Novel dnan-targeting, Anti-tubercular mycoplanecin derivatives

Abstract

The present invention relates to novel mycoplanecine derivatives of formula (I) and their use in the treatment of tuberculosis. (I)

Description

[0001] Novel DnaN-targeting, anti-tubercular mycoplanecin derivatives

[0002] The present invention relates to novel mycoplanecin derivatives and their use in the treatment of tuberculosis.

[0003] Tuberculosis (TB) continues to present a global health obstacle as indicated by the 2022 WHO Global TB Report, with an estimated incidence of 10.6 million new cases in 2021, an increase of 4.5% from 10.1 million in 2020. Additionally, the increasing prevalence of multidrug-resistant Mycobacterium tuberculosis (MDR-Mtb) necessitates the development of novel drugs against TB. Currently, only three recently developed novel anti-tubercular agents (bedaquiline (BDQ; Janssen Therapeutics), delamanid (Otsuka Pharmaceutical Co.), and pretomanid (TB Alliance)) have obtained marketing authorization, marking the initial approval of new TB drugs in more than four decades. However, even for these new TB drugs, resistance is on the rise and report showed that bedaquiline resistance acquisition was documented in > 15% of all MDR-TB patients receiving bedaquiline as part of their treatment regimen. Consequently, new drugs addressing novel targets in Mycobacterium tuberculosis (Mtb) are desired.

[0004] DNA polymerase III sliding clamp (DnaN) is considered as an attractive (and clinically validated) target in Mtb since compounds binding to the DNA sliding clamp have a very low risk of resistance development as mutants observed so far are highly impaired in their growth and show a reversible phenotype.

[0005] DnaN-targeting compounds have been disclosed in Fu, C., et al. Elucidation of unusual biosynthesis and DnaN-targeting mode of action of potent anti-tuberculosis antibiotics Mycoplanecins. Nat. Commun. 15, 791 (2024); https: / / doi.org / 10.1038 / s41467-024-44953-5 (Mycoplanecins), and in A. Kling et al. Targeting DnaN for tuberculosis therapy using novel griselimycins. Science 348, 1106-1112 (2015); DOI: 10.1126 / science.aaa4690 (Griselimycins). The total synthesis of Mycoplanecin A is disclosed in E. Papadopoulos et al. Org. Lett. 27, 9061–9065 (2025); https: / / doi.org / 10.1021 / acs.orglett.5c02803.Development of cyclohexyl griselimycin (CGM) was discontinued. One possible reason for this discontinuation is its potent inhibition of the bile salt export pump (BSEP) as a human off-target associated with the risk of e.g., liver toxicity. BSEP (ABCB11) is a key transporter located on the canalicular (bile-facing) membrane of hepatocytes that actively exports bile acids from liver cells into bile, thereby maintaining bile flow and preventing intracellular accumulation of bile acids. Inhibition of BSEP can result in bile acid retention, cholestasis, and may contribute to drug-induced liver injury.

[0006] In was an object of the present invention to provide novel compounds for use in the treatment of tuberculosis. It was a further object to provide compounds targeting DnaN.

[0007] The present invention provides compounds of formula (I):

[0008]

[0009] (I)

[0010] wherein

[0011] X is SO2 or CR4R4a;

[0012] X1is SO2 or CR7R7a;X2is SO2 or CR2R2a;

[0013] R1is a group of formula -C(=O)-C(=O)-R1a; -C(=O)-R1a; or -C(=O)-O-R1a;

[0014] R1ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted;

[0015] R2is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R2ais hydrogen; or fluorine; or R2and R2atogether with the carbon atom to which they are bound, form a cyclopropyl group;

[0016] R3is a C1-6 alkyl group;

[0017] R4is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R4ais hydrogen; or fluorine; or R4and R4atogether with the carbon atom to which they are bound, form a cyclopropyl group;

[0018] R5is a C1-6 alkyl group;

[0019] R6is a C1-6 alkyl group; a group of formula -(CH2)4-N3; or a group of the following formula:

[0020]

[0021] R6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; and

[0022] R7is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R7ais hydrogen; or fluorine; or R7and R7atogether with the carbon atom to which they are bound, form a cyclopropyl group;or a salt thereof.

[0023] According to a preferred embodiment, the present invention provides compounds of formula (la):

[0024]

[0025] (la)

[0026] wherein

[0027] R1is a group of formula -C(=O)-C(=O)-R1a; -C(=O)-R1a; or -C(=O)-O-R1a;

[0028] R1ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted;

[0029] R2is hydrogen; a C1-4 alkyl group; a C1-4 alkyloxy group; or a C1-4 alkenyloxy group;

[0030] R3is a C1-6alkyl group;

[0031] R4is hydrogen; a methyl group; a C1-4 alkyloxy group; or a cyclohexyl group;R5is a C1-6alkyl group;

[0032] R6is a C1-6alkyl group; a group of formula –(CH2)4-N3; or a group of the following formula:

[0033]

[0034] N=N

[0035] R6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; and

[0036] R7is hydrogen; or a methyl group;

[0037] or a salt thereof.

[0038] Preferably, R1ais a C1-12 alkyl group; a C1-12 alkenyl group; a C3-7 cycloalkyl group; a C1-6alkyl-C3-7cycloalkyl group; an optionally substituted phenyl group; a C1-6alkyl-phenyl group, wherein the phenyl may optionally be substituted; or a C1-6alkyl-heterocycloalkyl group, wherein the heterocycloalkyl group contains 5 or 6 ring atoms that are independently selected from C, N, 0 and S.

[0039] Further preferably, R1ais a C1-12 alkyl group; a C1-12 alkenyl group; a C3-7 cycloalkyl group; a C1-6alkyl-C3-7cycloalkyl group; or an optionally substituted phenyl group.

[0040] Further preferably, R1ais a C6-12alkyl group; or a C1-6alkyl-C3-7cycloalkyl group.

[0041] Moreover preferably, R1ais a C1-9 alkyl group; a cyclohexyl group; a group of formula -CH2-CH2-CH2-cyclohexyl; or a 4-fluorophenyl group.

[0042] Further preferably, R1ais a bicyclo(1.1.1)pentane group.Further preferably, R1ais an ethyl group.

[0043] Moreover preferably, R1ais group of formula -CH2-CH=CH2.

[0044] Further preferably, R1a group of formula -C(=O)-C(=O)-R1a.

[0045] Moreover preferably, R1a group of formula -C(=O)-C(=O)-R1a, wherein R1ais an ethyl group.

[0046] Further preferably, R1is a group of formula -C(=O)-R1a.

[0047] Moreover preferably, R1is a group of formula -C(=O)-R1a, wherein R1ais a C1-9 alkyl group; a cyclohexyl group; a group of formula -CH2-CH2-CH2-cyclohexyl; or a 4-fluorophenyl group.

[0048] Further preferably, R1is a group of formula -C(=O)-O-R1a.

[0049] Moreover preferably, R1is a group of formula -C(=O)-O-R1a, wherein R1ais group of formula -CH2-CH=CH2.

[0050] Further preferably, R2is hydrogen; a methyl group; an ethyl group; a methoxy group; or an ethoxy group.

[0051] Especially preferably, R2is a methyl group; or an ethyl group.

[0052] Further preferably, R2is an ethyl group; a methoxy group; or an ethoxy group.

[0053] Moreover preferably, R3is an isobutyl group.

[0054] Further preferably, R3is an isopropyl group.

[0055] Moreover preferably, R1is a group of formula -C(=O)-R1a, and R3is an isobutyl group.Further preferably, R5is a group of formula -CH(CH3)2 or -(CH2)3CH3.

[0056] Moreover preferably, R6is a C1-6 alkyl group.

[0057] Further preferably, R6is a group of formula -CH(CH3)2.

[0058] Moreover preferably, R6is a group of formula -(CH2)4-N3.

[0059] Further preferably, R6is a group of the following formula:

[0060]

[0061] Further preferably, R6ais a group of the following formula:

[0062]

[0063] Moreover preferably, R7is hydrogen.

[0064] Further preferably, R7is a methyl group.

[0065] Moreover preferably, R4is a methyl group; or a cyclohexyl group.

[0066] Further preferably, R4is a cyclohexyl group.

[0067] According to a further preferred embodiment, the present invention provides compounds of formula (lb):

[0068]

[0069] (lb) wherein

[0070] R1is a group of formula -C(=O)-R1a;

[0071] R1ais a C1-12alkyl group; or a C1-6alkyl-C3-7cycloalkyl group (especially a C6-12alkyl group; or a C1-6alkyl-C3-7cycloalkyl group);

[0072] R2is a C1-4 alkyl group; or a C1-4 alkyloxy group;

[0073] R3is an isobutyl group (i.e., a group of formula -CH2CH(CH3)2);

[0074] R4is a cyclohexyl group;

[0075] R5is an isopropyl group (i.e., a group of formula -CH(CH3)2);

[0076] R6is an isopropyl group; a group of formula -(CH2)4-N3; or a group of the following formula:

[0077]

[0078] R6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; and

[0079] R7is a methyl group;

[0080] or a salt thereof.

[0081] According to a further embodiment of the present invention, the following compounds are excluded from the present invention:

[0082] Pro-8

[0083]

[0084] H Griselimycin (GM)

[0085]

[0086] Me Methylgriselimycin (MGM)

[0087]

[0088] Cyclohexyl Cyclohexylgriselimycin (CGM)

[0089] According to a further embodiment of the present invention, the following compounds are excluded from the present invention: Mycoplanecin A, Mycoplanecin B, and Mycoplanecin D (disclosed in Fu, C., et al. Elucidation of unusual biosynthesis and DnaN-targeting mode of action of potent anti-tuberculosis antibiotics Mycoplanecins. Nat. Commun. 15, 791 (2024); https: / / doi.org / 10.1038 / s41467-024-44953-5).The most preferred compounds of the present invention are the compounds disclosed in the examples, or a salt thereof.

[0090] It is further preferred to combine the preferred embodiments of the present invention in any desired manner (e.g., any embodiment for R1amay be combined with any embodiment of R5).

[0091] The term "optionally substituted" refers to a group which is unsubstituted or substituted by one or more (especially by one, two or three; preferably by one or two) substituents.

[0092] If a group (e.g., group R1a) comprises more than one substituent, these substituents are independently selected, i.e., they may be the same or different.

[0093] If a group (e.g., group R1a) is substituted by a cyclic group, such as e.g., a cycloalkyl group or a heterocycloalkyl group, this cyclic group may be bonded to this group via a single or double bond or this cyclic group may be annulated or fused to said group.

[0094] Examples for substituents are fluorine, chlorine, bromine and iodine and OH, =0, SH, NH2, -SO3H, -SO2NH2, -COOH, -COOMe, -COMe (Ac), -NHS02Me, -SO2NMe2, -CH2NH2, -NHAc, -S02Me, -CONH2, -CN, -NHCONH2, -NHC(NH)NH2, –NOHCH3, -N3and -NO2 groups. Further examples of substituents are C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 heteroalkyl, C3-C18 cycloalkyl, C1-C17 heterocycloalkyl, C4-C20 alkylcycloalkyl, C1-C19 heteroalkylcycloalkyl, C6-C18aryl, C1-C17heteroaryl, C7-C20aralkyl and C1-C19 heteroaralkyl groups; especially C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6heteroalkyl, C3-C10cycloalkyl, C1-C9heterocycloalkyl, C4-C12alkylcycloalkyl, C1-C11heteroalkylcycloalkyl, C6-C10aryl, C1-C9heteroaryl, C7-C12aralkyl and C1-C11heteroaralkyl groups, further preferably C1-C6alkyl and C1-C6heteroalkyl groups.

[0095] Preferred substituents are halogen atoms (e.g., F, Cl, Br, I) and groups of formula -OH, =0, -O-C1-6 alkyl (e.g., -OMe, -OEt, -O-nPr, -O- / Pr, -0-nBu, -O- / Bu and -O-fBu), -NH2, -NHC1-6alkyl, -N(C1-6alkyl)2, -COOH, -COO-C1-6 alkyl (e.g., -COOMe), -CO-C1-6alkyl (e.g., -COMe), -COCF3, -NHSO2Me, -SO2NMe2, -SO3H, -SO2NH2, -CONH2, -CH2NH2, -CN, -C1-6alkyl (e.g., -Me, -Et, -nPr, -iPr, -nBu, -iBu, -tBu and -CF3), -C1-6heteroalkyl, -SH, –S-CO-Ci-6 alkyl, -S-C1-6 alkyl, -NHAc, -NO2, -C≡CH, -NHCONH2, -SO2Me, -SO2CF3, phenyl, -CO-4-fluorophenyl, -C3-6 cycloalkyl (e.g. cyclopropyl, cyclobutyl) and heterocycloalkyl containing 3 to 6 ring atoms selected from C, N, S and 0.

[0096] Further preferred substituents are halogen atoms (e.g., F, Cl, Br) and groups of formula -OH, =0, -O-C1-6 alkyl (e.g., -OMe, -OEt, -O-nPr, -O- / Pr, -0-nBu, -O- / Bu and -O-fBu), -NH2, -NHC1-6 alkyl, -N(C1-6alkyl)2, -COOH, -COO-C1-6 alkyl (e.g., -COOMe), -CO-C1-6 alkyl (e.g., -COMe), -NHSO2Me, -SO2NMe2, -SO3H, -SO2NH2, -CONH2, -CH2NH2, -CN, -C1-6 alkyl (e.g., -Me, -Et, -nPr, - / Pr, -nBu, - / Bu, -fBu and -CF3), -SH, -S-CO-C1-6 alkyl, -S-C1-6 alkyl, -NHCOOfBu, -NHAc, phenyl, -NO2, -C≡CH, -NHCONH2, -S02Me and cyclopropyl.

[0097] Especially preferred substituents are selected from halogen, -NH2, -N3, -N+(CH3)3, -CH2NH2, -CH3, -COCH3, -OCOCH3, -N(CH3)-CO-CH3, -NH-CO-CH3, -COOH, -NO2, -OH, -NHCH3, -N(CH3)2, -SCH3, and -CH2NHCOCH3.

[0098] The suffix "-ene" like e.g., in "phenylene" refers to the corresponding divalent group.

[0099] The expression alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 20 carbon atoms, preferably from 1 to 15 carbon atoms, especially from 1 to 10 (e.g., 1, 2, 3 or 4) carbon atoms, for example a methyl (Me, CH3), ethyl (Et), n-propyl ( / ? Pr), / so-propyl ( / Pr), n-butyl (nBu), / so-butyl ( / Bu), sec-butyl (sBu), fe / Y-butyl (fBu), n-pentyl, / so-pentyl, n-hexyl, 2,2-dimethylbutyl or n-octyl group.

[0100] Especially preferred alkyl groups are C1-6 alkyl groups; moreover preferred alkyl groups are C1-4 alkyl groups.

[0101] The expression C1-12 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 12 carbon atoms. The expression C1-9 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 9 carbon atoms. The expression C1-6 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1 to 6 carbon atoms. The expression C1-4 alkyl refers to a saturated, straight-chain or branched hydrocarbon group that contains from 1to 4 carbon atoms. Examples are a methyl, ethyl, n-propyl, / so-propyl, n-butyl, / so-butyl, sec-butyl or te / Y-butyl group.

[0102] The expressions alkenyl and alkynyl refer to at least partially unsaturated, straight-chain or branched hydrocarbon groups that contain from 2 to 20 carbon atoms, preferably from 2 to 15 carbon atoms, especially from 2 to 10 (e.g., 2, 3 or 4) carbon atoms, for example an ethenyl (vinyl), propenyl (allyl), isopropenyl, butenyl, ethynyl (acetylenyl), propynyl (e.g., propargyl), butynyl, isoprenyl or hex-2-enyl group. Preferably, alkenyl groups have one or two (especially preferably one) double bond(s), and alkynyl groups have one or two (especially preferably one) triple bond(s).

[0103] Furthermore, the terms alkyl, alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl) such as, for example, a 2,2,2-trichloroethyl or a trifluoromethyl group.

[0104] The expression heteroalkyl refers to an alkyl, alkenyl or alkynyl group in which one or more (preferably 1 to 8; especially preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or by a SO or a SO2 group. The expression heteroalkyl furthermore refers to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide or alkoxycarbonyloxy. Furthermore, the term heteroalkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl).

[0105] Preferably, a heteroalkyl group contains from 1 to 12 carbon atoms and from 1 to 8 heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen). Especially preferably, a heteroalkyl group contains from 1 to 6 (e.g., 1, 2, 3 or 4) carbon atoms and 1, 2, 3 or 4 (especially 1, 2 or 3) heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen). The term C1-C10 heteroalkyl refers to a heteroalkyl group containing from 1 to 10 carbon atoms and 1, 2, 3, 4, 5 or 6 heteroatoms selected from 0, S and / or N (especially 0 and / or N). The term C1-C6 heteroalkyl refers to a heteroalkyl group containing from 1 to 6 carbon atoms and 1, 2, 3 or 4 heteroatomsselected from 0, S and / or N (especially 0 and / or N). The term C1-C4 heteroalkyl refers to a heteroalkyl group containing from 1 to 4 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and / or N (especially 0 and / or N).

[0106] Further preferably, the expression heteroalkyl refers to an alkyl group as defined above (straight-chain or branched) in which one or more (preferably 1 to 6; especially preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen, sulfur or nitrogen atom or a CO group or a SO group or a SO2 group; this group preferably contains from 1 to 6 (e.g.

[0107] 1, 2, 3 or 4) carbon atoms and 1, 2, 3 or 4 (especially 1, 2 or 3) heteroatoms selected from oxygen, nitrogen and sulfur (especially oxygen and nitrogen); this group may preferably be substituted by one or more (preferably 1 to 6; especially preferably 1, 2, 3 or 4) fluorine, chlorine, bromine or iodine atoms or OH, =0, SH, =S, NH2, =NH, N3, CN or NO2 groups.

[0108] Examples of heteroalkyl groups are groups of formulae: Ra-0-Ya-, Ra-S-Ya-, Ra-S0-Ya-, Ra-S02-Ya-, Ra-N(Rb)-S02-Ya-, Ra-S02-N(Rb)-Ya-, Ra-N(Rb)-Ya-, Ra-C0-Ya- Ra-0-C0-Ya-, Ra-C0-0-Ya-, Ra-C0-N(Rb)-Ya-, Ra-N(Rb)-C0-Ya-, Ra-0-C0-N(Rb)-Ya-, Ra-N(Rb)-C0-0-Ya-, Ra-N(Rb)-C0-N(Rc)-Ya-, Ra-0-C0-0-Ya-, Ra-N(Rb)-C(=NRd)-N(Rc)-Ya-, Ra-CS-Ya-, Ra-0-CS-Ya-, Ra-CS-0-Ya-, Ra-CS-N(Rb)-Ya-Ra-N(Rb)-CS-Ya-, Ra-0-CS-N(Rb)-Ya-, Ra-N(Rb)-CS-0-Ya-, Ra-N(Rb)-CS-N(Rc)-Ya-Ra-0-CS-0-Ya-, Ra-S-C0-Ya-, Ra-C0-S-Ya-, Ra-S-C0-N(Rb)-Ya-, Ra-N(Rb)-C0-S-Ya-, Ra-S-C0-0-Ya-, Ra-0-C0-S-Ya-, Ra-S-CO-S-Ya-, Ra-S-CS-Ya-, Ra-CS-S-Ya- Ra-S-CS-N(Rb)-Ya-, Ra-N(Rb)-CS-S-Ya-, Ra-S-CS-O-Ya-, Ra-O-CS-S-Ya-, wherein Rabeing a hydrogen atom, a C1-C6alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; Rbbeing a hydrogen atom, a C1-C6alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; Rcbeing a hydrogen atom, a C1-C6alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group; Rdbeing a hydrogen atom, a C1-C6alkyl, a C2-C6 alkenyl or a C2-C6 alkynyl group and Yabeing a bond, a C1-C6alkylene, a C2-C6 alkenylene or a C2-C6 alkynylene group, wherein each heteroalkyl group contains at least one carbon atom and one or more hydrogen atoms may be replaced by fluorine or chlorine atoms.

[0109] Specific examples of heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, / so-propyloxy, n-butoxy, te / Y-butyloxy, methoxymethyl,ethoxymethyl, -CH2CH2OH, -CH2OH, -SC Me, -NHAc, methoxyethyl, 1 -methoxyethyl, 1-ethoxyethyl, 2-methoxyethyl or 2-ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropylethylamino, methylamino methyl, ethylamino methyl, diisopropylamino ethyl, methylthio, ethylthio, isopropylthio, enol ether, dimethylamino methyl, dimethylamino ethyl, acetyl, propionyl, butyryloxy, acetyloxy, methoxycarbonyl, ethoxycarbonyl, propionyloxy, acetylamino or propionylamino, carboxymethyl, carboxyethyl or carboxypropyl, / V-ethyl-ZV-methylcarbamoyl or A / -methyl-carbamoyl. Further examples of heteroalkyl groups are nitrile (-CN), isonitrile, cyanate, thiocyanate, isocyanate, isothiocyanate and alkylnitrile groups.

[0110] The expression cycloalkyl refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group that contains one or more rings (preferably 1 or 2), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms. The expression cycloalkyl refers furthermore to groups in which one or more hydrogen atoms have been replaced by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, N3 or NO2 groups, thus, for example, cyclic ketones such as, for example, cyclohexanone, 2-cyclohexenone or cyclopentanone. Further specific examples of cycloalkyl groups are a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, propellane (e.g., [1.1,1]propellane), bicyclo(1.1.1)pentane, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl group. Preferably, the expression cycloalkyl refers to a saturated cyclic group that contains one or more rings (preferably 1 or 2), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms.

[0111] The expression heterocycloalkyl refers to a cycloalkyl group as defined above in which one or more (preferably 1, 2 or 3) ring carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a SO group or a SO2 group. A heterocycloalkyl group has preferably 1 or 2 ring(s) and 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably selected from C, 0, N and S). The expression heterocycloalkyl refers furthermore to groups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, N3 or NO2 groups. Examples are a piperidyl, prolinyl, imidazolidinyl, piperazinyl,morpholinyl (e.g., -N(CH2CH2)2O), urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl group and also lactames, lactones, cyclic imides and cyclic anhydrides.

[0112] The expression alkylcycloalkyl refers to groups that contain both cycloalkyl and alkyl, alkenyl or alkynyl groups in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynylcycloalkyl groups. An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two rings and from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and one or two alkyl, alkenyl or alkynyl groups (especially alkyl groups) having 1 or 2 to 6 carbon atoms.

[0113] The expression heteroalkylcycloalkyl refers to alkylcycloalkyl groups as defined above in which one or more (preferably 1, 2 or 3) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a SO group or a SO2 group. A heteroalkylcycloalkyl group preferably contains 1 or 2 rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups (especially alkyl or heteroalkyl groups) having from 1 or 2 to 6 carbon atoms. Examples of such groups are alkylheterocycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcycloalkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, the cyclic groups being saturated or mono-, di- or tri-unsaturated.

[0114] The expression aryl refers to an aromatic group that contains one or more rings and from 6 to 14 ring carbon atoms, preferably from 6 to 10 (especially 6) ring carbon atoms. The expression aryl refers furthermore to groups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH2, N3 or NO2 groups. Examples are the phenyl (Ph), naphthyl, biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl or 4-hydroxyphenyl group.

[0115] The expression heteroaryl refers to an aromatic group that contains one or more rings and from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms, comprising one or more (preferably 1, 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N). The expression heteroaryl refers furthermore togroups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, SH, N3, NH2 or NO2 groups. Examples are pyridyl (e.g. 4-pyridyl), imidazolyl (e.g. 2-imidazolyl), phenylpyrrolyl (e.g., 3-phenylpyrrolyl), thiazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxadiazolyl, thiadiazolyl, indolyl, indazolyl, tetrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, 4-hydroxypyridyl (4-pyridonyl), 3,4-hydroxypyridyl (3,4-pyridonyl), oxazolyl, isoxazolyl, triazolyl, tetrazolyl, isoxazolyl, indazolyl, indolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzthiazolyl, pyridazinyl, quinolinyl, isoquinolinyl, pyrrolyl, purinyl, carbazolyl, acridinyl, pyrimidyl, 2,3'-bifuryl, pyrazolyl (e.g., 3-pyrazolyl) and isoquinolinyl groups.

[0116] The expression aralkyl refers to groups containing both aryl and also alkyl, alkenyl, alkynyl and / or cycloalkyl groups in accordance with the above definitions, such as, for example, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl groups. Specific examples of aralkyls are phenylcyclopentyl, cyclohexylphenyl as well as groups derived from toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, 1 / - / -indene, tetraline, dihydronaphthalene, indanone, cumene, fluorene and indane. An aralkyl group preferably contains one or two aromatic ring systems (especially 1 or 2 rings), each containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and / or alkynyl groups containing from 1 or 2 to 6 carbon atoms and / or a cycloalkyl group containing 3, 4, 5, 6 or 7 ring carbon atoms.

[0117] The expression heteroaralkyl refers to groups containing both aryl and / or heteroaryl groups and also alkyl, alkenyl, alkynyl and / or heteroalkyl and / or cycloalkyl and / or heterocycloalkyl groups in accordance with the above definitions. A heteroaralkyl group comprises one or more heteroatoms (preferably selected from 0, S or N). A heteroaralkyl group preferably contains one or two aromatic ring systems (especially 1 or 2 rings), each containing from 5 or 6 to 9 or 10 ring atoms (preferably selected from C, N, 0 and S) and one or two alkyl, alkenyl and / or alkynyl groups containing 1 or 2 to 6 carbon atoms and / or one or two heteroalkyl groups containing 1 to 6 carbon atoms and 1, 2 or 3 heteroatoms selected from 0, S and N and / or one or two cycloalkyl groups each containing 3, 4, 5, 6 or 7 ring carbon atoms and / or one or two heterocycloalkyl groups, each containing 3, 4, 5, 6 or 7 ring atoms comprising 1, 2, 3 or 4 oxygen, sulfur or nitrogen atoms.Examples are arylheteroalkyl, arylheterocycloalkyl, arylheterocycloalkenyl, arylalkylheterocycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocycloalkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroaryl-heterocycloalkyl, heteroarylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroaryl-alkylheterocycloalkenyl, heteroarylheteroalkylcycloalkyl, heteroarylheteroalkyl-cycloalkenyl and heteroarylheteroalkylheterocycloalkyl groups, the cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples are a tetrahydroisoquinolinyl, benzoyl, phthalidyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-, 3-or 4-methoxyphenyl, 4-ethoxyphenyl, 2-, 3- or 4-carboxyphenylalkyl group.

[0118] As already stated above, the expressions cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl also refer to groups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, =0, SH, =S, NH2, =NH, N3 or NO2 groups.

[0119] The term halogen refers to F, Cl, Br or I.

[0120] When an aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group contains more than one ring, these rings may be bonded to each other via a single or double bond or these rings may be annulated or fused or bridged.

[0121] The present invention further provides pharmaceutical compositions comprising one or more compounds described herein or a pharmaceutically acceptable salt thereof, optionally in combination with one or more carrier substances and / or one or more adjuvants.

[0122] The present invention furthermore provides compounds or pharmaceutical compositions as described herein for use in the treatment of bacterial infections, especially for use in the treatment of tuberculosis.The present invention further provides a compound as described herein or a pharmaceutical composition as defined herein for the preparation of a medicament for use in the treatment of bacterial infections, especially for use in the treatment of tuberculosis.

[0123] According to a moreover preferred embodiment, the present invention provides a method for treating a bacterial infection (especially for treating tuberculosis), which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound as described herein or a pharmaceutical composition as defined herein.

[0124] Examples of salts (especially of pharmacologically acceptable salts) of sufficiently basic compounds are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleic and salicylic acid. Further, a sufficiently acidic compound may form alkali or earth alkali metal salts, for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, meglumin, piperidine, morpholine, tris-(2-hydroxyethyl)amine, lysine or arginine salts; all of which are also further examples of salts of the compounds described herein.

[0125] The compounds described herein may be solvated, especially hydrated. The solvation / hydration may occur during the process of production or as a consequence of the hygroscopic nature of the initially water-free compounds. The solvates and / or hydrates may e.g. be present in solid or liquid form.

[0126] The therapeutic use of the compounds described herein, their pharmacologically acceptable salts, solvates and hydrates, respectively, as well as formulations and pharmaceutical compositions also lie within the scope of the present invention.

[0127] In general, the compounds and pharmaceutical compositions described herein will be administered by using the established and acceptable modes known in the art.For oral administration, such therapeutically useful agents can be administered by one of the following routes: oral, e.g. as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example soft and hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e.g. as an injectable solution or suspension, rectal as suppositories, by inhalation or insufflation, e.g. as a powder formulation, as microcrystals or as a spray (e.g., liquid aerosol), transdermal, for example via an transdermal drug delivery system (TDDS) such as a plaster containing the active ingredient or intranasal. For the production of such tablets, pills, semisolids, coated tablets, dragees and hard, e.g. gelatine, capsules the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like. For the production of soft capsules, one may use excipients as are e.g., vegetable, petroleum, animal or synthetic oils, wax, fat, and polyols. For the production of liquid solutions, emulsions or suspensions or syrups one may use as excipients e.g., water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils. Especially preferred are lipids and more preferred are phospholipids (preferred of natural origin; especially preferred with a particle size between 300 to 350 nm) preferred in phosphate buffered saline (pH = 7 to 8, preferred 7.4). For suppositories, one may use excipients as e.g. vegetable, petroleum, animal or synthetic oils, wax, fat and polyols. For aerosol formulations, one may use compressed gases suitable for this purpose, e.g., oxygen, nitrogen and carbon dioxide. The pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g., UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.

[0128] In general, in the case of oral or parenteral administration to adult humans weighing approximately 80 kg, a daily dosage of about 1 mg to about 10,000 mg, preferably from about 5 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion or subcutaneous injection.EXAMPLES

[0129] List of Abbreviations

[0130] Ac Acetyl

[0131] Bn Benzyl

[0132] Boc te / Y-Butyloxycarbonyl

[0133] Cbz Benzyloxycarbonyl

[0134] cHex Cyclohexane

[0135] Cl Chemical ionization

[0136] COMII (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino- carbenium-hexafluorophosphate

[0137] Cy Cyclohexyl

[0138] CyPro frans-4-Cyclohexylproline

[0139] DICHED 1,2-Dicyclohexyl-1,2-ethandiol

[0140] DIPA Diisopropylamine

[0141] DIPEA / V, / V4Diisopropylethylamine

[0142] DMAc Dimethylacetamide

[0143] DMBA Dimethylbarbituric acid

[0144] DMF Dimethylformamide

[0145] DMSO Dimethylsulfoxide

[0146] EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0147] ESI Electrospray ionization

[0148] Et Ethyl

[0149] EtOPro frans-4-Ethoxyproline

[0150] EtPro frans-4-Ethylproline

[0151] FDPP Pentafluorphenyl diphenylphosphinate

[0152] Fmoc Fluorenylmethoxycarbonyl

[0153] Gly Glycine

[0154] HATLI O-(7-Azabenzotriazol-1 -y l)- / , A / , / ‘ / '-tetramethy luron ium hexafluorphosphate

[0155] HOAt 1 -Hydroxy-7-azabenzotriazoleHOBt 1-Hydroxybenzotriazole

[0156] HoLeu Homoleucine

[0157] HPLC high performance liquid chromatography

[0158] HRMS high resolution mass spectrometry

[0159] IBCF / so-Butylchlorformiate

[0160] IPCF / so-Propylchloroformiate

[0161] LC liquid chromatography

[0162] LDA Lithiumdiisopropylamide

[0163] Leu Leucine

[0164] Lit. Literature value

[0165] Lys Lysine

[0166] Me Methyl

[0167] MeOPro frans-4-Methoxyproline

[0168] MePro frans-4-Methylproline

[0169] MNBA 2-Methyl-6-nitrobenzoic anhydride

[0170] MS mass spectrometry

[0171] n-Bu n-Butyl

[0172] NMI A / -Methylimidazole

[0173] NMM / V-Methylmorpholine

[0174] NMR nuclear magnetic resonance

[0175] Ph Phenyl

[0176] PPY 4-Pyrrolidinylpyridine

[0177] Pro Proline

[0178] PyAOP (7-Azabenzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate

[0179] Rf Retention factor

[0180] sat. saturated

[0181] TBS te / Y-Butyldimethylsilyl

[0182] TBTU O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium tetrafluoroborate f-Bu te / Y-Butyl

[0183] Tf Triflyl

[0184] TFA Trifluoroacetic acid

[0185] THF TetrahydrofuranThr Threonine

[0186] TLC Thin layer chromatography

[0187] Tren Tris(2-aminoethyl)amine

[0188] Trt Trityl

[0189] Ts Tosyl

[0190] Vai Valine

[0191] General Information

[0192] All air and moisture sensitive reactions were carried out in dried glassware (> 100 °C) under N2 or Ar atmosphere. Anhydrous solvents were purchased from Acros Organics or dried before use (THF was distilled over sodium / benzophenone) and stored under nitrogen atmosphere. The products were purified by column chromatography on silica gel columns (Machery-Nagel 60, 0.063-0.2 mm) or a Grace Reveleris PREP Chromatography system or a Buchi Pure C-815 Flash system using prepacked columns RediSep® R / Trom Teledyne Isco. For reverse-phase chromatography (indicated by C18-SiO2), a Grace Reveleris PREP Chromatography system was used with Buchi FlashPure Select C18 columns and MeCN / H₂O solvents. Analytical TLC was performed on precoated silica gel plates (Machery-Nagel, Polygram Sil G / UV254). Detection was accomplished with UV light (254 nm), KMnO4 solution, ninhydrin solution or cerium(IV) / ammonium molybdate solution. Melting points were determined with a MEL-TEMP II (Laboratory devices) apparatus and are uncorrected.1H and13C NMR spectra were recorded at 293 or 298 K on a Bruker Avance II 400 MHz spectrometer [1H 400 MHz and13C 100 MHz], a Bruker Avance I 500 MHz spectrometer [1H 500 MHz and13C 126 MHz] or a Bruker AV 500 Neo spectrometer [1H 500 MHz and13C 126 MHz], Chemical shifts (5) are reported in parts per million (ppm) relative to TMS or internal solvent signal. Peaks were assigned using (1H,1H)-COSY, (1H,13C)-HSQC and (1H,13C)-HMBC spectra. LC / MS measurements were performed on a Shimadzu system (system controller: SCL-10A, liquid chromatograph: LC-2030C ED Plus, autosampler: SCL-6B, mass spectrometer: LCMS-2020), using an Onyx C₁₈₍₂₎ column (50×4.6 mm, 3 µm particle size) from Phenomenex as the stationary phase. Detection was performed using a diode array detector (190-300 nm) and a mass detector (Shimadzu LCMS-2020) via ESI. Mass spectra were recorded with a Finnigan MAT 95 spectrometer (quadrupole) (Cl), a BrokerDaltonics maXis 4G (ESI) and a UHPLC Quadrupole Orbitrap (Q Exactive) mass spectrometer from Thermo Scientific (ESI). Optical rotations were measured with a Jasco P-2000 polarimeter or a Kruss P8000-T80 polarimeter in a thermostated (20 °C ± 1 °C) cuvette, using a sodium vapor lamp (A = 589 nm) as radiation source, [c ]^0values are given in 10’1deg cm2g-1. (R, R)-DICHED was prepared according to known literature protocols.!1,2]

[0193] Reaction Schemes

[0194] 1) TrtONa 1) LICHCI2, ZnCI2DMSO, 0 °C > RT THF, -98 °C -> RT 2) Pentaerythritol 2) RMgCI (R = Me, Et) Et2O / 1 M NaOH(aq )1:1, RT THF, 0 °C RT 3) (R, R)-DICHED, MgSO4P23 (R=Me) Et2O, RT P24 (R=Et) 1) LiCHCI2, ZnCI21) LiCHCI2, ZnCI2THF, -98 °C RT THF, -98 °C ->• RT 2) LiBHEt32) NaN3, DMF, RT THF, 0 °C — » RT P25 (R=Me) P26 (R=Me) P27 (R=Et) P28 (R=Et) 1) LiCHBr2, ZnCI21) AcOH / H2O 4:1, 55 °C THF, -82 °C -► RT (P29: R=Me; P33: R=Et) 2) NaCIO2, KH2PO4I2) TsCI, Et3N 2-Methyl-2-butene DCM, 0 °C > RT t-BuOH / H2O 2:1, RT P27 (R=Me) 3) t-BuBr, BnEt3NCI, K2CO3DMAc, 55 °C P32 (R=Et)

[0195] 1) H2Pd / C CHCI3 / TFA MeOH, RT 3:1 2) CbzCI, Et3N 50 °C MeOH, 0 °C — > RT R = Et P30 (R=Me) P31 (R=Me) P36

[0196]

[0197] P34 (R =Et) P35 (R=Et) Scheme 1: Synthesis of frans-4-methyl- and frans-4-ethyl-l-proline.

[0198] kat. AcCI TsCI, Et3N MeOH DCM 0 °C -> RT 0 °C -> RT

[0199]

[0200] P51 P52NaH LiOH(aq;

[0201] THF 1,4-Dioxane

[0202] 0 °C RT 0 °C — > RT Cbz

[0203]

[0204] P54 Scheme 2: Alternative route towards frans-4-methyl-l-proline (P54).

[0205] R4

[0206] 1 ) Boc- or Cbz- 1 ) Boc-cleavage cleavage 2) Boc-Pro-OH or Boc-ChPro-OH or 2) Boc- / V-Me-Val-OH Cbz-MePro-OH TBTU, DI PEA IPCF, DIPEA, kat. BnNMe2, MeCN, 0 °C > RT kat. HCI, kat. NMI 1,4-Dioxane / MeCN 3:1, 60 °C M5 (PG=Boc, R4=H) M10 (PG=Boc, R4=Cy) M66 (PG=Cbz, R4=Me)

[0207] R41 ) Boc-cleavage 2) Boc-HoLeu-OH or Boc-Leu-OH HATU, DIPEA DMF, 0 °C RT M6 (R4=H) M7 (R3= / -Bu, R4=H) M11 (R4=Cy) M12 (R3= / '-BU, R4=Cy) M67 (R4=Me) M64 (R3= / -Pr, R4=Cy)

[0208] M72 (R3= / -BU, R4=Me)

[0209] M48 (R3= / -BU, R4=H) LiOHjaq;

[0210] M49 (R3= / -BU, R4=Cy) 1,4-Dioxane M65 (R3= / -Pr, R4=Cy) RT M73 (R3= / -BU, R4=Me)

[0211]

[0212] Scheme 3: Synthesis of the tetrapeptide fragments M48, M49, M65 and M73.

[0213]

[0214] M55 M56LiOH(aq )

[0215] 1,4-Dioxane

[0216] RT

[0217]

[0218] M116

[0219] Scheme 4: Synthesis of the azide-functionalized tetrapeptide fragment M116.

[0220] 1) H2, Pd / C 1) H2, Pd / C, HCI MeOH, RT MeOH, RT 2) Cbz-Leu-OH 2) Cbz-O-TBS-N-Me-Thr-OH EDC, HOBt, NMM IBCF, NMM DMF, 0 °C — > RT THF / DCM 2.5:2

[0221] -20 °C ->■ RT P31

[0222] Cbz-EtPro-OH or Cbz-MePro-OH or Cbz-MeOPro-OH or cat. AcCI Cbz-EtOPro-OH MeOH MNBA, PPY 0 °C — > RT THF, 0 °C — > RT

[0223] M43

[0224] M46 (R2=Et) M40 (R2=Et) M68 (R2=Me) M74 (R2=Me) M94 (R2=OMe) M96 (R2=OMe) M95 (R2=OEt) M97 (R2=OEt)

[0225] Fmoc-Gly-OH M41 (R2=Et) MNBA, PPY M75 (R2=Me) THF M98 (R2=OMe) 0 °C — > RT M99 (R2=OEt)

[0226]

[0227] Scheme 5: Synthesis of the hexapeptide fragments M41, M75, M98 and M99.M42 (R2=Et, R3= / -BU, R4=H, R®= / -Pr) M44 (R2=Et, R3= / -Bu, R4=H, R®= / -Pr) M50 (R2=Et, R3= / -BU, R4=Cy, R®= / -Pr) M51 (R2=Et, R3= / -BU, R4=Cy, R®= / -Pr) M63 (R2=Et, R3= / -BU, R4=Cy, R®=(CH2)3CH2N3) M69 (R2=Et, R3= / -BU, R4=Cy, R6=(CH2)3CH2N3) M70 (R2=Et, R3= / -Pr, R4=Cy, R®= / -Pr) M76 (R2=Et, R3= / -Pr, R4=Cy, R®= / -Pr) M77 (R2=Me, R3= / -Bu, R4=Cy, R®= / -Pr) M79 (R2=Me, R3= / -Bu, R4=Cy, R6= / -Pr) M78 (R2=Me, R3= / -Bu, R4=Me, R®= / -Pr) M80 (R2=Me, R3= / -Bu, R4=Me, R®= / -Pr) M100 (R2=0Me, R3= / -Bu, R4=Cy, R®= / -Pr) M102 (R2=OMe, R3= / -Bu, R4=Cy, R®= / -Pr)

[0228]

[0229] M101 (R2=0Et, R3= / -BU, R4=Cy, R®= / -Pr) M103 (R2=OEt, R3= / -BU, R4=Cy, R®= / -Pr) Scheme 6: Synthesis of the open-chained decapeptide, subsequent global deprotection and macrocyclization.M44 (R2=Et, R3= / -BU, R4=H, R®= / -Pr) M45 (R1=(C=O)2CH2CH3, R2=Et, R3= / -Bu, R4=H, R6= / -Pr) M51 (R2=Et, R3= / -BU, R4=Cy, R®= / -Pr) M52 (R1=(C=O)2CH2CH3, R2=Et, R3= / -Bu, R4=Cy, R6= / -Pr) M69 (R2=Et, R3= / -Bu, R4=Cy, R®=(CH2)3CH2N3) M57 (R1=Ac, R2=Et, R3= / -Bu, R4=Cy, R6= / -Pr) M76 (R2=Et, R3= / -Pr, R4=Cy, R®= / -Pr) M58 (R1=Butyryl, R2=Et, R3= / -Bu, R4=Cy, R®= / -Pr) M79 (R2=Me, R3= / -BU, R4=Cy, R®= / -Pr) M59 (R1=Caproyl, R2=Et, R3= / -Bu, R4=Cy, R6= / -Pr) M80 (R2=Me, R3= / -BU, R4=Me, R®= / -Pr) M60 (R1=(C=O)p-F-Ph, R2=Et, R3= / -Bu, R4=Cy, R®= / -Pr) M102 (R2=0Me, R3= / -Bu, R4=Cy, R®= / -Pr) M61 (R1=(C=O)Cy, R2=Et, R3= / -Bu, R4=Cy, R6= / -Pr) M103 (R2=OEt, R3= / -BU, R4=Cy, R®= / -Pr) M62 (R1=(C=O)(CH2)3Cy, R2=Et, R3= / -Bu, R4=Cy, R®= / -Pr)

[0230] M106 (R1=Ac, R2=Et, R3= / -Bu, R4=Cy, R6=(CH2)3CH2N3) M81 (R1=(C=O)2CH2CH3, R2=Et, R3= / -Pr, R4=Cy, R®= / -Pr) M83 (R1=(C=O)2CH2CH3, R2=Me, R3= / -Bu, R4=Cy, R®= / -Pr) M82 (R1=(C=O)2CH2CH3, R2=Me, R3= / -Bu, R4=Me, R®= / -Pr) M104 (R1=(C=O)(CH2)3Cy, R2=OMe, R3= / -Bu, R4=Cy, R®= / -Pr) M105 (R1=(C=O)(CH2)3Cy, R2=OEt, R3= / -Bu, R4=Cy, R®= / -Pr) M114 (R1=Ac, R2=OMe, R3= / -Bu, R4=Cy, R®= / -Pr)

[0231]

[0232] M115 (R1=Ac, R2=OEt, R3= / -Bu, R4=Cy, R®= / -Pr) Scheme 7: Introduction of the R1substituent.

[0233]

[0234] Scheme 8: Synthesis of the triazole derivatives M107, M108, M109, M111 and M113.General Procedures (GP)

[0235] GP1: Matteson Homologation

[0236] Under N2 atmosphere, anhydrous DCM (3.0 eq.) was added to anhydrous THF (1.5 mL / mmol n-BuLi) and cooled to -98 °C (MeOH / N₂₍ₗ₎). Subsequently, n-BuLi (1.1-1.3 eq., 2.5 M in n-hexane) was added slowly along the inner wall of the flask and rinsed with anhydrous THF. After 20 min, a solution of the boronic acid ester (1.0 eq.) in anhydrous THF (1.4 mL / mmol boronic acid ester) was slowly added dropwise at -98 °C, rinsing twice with anhydrous THF. After another 20 min, a suspension of in a high vacuum oven-dried ZnCl2 (3.0 eq.) in anhydrous THF (0.6 mL / mmol ZnCl₂) was added, followed by warming to RT. The conversion to the a-chloroboronic ester was monitored via1H-NMR spectroscopy (usually 2-3 h).

[0237] Variant a): One-pot reaction of the a-chloroboronic ester: The reaction mixture was cooled to the indicated temperature and the corresponding nucleophile solution was added dropwise. After complete conversion was observed via1H-NMR spectroscopy, the mixture was quenched with sat. NH4CI solution and diluted with n-pentane. Next, the phases were then separated, and the aqueous phase was extracted once with n-pentane. The combined organic phases were dried over MgSO4, the solvent was removed in vacuo, and the resulting residue was purified by column chromatography.

[0238] Variant b): Reaction of the isolated a-chloroboronic ester: The reaction mixture was quenched with sat. NH4CI solution and diluted with n-pentane. Afterwards, the phases were then separated, and the aqueous phase was extracted once with n-pentane. The combined organic phases were dried over MgSO₄, and the solvent was removed in vacuo. Then the resulting residue was dissolved in anhydrous THF under N2 atmosphere and cooled to the indicated temperature, whereupon the nucleophile solution was added dropwise. After complete conversion was observed via1H-NMR spectroscopy, the mixture was quenched with sat. NH4CI solution and diluted with n-pentane. The phases were then separated, and the aqueous phase was extracted once with n-pentane. Lastly, the combined organic phases were dried over MgSO4, the solvent was removed in vacuo, and the resulting residue was purified by column chromatography.

[0239] GP2: Azide reduction and Cbz cleavage with Pd / C under H2 atmosphereIn a round-bottom flask, the corresponding azide or / V-Cbz-protected amino acid / peptide was dissolved in MeOH and Pd / C (10 wt%) was added. The reaction vessel was evacuated six times with a water aspirator vacuum pump until the black suspension began to boil, before the vessel was then flushed with H2 (balloon). The reaction mixture was stirred under H2 atmosphere (balloon pressure) until complete conversion was observed via TLC or LC / MS. Subsequently, it was filtered through Celite, rinsed with MeOH and the solvent was removed in vacuo.

[0240] GP3: Methyl Ester Saponification with LiOH

[0241] A solution of the corresponding methyl ester (1.0 eq.) in 1,4-dioxane was treated with 1 M LiOH(aq.) (1.1-1.5 eq.) at RT and stirred for 3-18.5 h until complete conversion was observed via LC / MS. Subsequently, the pH was adjusted to approx. 1 with 1 M HCI(aq.), causing a colorless solid to precipitate. Afterwards, the aqueous phase was extracted three times with EtOAc, before the combined organic phases were dried over MgSO4 and the solvent was removed in vacuo. The resulting residue was purified by column chromatography.

[0242] GP4: / V- or O-Methylation of Carbamate-Protected Amino Acids[3]

[0243] Under N2 atmosphere, the / V-protected amino acid (1.0 eq.) was dissolved in anhydrous THF and cooled to 0 °C. Subsequently, the solution was treated with methyl iodide (3-5 eq.), followed by portionwise addition of NaH (3-6 eq.). After the addition was complete, the reaction mixture was slowly warmed to RT overnight (for the introduction of an ethyl group, ethyl iodide was used instead of methyl iodide).

[0244] The reaction mixture was then carefully quenched with H2O until no further gas evolution was observed. Subsequently, the mixture was extracted once with n-pentane, and the aqueous phase was acidified to a pH of approx. 2-3 with 1 M KHSO4(aq), resulting in the formation of a colorless precipitate. Next, the aqueous phase was extracted three times with EtOAc, and the combined EtOAc phases were washed once with sat. Na2S2O3(aq.) and once with sat. NaCI(aq.) solution, before the now nearly colorless organic phase was dried over MgSO4 and the solvent was removed in vacuo. If necessary, the resulting residue was purified by column chromatography.

[0245] GP5: Boc-Cleavage of / V-Boc-Protected Amino Acid Esters and PeptidesIn a round-bottom flask, the / V-Boc-protected amine (1.0 eq.) was dissolved in MeOH and cooled to 0 °C. Acetyl chloride (7.5-10 eq.) was added dropwise, and the mixture was subsequently warmed to RT. Alternatively, 1,4-dioxane with the addition of MeOH (7.5-10 eq.) ora 4.0 M solution of HCI in 1,4-dioxane was used as the reaction medium instead of MeOH. After complete conversion was confirmed by TLC or LC / MS (1-6 h), the solvent was removed in vacuo, and the residue was dried under high vacuum.

[0246] GP6: Peptide Coupling with TBTU / HATU / HOBt+EDC / PyAOP / COMU

[0247] The carboxylic acid (1.0-2.5 eq.) and amine component (1.0 eq.) were dissolved in the indicated solvent under N2 or Ar atmosphere and cooled to 0 °C. Subsequently, HOBt / HOAt (1.0-1.4 eq., if necessary), the coupling reagent (1.1-2.5 eq.), and the tertiary amine base (2.2-4.0 eq.) were added sequentially, before the reaction mixture was slowly warmed to RT overnight.

[0248] Afterwards, the mixture was diluted with EtOAc and washed with 1 M KHSO4(aq ), followed by water, sat. NaHCO3 solution, and sat. NaCI solution, before the organic phase was dried over MgSO4 and the solvent was removed in vacuo. If DMF was used as the solvent, it was washed with 1 M LiCI(aq.) instead of water. Finally, the resulting residue was purified by column chromatography.

[0249] GP7: MN BA Esterification

[0250] A solution of the corresponding alcohol (1.0 eq.) and the corresponding carboxylic acid (1.4-2.0 eq.) in anhydrous THF was treated with PPY (1.1-1.4 eq.) followed by MNBA (1.4-2.0 eq.) under Ar atmosphere at 0 °C. After addition, the reaction mixture was warmed to RT overnight and then concentrated in vacuo. Finally, the resulting residue was purified by column chromatography.

[0251] GP8: Fmoc Deprotection with trenl4]

[0252] A solution of the corresponding Fmoc-protected peptide in DCM was treated with tren (10 eq.) at RT and stirred for 1-3 h. Subsequently, the now colorless suspension was diluted with EtOAc and washed twice with water and twice with a phosphate buffer (pH = 5.5). Afterwards, the organic phase was dried over MgSO4 and the solvent was removed in vacuo.GP9: Boc and t-Bu Ester Cleavage with Subsequent Macrocyclization The corresponding linear peptide (1.0 eq.) was dissolved in a 1:1 mixture of DCM and TFA and stirred at RT for 2-3 h. After complete conversion was observed via LC / MS, the yellow solution was concentrated in vacuo and the residue was co-evaporated three times with CHCh.

[0253] Subsequently, the globally deprotected peptide was dissolved in anhydrous DMF and added dropwise via syringe pump under N2 atmosphere to a solution of FDPP (5-10 eq.) and DIPEA (10-21 eq.) in anhydrous DMF at 70 °C (target concentration = 1 mM, V(DMFFDPP) / V(DMFpeptide) = 8:2). After complete conversion was observed via LC / MS, the mixture was cooled to RT and concentrated in vacuo. The resulting residue was taken up in EtOAc and washed successively with 1 M HCI(aq), 1 M LiCI(aq ), sat. NaHCO3, and sat. NaCI solution, before being dried over MgSO4. After the solvent was removed in vacuo, the crude product was purified by column chromatography.

[0254] GP10: Alloc Deprotection

[0255] A solution of Al loc-protected amine (1.0 eq.) and DMBA (5-25 eq.) in anhydrous DCM was treated with Pd(PPhs)4 (2-7 mol%) under N2 atmosphere at RT and stirred for 1.5-4 h. The yellow solution was then diluted with EtOAc, washed three times with sat. NaHCO3oq.) solution, and dried over MgSO4, before the solvent was removed in vacuo.

[0256] GP11: Acylation via Acid Chloride

[0257] To a solution of the corresponding carboxylic acid (6-20 eq.) in anhydrous DCM, oxalyl chloride (6-20 eq.), followed by one drop of anhydrous DMF, was added under N2 atmosphere, resulting in a vigorous gas evolution. After stirring for 2.5-4 h, this was slowly added dropwise at 0 °C to a solution of the amine deprotected according to GP10 (1.0 eq.) and DIPEA (10-40 eq.) in anhydrous DCM, forming a white smoke. The reaction mixture was then slowly warmed to RT overnight.

[0258] Subsequently, it was diluted with EtOAc and washed successively with 1 M H Cl(aq ), H2O, sat. NaHCO3oq.) and sat. NaCI(aq.) solution, before the organic phase was dried over MgS04. The solvent was then removed in vacuo and the residue was purified by column chromatography.

[0259] Synthesis of the Amino Acid Building BlocksSynthesis of the 4-Alkylprolinesl5!

[0260] (4 / ?,5 / ?)-4,5-Dicyclohexyl-2-[(trityloxy)methyl]-1,3,2-dioxaborolane [P22][6]Under N2 atmosphere, a solution of 49.5 g (190 mmol, 1.05 eq.) of triphenylmethanol in 360 mL anhydrous DMSO was treated portionwise with 8.72 g (60 wt% in mineral oil, 223 mmol, 1.2 eq.) of NaH. After stirring the reaction mixture for 16 h, 40.0 g (181 mmol, 1.0 eq.) of bromomethylboronic acid pinacol ester were added via a transfer cannula at 0 °C, warming to RT after complete addition. After 3 d, 380 mL of sat. Nh CIfaq.) solution were added to the reaction mixture (exothermic) and it was extracted twice with 250 mL of Et20. The combined organic phases were washed with water and the solvent was removed in vacuo. Next, the resulting residue was dissolved in 500 mL of Et20, treated with 500 mL of 1 M NaOH(aq ), followed by 61.6 g (452 mmol, 2.5 eq.) of pentaerythritol, and then stirred vigorously. After 21 h, the phases were separated and the aqueous phase was carefully neutralized with 1 M HCI(aq.) at 0 °C (pH = 6). The precipitated white solid was filtered off, washed with water, dried under high vacuum, and then suspended in 380 mL of n-pentane and treated with 24.3 g (107 mmol, 0.7 eq.) of (R, R)-DICHED, followed by 37.7 g (306 mmol, 2.0 eq.) of anhydrous MgSO4. After 4 h, an additional 3.46 g (15.3 mmol, 0.1 eq.) and after 16.5 h another 3.45 g (15.3 mmol, 0.1 eq.) of (R, R)~ DICHED were added to the mixture. After complete conversion was observed via TLC (25.5 h), the suspension was filtered and the filter cake was rinsed with Et20. The filtrate was freed from the solvent in vacuo and the residue was purified by column chromatography (n-Pentane / Et2O 9:1). In total, 66.6 g (131 mmol, 86 %) of the boronic ester P22 were isolated as a colorless, crystalline solid.

[0261] [c ]o° = +53.9 (c = 1.0, CHCI3); Lit.: [a] ° = +49.7 (c = 1.0, CHC )171

[0262] Melting range: 113-116 °C; Lit.: 108-110 °Cl7l

[0263] Rf = 0.55 (n-Pentane / EtOAc 95:5)

[0264]

[0265] HRMS (Cl): Calculated for C34H41BO3+[M]+: 508.3143, found: 508.3147.

[0266] (4 / ?,5 / ?)-4,5-Dicyclohexyl-2-[(S)-1 -(trityloxy)propan-2-yl]-1,3,2-dioxaborolane [P23] According to GP1a, 10.0 g (19.7 mmol, 1.0 eq.) of boronic acid ester P22 were reacted with 3.8 mL (59.1 mmol, 3.0 eq., p = 1.320 g / mL) anhydrous DCM, 9.0 mL (2.5 M in n-hexane, 22.5 mmol, 1.1 eq.) n-BuLi, and 8.06 g (59.1 mmol, 3.0 eq.) ZnCh After 2.5 h, the mixture was cooled to 0 °C and 16.4 mL (3.0 M in THF, 49.2 mmol, 2.5 eq.) MeMgCI were slowly added dropwise. After the addition was complete, the mixture was slowly warmed to RT. After 1.5 days, the mixture was cooled again to 0 °C, and another 3.2 mL (3.0 M in THF, 9.60 mmol, 0.5 eq.) of MeMgCI were slowly added dropwise before slowly warming to RT. After 4 h, the mixture was worked up according to GP1a. Automated column chromatography (SiO2, n-Pentane / Et2O 10:0 — 8:2) yielded 9.19 g (17.1 mmol, 87 %) of the boronic acid ester P23 as a colorless resin.

[0267] [c ]o° = +42.2 (c = 1.0, CHC ); Lit.: [a] ° = +36.4 (c = 1.0, CHCh)^

[0268] Rf = 0.55 (n-Pentane / Et2O 9:1)

[0269] 5 6

[0270]

[0271] HRMS (Cl): Calculated for C36H45BO3+[M]+: 536.3456, found: 536.3459.

[0272] (4 / ?,5 / ?)-4,5-Dicyclohexyl-2-[( / ?)-2-methyl-3-(trityloxy)propyl]-1,3,2-dioxaborolane [P25]

[0273] According to GP1b, 8.51 g (15.9 mmol, 1.0 eq.) of boronic acid ester P23 were reacted with 3.0 mL (46.6 mmol, 2.9 eq., p = 1.320 g / mL) anhydrous DCM, 7.5 mL (2.5 M in n-hexane, 18.8 mmol, 1.2 eq.) n-BuLi and 6.60 g (48.4 mmol, 3.1 eq.) ZnCL. After 3.5 h, aqueous work-up was carried out accordingly and the residue obtained was reacted according to GP1b in 40 mL anhydrous THF with 19.0 mL (1.0 M in THF, 19.0 mmol, 1.2 eq.) Superhydride® at 0 °C, adding after 1.5 h another 1.6 mL (1.0 M in THF, 1.60 mmol, 0.1 eq.) Superhydride® at 0 °C. After a further 1.5 h of stirring at RT, the reaction was worked up accordingly. Column chromatographic purification (SiO2, n-Pentane / Et2095:5) yielded 8.27 g (15.0 mmol, 95 %) of the boronic acid ester P25 as a colorless solid.

[0274] [a] ° = +20.4 (c = 1.0, CHCh)

[0275] Melting range: 82-85 °C

[0276] Rf = 0.54 (n-Pentane / Et2O 9:1 )

[0277]

[0278] HRMS (ESI): Calculated for C37H47BNaO3+[M+Na]+: 573.3510, found: 573.3511.

[0279] (4R,5R)-2-[(1R,3R)-1-Azido-3-methyl-4-(trityloxy)butyl]-4,5-dicyclohexyl-1,3,2-di-oxaborolane [P26]

[0280] According to GP1b, 8.25 g (15.0 mmol, 1.0 eq.) of boronic acid ester P25 were reacted with 3.0 mL (46.6 mmol, 3.1 eq., p = 1.320 g / mL) anhydrous DCM, 7.0 mL (2.5 M in n-hexane, 17.5 mmol, 1.2 eq.) n-BuLi and 6.23 g (45.7 mmol, 3.1 eq.) ZnCh. After 2.5 h, the reaction was worked up aqueously. The residue obtained was dissolved in 74 mL of anhydrous DMF under N2 atmosphere, to which 5.36 g (82.5 mmol, 5.5 eq.) NaNs were added and stirred for 19.5 h.

[0281] Contrary to GP1b, the yellow suspension was then quenched with sat. NH4CI(aq.) solution and diluted with n-pentane and a little H2O until two clear phases were obtained. After the phases were separated, the aqueous phase was extracted once with n-pentane and the combined organic phases were washed once with 1 M LiCI(aq.) before drying over MgSO4. The solvent was then removed in vacuo and the residue was purified by column chromatography (SiO2, n-Pentane / Et2O 9:1). A total of 7.44 g (12.3 mmol, 82 %) of the a-azidoboronic acid ester P26 could be isolated as a colorless, opaque resin.

[0282] [c ]o° = +16.0 (c = 1.0, CHCh)

[0283] Rf = 0.35 (n-Pentane / EtOAc 95:5)56

[0284]

[0285] HRMS (Cl): Calculated for C38H50BNO3+[M+2H-N2]+: 579.3878, found: 579.3850.

[0286] fert-Butyl (2S,4 / ?)-2-Azido-4-methyl-5-(trityloxy)pentanoate [P27] Homologation: Under N2atmosphere, 2.6 mL (18.4 mmol, 1.5 eq., p = 0.717 g / mL) DIPA was dissolved in 5.0 mL anhydrous THF and cooled to -40 °C, before 6.5 mL (2.5 M in n-hexane, 16.3 mmol, 1.3 eq.) n-BuLi was added. Stirring was then continued for 10 min at -40 °C and then for 20 min at RT. The LDA solution thus prepared was slowly added by transfer cannula to a solution of 7.44 g (12.3 mmol, 1.0 eq.) boronic acid ester P26 and 2.6 mL (37.2 mmol, 3.0 eq., p = 2.490 g / mL) CH2Br2in 17 mL anhydrous THF at -82 °C (EtOAc / N2(i)). After stirring further at -82 °C for 1 h, a suspension of 5.12 g (37.6 mmol, 3.1 eq.) of ZnCI2, heated beforehand under high vacuum, in 23 mL of anhydrous THF was added before subsequent slow warming to RT overnight. After 18 h, sat. NH4CI(aq.) solution was added to the reaction mixture and diluted with water and n-pentane. The phases were then separated, and the aqueous phase was extracted once with n-pentane before the combined organic phases were dried over MgSO4 and the solvent was removed in vacuo.

[0287] Oxidation: The previously obtained a-bromoboronic acid ester was dissolved in 238 mL f-BuOH and mixed with 52 mL (491 mmol, 40 eq., p = 0.662 g / mL) 2-methyl-2-butene. A solution of 13.9 g (80 wt%, 123 mmol, 10 eq.) NaCIO2and 16.8 g (123 mmol, 10 eq.) KH2PO4 in 118 mL water was then added dropwise. After 4 d, the yellow emulsion was concentrated in vacuo and acidified with 10 wt% aqueous citric acid solution to a pH of about 3 before extracting three times with Et2O. The combined etheric phases were washed with sat. Na2S2O3(aq.) solution, dried over MgSO4 and the solvent was removed in vacuo. The residue was then purified by automated column chromatography (SiO2, n-Pentane / EtOAc 10:0 7:3).

[0288] Esterification:!8! The previously isolated a-azidocarboxylic acid was dissolved in 122 mL DMAc and 56 mL (49.5 mmol, 40 eq., p = 1.210 g / mL) f-BuBr, 2.80 g (12.3 mmol,1.0 eq.) BnNEt3Cl and 44.1 g (319 mmol, 26 eq.) K2CO3 were added. The suspension was then stirred for 5 h at 55 °C. After cooling to RT, water was added to the mixture until a clear solution was obtained. This was extracted three times with Et20, the combined etheric phases were washed twice with water and once with sat. NaCI(aq.) solution before drying over MgSO4 and removing the solvent in vacuo. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — 8:2) afforded 3.60 g (7.63 mmol, 62 %) of the a-azido-te / Y-butyl ester P27 as a colorless oil.

[0289] [a] ° = -1.6 (c = 1.0, CHCI3)

[0290] Rf = 0.69 (n-Pentane / EtOAc 1:1)

[0291]

[0292] HRMS (Cl): Calculated for C29H33N3O3+[M-N2]+: 443.2455, found: 443.2475.

[0293] fert-Butyl (2S,4 / ?)-2-Azido-5-hydroxy-4-methylpentanoate [P29][9]

[0294] 3.59 g (7.61 mmol, 1.0 eq.) of the a-azido-te / Y-butyl ester P27 were dissolved in 30 mL AcOH, 7.5 mL water were added and the mixture was heated to 55 °C. After 2 h, the reaction was cooled to RT and carefully neutralized (strong gas evolution) with sat. NaHCO3oq.) solution. The aqueous phase was extracted three times with Et20, and the combined organic phases were dried over MgSO4. The solvent was then removed in vacuo and the residue was purified by automated column chromatography (cHex / EtOAc 1:0 — 6:4). 1.50 g (6.54 mmol, 86 %) of the alcohol P29 was isolated as a pale-yellow liquid.

[0295] [cr]o° = -12.0 (c = 1.0, CHCI3)

[0296] Rf = 0.30 (n-Pentane / EtOAc 1:1)

[0297] = N3

[0298] 8 5 || \

[0299]

[0300] 01

[0301] HRMS (Cl): Calculated for C10H19N3NaO3+[M+Na]+: 252.1319, found: 252.1318.

[0302] fert-Butyl (2S,4 / ?)-2-Azido-4-methyl-5-(tosyloxy)pentanoate [P30]To a solution of 1.50 g (6.52 mmol, 1.0 eq.) of alcohol P29 in 32 mL DCM, 2.73 mL (19.6 mmol, 3.0 eq., p = 0.726 g / mL) EtsN followed by 2.49 g (13.0 mmol, 2.0 eq.) tosyl chloride were added at 0 °C and slowly warmed to RT after the addition was complete. After 3 d, the reaction mixture was washed with sat. NaHCO3(aq.) solution and the aqueous phase was extracted three times with DCM before the combined organic phases were dried over MgSO4. The solvent was then removed in vacuo and the residue was purified by column chromatography (n-Pentane / Et2O 8:2). 2.48 g (6.47 mmol, 99 %) of the tosylate P30 was isolated as a yellow oil.

[0303] [cr]o° = -19.4 (c = 1.0, CHCI3)

[0304] Rf = 0.64 (n-Pentane / EtOAc 1:1)

[0305] N3

[0306] 10 6 •^3 / °x2

[0307] 8 5

[0308]

[0309] HRMS (Cl): Calculated for C17H26N3O5S+[M+H]+: 384.1588, found: 384.1587.

[0310] fert-Butyl (2S,4 / ?)- / V-Benzyloxycarbonyl-4-Methyl-prolinate [P31]

[0311] According to GP2, 1.11 g (2.89 mmol, 1.0 eq.) of the tosylate P30 were reacted in 12 mL MeOH with 111 mg Pd / C (10 wt%) under H2 atmosphere. After 23 h, the residue obtained was dissolved in 12 mL MeOH and cooled to 0 °C. Subsequently, 1.21 mL (8.67 mmol, 3.0 eq., p = 0.726 g / mL) EtsN and dropwise 0.46 mL (3.22 mmol, 1.1 eq., p = 1.195 g / mL) CbzCI were added to the solution and slowly warmed to RT after the addition was completed. After 3 d, the solvent was removed in vacuo and the residue was taken up in EtOAc. The organic phase was washed three times with 10 wt% aqueous citric acid solution and twice with water. It was then dried over MgSO4 and the solvent was removed in vacuo. The residue obtained was purified by automated column chromatography (cHex / EtOAc 1:0 ^ 8:2), resulting in the isolation of 787 mg (1.17 mmol, 85 %) of the proline derivative P31 as a colorless oil.

[0312] [

[0313]

[0314] cr]o° = -30.7 (c = 1.0, CHCI3)

[0315] Rf = 0.60 (n-Pentane / EtOAc 1:1)

[0316]

[0317] HRMS (Cl): Calculated for C18H26NO4+[M+H]+: 320.1856, found: 320.1868.

[0318] (4 / ?,5 / ?)-4,5-Dicyclohexyl-2-[(S)-1-(trityloxy)butan-2-yl]-1,3,2-dioxaborolane [P24] According to GP1a, 10.0 g (19.7 mmol, 1.0 eq.) of boronic acid ester P22 were reacted with 3.8 mL (59.1 mmol, 3.0 eq., p = 1.320 g / mL) anhydrous DCM, 9.0 mL (2.5 M in n-hexane, 22.5 mmol, 1.1 eq.) n-BuLi and 8.04 g (59.0 mmol, 3.0 eq.) ZnCh After 3 h, the reaction was cooled to 0 °C and 18.2 mL (2.7 M in THF, 49.2 mmol, 2.5 eq.) EtMgCI was slowly added dropwise. After the addition was completed, the reaction was warmed to RT and cooled back to 0 °C after 4.5 d to slowly add another 5.0 mL (2.0 M in THF, 10.0 mmol, 0.5 eq.) EtMgCI dropwise before warming to RT. After 2 d, the reaction was worked up accordingly. Column chromatographic purification (SiO2, n-Pentane / Et2O 9:1) afforded 9.60 g (17.4 mmol, 89 %) of the boronic acid ester P24 as a colorless, waxy solid.

[0319] [a] ° = +44.7 (c = 1.0, CHCh)

[0320] Rf = 0.37 (n-Pentane / EtOAc 95:5)

[0321] 56

[0322]

[0323] HRMS (Cl): Calculated for C37H48BO3+[M+H]+: 551.3691, found: 551.3676.

[0324] (4 / ?,5 / ?)-4,5-Dicyclohexyl-2-[( / ?)-2-methyl-3-(trityloxy)propyl]-1,3,2-dioxaborolane [P27]

[0325] According to GP1b, 9.59 g (17.4 mmol, 1.0 eq.) boronic acid ester P24 were reacted with 3.4 mL (52.8 mmol, 3.0 eq., p = 1.320 g / mL) anhydrous DCM, 8.5 mL (2.5 M in n-hexane, 21.3 mmol, 1.2 eq.) n-BuLi and 7.25 g (53.2 mmol, 3.1 eq.) ZnCL. After 4 h was worked up accordingly and the residue obtained was reacted according to GP1b in 44 mLanhydrous THF with 21.0 mL (1.0 M in THF, 21.0 mmol, 1.2 eq.) Superhydride® at 0 °C. After 2.5 h the reaction was worked up accordingly. Column chromatographic purification (SiO2, n-Pentane / Et2O 9:1) afforded 9.15 g (16.2 mmol, 93 %) of the boronic acid ester P27 as a colorless, opaque resin.

[0326] [c ]o° = +15.3 (c = 1.0, CHCh)

[0327] Rf = 0.41 (n-Pentane / EtOAc 95:5)

[0328]

[0329] HRMS (Cl): Calculated for C19H3BO3+[M+2H-Ci9His]+: 323.2752, found: 323.2767.

[0330] (4 / ?,5 / ?)-2-[(1 / ?,3 / ?)-1-Azido-3-((trityloxy)methyl)pentyl]-4,5-dicyclohexyl-1,3,2-dioxaborolane [P28]

[0331] According to GP1b, 9.14 g (16.2 mmol, 1.0 eq.) boronic acid ester P27 were reacted with 3.2 mL (49.7 mmol, 3.1 eq., p = 1.320 g / mL) anhydrous DCM, 8.0 mL (2.5 M in n-hexane, 20.0 mmol, 1.2 eq.) n-BuLi and 6.73 g (49.4 mmol, 3.1 eq.) ZnCh After2.5 h, the reaction was worked up aqueously. The residue obtained was dissolved in 80 mL anhydrous DMF under N2 atmosphere, to which 5.79 g (89.1 mmol, 5.5 eq.) NaN3was added and stirred for 16 h.

[0332] Contrary to GP1b, the yellow-orange suspension was quenched with sat. NF CIfaq.) solution and diluted with n-pentane and a little water until two clear phases were obtained. After the phases were separated, the aqueous phase was extracted once with n-pentane and the combined organic phases were washed once with 1 M LiCI(aq.) before drying over MgSO4. The solvent was then removed in vacuo and the residue was purified by column chromatography (SiO2, n-Pentane / Et2O 9:1). A total of 8.65 g (14.0 mmol, 86 %) of the a-azido-boronic acid ester P28 was isolated as a pale-yellow, opaque resin.

[0333] [a] ° = +10.5 (c = 1.0, CHCh)

[0334] Rf = 0.38 (n-Pentane / EtOAc 95:5)

[0335]

[0336] HRMS (ESI): Calculated for C39H50BN3NaO3+[M+Na]+: 642.3837, found: 642.3837.

[0337] fert-Butyl (2S,4 / ?)-2-Azido-4-((trityloxy)methyl)hexanoate [P32]

[0338] Homologation: Under N2 atmosphere, 2.5 mL DIPA (17.7 mmol, 1.5 eq., p = 0.717 g / mL) were dissolved in 4.9 mL anhydrous THF and cooled to -40 °C before 6.5 mL (2.5 M in n-hexane, 16.3 mmol, 1.3 eq.) of n-BuLi was added. Stirring was then continued for 10 min at -40 °C and then for 20 min at RT. The LDA solution thus prepared was slowly added by transfer cannula to a solution of 7.37 g (11.9 mmol, 1.0 eq.) of boronic acid ester P28 and 2.5 mL (35.8 mmol, 3.0 eq., p = 2.490 g / mL) CH2Br2 in 17 mL anhydrous THF at -82 °C (EtOAc / N2(i)). After stirring further at -82 °C for 1 h, a suspension of 4.96 g (36.4 mmol, 3.1 eq.) of ZnCL, heated under high vacuum beforehand, in 22 mL of anhydrous THF was added, followed by slow warming to RT overnight. After 16 h, sat. NH4CI(aq.) was added to the reaction mixture and diluted with n-pentane and a little H2O and. The phases were then separated, and the aqueous phase was extracted once with n-pentane before the combined organic phases were dried over MgSO4 and the solvent was removed in vacuo.

[0339] Oxidation: The previously obtained a-bromoboronic acid ester was dissolved in 225 mL f-BuOH and mixed with 50 mL (472 mmol, 40 eq., p = 0.662 g / mL) 2-methyl-2-butene. A solution of 13.5 g (80 wt%, 120 mmol, 10 eq.) NaCIO2 and 16.2 g (120 mmol, 10 eq.) KH2PO4 in 114 mL water was then added dropwise. After 4 d, the yellow emulsion was concentrated in vacuo and acidified with 10 wt% aqueous citric acid solution to a pH of about 3 before extracting three times with Et20. The combined etheric phases were washed with sat. Na2S2O3(aq.) solution, dried over MgSO4 and the solvent was removed in vacuo. The residue was then purified by automated column chromatography (SiO2, cHex / EtOAc 10:0 -^ 6:4).

[0340] Esterification:!81The previously isolated a-azidocarboxylic acid was dissolved in 106 mL DMAc and 48 mL (424 mmol, 36 eq., p = 1.210 g / mL) f-BuBr, 2.40 g (10.5 mmol, 0.9 eq.)BnNEt3Cl and 37.9 g (274 mmol, 23 eq.) K2CO3 were added. The suspension was then stirred for 4.5 h at 55 °C. After cooling to RT, water was added to the mixture until a clear solution was obtained. This was extracted three times with Et2O, the combined etheric phases were washed twice with water and once with sat. NaCI(aq.) solution before drying over MgSO4 and removing the solvent in vacuo. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — 8:2) afforded 2.83 g (5.83 mmol, 49 %) of the a-azido-te / Y-butyl ester P32 as a colorless oil.

[0341] [a]2° = -5.4 (C= 1.0, CHCI3)

[0342] Rf = 0.60 (n-Pentane / EtOAc 75:25)

[0343]

[0344] HRMS (ESI): Calculated for C30H35N3NaO3+[M+Na]+: 508.2571, found: 508.2566.

[0345] fert-Butyl (2S,4 / ?)-2-Azido-4-(hydroxymethyl)hexanoate [P33][9]

[0346] 2.77 g (5.70 mmol, 1.0 eq.) of the a-azido-te / Y-butyl ester P32 were dissolved in 23 mL AcOH, mixed with 5.8 mL H2O and heated to 55 °C. After 3.5 h, the reaction was cooled to RT and carefully neutralized (strong gas evolution) with sat. NaHCO3(aq.) solution. The aqueous phase was extracted three times with Et20, and the combined organic phases were dried over MgSO4. The solvent was then removed in vacuo and the residue was purified by automated column chromatography (cHex / EtOAc 1:0 — > 6:4). 1.14 g (4.69 mmol, 82 %) of the alcohol P33 was isolated as a pale-yellow liquid.

[0347] [a] ° = -38.6 (c = 1.0, CHCI3) Rf = 0.30 (n-Pentane / Et2O 1:1)

[0348]

[0349] HRMS (Cl): Calculated for C11H22N3O3+[M+H]+: 244.1656, found: 244.1650.

[0350] fert-Butyl (2S,4 / ?)-2-Azido-4-((tosyloxy)methyl)hexanoate [P34]To a solution of 1.13 g (4.65 mmol, 1.0 eq.) of the alcohol P33 in 23 mL DCM, 1.95 mL (14.0 mmol, 3.0 eq., p = 0.726 g / mL) Et3N followed by 1.79 g (9.37 mmol, 2.0 eq.) tosyl chloride were added at 0 °C and slowly warmed to RT after the addition was complete. After 3 d, the reaction mixture was washed with sat. NaHCO3(aq.) solution and the aqueous phase was extracted three times with DCM before the combined organic phases were dried over MgSO4. The solvent was then removed in vacuo and the residue was purified by column chromatography (n-Pentane / Et2O 9:1 — 8:2). 1.74 g (4.37 mmol, 94 %) of the tosylate P34 was isolated as a yellow oil.

[0351] [cr]o° = -28.8 (c = 1.0, CHCh)

[0352] Rf = 0.64 (n-Pentane / EtOAc 1:1)

[0353] 8

[0354] \7 M

[0355]

[0356] HRMS (Cl): Calculated for C18H28N3O5S+[M+H]+: 398.1744, found: 398.1748.

[0357] fert-Butyl (2S,4 / ?)- / V-benzyloxycarbonyl-4-ethyl-prolinate [P35]

[0358] According to GP2, 922 mg (2.32 mmol, 1.0 eq.) of the tosylate P34 were reacted in 12 mL MeOH with 99.1 mg Pd / C (10 wt%) under H2 atmosphere. After 1.5 d, the resulting residue was dissolved in 12 mL MeOH and cooled to 0 °C. Subsequently, 970 pL (6.96 mmol, 3.0 eq., = 0.726 g / mL) Et3N and 361 pL (2.53 mmol, 1.1 eq., p = 1.195 g / mL) CbzCI were added dropwise to the solution and slowly warmed to RT after the addition was completed. After 3 h, the solvent was removed in vacuo and the residue was taken up in EtOAc. The organic phase was washed three times with 10 wt% aqueous citric acid solution and twice with water. It was then dried over MgSO4, and the solvent was removed in vacuo. Lastly, the residue obtained was purified by automated column chromatography (cHex / EtOAc 100:0 ^ 75:25), resulting in the isolation of 702 mg (2.10 mmol, 91 %) of the proline derivative P35 as a colorless oil.

[0359] [a] ° = -28.0 (c = 1.0, CHCh)

[0360] Rf = 0.65 (n-Pentane / EtOAc 1:1)

[0361]

[0362] HRMS (Cl): Calculated for C19H28NO4+[M+H]+: 334.2013, found: 334.2004.

[0363] (2S,4 / ?)- / V-Benzyloxycarbonyl-4-ethyl-proline [P36]

[0364] 1.12 g (3.37 mmol, 1.0 eq.) of the proline derivative P35 were dissolved in a mixture of 17 mL CHCh and 5.6 mL TFA and heated to 50 °C. After 6 h, the yellow solution was cooled to RT, the solvent was removed in vacuo and the residue was co-evaporated three times with toluene. Automated column chromatography (cHex / EtOAc / AcOH 100:0:0 — 50:50:2) afforded 841 mg (3.03 mmol, 90 %) of the carboxylic acid P36 as a colorless oil.

[0365] [a]^0= -37.7 (c = 1.0, CHCh)

[0366] Rf = 0.43 (n-Pentane / EtOAc 1:1)

[0367]

[0368] HRMS (ESI): Calculated for C15H20NO4+[M+H]+: 278.1387, found: 278.1384.

[0369] Methyl (2S,4 / ?)-2-[(Benzyloxycarbonyl)amino]-5-hydroxy-4-methylpentanoate [P51][1°l

[0370] Under N2 atmosphere, 2.56 g (6.26 mmol, 1.0 eq.) of methyl (2S,4R)-2-[((benzyloxy)-carbonyl)amino]-5-((te / Y-butyldimethylsilyl)oxy)-4-methylpentanoatel11l was dissolved in 21 mL anhydrous MeOH and cooled to 0 °C before 66.8 pL (940 pmol, 0.15 eq., p = 1.104 g / mL) acetyl chloride was added. The solution was then slowly warmed to RT overnight and diluted with EtOAc after 18 h. It was then washed with sat. NaHCO3(aq.) solution and water. Next, it was dried over MgSO4, and the solvent was removed in vacuo. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 4:6) of the residue afforded 1.59 g (5.38 mmol, 86 %) of the alcohol P51 as a pale pink oil.[a] ° = +22.2 (c = 0.5, CHCh)

[0371] Rf = 0.18 (n-Pentane / EtOAc 1:1)

[0372] 12

[0373] 13

[0374]

[0375] HRMS (ESI): Calculated for C15H22NO5+[M+H]+: 296.1492, found: 296.1479.

[0376] Methyl (2S,4R)-2-[(Benzyloxycarbonyl)amino]-4-methyl-5-(tosyloxy)pentanoate [P52]

[0377] To a solution of 602 mg (2.04 mmol, 1.0 eq.) of the alcohol P51 in 6.8 mL DCM was added 881 pL (6.32 mmol, 3.1 eq., p = 0.726 g / mL) EtsN at 0 °C, followed by 777 mg (4.08 mmol, 2.0 eq.) tosyl chloride. The mixture was slowly warmed to RT, and after 21.5 h the now brown suspension was diluted with EtOAc and washed successively with 1 M HCI(aq), water, sat. NaHCO3(aq.) and sat. NaCI(aq.) solution. The organic phase was then dried over MgSO4, and the solvent was removed in vacuo. After automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 6:4) of the residue, 797 mg (1.77 mmol, 87 %) of the tosylate P52 could be isolated as a yellow oil.

[0378] [c ]o° = +10.0 (c = 0.5, CHCh)

[0379] Rf = 0.51 (n-Pentane / EtOAc 1:1)

[0380] 13

[0381]

[0382] HRMS (ESI): Calculated for C16H28NO5+[M+H]+: 450.1581, found: 450.1559.

[0383] Methyl (2S,4 / ?)- / V-Benzyloxycarbonyl-4-ethyl-prolinate [P53]

[0384] To a solution of 1.70 g (3.78 mmol, 1.0 eq.) of tosylate P52 in 13 mL anhydrous THF, 150 mg (60 wt%, 3.75 mmol, 1.0 eq.) NaH was added portionwise at 0 °C under N2 atmosphere. After complete addition, the mixture was stirred at RT for 2 h before being carefully quenched with water. Subsequently, the mixture was extracted three times with Et20, the combined organic phases were dried over MgSO4, and the solvent wasremoved in vacuo. Automated flash chromatography of the crude product afforded 877 mg (3.16 mmol, 84 %) of the proline derivative P53 as a colorless oil. In addition, 131 mg (291 pmol, 8 %) of the tosylate P52 could be recovered.

[0385] [α]D20 = –43.8 (c = 0.5, CHCl3)

[0386] Rf = 0.45 (n-Pentane / EtOAc 1:1)

[0387]

[0388] HRMS (ESI): Calculated for C16H28NO5+[M+H]+: 278.1387, found: 278.1383.

[0389] (2S,4 / ?)- / V-Benzyloxycarbonyl-4-methyl-proline [P54]

[0390] According to GP3, 420 mg (1.52 mmol, 1.0 eq.) of the proline derivative P53 were reacted with 1.67 mL (1.0 M, 1.67 mmol, 1.1 eq.) LiOH(aq.) in 5.2 mL 1,4-dioxane for 4 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 -^ 0:10) afforded 391 mg (1.49 mmol, 98 %) of carboxylic acid P54 as a colorless oil.

[0391] [α]D20 = –40.8 (c = 0.5, CHCl3)

[0392] Z',,4 3

[0393] r^\2 / P

[0394] 6;N / OH

[0395] jz"-O 10

[0396] °

[0397] 8\ \\\ 7? \ 12

[0398]

[0399] HRMS (ESI): Calculated for C14H18NO4+[M+H]+: 264.123, found: 264.1229.

[0400] Synthesis of the other Amino Acid Building Blocks

[0401] / V-tert-Butyloxycarbonyl-N-methyl-d-leucine [M2]

[0402] According to GP4, 5.81 g (25.1 mmol, 1.0 eq.) Boc-d-Leu-OH, 4.60 mL (73.6 mmol, 2.9 eq., p = 2.270 g / mL) methyl iodide and 6.00 g (60 wt% in mineral oil, 151 mmol, 6.0 eq.) NaH were reacted in 80 mL anhydrous THF for 19 h. Contrary to GP4, Et20 wasused for extraction instead of EtOAc. A total of 5.77 g (23.5 mmol, 94 %) of Boc- / V-Me-d-Leu-OH (M2) was isolated as a yellow oil.

[0403] [a] ° = +28.5 (c = 1.0, CHCh); Lit.: [a] ° = +33.5 (c = 0.86, CHCh)'12!

[0404]

[0405] HRMS (ESI): Calculated for C12H24NO4+[M+H]+: 246.1700, found: 246.1698.

[0406] Methyl / V-fert-butyloxycarbonyl- / V-methyl-d-leucinate [M3]

[0407] A solution of 5.73 g (23.4 mmol, 1.0 eq.) Boc- / V-Me-d-Leu-OH (M2) in 24 mL DMF was treated with 9.69 g (70.1 mmol, 3.0 eq.) K2CO3 and 3.65 mL (58.4 mmol, 2.5 eq., p = 2.270 g / mL) methyl iodide at 0 °C and slowly warmed to RT after addition. After 19 h, the mixture was diluted with EtOAc and washed twice with sat. NaHCO3(aq.) and once with sat. NaCI(aq.) solution. The mixture was then dried over MgSO4, and the solvent was removed in vacuo. Automated column chromatography (cHex / EtOAc 1:0 — 1: 1 ) afforded 5.59 g (21.6 mmol, 92 %) of Boc- / V-Me-d-Leu-OMe (M3) as a pale-yellow liquid.

[0408] [a] ° = +38.6 (c = 1.0, CHCh)

[0409] Rf = 0.50 (n-Pentane / EtOAc 8:2)

[0410]

[0411] HRMS (ESI): Calculated for C13H26NO4+[M+H]+: 260.1856, found: 260.1852.

[0412] N-ferf-butyloxycarbonyl-frans-4-Cyclohexyl -l-proline [M9]

[0413] To a suspension of 1.91 g (9.69 mmol, 1.0 eq.) frans-l-CyPro-OH and 2.45 g (29.2 mmol, 3.0 eq.) NaHCO3 in a mixture of 12 mL H2O and 10 mL THF was slowly added a solution of 2.33 g (10.7 mmol, 1.1 eq.) Boc2O in 2 mL THF at 0 °C. After the addition was complete, the reaction mixture was slowly warmed to RT and stirred for 14 h. The reaction mixture was then extracted twice with Et2O, and the aqueous phase was acidified with 1 M HCI(aq.) to a pH of 1, whereby a colorless solid precipitated. The suspension was extracted three times with Et2O, the combined etheric phases were dried over MgSO4,and the solvent was removed in vacuo. Drying in high vacuum afforded 2.69 g (9.06 mmol, 93 %) of the Boc-protected amino acid M9 as a colorless solid.

[0414] [α]D20 = –48.3 (c = 1.0, CHCl3) Melting range: 123-125 °C

[0415] \6

[0416] 5j 3

[0417] JO

[0418] \i 1

[0419]

[0420] / V-Benzyloxycarbonyl-O-tert-butyldimethylsilyl-l-threonine [M13]

[0013]

[0421] Under N2 atmosphere, a solution of 5.56 g (81.7 mmol, 3.0 eq.) imidazole in 7 mL anhydrous DMF was added to a solution of 6.89 g (27.2 mmol, 1.0 eq.) Cbz-I-Thr-OH in 16 mL anhydrous DMF at 0 °C, followed by a solution of 12.3 g (81.6 mmol, 3.0 eq.) TBS-Cl in 22 mL anhydrous DMF, and warmed to RT after complete addition. After 27 h, the yellow solution was poured into approx. 300 mL ice water and extracted three times with Et20 and dried over Na2SO4. After the solvent was removed in vacuo, the residue was dissolved in 54 mL THF, mixed with 54 mL 0.5 M KOH(aq.) and stirred for 4 h at 0 °C. The mixture was then extracted once with Et20, whereby this organic phase was discarded. The aqueous phase was acidified with 1 M HCI(aq.) solution to a pH of 2-3 and then extracted three times with Et20. Finally, the combined etheric phases were washed once with sat. NaCI(aq.) solution and dried over Na2SO4. After removing the solvent in vacuo, 9.38 g (25.5 mmol, 94 %) of Cbz-O-TBS-I-Thr-OH (M13) was isolated as a colorless solid.

[0422] [a] ° = +10.1 (c = 1.0, C

[0423]

[0424] HCI3); Lit.: [a]^3= +13.2 (c = 1.0, CHCh)'14!

[0425] Rf = 0.79 (DCM / MeOH 95:5)

[0426] Melting range: 157-159 °C (Lit.: 150.5-152.5 °C)I131

[0427]

[0428] O

[0429] HRMS (ESI): Calculated for C18H29NNaO5Si+[M+Na]+: 390.1707, found: 390.1707.

[0430] / V-Benzyloxycarbonyl-N-methyl-O-tert-butyldimethylsilyl-l-threonine [M15]According to GP4, 2.63 g (7.15 mmol, 1.0 eq.) of Cbz-O-TBS-I-Thr-OH (M13), 2.30 mL (36.8 mmol, 5.1 eq., p = 2.270 g / mL) methyl iodide and 1.44 g (60 wt% in mineral oil, 36.0 mmol, 5.0 eq.) NaH were reacted in 36 mL of anhydrous THF for 19 h. Contrary to GP4, the reaction mixture was acidified with 1 M HCI(aq.) for workup and washed twice with sat. Na2S2O3(aq.) solution. The residue obtained was purified by automated column chromatography (SiO2, cHex / EtOAc / AcOH 100:0:0 —> 50:50:1), whereby the isolated fraction was co-evaporated three times with toluene. A total of 2.20 g (5.78 mmol, 81 %) of A / -m ethylated amino acid M15 was isolated as a colorless, highly viscous oil.

[0431] [c ]o° = +13.4 (c = 1.0, CHCh)

[0432] Rf= 0.07 (n-Pentane / EtOAc / AcOH 80:20:1)

[0433] 13

[0434] ^ 12^

[0435] 11 / 'O o

[0436] 5 T3

[0437]

[0438] o

[0439] HRMS (Cl): Calculated for C19H32NO5Si+[M+H]+: 382.2044, found: 382.2059.

[0440] / V-Allyloxycarbonyl-l-valine [M14][15l

[0441] To a solution of 5.87 g (50.1 mmol, 1.0 eq.) I-Val-OH and 10.4 g (75.2 mmol, 1.5 eq.) K2CO3 in 110 mL water and 90 mL THF, a solution of 6.3 mL (59.1 mmol, 1.2 eq., p = 1.13 g / mL) allyl chloroformate in 20 mL THF was slowly added dropwise at 0 °C over a period of 15 min. After the addition was complete, the reaction was slowly warmed to RT and stirred for 3 d. The reaction mixture was concentrated in vacuo and extracted twice with Et20. The aqueous phase was acidified with 6 M HCI(aq.) to a pH of 1, whereby a colorless solid precipitated. After the suspension was extracted three times with DCM, the combined DCM phases were dried over MgSO4. Removal of the solvent in vacuo afforded 9.99 g (49.6 mmol, 99 %) of Alloc-I-Val-OH (M14) as a pale-yellow resin.

[0442] [a] ° = -17.3 (c = 1.0, CHCh); Lit.: [a] ° = -19.6 (c = 0.7, CHCI3)[161

[0443]

[0444] HRMS (ESI): Calculated for C9H16NO4 [M+H]+: 202.1074, found: 202.1073.N-Allyloxycarbonyl-N-methyl-l-valine [M16]

[0445] According to GP4, 4.82 g (24.0 mmol, 1.0 eq.) of Alloc-Val-OH (M14), 7.50 mL (120 mmol, 5.0 eq., p = 2.280 g / mL) methyl iodide and 2.88 g (60 wt% in mineral oil, 72.0 mmol, 3.0 eq.) NaH were reacted in 80 mL of anhydrous THF for 16.5 h. The crude product thus obtained was purified by automated column chromatography (SiO2, cHex / EtOAc / AcOH 100:0:0 — 50:50: 1 ) and the isolated fraction was co-evaporated three times with toluene. A total of 3.62 g (16.8 mmol, 70 %) of Alloc- / V-Me-Val-OH (M16) was isolated as a yellow oil.

[0446] [cr]o° = -79.5 (c = 1.0, CHCh) Rf = 0.16 (n-Pentane / EtOAc / AcOH 80:20:1)

[0447] 79I H

[0448] H=rvotN^oH

[0449]

[0450] Hb0 4

[0451] HRMS (Cl): Calculated for C10H18NO4+[M+H]+: 216.1230, found: 216.1234.

[0452] (2S,4 / ?)- / V-Benzyloxycarbonyl-4-methoxy-l-proline [M92]

[0453] According to GP4, 193 mg (728 pmol, 1.0 eq.) of Cbz-I-Hyp-OH was reacted with 0.23 mL (3.69 mmol, 5.1 eq., p = 2.280 g / mL) methyl iodide and 87.3 mg (60 wt% in mineral oil, 2.18 mmol, 3.0 eq.) NaH in 3.6 mL anhydrous THF for 23 h. Contrary to GP4, the aqueous work-up was carried out as follows: After adding H2O to the reaction mixture, it was acidified with 1 M HCI(aq.) to a pH of about 1. The aqueous phase was extracted three times with Et20 and the combined etheric phases were washed once with sat. Na2S2O3(aq.)- followed by sat. NaCI(aq.) solution. It was then dried over MgSO4, the solvent was removed in vacuo and the residue was purified by automated column chromatography (SiO2, cHex / EtOAc / AcOH 100:0:0 50:50:1). The isolated fraction was co-evaporated three times with toluene. A total of 203 mg (727 pmol, 100 %) of the proline derivative M92 was isolated as a colorless, viscous oil.

[0454] [

[0455]

[0456] cr]o° = -59.8 (c = 1.0, CHCh)

[0457] Rf= 0.38 (n-Pentane / EtOAc / AcOH 50:50:1)

[0458]

[0459] HRMS (ESI): Calculated for C14H18NO5+[M+H]+: 280.1179, found: 280.1185.

[0460] (2S,4 / ?)- / V-Benzyloxycarbonyl-4-ethoxy-l-proline [M93]

[0461] According to GP4, 116 mg (436 pmol, 1.0 eq.) of Cbz-L-Hyp-OH were reacted with 0.17 mL (2.10 mmol, 4.8 eq., p = 1.930 g / mL) of ethyl iodide and 53.7 mg (60 wt% in mineral oil, 1.34 mmol, 3.0 eq.) of NaH in 1.5 mL of anhydrous THF for 24 h. Contrary to GP4, the aqueous work-up was carried out as follows: After adding H2O to the reaction mixture, it was acidified with 1 M HCI(aq.) to a pH of about 1. The aqueous phase was extracted three times with Et20, and the combined etheric phases were washed once with sat. Na2S2O3(aq.)- followed by sat. NaCI(aq.) solution. It was then dried over MgSO4, the solvent was removed in vacuo and the residue was purified by automated column chromatography (SiO2, cHex / EtOAc / AcOH 100:0:0 —> 50:50:1; then Cis-SiO2, H2O / MeCN (+0.1 % HCOOH), 9:1 0:10). A total of 73.3 mg (250 pmol, 57 %) of the proline derivative M93 was isolated as a colorless, viscous oil.

[0462] [cr]o° = -61.4 (c = 1.0, CHCI3)

[0463] Rf= 0.46 (n-Pentane / EtOAc / AcOH 50:50:1)

[0464]

[0465] HRMS (ESI): Calculated for C15H20NO5+[M+H]+: 294.1336, found: 294.1332.

[0466] / Va-fert-Butyloxycarbonyl- / V£-azido-l-lysine [M53][17l

[0467] Triflylazide: A solution of 1.28 g (19.7 mmol, 1.6 eq.) NaNs in 3.1 mL H2O was mixed with 3.1 mL toluene and cooled to 0 °C before 2.1 mL (12.3 mmol, 1.0 eq., p = 1.670 g / mL) Tf20 was added. The two-phase mixture was first stirred vigorously for 30 min at 0 °C and then for 2 h at 10 °C. Sat. NaHCO3(aq.) solution was then added until no more gasevolution was observed. The phases were then separated, and the reaction vessel was rinsed with 1.0 mL toluene when the mixture was transferred. Next, the aqueous phase was extracted twice with 2.7 mL toluene, whereby the combined organic phases correspond to an approx. 1.3 M solution of TfNs.

[0468] Diazotransfer: To a solution of 412 mg (1.67 mmol, 1.0 eq.) Boc-I-Lys-OH in 2.0 mL water were added sequentially 561 mg (6.68 mmol, 4.0 eq.) NaHCO3, 18.0 mg (72.1 pmol, 4 mol%) CuSO4·5H2O, 4.0 mL (1.3 M in toluene, 5.20 mmol, 3.1 eq.) TfNs, and 15 mL MeOH. After the blue suspension was stirred vigorously for 17 h, the solvent was removed by rotary evaporation at RT(I). The resulting residue was purified by automated column chromatography (SiO2, cHex / EtOAc / AcOH 100:0:0 —> 50:50:1; then Ci8-SiO2, H2O / MeCN, 9:1 — 0:10). A total of 361 mg (1.33 mmol, 79 %) of the lysine derivative M53 was obtained as a colorless, highly viscous oil.

[0469] [α]D²⁰ = −27.4 (c = 0.5, CHCl3); Lit.: [α]D = −1.1 (c = 1.0, MeOH)

[0018]

[0470] Rf= 0.65 (n-Pentane / EtOAc / AcOH 50:50:1)

[0471] 8 6

[0472] 2

[0473] 7 |

[0474] HN^O

[0475] 3|

[0476]

[0477] HRMS (ESI): Calculated for C11H21N4O4+[M+H]+: 273.1557, found: 273.1552.

[0478] Synthesis of the Peptide Fragments

[0479] Synthesis of the Tetrapeptide Fragments

[0480] Boc-l-Pro-N-Me-d-Leu-OMe [M5]!19!

[0481] According to GP5, 4.71 g (18.2 mmol, 1.0 eq.) of Boc- / V-Me-d-Leu-OMe (M3) was reacted with 12.7 mL (179 mmol, 10 eq., p = 1.104 g / mL) acetyl chloride in 45 mL MeOH. After 2 h, 3.61 g (18.2 mmol, 100 %) of / V-Me-d-Leu-OMe HCI was isolated as a colorless solid.

[0482] Under N2 atmosphere, 3.09 g (14.4 mmol, 1.4 eq.) of Boc-I-Pro-OH was dissolved in 51 mL 1,4-dioxane and treated with 3.93 mL (22.5 mmol, 2.2 eq., p = 0.742 g / mL) DIPEA, followed by 152 pL (1.02 mmol, 0.1 eq., p = 0.910 g / mL) BnNMe2. The solution was heated to 60 °C and treated dropwise with 14.3 mL (1.0 M in toluene, 14.3 mmol,1.4 eq.) IPCF solution. After stirring at 60 °C for 15 min, a solution of 2.00 g (10.2 mmol, 1.0 eq.) A / -Me-d-Leu-OMe HCI, 163 pL (2.05 mmol, 0.2 eq., p = 1.030 g / mL) NMI, and 256 pL (4.0 M in 1,4-dioxane, 1.02 mmol, 0.1 eq.) HCI, dissolved in a mixture of 26 mL 1,4-dioxane and 42 mL MeCN, was added (rinsed twice with a total of 26 mL 1,4-dioxane). Stirring was continued at 60 °C for 17 h, and then the mixture was cooled to RT.

[0483] After concentration in vacuo, the residue was diluted with EtOAc. The organic phase was washed with 1 M KHSO4(aq), water, sat. NaHCO3(aq.) and sat. NaCI(aq.) solution. Then it was dried over MgSO4, the solvent was removed in vacuo, and the residue was purified by automated column chromatography (cHex / EtOAc 10:0 — > 4:6). A total of 3.02 g (8.48 mmol, 84 %) of dipeptide M5 was isolated as a colorless solid.

[0484] [α]D²⁰ = +12.4 (c = 1.0, CHCl3)

[0485] Melting range: 119-120 °C

[0486] Rf = 0.30 (n-Pentane / EtOAc 1:1)

[0487] 5

[0488]

[0489] 6

[0490] HRMS (Cl): Calculated for C18H32N2O5+[M]+: 356.2311, found: 356.2306.

[0491] Boc-N-Me-l-Val-l-Pro-N-Me-d-Leu-OMe [M6]

[0492] According to GP5, 1.52 g (4.26 mmol, 1.0 eq.) of dipeptide M5 was reacted with 2.30 mL (31.7 mmol, 7.5 eq., p = 1.104 g / mL) acetyl chloride in 11 mL MeOH for 1.5 h. Contrary to GP5, M5 was added only after acetyl chloride. The resulting peptide hydrochloride was reacted according to GP6 with 985 mg (4.26 mmol, 1.0 eq.) Boc- / V-Me-I-Val-OH, 1.51 g (4.70 mmol, 1.1 eq.) TBTU, and 1.64 mL (9.39 mmol, 2.2 eq., p = 0.742 g / mL) DIPEA in 29 mL anhydrous MeCN for 17 h. Afterwards, the mixture was concentrated in vacuo, the residue was taken up in EtOAc, and the following workup was performed accordingly. Instead of 1 M KHSO4(aq), 1 M HCI(aq.) was used in the first washing step. Automated column chromatography (SiO2, DCM / MeOH 100:0 — 97:3) of the crude product yielded 1.39 g (2.96 mmol, 70 %) of tripeptide M6 as a colorless resin.

[0493] [α]D²⁰ = −76.0 (c = 1.0, CHCl3)

[0494] Rf= 0.47 (DCM / MeOH 95:5)

[0495]

[0496] HRMS (Cl): Calculated for C24H44N3O6+[M+H]+: 470.3225, found: 470.3214.

[0497] Boc-l-HoLeu- / V-Me-l-Val-l-Pro- / V-Me-d-Leu-OMe [M7]

[0498] According to GP5, 99.9 mg (213 pmol, 1.0 eq.) of tripeptide M6 was reacted with 151 pL (2.13 mmol, 10 eq., p = 1.104 g / mL) acetyl chloride in 0.71 mL MeOH for 3 h. The resulting peptide hydrochloride was reacted according to GP6 with 70.0 mg (285 pmol, 1.3 eq.) Boc-l-HoLeu-OH, 29.5 mg (217 pmol, 1.0 eq.) HOAt, 162 mg (426 pmol, 2.0 eq.) HATU, and 148 pL (852 pmol, 4.0 eq., p = 0.742 g / mL) DIPEA in 2.1 mL anhydrous DMF for 16 h. Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 —> 0:10) of the crude product yielded 109 mg (183 pmol, 86 %) of the tetrapeptide M7 as a colorless, amorphous solid.

[0499] [α]D²⁰ = −85.9 (c = 1.0, CHCl3)

[0500] Rf = 0.25 (n-Pentane / EtOAc 1:1)

[0501]

[0502] HRMS (Cl): Calculated for C31H57N4O7+[M]+: 597.4222, found: 597.4234.

[0503] Boc-l-HoLeu-N-Me-l-Val-l-Pro-N-Me-d-Leu-OH [M48]

[0504] According to GP3, 102 mg (170 pmol, 1.0 eq.) of tetrapeptide M7 was reacted with 188 pL (1.0 M, 188 pmol, 1.1 eq.) LiOH(aq.) in 0.85 mL 1,4-dioxane for 3 h. The residue obtained after workup was lyophilized and used without further purification. A total of 94.4 mg (162 pmol, 95 %) of carboxy lie acid M48 was obtained as a colorless lyophilizate.

[0505] [

[0506]

[0507] cr]o° = -75.8 (c = 1.0, CHCh)

[0508]

[0509] HRMS (ESI): Calculated for C30H55N4O7 [M+H]+: 583.4065, found: 583.4074.

[0510] Boc-l-CyPro-N-Me-d-Leu-OMe [M10]I19l

[0511] According to GP5, 1.61 g (6.21 mmol, 1.0 eq.) of Boc- / V-Me-d-Leu-OMe (M3) was reacted with 4.41 mL (62.1 mmol, 10 eq., p = 1.104 g / mL) acetyl chloride and 2.51 mL (62.1 mmol, 10 eq., p = 0.792 g / mL) MeOH in 16 mL 1,4-dioxane for 2.5 h.

[0512] Under N2 atmosphere, 2.01 g (6.75 mmol, 1.1 eq.) of Boc-l-CyPro-OH was dissolved in 44 mL of 1,4-dioxane and treated with 2.38 mL (13.7 mmol, 2.2 eq., p = 0.742 g / mL) DIPEA, followed by 92.2 pL (621 pmol, 0.1 eq., p = 0.910 g / mL) BnNMe2. The solution was heated to 60 °C and treated dropwise with 6.8 mL (1.0 M in toluene, 6.80 mmol, 1.1 eq.) IPCF solution. After stirring at 60 °C for 10 min, a solution of the previously obtained / V-Me-d-Leu-OMe HCI, 99.0 pL (1.24 mmol, 0.2 eq., p = 1.030 g / mL) NMI, and 160 pL (4.0 M in 1,4-dioxane, 640 pmol, 0.1 eq.) HCI, dissolved in 22 mL 1,4-dioxane and 37 mL MeCN, was added (rinsed with a total 44 mL of 1,4-dioxane). Stirring was continued at 60 °C for 17 h, and then the mixture was cooled to RT.

[0513] After concentration in vacuo, the residue was taken up in DCM. The organic phase was washed with 1 M KHSO4(aq), H2O, sat. NaHCO3(aq.) and sat. NaCI(aq.) solution. Then it was dried over MgSO4, the solvent was removed in vacuo, and the residue was purified by automated column chromatography (cHex / EtOAc 10:0 — > 1:1). A total of 2.19 g (5.00 mmol, 81 %) of dipeptide M10 was isolated as a colorless solid.

[0514] [α]D²⁰ = +22.4 (c = 1.0, CHCl3)

[0515] Melting point: 149 °C

[0516] Rf = 0.59 (n-Pentane / EtOAc 1:1)15 14

[0517] 13

[0518]

[0519] HRMS (Cl): Calculated for C24H43N2O5+[M+H]+: 439.3166, found: 439.3173.

[0520] Boc-N-Me-l-Val-l-CyPro-N-Me-d-Leu-OMe [M11]

[0521] According to GP5, 543 mg (1.24 mmol, 1.0 eq.) of dipeptide M10 was treated with 3.0 mL (4.0 M in 1,4-dioxane, 12.0 mmol, 10 eq.) HCIfor3 h. The resulting peptide hydrochloride was reacted according to GP6 with 328 mg (1.42 mmol, 1.1 eq.) of Boc- / V-Me-I-Val-OH, 441 mg (1.38 mmol, 1.1 eq.) TBTU, and 544 pL (3.13 mmol, 2.5 eq., p = 0.742 g / mL) DIPEA in 4.2 mL anhydrous MeCN for 19 h. Before dilution with EtOAc for aqueous workup, the reaction mixture was concentrated in vacuo, and 1 M HCI(aq.) was used instead of 1 M KHSO4(aq.) in the first extraction step. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — 5:5) yielded 602 mg (1.09 mmol, 87 %) of tripeptide M11 as a pale-yellow resin.

[0522] [α]D²⁰ = −43.9 (c = 1.0, CHCl3)

[0523] Rf = 0.42 (n-Pentane / EtOAc 1:1)

[0524]

[0525] HRMS (ESI): Calculated for C30H54N3O6+[M+H]+: 552.4007, found: 552.3996.

[0526] Boc-l-HoLeu- / V-Me-l-Val-l-CyPro- / V-Me-d-Leu-OMe [M12]

[0527] According to GP5, 105 mg (176 pmol, 1.0 eq.) of tripeptide M11 was treated with 1.0 mL (4.0 M in 1,4-dioxane, 4.00 mmol, 23 eq.) HCI for 1 h. The resulting peptide hydrochloride was reacted according to GP6 with 55.5 mg (226 pmol, 1.3 eq.) Boc-l-HoLeu-OH,24.7 mg (181 pmol, 1.0 eq.) HOAt, 133 mg (349 pmol, 2.0 eq.) HATU, and 122 pL (703 pmol, 4.0 eq., p = 0.742 g / mL) DIPEA in 1.8 mL anhydrous DMF for 17 h. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — 5:5) of the crude product, followed by lyophilization, yielded 104 mg (153 pmol, 87 %) of the tetrapeptide M12 as a colorless lyophilizate.

[0528] [α]D²⁰ = −45.6 (c = 1.0, CHCl3)

[0529] Rf = 0.50 (n-Pentane / EtOAc 1:1)

[0530]

[0531] HRMS (ESI): Calculated for C37H67N4O7+[M+H]+: 679.5004, found: 679.5003.

[0532] Boc-l-HoLeu-N-Me-l-Val-l-CyPro-N-Me-d-Leu-OH [M49]

[0533] According to GP3, 594 mg (874 pmol, 1.0 eq.) of tetrapeptide M12 and 1.09 mL (1.0 M, 1.09 mmol, 1.2 eq.) LiOH(aq.) were reacted in 4.4 mL 1,4-dioxane for 3 h. After automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) and lyophilization, 495 mg (745 pmol, 85 %) of the carboxylic acid M49 were isolated as a colorless lyophilizate. [a] ° = -66.6 (c = 0.5, CHCh) Rf = 0.46 (n-Pentane / EtOAc / AcOH 50:50:1)

[0534]

[0535] HRMS (ESI): Calculated for C36H65N4O7 [M+H]+: 665.4848, found: 665.4828.

[0536] Boc-l-Leu-N-Me-l-Val-l-CyPro-N-Me-d-Leu-OMe [M64]According to GP5, 234 mg (424 pmol, 1.0 eq.) of tripeptide M11 was treated with 1.1 mL (4.0 M in 1,4-dioxane, 4.00 mmol, 10 eq.) HCI for 3.5 h. The resulting peptide hydrochloride was reacted according to GP6 with 140 mg (560 pmol, 1.3 eq.) Boc-I-Leu-OH, 210 mg (552 pmol, 1.3 eq.) HATU, and 185 pL (1.06 mmol, 2.5 eq., p = 0.742 g / mL) DIPEA for 16.5 h. Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10; then SiO2, cHex / EtOAc 10:0 — > 5:5) of the crude product, followed by lyophilization, yielded 201 mg (302 pmol, 71 %) of the tetrapeptide M64 as a colorless lyophilizate. [α]D²⁰ = −69.6 (c = 0.5, CHCl3)

[0537]

[0538] HRMS (ESI): Calculated for C36H64N4NaO7+[M+Na]+: 687.4667, found: 687.4673.

[0539] Boc-l-Leu- / V-Me-l-Val-l-CyPro- / V-Me-d-Leu-OH [M65]

[0540] According to GP3, 176 mg (264 pmol, 1.0 eq.) of tetrapeptide M64 was reacted with 290 pL (1.0 M, 290 pmol, 1.1 eq.) LiOH(aq.) in 0.9 mL 1,4-dioxane. After 5 h, additional 26.4 pL (1.0 M, 26.4 pmol, 0.1 eq.) of LiOH(aq.) was added. The reaction mixture was then worked up after 1 h and the crude product was purified by automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — 0:10). After lyophilization, a total of 153 mg (235 pmol, 89%) of carboxylic acid M65 was isolated as a colorless lyophilizate.

[0541] [α]D²⁰ = −65.4 (c = 0.5, CHCl3)

[0542]

[0543] HRMS (ESI): Calculated for C35H63N4O7 [M+H]+: 651.4691, found: 651.4676.Boc-l-MePro-N-Me-d-Leu-OMe [M66]l19]

[0544] According to GP5, 4.71 g (862 μmol, 1.0 eq.) of Boc-N-Me-d-Leu-OMe (M3) was reacted with 45 mL MeOH and 12.7 mL (179 mmol, 10 eq., p = 1.104 g / mL) acetyl chloride. After a reaction time of 2 h, 3.61 g (18.2 mmol, 100 %) of / V-Me-d-Leu-OMe HCI was isolated as a colorless solid.

[0545] Under N2 atmosphere, 227 mg (862 pmol, 1.1 eq.) of Cbz-l-MePro-OH (P54) was dissolved in 6 mL 1,4-dioxane and treated with 294 pL (1.69 mmol, 2.2 eq., p = 0.742 g / mL) DIPEA, followed by 11.4 pL (76.7 pmol, 0.1 eq., p = 0.910 g / mL) BnNMe2. The solution was heated to 60 °C and treated dropwise with 0.86 mL (1.0 M in toluene, 860 pmol, 1.1 eq.) IPCF solution. After stirring at 60 °C for 10 min, a solution consisting of 152 mg (778 pmol, 1.0 eq.) of the previously obtained / V-Me-d-Leu-OMe HCI, 12.4 pL (156 pmol, 0.2 eq., p = 1.030 g / mL) NMI, and 19.4 pL (4.0 M in 1,4-dioxane, 77.8 pmol, 0.1 eq.) HCI, dissolved in 3 mL 1,4-dioxane and 5 mL MeCN, was added (rinsed twice with a total of 6 mL of 1,4-dioxane). Stirring was continued at 60 °C for 14 h, and then the mixture was cooled to RT. After concentration in vacuo, the residue was taken up in EtOAc. The organic phase was washed with 1 M KHSCkfaq ), water, sat. NaHCO3oq.) and sat. NaCI(aq.) solution. Then it was dried over MgSO4, the solvent was removed in vacuo, and the residue was purified by automated column chromatography (Ci8-SiO2, H2O / MeCN 9:1 0:10). After lyophilization, a total of 247 mg (611 pmol, 79%) of dipeptide M66 was isolated as a colorless lyophilizate.

[0546] [α]D²⁰ = +32.0 (c = 0.5, CHCl3)

[0547]

[0548] HRMS (ESI): Calculated for C22H32N2NaO5+[M+Na]+: 427.2203, found: 427.2203.

[0549] Boc- / V-Me-l-Val-l-MePro- / V-Me-d-Leu-OMe [M67]

[0550] According to GP2, 224 mg (555 pmol, 1.0 eq.) of dipeptide M66 was reacted with 22.3 mg (10 wt%) Pd / C for 3.5 h. After addition of Pd / C, 0.14 mL (4.0 M in 1,4-dioxane, 560 pmol, 1.0 eq.) HCI was added before stirring under H2 atmosphere. The resulting peptide hydrochloride was reacted according to GP6 with 157 mg (678 pmol, 1.2 eq.) of Boc-N-Me-I-Val-OH, 214 mg (668 pmol, 1.2 eq.) TBTU, and 251 pL (1.44 mmol, 2.6 eq., p = 0.742 g / mL) DIPEA in 2.8 mL anhydrous MeCN for 16 h. Before dilution with EtOAc for aqueous workup, the reaction mixture was concentrated in vacuo. Automated column chromatography (SiO2, cHex / EtOAc 10:0 ^ 5:5) of the crude product yielded 219 mg (453 pmol, 82 %) of tripeptide M67 as a colorless resin.

[0551] [α]D²⁰ = −74.3 (c = 0.5, CHCl3)

[0552] '.11 10 7

[0553] (21 18 / 13V^T^

[0554] o 4 5

[0555] 6

[0556]

[0557] HRMS (ESI): Calculated for C25H46N3O6+[M+H]+: 484.3381, found: 484.3395.

[0558] Boc-l-HoLeu- / V-Me-l-Val-l-MePro- / V-Me-d-Leu-OMe [M72]

[0559] According to GP5, 215 mg (445 pmol, 1.0 eq.) of tripeptide M67 was reacted with 317 pL (4.45 mmol, 10 eq., p = 1.104 g / mL) acetyl chloride in 2.0 mL MeOH for6 h. The resulting peptide hydrochloride was reacted according to GP6 with 146 mg (595 pmol, 1.3 eq.) Boc-l-HoLeu-OH, 220 mg (577 pmol, 1.3 eq.) HATU, and 194 pL (1.11 mmol, 2.5 eq., p = 0.742 g / mL) DIPEA in 2.2 mL anhydrous DMF for 16.5 h. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 5:5) of the crude product, followed by lyophilization, yielded 216 mg (353 pmol, 79 %) of tetrapeptide M72 as a pale-yellow lyophilizate.

[0560] [α]D²⁰ = −88.4 (c = 0.5, CHCl3)

[0561] ^ 24 12 ',

[0562] < -.11 10 7

[0563] i2r\* i

[0564] \211813\

[0565] > J<19 M i 14 / n "

[0566] 20 04 5

[0567] 6

[0568]

[0569] HRMS (ESI): Calculated for C32H58N4NaO7+[M+Na]+: 633.4198, found: 633.4202.

[0570] Boc-l-HoLeu-N-Me-l-Val-l-MePro-N-Me-d-Leu-OH [M73]

[0571] According to GP3, 211 mg (345 pmol, 1.0 eq.) of tetrapeptide M72 was treated with 422 pL (1.0 M, 422 pmol, 1.2 eq.) LiOH(aq.) in 2.3 mL 1,4-dioxane. After 5 h, an additional 86.2 pL (1.0 M, 86.2 pmol, 0.25 eq.) of LiOH(aq.) was added. The reaction mixture wasthen worked up after 1 h and the crude product was purified by automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — 0:10). After lyophilization, a total of 194 mg (325 pmol, 94 %) of carboxylic acid M73 was isolated as a colorless lyophilizate.

[0572] [α]D²⁰ = −72.8 (c = 0.5, CHCl3)

[0573]

[0574] HRMS (ESI): Calculated for C31H57N4O7+[M+H]+: 597.4222, found: 597.4234.

[0575] Boc-l-Lys(N3)-l-CyPro-d-Leu-OMe [M54]

[0576] According to GP5, 205 mg (466 pmol, 1.0 eq.) of dipeptide M10 was treated with 1.2 mL (4.0 M in 1,4-dioxane, 4.80 mmol, 10 eq.) of HCI for 2 h. Subsequently, the resulting peptide hydrochloride was reacted according to GP6 with 156 mg (574 pmol, 1.2 eq.) Boc-l-Lys(N3)-OH (M53), 188 mg (585 pmol, 1.2 eq.) TBTU, and 203 pL (1.17 mmol, 2.5 eq., p = 0.742 g / mL) DIPEA in 1.6 mL anhydrous DMF for 22 h. After automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) and lyophilization, 242 mg (408 pmol, 88 %) of tripeptide M54 was isolated as a colorless lyophilizate.

[0577] [α]D²⁰ = −28.8 (c = 0.5, CHCl3)

[0578] 15 14

[0579] O

[0580] 25 0 19VVN3

[0581]

[0582] 2022

[0583] HRMS (ESI): Calculated for C30H52N6NaO6+[M+Na]+: 615.3841, found: 615.3836.

[0584] Boc- / Va-Me-l-Lys(N3)-l-CyPro-d-Leu-OMe [M55]

[0585] In a round-bottomed flask wrapped with aluminium foil, under an Ar atmosphere, 223 mg (377 pmol, 1.0 eq.) of tripeptide M54 was dissolved in 1.3 mL anhydrous DMF and treated with 350 mg (1.51 mmol, 4.0 eq.) Ag2O, followed by 0.38 mL (6.08 mmol, 16 eq.,p = 2.270 g / mL) methyl iodide. After 3 d, the black suspension was filtered through Celite and rinsed with EtOAc. The filtrate was then washed three times with H2O, dried over MgSO4, and concentrated in vacuo. Finally, the residue was purified by automated column chromatography (cHex / EtOAc 10:0 — > 5:5). After lyophilization, 222 mg (366 pmol, 97 %) of the A / -m ethylated tripeptide M55 was isolated as a colorless lyophilizate.

[0586] [a] ° = -39.2 (c = 0.5, CHCI3)

[0587] 15 14

[0588] Q13

[0589] "r' \ I0

[0590] 25 0'

[0591]

[0592] 2022

[0593] HRMS (ESI): Calculated for C31H55N6O6+[M+H]+: 607.4178, found: 607.4169.

[0594] Boc-l-HoLeu- / Va-Me-l-Lys(N3)-l-CyPro-d-Leu-OMe [M56]

[0595] According to GP5, 176 mg (290 pmol, 1.0 eq.) of tripeptide M55 was reacted with 0.73 mL (4.0 M in 1,4-dioxane, 2.90 mmol, 10 eq.) HCIfor2 h. Subsequently, the resulting peptide hydrochloride was reacted according to GP6 with 85.3 mg (348 pmol, 1.2 eq.) Boc-I-HoLeu-OH, 135 mg (354 pmol, 1.2 eq.) HATU, and 131 pL (753 pmol, 2.6 eq., p = 0.742 g / mL) DIPEA in 2.0 mL of anhydrous DMF for 17 h. After automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 4:6), 190 mg (258 pmol, 89 %) of tetrapeptide M56 was isolated as a colorless resin.

[0596] [cr]o° = -33.6 (c = 0.5, CHCI3)

[0597]

[0598] HRMS (ESI): Calculated for C38H67N7O7+[M+H]+: 734.5175, found: 734.5202.Boc-l-HoLeu- / Va-Me-l-Lys(N3)-l-CyPro-d-Leu-OH [M116]

[0599] According to GP3, 185 mg (252 pmol, 1.0 eq.) of the methyl ester M56 was reacted with 315 pL (1.0 M, 315 pmol, 1.25 eq.) LiOH(aq.) in 2.4 mL 1,4-dioxane for 18.5 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN 9:1 —> 0:10) and lyophilization yielded 153 mg (213 pmol, 84 %) of the carboxylic acid M116 as a colorless lyophilizate.

[0600] [a] ° = -28.8 (c = 0.5, CHCh)

[0601]

[0602] 1921

[0603] HRMS (ESI): Calculated for C37H65N7NaO7+[M+Na]+: 742.4838, found: 742.4836.

[0604] Synthesis of the Hexapeptide Fragments

[0605] Cbz-l-Leu-l-MePro-Ot-Bu [M35]

[0606] According to GP2, 1.01 g (3.17 mmol, 1.0 eq.) of Cbz-l-MePro-Of-Bu (P31) was hydrogenated with 100 mg (10 wt%) Pd / C in 13 mL MeOH for 3 h. The resulting free amine was coupled according to GP6 with 1.16 g (4.35 mmol, 1.4 eq.) Cbz-I-Leu-OH, 682 mg (4.45 mmol, 1.4 eq.) HOBt, 850 mg (4.44 mmol, 1.4 eq.) EDC, and 871 pL (7.92 mmol, 2.5 eq., p = 0.920 g / mL) NMM for 14.5 h. Automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 7:3) of the crude product yielded 1.24 g (2.87 mmol, 90 %) of dipeptide M35 as a colorless resin.

[0607] [a]2o = _48.7 (c = 1.0, CHCh)

[0608] Rf = 0.57 (n-Pentane / EtOAc 1:1)

[0609]

[0610] HRMS (ESI): Calculated for C24H37N2O5+[M+H]+: 433.2700, found: 433.2697.Cbz-O-TBS-N-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M37]

[0611] According to GP2, 1.12 g (2.58 mmol, 1.0 Aq.) of the dipeptide M35 was hydrogenated with 117 mg (10 wt%) Pd / C in 9.2 mL MeOH for 3 h. After addition of Pd / C, 650 pL (4.0 M in 1,4-dioxane, 2.60 mmol, 1.0 eq.) of HCI was added before stirring under H2 atmosphere.

[0612] Then, under an Ar atmosphere, 988 mg (2.59 mmol, 1.0 eq.) of Cbz-O-TBS- / V-Me-I-Thr-OH (M15) was dissolved in 14 mL of anhydrous THF and cooled to -20 °C before 625 pL (5.68 mmol, 2.2 eq., p = 0.920 g / mL) NMM was added. Subsequently, 340 pL (2.59 mmol, 1.0 eq., p = 1.040 g / mL) IBCF was added dropwise, and the resulting colorless suspension was stirred for 15 min at -20 °C. A solution of the previously obtained free amine in 8 mL DCM was then added dropwise, followed by two rinses with 1.5 mL of DCM each, and the mixture was slowly warmed to RT.

[0613] After 1.5 d, the reaction mixture was concentrated in vacuo and the residue was taken up in EtOAc before washing with 1 M KHSO4(aq), H2O, sat. NaHCO3(aq.) and sat. NaCI(aq.) solution. The organic phase was then dried over MgSO4 and concentrated in vacuo. Automated column chromatography of the residue (SiO2, cHex / EtOAc 100:0 —> 65:35) yielded 1.58 g (2.39 mmol, 92 %) of the tripeptide M37 as a colorless foam.

[0614] [a]2o = -42.7 (c = 1.0, CHCI3)

[0615] Rf = 0.57 (n-Pentane / EtOAc 1:1)

[0616]

[0617] HRMS (Cl): Calculated for C31H50N3O7Si+[M-C4H9]+: 604.3413, found: 604.3438.

[0618] Cbz-N-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M43][1°l

[0619] To a solution of 100 mg (152 pmol, 1.0 eq.) of tripeptide M37 in 1.5 mL of anhydrous MeOH, 4.80 pL (67.5 pmol, 0.45 eq., p = 1.104 g / mL) acetyl chloride was added under an Ar atmosphere at 0 °C. The colorless solution was slowly warmed to RT and after 17 h diluted with EtOAc. Subsequently, it was washed with saturated NaHCO3(aq.) solution and water before the organic phase was dried over MgSO4and concentrated in vacuo. Afterautomated column chromatography (Ci8-SiO2, FhO / MeCN 9:1 —> 0:10) and subsequent lyophilization of the residue, 76.0 mg (139 pmol, 92 %) of the alcohol M43 was isolated as a colorless lyophilizate.

[0620] [cr]o° = -108.4 (c = 0.5, CHCh)

[0621] Rf = 0.12 (n-Pentane / EtOAc 1:1)

[0622]

[0623] HRMS (ESI): Calculated for C29H46N3O7+[M+H]+: 548.3330, found: 548.3308.

[0624] Cbz-O-(EtPro-Cbz)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M46]

[0625] According to GP7, 127 mg (231 pmol, 1.0 eq.) of the tripeptide M43, 88.4 mg (319 pmol, 1.4 eq.) Cbz-l-EtPro-OH (P36), 43.6 mg (294 pmol, 1.3 eq.) PPY, and 112 mg (326 pmol, 1.4 eq) MNBA were reacted in 1.2 mL anhydrous THF for 15.5 h. After automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 4:6) and lyophilization, 179 mg (222 pmol, 96 %) of the depsipeptide M46 was isolated as a colorless lyophilizate.

[0626] [cr]o° = -79.4 (c = 0.5, CHCh)

[0627]

[0628] HRMS (ESI): Calculated for C44H63N4O10+[M+H]+: 807.4539, found: 807.4513.

[0629] Alloc-N-Me-l-Val-l-EtPro-N-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M40]

[0630] According to GP2, 1.38 g (1.71 mmol, 1.0 eq.) of the depsipeptide M46 was hydrogenated with 132 mg (10 wt%) Pd / C in 10 mL MeOH for 6 h. The resulting amine was then coupled according to GP6 with 917 mg (4.26 mmol, 2.5 eq.) Alloc-A / -Me-I-Val-OH (M16), 2.22 g (4.26 mmol, 2.5 eq.) PyAOP, and 658 pL (5.98 mmol, 3.5 eq.,p = 0.920 g / mL) NMM in 5.8 mL anhydrous DMF for 15 h. Automated column chromatography (SiO2, cHex / EtOAc 1:0 — > 0:1) yielded 1.10 g (1.50 mmol, 88 %) of the pentapeptide M40 as a colorless foam.

[0631] [cr]o° = -136.6 (c = 0.5, CHCh)

[0632] Rf = 0.04 (n-Pentane / EtOAc 1:1)

[0633]

[0634] HRMS (Cl): Calculated for C38H66N5O9+[M+H]+: 736.4855, found: 736.4831.

[0635] Alloc- / V-Me-l-Val-l-EtPro-O-(Gly-Fmoc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M41] According to GP7, 95.2 mg (129 pmol, 1.0 eq.) of alcohol M40, 78.3 mg (263 pmol, 2.0 eq.) Fmoc-Gly-OH, 21.6 mg (145 pmol, 1.1 eq.) PPY, and 89.6 mg (260 pmol, 2.0 eq) of MNBA were reacted in 1.3 mL anhydrous THF for 15 h. Automated column chromatography (Cis-SiO2; FhO / MeCN 9:1 -^ 0:10) and lyophilization yielded 126 mg (124 pmol, 96 %) of the depsipeptide M41 as a colorless lyophilizate.

[0636] [cr]o° = -114.2 (c = 0.5, CHCh) Rf = 0.04 (n-Pentane / EtOAc 1:1 )

[0637]

[0638] HRMS (ESI): Calculated for C55H79N6O12+[M+H]+: 1015.5750, found: 1015.5733.

[0639] Cbz-O-(MePro-Cbz)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M68]According to GP7, 187 mg (342 pmol, 1.0 eq) of tripeptide M43, 126 mg (479 pmol, 1.4 eq) Cbz-l-MePro-OH (P54), 58.4 mg (394 pmol, 1.2 eq) PPY, and 166 mg (483 pmol, 1.4 eq) MNBA were reacted for 20 h. After automated column chromatography (SiO2, cHex / EtOAc 10:0 — > 4:6) and lyophilization, 253 mg (319 pmol, 93 %) of depsipeptide M68 was isolated as a colorless lyophilizate.

[0640] [a] ° = -73.0 (c = 0.5, CHCh)

[0641]

[0642] HRMS (ESI): Calculated for C43H61N4O10+[M+H]+: 793.4382, found: 793.4407.

[0643] Alloc- / V-Me-l-Val-l-MePro- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M74]

[0644] According to GP2, 232 mg (292 pmol, 1.0 eq.) of depsipeptide M68 was hydrogenated with 23.5 mg (10 wt%) Pd / C in 1.5 mL MeOH for 4 h. The resulting amine was then coupled according to GP6 with 155 mg (721 pmol, 2.5 eq.) Alloc- / V-Me-I-Val-OH (M16), 373 mg (716 pmol, 2.5 eq.) PyAOP, and 113 pL (1.02 mmol, 3.5 eq., p = 0.920 g / mL) NMM in 1.0 mL anhydrous DMF for 16 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 1:9 —> 0:10; then SiO2, cHex / EtOAc 1:0 —> 0:1) and subsequent lyophilization yielded 138 mg (190 pmol, 65 %) of pentapeptide M74 as a colorless lyophilizate.

[0645] [cr]o° = -157.2 (c = 0.5, CHCh)

[0646]

[0647] HRMS (ESI): Calculated for C37H64N5O9+[M+H]+: 722.4699, found: 722.4704.

[0648] Alloc- / V-Me-l-Val-l-MePro-O-(Gly-Fmoc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M75]According to GP7, 115 mg (159 pmol, 1.0 eq.) of alcohol M74, 95.6 mg (322 pmol, 2.0 eq.) Fmoc-Gly-OH, 27.1 mg (183 pmol, 1.2 eq.) PPY, and 110 mg (321 pmol, 2.0 eq.) MNBA were reacted in 1.6 mL anhydrous THF for 17 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN 9:1 —> 0:10) and lyophilization yielded 155 mg (155 pmol, 97 %) of the depsipeptide M75 as a colorless lyophilizate.

[0649] [a] ° = -102.8 (c = 0.5, CHCh)

[0650]

[0651] HRMS (ESI): Calculated for C54H77N6O12+[M+H]+: 1001.5594, found: 1001.5608.

[0652] Cbz-O-(MeOPro-Cbz)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M94]

[0653] According to GP7, 160 mg (292 pmol, 1.0 eq.) of tripeptide M43, 116 mg (415 pmol, 1.4 eq.) Cbz-l-MeOPro-OH (M92), 60.3 mg (407 pmol, 1.4 eq.) PPY, and 145 mg (421 pmol, 1.4 eq.) MNBA were reacted in 1.9 mL anhydrous THF for 19 h. After automated column chromatography (Ci8-SiO2, H2O / MeCN 9:1 -^ 0:10) and lyophilization, 217 mg (269 pmol, 92 %) of depsipeptide M94 was isolated as a colorless lyophilizate.

[0654] [cr]o° = -98.2 (c = 0.5, CHCh)

[0655]

[0656] HRMS (ESI): Calculated for C43H61N4O11+[M+H]+: 809.4331, found: 809.4338.

[0657] Cbz-O-(Cbz-EtOPro)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M95]According to GP7, 171 mg (312 pmol, 1.0 eq.) of tripeptide M43, 125 mg (427 pmol, 1.4 eq.) Cbz-l-EtOPro-OH (M93), 60.3 mg (407 pmol, 1.4 eq.) PPY, and 145 mg (433 pmol, 1.4 eq) MNBA were reacted for 19 h in 1.4 mL anhydrous THF. After automated column chromatography (Ci8-SiO2, H2O / MeCN 9:1 —> 0:10) and lyophilization, 246 mg (299 pmol, 96 %) of depsipeptide M95 was isolated as a colorless lyophilizate.

[0658] [a] ° = -86.8 (c = 0.5, CHCh)

[0659]

[0660] HRMS (ESI): Calculated for C44H62N4NaO11+[M+Na]+: 845.4307, found: 845.4311.

[0661] Alloc- / V-Me-l-Val-l-MeOPro- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M96]

[0662] According to GP2, 201 mg (249 pmol, 1.0 eq.) of depsipeptide M94 was hydrogenated with 22.2 mg (10 wt%) Pd / C in 2.5 mL MeOH for 5 h. The resulting amine was then coupled according to GP6 with 135 mg (625 pmol, 2.5 eq.) Alloc- / V-Me-I-Val-OH (M16), 324 mg (621 pmol, 2.5 eq.) PyAOP, and 95.8 pL (871 pmol, 3.5 eq., p = 0.920 g / mL) NMM in 1.2 mL anhydrous DMF for 17 h. Automated column chromatography (Ci8-SiO2, H2O / MeCN 1:9 —> 9:1) and subsequent lyophilization yielded 141 mg (190 pmol, 76 %) of pentapeptide M96 as a colorless lyophilizate.

[0663] [a] ° = -138.6 (c = 0.5, CHCh)

[0664]

[0665] HRMS (ESI): Calculated for C37H63N5NaO10+[M+Na]+: 760.4467, found: 760.4454.

[0666] Alloc-N-Me-l-Val-l-EtOPro-N-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M97]According to GP2, 229 mg (278 pmol, 1.0 eq.) of depsipeptide M95 was hydrogenated with 23.2 mg (10 wt%) Pd / C in 2.8 mL MeOH for 5 h. The resulting amine was then coupled according to GP6 with 148 mg (688 pmol, 2.5 eq.) Alloc- / V-Me-I-Val-OH (M16), 360 mg (691 pmol, 2.5 eq.) PyAOP, and 107 pL (972 pmol, 3.5 eq., p = 0.920 g / mL) NMM in 1.4 mL anhydrous DMF for 20.5 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 1:9 —> 9:1) and subsequent lyophilization yielded 160 mg (213 pmol, 77 %) of pentapeptide M96 as a colorless lyophilizate.

[0667] [a] ° = -140.2 (c = 0.5, CHCh)

[0668]

[0669] HRMS (ESI): Calculated for C38H66N5O10+[M+H]+: 752.4804, found: 752.4806.

[0670] Alloc- / V-Me-l-Val-l-MeOPro-O-(Gly-Fmoc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M98] According to GP7, 122 mg (165 pmol, 1.0 eq.) of alcohol M96, 99.2 mg (334 pmol, 2.0 eq.) Fmoc-Gly-OH, 32.2 mg (218 pmol, 1.3 eq.) PPY, and 114 mg (330 pmol, 2.0 eq.) MNBA were reacted in 1.6 mL anhydrous THF for 18 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN 9:1 -^ 0:10) and lyophilization yielded 155 mg (153 pmol, 92 %) of depsipeptide M98 as a colorless lyophilizate.

[0671] [cr]o° = -100.8 (c = 0.5, CHCh)

[0672]

[0673] HRMS (ESI): Calculated for C54H77N6O13+[M+H]+: 1017.5543, found: 1017.5547.Alloc- / V-Me-l-Val-l-EtOPro-O-(Gly-Fmoc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M99] According to GP7, 139 mg (184 pmol, 1.0 eq.) of alcohol M97, 109 mg (366 pmol, 2.0 eq.) Fmoc-Gly-OH, 36.8 mg (248 pmol, 1.4 eq.) PPY, and 127 mg (369 pmol, 2.0 eq.) MNBA were reacted in 1.8 mL anhydrous THF for 18 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN 9:1 —> 0:10) and lyophilization yielded 157 mg (152 pmol, 83 %) of depsipeptide M99 as a colorless lyophilizate.

[0674] [a] ° = -107.8 (c = 0.5, CHCh)

[0675]

[0676] HRMS (ESI): Calculated for C55H79N6O13+[M+H]+: 1031.5700, found: 1031.5707.

[0677] Fragment Couplings and macrocyclizations

[0678] Alloc- / V-Me-l-Val-l-EtPro-O-(Gly- / V-Me-d-Leu-l-Pro- / V-Me-l-Val-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Ot-Bu [M42]

[0679] According to GP8, 426 mg (420 pmol, 1.0 eq.) of hexapeptide M41 was treated with 630 pL (4.20 mmol, 10 eq., p = 0.976 g / mL) tren in 4.2 mL DCM for 50 min. The resulting free amine was then coupled according to GP6 with 294 mg (505 pmol, 1.2 eq.) of carboxylic acid M48, 218 mg (509 pmol, 1.2 eq.) COMII, and 115 pL (1.05 mmol, 2.5 eq., p = 0.920 g / mL) NMM in 4.2 mL anhydrous DMF for 25 h. After automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — > 0:10), 461 mg (340 pmol, 81 %) of decapeptide M42 was isolated as a colorless foam.

[0680] [a] ° = -100.0 (c = 0.5, CHCh)61

[0681]

[0682] HRMS (ESI): Calculated for C70H120N10NaO10+[M+Na]+: 1379.8776, found: 1379.8768.

[0683] Alloc- / V-Me-l-Val-l-EtPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-Pro- / V-Me-l-Val-l-HoLeu-l-MePro-l-Leu)-l-Thr [M44]

[0684] According to GP9, 4.9 mg (3.61 pmol, 1.0 eq.) of decapeptide M42 was treated with 0.5 mL of a 1:1 mixture of DCM / TFA for 3 h. Deviating from GP9, macrocyclization was performed with 19.6 mg (37.6 pmol, 10 eq.) PyAOP and 4.40 pL (40.0 pmol, 11 eq., p = 0.920 g / mL) NMM, instead of FDPP and DIPEA, in a total of 3.6 mL of anhydrous DMF (addition time = 2.5 h, stirring time = 15 h). After automated column chromatography (Ci8-SiO2, H2O / MeCN 9:1 — 0:10) and lyophilization, 2.9 mg (2.45 pmol, 68 %) of the cyclic peptide M44 was isolated as a colorless lyophilizate.

[0685] [a] ° = -66.4 (c = 0.5, CHCh)

[0686]

[0687] HRMS (ESI): Calculated for C61H102N10NaO13+[M+Na]+: 1205.7520, found: 1205.7508.Alloc- / V-Me-l-Val-l-EtPro-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M50]

[0688] According to GP8, 426 mg (420 pmol, 1.0 eq.) of the hexapeptide M41 was treated with 630 pL (4.20 mmol, 10 eq., p = 0.976 g / mL) of tren in 4.2 mL of DCM for 1.5 h. The resulting free amine was then coupled according to GP6 with 336 mg (505 pmol, 1.2 eq.) of carboxylic acid M49, 216 mg (505 pmol, 1.2 eq.) COMII, and 115 pL (1.05 mmol, 2.5 eq., p = 0.920 g / mL) NMM in 4.7 mL anhydrous DMF for 17.5 h. After automated column chromatography (Ci8-SiO2, H2O / MeCN 9:1 — 0:10), 495 mg (343 pmol, 82 %) of decapeptide M50 was isolated as a colorless resin.

[0689] [cr]o° = -96.2 (c = 0.5, CHCh)

[0690] 65

[0691]

[0692] HRMS (ESI): Calculated for C76H130N10NaO16+[M+Na]+: 1461.9558, found: 1461.9543.

[0693] Alloc- / V-Me-l-Val-l-EtPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-HoLeu-l-MePro-l-Leu)-l-Thr [M51]

[0694] According to GP9, 55.8 mg (38.8 pmol, 1.0 eq.) of decapeptide M50 was treated with 2.0 mL of a 1:1 mixture of DCM / TFAfor3.5 h. The resulting fully deprotected peptide was then treated with 77.0 mg (200 pmol, 5.1 eq.) FDPP and 74.3 pL (427 pmol, 11 eq., p = 0.742 g / mL) DIPEA in a total of 38.5 mL anhydrous DMF (addition time = 20 min, stirring time = 40 min). Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — 0:10) of the crude product, followed by lyophilization, yielded 30.4 mg (24.0 pmol, 62 %) of the macrocycle M51 as a colorless lyophilizate.

[0695] [a] ° = -89.4 (c = 0.5, CHCh)

[0696]

[0697] HRMS (ESI): Calculated for C67H112N10O13+[M+H]+: 1265.8483, found: 1265.8475.

[0698] Alloc- / V-Me-l-Val-l-EtPro-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-Leu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M70]

[0699] According to GP8, 52.8 mg (52.0 pmol, 1.0 eq.) of hexapeptide M41 was treated with 78.0 pL (520 pmol, 10 eq., p = 0.976 g / mL) tren in 0.5 mL DCM for 3 h. The resulting free amine was then coupled according to GP6 with 37.4 mg (57.5 pmol, 1.1 eq.) of carboxylic acid M65, 25.3 mg (59.1 pmol, 1.1 eq.) COMII, and 12.0 pL (109 pmol, 2.5 eq., p = 0.920 g / mL) NMM in 1 mL of a 1:1 mixture of anhydrous DCM and anhydrous DMF for 20 h. After automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 -^ 0:10) and lyophilization, 59.4 mg (41.7 pmol, 80 %) of the decapeptide M70 was isolated as a colorless lyophilizate.

[0700] [a] ° = -84.6 (c = 0.5, CHCh)

[0701]

[0702] HRMS (ESI): Calculated for C75H128N10NaO16+[M+Na]+: 1447.9402, found: 1447.9404.

[0703] Alloc- / V-Me-l-Val-l-EtPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-Leu-l-MePro-l-Leu)-l-Thr [M76]

[0704] According to GP9, 35.4 mg (24.8 pmol, 1.0 eq.) of the decapeptide M70 was treated with 2.0 mL of a 1:1 mixture of DCM / TFA for 2 h. The resulting fully deprotected peptide was then treated with 49.9 mg (130 pmol, 5.2 eq.) FDPP and 47.4 pL (272 pmol, 11 eq., p = 0.742 g / mL) DIPEA in a total of 25 mL anhydrous DMF (addition time = 60 min, stirring time = 30 min). Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 —> 0:10) and purification by preparative HPLC (FhO / MeCN 5:5 -^ 0:10) of the crude product, followed by lyophilization, yielded 14.9 mg (11.9 pmol, 48 %) of the macrocycle M76 as a colorless lyophilizate.

[0705] [a] ° = -62.2 (c = 0.5, CHCh)

[0706]

[0707] HRMS (ESI): Calculated for C66H110N10O13+[M+H]+: 1251.8327, found: 1251.8329.

[0708] Alloc- / V-Me-l-Val-l-MePro-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M77]

[0709] According to GP8, 44.9 mg (44.8 pmol, 1.0 eq.) of hexapeptide M75 was treated with 67.2 pL (448 pmol, 10 eq., p = 0.976 g / mL) of tren in 1.0 mL of DCM for 2.5 h. The resulting free amine was then coupled according to GP6 with 42.3 mg (63.6 pmol, 1.4 eq.) of carboxylic acid M49, 27.5 mg (63.6 pmol, 1.4 eq.) COMII, and 12.8 pL (116 pmol, 2.6 eq., p = 0.920 g / mL) NMM in 1 mLof a 1:1 mixture of anhydrous DCM and anhydrous DMF for 20 h. After automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) and lyophilization, 51.0 mg (35.8 pmol, 80 %) of decapeptide M77 was isolated as a colorless lyophilizate.

[0710] [a] ° = -88.6 (c = 0.5, CHCh)

[0711]

[0712] HRMS (ESI): Calculated for C75H128N10NaO16+[M+Na]+: 1447.9402, found: 1447.9457.

[0713] Alloc- / V-Me-l-Val-l-MePro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-Leu-l-MePro-l-Leu)-l-Thr [M79]

[0714] According to GP9, 43.6 mg (30.6 pmol, 1.0 eq.) of decapeptide M77 was treated with 2.0 mL of a 1:1 mixture of DCM / TFA for 2 h. The resulting fully deprotected peptide was then treated with 58.8 mg (153 pmol, 5.0 eq.) FDPP and 53.3 pL (306 pmol, 10 eq., p = 0.742 g / mL) DIPEA in a total of 30 mL anhydrous DMF (addition time = 60 min, stirring time = 60 min). Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — 0:10) of the crude product, followed by preparative HPLC (FhO / MeCN 5:5 — 0:10) and lyophilization, yielded 17.1 mg (13.7 pmol, 45 %) of the macrocycle M79 as a colorless lyophilizate.

[0715] [cr]o° = -63.6 (c = 0.5, CHCh)

[0716]

[0717] 31HRMS (ESI): Calculated for C66H111N10O13+[M+H]+: 1251.8327, found: 1251.8334.

[0718] Alloc- / V-Me-l-Val-l-MePro-O-(Gly- / V-Me-d-Leu-l-MePro- / V-Me-l-Val-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M78]

[0719] According to GP8, 47.6 mg (47.5 pmol, 1.0 eq.) of hexapeptide M75 was treated with 71.2 pL (475 pmol, 10 eq., p = 0.976 g / mL) tren in 1.0 mL DCM for 3 h. The resulting free amine was then coupled according to GP6 with 39.7 mg (66.5 pmol, 1.4 eq.) of carboxylic acid M73, 26.6 mg (62.1 pmol, 1.3 eq.) COMU, and 13.6 pL (124 pmol, 2.6 eq., p = 0.920 g / mL) NMM in 1.0 mL of a 1:1 mixture of anhydrous DCM and anhydrous DMF for 15.5 h. After automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) and lyophilization, 50.9 mg (37.5 pmol, 79 %) of the decapeptide M78 was isolated as a colorless lyophilizate.

[0720] [cr]o° = -99.2 (c = 0.5, CHCh)

[0721]

[0722] HRMS (ESI): Calculated for C70H121N10O16+[M+H]+: 1357.8957, found: 1357.8957.

[0723] Alloc- / V-Me-l-Val-l-MePro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-MePro- / V-Me-l-Val-l-Leu-l-MePro-l-Leu)-l-Thr [M80]

[0724] According to GP9, 44.2 mg (32.6 pmol, 1.0 eq.) of decapeptide M78 was treated with 2.0 mL of a 1:1 mixture of DCM / TFA for 2 h. The resulting fully deprotected peptide was then treated with 63.6 mg (166 pmol, 5.1 eq.) FDPP and 56.8 pL (326 pmol, 10 eq., p = 0.742 g / mL) DIPEA in a total of 32.5 mL anhydrous DMF (addition time = 60 min, stirring time = 30 min). Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) of the crude product, followed by lyophilization, yielded 17.2 mg (14.5 pmol, 45 %) of the macrocycle M80 as a colorless lyophilizate.[a] ° = -66.8 (c = 0.5, CHCI3)

[0725]

[0726] - 28

[0727] HRMS (ESI): Calculated for C61H102N10O13+[M+H]+: 1183.7701, found: 1183.7690.

[0728] Alloc- / V-Me-l-Val-l-EtPro-O-(Gly- / V-Me-d-Leu-l-CyPro- / Va-Me-l-Lys(N3)-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M63]

[0729] According to GP8, 103 mg (101 pmol, 1.0 eq.) of hexapeptide M41 was treated with 151 pL (1.01 mmol, 10 eq., p = 0.976 g / mL) tren in 1.0 mL DCM for 1.5 h. The resulting free amine was then coupled according to GP6 with 82.2 mg (114 pmol, 1.1 eq.) of carboxylic acid M116, 50.2 mg (117 pmol, 1.2 eq.) COMII, and 27.8 pL (253 pmol, 2.5 eq., p = 0.920 g / mL) NMM in 1.0 mL of anhydrous DCM for 18.5 h. After automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) and lyophilization, 130 mg (87.2 pmol, 86 %) of the decapeptide M63 was isolated as a colorless lyophilizate.

[0730] [a] ° = -61.8 (c = 0.5, CHCI3)

[0731]

[0732] 13HRMS (ESI): Calculated for C77H131N13NaO16+[M+Na]+: 1516.9729, found: 1516.9798.

[0733] Alloc- / V-Me-l-Val-l-EtPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-CyPro- / Va-Me-l-Lys(N3)-l-HoLeu-l-MePro-l-Leu)-l-Thr [M69]

[0734] According to GP9, 38.9 mg (26.0 pmol, 1.0 eq.) of decapeptide M63 was treated with 1.0 mL of a 1:1 mixture of DCM / TFA for 2 h. The resulting fully deprotected peptide was then treated with 102 mg (265 pmol, 10 eq.) FDPP and 95.2 pL (547 pmol, 21 eq., p = 0.742 g / mL) DIPEA in a total of 25 mL anhydrous DMF (addition time = 30 min, stirring time = 40 min). Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 -^ 0:10) of the crude product, followed by lyophilization, yielded 22.8 mg (17.3 pmol, 66%) of macrocycle M69 as a colorless lyophilizate.

[0735] [a] ° = -58.7 (c = 0.3, CHCh)

[0736]

[0737] HRMS (ESI): Calculated for C68H114N13O13+[M+H]+: 1320.8654, found: 1320.8668.

[0738] Acetyl- / V-Me-l-Val-l-EtPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-CyPro- / Va-Me-l-Lys(N3)-l-HoLeu-l-MePro-l-Leu)-l-Thr [M106]

[0739] According to GP10, 50.0 mg (37.9 pmol, 1.0 eq.) of the Al loc-protected amine M69 was treated with 31.5 mg (201 pmol, 5.3 eq.) DMBA and 2.4 mg (2.08 pmol, 5 mol%) Pd(PPh3)4 in 2.5 mL anhydrous DCM for 3 h.The resulting free amine was dissolved under Ar atmosphere in 2.5 mL anhydrous DCM and cooled to 0 °C before 49.5 pL (284 pmol, 7.5 eq., p = 0.742 g / mL) DIPEA was added. Subsequently, the solution was treated with 13.4 pL (190 pmol, 5.0 eq., p = 1.104 g / mL) acetyl chloride and then slowly warmed to RT.

[0740] After 15.5 h, the mixture was diluted with EtOAc and washed with 1 M HCI(aq), H2O, sat. NaHCO3(aq ), and sat. NaCI(aq.) solution. The organic phase was dried over MgSO4 and concentrated in vacuo. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 0:10) of the crude product, followed by lyophilization, yielded 38.4 mg (30.0 pmol, 79 %) of cyclic peptide M106 as a colorless lyophilizate

[0741] [cr]o° = -65.4 (c = 0.5, CHCI3)

[0742]

[0743] HRMS (ESI): Calculated for C66H111N13NaO12+[M+Na]+: 1300.8367, found: 1300.8364.

[0744] Alloc- / V-Me-l-Val-l-MeOPro-O-(Gly- / V-Me-d-Leu-l-CyPro- / Va-Me-l-Lys(N3)-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M100]

[0745] According to GP8, 63.5 mg (62.4 pmol, 1.0 eq.) of hexapeptide M98 was treated with 93.5 pL (624 pmol, 10 eq., p = 0.976 g / mL) tren in 1.0 mL DCM for 50 min. The resulting free amine was then coupled according to GP6 with 48.5 mg (72.9 pmol, 1.2 eq.) of carboxylic acid M49, 34.1 mg (79.6 pmol, 1.3 eq.) COMU, and 17.2 pL (156 pmol, 2.5 eq., p = 0.920 g / mL) NMM in a mixture of 1.0 mL anhydrous DCM and 0.5 mL anhydrous DMF for 15 h. After automated column chromatography of the crude product(Cis-SiO2, H20 / MeCN 9:1 — > 0:10) and lyophilization, 75.8 mg (52.6 pmol, 84 %) of decapeptide M100 was isolated as a colorless lyophilizate.

[0746] [a] ° = -66.6 (c = 0.5, CHCh)

[0747]

[0748] HRMS (ESI): Calculated for C75Hi29NioOi7+[M+H]+: 1441.9532, found: 1441.9537.

[0749] Alloc- / V-Me-l-Val-l-MeOPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-Pro- / V-Me-l-Val-l-HoLeu-l-MePro-l-Leu)-l-Thr [M102]

[0750] According to GP9, 57.2 mg (39.7 pmol, 1.0 eq.) of decapeptide M100 was treated with 2.0 mL of a 1:1 mixture of DCM / TFAfor2.5 h. The resulting fully deprotected peptide was then treated with 79.5 mg (207 pmol, 5.2 eq.) FDPP and 76.1 pL (437 pmol, 11 eq., p = 0.742 g / mL) DIPEA in a total of 40 mL of anhydrous DMF (addition time = 60 min, stirring time = 15 min). Automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 —> 0:10) of the crude product, followed by lyophilization, yielded 27.0 mg (21.3 pmol, 54 %) of the macrocycle M102 as a colorless lyophilizate.

[0751] [a] ° = -53.4 (c = 0.5, CHCh)

[0752]

[0753] HRMS (ESI): Calculated for C66HiiiNioOi4+[M+H]+: 1267.8276, found: 1267.8280.

[0754] Alloc- / V-Me-l-Val-l-EtOPro-O-(Gly- / V-Me-d-Leu-l-CyPro- / V-Me-l-Val-l-HoLeu-Boc)- / V-Me-l-Thr-l-Leu-l-MePro-Of-Bu [M101]

[0755] According to GP8, 62.8 mg (60.9 pmol, 1.0 eq.) of hexapeptide M99 was treated with 95.0 pL (609 pmol, 10 eq., p = 0.976 g / mL) tren in 1.0 mL of DCM for 50 min. The resulting free amine was then coupled according to GP6 with 48.0 mg (72.2 pmol, 1.2 eq.) of carboxylic acid M49, 30.8 mg (71.9 pmol, 1.2 eq.) COMII, and 16.7 pL (152 pmol, 2.5 eq., p = 0.920 g / mL) NMM in a mixture of 1.0 mL of anhydrous DCM and 0.5 mL of anhydrous DMF for 25 h. After automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product and lyophilization, 76.6 mg (52.6 pmol, 86 %) of decapeptide M101 was isolated as a colorless lyophilizate.

[0756] [

[0757]

[0758] cr]o° = -83.0 (c = 0.5, CHCh)65

[0759]

[0760] HRMS (ESI): Calculated for C76Hi3iNioOi7+[M+H]+: 1455.9688, found: 1455.9705.

[0761] Alloc- / V-Me-l-Val-l-EtOPro- / V-Me-cyc / o-O-(Gly- / V-Me-d-Leu-l-Pro- / V-Me-l-Val-l-HoLeu-l-MePro-l-Leu)-l-Thr [M103]

[0762] According to GP9, 58.3 mg (40.0 pmol, 1.0 eq.) of decapeptide M101 was treated with 2.0 mL of a 1:1 mixture of DCM / TFA for 3 h. The resulting fully deprotected peptide was then treated with 79.0 mg (207 pmol, 5.1 eq.) FDPP and 76.6 pL (440 pmol, 11 eq., p = 0.742 g / mL) DIPEA in a total of 40 mL of anhydrous DMF (addition time = 60 min, stirring time = 15 min). Automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 —> 0:10) of the crude product, followed by lyophilization, yielded 23.6 mg (18.4 pmol, 46 %) of macrocycle M103 as a colorless lyophilizate.

[0763] [a] ° = -55.0 (c = 0.5, CHCh)

[0764]

[0765] HRMS (ESI): Calculated for C67Hu3NioOi4+[M+H]+: 1281.8432, found: 1281.8435.

[0766] Synthesis of Mycoplanecin A and its Derivatives

[0767] Mycoplanecin A [M45]

[0768] According to GP10, 208 mg (176 pmol, 1.0 eq.) of the Alloc-protected amine M44 was treated with 134 mg (860 pmol, 4.9 eq.) DMBA and 10.9 mg (9.41 pmol, 5 mol%) Pd(PPh3)4 in 2.3 mL anhydrous DCM for 2.5 h.

[0769] According to GP11, to prepare the acid chloride solution, 112 mg (1.09 mmol, 6.2 eq.) of 2-oxobutyric acid was treated with 94.0 pL (1.07 mmol, 6.1 eq., p = 1.450 g / mL) of oxalyl chloride and one drop of anhydrous DMF in 1.0 mL anhydrous DCM for 2.5 h. This solution was then treated with a solution of the previously obtained free amine and 306 pL (1.76 mmol, 10 eq., p = 0.742 g / mL) DIPEA in 1.0 mL anhydrous DCM for 19 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (FhO / MeCN 3:7 — > 0:10) and lyophilization yielded 145 mg (176 pmol, 70 %) of Mycoplanecin A (M45) as a colorless lyophilizate.

[0770] [c ]o° = -57.5 (c = 0.4, CHCh); Lit.: [a]^5= -66 (c = 0.4, CHCh)!20!

[0771]

[0772] HRMS (ESI): Calculated for C61H102N10NaO13+[M+Na]+: 1205.7520, found: 1205.7511.

[0773] Cyclohexylmycoplanecin A [M52]

[0774] According to GP10, 9.8 mg (7.74 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 30.0 mg (192 pmol, 25 eq.) DMBA and 0.5 mg (0.433 pmol, 6 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 4 h.

[0775] According to GP11, to prepare the acid chloride solution, 15.8 mg (155 pmol, 20 eq.) of 2-oxobutyric acid was treated with 13.0 pL (1.57 mmol, 20 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL anhydrous DCM for 2.5 h. This solution was then added to a solution of the previously obtained free amine and 40.0 pL (230 pmol, 30 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM and stirred for 16 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (FhO / MeCN 9:1 — > 0:10) and lyophilization yielded 6.9 mg (5.45 pmol, 70 %) of Cyclohexylmycoplanecin A (M52) as a colorless lyophilizate.

[0776] [a] ° = -50.0 (c = 0.3, CHCh)

[0777]

[0778] HRMS (ESI): Calculated for C67Hu2NioNaOi3+[M+Na]+: 1287.8303, found: 1287.8298.

[0779] Acetyl-cyclohexylmycoplanecin A [M57]

[0780] According to GP10, 237 mg (187 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 149 mg (954 pmol, 5.1 eq.) DMBA and 10.9 mg (9.41 pmol, 5 mol%) Pd(PPh3)4 in 2.5 mL anhydrous DCM for 3 h.

[0781] The resulting free amine was dissolved under an Ar atmosphere in 2.5 mL anhydrous DCM and cooled to 0 °C before 244 pL (1.40 mmol, 7.5 eq., p = 0.742 g / mL) DIPEA was added. Subsequently, 66.4 pL (934 pmol, 5.0 eq., p = 1.104 g / mL) acetyl chloride was added dropwise, and the reaction mixture was slowly warmed to RT.

[0782] After 19 h, the reaction mixture was diluted with EtOAc and washed with 1 M HCI(aq), H2O, sat. NaHCO3oq.) and sat. NaCI(aq.) solution. The organic phase was dried over MgSO4 and concentrated in vacuo. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (H2O / MeCN 3:7 — 0:10) and lyophilization yielded 169 mg (138 pmol, 74 %) of the Mycoplanecin derivative M57 as a colorless lyophilizate.

[0783] [

[0784]

[0785] cr]o° = -85.6 (c = 0.25, CHCI3)

[0786]

[0787] HRMS (ESI): Calculated for C65HiuNioOi2+[M+H]+: 1223.8377, found: 1223.8362.

[0788] Butyryl-cyclohexylmycoplanecin A [M58]

[0789] According to GP10, 14.0 mg (11.1 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 17.2 mg (111 pmol, 10 eq.) DMBA and 0.3 mg (0.260 pmol, 5 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2.5 h.

[0790] The resulting free amine was dissolved under N2 atmosphere in 0.5 mL anhydrous DCM and cooled to 0 °C before 58.0 pL (333 pmol, 30 eq., p = 0.742 g / mL) DIPEA was added. Subsequently, 23.2 pL (223 pmol, 20 eq, p = 1.025 g / mL) of butyryl chloride was added dropwise, and the reaction mixture was slowly warmed to RT.

[0791] After 17 h, the reaction mixture was diluted with EtOAc and washed with 1 M HCI(aq), H2O, sat. NaHCO3oq.) and sat. NaCI(aq.) solution. The organic phase was dried over MgSO4 and concentrated in vacuo. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (H2O / MeCN 2:8 — > 0:10) and lyophilization yielded 9.2 mg (7.35 pmol, 66 %) of the Mycoplanecin derivative M58 as a colorless lyophilizate.

[0792] [a] ° = -64.3 (c = 0.3, CHCI3)

[0793]

[0794] HRMS (ESI): Calculated for C67Hu5NioOi2+[M+H]+: 1251.8690, found: 1251.8656.

[0795] Caproyl-cyclohexylmycoplanecin A [M59]

[0796] According to GP10, 10.1 mg (7.98 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 9.9 mg (63.4 pmol, 8 eq.) DMBA and 0.7 mg (0.594 pmol, 7 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 3.5 h.

[0797] According to GP11, to prepare the acid chloride solution, 24.6 mg (143 pmol, 18 eq.) of capric acid was treated with 12.2 pL (139 pmol, 18 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL of anhydrous DCM for 2.5 h. This solution was then treated with a solution of the previously obtained free amine and 41.8 pL (240 pmol, 30 eq, p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 16 h. Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (H2O / MeCN (+0.1 % HCOOH) 2:8 — > 0:10) and lyophilization yielded 8.1 mg (6.06 pmol, 76 %) of the Mycoplanecin derivative M59 as a colorless lyophilizate.

[0798] [a] ° = -64.8 (c = 0.25, CHCh)

[0799]

[0800] HRMS (ESI): Calculated for C73Hi26NioNaOi2+[M+Na]+: 1357.9407, found: 1357.9449.

[0801] p-Fluorbenzoyl-cyclohexylmycoplanecin A [M60]

[0802] According to GP10, 12.5 mg (9.88 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 15.7 mg (101 pmol, 10 eq.) DMBA and 0.3 mg (0.260 pmol, 3 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 3 h.

[0803] According to GP11, to prepare the acid chloride solution, 27.7 mg (198 pmol, 20 eq.) of p-fluorobenzoic acid was treated with 17.3 pL (198 pmol, 18 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL anhydrous DCM for 2.5 h. This solution was then treated with a solution of the previously obtained free amine and 51.6 pL (296 pmol, 30 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 17 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (FhO / MeCN 4:6 — > 0:10) and lyophilization yielded 7.6 mg (5.83 pmol, 59 %) of Mycoplanecin derivative M60 as a colorless lyophilizate.

[0804] [a]2o=_44 8 (c =o.25, CHCh)

[0805]

[0806] HRMS (ESI): Calculated for C7oHmFNioNaOi2+[M+Na]+: 1325.8259, found: 1325.8248.

[0807] Cyclohexanoyl-cyclohexylmycoplanecin A [M61]

[0808] According to GP10, 12.5 mg (9.88 pmol, 1.0 eq.) of the Alloc-protected amine M51 was treated with 8.2 mg (52.5 pmol, 5.3 eq) DMBA and 0.3 mg (0.260 pmol, 3 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 3 h.

[0809] According to GP11, to prepare the acid chloride solution, 25.7 mg (200 pmol, 20 eq.) cyclohexanecarboxylic acid was treated with 17.3 pL (198 pmol, 20 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL anhydrous DCM for 3.5 h. This solution was then treated with a solution of the previously obtained free amine and 68.8 pL (395 pmol, 40 eq., p = 0.742 g / mL) DIPEA in 0.5 mL of anhydrous DCM for 19 h. Automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (FhO / MeCN 4:6 — > 0:10) and lyophilization yielded 6.4 mg (4.95 pmol, 50 %) of the Mycoplanecin derivative M61 as a colorless lyophilizate.

[0810] [

[0811]

[0812] cr]o° = -56.4 (c = 0.25, CHCh)

[0813]

[0814] HRMS (ESI): Calculated for C7oHu8NioNaOi2+[M+Na]+: 1313.8823, found: 1313.8766.

[0815] w-Cyclohexylbutyryl-cyclohexylmycoplanecin A [M62]

[0816] According to GP10, 10.2 mg (8.06 pmol, 1.0 eq.) of Alloc-protected amine M51 was treated with 8.2 mg (52.5 pmol, 6.5 eq.) DMBA and 0.5 mg (0.433 pmol, 5 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2.5 h.

[0817] According to GP11, to prepare the acid chloride solution, 27.0 mg (159 pmol, 20 eq.) 4-cyclohexylbutyric acid was treated with 13.8 pL (157 pmol, 20 eq., p = 1.450 g / mL) of oxalyl chloride and one drop of anhydrous DMF in 0.5 mL anhydrous DCM for 4 h. This solution was then treated with a solution of the previously obtained free amine and 42.2 pL (242 pmol, 30 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 17 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (H2O / MeCN (+0.1 % HCOOH) 4:6 — > 0:10) and lyophilization yielded 7.7 mg (5.77 pmol, 72 %) of Mycoplanecin derivative M62 as a colorless lyophilizate.

[0818] [

[0819]

[0820] cr]o° = -57.3 (c = 0.15, CHCh)

[0821]

[0822] HRMS (ESI): Calculated for C73Hi24NioNaOi2+[M+Na]+: 1355.9292, found: 1355.9352.

[0823] Di-l-Leucyl-cyclohexylmycoplanecin A [M81]

[0824] According to GP10, 10.7 mg (8.55 pmol, 1.0 eq.) of the Alloc-protected amine M76 was treated with 8.7 mg (55.7 pmol, 6.5 eq.) DMBA and 0.5 mg (0.433 pmol, 5 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 3 h.

[0825] According to GP11, a stock solution of the acid chloride was prepared from 33.1 mg (324 pmol, 1.0 eq.) 2-oxobutyric acid, 28.4 pL (324 pmol, 1.0 eq., p = 1.450 g / mL) oxalyl chloride, and one drop of anhydrous DMF in 2.5 mL anhydrous DCM. After 3 h, 0.66 mL (0.13 M, 85.5 pmol, 10 eq. relative to the free amine) of the freshly prepared acid chloride solution was treated with a solution of the previously obtained free amine and 29.8 pL (171 pmol, 20 eq., p = 0.742 g / mL) of DIPEA in 0.5 mL of anhydrous DCM for 20 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (FhO / MeCN 5:5 — > 0:10) and lyophilization yielded 5.1 mg (4.07 pmol, 48 %) of Mycoplanecin derivative M81 as a colorless lyophilizate. (EP745)

[0826] [a] ° = -61.0 (c = 0.3, CHCh)

[0827]

[0828] HRMS (ESI): Calculated for C66HuoNioNaOi3+[M+Na]+: 1273.8146, found: 1273.8119.

[0829] Di-l-Methylprolyl-cyclohexylmycoplanecin A [M83]

[0830] According to GP10, 7.6 mg (6.07 pmol, 1.0 eq.) of Alloc-protected amine M79 was treated with 5.3 mg (33.9 pmol, 5.6 eq.) DMBA and 0.4 mg (0.346 pmol, 6 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2.5 h.

[0831] According to GP11, a stock solution of the acid chloride was prepared from 33.1 mg (324 pmol, 1.0 eq.) 2-oxobutyric acid, 28.4 pL (324 pmol, 1.0 eq., p = 1.450 g / mL) oxalyl chloride, and one drop of anhydrous DMF in 2.5 mL anhydrous DCM. After 3 h, 0.47 mL (0.13 M, 60.1 pmol, 10 eq. relative to the free amine) of the freshly prepared acid chloride solution was treated with a solution of the previously obtained free amine and 21.0 pL (121 pmol, 20 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 19 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by preparative HPLC purification (H2O / MeCN 5:5 — > 0:10) and lyophilization yielded 4.4 mg (3.51 pmol, 58 %) of Mycoplanecin derivative M83 as a colorless lyophilizate. [

[0832]

[0833] cr]o° = -61.3 (c = 0.3, CHCh)

[0834]

[0835] HRMS (ESI): Calculated for C66HI 11 N 0i3+[M+H]+: 1251.8345, found: 1251.8327.

[0836] Di-I-Methylprolyl-Methylmycoplanecin A [M82]

[0837] According to GP10, 7.3 mg (6.16 pmol, 1.0 eq.) of Alloc-protected amine M80 were reacted with 6.0 mg (38.4 pmol, 5.6 eq.) DMBA and 0.4 mg (0.346 pmol, 6 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2 h.

[0838] According to GP11, a stock solution of the acid chloride was prepared from 33.1 mg (324 pmol, 1.0 eq.) 2-oxobutyric acid, 28.4 pL (324 mmol, 1.0 eq., p = 1.450 g / mL) oxalyl chloride, and one drop of anhydrous DMF in 2.5 mL anhydrous DCM. After 3 h, 0.48 mL (0.13 M, 61.6 pmol, 10 eq. based on the free amine) of the freshly prepared acid chloride solution was reacted with a solution of the previously obtained free amine and 22.0 pL (126 pmol, 20 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 21.5 h.

[0839] Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by purification via preparative HPLC (H2O / MeCN 5:5 -^ 0:10) yielded, after lyophilization, 3.5 mg (2.96 pmol, 48 %) of mycoplanecin derivative M82 as a colorless lyophilizate.

[0840] [a] ° = -64.3 (c = 0.3, CHCh)

[0841]

[0842] HRMS (ESI): Calculated for Cei Hio3NioOi3+[M+H]+: 1183.7701, found: 1183.7722.

[0843] w-Cyclohexylbutyryl-l-methoxyprolyl-cyclohexylmycoplanecin A [M104] According to GP10, 9.4 mg (7.42 pmol, 1.0 eq.) of the Alloc-protected amine M102 were reacted with 6.6 mg (42.2 pmol, 5.7 eq.) DMBA and 0.4 mg (0.346 pmol, 5 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2 h.

[0844] According to GP11, to prepare the acid chloride solution, 24.3 mg (143 pmol, 19 eq.) 4-cyclo-hexylbutyric acid were reacted with 12.3 pL (141 pmol, 19 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL of anhydrous DCM for 3 h. This solution was then reacted with a solution of the previously obtained free amine and 38.7 pL (222 pmol, 30 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 17 h. Automated column chromatography (Ci8-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 —> 0:10) of the crude product followed by purification via preparative HPLC (H2O / MeCN 5:5 -^ 0:10) yielded, after lyophilization, 8.2 mg (6.14 pmol, 83 %) of the mycoplanecin derivative M 104 as a colorless lyophilizate.

[0845] [

[0846]

[0847] cr]o° = -68.0 (c = 0.5, CHCh)

[0848]

[0849] HRMS (ESI): Calculated for C72Hi22NioNaOi3+[M+Na]+: 1357.9085, found: 1357.9094.

[0850] w-Cyclohexylbutyryl-l-ethoxyprolyl-cyclohexylmycoplanecin A [M105] According to GP10, 10.4 mg (8.11 pmol, 1.0 eq.) of the Alloc-protected amine M103 was treated with 6.9 mg (44.2 pmol, 5.4 eq.) DMBA and 0.3 mg (0.260 pmol, 3 mol%) Pd(PPh3)4 in 0.5 mL anhydrous DCM for 2 h.

[0851] According to GP11, to prepare the acid chloride solution, 27.4 mg (161 pmol, 20 eq.) 4-cyclo-hexylbutyric acid were reacted with 14.0 pL (160 pmol, 20 eq., p = 1.450 g / mL) oxalyl chloride and one drop of anhydrous DMF in 0.5 mL of anhydrous DCM for 3 h. This solution was then reacted with a solution of the previously obtained free amine and 42.4 pL (243 pmol, 30 eq., p = 0.742 g / mL) DIPEA in 0.5 mL anhydrous DCM for 18 h. Automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 —> 0:10) of the crude product followed by purification via preparative HPLC (H2O / MeCN 2:8 -^ 0:10) yielded, after lyophilization, 9.3 mg (6.89 pmol, 85 %) of the mycoplanecin derivative M105 as a colorless lyophilizate.

[0852] [

[0853]

[0854] cr]o° = -57.6 (c = 0.5, CHCh)

[0855]

[0856] HRMS (ESI): Calculated for C73Hi24NioNaOi3+[M+Na]+: 1371.9258, found: 1371.9242.

[0857] Acetyl-l-methoxyprolyl-cyclohexylmycoplanecin A [M114]

[0858] According to GP10, 13.0 mg (10.3 pmol, 1.0 eq.) of the Alloc-protected amine M102 were reacted with 8.8 mg (56.4 pmol, 5.5 eq.) DMBA and 0.3 mg (0.260 pmol, 3 mol%) Pd(PPh3)4 in 0.6 mL anhydrous DCM for 1.5 h.

[0859] The resulting free amine was dissolved under Ar atmosphere in 0.5 mL anhydrous DCM and cooled to 0 °C before 13.5 pL (77.3 pmol, 7.5 eq., p = 0.742 g / mL) DIPEA were added. Subsequently, the solution was treated with 3.67 pL (51.5 pmol, 5.0 eq., p = 1.104 g / mL) acetyl chloride and then slowly warmed to RT. After 17 h, it was diluted with EtOAc and washed with 1 M HCI(aq), followed by H2O, sat. NaHCO3(aq), and sat. NaCI(aq.) solution, the organic phase was dried over MgSO4 and the solvent was removed in vacuo. Automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 —> 0:10) of the crude product and subsequent purification via preparative HPLC (H2O / MeCN (+0.1 % HCOOH) 3:7 ^ 0:10) yielded, after lyophilization, 10.1 mg (8.24 pmol, 80 %) of the mycoplanecin derivative M114 as a colorless lyophilizate.

[0860] [

[0861]

[0862] cr]o° = -58.5 (c = 1.0, CHCI3)

[0863]

[0864] HRMS (ESI): Calculated for C65Hio9N9NaOi3+[M+Na]+: 1247.8071, found: 1247.8024.

[0865] Acetyl-l-ethoxyprolyl-cyclohexylmycoplanecin A [M115]

[0866] According to GP10, 14.8 mg (11.5 pmol, 1.0 eq.) of the Alloc-protected amine M103 were reacted with 9.0 mg (57.7 pmol, 5.0 eq.) DMBA and 0.7 mg (0.606 pmol, 5 mol%) Pd(PPh3)4 in 0.6 mL anhydrous DCM for 3 h.

[0867] The resulting free amine was dissolved under Ar atmosphere in 0.6 mL anhydrous DCM and cooled to 0 °C before 15.0 pL (86.3 pmol, 7.5 eq., p = 0.742 g / mL) DIPEA were added. Subsequently, the solution was treated with 4.12 pL (57.9 pmol, 5.0 eq., p = 1.104 g / mL) acetyl chloride and then slowly warmed to RT. After 18 h, it was diluted with EtOAc and washed with 1 M HCI(aq), followed by H2O, sat. NaHCO3(aq), and sat. NaCI(aq.) solution, the organic phase was dried over MgSO4 and the solvent was removed in vacuo. Automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 —> 0:10) of the crude product and subsequent purification via preparative HPLC (H2O / MeCN (+0.1 % HCOOH) 3:7 ^ 0:10) yielded, after lyophilization, 4.9 mg (3.95 pmol, 34 %) of the mycoplanecin derivative M115 as a colorless lyophilizate.

[0868] [a] ° = -51.0 (c = 0.5, CHCI3)

[0869]

[0870] HRMS (ESI): Calculated for C66HI 11 N9NaOi3+[M+Na]+: 1261.8228, found: 1261.8198.

[0871] Acetyl-w-[4-(morpholinomethyl)-1 H-1,2,3-triazol-1 -yl]-l-lysyl-cyclohexyl-mycoplanecin A [M107]

[0872] 4.9 mg (3.83 pmol, 1.0 eq.) of azide M106 were dissolved in 0.6 mL of a 1:1 mixture of THF / H2O and treated with 2.30 pL (2.30 pmol, 0.6 eq., 1.0 M in H2O) of a freshly prepared sodium ascorbate solution, 1.90 pL (1.0 M, 1.90 pmol, 0.5 eq.) of CuSO4(aq.), and 0.57 pL (4.63 pmol, 1.2 eq., p = 1.002 g / mL) of / V-propargylmorpholine at RT.

[0873] After 15.5 h, the solvent was removed in vacuo and the residue was purified by automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 -^ 0:10), followed by preparative HPLC (H2O / MeCN (+0.1 % HCOOH) 9:1 -^ 0:10). After lyophilization of the isolated fraction, 3.5 mg (2.49 pmol, 65 %) of the mycoplanecin derivative M107 were obtained as a colorless lyophilizate.

[0874] [a]2o=_440 (C= 0.25, CHCI3)

[0875]

[0876] HRMS (ESI): Calculated for C73Hi23Ni4Oi3+[M+H]+: 1403.9389, found: 1403.9385.

[0877] Acetyl-w-[4-ethinylestradiolyl-1 H-1,2,3-triazol-1 -yl]-l-lysyl-cyclohexyl-mycoplanecin A [M108]

[0878] 5.3 mg (4.14 pmol, 1.0 eq.) of azide M106 were dissolved in 0.6 mL of a 1:1 mixture of THF / H2O and treated with 2.50 pL (1.0 M in H2O, 2.50 pmol, 0.6 eq.) of a freshly prepared sodium ascorbate solution, 2.10 pL (1.0 M, 2.10 pmol, 0.5 eq.) CuSO4(aq.), and 1.7 mg (5.74 pmol, 1.4 eq.) ethinylestradiol at RT.

[0879] After 16 h, the solvent was removed in vacuo and the residue was purified by automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 -^ 0:10), followed by preparative HPLC (H2O / MeCN (+0.1 % HCOOH) 7:3 —> 0:10). After lyophilization of the isolated fraction, 5.3 mg (3.36 pmol, 81%) of the mycoplanecin derivative M108 were obtained as a colorless lyophilizate.

[0880] [a]o° = -31.6 (c = 0.25, CHCI3)

[0881]

[0882] HRMS (ESI): Calculated for C86H136N13O14+[M+H]+: 1575.0324, found: 1575.0326.

[0883] Acetyl-w-[4-cyclopropyl-1H-1,2,3-triazol-1-yl]-l-lysyl-cyclohexylmycoplanecin A [M109]

[0884] 5.0 mg (3.91 pmol, 1.0 eq.) of azide M106 and 0.50 pL (5.92 pmol, 1.5 eq., p = 0.783 g / mL) cyclopropylacetylene were dissolved in 0.6 mL of a 1:1 mixture of THF / H2O and treated with 2.30 pL (1.0 M in H2O, 2.30 pmol, 0.6 eq.) of a freshly prepared sodium ascorbate solution and 2.00 pL (1.0 M, 2.00 pmol, 0.5 eq.) of CuSO4(aq.) at RT. After 17 h, an additional 2.30 pL (1.0 M in H2O, 2.30 pmol, 0.6 eq.) of a freshly prepared sodium ascorbate solution and 1.00 pL (11.8 pmol, 3.0 eq., p = 0.783 g / mL) of cyclopropylacetylene were added.

[0885] After 15.5 h, the solvent was removed in vacuo and the residue was purified by automated column chromatography (Cis-SiO2, FhO / MeCN (+0.1 % HCOOH) 9:1 -^ 0:10), followed by preparative HPLC (H2O / MeCN (+0.1 % HCOOH) 4:6 — 0:10). After lyophilization of the isolated fraction, 3.5 mg (2.60 pmol, 67 %) of the mycoplanecin derivative M109 were obtained as a colorless lyophilizate.

[0886] [a] ° = -63.5 (c = 0.4, CHCI3)

[0887]

[0888] HRMS (ESI): Calculated for C71H117N13O12+[M+H]+: 1344.9017, found: 1344.9015.

[0889] 1-O-Propargyl-2,3,4,6-tetra-O-acetyl-p-d-glucopyranoside [M110]l21J

[0890] To a solution of 203 mg (521 pmol, 1.0 eq.) [3-d-glucose pentaacetate in 5.0 mL anhydrous DCM, 99.0 pL (781 pmol, 1.5 eq., = 1.120 g / mL) BF3'OEt2 followed by 45.1 pL (781 pmol, 1.5 eq., p = 0.972 g / mL) propargyl alcohol were added under N2 atmosphere at RT. The slightly brownish solution was stirred for 22 h and then treated with 110 mg (796 pmol, 1.5 eq.) of K2CO3 and stirred for an additional 17.5 h.

[0891] After that, it was diluted with EtOAc, washed three times with H2O and once with sat. NaCI(aq.) solution, before the organic phase was dried over MgSO4 and the solvent was removed in vacuo. Finally, the resulting residue was purified by automated column chromatography (Cis-SiO2, FhO / MeCN 9:1 — > 0:10) and the isolated fraction was lyophilized. In total, 149 mg (385 pmol, 74 %) of the glucose derivative M110 were isolated as a colorless lyophilizate.

[0892] [

[0893]

[0894] a]2o=_437 (C=10 CHCI3); Lit.: [a]^5= -42.6 (c = 1.0, CHCh)!22!

[0895] Rf = 0.38 (n-Pentane / EtOAc 1:1)o

[0896]

[0897] o

[0898] HRMS (ESI): Calculated for C17H22NaO10+[M+Na]+: 409.1105, found: 409.1103.

[0899] Acetyl-w-[4-tetraacetyl-methyl-a-d-glucopyranosidyl-1H-1,2,3-triazol-1-yl]-l-lysyl-cyclohexylmycoplanecin A [M111]

[0900] 5.0 mg (3.91 pmol, 1.0 eq.) of azide M106 and 2.0 mg (5.18 pmol, 1.3 eq.) of glucose derivative M110 were dissolved in 0.6 mL of a 1:1 mixture of THF / H2O and treated with 2.30 pL (1.0 M in H2O, 2.30 pmol, 0.6 eq.) of a freshly prepared sodium ascorbate solution and 2.00 pL (1.0 M, 2.00 pmol, 0.5 eq.) CuSO4(aq.) at RT.

[0901] After 20.5 h, the solvent was removed in vacuo and the residue was purified by automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 — > 0:10), followed by preparative HPLC (H2O / MeCN 4:6 ^ 0:10). After lyophilization of the isolated fraction, 3.9 mg (2.34 pmol, 60 %) of the mycoplanecin derivative M111 were obtained as a colorless lyophilizate.

[0902]

[0903] HRMS (ESI): Calculated for C83H134N13O22+[M+H]+: 1664.9761, found: 1664.9758.Propargyl-p-d-glucopyranoside [M 112]

[0023]

[0904] To a suspension of 72.3 mg (187 pmol, 1.0 eq.) of peracetylated sugar M110 in 0.62 mL of MeOH, 3.1 mg (57.4 pmol, 0.3 eq.) of NaOMe was added. After 23 h, the mixture was neutralized with Amberlite IR120 and filtered, rinsing with MeOH. Finally, the solvent was removed in vacuo, yielding 40.9 mg (187 pmol, 100 %) of the glucose derivative M112 as a colorless oil.

[0905] [α]20D= -80.1 (c = 1.0, MeOH); Lit.: [α]26D= -91.9 (c = 0.66, MeOH)

[0906] Rf = 0.03 (n-Pentane / EtOAc 1:1)

[0907] / Ox

[0908] 8 4

[0909] 53

[0910] 6 OH

[0911]

[0912] OH

[0913] HRMS (ESI): Calculated for C9H14NaO6+[M+Na]+: 241.0683, found: 241.0685.

[0914] Acetyl-w-[4-methyl-p-d-glucopyranosidyl-1 H-1,2,3-triazol-1 -y I] -I -lysyl -cyclohexylmycoplanecin A [M113]

[0915] 8.5 mg (6.65 pmol, 1.0 eq.) of azide M106 and 2.1 mg (9.62 pmol, 1.5 eq.) of the glucopyranoside M112 were dissolved in 0.6 mL of a 1:1 mixture of THF / H2O and treated with 4.00 pL (1.0 M in H2O, 4.00 pmol, 0.6 eq.) of a freshly prepared sodium ascorbate solution and 3.30 pL (1.0 M, 3.30 pmol, 0.5 eq.) of CuSO4(aq.) at RT.

[0916] After 20.5 h, the solvent was removed in vacuo and the residue was purified by automated column chromatography (Cis-SiO2, H2O / MeCN 9:1 — > 0:10), followed by preparative HPLC (H2O / MeCN 9:1 — > 0:10). After lyophilization of the isolated fraction, 4.2 mg (2.81 pmol, 42 %) of the mycoplanecin derivative M113 were obtained as a colorless lyophilizate.

[0917] [α]20D= –46.4 (c = 0.25, CHCl3)

[0918]

[0919] HRMS (ESI): Calculated for C75H126N13O18+[M+H]+: 1496.9338, found: 1496.9333.

[0920] Literature

[0921] [1] Z. M. Wang, K. B. Sharpless, J. Org. Chem. 1994, 59, 8302-8303.

[0922] [2] W. C. Hiscox, D. S. Matteson, J. Org. Chem. 1996, 61, 8315-8316.

[0923] [3] S. T. Cheung, N. L. Benoiton, Can. J. Chem. 1977, 55, 906-910.

[0924] [4] L. A. Carpino, D. Sadat-Aalaee, M. Beyermann, J. Org. Chem. 1990, 55, 1673- 1675.

[0925] [5] U. Kazmaier, A. Horn, E. Papadopoulos, T. Kinsinger, J. Greve, E. Bickel, S.

[0926] Pachoula, Z. Für Anorg. Allg. Chem. 2024, e202400113.

[0927] [6] O. C. Ho, R. Soundararajan, J. Lu, D. S. Matteson, Z. Wang, X. Chen, M. Wei, R.

[0928] D. Willett, Organometallics 1995, 14, 2855-2860.

[0929] [7] A. Horn, U. Kazmaier, Org. Lett. 2022, 24, 7072-7076.

[0930] [8] F. Brackmann, H. Schill, A. De Meijere, Chem. - Eur. J. 2005, 11, 6593-6600.

[0931] [9] G. Franck, K. Brödner, G. Helmchen, Org. Lett. 2010, 12, 3886-3889.

[0932]

[0010] A. T. Khan, E. Mondal, Synlett 2003, 694-698.

[0933]

[0011] P. Barbie, U. Kazmaier, Org. Lett. 2016, 18, 204-207.

[0934]

[0012] R. M. Wenger, Helv Chim Acta 1983, 66, 2672-2702.

[0935]

[0013] N. Kurokawa, Y. Ohfune, Tetrahedron 1993, 49, 6195-6222.

[0936]

[0014] N. Kurokawa, Y. Ohfune, J. Am. Chem. Soc. 1986, 108, 6043-6045.

[0015] F. Gille, A. Kirschning, Beilstein J. Org. Chem. 2016, 12, 564-570.

[0937]

[0016] P. M. E. Hawkins, W. Tran, G. Nagalingam, C. Y. Cheung, A. M. Giltrap, G. M.

[0938] Cook, W. J. Britton, R. J. Payne, Chem. - Eur. J. 2020, 26, 15200-15205.

[0939]

[0017] A. Titz, Z. Radic, 0. Schwardt, B. Ernst, Tetrahedron Lett. 2006, 47, 2383-2385.

[0940]

[0018] A. Le Chevalier Isaad, F. Barbetti, P. Rovero, A. M. D’llrsi, M. Chelli, M. Chorev, A. M. Papini, Eur. J. Org. Chem. 2008, 2008, 5308-5314.

[0941]

[0019] Y. Otake, Y. Shibata, Y. Hayashi, S. Kawauchi, H. Nakamura, S. Fuse, Angew.

[0942] Chem. 2020, 132, 13025-13030.

[0943]

[0020] M. Nakajima, A. Torikata, Y. Ichikawa, T. Katayama, A. Shiraishi, T. Haneishi, A.

[0944] Mamoru, J. Antibiot. (Tokyo) 1983, 36, 961-966.

[0945]

[0021] O. Rasheed, A. Lawrence, P. Quayle, P. Bailey, Synlett 2015, 27, 905-911.

[0946]

[0022] W. Ma, J. Bi, C. Zhao, Y. Gao, G. Zhang, Carbohydr. Res. 2020, 491, 107977.

[0947]

[0023] K. Jiang, D. Fan, Y. Belabassi, G. Akkaraju, J.-L. Montchamp, J. L. Coffer, ACS Appl. Mater. Interfaces 2009, 1, 266-269.

[0948] Biological evaluation

[0949] 1. Minimal Inhibitory concentration (MIC) determination

[0950] To determine the inhibitory activity of the mycoplanecins, the minimum inhibitory concentration (MIC) assay was conducted against Mycobacterium tuberculosis (Mtb) strains H37Ra (in-house) and H37Rv (Evotec ID, Lyon, France). Mtb cultures were grown to mid-log phase (optical density at 600 nm [OD600] of 0.6) in Middlebrook 7H9 complete medium (BD Difco; Becton Dickinson) supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC, BD), 0.4% glycerol, and 0.05% Tween 80 (Merck) at °C and 5% CO2. Single cells were prepared by thoroughly suspending the culture using a 26-gauge syringe needle in Middlebrook 7H9 medium. The cultures were harvested and centrifuged at 3,700 x g for 10 minutes. The resulting pellet was reconstituted in phosphate buffered saline (PBS) and stored at -80°C in 1.5 mL aliquots prior to testing. The number of colony forming units (CFU) was determined by culturing on Middlebrook 7H11 complete medium (BD Difco; Becton Dickinson) supplemented with 10% OADC.

[0951] Compounds were tested in 2-fold serial dilutions starting from 128 pM and 1.28 pM as the highest concentrations. MIC values were validated using a Resazurin microtiter assay(REMA) by adding 45 pL of 0.01% Resazurin (Cayman) solution to each well, followed by 20 hours of incubation without agitation. Fluorescence was measured with a microplate reader (Tecan Infinite M200Pro) at an excitation wavelength of 570 nm and an emission wavelength of 590 nm.

[0952] 2. Cytotoxicity evaluation

[0953] HepG2 cells (human hepatoblastoma cell line; ACC 180, DSMZ) and CHO-K1 cells (Chinese hamster (Cricetulus griseus) ACC 110) were handled under conditions recommended by the depositor. The cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). To determine the anti-proliferative activity of test compounds, cells were seeded at 6 x 103cells per well in 96-well plates containing 120 pL of complete medium. After 2 hours of equilibration, compounds were added in serial dilutions in 60 pL of complete medium. Compounds, the solvent control, and doxorubicin (reference compound) were tested in duplicate across two independent experiments.

[0954] Following a 5-day incubation period, 20 pL of 5 mg / mL MTT (thiazolyl blue tetrazolium bromide) in PBS was added to each well, and cells were incubated for an additional 2 hours at 37 °C. The medium was then discarded, and cells were washed with 100 pL of PBS before adding 100 pL of 2-propanol / 10 N HCI (250: 1 ) to dissolve formazan granules. Absorbance at 570 nm was measured using a microplate reader (Tecan Infinite M200Pro). Cell viability was expressed as a percentage relative to the solvent control. ICso values were determined by sigmoidal curve fitting using GraphPad PRISM 8 (GraphPad Software, San Diego, CA, USA).

[0955] 3. Vesicle Based Efflux (ABC) transporter screening inhibition

[0956] The assay was conducted at Cyprotex (United Kingdom) to evaluate the ability of mycoplanecis to inhibit the active uptake of a known probe substrate into inside-out vesicles that express a single ATP-binding cassette (ABC) efflux transporter, specifically BSEP (Bile Salt Export Pump). The below described protocol is adopted and summarized from the Proposal for ADMET services provided by Cyprotex.The vesicle inhibition assay was performed in a 96-well plate using a rapid filtration system (vacuum manifold). A reaction mixture containing a specified amount of vesicles per well and radiolabeled probe substrate in assay buffer (10 mM HEPES-Tris pH 7.4, 100 mM KNO3, 12.5 mM Mg(NO3)2, and 50 mM sucrose) was pre-incubated at 37 °C for 5 minutes at 300 rpm with solvent (DMSO), mycoplanecins, or positive control inhibitor. Uptake was initiated by adding 5 mM ATP or AMP (solvent control 0 pM only) and incubating at 37 °C for a set time with gentle shaking (300 rpm). The assay was terminated by transferring the mixture to a 96-well filter plate pre-treated with 100 pM unlabeled taurocholic acid and containing cold washing buffer (10 mM HEPES-Tris pH 7.4, 100 mM KNO3, and 50 mM sucrose). The filter plate was washed with cold washing buffer, and after washing and filtration, Mammalian Protein Extraction Reagent (MPER) was added to the sample wells. After a 10-minute incubation at room temperature with shaking (350 rpm) to lyse the vesicles, samples were eluted into a collection plate by centrifugation. An aliquot of the supernatant was transferred to a white-walled, clear-bottomed multiwell plate, and the released radiolabeled probe substrate was analyzed by liquid scintillation counting to measure disintegrations per minute (dpm). All assay conditions were tested in duplicate wells.

[0957] For data analysis, corrected uptake (pmol / mg) was calculated by subtracting solvent uptake in the presence of AMP from uptake in the presence of ATP. The results from solvent wells (0 pM test article) were defined as 100% uptake, which was then used to calculate the percentage control transport activity for all other test article concentrations. These results were plotted against test article concentration and fitted to determine an IC50 value (the concentration that produces 50% inhibition of transport activity).

[0958] 4. Metabolic Stability in human and mouse liver microsomes

[0959] For the evaluation of phase I metabolic stability, the compound (1 pM) was incubated with either 0.5 mg / mL pooled human or mouse liver microsomes (Xenotech, Kansas City, USA), 2 mM NADPH, 10 mM MgCI2 at 37 °C for 120 min on a microplate shaker (Eppendorf, Hamburg, Germany). The metabolic stability (of testosterone, verapamil and ketoconazole for mouse microsome, and of testosterone, diclofenac and propranolol for human microsomes) was determined in parallel to confirm the enzymatic activity of liver microsomes. The incubation was stopped after defined time points by precipitation ofaliquots of enzymes with 2 volumes of cold internal standard solution (15 nM diphenhydramine in 10% methanol / acetonitrile). Samples were stored on ice until the end of the incubation and precipitated protein was removed by centrifugation (15 min, 4 °C, 4,000 g). Concentration of the remaining test compound at the different time points was analyzed by HPLC-MS / MS (Vanquish Flex coupled to a TSQ Altis Plus, Thermo Fisher, Dreieich, Germany) and used to determine half-life (t1 / 2).

[0960] Table 1: Bioactivity data

[0961] MIC (pM) IC50 (pM) Compound Mtb H37 Ra Mtb H37 Rv Mtb H37 Rv HepG2 ACC CHO-K1 (IC50) (IC90) 180 (DSMZ) ACC 110 (DSMZ) M57 1.563 nM 12 nM 27 nM 31.995±3.8 7.94±4.8 M58 3.906 nM 10 nM 20 nM > 37 7.41±2.3 M60 7.813 nM 13 nM 23 nM > 37 9.51±0.1 M61 31.25 nM 58 nM 100 nM > 37 11.19±0.6 M59 0.5 0.31 0.53 > 37 > 37 M62 0.25 40 nM 73 nM > 37 > 37 M83 30 nM 19 nM 36 nM > 37 > 37 M82 30 nM 29 nM 98 nM > 37 > 37 M81 30 nM 14 nM 25 nM > 37 > 37 M104 0.64 0.4 0.7 > 37 19.6 M105 0.64 0.34 0.6 > 37 21.8 M107 0.16 0.14 0.78 26.9 34.6 M108 0.32 0.42 0.73 > 37 23.5 M109 80 nM 0.1 0.23 11.2 6.5 M111 0.32 0.15 0.85 > 37 > 37 M113 0.64 0.25 0.4 > 37 > 37 M114 40 nM 25 nM 67 13.76±1.11 17.15±4.18 M115 20 nM 23 nM 67 > 37 14.2

[0962]

[0963] Table 2: Metabolic stability

[0964] Compound MLM Mouse HLM Mouse Human t1 / 2 [min] / Hepatocytes t1 / 2 [min] 1 Clint Plasma Plasma Clint [pl / mg / min] t1 / 2 [min] [pl / mg / min] t1 / 2 [min] t1 / 2 [min] M57 >120 / <11.6 (n=1) > 120 (n=1) >120 / <11.6 > 240 > 240

[0965] 43 ± 18 / 36 ± 15 (n=1)

[0966] M58 36.8 ± 2.8 / 37.9 ± 2.9 >120 / <11.6 > 240 > 240 M60 >120 / <11.6 >120 / <11.6 >240 >240 M61 >120 / <11.6 >120 / <11.6 >240 >240 M59 >120 / <11.6 (n=1) >120 / <11.6 >240 >240

[0967] 104 / 11.3 (n=1)

[0968] M62 >120 / <11.6 >120 / <11.6 >240 >240 M83 65.7 ± 0.28 / 21.3 ±

[0969] 0.28

[0970] M82 >120 / <11.6 (n=1)

[0971] M81 16.0 / 86.5 (n=1)

[0972] M104 17.8 ± 3.2 / 79 ± 13

[0973] M105 >120 / <11.6

[0974] M107 >120 / <11.6

[0975] M108 >120 / <11.6

[0976] M109 >120 / <11.6

[0977] M111 4.4 ± 0.42 / 314 ± 28

[0978] M113 >120 / <11.6

[0979] M114

[0980] M115

[0981]

[0982] Table 3: BSEP

[0983] Compound % HEK-BSEP Activity Remaining at 30 pM

[0984] M57 5,2

[0985] M58 9,2

[0986] M60 27

[0987] M61 19.6

[0988] M59 43,45

[0989] M62 41

[0990] M83 30

[0991]

[0992] M82 14.2

[0993] M81 15,05

[0994] M104 31,85

[0995] M105 32,8

[0996] M107 2,2

[0997] M108 36,55

[0998] M109 5,05

[0999] M111 4,3

[1000] M113 2,8

[1001] M114 1,7

[1002] M115 1,7

[1003]

[1004] 5. Mitochondrial Function Assay (Seahorse Extracellular Flux Analysis)

[1005] To determine the effects of a test compound on mitochondrial respiration, glycolytic activity, and mitochondrial reserve capacity in primary human hepatocytes.

[1006] Primary human hepatocytes were plated in multiwell microplates compatible with extracellular flux analysis and cultured overnight. Prior to the assay, growth medium was replaced with unbuffered assay medium supplemented with metabolic substrates, and cells were equilibrated under CO2-free conditions. Test compounds were administered at increasing concentrations (0.1- 100 pM), typically across a multi-point concentration range, and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an extracellular flux analyzer. Baseline OCR and ECAR measurements were obtained prior to compound addition. Mitochondrial stress was induced by sequential addition of oligomycin, an uncoupler, and electron transport chain inhibitors to determine parameters including ATP-linked respiration, proton leak, maximal respiration, reserve capacity, and non-mitochondrial respiration. Results were normalized to vehicle control wells. Changes in OCR relative to baseline, including both increases and decreases, were interpreted as indicative of mitochondrial perturbation. Concentration-response analysis was performed to determine the minimum effective concentration (MEC) and AC50values where a dose-dependent effect was observed.6. P-glycoprotein (P-gp) Inhibition Assay (Caco-2 Permeability)

[1007] To determine the inhibitory effect of a test compound on P-glycoprotein (P-gp)-mediated transport by assessing inhibition of basolateral-to-apical permeability of a P-gp probe substrate across Caco-2 cell monolayers and calculating the IC50value.

[1008] Caco-2 cells were cultured on permeable support inserts under conditions permitting formation of confluent, polarized monolayers. On the day of assay, cells were equilibrated in transport buffer and pre-incubated with test compound or reference inhibitor (verapamil). Transport studies were initiated by adding a radiolabeled P-gp probe substrate to the basolateral compartment in the presence of increasing concentrations of test compound, typically across a multi-point concentration range. After incubation at 37°C, samples were collected from the apical compartment and quantified by liquid scintillation counting. Monolayer integrity was monitored using a membrane integrity marker to confirm barrier function, and data from compromised monolayers were excluded where appropriate. Apparent permeability (Papp) in the basolateral-to-apical direction was calculated and corrected for passive permeability using values obtained in the presence of a reference inhibitor producing complete transporter inhibition. Transport activity at each concentration was expressed relative to solvent control (defined as 100%), and concentration-response curves were generated to determine IC50values using nonlinear regression analysis.

[1009] 7. Human Hepatocyte Cellular Biliary Flux Assay (Cholestasis Assay)

[1010] To evaluate the effect of a test compound on cellular biliary flux in human primary hepatocytes and determine the concentration associated with inhibition of bile canalicular transport.

[1011] Cryopreserved human primary hepatocytes were seeded onto collagen-coated multiwell plates and cultured under sandwich conditions with an extracellular matrix overlay to allow formation of functional bile canaliculi. After establishment of the hepatocyte sandwich culture, cells were incubated with compounds or control solutions for approximately 30 minutes at 37°C. A fluorescent bile acid analog, cholyllysyl fluorescein (CFL), was then added and allowed to accumulate within bile canaliculi during a further incubation period. Cells were subsequently washed and imaged using high-content fluorescencemicroscopy to quantify canalicular accumulation of CFL. Bile salt accumulation was determined by image-based analysis of fluorescent canalicular structures. Results were expressed relative to vehicle control, and concentration-response analysis was performed to determine EC50values, defined as the extracellular concentration of compounds producing a 50% reduction in canalicular CFL accumulation.

[1012] Comparison of new mycoplanecin derivatives to cyclohexyl griselimycin (CGM), mycoplanecin A, and cyclohexyl mycoplanecin A

[1013] Table 4: Human BSEP inhibition for CGM, Mycoplanecin A, and mycoplanecin (M) derivatives of the present invention.

[1014] Compound HEK-BSEP remaining x-fold increase in BSEP (tested at 30 pM) activity in % safety compared to CGM Benzbromarone (control inhibitor) -0.7 n / a

[1015] CGM 2.2 - Mycoplanecin A 10.05 4.6 Cyclohexyl mycoplanecin A 1.85 0.8

[1016] M59 43.45 19.8

[1017] M62 41 18.6

[1018] M104 31.85 14.5

[1019] M105 32.8 14.9

[1020] M108 36.55 16.6

[1021]

[1022] Beyond BSEP assessment, the in vitro toxicity profiles of the MP derivatives given in Table 4 were evaluated and benchmarked against CGM, with a particular focus on liver-injury-relevant mechanisms. The objective was to identify complementary assay systems capable of flagging CGM-associated liabilities and supporting hepatotoxicity risk mitigation. BSEP inhibition is associated with intracellular bile acid accumulation, which may trigger oxidative stress, mitochondrial membrane depolarization, and reduced ATP production. Accordingly, mitochondrial function was evaluated using a Seahorse extracellular flux assay. This assay detects mitochondrial impairment by measuring oxygen consumption rate (OCR) (indicative of mitochondrial respiration) and extracellular acidification rate (ECAR) (indicative of compensatory glycolytic shifts). Under the tested conditions, three of the four MP derivatives exhibited no measurable mitochondrial toxicityat concentrations up to 100 pM, whereas CGM generated a positive signal consistent with electron transport chain (ETC) inhibition (Table 5).

[1023] Table 5: Functional Mitochondrial Toxicity Assay (Seahorse assay) to provide insights into cellular dysfunction caused by BSEP inhibitors. The compounds of the present invention (mycoplanecins, M) show no mitochondrial toxicity up to 100 pM Compound Alert OCR Reverse ECAR AC50 Mechanism AC50 [pM] Capacity AC50 [pM]

[1024] [pM]

[1025] CGM Yes (+) > 31.6* 34.2 > 100* ETC inhibitor M59 No (-) NR NR NR No effect M62 No (-) NR NR NR No effect M105 Yes (+ / -) > 100* > 100* > 100* ETC inhibitor M108 No (-) NR NR > 100* No effect

[1026]

[1027] NR: no response observed

[1028] OCR: oxygen consumption rate

[1029] ECAR: extracellular acidification rate

[1030] Yes (+ / -) a feature deviates outside the vehicle control however the AC50 is above 50 pM *If the AC50 values of all responding features are greater than 100x plasma total Cmax this is considered to have lower potential to exhibit mitochondrial toxicity in vivo. In the absence of Cmax data then this cut-off is 50 pM (as described by Eakins et al., (2016), TIV, 34, 161-170).

[1031] Potential interactions with the efflux transporter P-glycoprotein (P-gp) were examined using a Caco-2 monolayer assay measuring inhibition of transporter-mediated basolateral-to-apical (B-A) apparent permeability (Papp) of the radiolabeled P-gp substrate. CGM inhibited transport with an IC50 of 9.3 pM. In contrast, compounds M59, M62, M105, and M108 showed no detectable inhibition at concentrations up to 100 pM. These results are consistent with a reduced propensity of the tested MP derivatives to inhibit P-gp under the assay conditions (Table 6).

[1032] Table 6: P-gp (glycoprotein) Transporter Inhibition Screening. The MP derivatives of the present invention show no inhibition across the concentrations tested.Compound IC50

[1033] CGM 9.29

[1034] MP59 > 100

[1035] MP62 > 100

[1036] MP105 > 100

[1037] MP108 > 100

[1038]

[1039] Cholestatic liability was further assessed using a high-content imaging assay in human hepatocytes following 30-minute compound exposure, with automated quantification of parameters including bile canaliculi formation, cellular morphology, and intracellular trafficking. The cholestasis high content screening assay is a 2D cellular imaging assay used to evaluate the potential of compounds to induce cholestasis. CGM inhibited cellular biliary flux with an EC50of 2.4 pM. In contrast, mycoplanecin derivative M62 showed no inhibition of cellular biliary flux under the same assay conditions. Collectively, these data support that the MP derivatives of the present invention exhibit an improved in vitro safety profile relative to CGM, reduced liability in BSEP-related assays, absence of mitochondrial toxicity in the Seahorse assay, and lack of cholestasis-associated inhibition of biliary flux.

[1040] In summary, the mycoplanecin derivatives of the present invention provide a differentiated and advantageous profile versus CGM, combining reduced hBSEP liability with improved performance across complementary hepatotoxicity de-risking assays (including mitochondrial function and cholestasis-related endpoints), absence of P-gp-mediated transporter inhibition, and supportive in vivo mouse PK, while retaining or enhancing antimycobacterial activity in intramacrophage and caseum models.

Claims

Claims1. A compound of formula (I):(I) whereinX is SO2 or CR4R4a;XIis SO2 or CR7R7a;X2is SO2 or CR2R2a;R1is a group of formula -C(=O)-C(=O)-R1a; -C(=O)-R1a; or -C(=O)-O-R1a;R1ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted;R2is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R2ais hydrogen; or fluorine; or R2and R2atogether with the carbon atom to which they are bound, form a cyclopropyl group;R3is a C1-6alkyl group;R4is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R4ais hydrogen; or fluorine; or R4and R4atogether with the carbon atom to which they are bound, form a cyclopropyl group;R5is a Ci-6 alkyl group;R6is a Ci-6 alkyl group; a group of formula -(CH2)4-N3; or a group of the following formula:NN=NR6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; andR7is hydrogen; fluorine; a C1-4 alkyl group; a C1-4 alkyloxy group; a C1-4 alkenyloxy group; or a cyclohexyl group; and R7ais hydrogen; or fluorine; or R7and R7atogether with the carbon atom to which they are bound, form a cyclopropyl group;or a salt thereof.

2. A compound of formula (la):(la) whereinR1is a group of formula -C(=O)-C(=O)-R1a; -C(=O)-R1a; or -C(=O)-O-R1a;R1ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted;R2is hydrogen; a C1-4 alkyl group; a C1-4 alkyloxy group; or a C1-4 alkenyloxy group;R3is a C1-6alkyl group;R4is hydrogen; a methyl group; a C1-4 alkyloxy group; or a cyclohexyl group;R5is a Ci-6 alkyl group;R6is a Ci-6 alkyl group; a group of formula -(CH2)4-N3; or a group of the following formula:R6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; andR7is hydrogen; or a methyl group;or a salt thereof.

3. A compound according to claim 1 or 2, wherein R1ais a C1-12 alkyl group; a C1-12 alkenyl group; a C3-7 cycloalkyl group; a C1-6alkyl-C3-7cycloalkyl group; an optionally substituted phenyl group; a C1-6alkyl-phenyl group, wherein the phenyl may optionally be substituted; or a C1-6alkyl-heterocycloalkyl group, wherein the heterocycloalkyl group contains 5 or 6 ring atoms that are independently selected from C, N, 0 and S; especially wherein R1ais a C1-12 alkyl group; a C1-12 alkenyl group; a C3-7 cycloalkyl group; a C1-6alkyl-C3-7cycloalkyl group; or an optionally substituted phenyl group.

4. A compound according to claim 1, 2 or 3, wherein R1a group of formula -C(=O)- C(=O)-R1a; especially wherein R1ais an ethyl group.

5. A compound according to claim 1, 2 or 3, wherein R1is a group of formula - C(=O)-R1a; especially wherein R1ais a C1-9 alkyl group; a cyclohexyl group; a group of formula -CH2-CH2-CH2-cyclohexyl; or a 4-fluorophenyl group.

6. A compound according to claim 1, 2 or 3, wherein R1is a group of formula - C(=O)-O-R1a; especially wherein R1ais group of formula -CH2-CH=CH2.

7. A compound according to any one of the preceding claims, wherein R2is hydrogen; a methyl group; an ethyl group; a methoxy group; or an ethoxy group; especially wherein R2is an ethyl group; a methoxy group; or an ethoxy group.

8. A compound according to any one of the preceding claims, wherein R3is an isobutyl group; or an isopropyl group.

9. A compound according to any one of claims 1, 2, 3, 5, 7 and 8, wherein R3is an isobutyl group, and R1is a group of formula -C(=O)-R1a.

10. A compound according to any one of the preceding claims, wherein R5is a group of formula -CH(CH3)2or -(CH2)3CH3.

11. A compound according to any one of the preceding claims, wherein R6is a C1-6 alkyl group; especially a group of formula -CH(CH3)2.

12. A compound according to any one of the preceding claims 1 to 10, wherein R6is a group of formula -(CH2)4-N3; or a group of the following formula:

13. A compound according to any one of the preceding claims, wherein R7is hydrogen.

14. A compound according to any one of the preceding claims 1 to 12, wherein R7is a methyl group.

15. A compound according to any one of the preceding claims, wherein R4is a methyl group; or a cyclohexyl group; especially wherein R4is a cyclohexyl group.

16. A compound of formula (lb):(lb)whereinR1is a group of formula -C(=O)-R1a;R1ais a C1-12alkyl group; or a C1-6alkyl-C3-7cycloalkyl group (especially a C6-12alkyl group; or a C1-6alkyl-C3-7cycloalkyl group);R2is a C1-4 alkyl group; or a C1-4 alkyloxy group;R3is an isobutyl group;R4is a cyclohexyl group;R5is an isopropyl group;R6is an isopropyl group; a group of formula -(CH2)4-N3; or a group of the following formula:NN=NR6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted; andR7is a methyl group;or a salt thereof.

17. A compound according to claim 16, wherein R2is an ethyl group; a methoxy group; or an ethoxy group18. A compound according to claim 16 or 17, wherein R6is an isopropyl group.

19. A compound according to claim 16 or 17, wherein R6is a group of formula - (CH2)4-N3; or a group of the following formula:NN=Nwherein R6ais an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group, all of which may optionally be substituted20. A compound selected from the following compounds or a salt thereof:M59:M62:M104:M105:

21. Pharmaceutical composition comprising a compound according to anyone of the preceding claims and optionally one or more carrier substances and / or one or more adjuvants.

22. Compound according to any one of claims 1 to 20 or pharmaceutical composition according to claim 21 for use in the treatment of a bacterial infection; especially for use in the treatment of tuberculosis.

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