1 Hz, H-6), 5.77 (1H, d, J = 3.0 Hz, H-1’), 5.64 (1H, d, J= 8.1 Hz, H-5), 5.11-5.04 (1H, m, DABA1-p-CH), 4.64-4.37 (2H, m, DABA1-a-CH + Pal2-oc-CH), 3.40-3.32 (1H, m, Pal2-3-CH2), 3.11-3.05 (1H, m, Pal2-3-CH2), 3.01 (3H, s, NCH3); ¹³C NMR (125 MHz, CD3OD, ratio 1.3:1, rotamer 2)-. δ 165.7 (C=O), 146.7,

141.7, 51.9, 33.4, 30.6, 12.2. LRMS [/W+H⁺] 833.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₄oH₅₂NioOio 833.3940, found 833.3938.

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PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-((S)-2-amino-A/-methyl-3-(naphthalen-2-yl)propanamido)1 -((((2/7,4/7,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)amino)-1-oxobutan-2-yl)amino)-4-methyl-1oxopentan-2-yl)carbamoyl)-L-tryptophan (9)

Depsipeptide S27 (30 mg, 33 pmol) was reacted with amine 18 (9.8 mg, 40 pmol) in CH2CI2: DMF (1:1 v/v, 320 pL) in the presence of HOAt (23 mg, 165 pmol) and DIC (5.3 pL, 33 pmol) 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 /Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (10 mg, 32% over 2 steps).

IR (ATR): 3264, 3056, 2954, 2918, 2851, 1672, 1636 cm’¹. ¹H NMR (500 MHz, DMSO-cfe, rotamers in 1.8:1 ratio, major rotamer): 10.78 (1H, s), 8.52 (1H, s), 8.32 (1H, s), 8.20 (2H, s), 7.88-7.77 (3H, m, Ar-H), 7.65 (1H, app. s, Ar-H), 7.58 (1H, d, J = 8.1 Hz, H-6), 7.54-7.39 (1H, m, Ar-H), 7.47-7.35 (3H, m, Ar-H), 7.32-7.27 (1H, m, Ar-H), 7.12-7.07 (1H, m, Ar-H), 7.05-6.99 (1H, m, Ar-H), 6.97-6.88 (1H, m, Ar-H), 6.46 (1H, d, J= 8.0 Hz), 6.39 (1H, d, J = 8.0 Hz), 6.13 (1H, s), 5.64 (1H, d, Η-Γ), 5.58 (1H, d, H-5), 4.46 (1H, app. t, J = 9.2 Hz, DABA1-a-CH), 4.39-4.24 (3H, m,

H-2’ + H-4’ + Trp4-oc-CH), 4.24-4.08 (3H, m, DABA1-p-CH + Nal2-oc-CH + Leu3-oc-CH), 3.34-3.28 (1H, m, H-5’), 3.12-2.96 (3H, H-5’ + Trp4-3-CH₂), 2.91-2.86 (1H,

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PCT/AU2017/051394 m, Nal2-3-CH₂), 2.81 (1H, dd, J = 13.2, 6.7 Hz, Nal2-3-CH₂), 2.60 (3H, s, NCH₃), 2.58-

2.52 (1H, m, Nal2-3-CH₂), 2.10 (1H, app. dt, J = 13.4, 6.8 Hz, H-3’), 1.68-1.50 (2H, m, H-3’ + Leu3-y-CH), 1.36-1.28 (2H, m, Leu3-3-CH₂), 0.89-0.76 (6H, m, 2x Leu3-5-CH₃), 0.63 (3H, d, J = 6.5 Hz, DABA1-y-CH₃). ¹³C NMR (125 MHz, DMSO-d₆, rotamers in 1.8:1 ratio, major rotamer)·. δ 174.3 (C=O), 172.8 (C=O), 169.9 (C=O), 164.6 (C=O),

163.2 (C=O), 157.3 (C=O), 150.4 (C=O), 141.8, 136.2, 135.9, 135.7, 132.0, 131.7, 128.0, 127.8, 127.6, 127.5, 127.4 (x2), 127.3, 125.4, 123.4, 120.4, 118.4, 118.0, 111.0, 110.0, 101.4, 92.1, 78.8, 74.0, 55.2, 54.1, 51.8, 51.6, 43.4, 41.2, 40.1, 35.2, 27.9, 27.3,

23.7, 21.8, 14.5. LRMS [/W+H⁺] 882.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C45H55N9O10 882.4144, found 882.4140.

(((S)-1-(((2S,3S)-3-((S)-2-amino-3-cyclohexyl-A/-methylpropanamido)-1((((2R, 4R, 5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)amino)-1-oxobutan-2-yl)amino)-4-methyl-1oxopentan-2-yl)carbamoyl)-L-tryptophan (10)

Depsipeptide S28 (22 mg, 25 pmol) was reacted with amine 18 (6.8 mg, 30 pmol) in CH₂CI₂: DMF (1:1 v/v, 240 pL) in the presence of HOAt (17 mg, 123 pmol) and EDC.HCI (5.2 mg, 25 pmol) and NMM (3 pL, 25 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 2.5 mL) according to general procedure 8 to afford 10 (as a TFA salt) after

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PCT/AU2017/051394 reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100% H₂O) as an amorphous white solid (10.4 mg, 44% over 2 steps).

IR (ATR): 3346, 2924, 1728, 1627 cm’¹. ¹H NMR (500 MHz, CD₃OD, rotamers in 1.6:1 ratio, major rotamer)·. δ 8.67 (1H, t, J = 6.0 Hz), 8.45 (1H, d, J = 9.0 Hz), 8.16-8.05 (1H, m), 7.62 (1H, d, J = 8.1 Hz, H-6), 7.58 (1H, dd, J = 7.9, 4.0 Hz, Ar-H), 7.33 (1H, dd, J = 8.1, 5.6 Hz, Ar-H), 7.14-7.05 (2H, m, Ar-H), 7.03-6.98 (1H, m, Ar-H), 5.74 (1H, d, J =

2.7 Hz, H-1 ’), 5.70 (1H, d, J = 8.0 Hz, H-5), 4.88-4.78 (1H, m, DABA1-p-CH), 4.67-4.54 (3H, m, DABA1-a-CH + Cha2-oc-CH + Trp4-oc-CH), 4.49-4.38 (2H, m, H-2’ + H-4’), 4.194.08 (1H, m, Leu3-oc-CH), 3.52-3.46 (1H, m, H-5’), 3.29-3.23 (2H, m, H-5’ major + Trp4-

3-CH₂), 3.24-3.14 (1H, m, Trp4-p-CH₂), 2.85 (3H, s, NCH₃), 2.30-2.18 (1H, m, H-3’), 1.84-1.55 (10H, m, 4x CH₂ + H-3’ + Leu3-y-CH), 1.52-1.40 (3H, CH₂ + Cha2-y-CH),

1.36-1.26 (2H, m, Leu3-3-CH₂), 1.24-1.10 (3H, m, DABA1-y-CH₃), 1.07-0.95 (2H, m, CH₂), 0.94-0.86 (6H, m, 2x Leu3-5-CH₃). ¹³C NMR (125 MHz, CD₃OD, rotamers in 1.6:1 ratio, major rotamer)·. δ 176.2 (C=O), 175.6 (C=O), 171.8 (C=O), 171.0 (C=O), 166.3 (C=O), 159.8 (C=O), 152.3 (C=O), 142.2, 137.9, 128.6, 124.4, 122.0, 119.4, 119.2,

111.9, 110.6, 102.2, 95.0, 80.9, 76.1, 54.9, 54.0, 52.6, 50.3, 44.8, 41.8, 39.6, 36.0, 34.8,

34.2, 29.0, 28.3, 26.9, 25.6, 22.9, 14.6. LRMS [/W+H⁺] 838.4. HRMS (ESI m/z) [/W+Na⁺] calcd. for C46H₇2N₆0iiNa 907.5151, found 907.5150.

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PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-((S)-2-amino-2-cyclohexyl-A/-methylacetamido)-1((((2/?,4fl,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)amino)-1-oxobutan-2-yl)amino)-4-methyl-1oxopentan-2-yl [carbamoyl )-L-try ptophan (11)

Depsipeptide S29 (19 mg, 22 pmol) was reacted with amine 18 (5.8 mg, 26 pmol) in CH2CI2: DMF (1:1 v/v, 200 pL) in the presence of HOAt (14 mg, 107 pmol) and DIC (5.2 pL, 33 pmol) 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 /Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (5.4 mg, 26% over 2 steps).

IR (ATR): 3261, 2931, 2856, 1665 cm’¹. ¹H NMR (400 MHz, CD₃OD, rotamers in 1.6:1 ratio, majorrotamer): δ 10.30 (1H, s, NH), 8.67 (1H, s, NH), 8.12 (1H, s, NH), 7.62 (1H, d, J= 8.1 Hz, H-6), 7.58 (1H, dd, J = 8.0, 3.0 Hz, Ar-H), 7.39-7.24 (1H, m, Ar-H), 7.18-7.03 (2H, m, Ar-H), 7.04-6.98 (1H, m, Ar-H), 5.74 (1H, d, J = 2.7 Hz, Η-Γ), 5.71 (1H, d, J = 8.0 Hz, H-5), 4.91-4.78 (1H, m, DABAI-β-ΟΗ), 4.71-4.53 (1H, Trp4-oc-CH), 4.51-4.37 (2H, m, H-2’ + H-4’), 4.30-4.22 (1H, m, DABA1-oc-CH), 4.20-4.08 (1H, m, Leu3-oc-CH), 4.03 (1H, d, J = 4.6 Hz, Chg2-oc-CH), 3.55-3.45 (1H, m, H-5’),

3.29-3.25 (2H, m, H-5’ major + Trp4-3-CH₂), 3.22-3.09 (1H, m, Trp4-3-CH₂), 2.86 (3H, s, NCH₃), 2.35-2.16 (1H, m, H-2’), 1.87-1.54 (6H, m, H-2’ + Chg2-p-CH + 1.5 x Chg2CH₂ + Leu3-y-CH), 1.53-1.42 (2H, m, Leu3-3-CH₂), 1.35-1.02 (6H, m, DABA1-y-CH₃ +

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1.5 x Chg2-CH₂, major), 0.91 (6H, m, 2x Leu3-5-CH₃ major). ¹³C NMR (100 MHz, CD₃OD, major rotamer): δ 176.1 (C=O), 175.1 (C=O), 171.4 (C=O), 169.7 (C=O), 166.0 (C=O), 159.5 (C=O), 151.9 (C=O), 142.3, 137.8, 128.6, 124.7, 122.0, 119.6, 119.2,

111.9, 110.5, 109.1, 102.3, 95.0, 80.8, 76.2, 56.4, 55.0, 54.4, 53.8, 52.8, 44.6, 43.6, 42.0, 40.5, 35.8, 30.2, 29.0, 28.3, 26.7, 25.7, 23.4, 15.2. LRMS [/W+H⁺] 824.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C40H57N9O10 824.4301, found 824.4294.

(((2S,3/?)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-methyl-1-oxopentan-2-yl)carbamoyl)L-tryptophan (21)

Depsipeptide S36 (17 mg, 22 pmol) was reacted with amine 18 (7.4 mg, 32 pmol) in CH₂CI₂: DMF (1:1 v/v, 210 pL) in the presence of HOAt (15 mg, 108 pmol) and EDC.HCI (5.4 mg, 28 pmol) and NMM (3.0 pL, 28 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 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% H₂O, flow rate 9 mL/min) as an amorphous white solid (7.4 mg, 44% over 2 steps).

IR (ATR): 3374, 2979, 2932, 1731, 1666 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 1): δ 10.77 (s, 1H), 8.37 (s, 1H), 8.05 (s, 1H), 7.62-7.47 (2H, m, Ar-H + H-6), 7.30 (1H, dd, J= 8.1, 5.0 Hz, Ar-H), 7.13 (1H, s, Ar-H), 7.09 (1H, s, Ar-H), 7.04-6.99 (1H, m, Ar-H), 6.94 (1H, app. td, J = 7.2, 3.0 Hz, Ar-H), 6.40 (1H, d, J =

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8.6 Hz), 6.22 (1H, s), 5.70 (1H, d, J = 3.6 Hz, H-1’), 5.60-5.56 (1H, m, H-5), 4.59-4.52 (1H, m, DABA1-a-CH), 4.40-4.16 (3H, m, H-2’ + H-4’ + Trp4-oc-CH), 4.04-3.89 (2H, m, DABAI-β-ΟΗ + He3-oc-CH), 3.67 (1H, d, J = 16.2 Hz, Gly2-oc-CH₂), 3.62-3.54 (1H, m, Gly2-a-CH₂), 3.41 (1H, J = 14.5 Hz), 3.28-3.04 (3H, m, H-5’ + Trp4-p-CH₂), 3.00 (1H, dd, J = 14.5, 6.3 Hz, Trp4-p-CH₂), 2.70 (3H, s, NCH₃), 2.20-2.07 (1H, m, H-3’), 1.80-

1.56 (2H, m, H-3’ + lle3-p-CH), 1.47-1.34 (1H, m, lle3-y-CH₂), 1.11 (3H, d, J = 6.5 Hz, DABA1-y-CH₃), 1.08-0.98 (1H, m, lle3-y-CH₂), 0.78 (6H, m, 2x CH₃). ¹³C NMR (125 MHz, DMSO-cfe, rotamers in 1:1 ratio, rotamer 1, one ¹³C signal is obscure): δ 174.8 (C=O), 172.2 (C=O), 169.8 (C=O), 169.3 (C=O), 163.2 (C=O), 150.6 (0=0), 141.1, 136.0, 127.5, 123.4, 120.6, 118.4, 117.8, 111.5, 110.3, 101.5, 91.9, 78.2, 72.7, 57.4,

55.3, 54.5, 52.3, 43.2, 40.4, 39.6, 36.8, 35.2, 27.7, 24.2, 14.9, 14.5, 10.9. ¹H NMR (500 MHz, DMSO-c/6, rotamers in 1:1 ratio, rotamer 2)·. δ 8.52 (s, 1H), 8.18 (d, J = 9.0 Hz, 1H), 5.65 (1H, d, J = 3.6 Hz, H-1’), 4.82-4.74 (1H, m, DABAI-β-ΟΗ), 4.51-4.44 (1H, m, DABA1-a-CH), 2.72 (3H, s, NCH3). ¹³C NMR (125 MHz, DMSO-d6, rotamers in 1:1 ratio, rotamer 2): δ 50.0. LRMS [M+H⁺] 742.4. HRMS (ESI m/z) [/W+H⁺] calcd. for 0₃₄Η48Ν₉Οιο 742.3518, found 742.3516.

(2S,6S,9S,10S)-2-((1H-indol-3-yl)methyl)-13-amino-9-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)carbamoyl)-6-isopropyl-10,11-dimethyl-4,7,12-trioxo-3,5,8,11tetraazatridecan-1-oic acid (22)

Depsipeptide S37 (17.8 mg, 23.4 pmol.) was reacted with amine 18 (10.5 mg, pmol) in CH₂CI₂: DMF (1:1 v/v, 240 pL) in the presence of HOAt (15.7 mg,

115 pmol), EDO.HCI (5.8 mg, 30 pmol) and NMM (3.3 pL, 30 pmol) for 4 h at rt

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PCT/AU2017/051394 according to general procedure 8 condition B to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /-Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 22 as a 9:1 mixture of diastereomers which were inseparable* by reverse-phase HPLC purification (0 to 50% MeCN over 40 min, 0.1 vol.% formic acid) which yielded 22 as a fluffy white solid (8.5 mg, 62%, yield calculated based on both diastereomers as formate salts after HPLC purification). * The inseparable 9:1 mixture of diastereomers was thus submitted for characterisation and biological assays.

IR (ATR): 3316, 2925, 2872, 1672, 1558 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 1.4:1 ratio, rotamer 1\. δ 7.52 (2H, m, H-6 + Ar-H), 7.30 (1H, m, Ar-H), 7.09 (1H, m, Ar-H), 7.02 (1H, m, Ar-H), 6.92 (1H, m, Ar-H), 5.64 (1H, m, H-T), 5.59 (1H, m, H-5), 4.57 (1H, m, DABA1-O-CH), 4.30 (2H, m, H-2’ + H-4’ + Val3-a-CH + Trp4-a-CH),

3.98 (1H, m, ΟΑΒΑΙ-β-CH), 3.89 (2H, m, Gly2-a-CH₂), 3.22-3.00 (4H, m, Trp4^-CH₂ + 2x H-5’), 2.73 (3H, s, NCH₃), 2.14-2.11 (1H, m,

H-3’), 1.63-1.60 (1H, m, H-3’), 1.11 (3H, m, DABA1-y-CH₃), 1.04 (1H, m, Val3^-CH), 0.79 (6H, m, 2 x Val-y-CH₃). ¹³C NMR (126 MHz, DMSO-d₆, rotamers in 1.4:1 ratio, rotamer 1\. δ 173.7 (C=O), 172.4 (C=O), 170.1 (C=O), 167.2 (C=O), 163.3 (C=O), 157.9 (C=O), 151.0 (C=O), 141.4, 136.5, 128.0, 123.6, 120.9, 118.9, 111.7, 110.7, 101.4,

91.9, 78.6, 74.1, 58.4, 55.0, 54.6, 52.9, 43.7, 40.0, 35.9, 31.4, 29.1, 27.6, 19.8, 18.1,

15.1, 14.5. ¹H NMR (500 MHz, DMSO-d6, rotamers in 1.4:1 ratio, rotamer 2)·. δ 5.67 (1H, m, H-T). ¹³C NMR (126 MHz, DMSO-d6, rotamers in 1.4:1 ratio, rotamer 2)·. δ 92.3. LRMS [M+H⁺] 728.4. HRMS (ESI m/z) [M+H⁺] calcd. for C₃₃H₄5N₉Oi₀ 728.3362, found: 728.3359.

WO 2018/107236

PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-1-oxoheptan-2-yl)carbamoyl)-Ltryptophan (23) h₂n-^Y°

Depsipeptide S38 (30 mg, 37 pmol) was reacted with amine 18 (20 mg, 92 pmol) in CH2CI2: DMF (1:1 v/v, 370 pL) in the presence of HOAt (25 mg, 185 pmol) and EDC.HCI (9.2 mg, 48 pmol) and NMM (5.0 pL, 48 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (8.1 mg, 27% over 2 steps).

IR (ATR): 3356, 2977, 2932, 1731, 1638 cm’¹. ¹H NMR (400 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 1\. δ 10.79 (1H, s), 8.64 (s, 1H), 8.37 (s, 1H), 8.23 (s, 1H), 8.16 (s, 1H), 7.54 (2H, m, H-6 + Ar-H), 7.33-7.28 (1H, m, Ar-H), 7.13 (1H, s, Ar-H), 6.976.91 (1H, m, Ar-H), 6.45 (1H, d, J = 7.8 Hz), 6.15 (1H, s), 5.71 (1H, d, J = 3.3 Hz, H-T), 5.58 (1H, app. dd, J = 8.0, 2.5 Hz, H-5), 4.55 (1H, app. t, J = 9.4 Hz, DABA1-oc-CH), 4.24-4.17 (3H, m, H-2’ + H-4’ + Trp4-oc-CH), 4.09-4.00 (1H, m, Aha3-ocCH), 3.99-3.88 (1H, m, DABA1-p-CH), 3.32-3.00 (3H, m, H-5’ + Trp4-p-CH₂), 3.70-3.61 (1H, m, Gly2-a-CH₂), 3.46 (1H, d, J = 14.9 Hz, Gly2-oc-CH₂), 2.99 (1H, dd, J =14.6, 6.5

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Hz, Trp4-3-CH₂), 2.72 (3H, s, NCH₃), 2.71 (3H, s, NCH₃), 2.15-2.05 (1H, m, H-3’), 1.711.63 (1H, m, H-3’), 1.56-1.37 (2H, m, Aha3-3-CH₂), 1.30-1.14 (6H, m), 1.10 (3H, d, J =

6.5 Hz, DABA1-y-CH₃), 0.86-0.77 (3H, m). ¹³C NMR (100 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 1\. δ 175.2 (C=O), 173.9 (C=O), 170.1 (C=O), 169.8 (C=O), 168.8 (C=O), 163.3 (C=O), 150.9 (C=O), 141.5, 136.4, 128.0, 124.2, 123.8, 120.7, 117.8,

111.6, 110.6, 101.2, 92.0, 78.4, 73.8, 54.3, 53.4, 50.7, 42.9, 40.2, 34.7, 31.2, 28.6, 28.4,

24.9, 21.8, 15.3, 13.1. ¹H NMR (400 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 2)·. δ 8.06 (s, 1H), 7.06-6.99 (1H, m, Ar-H), 7.10 (1H, app. d, J = 2.3 Hz, Ar-H), 5.65 (1H, d, J = 3.4 Hz, H-T), 4.82-4.73 (1H, m, DABA1-p-CH), 4.49-4.42 (1H, m, DABA1-a-CH),

4.41-4.34 (1H, m, H-4’), 2.15-2.05 (1H, m, H-3’), H-3’ minor), 1.77 (1H, app. d, J = 13.0 Hz, H-3’). ¹³C NMR (100 MHz, DMSO-cfe, rotamers in 1:1 ratio, rotamer 2\. δ 27.0. LRMS [M+H⁺] 756.4. HRMS (ESI m/z) [/W+H⁺] calcd. for CssHsoNgOw 756.3675, found 756.3674.

(((S)-2-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-1-cyclohexyl-2-oxoethyl)carbamoyl)-Ltryptophan (24)

O

Depsipeptide S39 (24 mg, 30 pmol) was reacted with amine 18 (14 mg, 60 pmol) in CH₂CI₂: DMF (1:1 v/v, 300 pL) in the presence of HOAt (20 mg, 150 pmol) and EDC.HCI (7.4 mg, 39 pmol) and NMM (4.0 pL, 39 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.%

WO 2018/107236

PCT/AU2017/051394 /Pr₃SiH, 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%

H₂O) as an amorphous white solid (13.7 mg, 60% over 2 steps).

IR (ATR): 3323, 2979, 2932, 1626 cm’¹. ¹H NMR (400 MHz, CD₃OD, rotamers in 1.6:1 ratio, major rotamer)·. δ 8.78 (1H, t, J = 5.9 Hz), 8.37 (1H, d, J = 8.9 Hz), 8.22 (1H, d, J = 8.5 Hz), 8.15 (1H, t, J = 5.8 Hz), 7.62 (1H, d, J = 8.1 Hz, H-6), 7.58-7.54 (1H, m, Ar-H), 7.38-7.26 (1H, m, Ar-H), 7.16-7.04 (2H, m, Ar-H), 7.04-6.95 (1H, m, Ar-H), 5.77-5.70 (2H, m, H-1 ’ + H-5), 4.62-4.56 (2H, m, DABA1-a-CH + Trp4-oc-CH), 4.50-4.42 (2H, m, H-2’), 4.39-4.31 (1H, m, H-4’), 4.16 (1H, d, J = 16.0 Hz, Gly2-oc-CH₂), 4.03-3.88 (3H, m, DABA1-p-CH + Gly2-oc-CH₂ + Chg3-oc-CH), 3.54-3.46 (1H, m, H-5’),

3.29-3.13 (3H, m, H-5’ + Trp4-3-CH₂), 2.87 (3H, s, NCH₃), 2.78 (3H, s, NCH₃), 2.21 (1H, ddd, J = 13.6, 7.6, 6.1 Hz, H-3’), 1.84-1.48 (6H, m, H-3’ + 2.5x CH₂ + Chg3-p-CH), 1.25-0.95 (8H, m, DABA1-y-CH₃ + 2.5x CH₂). ¹³C NMR (100 MHz, CD₃OD, rotamers in 1.6:1 ratio, major rotamer)·. δ 176.2 (C=O), 175.0 (C=O),

171.3 (C=O), 167.6 (C=O), 167.2 (C=O), 160.0 (C=O), 152.2 (C=O), 142.4, 137.9,

128.8, 124.2, 122.1, 119.6, 119.4, 111.8, 110.4, 102.0, 94.7, 81.2, 75.8, 60.0, 56.6,

54.6, 54.1, 45.0, 41.5, 40.8, 35.6, 30.6, 29.6, 29.0, 27.8, 26.8, 14.9. LRMS [M+H⁺]

768.3. HRMS (ESI m/z) [/W+H⁺] calcd. for CseHsoNgOw 768.3875, found 768.3867.

100

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PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)carbamoyl)-L-tryptophan (25)

Depsipeptide S40 (17 mg, 20 pmol) was reacted with amine 18 (9.5 mg, 42 pmol) in CH2CI2: DMF (1:1 v/v, 200 pL) in the presence of HOAt (14 mg, 103 pmol) and EDC.HCI (5.1 mg, 27 pmol) and NMM (3.0 pL, 27 pmol) 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 /Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (6.0 mg, 35% over 2 steps).

IR (ATR): 3368, 2926, 1647, 1549 cm’¹. ¹H NMR (600 MHz, CD₃OD, rotamers in

l. 5:1 ratio, major rotamer): δ 7.62 (1H, d, J = 8.1 Hz, H-6), 7.60-7.54 (1H, m, Ar-H), 7.33 (1H, dd, J = 9.1, 8.2 Hz, Ar-H), 7.11 (1H, s, Ar-H), 7.10-7.05 (1H, m, Ar-H), 7.04-6.98 (1H, m, Ar-H), 5.73 (1H, d, J = 2.6 Hz, Η-Γ), 5.71 (1H, d, J = 8.0 Hz, H-5), 4.65 (1H, d, J = 7.6 Hz, DABA1-oc-CH), 4.60-4.52 (2H, m, H-4’ + Trp4-oc-CH), 4.49-4.44 (1H, m, H-2’), 4.21-4.12 (2H, m, Gly2-CH₂ + Cha3-oc-CH), 3.99 (1H, dq,

J= 10.1, 6.6 Hz, DABA1-3-CH), 3.91 (1H, d, J = 16.0 Hz, Gly2-CH₂), 3.55-3.49 (1H, dd, m, H-5’), 3.29-3.16 (2H, m, Trp4-3-CH₂), 2.88 (3H, s, NCH₃), 2.22 (1H, ddd, J = 13.8,

7.7, 6.2 Hz, H-3’), 1.84-1.60 (5H, m, 2x Cha3-CH₂ + H-3’), 1.49-1.41 (2H, m, Cha3-p101

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CH₂), 1.36-1.31 (1H, m, Cha3-y-CH), 1.26-1.09 (7H, m, 2x Cha3-CH₂ + DABA1-y-CH₃), 1.03-0.79 (2H, m, Cha3-CH₂). ¹³C NMR (150 MHz, CD₃OD, rotamers in 1.5:1 ratio, major rotamer): δ 176.3 (C=O), 175.9 (C=O), 171.9 (C=O), 167.4 (C=O), 167.1 (C=O),

160.2 (C=O), 152.5 (C=O), 142.6, 138.1, 129.1, 124.5, 122.0, 119.3, 119.0, 112.0,

110.4, 102.3, 95.1, 80.6, 76.2, 56.6, 54.8, 54.4, 53.0, 44.8, 40.9, 40.6, 36.2, 34.9, 34.4,

28.5, 28.4, 27.5, 26.8, 13.9. LRMS [/W+H⁺] 782.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₃7H5₂N₉Oio 782.3831, found 782.3824.

(((2S,3S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-hydroxy-1-oxobutan-2-yl)carbamoyl)L-tryptophan (26)

O

Depsipeptide S41 (18.0 mg, 23.2 pmol.) was reacted with amine 18 (10.5 mg,

46.4 pmol) in CH₂CI₂: DMF (1:1 v/v, 240 pL) in the presence of HOAt (15.8 mg, 116.0 pmol), EDC.HCI (5.8 mg, 30.1 pmol) and NMM (3.3 pL, 30.1 pmol) 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 /-Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /-Pr₃SiH, 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).

IR (ATR): 3293, 2922, 1677, 1594, 1436 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 1.3:1 ratio, rotamer 1): δ 7.53 (2H, m, H-6 + Ar-H), 7.30 (1H, m, Ar-H), 7.12 (1H, m, Ar-H), 7.03 (1H, m, Ar-H), 6.94 (1H, m, Ar-H), 6.55 (1H, br s, Thr3-y-OH), 5.64

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PCT/AU2017/051394 (1H, d, J= 3.1 Hz, H-1’), 5.57 (1H, d, J = 6.4 Hz, H-5), 4.60 (1H, m, DABA1-0-CH), 4.30 (1H, m, Trp4-a-CH), 4.27-4.23 (2H, m, H-2’ + H-4’), 4.00-3.94 (3H, m, Thr3-a-CH + ΤήΓ3-β-0Η + DABAI-β-ΟΗ), 3.36-3.00 (6H, m, Gly2-a-CH₂ + 2x H-5’ + ΤΓρ4-β-0Η₂), 2.49 (3H, s, NCH₃), 2.14-2.11 (1H, m, H-3’), 1.71-1.62 (1H, m, H-3’), 1.15 (3H, d, J =

4.7 Hz,Thr3-Y-CH₃), 0.99 (3H, d, J = 5.5 Hz, DABA1-y-CH₃). ¹³C NMR (126 MHz, DMSO-de, rotamers in 1.3:1 ratio, rotamer 1): δ 179.3 (C=O), 173.7 (C=O), 171.8 (C=O), 170.0 (C=O), 164.5 (0=0), 163.6 (0=0), 150.8 (0=0), 141.7, 136.4, 127.9, 124.3,

121.2, 119.2, 118.5, 111.7, 110.0, 102.1, 92.6, 79.0, 78.9, 74.1, 66.9, 59.4, 55.3, 55.2,

44.1, 40.3, 35.8, 29.2, 27.6, 20.1, 15.1. ¹H NMR (500 MHz, DMSO-d₆; rotamers in 1.3:1 ration, rotamer 1): δ 5.68 (1H, m, H-1’), 5.58 (1H, m, H-5). LRMS [/W+H⁺] 730.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C32H44N9O11 730.3154, found 730.3150.

(2S,6S,9S,10S)-2-((1H-indol-3-yl)methyl)-13-amino-6-(carboxymethyl)-9((((2/?,4/?,5/?)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)carbamoyl)-10,11-dimethyl-4,7,12-trioxo-

3,5,8,11-tetraazatridecanoic acid (27)

Depsipeptide S42 (19.2 mg, 21.6 pmol) was reacted with amine 18 (12.2 mg,

53.9 pmol) in CH₂CI₂: DMF (1:1 v/v, 220 pL) in the presence of HOAt (14.7 mg, 107.8 pmol), EDC'HCI (5.4 mg, 28.0 pmol) and NMM (3.1 pL, 28.0 pmol) for 4 h according to general procedure 8 condition B to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /-Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 27 (as a

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IR(ATR): 3304, 2952, 2844, 1643, 1450, 1411 cm’¹. ¹H NMR (500 MHz, DMSOde, rotamers in 1.1:1 ratio, rotamer 1\. 5 7.52 (2H, m, H-6 + Ar-H), 7.31 (1H, m, Ar-H),

7.12 (1H, s, Ar-H), 6.99 (1H, m, Ar-H), 6.93 (1H, m, Ar-H), 5.65 (1H, m, H-5), 5.57 (1H, m, H-T), 4.41 (1H, m, Asp3-a-CH), 4.33-4.28 (4H, m, H-2’ + H-4’ + DABA1-a-CH + Trp4-aCH), 3.94-3.55 (2H, m, Gly2-a-CH₂), 3.27 (1H, m, ϋΑΒΑΙ-β-CH), 3.12 (1H, m, H-5’), 2.99 (1H, m, H-5’), 2.71 (3H, m, NMe), 2.44 (4H, m, Asp3^-CH₂ + Τφ4-β-ΟΗ₂),

2.15-2.11 (1H, m, H-3’), 1.68-1.65 (1H, m, H-3’), 1.08 (3H, m, DABA1-y-CH₃). ¹H NMR (500 MHz, DMSO-de, rotamers in 1.1:1 ratio, rotamer 2)·. 5 5.59 (1H, m, H-1 ’), 5.66 (1H, m, H-5), 2.65 (3H, m, NMe), 1.75 - 1.68 (1H, m, H-3’). ¹³C NMR: (126 MHz, DMSO-de): δ 179.5 (C=O), 172.1 (C=O), 167.8 (C=O), 166.8 (C=O), 166.1 (C=O), 163.8 (C=O),

163.7 (C=O), 150.9 (C=O), 141.5, 136.3, 128.0, 124.0, 123.9, 121.0, 118.9, 111.6,

110.7, 110.9, 101.9, 92.5, 78.8, 74.4, 55.6, 55.0, 51.2, 43.9, 40.2, 40.0, 35.9, 29.4, 28.5,

27.8, 15.0. Rotamer 2: not distinguishable. LRMS [/W+H⁺] 744.4. HRMS (ESI m/z) [/W+H]⁺ calcd. for C₃₂H₄₂N₉Oi₂ 744.2947, found 744.2943.

(((S)-6-amino-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-1-oxohexan-2-yl)carbamoyl)-Ltryptophan (28)

Depsipeptide S43 (13.4 mg, 14.8 pmol.) was reacted with amine 18 (11 mg,

48.7 pmol) in CH₂CI₂: DMF (1:1 v/v, 180 pL) in the presence of HOAt (10 mg,

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74.0 pmol), EDC.HCI (3.7 mg, 19.2 pmol) and NMM (2.1 pL, 19.2 pmol) 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 /-Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /-Pr₃SiH, 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).

IR (ATR): 3285, 3083, 2941, 1672, 1555 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 2.2:1 ratio, major rotamer): 5 7.55-7.49 (2H, m, H-6 + Ar-H), 7.32 (1H, d, J = 8.0 Hz, Ar-H), 7.11 (1H, s, Ar-H), 7.04 (1H, m, Ar-H), 6.95 (1H, m, Ar-H), 5.68-5.57 (2H, m, H-5 + H-T), 4.79-4.73 (1H, m, Lys3-a-CH), 4.36-4.32 (3H, m,

H-2’ + DABAI-a-CH + H-4’), 4.11-4.09 (1H, m, Trp4-a-CH), 3.89-3.86 (1H, m, DABA1β-CH), 3.86-3.65 (2H, m, Gly2-a-CH₂), 3.25-3.24 (1H, m, H-5’), 3.19-2.98 (3H, m, H5’+ 2 x Trp4-3-CH₂), 2.74 (5H, m, NMe + Lys3-s-CH₂), 2.21-2.08 (1H, m, H-3’), 1.51-1.48 (3H, m, H-3’+ Lys3-5-CH₂), 1.27-1.24 (2H, m, Lys3-y-CH₂), 1.11 (3H, d, J =

6.4 Hz, DABA1-y-CH₃), 1.02 (2H, d, J = 6.7 Hz, Lys3^-CH₂). ¹³C NMR (126 MHz, DMSO-cfe, rotamers in 2.2:1 ratio, major rotamer): δ 173.7 (C=O), 172.0 (C=O), 169.8 (C=O), 169.0 (C=O), 165.6 (C=O), 162.8 (C=O), 150.0 (C=O),

141.7, 135.6, 126.9, 124.0, 121.2, 118.7, 111.7, 109.0, 79.0, 74.3, 55.4, 55.1, 54.8,

53.7, 53.1, 44.0, 40.1, 39.1, 36.0, 32.2, 29.0, 28.1, 27.7, 27.0, 22.7, 15.0, 14.3. LRMS [/W+H⁺] 757.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₃₄H₄9NioOio 757.3627, found 757.3620.

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PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1 -oxobutan-2-yl)amino)-1 -oxo-3-(4(trifluoromethyl)phenyl)propan-2-yl)carbamoyl)-L-tryptophan (30)

Depsipeptide S45 (28 mg, 32 pmol) was reacted with amine 18 (15 mg, 64 pmol) in CH2CI2: DMF (1:1 v/v, 320 pL) in the presence of HOAt (22 mg, 161 pmol) and EDC.HCI (8.0 mg, 42 pmol) and NMM (4.4 pL, 42 pmol) 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 /Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (5.6 mg, 20% over 2 steps).

IR (ATR): 3331, 2948, 2837, 1668 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 1): δ 10.73 (1H, s), 8.64 (1H, s), 8.45-8.29 (1H, m), 8.21 (1H, s), 7.66-7.42 (4H, H-6 + Ar-H), 7.37-7.29 (2H, m, Ar-H), 7.30-7.16 (1H, m, Ar-H), 7.05 (1H, s, Ar-H), 6.97 (1H, app. t, J = 7.5 Hz, Ar-H), 6.88 (1H, app. t, J = 7.5 Hz, Ar-H), 6.52 (1H, s), 6.22 (1H, s), 5.76-5.59 (1H, m, H-1’), 5.55 (1H, d, J = 8.0, H-5), 4.54 (1H, app. t, J = 9.1 Hz, DABA1-oc-CH), 4.43-4.20 (3H, m, H-2’ + H-4’ + p-CF₃-Pal3-a-CH), 4.19-4.08 (1H, m, Trp4-oc-CH), 3.94-3.82 (1H, m, DABA1-p-CH), 3.79-3.86 (1H, m, Gly2-oc-CH₂), 3.60-3.34 (1H, m, Gly2-oc-CH₂), 3.61-3.39 (m, 1H), 3.342.89 (5H, m, H-5’ + p-CF₃-Pal3-3-CH₂ + Trp4-p-CH₂), 2.88-2.78 (1H, m, p-CF₃-Pal3-3CH₂), 2.70 (3H, s, NCH₃), 2.19-2.04 (1H, m, H-3’), 1.73-1.53 (1H, m, H-3’), 1.03 (3H, d,

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J = 6.0 Hz, DABA1-y-CH₃). ¹³C NMR (125 MHz, DMSO-d₆, rotamers in 1:1 ratio, rotamer 1): δ 175.1 (C=O), 173.0 (C=O), 170.9 (C=O), 169.0 (C=O), 167.0 (C=O), 163.1 (C=O), 150.4 (C=O), 142.1, 140.6, 135.3, 129.4, 127.4, 126.6, 123.9, 123.6, 119.7, 118.0, 117.4, 117.1, 110.4, 110.3, 100.9, 91.3, 78.6, 73.4, 54.4, 54.1, 53.5, 52.1, 42.8,

39.3, 36.8, 34.6, 27.2, 26.5, 13.8. ¹H NMR (500 MHz, DMSO-d6, rotamers in 1:1 ratio, rotamer 2): δ 4.77-4.70 (1H, m, DABA1-p-CH), 4.46 (1H, app. t, J = 8.7 Hz, DABA1-aCH), 2.68 (3H, s, NCH3). ¹³C NMR (125 MHz, DMSO-d6, rotamers in 1:1 ratio, rotamer 2): δ 53.5, 49.6, 28.0, 13.2. LRMS [/W+H⁺] 844.3. HRMS (ESI m/z) [/W+H⁺] calcd. for C₃₈H45F₃N₉Oio 844.3260, found 844.3234.

(2S,6S,9S,10S)-2-((1H-indol-3-yl)methyl)-13-amino-9-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)carbamoyl)-6-(4-hydroxybenzyl)-10,11-dimethyl-4,7,12-trioxo-3,5,8,11tetraazatridecan-1-oic acid (31)

Depsipeptide S46 (29.5 mg, 33.0 pmol.) was reacted with amine 18 (18.7 mg,

82.4 pmol) in CH2CI2: DMF (1:1 v/v, 340 pL) in the presence of HOAt (22.5 mg, 165.0 pmol), EDC’HCI (8.2 mg, 42.9 pmol) and NMM (4.7 pL, 42.9 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 31 (as a formate salt) after reverse-phase HPLC purification (0 to 50% MeCN over 40 min) as a fluffy white solid (15.7 mg, 50%, yield over two steps).

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IR (ATR): 3325, 2928, 1676, 1541, 1498 cm’¹. ¹H NMR (400 MHz, DMSO-d6, rotamers in 1.5:1 ratio, major rotamer): δ 7.53 (2H, m, H-6 + Ar-H), 7.30 (1H, d, J = 8.0 Hz Ar-H), 7.10 (1H, app. dd, J = 6.2 Hz, Ar-H), 7.03 (1H, m, Ar-H), 6.95 (3H, m, 3x Ar-H), 6.61 (2H, m, 2x Ar-H), 5.67 (1H, d, J = 3.2 Hz, H-1’), 5.59 (1H, d, J = 2.3 Hz, H-5), 4.55 (1H, m, DABA1-O-CH), 4.32-4.24 (4H, m, Trp4-a-CH + Tyr3-a-CH, H-2’ + H-4’), 3.91 (1H, m, DABAI-β-ΟΗ), 3.33-2.96 (6H, m, Τφ4-β-ΟΗ2 + Tyr3^-CH2+ 2x H-5’), 2.82-2.60 (5H, m, Gly2-a-CH2+ NCH3), 2.14-2.11 (1H, m, H-3’), 1.70-1.62 (1H, m, H-3’), 1.10 (3H, d, J 6.5 Hz, DABA1-y-CH3). ¹³C NMR (101 MHz, DMSO-c/6, rotamers in 1.5:1 ratio, major rotamer): δ 175.0 (C=O),

172.3 (C=O), 169.1 (C=O), 168.3 (C=O), 163.6 (C=O), 159.9, 156.2 (0=0), 156.0 (0=0), 141.3, 135.6, 130.4, 127.3, 127.1, 124.0, 121.1, 118.8, 118.5, 116.2, 115.2,

109.9, 108.8, 92.3, 79.0, 74.4, 55.3, 54.2, 50.8, 43.9, 40.6, 35.8, 29.3, 28.3, 27.6, 15.0. LRMS [/W+H⁺] 792.5. HRMS (ESI m/z) [/W+H⁺] calcd. for C^eNgOn 792.3311, found 792.3304.

(((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-1-oxo-3-(pyridin-3-yl)propan-2yl)carbamoyl)-L-tryptophan (32)

O

Depsipeptide S47 (14 mg, 17 pmol) was reacted with amine 18 (10 mg, 44 pmol) in CH₂CI₂: DMF (1:1 v/v, 170 pL) in the presence of HOAt (12 mg, 86 pmol) and EDO.HCI (4.1 mg, 22 pmol) and NMM (2.3 pL, 22 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.%

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PCT/AU2017/051394 /Pr₃SiH, 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%

H₂O) as an amorphous white solid (8.2 mg, 54% over 2 steps).

IR (ATR): 3367, 3063, 1666 cm’¹. ¹H NMR (400 MHz, CD₃OD, rotamers in 1.4:1 ratio, rotamer 1): δ 8.74 (1H, t, J = 6.0 Hz), 8.60-8.54 (1H, m, Ar-H), 8.41 (1H, d, J = 8.8 Hz), 8.25-8.16 (1H, m, Ar-H), 7.77-7.67 (1H, m, Ar-H), 7.63 (1H, d, J = 8.1 Hz, H-6), 7.57-7.47 (1H, m, Ar-H), 7.35-7.30 (1H, m, Ar-H), 7.11-7.04 (2H, m, Ar-H), 7.036.97 (1H, m, Ar-H), 5.76-5.74 (1H, m, H-1’), 5.71 (1H, d, J = 8.0 Hz, H-5), 4.74-4.62 (1H, m, DABA1-a-CH), 4.61-4.43 (4H, m, H-2’ + DABA1-a-CH +

3-Pal3-oc-CH + Trp4-oc-CH), 4.41-4.32 (1H, m, H-4’), 4.14 (1H, d, J = 16.0 Hz, Gly2-ocCH₂), 4.01-3.88 (2H, m, DABA1-p-CH + Gly2-oc-CH₂), 3.55-3.46 (1H, m, H-5’), 3.30-3.10 (4H, m, H-5’, 3-Pal3-3-CH₂ + Trp4-p-CH₂), 3.07-2.97 (1H, m, 3-Pal3-3-CH₂ major), 2.88 (3H, s, NCH₃), 2.28-2.18 (1H, m, H-3’), 1.84-1.69 (1H, m, H-3’), 1.25-1.17 (3H, m, DABA1-y-CH₃). ¹³C NMR (100 MHz, CD₃OD, rotamers in 1.4:1 ratio, rotamer 1): δ 176.3 (C=O), 172.8 (C=O), 171.4 (C=O), 167.3 (C=O), 166.8 (C=O), 165.9 (C=O), 152.2 (C=O), 145.8, 145.4, 142.8, 142.2, 137.9 (x2), 128.8, 126.6, 124.3, 122.1, 119.6, 119.2,

111.8, 110.5, 102.2, 94.8, 81.0, 76.1, 56.8, 55.0, 54.8, 54.2, 44.9, 41.0, 36.1, 36.0, 28.6,

27.8, 14.8. ¹H NMR (400 MHz, CD₃OD, rotamers in 1.4:1 ratio, rotamer 2): δ 8.33 (1H, d, J = 8.2 Hz), 8.15-8.10 (1H, m, Ar-H), 7.57-7.47 (1H, m, Ar-H, H-6), 5.67 (1H, d, J =

8.1 Hz, H-5), 4.74-4.62 (1H, m, DABA1-p-CH), 4.61-4.43 (1H, m, H-4’), 3.83 (1H, d, J =

16.2 Hz, Gly2-a-CH₂), 3.75 (1H, d, J = 16.2 Hz, Gly2-oc-CH₂), 3.46-3.35 (1H, m, H-5’),

3.30-3.10 (4H, m, H-5’, 3-Pal3-3-CH₂ + Trp4-p-CH₂), 3.07-2.97 (1H, m, 3-Pal3-3-CH₂),

2.81 (3H, s, NCH₃), 2.35 (1H, app. dt, J = 13.6, 7.0 Hz, H-3’), 1.84-1.69 (1H, m, H-3’), 1.25-1.17 (3H, m, DABA1-y-CH₃). ¹³C NMR (100 MHz, CD3OD, rotamers in 1.4:1 ratio, rotamer 2): δ 145.2, 102.5, 80.4, 75.9, 56.4, 53.3, 44.6, 41.2, 36.4, 30.4, 13.6. LRMS [/W+H⁺] 777.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C36H₄5NioOio 777.3314, found 777.3311.

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PCT/AU2017/051394 (2S,6S,9S,10S)-2,6-bis((1H-indol-3-yl)methyl)-13-amino-9-((((2/?,4/?,5/?)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)carbamoyl)-10,11-dimethyl-4,7,12-trioxo-3,5,8,11-tetraazatridecan-1-oic acid (33)

Depsipeptide S48 (36.0 mg, 37.4 pmol.) was reacted with amine 18 (21.3 mg,

93.5 pmol) in CH2CI2: DMF (1:1 v/v, 380 pL) in the presence of HOAt (25.5 mg, 187.0 pmol), EDC.HCI (9.3 mg, 48.6 pmol) and NMM (5.4 pl_, 48.6 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 33 (as a formate salt) after reverse phase HPLC (0 to 50% MeCN over 40 min) as a fluffy white solid (9.1 mg, 32% over two steps).

IR (ATR): 3327, 2964, 2928, 1680, 1554 cm’¹. ¹H NMR (400 MHz, DMSO-d6, rotamers in 1.1:1 ratio, rotamer 1): δ 7.54 (2H, m, H-6 + 2x Ar-H), 7.30 (2H, d, J = 8.0 Hz, 2x Ar-H), 7.11-6.92 (6H, m, 6x Ar-H), 7.02 (1H, m, Ar-H), 6.92 (1H, m, Ar-H), 5.67 (1H, d, J = 3.4 Hz, H-1’), 5.59 (1H, app. d, H-5), 4.56 (1H, m, DABA1-O-CH), 4.48-4.25 (4H, m, H-2’ + H-4’ + Trp3-a-CH + Trp4-a-CH), 3.88 (1H, m, ΟΑΒΑΙ-β-CH), 3.71 (2H, m, Gly2-a-CH2), 3.34-2.88 (6H, m, ΤΓρ3-β-ΰΗ2 + ΤΓρ4-β-ΰΗ2 + 2x H-5’), 2.73 (3H, s, NCH3), 2.14-2.11 (1H, m, H-3’), 1.76-1.65 (1H, m, H-3’), 1.11 (3H, m, DABA1-y-CH3). ¹³C NMR (101 MHz, DMSO-d6, rotamers in 1.1:1 ratio, rotamer 1): δ 173.7 (C=O), 169.0 (C=O), 168.0 (C=O), 167.3 (C=O), 163.7 (C=O), 163.6 (C=O), 150.8 (C=O), 141.2,

136.4, 136.3, 127.9, 127.8, 124.3, 123.7, 121.9, 120.8, 119.7, 119.5, 118.6, 118.5,

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112.2, 111.5, 111.4, 110.2, 101.8, 92.4, 78.8, 74.3, 55.2, 54.5, 51.0, 44.0, 40.4, 39.4,

35.8, 29.3, 28.3, 27.2, 14.9. ¹H NMR (400 MHz, DMSO-d₆, rotamers in 1.1:1 ratio, rotamer 2): δ 5.72 (1H, m, H-Γ). ¹³C NMR (101 MHz, DMSO-d₆, rotamers in 1.1:1 ratio, rotamer 2\. 93.4, 102.8. LRMS [M⁺H⁺] 815.4. HRMS (ESI m/z) [M+H⁺] calcd. for

C39H47N10O10 815.3471, found 815.3465.

(2S,6S,9S,10S)-2-((1H-indol-3-yl)methyl)-13-amino-9-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)carbamoyl)-10,11-dimethyl-4,7,12-trioxo-6-phenyl-3,5,8,11tetraazatridecan-1-oic acid (34)

Depsipeptide S49 (25.3 mg, 31.3 pmol.) was reacted with amine 18 (17.8 mg,

78.2 pmol) in CH2CI2: DMF (1:1 v/v, 320 pL) in the presence of HOAt (21.3 mg, 156.5 pmol), EDC.HCI (7.8 mg, 40.7 pmol) and NMM (4.5 pL, 40.7 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH2CI2 (1:1 v/v TFA: CH2CI2, 2.5 vol.% /Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 34 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min) as a fluffy white solid (12.0 mg, 47% over two steps).

IR (ATR): 3407, 2923, 2853, 1679, 1437 cm’¹. ¹H NMR (400 MHz, DMSO-d₆, rotamers in 1.3:1 ratio, rotamer 1): δ 7.52 (2H, m, H-6 + Ar-H), 7.35-7.29 (6H, m, 6x ArH), 7.11-6.93 (3H, m, 3x Ar-H), 5.64-5.59 (2H, m, H-T + H-5), 4.81 (1H, m, Phg3-aCH), 4.34 (1H, m, DABAI-a-CH), 4.32-4.25 (3H, m, H-2’ + H-4’ + Trp4-a-CH), 3.90 (1H,

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PCT/AU2017/051394 m, DABAI-β-ΟΗ), 3.71-3.50 (4H, m, Gly2-a-CH₂ + ΤΓρ4-β-0Η₂), 3.24-2.99 (4H, m,

ΤΓρ4-β-0Η₂ + 2x H-5’), 2.73 (3H, s, NCH₃), 2.06-1.98 (1H, m,

H-3’), 1.76 - 1.65 (1H, m, H-3’), 1.13 (3H, m, DABA1-y-CH₃). ¹H NMR (400 MHz, DMSO-de, rotamers in 1.3:1 ratio, rotamer 2)·. δ 2.17-2.08 (1H, m, H-3’). ¹³C NMR (101 MHz, DMSO-de; rotamers in 1.3:1 ratio, rotamer 1\. δ 175.1 (C=O), 174.8 (C=O), 173.0 (C=O), 169.9 (C=O), 168.7 (C=O), 167.9 (C=O), 163.6, 150.8 (C=O), 141.5, 136.2,

128.5, 128.0, 127.7, 123.8, 121.0, 118.9, 118.5, 111.5, 110.6, 101.9, 92.4, 78.7, 74.4, 57.0, 55.4, 54.6, 53.3, 50.8, 44.0, 40.5, 36.5, 29.1, 28.3, 27.3, 14.9. ¹³C NMR (101 MHz, DMSO-de, rotamers in 1.3:1 ratio, rotamer 2): δ 35.7. LRMS [/W+H⁺] 762.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C₃₆H44N₉Oio 762.3206, found 762.3200.

(2S,6S,9S,10S)-2-((1H-indol-3-yl)methyl)-13-amino-9-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)carbamoyl)-10,11-dimethyl-4,7,12-trioxo-6-phenethyl-3,5,8,11tetraazatridecan-1-oic acid (35)

Depsipeptide S50 (29.3 mg, 35.1 pmol.) was reacted with amine 18 (19.9 mg, 87.7 pmol) in CH₂CI₂: DMF (1:1 v/v, 360 pL) in the presence of HOAt (23.9 mg, 175.5 pmol), EDC.HCI (8.7 mg, 45.6 pmol) and NMM (5.0 pL, 45.6 pmol) for 4 h according to general procedure 8 to obtain the fully protected analogue. This compound was then treated with a mixture of TFA and /-Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /-Pr₃SiH, 1.2 mL) for 16 h according to general procedure 8 to afford 35 (as a formate salt) after reverse phase HPLC purification (0 to 50% MeCN over 40 min) as a fluffy white solid (7.0 mg, 24% over two steps).

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IR (ATR): 3377, 2960, 2929, 1680, 1435 cm’¹. ¹H NMR (500 MHz, DMSO-d₆, rotamers in 1.5:1 ratio, major rotamer): δ 7.53 (2H, m, H-6 + Ar-H), 7.30 (7H, m, 7x Ar-H), 7.01 (2H, m, 2x Ar-H), 5.64 (1H, m, Η-Γ), 5.55 (1H, m, H-5), 4.56 (1H, m, DABA1-O-CH), 4.28 (3H, m, Trp4-a-CH + H-2’ + H-4’), 4.10-4.04 (2H, m, HPhe3-a-CH + DABAI-β-ΟΗ), 3.69 (2H, m, Gly2-a-CH₂), 3.26-3.14 (4H, m, Τφ4-β-ΟΗ₂ + 2x H-5’),

2.73 (3H, s, NCH₃), 2.50 (2H, obscure, HPhe3-y-CH₂), 2.09 (1H, m, H-3’), 1.83-1.66 (3H, m, H-3’+ ΗΡήβ3-β-ΟΗ₂), 1.12 (3H, m, DABA1-y-CH₃). ¹³C NMR (126 MHz, DMSOd&, rotamers in 1.5:1 ratio, major rotamer): δ 177.7 (C=O), 172.7 (C=O), 171.4 (C=O),

169.9 (C=O), 169.0 (C=O), 163.7 (C=O), 150.8 (C=O), 141.4, 136.3, 128.8, 128.7,

126.2, 126.0, 125.1, 124.1, 121.0, 118.9, 111.4, 110.3, 101.6, 92.5, 79.0, 78.9, 74.2,

55.5, 53.4, 52.9, 50.8, 43.8, 40.9, 40.3, 35.7, 31.4, 29.2, 27.5, 15.1. LRMS [/W+H⁺]

790.5. HRMS (ESI m/z) [/W+H⁺] calcd. for 0₃₈Η48Ν₉Οιο 790.3518, found 790.3509.

(((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2yl)carbamoyl)-L-tryptophan (36)

HO

HN

Depsipeptide S51 (21 mg, 25 pmol) was reacted with amine 18 (14 mg, 63 pmol) in CH₂CI₂: DMF (1:1 v/v, 250 pL) in the presence of HOAt (17 mg, 123 pmol) and EDO.HCI (6.1 mg, 32 pmol) and NMM (3.5 pL, 32 pmol) 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 /Pr₃SiH in CH₂CI₂ (1:1 v/v TFA: CH₂CI₂, 2.5 vol.% /Pr₃SiH, 2.5 mL) for 4 h according to general procedure 8 to afford 36 (as a formate salt)

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PCT/AU2017/051394 after reverse phase HPLC purification (0 to 50% MeCN over 40 min, 10 min at 100%

H₂O, flow rate 9 mL/min) as an amorphous white solid (6.0 mg, 28% over 2 steps).

IR (ATR): 3390, 2977, 2936, 1730, 1644 cm’¹. ¹H NMR (500 MHz, CD₃OD, major rotamer)·. δ 8.56 (1H, t, J = 6.0 Hz, 1H), 8.16 (1H, d, J = 9.1 Hz), 8.13 (0.4H, d, J = 8.6 Hz), 7.84 (1H, t, J = 5.9 Hz), 7.81-7.69 (2H, m, Ar-H), 7.65-7.60 (1H, m, Ar-H), 7.58-7.54 (2H, m, Ar-H + H-6), 7.45-7.40 (3H, m, Ar-H), 7.36-7.29 (2H, m, Ar-H), 7.117.04 (2H, m, Ar-H), 7.03-6.92 (1H, m, Ar-H), 5.72-5.66 (2H, m, H-1’ + H-5), 4.64-4.53 (1H, m, Trp-a-CH), 4.53-4.48 (1H, m, DABA1-a-CH), 4.48-4.34 (2H,

H-2’ + Nal3-oc-CH), 4.25-4.16 (1H, m, H-4’), 4.07 (1H d, J= 15.9 Hz, Gly2-oc-CH₂), 3.88-

3.81 (2H, m, DABA1-p-CH + Gly2-oc-CH₂), 3.30-3.23 (1H, m, Trp4-p-CH₂), 3.22-3.04 (4H, m, H-5’ + Trp4-3-CH₂+ Nal3-3-CH₂), 3.13-3.03 (1H, m, H-5’), 2.91 (1H, ddd, J =

13.6, 8.6, 4.8 Hz, H-5’), 2.83 (3H, s, NCH₃), 2.14 (1H, ddd, J = 13.6, 7.5, 6.0 Hz, H-3’), 1.66-1.59 (1H, m, H-3’), 1.17 (3H, d, J = 6.6 Hz, DABA1-y-CH₃). ¹³C NMR (125 MHz, CD₃OD, major rotamer, two ¹³C signals are obscure): δ 176.0 (C=O), 174.4 (C=O), 171.0 (C=O), 167.6 (C=O), 166.3 (C=O), 159.5 (C=O), 152.4 (C=O), 142.6, 142.4, 137.6, 135.8, 134.7, 134.0, 128.8 (x2), 128.6, 128.2, 126.6, 124.4, 122.0, 119.6, 119.2, 112.0, 110.6, 102.3, 94.8, 81.1, 76.0, 56.6 (x2), 55.0, 54.8, 45.0,

40.9, 39.2, 35.9, 29.1, 27.7, 14.5. LRMS [M+H⁺] 826.4. HRMS (ESI m/z) [/W+H⁺] calcd. for C4iH₄₈N₉Oio 826.3518, found 826.3515.

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PCT/AU2017/051394 (((S)-1-(((2S,3S)-3-((S)-2-amino-3-(3-hydroxyphenyl)-A/-methylpropanamido)1 -((((2R,4R,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl1 -oxopropan-2-yl)carbamoyl)-L-tryptophan (37)

Depsipeptide S52 (18 mg, 17 pmol) was reacted with amine 18 (12 mg, 52 pmol) in CH2CI2: DMF (1:1 v/v, 180 pL) in the presence of HOAt (11 mg, 150 pmol) and EDC.HCI (4.4 mg, 22 pmol) and NMM (2.6 pL, 22 pmol) 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 /Pr₃SiH in H₂O (9:1 v/v TFA: CH2CI2, 2 vol.% /Pr₃SiH, 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% H₂O) as an amorphous white solid (8.0 mg, 53% over 2 steps).

IR (ATR): 3388, 2926, 1647 cm’¹. ¹H NMR (500 MHz, CD₃OD, rotamers in 2.6:1 ratio, major rotamer)·. δ 7.62 (1H, d, J = 8.1 Hz, H-6), 7.54 (1H, app. t, J = 7.8 Hz, Ar-H),

7.32 (1H, app. d, J = 8.2 Hz, Ar-H), 7.18 (1H, app. t, J = 7.9 Hz, Ar-H), 7.12-7.04 (2H, m, Ar-H), 7.02-6.96 (1H, m, Ar-H), 6.78-6.71 (2H, m, Ar-H), 6.69-6.66 (1H, m, Ar-H),

5.74 (1H, d, J = 2.5 Hz, H-1’), 5.71 (1H, d, J = 8.1 Hz, H-5), 4.91-4.75 (1H, m, m-Tyr2-ocCH), 4.59-4.49 (2H, m, DABA1-a-CH + Trp4-oc-CH), 4.48-4.35 (2H, H-2’ + H-4’), 4.234.07 (2H, m, DABA1-p-CH + Cha3-oc-CH), 3.52 (1H, dd, J = 13.9, 3.9 Hz, H-5’), 3.29115

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3.23 (2H, m, H-5’ + Trp4-p-CH₂), 3.23-3.11 (1H, m, Trp4-3-CH₂), 3.02-2.97 (2H, m, mTyr2-3-CH₂), 2.78 (3H, s, NCH₃), 2.23 (1H, app. dt, J = 13.7, 7.0 Hz, H-3’), 1.82-1.58 (5H, m, H-3’ + 2x CH₂), 1.48-1.40 (2H, m, Cha3-3-CH₂), 1.38-1.11 (6H, m, Cha3-y-CH + 2.5x CH₂), 0.98-0.85 (1H, m, 0.5x CH₂), 0.82 (3H, d, J = 6.5 Hz, DABA1-y-CH₃). ¹³C NMR (125 MHz, CD₃OD, rotamers in 2.6:1 ratio, major rotamer)·. δ 175.8 (C=O), 175.4 (C=O), 171.3 (C=O), 170.2 (C=O), 165.8 (C=O), 159.8 (C=O), 159.0, 151.6 (C=O),

142.1, 137.6, 136.1, 131.2, 128.6, 124.4, 122.2, 121.7, 119.7, 119.3, 116.9, 115.7,

112.1, 110.5, 102.3, 94.9, 80.9, 76.2, 56.9, 54.6, 54.5, 52.9, 52.8, 44.8, 40.4, 38.3, 35.7, 35.0, 33.8, 28.8, 28.3, 27.1, 14.2. LRMS [M+H⁺] 888.3. HRMS (ESI m/z) [/W+H⁺] calcd. for C44H58N9O11 888.4250, found 888.4244.

iert-Butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(allyloxy)3-oxopropyl)-1 H-indole-1 -carboxylate (38)

FmocHN

O

To a solution of Fmoc-Trp(Boc)-OH (1.0 g, 1.9 mmol, 1 eq.) in DMF (2.8 mL) at 0 °C was added dropwise /Pr₂EtN (660 pL, 3.8 mmol, 2 eq.), followed by allyl bromide (330 pL, 3.8 mmol, 2 eq.). The reaction was allowed to warm to room temperature and stirred for 16 h before being diluted with EtOAc (80 mL). The organic layer was then washed with H₂O (6 x 40 mL), brine (40 mL), dried (MgSO₄) and concentrated in vacuo to yield 38 as a white foam (1.1 g, 2.0 mmol, quantitative).

IR (ATR): 2923, 2853, 1731, 1512 cm’¹; [αβ° = +6° (c = 0.1, CH2CI2); ¹H NMR (500 MHz, CDCI3) δ 8.13-8.12 (m, 1H, Ar-H), 7.76-7.75 (m, 2H, Ar-H), 7.57-7.51 (m, 3H, Ar-H), 7.41-7.37 (m, 3H, Ar-H), 7.33-7.27 (m, 3H, Ar-H), 7.24-7.21 (m, 1H, Ar-H), 5.89-

5.81 (m, 1H, OCH₂CH=CH₂) 5.41 (d, J = 8.4 Hz, 1H, NH), 5.31-5.23 (m, 2H, OCH₂CH=CH₂), 4.80-4.77 (m, 1H, a-CH), 4.59 (d, J = 5.5 Hz, 2H, OCH₂CH=CH₂),

4.42-4.34 (m, 2H, Fmoc-CH₂), 4.21 (t, J= 7.1 Hz, 1H, Fmoc-CH), 3.52 (d, J = 4.6 Hz, 2H, β-ΟΗ₂), 1.66 (s, 9H, CO₂/-Bu); ¹³C NMR (125 MHz, CDCI₃) δ 171.5, 155.9, 149.7, 144.0, 143.9, 141.4, 138.2, 135.5, 131.5, 130.7, 128.9, 127.9, 127.2, 127.2, 125.3,

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124.8, 124.4, 122.8, 122.8, 121.2, 120.1, 120.0, 119.4, 119.0, 115.5, 114.9, 107.9, 83.9,

67.4, 66.4, 54.4, 47.3, 28.4, 28.1; LRMS [M+Na]⁺589.4.

iert-Butyl (S)-3-(3-(allyloxy)-2-amino-3-oxopropyl)-1 H-indole-1 -carboxylate (39)

Allyl ester 38 (1.1 g, 2.0 mmol, 1 eq.) was treated with 20% v/v piperidine in MeCN (9.5 mL) and stirred for 30 min. The solvent was removed in vacuo and the resulting residue was purified by column chromatography (eluent: 1:1 v/v hexane: EtOAc, 0.1% v/v diethylamine to neat EtOAc, 0.1% v/v diethylamine) to yield 39 as a yellow oil (0.56 g, 1.6 mmol, 85%).

IR (ATR): 2924, 2854, 1732, 1454 cm’¹; [αβ° = +0.5° (c = 0.1 in CH2CI2); ¹H NMR (400 MHz, CDCI3) δ 8.13-8.11 (m, 1H, Ar-H), 7.57-7.55 (m, 1H, Ar-H), 7.51 (m, 1H, Ar-H), 7.33-7.29 (m, 1H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 5.91-5.83 (dddd, 1H, OCH₂CH=CH₂), 5.29 (dq, J = 17.2, 1.5 Hz, 1H, OCH₂CH=CH₂), 5.23 (dq, J = 10.4,

1.2 Hz, 1H, OCH₂CH=CH₂), 4.59 (ddd, J = 6.0, 5.9, 1.3 Hz, 2H, OCH₂CH=CH₂), 3.94 (dd, J = 7.5, 5.3 Hz, 1H, a-CH), 3.26 (dd, J = 14.3, 5.1 Hz, 1H, p-CH₂) 3.08 (dd, J =

14.4, 7.4 Hz, 1H, p-CH₂), 1.66 (s, 9H, CO₂f-Bu); ¹³C NMR (100 MHz, CDCI3) δ 174.7,

149.6, 135.5, 131.8, 130.4, 124.5, 124.2, 122.5, 118.9, 118.8, 116.0, 115.3, 83.6, 65.7,

54.5, 30.5, 28.2; LRMS [M+H]⁺345.1.

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PCT/AU2017/051394 iert-Butyl (S)-3-(3-(allyloxy)-2-(((4-nitrophenoxy)carbonyl)amino)-3-oxopropyl)-1 H-indole-1 -carboxylate (40)

To a solution of 39 (0.56 g, 1.6 mmol, 1 eq.) and /Pr₂EtN (280 μΙ_, 1.6 mmol, 1 eq.) in CH₂CI₂ (8 mL) was added dropwise p-nitrophenyl chloroformate (0.33 g,

1.6 mmol, 1 eq.) in CH₂CI₂ (4 mL) and stirred for 1 h. The solvent was removed in vacuo to yield a crude yellow oil 40 (1.3 g) which was used directly in the subsequent step.

tert-Butyl (S)-3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3oxopropyl)-1 H-indole-1 -carboxylate (41)

NBoc

FmocHN

To a solution of FmocTrp(Boc)-OH (0.20 mg, 0.38 mmol, 1 eq.) in MeOH (3 mL) was added dropwise SOCI₂ (40 pL, 0.53 mmol, 1.4 eq.) at 0 °C. The reaction was subsequently allowed to warm to room temperature and stirred for 30 min prior to heating at 40 °C for 4 h. The reaction was then allowed to cool to room temperature before being concentrated in vacuo. The crude residue was redissolved in MeOH (1.6 mL) and concentrated in vacuo once more. The flask was cooled to 0 °C prior to dissolving the residue in a saturated aqueous solution of Na₂CO₃ (5 mL). The reaction was partitioned between CHCI₃ (10 mL) and H₂O (5 mL). The aqueous phase was extracted once with CHCI₃ (10 mL). The combined organic extracts were dried (MgSO4) 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 (0.11 mg, 0.20 mmol, 52%).

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IR (ATR): 3333, 2924, 1730 cm’¹; [αβ° = +21.3° (c = 0.3 in CH2CI2); ¹H NMR (300 MHz, CDCIs) δ 8.12 (m, 1H, Ar-H), 7.78-7.74 (m, 2H, Ar-H), 7.58-7.49 (m, 3H, ArH), 7.43-7.32 (m, 7H, Ar-H), 5.38 (m, 1H, NH), 4.83-4.71 (m, 1H, a-CH), 4.40-4.36 (m, 2H, Fmoc-CH2), 4.28-4.21 (m, 1H, Fmoc-CH), 3.71 (s, 3H, CH₃), 3.28-3.26 (m, 2H, 3-CH₂), 1.65 (s, 4.5H, CO₂ftBu), 1.57 (s, 4.5H, CO₂ftBu); ¹³C NMR (75.5 MHz, CDCI₃) δ

172.1, 155.7, 149.5, 143.7, 141.2, 135.3, 130.4, 127.6, 127.0, 125.1, 124.5, 124.1,

122.6, 119.9, 118.7, 115.3, 114.9, 83.6, 67.1, 52.3, 47.0, 28.1, 27.7; LRMS [M+Na]⁺563.5; HRMS Calcd for C₃₂H₃₂N₂O₆: MNa⁺, 563.21581. Found: MNa⁺, 563.21531.

iert-Butyl (S)-3-(3-methoxy-2-(((4-nitrophenoxy)carbonyl)amino)-3oxopropyl)-1 H-indole-1 -carboxylate (42)

NBoc

Compound 41 (0.69 g, 1.3 mmol, 1 eq.) was treated with 20% v/v piperidine in MeCN (6.5 mL) for 1 h. The crude mixture was purified by column chromatography (eluent: 2:1 v/v hexane: EtOAc, 0.1% v/v diethylamine to neat EtOAc, 0.1% v/v diethylamine) to yield a yellow oil (0.27 g). This was redissolved in CH₂CI₂(6.6 mL) and formation of the carbamate was achieved through treatment with p-nitrophenyl chloroformate (0.21 g, 1 mmol, 1.2 eq.) in the presence of/Pr₂EtN (0.15 mL, 0.86 mmol, 1 eq.) and allowed to stir for 20 h. The crude residue was purified by column chromatography (eluent: 3:1 v/v hexane: EtOAc) to yield a white foam (0.37 g, 0.77 mmol, 60% over 2 steps).

IR (ATR): 3346, 2923, 1733, 1595, 1371 cm’¹; [αβ° = +70.3° (c = 0.3 in CH2CI2); ¹H NMR (300 MHz, CDCI3) δ 8.23-8.20 (m, 2H, Ar-H), 8.15-8.12 (1H, Ar-H), 7.55-7.49 (m, 2H, Ar-H), 7.37-7.32 (m, 1H, Ar-H), 7.24 (m, 3H, Ar-H), 5.91 (m, 1H, NH), 4.79-4.77 (m, 1H, a-CH), 3.78 (s, 3H, CH₃), 3.41-3.27 (m, 2H, p-CH₂), 1.69 (s, 9H, CO₂ftBu); ¹³CNMR (75.5 MHz, CDCI₃) δ 171.6, 155.6, 152.7, 149.6, 144.9, 135.4, 130.4, 125.1,

124.8, 124.3, 122.7, 122.0, 118.7, 115.5, 114.5, 84.0, 54.4, 52.8, 28.2; LRMS [M+Na]⁺506.13; HRMS Calcd for C₂4H₂₅N₃O₈: MNa⁺, 506.15393. Found: MNa⁺, 506.15349.

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3-ethoxy-3-oxopropanoic acid (43)

To a solution of diethyl malonate (3.1 mL, 20 mmol, 1 eq.) in 1:10 v/v THF: H₂O (400 mL) was added dropwise KOH(_aq) (64 mL, 0.25 M, 16 mmol, 0.8 eq.) at 0 °C. The reaction was allowed to stir for 1 h before being acidified with 1 M HCI(_aq) to pH 2 at 0 °C. The aqueous solution was then diluted with saturated aqueous NaCI solution (50 mL) and extracted with EtOAc (3 x 200 mL), dried (MgSO4) and concentrated in vacuo. The resulting residue was purified by column chromatography (2:1 v/v hexane: EtOAc) to yield 43 as a colourless oil (1.2 g, 8.9 mmol, 45%).

IR (ATR): 3000, 1714 cm’¹; ¹H NMR (300 MHz, CDCI3) δ 4.24 (q, J = 7.1 Hz, 2H, OCH2), 3.43 (s, 2H, CH2), 1.30 (t, J = 7.2 Hz, 3H, CH3); ¹³C NMR (75.5 MHz, CDCI3) δ

170.6, 167.2, 62.0, 40.6, 14.0; LRMS [M+H]⁺ 133.1. These data are in agreement with those reported by Niwayama, et al, Tetrahedron Lett. 2008, 49, 4434-4436.

Monoethyl magnesium malonate (44)

Mg

Compound 43 was added to a solution of magnesium ethoxide (0.47 g, 4.1 mmol, 1 eq.) in anhydrous THF (11 mL) and stirred for 2 h. The resulting yellow solution was concentrated in vacuo and used in the subsequent step without further purification. Procedure adapted from Kuhn, S. et al, J. Am. Chem. Soc. 2011, 133, 3708-3711.

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-(tert-Butyl) 6-ethyl (S)-2-((tert-butoxycarbonyl)amino)-4-oxohexanedioate (45)

To a solution of Boc-Asp-OtBu (2.2 g, 7.5 mmol, 1 eq.) in anhydrous THF (30 mL) was added carbonyldiimidazole (1.3 g, 8.3 mmol, 1.1 eq.) and stirred for 2 h. The reaction was then cooled to 0 °C before adding 44 in anhydrous THF (5.2 mL). The reaction was then allowed to warm to room temperature and stirred for 20 h before being diluted with H₂O (30 mL) and acidified to pH 2 with 1 M HCI(_aq). The aqueous solution was then extracted with Et₂O (3 x 30 mL). The organic phase was washed with sat. NaHCO_3(aq) (30 mL), dried (MgSO₄) and concentrated in vacuo. The resulting residue was purified by column chromatography (2:1 v/v hexane: EtOAc) to yield 45 as a yellow oil (1.9 g, 5.3 mmol, 71%).

IR (ATR): 2979, 2923, 2852, 1718 cm’¹; [αβ°= +17.3° (c = 0.1 in CH2CI2) ¹H NMR (500 MHz, CDCI3) δ 5.41 (d, J = 8.1 Hz, 1H, NH), 4.40-4.37 (m, 1H, a-CH),

4.19 (q, J= 7.2 Hz, 2H, OCH₂), 3.44 (s, 2H, CH₂), 3.21 (dd, J = 18.2, 4.4 Hz, 1H, βCH₂), 3.04 (dd, J = 18.1, 4.3 Hz, 1H, β-ΟΗ₂); 1.43 (s, 18H, 9 x CO₂f-Bu + 9 x Of-Bu),

1.27 (t, J = 7.1 Hz, 3H, CH₃); ¹³C NMR (75.5 MHz, CDCI₃) δ 200.7, 169.9, 166.5, 155.5,

82.2, 79.8, 61.4, 50.0, 49.2, 45.0, 28.3, 27.8, 14.0; LRMS [M+Na]⁺ 382.2. These data are in agreement with those reported by Brun, M. et al., Angew. Chem. Int. Ed. Engl. 2004, 43, 3432-3436.

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PCT/AU2017/051394 (S)-1-carboxy-2-(6,7-dimethoxy-2-oxo-2H-chromen-3-yl)ethan-1-aminium methanesulfonate (46)

© A

CH3SO3 (J OH

Compound 45 (1.5 g, 4.2 mmol, 1 eq.) was treated with 1:1 v/v TFA: CH2CI2 (14 mL) for 1 h at room temperature. The solvent was then removed in vacuo and azeotroped with toluene (3 x 20 mL) before the addition of 3,4-dimethoxyphenol (0.97 g,

6.3 mmol, 1.5 eq.). Methanesulfonic acid (6.8 mL, 100 mmol, 25 eq.) was then added at 0 °C and the reaction was subsequently allowed to warm to room temperature and stirred for 2 h. The product was precipitated out in diethyl ether and centrifuged at 3600 g for 20 min. The precipitate was redissolved in H₂O and lyophilised to yield a 46 as a blue oil (3.2 g, 8.1 mmol, quantitative).

IR (ATR): 2954, 2923, 1707, 1613 cm’¹; [αβ° = +0.9° (c = 0.2 in MeOH) ¹H NMR (300 MHz, (CH₃)₂SO) δ 8.42 (s, 3H, NH₃), 7.15-7.06 (m, 2H, Ar-H), 6.28 (s, 1H, H-3),

4.30-4.25 (m, 1H, a-CH), 3.84 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.36 (dd, J = 14.7,

6.4 Hz, 1H, 3-CH₂), 3.26 (dd, J = 14.7, 8.0 Hz, 1H, p-CH₂); ¹³C NMR δ 173.0, 160.7, 156.4, 153.3, 149.5, 146.4, 112.5, 111.5, 106.2, 100.9, 56.6, 56.5, 53.4, 47.0, 33.3; LRMS [M+H]⁺ 294.0. These data are in agreement with those reported by Brun, M. et al., Angew. Chem. Int. Ed. Engl. 2004, 43, 3432-3436.

(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(6,7-dimethoxy-2-oxo2H-chromen-3-yl)propanoic acid (47)

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To a solution of 46 (3.0 g, 7.7 mmol, 1 eq.) in 1.5:1 v/v THF: sat. NaHCO3(_aq) (77 mL) was added Fmoc-OSu (2.8 mg, 8.4 mmol, 1.1 eq.) and allowed to stir for 17 h. The reaction was then diluted with H₂O (80 mL) and washed with diethyl ether (3 x 80 mL). The aqueous phase was then acidified to pH 2 with 1 M HCI(_aq), extracted with EtOAc (3 x 100 mL), dried (MgSO₄) and concentrated in vacuo to yield 25 as a green powder (2.0 g, 3.8 mmol, 50%).

IR (ATR): 3368, 1701, 1614 cm’¹; [αβ° = +2° (c = 0.1 in CH2CI2) ¹H NMR (500 MHz, (CH3)₂SO) δ 7.88-7.86 (m, 2H, Ar-H), 7.63-7.60 (m, 2H, Ar-H), 7.40-7.38 (m, 2H, Ar-H), 7.29-7.25 (m, 3H, Ar-H), 7.07 (m, 1H, Ar-H), 6.24 (s, 1H, H-3), 4.37-4.32 (m, 1H, a-CH), 4.24-4.22 (m, 2H, Fmoc-CH₂), 4.16 (m, 1H, Fmoc-CH), 3.85 (s, 3H, OMe), 3.84 (s, 3H, OMe), 3.32 (dd, J = 14.5, 4.0 Hz, 1H, β-ΟΗ₂) 3.10 (dd, J = 14.4, 9.9 Hz, 1H, β-ΟΗ₂); ¹³C NMR δ 173.0, 160.7, 156.4, 153.3, 149.5, 146.4, 144.2, 144.1, 144.1, 144.1,

128.1, 127.5, 125.6, 125.6, 120.6, 112.5, 111.5, 106.2, 100.9, 66.2, 56.6, 56.5, 53.4, 47.0, 33.3, 25.7; LRMS [M+H]⁺ 516.3. These data are in agreement with those reported by Brun, M. et al., Angew. Chem. Int. Ed. Engl. 2004, 43, 3432-3436.

General Procedure 7.1

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 CH₂CI₂ (0.04 M) with shaking for 15 min. The resin was then washed with DMF (5x5 mL), CH₂CI₂ (5x5 mL) and DMF (5x5 mL). Complete removal of the Alloc-protecting group was determined through LCMS of the minicleavage solution.

General Procedure 7.2

Condition A:

Resin-bound pseudo peptide 51 - 54, 58 - 62 was activated with A/,A/-diispropylcarbodiimide (DIC) (0.62 - 1.3 mmol, 5 eq.) in anhydrous CH₂CI₂ (0.5 M) for 5 min with shaking prior to the addition of alcohol (0.62 - 1.3 mmol, 5 eq.) and 4dimethylaminopyridine (DMAP) (12-26 pmol, 0.1 eq) in anhydrous CH₂CI₂ (0.5 M). The resin was shaken for 16 - 20 h, followed by washing with DMF (5x5 mL), CH₂CI₂

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Condition B:

Resin-bound pseudo peptide 55 - 57, 63 - 65 was activated with Λ/,Λ/’-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 (5x5 mL), CH2CI2 (5x5 mL) and DMF (5x5 mL). Successful coupling was determined by LCMS of the minicleavage solution.

General Procedure 8

Condition C:

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 CH2CI2 (6x10 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.) was preactivated with NMM (46 - 65 pmol, 1.3 eq.) in 2:1 v/v CH2CI2: DMF (190 - 260 pL) for 15 min with shaking at rt. The solution was then added dropwise to a solution of the crude residue 51 - 65 (36 - 50 pmol, 1 eq.), uridyl amine 18 (0.11 - 0.15 mmol, 3 eq.) and HOAt (0.18 - 0.25 mmol, 5 eq.) in 1:2 v/v CH2CI2: DMF (190 - 260 pL) at 0 °C. 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 (11-16 mL), saturated aqueous NaHCO₃ solution (11 - 16 mL), H₂O (11 - 16 mL), brine (11 - 16 mL), and dried (MgSO₄). 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.% /Pr₃SiH (0.11 - 0.16 mL). The reaction was allowed to stir for 14 17 h and was then concentrated in vacuo. The resulting residue was redissolved in 1% TFA in H₂O (25 mL) and blown off under N₂(₉) flow. The resulting residue was then redissolved in 20 - 30% v/v MeCN in H₂O and purified by reverse-phase HPLC.

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Resin-bound pseudo peptide (48)

Amino acid 20 (240 mg, 0.55 mmol, 1.1 eq.) in CH2CI2 (0.1 M, 5.5 mL) with /Pr₂EtN (350 pL, 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, 11 mL) with HATU (420 mg, 1.1 mmol, 2 eq.) and /Pr₂EtN (290 pL, 1.7 mmol, 3 eq.) as described in general procedure 5 and 6. Subsequent Fmoc deprotection and coupling of Fmoc-Cha-OH (0.47 g, 1.2 mmol, 4 eq.) in DMF (0.1 M, 12 mL) with PyBOP (0.62 g,

1.2 mmol, 4 eq.) and NMM (0.27 mL, 2.4 mmol, 8 eq.) was achieved as described in general procedure 3 condition A. Following general procedure 4, crude allyl carbamate (0.18 g, 0.35 mmol, 2 eq.) with /Pr₂EtN (0.12 mL, 0.70 mmol, 4 eq.) in anhydrous DMF (0.1 M, 3.5 mL) was then coupled to the Fmoc deprotected resin bound tripeptide.

Allyl deprotection was achieved as described in general procedure 7.1 to yield the resinbound pseudo peptide 48.

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Resin-bound pseudo peptide (49)

H °^<5]A'NHBoc

Amino acid 20 (240 mg, 0.55 mmol, 1.1 eq.) in CH2CI2 (0.1 M, 5.5 mL) with /Pr₂EtN (350 pL, 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, 11 mL) with HATU (420 mg, 1.1 mmol, 2 eq.) and /Pr₂EtN (290 pL, 1.7 mmol, 3 eq.) as described in general procedure 5 and 6. Subsequent Fmoc deprotection and coupling of Fmoc-2-Nal-OH (0.46 g, 1.1 mmol, 4 eq.) in DMF (0.1 M, 11 mL)with PyBOP (0.54 g,

1.1 mmol, 4 eq.) and NMM (0.23 mL, 2.1 mmol, 8 eq.) was achieved as described in general procedure 3 condition A. Following general procedure 4, crude allyl carbamate 40 (0.18 g, 0.35 mmol, 2 eq.) with /Pr₂EtN (0.12 mL, 0.70 mmol, 4 eq.) in anhydrous DMF (0.1 M, 3.5 mL) was then coupled to the Fmoc deprotected resin bound tripeptide. Allyl deprotection was achieved as described in general procedure 7.1 to yield the resinbound pseudo peptide 49.

Resin-bound pseudopeptide (50)

Amino acid 20 (240 mg, 0.55 mmol, 1.1 eq.) in CH2CI2 (0.1 M, 5.5 mL) with /Pr₂EtN (350 pL, 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

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PCT/AU2017/051394 deprotection and coupling of Boc-Gly-OH (190 mg, 1.1 mmol, 2 eq.) in DMF (0.1 M, 11 mL) with HATU (420 mg, 1.1 mmol, 2 eq.) and /Pr₂EtN (290 pL, 1.7 mmol, 3 eq.) as described in general procedure 5 and 6. A solution of compound 47 (0.13 g, 0.25 mmol,

1.5 eq.), HATU (95 mg, 0.25 mmol, 1.5 eq.), HOAt (34 mg, 0.25 mmol, 1.5 eq.) and /Pr₂EtN (89 pL, 0.51 mmol, 3 eq.) in anhydrous DMF (0.1 M, 1.7 mL) was added to the Fmoc deprotected resin-bound isopeptide and shaken for 2 h. The resin was washed with DMF (5x5 mL), CH₂CI₂(5 x 5 mL) and DMF (5x5 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), CH₂CI₂(5 x 5 mL) and DMF (5x5 mL). Following general procedure 4, crude allyl carbamate 40 (0.18 g, 0.35 mmol, 2 eq.) with /Pr₂EtN (0.12 mL, 0.70 mmol, 4 eq.) in anhydrous DMF (0.1 M, 3.5 mL) was then coupled to the Fmoc deprotected resin bound tripeptide. Allyl deprotection was achieved as described in general procedure 7.1. The resin-bound pseudo peptide was then preactivated with DIC (150 pL, 0.96 mmol, 5 eq.) in CH₂CI₂ (1.9 mL) prior to the addition of benzyl alcohol (99 pL, 0.96 mmol, 5 eq.) and DMAP (2.3 mg, 19 pmol, 0.1 eq.) in CH₂CI₂ (1.9 mL) according to general procedure 7.2 condition A to yield the resin-bound pseudo peptide 50

Resin-bound pseudo peptide (51)

Resin-bound pseudo peptide 48 was preactivated with DIC (150 pL, 0.97 mmol, 5 eq.) in CH₂CI₂ (1.9 mL) prior to the addition of methanol (39 pL, 0.97 mmol, 5 eq.) and DMAP (2.4 mg, 19 pmol, 0.1 eq.) in CH₂CI₂ (1.9 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 51.

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Resin-bound pseudo peptide (52)

Resin-bound pseudo peptide 48 was preactivated with DIC (140 pL, 0.88 mmol, 5 eq.) in CH2CI2 (1.8 mL) prior to the addition of hexanol (110 pL, 0.88 mmol, 5 eq.) and

DMAP (2.14 mg, 17.5 pmol, 0.1 eq.) in CH₂CI₂ (1.74 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 52.

Resin-bound pseudo peptide (53)

Resin-bound pseudo peptide 48 was preactivated with DIC (110 pL, 0.69 mmol, 10 5 eq.) in CH₂CI₂ (1.4 mL) prior to the addition of dodecanol (150 pL, 0.69 mmol, 5 eq.) and DMAP (1.7 mg, 14 pmol, 0.1 eq.) in CH₂CI₂ (1.4 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 53.

Resin-bound pseudo peptide (54)

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Resin-bound pseudo peptide 48 was preactivated with DIC (96 pL, 0.62 mmol, 5 eq.) in CH2CI2 (1.2 mL) prior to the addition of benzyl alcohol (64 pL, 0.62 mmol, 5 eq.) and DMAP (1.5 mg, 12 pmol, 0.1 eq.) in CH2CI2 (1.2 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 54.

Resin-bound pseudo peptide (55)

Resin-bound pseudo peptide 48 was preactivated with DIC (150 pL, 0.98 mmol, 5 eq.) in CH2CI2 (2 mL) prior to the addition of hexyl amine (52 pL, 0.39 mmol, 2 eq.) in CH2CI2 (2 mL) according to general procedure 7.2 condition B to yield the resin bound 10 pseudo peptide 55.

Resin-bound pseudo peptide (56)

H ‘G'GhBoc

Resin-bound pseudo peptide 48 was preactivated with DIC (83 pL, 0.54 mmol, 5 eq.) in CH2CI2 (1.1 mL) prior to the addition of neopentyl amine (25 pL, 0.21 mmol, 2 eq.) in CH2CI2 (1.1 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 56.

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Resin-bound pseudo peptide (57)

H θΥ^ΝΗΒοο

Resin-bound pseudo peptide 48 was preactivated with DIC (90 pL, 0.58 mmol, 5 eq.) in CH2CI2 (1.2 mL) prior to the addition of benzyl amine (25 pL, 0.23 mmol, 2 eq.) in CH₂CI₂ (1.2 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 57.

Resin-bound pseudo peptide (58)

H θΥ^ΝΗΒοο

Amino acid 20 (240 mg, 0.55 mmol, 1.1 eq.) in CH₂CI₂ (0.1 M, 5.5 mL) with /Pr₂EtN (350 pL, 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, 11 mL) with HATU (420 mg, 1.1 mmol, 2 eq.) and /Pr₂EtN (290 pL, 1.7 mmol, 3 eq.) as described in general procedure 5 and 6. Subsequent Fmoc deprotection and coupling of Fmoc-2-Nal-OH (0.46 g, 1.1 mmol, 4 eq.) in DMF (0.1 M, 11 mL)with PyBOP (0.54 g,

1.1 mmol, 4 eq.) and NMM (0.23 mL, 2.1 mmol, 8 eq.) was achieved as described in general procedure 3 condition A. Following general procedure 4, allyl carbamate 42 (78 mg, 0.16 mmol, 1.9 eq.) with /Pr₂EtN (60 pL, 0.35 mmol, 4 eq.) in anhydrous DMF (0.1 M, 1.6 mL) was then coupled to the Fmoc deprotected resin bound tripeptide to yield the pseudo peptide 58.

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Resin-bound pseudo peptide (59)

Resin-bound pseudo peptide 49 was preactivated with DIC (190 pL, 1.3 mmol, 5 eq.) in CH2CI2 (2.5 mL) prior to the addition of hexanol (160 pL, 1.3 mmol, 5 eq.) and 5 DMAP (3.1 mg, 25 pmol, 0.1 eq.) in CH2CI2 (2.5 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 59.

Resin-bound pseudo peptide (60)

H °Y^NHBoc

Resin-bound pseudo peptide 49 was preactivated with DIC (200 pL, 1.3 mmol, 5 eq.) in CH2CI2 (2.6 mL) prior to the addition of dodecanol (290 pL, 1.3 mmol, 5 eq.) and

DMAP (3.2 mg, 26 pmol, 0.1 eq.) in CH2CI2 (2.6 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 60.

Resin-bound pseudo peptide (61)

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Resin-bound pseudo peptide 49 was preactivated with DIC (130 pL, 0.81 mmol, eq.) in CH2CI2 (1.6 mL) prior to the addition of neopentyl alcohol (88 pL, 0.81 mmol, 5 eq.) and DMAP (2.0 mg, 16 pmol, 0.1 eq.) in CH2CI2 (1.6 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 61.

Resin-bound pseudo peptide (62)

Resin-bound pseudo peptide 49 was preactivated with DIC (160 pL, 1.1 mmol, 5 eq.) in CH2CI2 (2.1 mL) prior to the addition of benzyl alcohol (110 pL, 1.1 mmol, 5 eq.) and DMAP (2.6 mg, 21 pmol, 0.1 eq.) in CH2CI2 (2.1 mL) according to general 10 procedure 7.2 condition A to yield the resin bound pseudo peptide 62.

Resin-bound pseudo peptide (63)

Resin-bound pseudo peptide 49 was preactivated with DIC (150 pL, 0.98 mmol, 5 eq.) in CH2CI2 (2 mL) prior to the addition of hexyl amine (52 pL, 0.39 mmol, 2 eq.) in

CH2CI2 (2 mL) according to general procedure 7.2 condition B to yield the resin bound pseudo peptide 63.

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Resin-bound pseudo peptide (64)

Resin-bound pseudo peptide 49 was preactivated with DIC (85 pL, 0.55 mmol, 5 eq.) in CH2CI2 (1.1 mL) prior to the addition of neopentyl amine (26 pL, 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 (65)

Resin-bound pseudo peptide 49 was preactivated with DIC (130 pL, 0.8 mmol, 5 eq.) in CH2CI2 (1.6 mL) prior to the addition of benzyl amine (40 pL, 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.

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Methyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (66)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 51 (32 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 66. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 98:2 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 66 as a white fluffy solid (8.0 mg, 8.8 pmol, 22%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3279, 2926, 1680 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.90 (s, 1H, Trp-NH), 8.56 (t, J = 5.5 Hz, 0.5H, CONHCH₂), 8.23 (d, J = 9.0 Hz, 0.5H, CONH), 8.17 (d, J = 9.1 Hz, 0.5H, CONH), 8.08 (t, J = 5.3 Hz, 0.5H, CONHCH₂), 8.02 (br s, 3H, NH₃), 7.53 (d, J = 8.1 Hz, 0.5H, H-6),

7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.45-7.43 (m, 1H, Ar-H), 7.34-7.33 (m, 1H, Ar-H), 7.12-7.11 (m, 1H, Ar-H), 7.07-7.04 (m, 1H, Ar-H), 6.99-6.96 (m, 1H, Ar-H), 6.38 (d, J = 8.0 Hz, 1H, NHCONH), 6.34-6.31 (m, 1H, NHCONH), 5.71 (d, J = 3.4 Hz, 0.5H, Η-Γ),

5.66 (d, J = 3.4 Hz, 0.5H, Η-Γ), 5.61-5.58 (m, 1H, H-5), 4.82-4.76 (m, 0.5H, DABA-βCH), 4.58 (t, J = 9.2 Hz, 0.5H, DABA-a-CH), 4.49 (t, J = 9.2 Hz, 0.5H, DABA-a-CH),

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4.42-4.36 (m, 1.5H, 1 x Trp-a-CH + 0.5 x H-4’), 4.35-4.30 (m, 1H, H-2’), 4.28-4.25 (m, 0.5H, H-4’), 4.16-4.12 (m, 1H, Cha-a-CH), 3.94-3.77 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x DABA-β-ΟΗ), 3.59-3.57 (m, 0.5H, Gly-a-CH₂), 3.55 (s, 3H, OCH₃), 3.28-3.22 (m, 1H, H5’), 3.18-3.13 (m, 1H, H-5’), 3.06-3.05 (m, 2H, ΤΓρ-β-ΟΗ₂), 2.77 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.21-2.10 (m, 1H, H-3’), 1.69-1.60 (m, 6H, 1 x H-3’+ 5 x Cy), 1.41-1.28 (m,2H, Cha^-CH₂), 1.23 (m, 1H, Cy), 1.15-1.08 (m, 3.5H, 1.5 x CH₃ + 2 x Cy), 1.02 (d, J= 7.1 Hz, 1.5H, CH₃), 0.87-0.76 (m, 3H, Cy); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.8, 173.5, 170.4, 170.0, 166.7, 166.3, 163.9, 163.8, 158.6, 158.4, 158.1, 157.7, 157.6, 151.2, 141.7, 141.5, 136.7, 127.8, 124.5, 121.6, 119.0,

118.7, 112.0, 109.6, 102.3, 102.0, 92.6, 92.5, 79.2, 74.6, 74.5, 55.4, 55.0, 54.2, 53.4,

52.2, 51.7, 51.0, 44.4, 44.3, 36.2, 36.0, 34.1, 33.8, 32.8, 32.7, 29.4, 28.3, 27.8, 26.6,

26.5, 26.3, 15.2, 14.4; LRMS [M+H]⁺ 796.7; HRMS Calcd for C₃8H₅₃N₉Oio: MH⁺, 796.39882. Found: MH⁺, 796.39969.

Hexyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (67)

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 pl_, 46 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (180 pL) was added dropwise to the crude residue 52 (30 mg, 35 pmol, 1 eq.), uridyl amine 18 (24 mg, 110 pmol, 3 eq.) and HOAt (24 mg, 180 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (180 pL) according to general procedure 8 condition C, yielding the protected analogue 67.

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Deprotection was achieved through treatment with 1:1 v/v CH2CI2: TFA (4.4 mL) and 2.5 vol.% /Pr₃SiH (0.11 mL) to afford a 98:2 mixture of separable diastereoisomers.

Purification by reverse-phase HPLC yielded 67 as a white fluffy solid (9.3 mg, 9.5 pmol,

27%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3306, 2925, 2854, 1664, 1555 cm’¹; (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.56 (t, J = 5.5 Hz, 0.5H, CONHCH2), 8.23 (d, J = 9.0 Hz, 0.5H, CONH), 8.18 (d, J = 9.1 Hz, 0.5H, CONH), 8.08 (t, J = 5.3 Hz, 0.5H, CONHCH₂), 8.01 (br s, 3H, NH₃), 7.53 (d, J = 8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.45-7.44 (m, 1H, Ar-H), 7.34-7.32 (m, 1H, Ar-H), 7.12-7.11 (m, 1H, Ar-H), 7.07-7.04 (m, 1H, Ar-H), 6.98-6.95 (m, 1H, Ar-H), 6.37 (d, J = 8.0 Hz, 1H, NHCONH), 6.35-6.32 (m, 1H, NHCONH), 5.70 (d, J = 3.5 Hz, 0.5H, H-1’), 5.66 (d, J = 3.5 Hz, 0.5H, H-1’), 5.61-5.58 (m, 1H, H-5), 4.82-4.76 (m, 0.5H, ΟΑΒΑ-β-CH), 4.59 (t, J = 9.2 Hz, 0.5H, DABA-a-CH), 4.49 (t, J = 9.2 Hz, 0.5H, DABA-a-CH), 4.41-4.36 (m, 1.5H, 1 x Trp-a-CH + 0.5 x H-4’), 4.35-4.30 (m, 1H, H-2’),

4.28- 4.25 (m, 0.5H, H-4’), 4.16-4.12 (m, 1H, Cha-a-CH), 3.92-3.77 (m, 4H, 1.5 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH + 2 x OCH₂(CH₂)₄CH₃), 3.59-3.54 (m, 0.5H, Gly-a-CH₂),

3.28- 3.21 (m, 1H, H-5’), 3.18-3.13 (m, 1H, H-5’), 3.04 (d, J = 6.4 Hz, 2H, Τφ-β-0Η₂),

2.77 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.21-2.10 (m, 1H, H-3’), 1.69-1.60 (m, 6H, 1 x H-3’ + 5 x Cy), 1.44-1.38 (m, 2H, OCH₂(CH₂)₄CH₃), 1.35-1.31 (m, 2H, Cha^-CH₂), 1.24-1.08 (m, 10.5H, 3 x Cy + 1.5 CH₃, 6 x OCH₂(CH₂)₄CH₃), 1.02 (d, J = 6.8 Hz, 1,5H, CH₃), 0.87-0.77 (m, 6H, 3 x Cy + 3 x OCH₂(CH₂)₄CH₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.5, 173.4, 170.4, 170.0, 166.7, 166.3, 163.9, 163.8,

158.8, 158.6, 158.4, 158.1, 157.7, 157.6, 151.2, 141.7, 141.5, 136.7, 127.8, 124.3,

121.5, 119.0, 118.7, 116.6, 112.0, 109.7, 102.3, 102.0, 92.6, 92.5, 79.2, 74.6, 74.5,

64.8, 55.4, 55.0, 54.3, 53.3, 51.7, 44.4, 44.3, 36.2, 36.0, 34.0, 33.9, 33.8, 32.8, 32.7,

31.4, 29.4, 28.5, 28.4, 27.8, 26.6, 26.5, 26.3, 25.5, 22.5, 15.2, 14.5, 14.4; LRMS [M+H]⁺866.8; HRMS Calcd for C₄₃H₆₃N₉Oio: MH⁺, 866.47707. Found: MH⁺, 866.47737.

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Dodecyl (((S)-1 -(((2S,3S)-3-(2-amino-/V-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (68)

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 pl_, 48 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (190 pL) was added dropwise to the crude residue 53 (35 mg, 37 pmol, 1 eq.), uridyl amine 18 (25 mg, 110 pmol, 3 eq.) and HOAt (25 mg, 190 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (190 pL) according to general procedure 8 condition C, yielding the protected analogue 68. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 97:3 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 68 as a white fluffy solid (12 mg, 12 pmol, 29%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3310, 2924, 2853, 1671, 1556 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.30 (s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.57 (t, J =5.5 Hz, 0.5H, CONHCH₂), 8.24 (d, J = 9.1 Hz, 0.5H, CONH), 8.19 (d, J = 9.1 Hz, 0.5H, CONH), 8.09 (t, J = 5.1 Hz, 0.5H, CONHCH₂), 8.01 (br s, 3H, NH₃), 7.53 (d, J =

8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.45-7.43 (m, 1H, Ar-H), 7.34-7.32 (m, 1H, Ar-H) 7.11-7.10 (m, 1H, Ar-H), 7.07-7.03 (m, 1H, Ar-H), 6.98-6.95 (m, 1H, Ar-H),

6.56, (s, 1H, OH), 6.34-6.31 (m, 2H, NHCONH), 5.71 (d, J = 3.4 Hz, 0.5H, H-1’), 5.66 (d, J = 3.3 Hz, 0.5H, H-T), 5.61-5.58 (m, 1H, H-5), 4.82-4.75 (m, 0.5H, ΟΑΒΑ-β-CH),

4.57 (t, J = 9.5 Hz, 0.5H, DABA-a-CH), 4.49 (t, J = 9.1 Hz, 0.5H, DABA-a-CH) 4.41-4.35 (m, 1.5H, 1 x Trp-a-CH + 0.5 x H-4’), 4.32-4.30 (m, 1H, H-2’), 4.28-4.24 (m, 0.5H, H-4’)

4.15-4.12 (m, 1H, Cha-a-CH), 3.92-3.77 (m, 4H, 1.5 x Gly-a-CH₂ + 2 x OCH₂(CH₂)i₀CH₃

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136.7, 127.8, 124.4, 121.5, 118.9, 118.7, 116.6, 112.0, 109.7, 102.3, 102.0, 92.6, 92.5,

79.2, 74.6, 74.5, 64.8, 55.4, 55.0, 54.3, 53.3, 51.7, 51.0, 44.4, 44.3, 36.2, 36.0,34.1,

33.9, 33.8, 32.9, 32.8, 31.9, 29.6, 29.6, 29.6, 29.5, 29.3, 29.2, 28.6, 28.5, 26.6,26.5,

26.3, 25.8, 22.7, 15.1, 14.6; LRMS [M+H]⁺951.0; HRMS Calcd for C49H75N9O10:MH

950.57097. Found: MH⁺, 950.57081.

Benzyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (69)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 54 (35 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 69. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 97:3 mixture of separable diastereoisomers.

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Purification by reverse-phase HPLC yielded 69 as a white fluffy solid (12 mg, 12 pmol,

29%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3316, 2926, 2851, 1665, 1552 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.90 (s, 1H, Trp-NH), 8.55 (t, J =5.2 Hz, 0.5H, CONHCH₂), 8.24 (d, J = 9.0 Hz, 0.5H, CONH), 8.19 (d, J = 8.9 Hz, 0.5H, CONH), 8.07 (t, J = 5.4 Hz, 0.5H, CONHCH₂), 8.00 (br s, 3H, NH₃), 7.53 (d, J =

8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.49-7.44 (m, 1H, Ar-H), 7.36-7.34 (m, 1H, Ar-H), 7.32-7.30 (m, 3H, Ar-H) 7.19-7.17 (m, 2H, Ar-H), 7.10-7.09 (m, 1H, Ar-H), 7.07-7.05 (m, 1H, Ar-H), 6.99-6.95 (m, 1H, Ar-H), 6.40-6.36 (m, 2H, NHCONH), 5.71 (d, J = 3.4 Hz, 0.5H, H-1’), 5.66 (d, J = 3.6 Hz, 0.5H, H-1’), 5.61-5.57 (m, 1H, H-5), 5.00 (q, J = 12.7 Hz, 2H, OC/-/₂(C₆H₅)), 4.82-4.75 (m, 0.5H, ΟΑΒΑ-β-CH), 4.58 (t, J = 9.6 Hz, 0.5H, DABA-a-CH), 4.52-4.43 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH) 4.40-4.36 (m, 0.5H, H-4’), 4.33-4.31 (m, 1H, H-2’), 4.29-4.24 (m, 0.5H, H-4’) 4.18-4.13 (m, 1H, Cha-a-CH), 3.95-3.76 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH), 3.59-3.53 (m, 0.5H, Gly-a-CH₂), 3.29-3.21 (m, 1H, H-5’), 3.19-3.12 (m, 1H, H-5’), 3.08 (d, J = 6.4 Hz, 2H, ΤΓρ-β-ΟΗ₂), 2.77 (s, 1.5H, NCH₃), 2.74 (s, 1.5H, NCH₃), 2.21-2.09 (m, 1H, H-3’), 1.69-1.60 (m, 6H, 1 x H-3’ + 5 x Cy), 1.39-1.29 (m, 2H, Cha^-CH₂), 1.28-1.22 (m, 1H, Cy), 1.13-1.07 (m, 3.5H, 2 x Cy + 1.5 x CH₃), 1.02 (d, J = 6.9 Hz, 1.5H, CH₃), 0.87-0.78 (m, 3H, Cy); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.3, 173.2,

170.3, 169.9, 166.5, 166.2, 163.7, 163.7, 158.5, 158.1, 157.6, 157.5, 151.0, 141.5,

141.3, 136.6, 136.3, 128.8, 128.4, 128.1, 127.6, 124.3, 121.4, 118.9, 118.6, 111.9,

109.5, 102.2, 101.9, 92.5, 92.3, 79.0, 74.5, 74.4, 66.2, 55.2, 54.9, 54.3, 53.2, 51.6, 44.2, 44.2, 36.0, 35.9, 33.9, 33.6, 32.7, 32.6, 29.2, 28.3, 27.7, 26.5, 26.3, 26.1, 15.0, 14.3; LRMS [M+H]⁺ 872.8; HRMS Calcd for C44H57N9O10: MH⁺, 872.43012. Found: MH⁺, 872.43013.

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PCT/AU2017/051394 (2S,3S)-3-(2-amino-A/-methylacetamido)-2-((S)-3-cyclohexyl-2-(3-((S)-1(hexylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl)ureido)propanamido)-A/(((2/?,4/?,5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)butanamide (70)

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 pL, 52 pmol, 1.3 eq.) in 2:1 ν/ν CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 55 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 70. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 99:1 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 70 as a white fluffy solid (18 mg, 19 pmol, 46%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3293, 2926, 2853, 1650, 1554 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO) δ , ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.79 (s, 1H, Trp-NH), 8.56 (t, J =5.5 Hz, 0.5H, CONHCH₂), 8.24 (d, J = 9.0 Hz, 0.5H, CONH), 8.13-8.09 (m, 1H, 0.5 x CONH + 0.5 x CONHCH₂), 8.05 (br s, 3H, NH₃), 7.75-7.72 (m, 1H,

CONHCH₂(CH₂)₄CH₃), 7.55-7.52 (m, 1.5H, 1 x H-6 + 0.5 x Ar-H), 7.52-7.50 (m, 0.5H, Ar-H), 7.32-7.30 (m, 1H, Ar-H), 7.08-7.02 (m, 2H, Ar-H), 6.97-6.93 (m, 1H, Ar-H), 6.35-

6.33 (m, 1H, NHCONH), 6.24-6.22 (m, 1H, NHCONH), 5.71 (d, J = 3.7 Hz, 0.5H, Η-Γ),

5.67 (d, J = 3.4 Hz, 0.5H, Η-Γ), 5.62-5.58 (m, 1H, H-5), 4.83-4.76 (m, 0.5H, DABA-βCH), 4.59 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.51 (t, J = 9.3 Hz, 0.5H, DABA-a-CH), 4.41-4.25 (m, 3H, 1 x Trp-a-CH + 1 x H-4’ + 1 x H-2’), 4.17-4.11 (m, 1H, Cha-a-CH),

3.90-3.77 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH), 3.58-3.55 (m, 0.5H,

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Gly-a-CH₂), 3.30-3.24 (m, 1H, H-5’), 3.18-3.12 (m, 1H, H-5’), 3.03-2.88 (m, 4H, 2 x ΤΓρ-β-0Η_{2 +} 2 x CONHCH₂(CH₂)₄CH₃), 2.76 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.222.09 (m, 1 Η, H-3’), 1.69-1.60 (m, 6H, 1 x H-3’ + 5 x Cy), 1.44-1.29 (m, 2H, Cha^-CH₂),

1.28-1.12 (m, 12.5H, 3 x Cy + 1.5 x CH₃ + 8 x CONHCH₂(CH₂)₄CH₃), 1.03 (d, J = 6.9 Hz, 1.5H, CH₃), 0.89-0.70 (m, 6H, 3 x Cy + 3 x CONHCH₂(CH₂)₄CH₃); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.4, 172.3, 170.3, 169.9, 166.5, 166.1,

163.7, 163.7, 158.9, 158.6, 158.3, 157.6, 157.6, 151.0, 141.5, 141.4, 136.5, 128.0,

123.9, 121.2, 119.2, 119.0, 118.6, 116.2, 111.6, 110.5, 102.2, 101.9, 92.5, 92.3, 79.1,

74.5, 74.4, 55.2, 54.8, 54.5, 53.2, 51.7, 51.6, 50.9, 44.2, 44.1, 36.0, 35.9, 33.9, 33.7,

32.7, 32.6, 31.4, 29.3, 29.2, 27.7, 26.5, 26.4, 26.3, 26.1, 22.5, 15.0, 14.4, 14.2; LRMS [M+H]⁺865.8;HRMS Calcd for C43H64N10O9: MH⁺, 865.49305. Found: MH⁺, 865.49207.

(2S,3S)-2-((S)-2-(3-((S)-3-(1 H-indol-3-yl)-1 -(neopentylamino)-l -oxopropan-2yl)ureido)-3-cyclohexylpropanamido)-3-(2-amino-A/-methylacetamido)-A/(((2R, 4R, 5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)butanamide (71)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 56 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 71. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 98:2 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 71 as a white fluffy solid (20 mg, 21 pmol, 53%, yield calculated as TFA salt after HPLC purification).

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IR (ATR): 3305, 2925, 1654, 1551 cm’¹;¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.80 (s, 1H, Trp-NH), 8.56 (t, J =5.5 Hz, 0.5H, CONHCH₂), 8.18 (d, J = 9.3 Hz, 0.5H, CONH), 8.12 (d, J = 9.3 Hz, 0.5H, CONH), 8.09-8.05 (m, 3.5H, 3 x NH₃ + 0.5 x CONHCH₂), 7.65-7.61 (m, 1H, CONHCH₂C(CH₃)₃), 7.57-7.50 (m, 2H, 1 x H-6 + 1 x Ar-H), 7.33-7.30 (m, 1H, Ar-H),

7.11-7.10 (m, 1H, Ar-H), 7.07-7.03 (m, 2H, Ar-H), 6.98-6.94 (m, 1H, Ar-H), 6.34-6.27 (m, 2H, NHCONH), 5.71 (d, J = 3.7 Hz, 0.5H, H-1 ’), 5.67 (d, J = 3.5 Hz, 0.5H, H-1 ’), 5.62-5.58 (m, 1H, H-5), 4.82-4.75 (m, 0.5H, ΟΑΒΑ-β-CH), 4.57 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.49 (t, J = 9.2 Hz, 0.5H, DABA-a-CH), 4.44-4.38 (m, 1H, Trp-a-CH),

4.36-4.24 (m, 2H, 1 x H-4’ + 1 x H-2’), 4.17-4.12 (m, 1H, Cha-a-CH), 3.93-3.75 (m, 2H,

1.5 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH), 3.59-3.53 (m, 0.5H, Gly-a-CH₂), 3.29-3.22 (m, 1H, H-5’), 3.18-3.11 (m, 1H, H-5’), 3.02 (dd, J= 14.6, 6.3 Hz, 1H, ΤΓρ-β-ΟΗ₂), 2.93-2.85 (m, 2H, 1 x ΤΓρ-β-ΟΗ₂ + 1 x CONHCH₂C(CH₃)₃), 2.80 (dd, J = 13.2, 6.3 Hz, 1H, CONHCH₂C(CH₃)₃), 2.75 (s, 1.5H, NCH₃), 2.74 (s, 1.5H, NCH₃), 2.22-2.10 (m, 1H, H3’), 1.69-1.60 (m, 6H, 1 x H-3’ + 5 x Cy), 1.43-1.30 (m, 2H, Cha^-CH₂), 128-1.25 (m, 1H, 1 x Cy) 1.16-1.10 (m, 3.5H, 2 x Cy + 1.5 x CH₃), 1.00 (d, J= 6.9 Hz, 1.5H, CH₃), 0.92-0.82 (m, 2H, Cy), 0.76 (s, 10H, 9 x CONHCH₂C(CH₃)₃ + 1 x Cy); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.4, 172.8, 170.3, 169.9, 166.5, 166.1,

163.7, 163.7, 159.0, 158.7, 158.4, 158.1, 157.7, 157.6, 151.0, 141.5, 141.4, 136.5,

127.9, 123.9, 121.8, 121.2, 119.0, 118.8, 118.6, 115.8, 112.9, 111.6, 110.6, 102.2,

101.9, 92.5, 92.3, 79.1, 74.5, 74.4, 55.2, 54.9, 54.5, 53.2, 51.6, 51.5, 50.9, 50.1, 44.2,

44.1, 36.0, 35.9, 33.9, 33.7, 32.6, 32.4, 29.2, 29.0, 27.6, 26.5, 26.3, 26.1, 15.0, 14.2; LRMS [M+H]⁺ 851.8; HRMS Calcd for C42H62N10O9: MH⁺, 851.47740. Found: MH⁺, 851.7703.

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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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 57 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 72. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 99:1 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 72 as a white fluffy solid (13 mg, 14 pmol, 34%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3292, 2924, 1672, 1550 cm’¹;¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.82 (s, 1H, Trp-NH), 8.55 (t, J =5.3 Hz, 0.5H, CONHCH₂), 8.34-8.31 (m, 1H, CONHCH₂(C₆H₅)), 8.19 (d, J = 9.2 Hz, 0.5H, CONH), 8.14 (d, J = 9.2 Hz, 0.5H, CONH), 8.09-8.03 (m, 3.5H, 3 x NH₃ + 0.5 CONHCH₂), 7.58-7.49 (m, 2H, 1 x Ar-H + 1 x H-6), 7.35-7.30 (m, 1H, Ar-H), 7.25-7.17 (m, 3H, Ar-H), 7.08-7.03 (m, 4H, Ar-H), 6.98-6.94 (m, 1H, Ar-H), 6.35-6.33 (m, 1H, NHCONH), 6.30-6.27 (m, 1H, NHCONH), 5.70 (d, J = 3.4 Hz, 0.5H, H-T), 5.66 (d, J =3.6 Hz, 0.5H, H-T), 5.61-5.57 (m, 1H, H-5), 4.83-4.75 (m, 0.5H, ϋΑΒΑ-β-CH), 4.58 (t, J = 9.5 Hz, 0.5H, DABA-a-CH), 4.50 (t, J = 9.2 Hz, 0.5H, DABA-a-CH), 4.44-4.36 (m, 1H, Trp-a-CH), 4.32-4.26 (m, 2H, 1 x H-4’ + 1 x H-2’), 4.24-4.20 (m, 2H, CONHCH₂(C₆H₅)), 4.17-4.12 (m, 1H, Cha-a-CH), 3.88-3.76 (m, 2H, 1.5 x Gly-a-CH₂ +

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0.5 x ΟΑΒΑ-β-CH), 3.58-3.54 (m, 0.5H, Gly-a-CH₂), 3.29-3.22 (m, 1H, H-5’), 3.17-3.11 (m, 1H, H-5’), 3.04 (dd, J = 14.5, 6.6 Hz, 1 Η, ΤΓρ-β-ΟΗ₂), 2.95 (dd, J = 14.3, 6.7 Hz, 1H, ΤΓρ-β-ΟΗ₂), 2.75 (s, 1.5H, NCH₃), 2.74 (s, 1.5H, NCH₃), 2.21-2.19 (m, 1H, H-3’), 1.68-1.58 (m, 6H, 1 x H-3’ + 5 x Cy), 1.44-1.29 (m, 2H, Cha^-CH₂), 128-1.23 (m, 1H, 1 x Cy) 1.16-1.09 (m, 3.5H, 2 x Cy + 1.5 x CH₃), 1.02 (d, J= 6.9 Hz, 1.5H, CH₃), 0.880.76 (m, 3H, Cy); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.4,

172.7, 170.3, 169.9, 166.5, 166.1, 163.7, 158.6, 158.3, 157.6, 151.0, 141.5, 141.4,

139.7, 136.6, 128.6, 127.9, 127.4, 127.0, 124.1, 121.3, 119.1, 118.7, 116.3, 111.7,

110.4, 102.2, 101.9, 92.5, 92.3, 79.1, 74.5, 74.4, 55.2, 54.6, 53.2, 51.7, 50.9, 44.2, 44.1,

42.5, 36.0, 35.9, 33.9, 33.7, 32.7, 32.6, 29.3, 27.7, 26.5, 26.3, 26.1, 15.0, 14.2; LRMS [M+H]⁺871.8; HRMS Calcd for C44H58N10O9: MH⁺, 871.44610. Found: MH⁺, 871.44632.

Methyl (((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-(naphthalen-2-yl)-1-oxopropan-2-yl)carbamoyl)L-tryptophanate (73)

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 pL, 65 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (160 pL) 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 CH₂CI₂: DMF (161 pL) according to general procedure 8 condition B, yielding the protected analogue 73. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (6.5 mL) and 2.5 vol.% /Pr₃SiH (0.17 pL) to afford a 97:3 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 73 as a single diastereoisomer as a white fluffy solid (13 mg, 14 pmol, 27%, yield calculated as TFA salt after HPLC purification).

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IR (ATR): 3213, 1673, 1549, 1440 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.61 (t, J = 5.1 Hz, 0.5H, CONHCH₂), 8.43 (d, J = 9.1 Hz, 0.5H, CONH), 8.38 (d, J = 9.1 Hz, 0.5H, CONH), 8.13 (t, J = 5.1 Hz, , 0.5H, CONHCH₂), 8.05 (br s, 3H, NH₃), 7.84-7.83 (m, 1H, Ar-H), 7.77-7.75 (m, 2H, Ar-H), 7.62 (m, 1H, Ar-H), 7.55 (d, J = 8.1 Hz, 0.5H, H6), 7.51 (d, J = 8.1 Hz, 0.5H, H-6), 7.48-7.42 (m, 3H, Ar-H), 7.33-7.30 (m, 2H, Ar-H),

7.11-7.04 (m, 2H, Ar-H), 6.98-6.95 (m, 1H, Ar-H), 6.56-6.54 (m, 1H, NHCONH), 6.43-

6.39 (m, 1H, NHCONH), 5.72 (d, J = 3.6 Hz, 0.5H, H-1’), 5.69 (d, J = 3.4 Hz, 0.5H, H1’), 5.60-5.57 (m, 1H, H-5), 4.81-4.78 (m, 0.5H, ΟΑΒΑ-β-CH), 4.61 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.54-4.48 (m, 1.5H, 1 x DABA-a-CH + 1 x Trp-a-CH), 4.41-4.33 (m, 2.5H, 0.5 x H-4’ + 1 x H-2’ + 1 x Nal-a-CH), 4.28-4.26 (m, 0.5H, H-4’), 3.96-3.78 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH), 3.59-3.55 (m, 0.5H, Gly-a-CH₂), 3.52 (s, 1.5H, OCH₃),

3.51 (s, 1.5H, OCH₃), 3.26-3.12 (m, 2H, H-5’), 3.11-3.07 (m, 1H, Τφ-β-ΟΗ₂), 3.05-2.98 (m, 2H, Nal^-CH₂), 2.93-2.89 (m, 1H, Τφ-β-ΟΗ₂), 2.78 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.22-2.12 (m, 1H, H-3’), 1.70-1.62 (m, 1H, H-3’), 1.14 (d, J = 6.5 Hz, 1.5H, CH₃), 1.04 (d, J = 6.8 Hz, 1.5H, CH₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.8, 172.1, 170.3, 170.0, 166.7, 166.3, 163.9, 163.8, 158.7, 158.5, 157.5, 151.2, 141.8, 141.5, 136.7, 135.8, 135.7, 133.5, 132.4, 128.6, 128.5, 128.3, 128.0, 128.0, 127.9, 127.9, 127.8, 126.4, 125.9, 124.4, 121.5, 119.0, 118.7, 112.0, 109.7,

102.4, 102.0, 92.6, 92.5, 79.2, 79.2, 74.6, 74.5, 55.4, 55.0, 54.9, 54.8, 54.2, 53.6, 52.2, 51.0, 44.5, 44.3, 38.9, 38.7, 36.3, 36.1, 29.4, 28.4, 27.9, 15.2, 14.4; LRMS [M+H]⁺840.4; HRMS Calcd for C42H49N9O10: MH⁺, 840.36752. Found: MH⁺, 840.36862.

Hexyl (((S)-1 -(((2S,3S)-3-(2-amino-/V-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (74)

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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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH2CI2: DMF (210 pL) was added dropwise to the crude residue 59 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH2CI2: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 74. Deprotection was achieved through treatment with 1:1 v/v CH2CI2: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 99:1 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 74 as a white fluffy solid (12 mg, 12 pmol, 30%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3350, 2931, 1679, 1555 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.88 (s, 1H, Trp-NH), 8.61 (t, J =5.3 Hz, 0.5H, CONHCH2), 8.44 (d, J = 9.1 Hz, 0.5H, CONH), 8.40 (d, J = 9.2 Hz, 0.5H, CONH), 8.13 (t, J = 5.6 Hz, 0.5H, CONHCH₂), 8.05 (br s, 3H, NH₃), 7.85-7.83 (m, 1H, Ar-H), 7.78-7.75 (m, 2H, Ar-H), 7.63 (m, 1H, Ar-H), 7.55 (d, J = 8.1 Hz, 0.5H, H-6),

7.52 (d, J = 8.1 Hz, 0.5H, H-6), 7.49-7.42 (m, 3H, Ar-H), 7.34-7.30 (m, 2H, Ar-H), 7.11 (m, 1H, Ar-H), 7.08-7.04 (m, 1H, Ar-H), 6.99-6.95 (m, 1H, Ar-H), 6.59-6.56 (m, 1H, NHCONH), 6.43-6.39 (m, 1H, NHCONH), 5.73 (d, J = 3.6 Hz, 0.5H, H-T), 5.70 (d, J =3.5 Hz, 0.5H, H-T), 5.61-5.58 (m, 1H, H-5), 4.84-4.77 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.55-4.49 (m, 1.5H, 1 x DABA-a-CH + 1 x Trp-a-CH), 4.41-4.32 (m, 2.5H, 0.5 x H-4’ + 1 x H-2’ + 1 x Nal-a-CH), 4.29-4.25 (m, 0.5H, H-4’),

3.96-3.79 (m, 4H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH + 2 x OCH₂(CH₂)₄CH₃), 3.64-3.51 (m, 0.5H, Gly-a-CH₂), 3.27-3.12 (m, 2H, H-5’), 3.10-3.06 (m, 1H, Τφ-β-0Η₂), 3.02-3.00 (m, 2H, Nal^-CH₂), 2.97-2.90 (m, 1H, Τφ-β-CH;,), 2.79 (s, 1.5H, NCH₃), 2.76 (s, 1.5H, NCH₃), 2.24-2.12 (m, 1H, H-3’), 1.71-1.62 (m, 1H, H-3’), 1.39-1.37 (m, 2H, OCH₂(CH₂)₄CH₃), 1.24-1.13 (m, 7.5H, 6 x OCH₂(CH₂)₄CH₃ + 1.5 x CH₃), 1.04 (d, J = 6.9 Hz, 1.5H, CH₃), 0.83 (t, J = 6.8 Hz, 3H,CH₃); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.3, 171.9, 170.2, 169.8, 166.5, 166.2, 163.7, 158.6, 158.2,

157.9, 157.4, 151.0, 141.6, 141.4, 136.6, 135.6, 135.5, 133.4, 132.3, 128.4, 128.1,

127.9, 127.8, 127.8, 127.7, 127.6, 126.2, 125.8, 124.2, 121.4, 118.8, 118.5, 111.8,

109.6, 102.2, 101.9, 92.5, 92.3, 79.1, 74.5, 74.3, 64.7, 55.3, 54.9, 54.7, 54.6, 54.1, 53.4,

50.9, 44.3, 44.2, 38.8, 38.6, 36.1, 35.9, 31.3, 29.2, 28.4, 27.7, 25.3, 22.4, 15.0, 14.3;

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LRMS [M+H]⁺ 910.9; HRMS Calcd for C47H59N9O10: MH⁺, 910.44577. Found: MH⁺,

910.44614.

Dodecyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (75)

NH

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 pl_, 52 pmol, 1.3 eq.) in 2:1 v/v CH2CI2: DMF (210 pL) was added dropwise to the crude residue 60 (39 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH2CI2: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 75. Deprotection was achieved through treatment with 1:1 v/v CH2CI2: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 99:1 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 75 as a white fluffy solid (14 mg, 13 pmol, 31%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3305, 2924, 2854, 1677, 1557 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.86 (s, 1H, Trp-NH), 8.60 (t, J=5.5Hz, 0.5H, CONHCH2), 8.44-8.39 (m, 1H, CONH), 8.13 (t, J = 5.1 Hz, 0.5H, CONHCH2), 8.01 (brs, 3H, NH₃), 7.84-7.82 (m, 1H, Ar-H), 7.77-7.74 (m, 2H, Ar-H), 7.62 (m, 1H, Ar-H), 7.54 (d, J= 8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.48-7.42 (m, 3H, Ar-H), 7.33-7.29 (m, 2H, Ar-H), 7.10 (m, 1H, Ar-H), 7.06-7.03 (m, 1H, Ar-H),

6.97-6.94 (m, 1H, Ar-H), 6.58-6.55 (m, 1H, NHCONH), 6.41-6.36 (m, 1H, NHCONH),

5.72 (d, J = 3.6 Hz, 0.5H, H-1 ’), 5.69 (d, J = 3.6 Hz, 0.5H, H-1 ’), 5.64-5.57 (m, 1H, H-5),

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4.82-4.77 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.55-4.49 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.41-4.33 (m, 2.5H, 0.5 x H-4’ + 1 x H-2’ + 1 x Nal-a-CH), 4.30-4.25 (m, 0.5H, H-4’), 3.95-3.78 (m, 4H, 1.5 x Gly-a-CH₂ + 0.5 x DABAβ-CH + 2 x OCH₂(CH₂)i₀CH₃), 3.59-3.54 (m, 0.5H, Gly-a-CH₂), 3.27-3.12 (m, 2H, H-5’), 3.10-3.05 (m, 1H, ΤΓρ-β-ΰΗ₂), 3.04-2.97 (m, 2H, Nal^-CH₂), 2.94-2.90 (m, 1H, ΤΓρ-β-ΰΗ₂), 2.78 (s, 1.5H, NCH₃), 2.76 (s, 1.5H, NCH₃), 2.22-2.12 (m, 1H, H-3’), 1.70-

1.62 (m, 1H, H-3’), 1.41-1.36 (m, 2H, OCH₂(CH₂)i₀CH₃), 1.26-1.12 (m, 19.5H, 18 x OCH₂(CH₂)i₀CH₃ + 1.5 x CH₃), 1.03 (d, J = 6.9 Hz, 1,5H, CH₃), 0.84 (t, J = 7.0 Hz, 3H,

CH₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 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, 118.9, 118.7, 112.0, 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 Calcd for C53H71N9O10: MH⁺, 994.53967. Found: MH⁺, 994.54051.

Neopentyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-

5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2yl)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 pl_, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 61 (35 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 76. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 97:3 mixture of separable diastereoisomers.

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Purification by reverse-phase HPLC yielded 76 as a white fluffy solid (13 mg, 13 pmol,

32%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3285, 3063, 2957, 1676, 1552 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.86 (s, 1H, Trp-NH), 8.59 (t, J =5.5 Hz, 0.5H, CONHCH₂), 8.43 (d, J = 9.0 Hz, 0.5H, CONH), 8.39 (d, J = 9.3 Hz, 0.5H, CONH), 8.12 (t, J = 5.2 Hz, 0.5H, CONHCH₂), 8.02 (br s, 3H, NH₃), 7.84-7.82 (m, 1H, Ar-H), 7.77-7.74 (m, 2H, Ar-H), 7.62 (m, 1H, Ar-H), 7.54 (d, J = 8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.46-7.42 (m, 3H, Ar-H), 7.33-7.29 (m, 2H, Ar-H), 7.10 (m, 1H, Ar-H), 7.07-7.04 (m, 1H, Ar-H), 6.98-6.95 (m, 1H, Ar-H), 6.57-6.54 (m, 1H, NHCONH), 6.43-6.39 (m, 1H, NHCONH), 5.72 (d, J = 3.7 Hz, 0.5H, H-1’), 5.69 (d, J =3.5 Hz, 0.5H, H-1’), 5.60-5.57 (m, 1H, H-5), 4.81-4.77 (m, 0.5H, ΟΑΒΑ-β-CH), 4.60 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.55-4.49 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.45-4.41 (m, 1H, Nal-a-CH), 4.38-4.31 (m, 1.5H, 0.5 x H-4’ + 1 x H-2’),

4.29-4.24 (m, 0.5H, H-4’), 3.96-3.77 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH), 3.61 -

3.57 (m, 2.5H, 2 x OCH₂C(CH₃)₃ + 0.5 x Gly-a-CH₂), 3.26-3.11 (m, 2H, H-5’), 3.09-3.01 (m, 3H, 1 x ΤΓρ-β-ΟΗ₂ + 2 x Nal^-CH₂), 2.94-2.89 (m, 1H, ΤΓρ-β-ΟΗ₂), 2.78 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.22-2.12 (m, 1H, H-3’), 1.70-1.62 (m, 1H, H-3’), 1.12 (d, J = 6.6 Hz, 1.5H, CH₃), 1.03 (d, J= 6.9 Hz, 1.5H, CH₃), 0.75 (s, 4.5H, OCH₂C(CH₃)₃), 0.74 (s, 4.5H, OCH₂C(CH₃)₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.4, 172.1, 170.3, 170.0, 166.7, 166.3, 163.9, 163.8, 158.7, 158.4, 157.5,

151.2, 141.7, 141.5, 136.8, 135.7, 135.6, 133.5, 132.5, 128.5, 128.5, 128.2, 128.0,

127.9, 127.8, 126.4, 125.9, 124.3, 121.5, 119.0, 118.7, 112.0, 109.8, 102.4, 102.0, 92.6,

92.5, 79.2, 74.6, 74.5, 73.8, 55.4, 55.0, 54.8, 54.7, 54.3, 53.5, 51.0, 44.5, 44.3, 39.0,

38.8, 36.2, 36.1, 31.5, 29.4, 28.6, 27.9, 26.6, 15.2, 14.4; LRMS [M+H]⁺896.8; HRMS Calcd for C46H57N9O10: MH⁺, 896.43012. Found: MH⁺, 896.43189.

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Benzyl (((S)-1 -(((2S,3S)-3-(2-amino-A/-methylacetamido)-1 -((((2R,4R,5R)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2yl)carbamoyl)-L-tryptophanate (77)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 62 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 77. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 99:1 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 77 as a white fluffy solid (11 mg, 11 pmol, 27%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3331, 3057, 2945, 1677, 1550 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.61 (t, J =5.0 Hz, 0.5H, CONHCH₂), 8.44 (d, J = 9.0 Hz, 0.5H, CONH), 8.41 (d, J = 9.2 Hz, 0.5H, CONH), 8.13 (t, J = 5.6 Hz, 0.5H, CONHCH₂), 8.03 (br s, 3H, NH₃), 7.83-7.82 (m, 1H, Ar-H), 7.75-7.72 (m, 2H, Ar-H), 7.60 (m, 1H, Ar-H), 7.55 (d, J = 8.0 Hz, 0.5H, H-6),

7.51 (d, J = 8.1 Hz, 0.5H, H-6), 7.47-7.42 (m, 3H, Ar-H), 7.35-7.34 (m, 1H, Ar-H), 7.307.23 (m, 4H, Ar-H), 7.16-15 (m, 2H, Ar-H), 7.08-7.05 (m, 2H, Ar-H), 6.98-6.95 (m, 1H, Ar-H), 6.64-6.61 (m, 1H, NHCONH), 6.42-6.38 (m, 1H, NHCONH), 5.72 (d, J = 3.5 Hz, 0.5H, H-1’), 5.69 (d, J = 3.3 Hz, 0.5H, H-1’), 5.60-5.57 (m, 1H, H-5), 5.02-4.95 (m, 2H, OCH₂(C₆H₅)), 4.83-4.79 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.3 Hz, 0.5H, DABA-a-CH), 4.56-4.50 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.48-4.44 (m, 1H, Nal-a-CH),

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4.39-4.33 (m, 1.5H, 0.5 x H-4’ + 1 x H-2’), 4.30-4.25 (m, 0.5H, H-4’), 3.96-3.78 (m, 2H,

1.5 x Gly-a-CH₂ + 0.5 x DABA-β-ΟΗ), 3.60-3.55 (m, 0.5H, Gly-a-CH₂), 3.25-3.11 (m, 2H, H-5’), 3.10-3.01 (m, 3H, 1 x ΤΓρ-β-ΟΗ₂ + 2 x ΝθΙ-β-ΟΗζ), 2.94-2.90 (m, 1H, ΤΓρ-β-ΟΗ₂), 2.78 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.22-2.12 (m, 1H, H-3’), 1.70-

1.62 (m, 1H, H-3’), 1.13 (d, J = 6.4 Hz, 1.5H, CH₃), 1.03 (d, J= 6.8 Hz, 1.5H, CH₃); ¹³CNMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) 5 173.3, 172.0, 170.3,

170.0, 166.7, 166.3, 163.9, 163.8, 158.9, 158.7, 158.4, 157.5, 151.2, 141.7, 141.5,

136.8, 136.4, 135.7, 135.6, 133.5, 132.4, 129.5, 128.9, 128.8, 128.5, 128.3, 128.3,

128.0, 128.0, 127.9, 127.9, 127.8, 126.4, 125.9, 124.5, 121.6, 119.1, 118.7, 112.0,

109.7, 102.4, 102.0, 92.6, 92.5, 79.2, 74.6, 74.5, 67.6, 66.4, 55.4, 55.0, 54.8, 54.7, 54.4,

53.6, 51.0, 44.5, 44.3, 39.0, 38.8, 36.2, 36.1, 31.3, 29.4, 28.5, 27.9, 25.7, 15.2, 14.5; LRMS [M+H]⁺ 916.8; HRMS Calcd for C48H53N9O10: MH⁺, 916.39882. Found: MH⁺, 916.39964.

(2S, 3S)-3-(2-amino-A/-methylacetamido)-A/-(((2/?, 4R, 5/7)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)methyl)-2-((S)-2-(3-((S)1 -(hexylamino)-3-(1 H-indol-3-yl)-1 -oxopropan-2-yl)ureido)-3-(naphthalen-2yl)propanamido)butanamide (78)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 63 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 78. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 98:2 mixture of separable diastereoisomers.

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Purification by reverse-phase HPLC yielded 78 as a white fluffy solid (22 mg, 22 pmol,

54%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3292, 2930, 1663, 1551 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.78 (s, 1H, Trp-NH), 8.59 (t, J =5.2 Hz, 0.5H, CONHCH₂), 8.37 (d, J = 9.1 Hz, 0.5H, CONH), 8.33 (d, J = 9.1 Hz, 0.5H, CONH), 8.10 (t, J = 5.2 Hz, , 0.5H, CONHCH₂), 8.05 (br s, 3H, NH₃), 7.84-7.74 (m, 3H, Ar-H), 7.68 (t, J = 5.5 Hz, 1H, CONHCH₂(CH₂)₄CH₃), 7.63 (m, 1H, Ar-H), 7.55-

7.51 (m, 2H, 1 x H-6 + 1 x Ar-H), 7.49-7.42 (m, 2H, Ar-H), 7.33-7.29 (m, 2H, Ar-H), 7.06-7.01 (m, 2H, Ar-H), 6.96-6.92 (m, 1H, Ar-H), 6.42-6.40 (m, 2H, NHCONH), 5.72 (d, J = 3.7 Hz, 0.5H, H-1’), 5.69 (d, J = 3.5 Hz, 0.5H, H-1’), 5.61-5.57 (m, 1H, H-5), 4.82-

4.73 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.3 Hz, 0.5H, DABA-a-CH), 4.55-4.41 (m, 1.5H, 1 x DABA-a-CH + 1 x Trp-a-CH), 4.38-4.24 (m, 3H, 1 x H-4’ + 1 x H-2’ + 1 x Nal-a-CH),

3.91-3.77(m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH), 3.59-3.54 (m, 0.5H, Gly-a-CH₂), 3.27-3.07 (m, 3H, 2 x H-5’ + 1 x CONHCH₂(CH₂)₄CH₃), 3.00-2.85 (m, 5H, 2 x

Trp-p-CH₂ + 2 x Nal-p-CH₂ + 1 x CONHCH₂(CH₂)₄CH₃), 2.77 (s, 1.5H, NCH₃), 2.76 (s, 1.5H, NCH₃), 2.22-2.11 (m, 1H, H-3’), 1.70-1.61 (m, 1H, H-3’), 1.24-1.13 (m, 9.5H, 8x CONHCH₂(CH₂)₄CH₃ + 1.5 x CH₃), 1.03 (d, J = 6.8 Hz, 1,5H, CH₃), 0.81 (t, J = 6.9 Hz, 3H, CONHCH₂(CH₂)₄CH₃); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 172.3, 172.1, 170.2, 169.8, 166.5, 166.1, 163.7, 163.7, 159.1, 158.8, 158.6, 158.5,

158.1, 157.4, 151.0, 141.6, 141.4, 136.5, 135.8, 135.7, 135.0, 133.4, 132.3, 128.4,

128.1, 127.9, 127.9, 127.8, 127.8, 126.3, 125.8, 123.9, 121.2, 119.0, 118.7, 118.5,

115.7, 113.9, 111.6, 110.5, 102.2, 101.9, 92.5, 92.3, 79.1, 79.0, 74.5, 74.3, 63.0, 55.5,

55.3, 54.9, 54.8, 54.5, 53.4, 50.9, 44.3, 44.1, 38.9, 38.7, 36.1, 35.9, 31.4, 29.3, 29.2,

27.7, 26.4, 22.4, 15.0, 14.4, 14.2; LRMS [M+H]⁺909.9; HRMS Calcd for C₄₇H6oNio0₉: MH⁺, 909.47175. Found: MH⁺, 909.46152.

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PCT/AU2017/051394 (2S,3S)-2-((S)-2-(3-((S)-3-(1 H-indol-3-yl)-1 -(neopentylamino)-l -oxopropan-2yl)ureido)-3-(naphthalen-2-yl)propanamido)-3-(2-amino-A/-methylacetamido)-A/(((2R, 4R, 5/7)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)butanamide (79)

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 pl_, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 64 (35 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 79. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 98:2 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 79 as a white fluffy solid (13 mg, 13 pmol, 32%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3292, 2935, 1672, 1556 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.31 (s, 1H, Uracil-NH), 10.79 (s, 1H, Trp-NH), 8.60 (t, J =5.6 Hz, 0.5H, CONHCH₂), 8.40 (d, J = 8.9 Hz, 0.5H, CONH), 8.34 (d, J = 9.1 Hz, 0.5H, CONH), 8.13 (t, J = 5.6 Hz, 0.5H, CONHCH₂), 8.03 (br s, 3H, NH₃), 7.85-7.83 (m, 1H, Ar-H), 7.79-7.74 (m, 2H, Ar-H), 7.64-7.61 (m, 2H, 1 x Ar-H + 1 x

CONHCH₂C(CH₃)₃), 7.57-7.54 (m, 1.5H, 0.5 x H-6 + 1 x Ar-H), 7.51 (d, J = 8.1 Hz, 0.5H, H-6), 7.49-7.42 (m, 2H, Ar-H), 7.33-7.30 (m, 2H, Ar-H), 7.10-7.09 (m, 1H, Ar-H), 7.06-7.02 (m, 1H, Ar-H), 6.97-6.94 (m, 1H, Ar-H), 6.47-6.44 (m, 1H, NHCONH), 6.406.37 (m, 1H, NHCONH), 5.72 (d, J = 3.6 Hz, 0.5H, H-1’), 5.69 (d, J = 3.5 Hz, 0.5H, H1’), 5.62-5.58 (m, 1H, H-5), 4.84-4.76 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.55-4.48 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.43-4.38 (m, 1H,

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Nal-a-CH), 4.37-4.31 (m, 1.5H, 0.5 x H-4’ + 1 x H-2’), 4.29-4.24 (m, 0.5H, H-4’),

3.92-3.77 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ϋΑΒΑ-β-CH), 3.59-3.54 (m, 0.5H, Gly-a-CH₂), 3.28-3.14 (m, 2H, H-5’), 3.11-3.06 (m, 1H, ΤΓρ-β-ΟΗ₂), 3.03-2.98 (m, 1H, Nal^-CH₂), 2.94-2.85 (m, 2H, 1 x ΤΓρ-β-ΟΗ₂ + 1 x Nal^-CH₂), 2.83-2.78 (m, 2H, CONHCH₂C(CH₃)₃), 2.77 (s, 1.5H, NCH₃), 2.76 (s, 1.5H, NCH₃), 2.23-2.11 (m, 1H, H-3’), 1.70-1.62 (m, 1H, H-3’), 1.13 (d, J = 6.5 Hz, 1.5H, CH₃), 1.02 (d, J = 6.9 Hz, 1.5H, CH₃), 0.72 (s, 4.5H, CONHCH₂C(CH₃)₃), 0.71 (s, 4.5H, CONHCH₂C(CH₃)₃); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 172.8, 172.0, 170.2, 169.8, 166.5,

166.1, 163.7, 159.0, 158.7, 158.3, 158.0, 157.5, 151.0, 141.6, 141.4, 136.5, 135.7,

135.6, 133.4, 132.3, 128.4, 128.1, 127.9, 127.8, 127.8, 127.7, 126.2, 125.8, 123.9,

121.2, 119.0, 118.7, 118.6, 115.7, 111.6, 110.6, 102.2, 101.9, 92.4, 92.3, 79.0, 79.0,

74.5, 74.3, 55.3, 54.8, 54.7, 54.4, 53.4, 50.9, 50.1, 44.3, 44.1, 38.8, 38.5, 36.1, 35.9,

32.3, 29.2, 29.1, 27.7, 27.6, 15.0, 14.2; LRMS [M+H]⁺ 895.8; HRMS Calcd for C46H58N10O9: MH⁺, 895.44610. Found: MH⁺, 895.44543.

(2S,3S)-3-(2-amino-N-methylacetamido)-2-((S)-2-(3-((S)-1-(benzylamino)-3(1H-indol-3-yl)-1-oxopropan-2-yl)ureido)-3-(naphthalen-2-yl)propanamido)-N(((2R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4hydroxytetrahydrofuran-2-yl)methyl)butanamide (80)

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 65 (36 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 80. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5

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Purification by reverse-phase HPLC yielded 80 as a white fluffy solid (12 mg, 12 pmol,

29%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3293, 3041, 2937, 1655, 1549 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.81 (s, 1H, Trp-NH), 8.59 (t, J =5.5 Hz, 0.5H, CONHCH₂), 8.38-8.28 (m, 2H, 1 x CONH + 1 x CONHCH₂(C₆H₅)), 8.10 (t, J = 5.5 Hz, 0.5H, CONHCH₂), 8.03 (br s, 3H, NH₃), 7.84-7.82 (m, 1H, Ar-H), 7.77-7.73 (m, 2H, Ar-H), 7.62 (m, 1H, Ar-H), 7.58-7.56 (m, 1H, Ar-H), 7.54 (d, J =

8.1 Hz, 0.5H, H-6), 7.50 (d, J = 8.1 Hz, 0.5H, H-6), 7.47-7.42 (m, 2H, Ar-H), 7.34-7.30 (m, 2H, Ar-H), 7.22-7.14 (m, 3H, Ar-H), 7.07-7.03 (m, 4H, Ar-H), 6.97-6.93 (m, 1H, Ar-H), 6.48-6.45 (m, 1H, NHCONH), 6.40-6.37 (m, 1H, NHCONH), 5.71 (d, J = 3.7 Hz, 0.5H, Η-Γ), 5.69 (d, J = 3.5 Hz, 0.5H, Η-Γ), 5.61-5.57 (m, 1H, H-5), 4.84-4.76 (m, 0.5H, ΟΑΒΑ-β-CH), 4.61 (t, J = 9.3 Hz, 0.5H, DABA-a-CH), 4.55-4.46 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.44-4.38 (m, 1H, Nal-a-CH), 4.37-4.30 (m, 1.5H, 0.5 x H4’ + 1 x H-2’), 4.28-4.24 (m, 0.5H, H-4’), 4.21-4.14 (m, 2H, CONHCH₂(C₆H₅)), 3.95-3.76 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH), 3.57-3.54 (m, 0.5H, Gly-a-CH₂), 3.263.14 (m, 2H, H-5’), 3.12-3.07 (m, 1H, ΤΓρ-β-ΟΗ₂), 3.06-3.01 (m, 1H, Nal^-CH₂), 2.952.90 (m, 2H, 1 x ΤΓρ-β-ΟΗ₂ + 1 x Nal^-CH₂), 2.77 (s, 1.5H, NCH₃), 2.75 (s, 1.5H, NCH₃), 2.23-2.11 (m, 1H, H-3’), 1.69-1.61 (m, 1H, H-3’), 1.13 (d, J = 6.5 Hz, 1.5H, CH₃), 1.03 (d, J= 6.9 Hz, 1.5H, CH₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 172.7, 172.0, 170.2, 166.1, 163.7, 163.7, 158.5, 158.2, 157.9, 157.5, 151.0,

141.6, 139.7, 136.5, 135.7, 133.4, 132.3, 128.6, 128.1, 127.9, 127.8, 127.4, 127.0,

126.3, 125.8, 124.1, 121.3, 119.1, 118.6, 111.7, 110.4, 102.2, 101.9, 92.5, 92.3, 79.0,

74.5, 74.3, 55.3, 54.9, 54.9, 54.6, 53.4, 50.9, 44.3, 44.1, 42.4, 38.7, 38.5, 36.1, 35.9,

29.2, 27.7, 15.0, 14.2; LRMS [M+H]⁺ 915.8; HRMS Calcd for C48H54N10O9: MH⁺, 915.41480. Found: MH⁺, 915.41373.

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Benzyl (((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-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-(6,7-dimethoxy-2-oxo-2H-chromen-4yI)-1 -oxopropan-2-yl)carbamoyl)-L-tryptophanate (81)

OMe

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 pL, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 50 (39 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 81. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 95:5 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 81 as a white fluffy solid (9.2 mg, 8.3 pmol, 21%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3332, 2929, 1676, 1556 cm’¹; ¹H NMR (500 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.88 (s, 1H, Trp-NH), 8.57 (t, J =5.7 Hz, 0.5H, CONHCH₂), 8.42-8.38 (m, 2H, CONH), 8.12 (t, J = 5.8 Hz, 0.5H, CONHCH₂), 7.99-7.97 (m, 3H, NH₃), 7.53 (d, J = 8.1 Hz, 0.5H, H-6), 7.49 (d, J = 8.1 Hz, 0.5H, H-6),7.45-7.42 (m, 2H, Ar-H), 7.35-7.34 (m, 1H, Ar-H), 7.26-7.24 (m, 3H, Ar-H),

7.16-7.13 (m, 2H, Ar-H), 7.08-7.05 (m, 2H, Ar-H), 7.01 (m, 1H, Ar-H), 6.98-6.95 (m, 1H, Ar-H), 6.70-6.65 (m, 2H, NHCONH), 5.95 (s, 0.5H, H-3”), 5.94 (s, 0.5H, H-3”), 5.69 (d, J = 3.6 Hz, 0.5H, H-1 ’), 5.65 (d, J = 3.4 Hz, 0.5H, H-1 ’), 5.61 -5.57 (m, 1H, H-5), 4.99-4.98 (m, 2H, OCH₂(C₆H₅)), 4.70-4.67 (m, 0.5H, ϋΑΒΑ-β-CH), 4.56-4.47 (m, 3H, 1 x DABA-a-CH + 1 x Trp-a-CH + 1 x H-10”), 4.37-4.28 (m, 1.5H, 0.5 x H-4’ + 1 x H-2’),

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4.22-4.17 (m, 0.5H, H-4’), 3.84-3.73 (m, 8.5H, 2 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH + 6 x OCHs), 3.33-3.21 (m, 1H, H-5’), 3.13-2.99 (m, 4H, 2 χΤφ-β-ΟΗ₂+ 1 x H-5’ + 1 x H-9”), 2.94-2.86 (m, 1H, H-9”), 2.74 (s, 1.5H, NCH₃), 2.72 (s, 1.5H, NCH₃), 2.22-2.17 (m, 0.5H, H-3’), 2.15-2.10 (m, 0.5H, H-3’), 1.67-1.59 (m, 1H, H-3’), 1.08 (d, J = 6.5 Hz, 1.5H, CH₃), 0.98 (d, J= 6.9 Hz, 1.5H, CH₃); ¹³C NMR (125 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 173.1, 171.0, 170.8, 170.0, 169.6, 166.6, 166.6, 162.2, 164.0, 163.9, 160.9,

160.8, 158.8, 158.6, 157.6, 153.0, 152.6, 151.2, 149.6, 146.5, 141.7, 136.8, 136.3,

128.9, 128.6, 128.3, 128.3, 127.8, 124.4, 121.6, 119.1, 118.7, 112.7, 112.1, 111.9,

109.5, 106.9, 102.4, 102.0, 100.7, 92.5, 79.3, 79.2, 74.7, 74.5, 66.5, 56.7, 56.5, 55.4,

54.9, 54.5, 53.5, 44.3, 36.2, 35.9, 31.3, 28.5, 27.9, 15.1, 14.4; LRMS [M+H]⁺ 994.9; HRMS Calcd for C49H55N9O14: MH⁺, 994.39412. Found: MH⁺, 994.39394.

Resin-bound pseudo peptide (82)

H °Υ^ΝΗΒοο

Resin-bound pseudo peptide 48 was preactivated with DIC (150 pL, 0.94 mmol, 5 eq.) in CH₂CI₂ (1.9 mL) prior to the addition of neopentyl alcohol (83 pL, 0.94 mmol, 5 eq.) and DMAP (2.3 mg, 19 pmol, 0.1 eq.) in CH₂CI₂ (1.9 mL) according to general procedure 7.2 condition A to yield the resin bound pseudo peptide 82.

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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-2yl)methyl)amino)-1-oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2yl)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 pl_, 52 pmol, 1.3 eq.) in 2:1 v/v CH₂CI₂: DMF (210 pL) was added dropwise to the crude residue 82 (34 mg, 40 pmol, 1 eq.), uridyl amine 18 (27 mg, 120 pmol, 3 eq.) and HOAt (27 mg, 200 pmol, 5 eq.) in 1:2 v/v CH₂CI₂: DMF (210 pL) according to general procedure 8 condition C, yielding the protected analogue 83. Deprotection was achieved through treatment with 1:1 v/v CH₂CI₂: TFA (5.2 mL) and 2.5 vol.% /Pr₃SiH (0.13 mL) to afford a 96:4 mixture of separable diastereoisomers. Purification by reverse-phase HPLC yielded 83 as a white fluffy solid (15 mg, 16 pmol, 39%, yield calculated as TFA salt after HPLC purification).

IR (ATR): 3293, 2953, 2852, 1668, 1557 cm’¹; ¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.54 (t, J =

5.6 Hz, 0.5H, CONHCH₂), 8.22 (d, J = 8.9 Hz, 0.5H, CONH), 8.17 (d, J = 9.2 Hz, 0.5H, CONH), 8.03 (br s, 3.5H, 0.5 x CONHCH₂ + 3 x NH₃), 7.54-7.50 (m, 1H, H-6), 7.46-7.44 (m, 1H, Ar-H), 7.34-7.32 (m, 1H, Ar-H), 7.12-7.11 (m, 1H, Ar-H), 7.07-7.04 (m, 1H, ArH), 6.99-6.96 (m, 1H, Ar-H), 6.38 (d, J = 7.9 Hz, 1H, NHCONH), 6.35-6.31 (m, 1H, NHCONH), 5.70 (d, J = 3.5 Hz, 0.5H, H-1’), 5.66 (d, J = 3.5 Hz, 0.5H, H-1’), 5.61-5.58 (m, 1H, H-5), 4.82-4.75 (m, 0.5H, ΟΑΒΑ-β-CH), 4.56 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.45-4.41 (m, 1.5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.39-4.24 (m, 2H, 1 x H-4’ + 1 x H-2’), 4.14-4.10 (m, 1H, Cha-a-CH), 3.94-3.76 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-βCH), 3.66-3.54 (m, 2.5H, 0.5 x Gly-a-CH₂ + 2 x OCH₂C(CH₃)₃), 3.29-3.21 (m, 1H, H-5’),

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3.18-3.11 (m, 1H, H-5’), 3.09-3.01 (m, 2H, Τφ-β-ΟΗ₂), 2.76 (s, 1.5H, NCH₃), 2.74 (s, 1.5H, NCH₃), 2.21-2.11 (m, 1H, H-3’), 1.68-1.59 (m, 6H, 1 x H-3’ + 5 x Cy), 1.37-1.28 (m, 2H, Cha^-CH₂), 1.26-1.23 (m, 1H, Cy), 1.14-1.10 (m, 3.5H, 1.5 x CH₃ + 2 x Cy), 1.01 (d, J= 6.8 Hz, 1.5H, CH₃), 0.87-0.80 (m, 12H, 3 x Cy + 9 x OCH₂C(CH₃)₃); ¹³CNMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) 5173.4, 173.3, 170.3,

169.9, 166.5, 166.2, 163.7, 163.7, 158.9, 158.6, 158.3, 158.0, 157.6, 157.6, 151.0,

141.6, 141.4, 136.6, 127.6, 124.2, 121.4, 118.8, 118.5, 111.9, 109.6, 102.2, 101.9, 92.5,

92.3, 79.1, 74.5, 74.4, 73.7, 55.2, 54.9, 54.2, 53.2, 51.5, 50.8, 44.2, 44.1, 36.0, 35.9,

33.8, 33.7, 33.7, 32.7, 32.6, 31.4, 29.2, 28.4, 27.7, 26.5, 26.3, 26.1, 15.0, 14.3; LRMS [M+H]⁺852.8; HRMS Calcd for C42H61N9O10: MH⁺, 852.46142. Found: MH⁺, 852.46191.

General procedure 8

Condition D:

The resin bound peptide was treated with 95:2.5:2.5 v/v/v TFA:H₂O:TIS for 1 h with shaking. The resin was subsequently washed with TFA:H₂O:TIS (x 10), combined with the cleavage solution and concentrated. The resulting crude residue was purified via reverse-phase HPLC.

(((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)methyl)amino)-1oxobutan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2-yl)carbamoyl)-L-tryptophan (99)

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 pL, 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. Following Fmoc deprotection, 20 (18 mg, 0.040 mmol, 2 eq.)

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PCT/AU2017/051394 was coupled using HATU (15 mg, 0.040 mmol, 2 eq.) and /Pr₂EtN (11 pL, 0.060 mmol, 3 eq.) in DMF (0.40 mL) according to procedure 6 condition A. The resin was then treated with a solution of Pd(PPh₃)₄ (4.6 mg, 4.0 pmol, 0.2 eq.) in CH2CI2 (0.50 mL) with PhSiH₃ (49 pL, 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 /Pr₂EtN (11 pL, 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 pL, 0.16 mmol, 8 eq.) in DMF (0.8 mL) as described in general procedure 6 condition B. Following Fmoc deprotection, coupling of carbamate S12 (21 mg, 0.040 mmol, 2 eq.) was achieved using /Pr₂EtN (14 pL, 0.080 mmol, 4 eq.) in DMF (0.40 mL) according to general procedure 4. The resin bound peptide was then cleaved off resin as described in general procedure 8 condition E to yield a crude residue that was purified via reversephase HPLC to afford 99 as a fluffy white solid (8.8 mg, 11 pmol, 53%, yield calculated as FA salt after HPLC purification).

¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.29 (br s, 1H, Uracil-NH), 10.89 (s, 1H, Trp-NH), 8.57 (t, J = 5.5 Hz, 0.5H, CONHCH₂), 8.21 (d, J = 9.0 Hz, 0.5H, CONH), 8.15 (d, J= 9.1 Hz, 0.5H, CONH), 8.08 (t, J = 5.4 Hz, 0.5H, CONHCH2), 7.54 (d, J = 8.1 Hz, 1H, H-6), 7.52-7.50 (m, 1H, Ar-H), 7.34-7.32 (m, 1H, Ar-H), 7.12-7.11 (m, 1H, Ar-H), 7.07-7.03 (m, 1H, Ar-H), 6.98-6.94 (m, 1H, Ar-H), 6.38 (d, J = 7.9 Hz, 1H, NHCONH), 6.24-6.21 (m, 1H, NHCONH), 5.70 (d, J = 3.7 Hz, 0.5H, H-1’), 5.66 (d, J = 3.4 Hz, 0.5H, H-1’), 5.61 (d, J = 8.0 Hz, 0.5H, H-5), 5.58 (d, J = 8.0 Hz, 0.5H, H-5), 4.82-4.74 (m, 0.5H, ϋΑΒΑ-β-CH), 4.57 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.49 (t, J = 9.3 Hz, 0.5H, DABA-a-CH), 4.40-4.24 (m, 3H, 1 x Trp-a-CH + 1 x H-2’ + 1 x H-4’), 4.17-4.11 (m, 1H, Cha-a-CH), 3.92-3.84 (m, 1.5H, 1 x Gly-a-CH₂ + 0.5 x ΟΑΒΑ-β-CH), 3.76 (d, J = 16.4 Hz, 0.5H, Gly-a-CH₂), 3.55 (d, J = 16.0 Hz, 0.5H, Gly-a-CH₂), 3.26-3.25 (m, 1H, H-5’), 3.18-3.00 (m, 3H, 1 x H-5’ + 2 x Τφ-β-ΟΗ₂), 2.75 (s, 1.5H, NCH₃), 2.74 (s, 1.5H, NCH₃), 2.22-2.10 (m, 1H, H-3’), 1.69-1.59 (m, 6H, 1 x H3’ + 5x Cy), 1.41-1.27 (m, 3H, 1 x Cy + 2 x Cha^-CH₂), 1.16-1.01 (m, 3.5H, 2 x Cy +

1.5 CH₃), 1.02 (d, J = 6.9 Hz, 1.5H, CH₃), 0.88-0.76 (m, 3H, 3 x Cy); ¹³C NMR (100 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 174.1, 172.9, 169.8, 169.4, 166.0,

165.7, 163.3, 163.2, 157.7, 157.2, 157.1, 150.5, 141.1, 141.0, 136.0, 127.4, 127.3,

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123.8, 120.8, 118.4, 118.3, 111.3, 109.4, 101.7, 101.4, 91.9, 91.8, 78.6, 74.0, 73.9,

54.8, 54.4, 53.4, 52.8, 51.1, 50.4, 43.8, 43.6, 41.4, 35.6, 35.4, 33.4, 33.2, 32.1, 28.7,

27.2, 26.0, 25.8, 25.7, 22.9, 14.5, 13.8; LRMS [M+H]⁺ 782.0. These data are in agreement with those reported by Tran et al^[4] (((S)-1-(((2S,3S)-3-(2-amino-A/-methylacetamido)-1-((((2/?,4/?,5/?)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)methyl)amino)-1 oxobutan-2-yl)amino)-3-(naphthalen-2-yl)-1-oxopropan-2-yl)carbamoyl)-Ltryptophan (100)

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 pL, 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. Following Fmoc deprotection, 20 (9.2 mg, 0.021 mmol, 2 eq.) was coupled using HATU (8 mg, 0.021 mmol, 2 eq.) and /Pr₂EtN (5.5 pL, 0.032 mmol, 3 eq.) in DMF (0.20 mL) according to procedure 6 condition A. The resin was then treated with a solution of Pd(PPh₃)₄ (2.4 mg, 2.1 pmol, 0.2 eq.) in CH₂CI₂ (0.26 mL) with PhSiH₃ (26 pL, 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 /Pr₂EtN (5.5 pL, 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 pL, 0.084 mmol, 8 eq.) in DMF (0.40 mL) as described in general procedure 6 condition B. Following Fmoc deprotection, coupling of carbamate S12 (11 mg,

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0.021 mmol, 2 eq.) was achieved using /Pr₂EtN (7 pL, 0.042 mmol, 4 eq.) in DMF (0.20 mL) according to general procedure 4. The resin bound peptide was then cleaved off resin as described in general procedure 8 condition E to yield a crude residue that was purified via reverse-phase HPLC to afford 100 as a fluffy white solid (5.8 mg,

6.2 pmol, 59%, yield calculated as TFA salt after HPLC purification).

¹H NMR (400 MHz, (CD₃)₂SO, ca. 1:1 mixture of rotamers) δ 11.30 (s, 1H, Uracil-NH),

10.86 (s, 1H, Trp-NH), 8.60 (t, J = 5.6 Hz, 0.5H, CONHCH₂), 8.41 (d, J = 9.1 Hz, 0.5H, CONH), 8.37 (d, J = 9.2 Hz, 0.5H, CONH), 8.12 (t, J = 5.4 Hz, 0.5H, CONHCH₂), 8.04 (br s, 3H, NH₃), 7.84-7.82 (m, 1H, Ar-H), 7.79-7.74 (m, 2H, Ar-H), 7.64 (m, 1H, Ar-H), 7.55 (d, J = 8.1 Hz, 0.5H, H-6), 7.52-7.50 (m, 1.5H, 1 x Ar-H + 0.5H x H-6), 7.47-7.42 (m, 2H, Ar-H), 7.33-7.30 (m, 2H, Ar-H), 7.11 (m, 1H, Ar-H), 7.06-7.03 (m, 1H, Ar-H),

6.98-6.95 (m, 1H, Ar-H), 6.47-6.44 (m, 1H, NHCONH), 6.42-6.38 (m, 1H, NHCONH),

5.72 (d, J = 3.7 Hz, 0.5H, H-1 ’), 5.69 (d, J = 3.5 Hz, 0.5H, H-1 ’), 5.60-5.57 (m, 1H, H-5), 4.82-4.77 (m, 0.5H, ϋΑΒΑ-β-CH), 4.61 (t, J = 9.4 Hz, 0.5H, DABA-a-CH), 4.55-4.48 (m, 1,5H, 0.5 x DABA-a-CH + 1 x Trp-a-CH), 4.39-4.33 (m, 2.5H, 0.5 x H-4’ + 1 x H-2’ + 1 x Nal-a-CH), 4.30-4.25 (m, 0.5H, H-4’), 3.96-3.78 (m, 2H, 1.5 x Gly-a-CH₂ + 0.5 x DABA- β-CH), 3.58-3.55 (m, 0.5H, Gly-a-CH₂), 3.26-3.12 (m, 2H, ΤΓρ-β-ΟΗ₂+ 1 x Nal^-CH₂), 2.99 (dd, J = 14.7, 6.9 Hz, 1H, 1H, ΤΓρ-β-ΟΗ₂), 2.78 (s, 1.5H, NCH₃), 2.76 (s, 1.70-1.62 (m, 1H, H-3’), 1.14 (d, J = 6.5 Hz, CH₃); ¹³CNMR (100 MHz, (CD₃)₂SO, ca.

170.3, 170.0, 166.7, 166.3, 163.9, 163.8,

141.8, 141.5, 136.7, 135.8, 135.7, 133.6,

127.9, 127.8, 126.4, 125.9, 124.4, 124.3,

H-5’), 3.15-3.06 (m, 2H, 1 x

Nal^-CH₂), 2.94-2.90 (m,

1.5H, NCH₃), 2.22-2.12 (m, 1H, H-3’),

1.5H, CH₃), 1.04 (d, J= 6.9 Hz, 1.5H,

1:1 mixture of rotamers) δ 174.8, 172.1,

158.9, 158.7, 158.4, 158.2, 157.6, 151.2,

132.4, 128.6, 128.5, 128.3, 128.1, 128.0,

121.4, 119.1, 119.0, 116.7, 111.9, 110.1,

102.4, 102.0, 92.6, 92.5, 79.2, 79.1, 74.6, 74.5, 55.4, 55.0, 54.9, 54.8, 54.0, 53.9, 53.6,

51.1, 44.5, 44.3, 38.8, 38.6, 36.3, 36.1, 29.4, 28.4, 27.9, 15.2, 14.4; LRMS [M+H]⁺

940.0. These data are in agreement with those reported by Tran et a/.^[4]

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Results of assays/pharmacoloqical testing

Whole cell inhibition assays Mtb

Materials and methods

Mtb H37Rv (ATCC 27294) and 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 (OD₆oo 0.4-0.8) for use in the inhibition assays.

The effect of these analogues against the growth of Mtb H37Rv was measured by a resazurin reduction microplate assay, using the procedure previously described by Taneja and Tyagi.² Mtb grown to mid-exponential phase (OD₆oo 0.4-0.8) was diluted to OD₆oo 0.002 in 7H9S media (Middlebrook 7H9 with 10% ADC, 0.05% glycerol, 0.05% Tween80, 1% tryptone); 96-well microtiter plates were set up with 100 pL inhibitors, serially diluted into 7H9S. Diluted Mtb (100 pL, representing ~2 *10⁴ 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 pL) and 20% Tween-80 (12.5 pL) 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 MIC50 values.

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Results for these assays are shown in Figures 1 to 21, and below in Table 1.

Table 1

Structure	MICso(nM)	MIC₉₉(nM)
Vnh₂ Q^h ° Η H °'Ύ% ?5·ΌΗ HO'^Y ^N γ ^N N*y ^N ^hCT o	90

°Ynh₂ 9 η h 9 Ύ% ?Λ.„_Οη 0 0 ^ΗΝΛ O	40	200

°Ϋνη₂ Ct 0 Η H ftV'h oh 0 0 ^HNA O	22	89
''J
°ν^ΛΝΗ₂ ζΥ γ_ΝΧγ,..<5 ^HNCX O	115	> 500

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«Λ A 9 η h 9 9A.„ ° A 0 0 o	200	625
cA HN fl	Vnh₂ A •VX^A·'⁰ ⁰ X)°	9	166
o
erf HN T	ofl Ynh °Y NH₂ <_νΛ ⁰ A°	57	> 500
o
	Η P VnH₂ r?NH H H ° T% oft ° ⁿ..ⁿ _n-..ⁿ .oh 0 / ^H 0
oA HN JO	100	312
o	°A WOMe
	OMe

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oUt ^nh 0 Η Η ξΎ% ?AOH vAⁿtⁿMⁿ-^{v oh} /L/* ° A ° ^HNX O	22	333
Anh₂ A ° H H °'A ?Λ ° η ο Aj-^N γ ^N n *qf ^N A/ A 0 ^Ξ\Η 0 HN/T (Γ	180	800
οΛ Ah '_ν_4 0 \|_\| LI 0 Υ% ^ΟΛ ⁰ z\J O \J( O ^HNJT c/S	60	160
°A Cr 0 Η Η ?Λ. ° N N A _N*Y ^N X'^ A ° ΑΛ ⁰ HN/T (Γ A\	30	76

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-·ν -^γ °'^0H ^Hrsp) R)	89	229
i A° Vnh₂ Q^H Ο Η H 9'T% ίΛ-ΟΗ ^N γ ^N _N Αγ N A/ A 0 Κΰ 0 ^HN ΖΓ Ϊ/Λ	250	300
η Λ Vnh. Q^h ° Η Η θ'Λ o\oh rA^V^O'^Y' Ν N N*]f ^''' o kJt o ^hnCa TYy	15	39
°Λη₂ A o _H η ⁰ Ύ% °Λ ⁰ _(Γ^Ν-\'^ΝΥ^Νυ¹ΝΑ^Ν-''^^> ^0ΗU hJ o kA o ^hnCa	99	328

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A Cr 9 Η H 9 ? >..,_Oh ° χΛ ⁰ ^HNu T / S	11	44
A c? Ο Η Η ?A. ° ΥΥ^Ο^Υ ^N γ ^N A Ν*γ ^N ^⁷/A ^{0 0} ^HNX O	3	15.6

Intracellular Killing Assay

Materials

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; SigmaAldrich).

Susceptibility testing using Mtb-infected macrophage culture

Mtb H37Ra strain (ATCC 25177) was used to infect a human macrophage-like cell line (THP-1; ATCC TIB-202). Briefly, THP-1 stocks were maintained at a culture density between 1x10⁵ and 1x10⁶ cells/mL in RPM 1-1640 media (with phenol red, 25 mM HEPES and 2 mM L-glutamine; Gibco) supplemented with 10% FBS (FBS-500; Scientifix-life) and 0.05 mM β-mercaptoethanol (M7522; Sigma-Aldrich).

THP-1 cells were plated in 96 well tissue culture plates (Costar 3903; Coming) at a density of 1x10⁵ 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% CO2.

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A cell suspension of sonicated Mtb H37Ra in RPM 1-1640 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% CO₂. Supernatant was then removed from all wells, THP-1 cells were washed with 200 pl_ 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% CO₂.

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% CO₂, tissue culture medium containing the test compound was removed from the wells; the cells were washed with 200 μΙ_ 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 7H11/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 Figures 22-24.

Growth Inhibition Screen Against a Panel of Gram Positive and Gram Negative Bacterial Strains

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

Materials and methods

The effect of analogues against 15 clinically relevant Gram-positive and Gramnegative bacteria strains was evaluated using previously published procedure by Wong et al³ Specifically, the screening panel consisted of six Gram-positive strains (BSL1:

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Bacillus subtilis 168, Staphylococcus epidermis [ATCC 14990], Enterococcus faecium [ATCC 6569], Listeria ivanovii [BAA-139]; BSL2: S. aureus [ATCC 29213], methicillinresistant S. aureus (MRSA) [BAA-44] and nine Gram-negative strains (BSL1: Escherichia coli K12 [BW25113], Acinetobacter baumanii [NCIMB 12457], Enterobacter aerogenes [ATCC 35029], Ochrobactrum anthropi [ATCC 49687], Providencia alcalifaciens [ATCC 9886]; BSL2: Yersinia pseudotuberculosis [IP2666 pl Bl], Pseudomonas aeruginosa [ATCC 27835], Salmonella typhimurium LT2, Vibrio cholerae 01 [biotype El Tor A1552],

All staphylococcal strains, L. ivanovii and E. faecium were cultured in 10 mL of tryptic soy broth (17 g tryptone, 3 g soytone, 2.5 g dextrose, 5 g NaCI and 2.5 g dipotassium phosphate in 1 L distilled water; pH 7.5). P. alcalifaciens, 0. anthropi, E. aerogenes and A. baumanii were grown in nutrient broth (Difco, USA) while B. subtilis, E. coli, V. cholerae, S. typhimurium, P. aeruginosa and Y. 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).

Overnight saturated cell cultures of pathogenic strains were diluted 1:1000 with fresh media and 30 pL of culture dispensed into each well of sterile clear 384-well plates. 300 nL of DMSO prefraction stock solutions were pinned into screening plates using a Perkin Elmer Janus MDT robot. After inoculation, screening plates were stacked in a plate reader/shaker (Perkin Elmer EnVision) and OD₆oo readings taken once per hour for 24 hr. Computer generated growth curves for serially diluted pure compounds (with the top final screening concentration of 100 pM) were used to determine MIC values by correlating the OD₆oo reading at the pre-exponential phase of the bacteria to the concentrations in individual wells.

Activity of analogues against a panel of Gram-positive and Gram-negative bacteria

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Table 2. Preliminary data on activity of analogues 4-17 against a panel of Grampositive and Gram-negative bacteria.

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| Y. pseudotuberculosis

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Table 3. Preliminary data on activity of analogues against a panel of Grampositive and Gram-negative bacteria.

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MurX enzyme inhibition

Materials

UDP-/V-acetyl-D-glucosamine [glucosamine-¹⁴C(U)] ([¹⁴C] UDP-GIcNAc, Specific activity 300 mCi/mmol) was obtained from American Radio Chemicals, UDP-MurNAcpentapeptide was purchased from BacWAN, University of Warwick, Coventry, UK (www.warvfeick.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 F254 plates were procured from Merck (Germany). All other chemicals used were at least analytical grade and were obtained from Sigma-Aldrich. Mtb me² 6230 was a generous gift from Dr. William Jacobs, Albert Einstein College of Medicine, New York.

Methods: membrane preparation from Mtb me² 6230

Mtb me² 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 MgCl2, 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). 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).

Preparation of dansylated UDP-MurNAc pentapeptide

Synthesis and purification UDP-MurNAc-pentapeptide was achieved as described⁴ 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.Cl pH 9 to dansyl chloride before rotary evaporation to remove solvents. Dried products were resuspended in 1 mL sterile water and purified by size exclusion chromatography

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PCT/AU2017/051394 on a Superdex peptide 10/300 column (GE Healthcare) pre equilibrated with 1.5 CV 0.1 M ammonium bicarbonate. Dansylated UDP-MurNAc-pentapeptide is the first peak to elute in this procedure with a characteristic absorbance at 340 nm and 280nm. Fractions containing the required product were freeze dried 4 times and resuspended in a small volume of sterile water. Quantification and purity were confirmed by absorbance and mass spectrometry respectively.

Radiochemical MurX inhibition assay

Assay mixtures (200 pL) contained 50 mM MOPS pH 7.9, 5 mM MgCh 5 mM DTT, 10% glycerol (v/v), 0.1% CHAPS, 100 μΜ ATP, 25 μΜ UDP-MurNAcpentapeptide, 0.5 μΜ [¹⁴C] UDP-GIcNAc, 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 pg of Mtb me² 6230 membrane protein and incubated at 37 °C for 1 h. Reactions were stopped by the addition of 6 mL of chloroform/MeOH (2:1), followed by low speed centrifugation and the organic extract was moved to a second tube. Extracts were back washed twice [once with water (800 pL) and then with chloroform/MeOH/water (3:47:48)], evaporated to dryness under a nitrogen stream, and dissolved in chloroform/MeOH (2:1 v/v). An aliquot was subjected to liquid scintillation counting (LS 6500, Beckman Coulter); a second aliquot was subjected to thin layer chromatography (Silica gel 60 F254) developed in chloroform/MeOH/water/ammonium hydroxide (88:48:10:1). Distribution of radioactivity was detected by phosphorimaging (Typhoon TRIO, Amersham Biosciences) and quantified with ImageQuant TL v2005 software (Amersham Biosciences). IC₅₀ values were calculated by using GraFit Software (Version 5.0.13).

Inhibition of MurX activity by analogues at 200 nM

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Table 4. Inhibition of MurX by analogues 7-11

Compound	Ri	% MurX inhibition at 200 nM
7		27%
8		5%
9		27%
10	/J	41%
11		14%

Table 5. Inhibition of MurX by analogues 21-28 and 30-37

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Compound	Ri	r₂	% MurX inhibition at 200 nM
21	H	T	46%
22	H	X 1	26%
23	H		59%
24	H	-Kl·	41%
25	H	Hl·	100%
26	H	^HOx	40%
27	H	^co₂h	34%
28	H	nh₂	47%
30	H	AZA¹ ³	100%
31	H		40%
32	H		52%
33	H	^NH	52%
34	H		51%
36	H		100%
37	hAAv		100%

Refer to Figures 25-28.

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Fluorescence-based MurX inhibition assay: inhibition of MurX activity by analogues 25, 36 and 37

Continuous fluorescence MurX assays, typically 100 pL in volume, were carried out as previously described⁵ (with minor modification) in vitro at 25 °C in an assay buffer consisting of 83 mM Tris pH 7.5, 21 mM MgCk, 6% glycerol, 0.1% TritonX-100, 15 μΜ dansylated UDP-MurNAc-pentapeptide, 40 pg/mL polyisoprenyl phosphate and varying concentrations of inhibitor. Reactions were initiated by the addition of 60-70 pg of Mtb me² 6230 membrane protein and fluorescence was monitored at 340 and 530 nm for excitation and emission, respectively. Assays were carried out in duplicate and IC₅₀values were calculated using GraFit Software (Version 5.0.13).

Refer to Figures 29 to 33.

Preliminary in vitro DMPK studies

Stability in human and mouse plasma

Methods: Pooled human and Swiss outbred mouse plasma samples were thawed and spiked with test compound solutions prepared in DMSO/acetonitrile/water (20:40:40) to provide a final compound concentration of 1000 ng/mL and final DMSO and acetonitrile concentrations of 0.2% and 0.4% (v/v), respectively. Plasma was vortex mixed and aliquots (50 pL) were transferred to fresh microcentrifuge tubes and incubated 37 °C. At various time points over the 6 h incubation period, duplicate plasma samples were removed and immediately snap-frozen in dry ice. All samples were stored frozen at -80 °C until analysis by LC-MS. Samples were processed by protein precipitation using a 2-fold excess of acetonitrile followed by centrifugation. Analysis of the supernate was conducted using a Waters (Milford, MA) Acquity UPLC coupled to a Waters Micromass Quattro Premier mass spectrometer operated in positive electrospray ionisation mode with multiple reaction monitoring. The cone voltage was 45 V, collision energy was 30, 30 and 40 V for 25, 36, and 37, respectively, and m/z transitions were 782.30 > 126.06 (25), 826.32 > 170.16 (36) and 888.39 > 126.06 (37). Processed samples (3 pL) were injected onto a Supelco Ascentis Express RP Amide column (50x2.1 mm, 2.7 pm) and analytes eluted using a water/acetonitrile (each containing 0.05% formic acid) gradient over 4 min with a flow rate was 0.4

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Table 6. Measured concentrations of 25, 36, 37 in human and mouse plasma over a 6 hour incubation at 37°C

Human Plasma
Sampling time	Replicate	Measured cone. (ng/mL)
iiiiiiiiiii		llllllllillllll
2 min	1	826.7	808.7	964.3
2	818.8	928.1	1092.6
0.5 h	1	943.7	937.9	1066.6
2	853.3	908.9	1098.7
1 h	1	852.1	899.6	1014.9
2	898.0	954.7	1058.1
2h	1	803.9	916.8	1025.1
2	735.1	890.9	1034.6
4h	1	841.9	883.3	1024.2
2	922.5	892.2	1117.3
6h	1	871.0	893.7	1002.9
2	904.5	900.1	1014.2
Mouse Plasma
Sampling time	Replicate	Measured cone. (ng/mL)
llllllilllllll	llllllilllllll	llllllllillllll!
2 min	1	849.7	960.5	948.7
2	902.2	985.7	859.0
0.5 h	1	931.1	980.2	948.2
2	867.8	1030.6	996.1
1 h	1	899.8	1043.2	933.2
2	926.8	1016.3	938.7
2h	1	871.1	1019.1	996.2
2	880.9	962.5	958.0
4h	1	886.1	957.5	918.2
2	889.4	996.6	895.1
6h	1	881.5	929.1	878.5
2	878.0	928.4	905.1

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Table 7. In vitro stability data for 71, 78, 79 in human and mouse plasma.

Compound	Sample (min)	Human Plasma	Mouse Plasma
Mean ± SD (ng/mL)	% Remaining*	Mean ± SD (ng/mL)	% Remaining*
71	2	873 ± 95	100	845 ± 65	100
10	782 ± 57	89.6	796 ± 65	94.2
30	805 ±81	92.2	858 ± 63	101.5
60	785 ± 25	90.0	856 ± 78	101.3
120	813	93.1	780 ± 73	92.3
240	805 ±112	92.3	826 ± 36	97.7
78	2	1018.4 ± 12.8	115.2	912.9 ±7.5	95.8
10	1016.7 ±24.9	115	899.2 ±11.8	94.4
30	971 ±29.1	109.8	856.4 ± 19.0	89.9
60	1001.9 ±63.8	113.3	849.1 ± 14.4	89.1
120	958.5 ±26.3	108.4	809.0 ±6.7	84.9
240	958.5 ±26.3	108.4	809.0 ±6.7	84.9
79	2	790.5 ±97.1	100	957.0 ±26.5	100
10	869.3 ±35	110	943.0”	98.5
30	854.2 ±35.3	108.1	933.8 ± 17.9	97.6
60	846.2 ±46.0	107	897.1 ±28.8	93.7
120	880.0 ± 10.6	111.3	924.1 ±43.7	96.6
240	863.0 ±37.1	109.2	975.6 ±74.8	101.9

Data presented were mean and standard deviation from triplicate samples at each time 5 point.

* Percentage remaining was calculated relative to the measured concentration at the 2 minute time point # Mean of n=2 samples; one replicate was omitted as an outlier

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Stability in human and mouse liver microsomes

Methods: The metabolic stability was assessed by incubating each test compound (0.5 μΜ) in duplicate with human and mouse liver microsomes (XenoTech, Kansas City, KS) at 37 °C and 0.4 mg/mL microsomal protein. The metabolic reaction 5 was initiated by the addition of an NADPH-regenerating system and quenched at various time points over a 60 minute incubation period by the addition of acetonitrile containing diazepam as internal standard. Control samples (containing no NADPH) were included and quenched at 2, 30 and 60 minutes to monitor for potential degradation in the absence of cofactor. Samples were centrifuged and the supernatant 10 analysed by LCMS. Analysis was conducted using a Waters Acquity UPLC coupled to a Waters Xevo G2 QTOF mass spectrometer operated in positive electrospray ionisation MS^e mode with a cone voltage of 30V. Samples (5 pL) were injected onto an Ascentis Express Amide column (50 x 2.1 mm, 2.7 pm) and eluted using a water/acetonitrile (both containing 0.05% formic acid) gradient over 4 min at a flow rate of 0.4 mL/min. 15 Degradation rate half-lives and in vitro intrinsic clearance values were determined from the first order degradation profiles.

Table 8. Stability in human and mouse liver micosomes

Compound	Species	Average half-life (min)	Intrinsic Clearance (pL/min/mg protein)
25	Human	>247	<7
Mouse	>247	<7
36	Human	>247	<7
Mouse	166	10
37	Human	174	10
Mouse	233	7

Table 9. Stability in human and mouse liver microsomes.

Compound	Species	Average Half-life (min)	Intrinsic Clearance (pL/min/mg protein)
71	Human	50	35
Mouse	>255	< 7
78	Human	45	38
Mouse	208	8
79	Human	45	38
Mouse	>255	< 7

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Table 10. MICs of 71 and 79 against other mycobacteria.

Strain	N	IC(nM)
71	79
M. abscessus (RA 16)	22528	12800
M. abscessus subsp. abscessus	22528	25600
M. fortuitum (RA 17)	1408	800
M fortuitum PN	704	1600
M. avium (CA 6)	704	800

1. Boojamra, C. G. et al. J. Am. Chem. Soc. 123, 870-874 (2001).

2. Taneja, N. K.; Tyagi, J. S. J. Antimicrob. Chemother. 60, 288-293 (2007).

3. Wong, Weng R.; etal. Chem. Biol. 19, 1483-1495 (2012).

4. Lloyd, A. J. et al J. Biol. Chem. 283, 6402-6417 (2008).

5. Mihalyi, A. PhD Thesis, University of Warwick (2014).

Claims

1. A compound according to Formula I

Formula I

5 or a salt, solvate, polymorph or prodrug thereof;

wherein

Ri is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl,

10 carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; C1-C6 alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C-i-C₆ alkylcarboxyamide, C₃-C₇ cycloalkyl; (C1-C4 alkyl)C₃-C₇ cycloalkyl, C₃-C₇ heterocyclyl; (C-i-C4alkyl)C₃-C₇ heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-r C₄aralkyl, Ci-C₄aralkoxy, C1-C4 aralkylamino, heteroaryl, (Ci-C₄alkyl)heteroaryl,

15 amino, carbamoyl, aminosulfonyl, ureido and aroyl;

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R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, Ο-Οβ alkylamino; C1-C6 alkoxy; Ci-C₆ alkylthio; Ci-C₆ haloalkyl, Ci-C₆ haloalkoxy, Ci-C₆ hydroxyalkyl, Ci-C₆alkylcarboxy, Ο-Οβ alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C? cycloalkyl, C3-C7 heterocyclyl; (C-i-C4alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-r C₄aralkyl, C-i-C₄aralkoxy, C1-C4 aralkylamino, heteroaryl, (Ci-C₄alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R3 is -C(O)R₄ or -CH₂R_4;; and

R₄ is selected from the group consisting of hydroxyl or optionally substituted: C1-C15 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; (C1-C4 alkyl)C3-C7 cycloalkyl, C3-C7 heterocyclyl; (C-i-C₄alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C1-C4 aralkyl, C1-C4 aralkoxy, C1-C4 aralkylamino, heteroaryl, (C-i-C₄alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl.

with the proviso that:

when------is a double bond, R₃ is not -COOH, and when------is a single bond and R1 is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C3-C4 alkylamino, -CH(CH₃)OH, -CH₂COOH, CH₂C(O)C(CH₃)3, -CH(OH)CH₃ or-CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl, -(CH₂)₄NH₂, -CH₂CH₂SCH₃, -CH₂CH₂S(O)CH₃, Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₃ is not COOH.

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2. A compound according to Formula II:

Formula II or a salt, solvate, polymorph or prodrug thereof;

5 wherein

R-ι is selected from the group consisting of: hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; C1-C6

10 alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6 alkylcarboxy, C1-C6 alkylcarboxyamide, C3-C7 cycloalkyl; (C1-C4 alkyl)C3-C? cycloalkyl, C3-C7 heterocyclyl; (Ci-C₄alkyl)C₃-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-r C₄aralkyl, C-i-C₄aralkoxy, C-i-C₄ aralkylamino, heteroaryl, (C-i-C₄alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

15 R₂ is selected from the group consisting of hydrogen, halo, mercapto, hydroxyl, acyl, carboxy, nitro, cyano, or optionally substituted: C1-C6 alkyl, C1-C6 alkylamino; C1-C6 alkoxy; C1-C6 alkylthio; C1-C6 haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, C1-C6

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C3-C7 heterocyclyl; (C-i-C₄alkyl)C3-C7 heterocyclyl, aryl, aryloxy, arylamino, arylthio, C-r

C₄aralkyl, C-i-C₄aralkoxy, C1-C4 aralkylamino, heteroaryl, (C-i-C₄alkyl)heteroaryl, amino, carbamoyl, aminosulfonyl, ureido and aroyl;

R₃ is -C(O)R₄ or -CH₂R_4;; and

with the proviso that:

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3. A compound according to claim 2, wherein the compound has the Formula III:

Formula III or a salt, solvate, polymorph or prodrug thereof;

5 wherein R-ι, R₂ and R₄ are defined as in claim 2, with the proviso that:

when------is a double bond, R₄ is not -OH, and when------is a single bond and Ri is hydrogen, methyl, isobutyl, benzyl, fluoro- or hydroxy-substituted benzyl, -C3-C₄ alkylamino, -CH(CH₃)OH, -CH₂COOH, 10 CH₂C(O)C(CH₃)₃, -CH(OH)CH₃ or-CH₂CH₂SCH₃; and R₂ is methyl, isopropyl, isobutyl,

-(CH₂)₄NH₂, -CH₂CH₂SCH_3i -CH₂CH₂S(O)CH_3i Ci-C₂ aralkyl, fluoro-substituted benzyl, -CF₃-substituted benzyl, aryl-substituted benzyl, naphthyl or -CH₂-cyclohexyl; R₄ is not OH.

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4. A compound according to claim 2, wherein the compound has the Formula IV:

Formula IV or a salt, solvate, polymorph or prodrug thereof;

5 wherein R-ι, R₂ and R₄ are defined as in claim 2.

5. A compound according to any one of claims 1 to 4, wherein is a double bond.

6. A compound according to any one of claims 1 to 4, wherein Ri is selected from the group consisting of hydrogen or optionally substituted: C-i-C₆ alkyl, C-i-C₂ aralkyl, optionally substituted (C-i-C₂alkyl)heteroaryl, C1-C6 alkylamino; C1-C6 alkyloxy, C1-C6 10 alkylcarboxy, C1-C6 hydroxyalkyl.

7. A compound according to claim 6, wherein Ri is selected from the group consisting of hydrogen, methyl, hydroxyl-substituted benzyl, naphthyl, C₂-C₄ alkylamino, cyclohexyl, CH₂-cyclohexyl or-CH(OH)CH₃.

8. A compound according to any one of claims 1 to 7, wherein R₂ is selected from the

15 group consisting of optionally substituted: C1-C6 alkyl, (C-i-C₂ alkyl)C₃-C? cycloalkyl, C-r C₄ aralkyl and C-i-C₆ alkylthio.

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9. A compound according to claim 8, wherein R₂ is selected from the group consisting of isopropyl, -CH₂-naphthyl, -CH₂-cyclohexyl, -CH₂CH₂SCH₃; -CH₂CH₂S(O)CH₃ and

10. A compound according to any one of claims 1 to 9, wherein R₃ is -C(O)R4.

5

11. A compound according to any one of claims 1 to 10, wherein R₄ is selected from the group consisting of optionally substituted: C1-C15 alkyloxy, C1-C4 aralkyl; C1-C15 alkylamino; C1-C4 aralkoxy, C1-C4 aralkylamino, C1-C15 alkylamino.

12. A compound according to claim 11, wherein R₄ is selected from the group consisting of methoxy, hexoxy, dodecanyloxy, hydroxy, -CH₂C(CH₃)₃, -O-benzyl, -NH-benzyl, -

10 NH-benzyl, hexylamino.

13. A compound according to claim 12, wherein R₄ is selected from the group consisting of -CH₂C(CH₃)₃, -O-benzyl, -NH-benzyl and hexylamino.

14. A compound according to claim 1 or claim 2, wherein the compound is selected from the group consisting of: