Synthesis of disorazole z1 derivatives, stereoisomers, analogues and derivatives thereof

Abstract

The present invention discloses novel compounds according to general formula I and the method for preparation of the novel compounds according to the invention. One part of the method for preparation is the total synthesis of disorazole Z1, its isomers and analogues thereof. The novel compounds according to the invention are various linkers of disorazole Z1 that can be used in targeted cancer therapy as payloads for Antibody-Drug-Conjugates (ADCs).

Description

[0001] Synthesis of disorazole Z1, stereoisomers, analogues and derivatives thereof

[0002] The present invention discloses novel compounds that are derivatives of disorazole Z1, the synthesis and use thereof.

[0003] The present invention discloses the total synthesis of disorazole Z1 , its isomers and analogues thereof. Furthermore, the present invention discloses the synthesis of various linkers of disorazole Z1 that can be used in targeted cancer therapy as pay¬ loads for Antibody-Drug-Conjugates (ADCs).

[0004] Ten members of a disorazole Z1 subclass were discovered in the culture broth of Sorangium cellulosum strain So ce 1875. The main component disorazole Z1 gained outstanding interest, because it interacts with tubulin, arrests the cells in G2 / M phase and induces apoptosis at sub-nanomolar concentrations. In addition, it enhances phosphorylation of ezrin. Since sub-nanomolecular concentrations of dis¬ orazole Z1 cause death of human cells cultured in vitro, extreme toxicity of this sub¬ stance can be assumed and severe adverse effects may be expected, if the sub¬ stance is applied in vivo without further modification. Recent studies suggest ways to make disorazole Z1 to a very potent anti-cancer drug and high amounts of the substance will be required, which can only be provided by chemical synthesis of dis¬ orazole Z1. In order to reduce the general toxicity, disorazole Z1 and its analogues can be used as payloads for Antibody-Drug-Conjugates (ADCs). In general, the dis- orazoles possess all the basic criteria to be exploited in targeted cancer therapy: they are all very potent cytotoxic compounds showing up to picomolar IC50 and their free alcohol functionalities confer them a sufficient solubility and allow easy conjuga¬ tion to the linker. Many potent drugs lack chemical groups that are necessary for conjugation, and modification to incorporate such moieties can have deleterious ef¬ fects on drug action, hence, the disorazoles represent the perfect candidates for the development of an ADC.

[0005] Disorazole Z1 consists of a 24 membered macrocycle with two lateral chains of which each includes a chiral quaternary center (Formula A). Natural disorazole Z1 has not been synthesized yet. Synthesis of natural disorazole Z1 is hampered be¬ cause of three consecutive stereocenters, of which the middle consists of a chiral

[0006] P9641PC00quaternary center with an ester moiety and a methyl group. To prepare natural diso-razole Z1 and its analogues it is necessary to know how the marked part of disora-zole Z1 is synthesized. This is shown in the below disclosed structure of disorazole Z1.

[0007]

[0008] disorazole Z1

[0009] The compounds according to the invention can be considered as derivatives and an¬ alogues of disorazole Z1 and these compounds are prepared by using disorazole Z1 as starting material.

[0010] Therefore, it is an object of the present invention to provide a method for the synthe¬ sis of natural disorazole Z1 and its analogues and stereoisomers. In addition, the in¬ vention covers the strategy and synthesis of various linker conjugates of disorazole Z1 according to formula I for the use in targeted cancer therapy as payloads for ADCs.

[0011] Therefore, it is an object of the present invention

[0012] to provide a method for the synthesis of natural disorazole Z1 (totals synthe¬ sis) and for the synthesis of its analogues and stereoisomers;

[0013] to provide novel analogues of disorazole Z1 ,

[0014] to provide novel stereoisomers of disorazole Z1 and its analogues;

[0015] to provide novel derivatives of disorazole Z1 and

[0016] to provide the strategy and synthesis of various linkers (linker conjugates) of disorazole Z1 and of its analogues, derivatives and stereoisomers for the use in targeted cancer therapy as payloads for ADCs.

[0017] P9641PC00The objects of the present invention are fulfilled by a novel strategy and synthesis to provide natural disorazole Z1, its analogues and derivatives, E / Z-isomers and stere¬ oisomers (diastereomers and enantiomers).

[0018] The novel compounds are compounds according to general formula I and the syn¬ thesis of these compounds is based on the starting material disorazole Z1. It is of course possible to use disorazole Z1 that is isolated from natural materials to pre¬ pare the compounds of formula I. But to allow to provide the compounds according to formula I in sufficient amounts it is crucial to provide a synthesis of disorazole Z1.

[0019] The objects of the present invention are fulfilled by providing compounds according to general formula I

[0020]

[0021] wherein Ri is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo¬ alkyl, cyclo-alkenyl, aryl, hetero-aryl; R2 is independently selected from H, alkyl, iso¬ alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, hetero-aryl; R3 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, het- ero-aryl; R4is independently selected from the group comprising H, carbonyl-N-imid-azolyl, carbonyl-N,N-dimethylaminoethan, silyl ether linkers, dipeptide linkers and tetrapeptide linkers; X is independently selected from O, S and N,

[0022] and the E / Z-isomers, diastereomers and enantiomers of the compound according to formula I, under the provision that the compound according to formula I is not diso¬ razole Z1. This means that a compound is excluded wherein in the general formula I R1, R2, and R3 are methyl, R4is H and X is O.

[0023] In a preferred embodiment of the invention in the compound according to general formula I R4is independently selected from silyl ether linkers, dipeptide linkers and

[0024] P9641PC00tetrapeptide linkers of the group comprising 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyr- rol-1-yl)ethoxy)ethoxy)diisopropylsilyl)oxy)benzyl hydrogen carbonate (L1 ), 4-((R)-2- ((R)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl hydrogen carbonate-methane (1 / 1) (L2), (6-(2,5-dioxo-2, 5-dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-D-phenylalanylglycine (L3), (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxa- 5,8,11 ,14,17-pentaazatricosanoic acid (L4), (R)-4-(5-benzyl-18-(2,5-dioxo-2,5-dihy-dro-1 H-pyrrol-1 -yl)-4, 7, 10,13-tetraoxo-3, 6, 9, 12-tetraazaoctadecanamido)benzyl hy¬ drogen carbonate (L5). The respective linkers are shown in the detailed part of the description where the preparation steps are disclosed.

[0025] Especially preferred is a compound according to the invention wherein in general formula I Ri, R2 and R3 are methyl, and X is O. In this embodiment according to the invention the compound of general formula I includes the structure element of diso-razole Z1.

[0026] Very preferred is a compound according to the invention wherein in general formula I R1, R2 and R3 are methyl, X is O, and one of R4 or both of R4 is a linker selected from the above disclosed linkers, namely:

[0027] 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)diisopropylsi-lyl)oxy)benzyl hydrogen carbonate (L1), 4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro- 1 H-pyrrol-1 -yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl hy¬ drogen carbonate-methane (1 / 1) (L2), (6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexa- noyl)glycylglycyl-D-phenylalanylglycine (L3), (S)-10-benzyl-23-(2,5-dioxo-2,5-dihy- dro-1 H-pyrrol-1 -yl)-6, 9, 12,15, 18-pentaoxo-3-oxa-5, 8,11,14,17-pentaazatricosanoic acid (L4), (R)-4-(5-benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-4 , 7 , 10, 13- tetraoxo-3,6,9,12-tetraazaoctadecanamido)benzyl hydrogen carbonate (L5).

[0028] In this embodiment where only one of R4 is a linker, the other of R4 is H.

[0029] The preferred compound is compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8 or compound 9, namely:

[0030] P9641PC00

[0031]

[0032] P9641PC00, or

[0033]

[0034] P9641PC00

[0035]

[0036] P9641PC00

[0037]

[0038] P9641PC00

[0039]

[0040] The object of the present invention is furthermore fulfilled by the method for the preparation of a compound according to general formula I. The method is performed as a method of three parts, wherein in a first part a) disorazole Z1 is synthesized. Only with now being able to perform the total synthesis of disorazole Z1 for the first time it is possible to provide disorazole Z1 in sufficient amounts without the need to isolate the compound from natural materials. It has to be understood that from the technical point of view in the procedure for preparing the compounds according to the invention as starting material disorazole Z1 isolated from natural sources might have been used. But with the now disclosed totals synthesis other dimensions of availability of the starting material disorazole Z1 are reached.

[0041] In the method according to the invention in a first part a)

[0042]

[0043] (+)-Dlsorazole Z1

[0044] is prepared by reacting a compound of formula SA-8

[0045] P9641PC00with a compound of formula 20

[0046] TBSO OH

[0047]

[0048] 20

[0049] to yield a compound of formula SA-9

[0050]

[0051] SA-9

[0052] that is reacted to a compound of formula SA-10

[0053]

[0054] that is reacted to (+)-disorazole Z1 ,

[0055] P9641PC00and wherein in a part b1) after part a) (+)-disorazole Z1 is reacted to compound S1

[0056]

[0057] that is converted to yield compound S2

[0058]

[0059] or wherein in a part b2) after part a) (+)-Disorazole Z1 is reacted to compound S3

[0060]

[0061] S3

[0062] that is converted to yield compound S4

[0063] P9641PC00

[0064]

[0065] and wherein in a third part c) of the method after part b1 ) or after part b2) compound S2 or compound S4 are reacted with silyl ether linkers, dipeptide linkers and tetrapeptide linkers of the group comprising

[0066] 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)diisopropylsi-lyl)oxy)benzyl hydrogen carbonate (L1),

[0067] 4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3-methyl-butanamido)-5-ureidopentanamido)benzyl hydrogen carbonate-methane (1 / 1) (L2), (6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-D-phenylalanylglycine (L3), (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-6 , 9 , 12,15,18-pentaoxo-3-oxa-5, 8,11 ,14,17-pentaazatricosanoic acid (L4),

[0068] (R)-4-(5-benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-4 , 7 , 10,13-tetraoxo- 3,6,9, 12-tetraazaoctadecanamido)benzyl hydrogen carbonate (L5),

[0069] to yield a compound according to claim 4 that is compound 1, 2, 3, 4, 5, 6, 7, 8, or 9.

[0070] Furthermore the object of the present invention is fulfilled by providing the com¬ pounds according to the invention for use in the preparation of payloads for Anti- body-Drug-Conjugates (ADCs).

[0071] And especially preferred is the use of a compound according to the invention as payloads for Antibody-Drug-Conjugates (ADCs).

[0072] P9641PC00Formula B below shows a general structure of starting materials that can be used beside disorazole Z1 to prepare the compounds according to the invention.

[0073]

[0074] wherein Ri is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo¬ alkyl, cyclo-alkenyl, aryl, hetero-aryl;

[0075] R2 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cy- clo-alkenyl, aryl, hetero-aryl;

[0076] R3 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cy- clo-alkenyl, aryl, hetero-aryl;

[0077] X is independently selected from O, S and N,

[0078] and the E / Z-isomers, diastereomers and enantiomers of the compound according to formula B.

[0079] The compounds according to the invention are the compounds according to formula I

[0080]

[0081] P9641PC00wherein Ri is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo¬ alkyl, cyclo-alkenyl, aryl, hetero-aryl; R2 is independently selected from H, alkyl, iso¬ alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, hetero-aryl; R3 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, het- ero-aryl; R4 is independently selected from the group comprising H, carbonyl-N-imid-azolyl, carbonyl-N,N-dimethylaminoethan, silyl ether linkers, dipeptide linkers and tetrapeptide linkers, X is independently selected from O, S and N, and the E / Z-iso- mers, diastereomers and enantiomers of the compound according to formula I, under the provision that the compound according to formula I is not disorazole Z1.

[0082] In preferred embodiments R4 is independently selected from silyl ether linkers, di¬ peptide linkers and tetrapeptide linkers of the group comprising 4-(((2-(2-(2,5-dioxo-2, 5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)diisopropylsilyl)oxy)benzyl hydrogen car¬ bonate (L1 ), 4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl hydrogen carbonate-methane (1 / 1) (L2), (6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-D-phenyl- alanylglycine (L3), (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)- 6,9,12,15,18-pentaoxo-3-oxa-5,8,11 ,14,17-pentaazatricosanoic acid (L4), (R)-4-(5- benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-4, 7, 10,13-tetraoxo-3, 6, 9,12- tetraazaoctadecanamido)benzyl hydrogen carbonate (L5).

[0083] In a preferred embodiment the compounds according to the invention have the gen¬ eral Y2:

[0084]

[0085] wherein R1 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo¬ alkyl, cyclo-alkenyl, aryl, hetero-aryl;

[0086] P9641PC00R2 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cy- clo-alkenyl, aryl, hetero-aryl; R3 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, hetero-aryl; R4 is independently se¬ lected from the group comprising H, carbonyl-N-imidazolyl, carbonyl-N,N-dimethyla-minoethan, silyl ether linkers, dipeptide linkers and tetrapeptide linkers,

[0087] X is independently selected from O, S and N, and the E / Z-isomers, diastereomers and enantiomers thereof and

[0088] under the provision that Y2 is not disorazole Z1.

[0089] In preferred embodiments the compounds according to formula I are S2 and S4.

[0090]

[0091] P9641PC00List of Abbreviations

[0092] Bis-MAEC Dis-Ci (Bis-Methylaminoethyl Carbamate) Disorazole Ci Bis-MAEC Dis-Z (Bis-Methylaminoethyl Carbamate) Disorazole Z1 CDI Carbonyldiimidazole

[0093] CH2CI2 Dichloromethane

[0094] DIPEA N,N-Diisopropylethylamine

[0095] DMAP 4-Dimethylaminopyridine

[0096] DMEDA N,N'-Dimethylethylenediamine

[0097] DMF Dimethylformamid

[0098] DMSO Dimethyl sulfoxide

[0099] DMTMM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholini- umchlorid

[0100] eq Equivalents

[0101] EA Ethyl Acetate

[0102] Et2O Diethyl ether

[0103] h Hour(s)

[0104] H2O Water

[0105] H2SO4 Sulphuric acid

[0106] HCI Hydrochloric acid

[0107] MeCN Acetonitrile

[0108] MeOH Methanol

[0109] MgSC Magnesium sulphate

[0110] ml Milliliter(s)

[0111] mol mole(s)

[0112] MS Mass Spectroscopy

[0113] N2Nitrogen

[0114] NaHCO3Sodium hydrogen carbonate

[0115] Rr Retention factor

[0116] r.t Room temperature

[0117] TEA Triethylamine

[0118] TLC Thin Layer Chromatography

[0119] P9641PC00Overview

[0120] Scheme 1 : Synthesis of the starting material (1-5) and synthesis of the natural disorazole Z1 lateral chain (6-11).

[0121] ’OTBS

[0122]

[0123] P9641PC00Scheme 2: Synthesis of the analog lateral chain.

[0124] 5

[0125]

[0126] Scheme 3: Synthesis of the aldehydes

[0127] 16

[0128] HO'^X- / X'OTBS05^X^QTBS

[0129]

[0130] 17 18

[0131] Experimental Part

[0132] (S)-thiazolidinethione (2)

[0133]

[0134] 2

[0135] (S)-phenylalanine (16.52 g, 100 mmol, 1.0 eq.) and sodium borohydride (9.45 g, 250 mmol, 2.5 eq.) are mixed in anhydrous THF (250 ml) and cooled to 0 °C. Then a

[0136] P9641PC00solution of iodine (25.38 g, 100 mmol, 1.0 eq.) in anhydrous THF (130 ml) is added dropwise over 2.5 h. The cooling bath is removed and stirring is continued for 20 h at room temperature. Then the white suspension is cooled again to 0 °C, quenched slowly with methanol (100 ml) and allowed to stir for 1 h at room temperature. After removing of the solvent under reduced pressure, 2M aqueous sodium hydroxide solution (250 ml) is added to the white pasty residue. This mixture is stirred for 1.5 h at room temperature. Then the aqueous solution is extracted with dichloromethane (3x200 ml), the combined organic layers are dried over MgSC , filtered and concen¬ trated under reduced pressure to afford (S)-phenylalaninol (1) (14.69 g) as a color¬ less solid, which is dissolved in 1M aqueous potassium hydroxide solution (600 ml) for the second step. Carbon disulfide (30 ml, 500 mmol, 5.14 eq.) is added and the reaction mixture is refluxed for 18 h. The green solution is cooled to room tempera¬ ture and extracted with dichloromethane (3x200 ml). The combined organic layers are washed with brine (250 ml), dried over MgSC , filtered and concentrated under reduced pressure to obtain (S)-thiazolidinethione (2) (16.52 g), which shows suffi¬ cient purity for the next step.

[0137] 1H NMR (CDCI3, 400 MHz): 57.37 - 7.10 (m, 6H), 4.47 - 4.35 (m, 1H), 3.56 (dd, 1H, J = 11.2, 7.7 Hz), 3.29 (dd, 1 H, J = 11.2, 6.9 Hz), 3.03 - 2.89 (m, 2H).

[0138] (S)-W-propionyl-thiazolidinethione (3)

[0139]

[0140] (S)-thiazolidinethione (2) (11 g, 52.6 mmol, 1.0 eq.) is dissolved in anhydrous di¬ chloromethane (213 ml). After addition of triethylamine (10.2 ml, 73.64 mmol, 1.4 eq.) and 4-dimethylaminopyridine (1.29 g, 10.52 mmol, 0.2 eq.) the stirred solution is cooled to -78 °C. Then propionyl chloride (6 ml, 68.38 mmol, 1.3 eq.) is added, the resulting yellow solution is stirred for 45 min at -78 °C and for additional 3 h at room temperature. The reaction mixture is quenched with saturated aqueous NH4CI solu¬ tion (60 ml) and the organic phase is separated. The aqueous layer is extracted with

[0141] P9641PC00dichloromethane (2x150 ml). The combined organic layers are dried over MgSC , filtered, concentrated under reduced pressure and the residue is purified by column chromatography (n-pentane / Et?©, 9:1) to provide compound 3 (11.7 g, 44.15 mmol, 84 %) as yellow crystalline needles.

[0142] 1H NMR (CDCI3, 400 MHz): 57.39 - 7.23 (m, 5H), 5.41 - 5.34 (m, 1H), 3.41 (dq, 1H, J = 18.3, 7.3 Hz), 3.37 (dd, 1H, J = 11.5, 7.3 Hz), 3.21 (dd, 1H, J = 13.2, 3.8 Hz), 3.12 (dq, 1H, J = 18.3, 7.2 Hz), 3.04 (dd, 1H, J = 13.1, 10.5 Hz), 2.87 (d, 1H, J = 11.5 Hz), 1.19 (t, 3H, J = 7.2 Hz).

[0143] 13C{1H} NMR (CDCI3, 101 MHz): 5201.12, 174.92, 136.62, 129.49, 128.92, 127.22, 68.69, 36.79, 32.38, 31.96, 8.86.

[0144] (2R,3S)-1-((4S)-4-benzyl-2-thioxo-1,3-thiazolidin-3-yl)-5-((tert-butyl(dimethyl)si-lyl)oxy)-3-hydroxy-2-methylpentan-1 -one (4)

[0145]

[0146] Under nitrogen atmosphere (S)- / V-propionyl-thiazolidinethione (3) (2 g, 7.55 mmol, 1.0 eq.) is dissolved in anhydrous dichloromethane (60 ml) and cooled to 0 °C. Tita¬ nium tetrachloride (0.96 ml, 8.74 mmol, 1.16 eq.) is added slowly and the orange suspension is stirred for 5 min. Then DIPEA (1.49 ml, 8.74 mmol, 1.16 eq.) is intro¬ duced slowly which leads to a red / black solution. After stirring for 20 min at 0 °C the reaction mixture is cooled to -78 °C. 3-((fertbutyl(dimethyl)silyl)oxy)propanal (18) (1.7 g, 9.0 mmol, 1.2 eq.) is injected within two minutes and stirred for further 1 h at -78 °C. The reaction is quenched under vigorous stirring with saturated aqueous NH4CI solution (28 ml) at -78 °C. While warming to room temperature water (28 ml) is poured in. The organic layer is separated and the aqueous layer is extracted with dichloromethane (3x50 ml). The combined organic layers are dried over MgSO4, fil¬ tered and the solvent is removed under reduced pressure. The residue is purified by column chromatography (n-pentane / Et2O, 9:1) to afford product 4 (2.7 g, 5.95 mmol, 79 %) as a yellow sticky oil.

[0147] P9641PC001H NMR (CDCI3, 600 MHz): 57.36 - 7.32 (m, 2H), 7.30 - 7.27 (m, 3H), 5.40 (dddd, 1 H, J = 10.5, 7.3, 4.0, 1.0 Hz), 4.70 (qd, 1H, J = 7.0, 3.9 Hz), 4.28 (ddd, 1H, J = 9.4, 3.9, 2.8 Hz), 3.87 (ddd, 1H, J = 10.6, 6.0, 4.9 Hz), 3.82 (ddd, 1H, J = 10.2, 7.7, 4.6 Hz), 3.43 (s, 1H), 3.36 (ddd, 1H, J = 11.5, 7.2, 1.0 Hz), 3.24 (dd, 1H, J = 13.2, 4.0 Hz), 3.04 (dd, 1H, J = 13.2, 10.5 Hz), 2.88 (dd, 1H, J = 11.5, 1.0 Hz), 1.80 (dddd, 1H, J = 14.2, 9.4, 7.7, 4.9 Hz), 1.64 (dddd, 1H, J = 14.1, 6.0, 4.6, 2.7 Hz), 1.24 (d, 3H, J = 6.9 Hz), 0.90 (s, 9H), 0.08 (s, 6H).

[0148] 13C{1H} NMR (CDCI3, 151 MHz): 5201.49, 177.48, 136.61, 129.58, 129.03, 127.35, 70.95, 69.10, 62.04, 43.64, 37.15, 36.11, 31.89, 26.05, 18.35, 11.63, -5.30, -5.31.

[0149] (3R,4S)-4-((tert-butyl(dimethyl)silyl)oxy)-3-methyloxan-2-one (5)

[0150] O

[0151]

[0152] The aldol product 4 (2.595 g, 5.72 mmol, 1.0 eq.) is dissolved in dry dichloro¬ methane (19.1 ml) and cooled to 0 °C. Para-toluenesulfonic acid monohydrate (598.4 mg, 3.146 mmol, 0.55 eq.) is added in portions and the solution is stirred for 1 h at 0 °C. After 23 h stirring at room temperature the colorless solution is again cooled to 0 °C and imidazole (1.17 g, 17.16 mmol, 3.0 eq.) and TBSCI (1.3 g, 8.58 mmol, 1.5 eq.) are introduced sequentially in portions. The reaction mixture is al¬ lowed to stir 10 min at 0 °C before it is warmed to room temperature and stirred for another 25 h. Under intense stirring saturated aqueous NaHCOs solution (15 ml) and dichloromethane (20 ml) are added. The organic phase is separated and the aqueous layer is extracted with dichloromethane (3x20 ml). The combined organic layers are washed with water (16 ml), dried over MgSC , filtered and lactone 5 is provided (990 mg, 4.05 mmol, 71 %) as a colorless crystalline solid.

[0153] 1H NMR (CDCI3, 400 MHz): 54.48 (ddd, 1 H, J = 11.3, 8.9, 4.0 Hz), 4.23 (ddd, 1 H, J = 11.3, 5.7, 4.7 Hz), 3.79 (td, 1H, J = 6.3, 4.5 Hz), 2.54 (p, 1H, J = 7.1 Hz), 2.12 (ddt, 1H, J = 14.2, 9.0, 4.6 Hz), 1.80 (dtd, 1H, J = 14.3, 5.8, 4.0 Hz), 1.29 (d, 3H, J = 7.2 Hz), 0.89 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).

[0154] 13C{1H} NMR (CDCI3, 101 MHz): 5 174.07, 70.32, 64.88, 44.75, 31.39, 25.79, 18.03, 14.73, -4.37, -4.73.

[0155] P9641PC00Preparation of the natural fragment

[0156] (3R,4S)-4-((tert-butyl(dimethyl)silyl)oxy)-3-((1S)-1 -hydroxy -3 -((4-methoxyben-zyl)oxy)propyl)-3-methyloxan-2-one (6)

[0157] O OH

[0158] 0A,

[0159]

[0160] ^^OTBS

[0161] 6

[0162] To a solution of lactone 5 (488 mg, 2.0 mmol, 1.0 eq.) in dry dichloromethane (9.2 ml) 1 M Bu2BOTf in dichloromethane (2.2 ml, 2.2 mmol, 1.1 eq.) is injected at -78 °C under nitrogen atmosphere. After stirring of the yellow solution for 20 min at -78 °C triethylamine (0.36 ml, 2.6 mmol, 1.3 eq.) is added slowly which leads to a colorless solution. The reaction mixture is allowed to stir 1 h at this temperature. Then a mix¬ ture of 3-((4-methoxybenzyl)oxy)propanal (16) (777 mg, 4.0 mmol, 2.0 eq.) with an¬ hydrous dichloromethane (1.0 ml) is added slowly dropwise within 20 min. After stir¬ ring of the colorless solution for 3 h at -78 °C the cold reaction mixture is quenched sequentially with 20 mM aqueous phosphate buffer (8 ml), methanol (4 ml) and 30 % H2O2 (2 ml) at -78 °C. The cooling bath is removed and vigorous stirring is continued for additional 1.5 h. Dichloromethane (10 ml) is added and the organic layer is separated. The aqueous layer is extracted with dichloromethane (3x20 ml) and the combined organic layers are washed once with an aqueous saturated Na2SO3 solution (14 ml). After drying over MgSC , filtration and removing of the sol¬ vent the residue is purified by column chromatography (n-pentane / Et2O, 2:1) to af¬ ford the desired product 6 (712 mg, 1.62 mmol, 81.2 %) as a colorless oil.

[0163] 1H NMR (CDCh, 600 MHz): 57.24 - 7.21 (m, 2H), 6.88 - 6.85 (m, 2H), 4.48 - 4.40 (m, 2H), 4.40 - 4.37 (m, 1 H), 4.33 (dd, 1 H, J = 8.8, 3.4 Hz), 4.23 (ddd, 1 H, J = 11.1 , 8.5, 4.1 Hz), 3.88 (dd, 1H, J = 10.5, 1.9 Hz), 3.80 (s, 3H), 3.72 (ddd, 1H, J = 9.6, 5.5, 4.4 Hz), 3.60 (td, 1H, J = 9.0, 3.8 Hz), 3.35 (s, 1H), 2.16 (dddd, 1H, J = 14.7, 10.5, 8.8, 4.3 Hz), 2.03 (dddd, 1H, J = 13.7, 6.1, 4.1, 3.4 Hz), 1.96 - 1.87 (m, 1H), 1.79 (dddd, 1H, J = 14.6, 5.7, 3.8, 1.9 Hz), 1.20 (s, 3H), 0.88 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H).

[0164] 13C{1H} NMR (CDCh, 151 MHz): 5 175.12, 159.38, 130.01, 129.46, 113.94, 75.03, 73.05, 69.61, 68.87, 65.23, 55.38, 54.07, 31.83, 29.49, 25.84, 18.10, 16.90, -4.16, -4.85.

[0165] P9641PC00(3R,4S)-4-((tert-butyl(dimethyl)silyl)oxy)-3-((1S)-3-((4-methoxybenzyl)oxy)-1- ((triethylsilyl)oxy)propyl)-3-methyloxan-2-one (7)

[0166] O OTES OPMB

[0167]

[0168] OTBS

[0169] 7

[0170] A solution of the previous product 6 (630 mg, 1.44 mmol, 1.0 eq.) in anhydrous di¬ chloromethane (5.3 ml) is cooled to -78 °C under nitrogen atmosphere. DIPEA (0.50 ml, 2.88 mmol, 2.0 eq.) and TESOTf (0.50 ml, 2.16 mmol, 1.5 eq.) are added se¬ quentially. After stirring for 2 h at -78 °C the reaction mixture is quenched with a sat¬ urated aqueous NaHCOa solution (3.5 ml) at -78 °C and diluted with dichloro¬ methane (10 ml). The mixture is allowed to warm to ambient temperature, the or¬ ganic phase is separated and the aqueous layer is extracted with dichloromethane (3x20 ml). The combined organic layers are dried over MgSC , filtered and concen¬ trated under reduced pressure. After column chromatography (n-pentane / Et2O, 9:1) compound 7 (539 mg, 0.97 mmol, 68 %) is obtained as a colorless oil.

[0171] 1H NMR (C6D6, 600 MHz): 57.28 - 7.23 (m, 2H), 6.85 - 6.80 (m, 2H), 4.39 (dd, 1 H, J = 8.4, 3.0 Hz), 4.37 - 4.30 (m, 2H), 4.18 (ddd, 1H, J = 11.0, 9.4, 4.3 Hz), 4.06 (dd, 1H, J = 6.0, 2.6 Hz), 3.86 (dt, 1H, J = 10.5, 5.0 Hz), 3.55 (td, 1H, J = 9.2, 4.7 Hz), 3.44 (ddd, 1 H, J = 9.2, 5.9, 4.4 Hz), 3.31 (s, 3H), 2.06 - 1.99 (m, 1 H), 1.82 - 1.72 (m, 2H), 1.40 (s, 3H), 1.32 (ddt, 1H, J = 14.1, 6.0, 4.5 Hz), 1.06 (t, 9H, J = 8.0 Hz), 0.87 (s, 9H), 0.74 (qd, 6H, J = 7.9, 2.2 Hz), 0.01 (s, 3H), -0.10 (s, 3H).

[0172] 13C{1H} NMR (C6D6, 151 MHz): 5 173.45, 159.86, 130.95, 129.75, 128.35, 114.11, 73.97, 72.94, 69.98, 67.17, 64.62, 55.07, 54.80, 34.55, 29.36, 25.93, 18.21, 17.73, 7.44, 5.95, -4.23, -4.75.

[0173] Methyl (2R,3S)-3-((tert-butyl(dimethyl)silyl)oxy)-5-hydroxy-2-((1S)-3-((4-meth-oxybenzyl)oxy)-1-((triethylsilyl)oxy)propyl)-2-methylpentanoate (8)

[0174]

[0175] P9641PC00In a 25 ml pointed flask lactone 7 (529 mg, 0.957 mmol, 1.0 eq.) is dissolved in an¬ hydrous THF (0.56 ml) and anhydrous methanol (0.19 ml). A 1.04 M KOH solution in anhydrous methanol (1.26 ml, 1.31 mmol, 1.37 eq.) is added slowly at 0 °C and the reaction solution is allowed to stir for further 50 min at 0 °C. After stirring for 23 h at room temperature the clear bright yellow solution is monitored by TLC (n-pen-tane / Et2O, 1:1). Another portion of the same 1.04 M KOH solution (0.2 ml, 0.208 mmol, 0.22 eq.) is injected slowly at room temperature and again the reaction solu¬ tion is stirred for additional 27 h. A solution of camphorsulfonic acid (351.6 mg, 1.514 mmol, 1.58 eq.) in anhydrous methanol (6 ml) is added slowly dropwise at 0 °C. After stirring for 20 min at 0 °C diethyl ether (15 ml) is added. Then the reac¬ tion mixture is allowed to stir further 10 min at 0 °C before it is warmed to room tem¬ perature. The reaction solution becomes a white suspension which must be stirred more vigorous to keep it homogeneous. TMSCH2N2 solution in hexane is injected in several portions until TLC shows full conversion of the carboxylic acid intermediate (Rf: 0.6 (n-pentane / Et2O, 1:1)). Excess of TMSCH2N2 represented by a yellow sus¬ pension must be destroyed by slowly dropwise addition of acetic acid glacial at 0 °C. To the resulting white suspension triethylamine (2.5 ml) is added immediately. After warm up the suspension, dichloromethane (20 ml) and a saturated aqueous NaHCOa solution (4 ml) are poured in a separating funnel. After shaking water (6 ml) is added and the organic layer is separated. The aqueous layer is extracted with di¬ chloromethane (3x20 ml) and the combined organic layers are dried over MgSC , filtered and concentrated under reduced pressure. The residue is purified by column chromatography (n-pentane / ethyl acetate, 95:5 — 9:1) to afford product 8 (390 mg, 0.667 mmol, 70 %) as a colorless oil.

[0176] 1H NMR (C6D6, 600 MHz): 57.25 - 7.21 (m, 2H), 6.83 - 6.78 (m, 2H), 4.75 (d, 1H, J = 9.2 Hz), 4.36 - 4.26 (m, 2H), 3.96 (dd, 1 H, J = 8.6, 1.8 Hz), 3.60 - 3.53 (m, 3H), 3.49 (s, 3H), 3.40 (ddd, 1 H, J = 8.8, 4.9, 3.5 Hz), 3.31 (s, 3H), 2.34 - 2.23 (m, 1 H), 1.96 - 1.87 (m, 2H), 1.50 (s, 3H), 1.46 (ddt, 2H, J = 14.8, 9.4, 3.4 Hz), 1.07 (t, 9H, J = 8.0 Hz), 0.99 (s, 9H), 0.79 - 0.63 (m, 6H), 0.17 (s, 3H), 0.12 (s, 3H).

[0177] 13C{1H} NMR (C6D6, 151 MHz): 5 174.70, 159.96, 130.52, 129.84, 128.35, 114.21, 75.31, 73.17, 72.62, 67.15, 59.29, 57.84, 54.80, 50.97, 36.14, 33.35, 26.33, 18.70, 15.31, 7.36, 5.96, -3.44, -3.92.

[0178] P9641PC00Methyl (2R,3S)-3-((tert-butyl(dimethyl)silyl)oxy)-2-((1S)-3-((4-methoxyben-zyl)oxy)-1-((triethylsilyl)oxy)propyl)-2-methyl-5-oxopentanoate (9)

[0179] TBSO OTES

[0180]

[0181] To a solution of the opened lactone 8 (354 mg, 0.605 mmol, 1.0 eq.) in anhydrous dichloromethane (6 ml) NaHCCh (133 mg, 1.574 mmol, 2.6 eq.) and DMP (334 mg, 0.787 mmol, 1.3 eq.) are added sequentially at 0 °C. Before removing of the cooling bath the suspension is allowed to stir for further 15 min at 0 °C. After stirring for 2 h at room temperature the slightly yellow suspension is quenched by addition of satu¬ rated aqueous Na2S2O3 solution (5 ml) and saturated aqueous NaHCOa solution (5 ml). The mixture is diluted with diethyl ether (10 ml) and vigorously stirred until two clear layers are visible. After separating of the organic phase the aqueous layer is extracted with diethyl ether (3x10 ml). The combined organic layers are dried over MgSC , filtered, concentrated under reduced pressure and purified by column chro¬ matography (n-pentane / Et2O, 6:1) to obtain the aldehyde 9 (338 mg, 0.58 mmol, 96 %) as a colorless oil.

[0182] 1H NMR (C6D6, 600 MHz): 59.44 (d, 1H, J = 1.7 Hz), 7.23 - 7.18 (m, 2H), 6.85 - 6.80 (m, 2H), 4.58 (dd, 1H, J = 9.5, 1.3 Hz), 4.32 - 4.30 (m, 1H), 4.30 - 4.23 (m, 2H), 3.50 (ddd, 1H, J = 10.9, 9.2, 3.2 Hz), 3.47 (s, 3H), 3.31 (s, 3H), 3.29 (ddd, 1H, J = 9.1, 4.9, 3.5 Hz), 3.03 (dd, 1H, J = 18.5, 2.4 Hz), 2.81 (ddd, 1H, J = 18.5, 7.4, 1.9 Hz), 1.59 (dddd, 1H, J = 14.6, 10.8, 4.9, 1.2 Hz), 1.46 (s, 3H), 1.40 (ddt, 1H, J = 14.6, 9.4, 3.3 Hz), 1.06 (t, 9H, J = 8.0 Hz), 0.93 (s, 9H), 0.75 - 0.61 (m, 6H), 0.19 (s, 3H), -0.01 (s, 3H).

[0183] 13C{1H} NMR (C6D6, 151 MHz): 5200.31, 174.60, 159.89, 130.77, 129.59, 128.35, 114.16, 72.99, 72.24, 72.22, 66.39, 57.39, 54.81, 51.06, 48.25, 33.42, 26.11, 18.50, 14.95, 7.32, 5.89, -3.74, -4.79.

[0184] P9641PC00Methyl (2S,3S)-3-((tert-butyl(dimethyl)silyl)oxy)-2-((1S)-3-((4-methoxyben-zyl)oxy)-1 -((triethylsilyl)oxy)propyl)-2-methylhex-5-enoate (10)

[0185] TBSO OTES

[0186]

[0187] Methyltriphenylphosphonium bromide (239 mg, 0.667 mmol, 1.2 eq.) is suspended in anhydrous THF (1.8 ml) under nitrogen atmosphere. Then 2.5 M n-butyllithium so¬ lution in hexane (0.25 ml, 0.625 mmol, 1.12 eq.) is added at 0 °C. After stirring of the red mixture for 1 h at room temperature the orange clear solution is cooled to -78 °C. A solution of aldehyde 9 (324 mg, 0.556 mmol, 1.0 eq.) in anhydrous THF (3.8 ml) is injected. The reaction mixture is allowed to warm to -30 °C over 1 h be¬ fore the cooling bath is removed. Then the yellow suspension is stirred for 4.5 h at room temperature. The resulting orange suspension is cooled to -20 °C and quenched with saturated aqueous NH4CI solution (5 ml). After removing of the cool¬ ing bath the reaction is diluted with diethyl ether (15 ml). The organic phase is sepa¬ rated and the aqueous layer is extracted with diethyl ether (3x15 ml). The combined organic layers are dried over MgSC , filtered and concentrated under reduced pres¬ sure. After purification by column chromatography (n-pentane / Et?©, 98:2) the de¬ sired compound 10 is obtained (273 mg, 0.47 mmol, 85 %) as a colorless oil.

[0188] 1H NMR (CDCI3, 400 MHz): 57.29 - 7.21 (m, 2H), 6.91 - 6.83 (m, 2H), 5.77 (ddt, 1H, J = 17.2, 10.1, 7.1 Hz), 5.05 - 4.93 (m, 2H), 4.48 - 4.39 (m, 2H), 4.37 (dd, 1H, J = 9.5, 1.6 Hz), 3.80 (s, 3H), 3.64 (s, 3H), 3.55 (dd, 1 H, J = 8.1 , 3.4 Hz), 3.53 - 3.43 (m, 2H), 2.53 (dddt, 1H, J = 14.9, 6.9, 3.0, 1.4 Hz), 2.34 (dddt, 1H, J = 14.9, 8.2, 7.0, 1.3 Hz), 1.67 (dtd, 1H, J = 14.0, 8.0, 1.6 Hz), 1.56 (dddd, 1H, J = 13.9, 9.5, 6.5, 4.4 Hz), 1.23 (s, 3H), 0.90 (t, 9H, J = 7.9 Hz), 0.86 (s, 9H), 0.51 (qd, 6H, J = 8.0, 1.6 Hz), 0.06 (s, 3H), 0.01 (s, 3H).

[0189] 13C{1H} NMR (CDCI3, 101 MHz): 5 174.61, 159.29, 137.04, 130.64, 129.39, 116.77, 113.87, 78.31, 72.85, 71.86, 67.22, 58.22, 55.40, 51.24, 37.76, 33.17, 26.10, 18.37, 14.93, 7.07, 5.55, -2.86, -3.91.

[0190] P9641PC00Methyl (2R,3S,4E)-3-((tert-butyl(dimethyl)silyl)oxy)-2-((1S)-3-((4-rnethoxyben-zyl)oxy)-1 -((triethylsilyl)oxy)propyl)-2-methylhex-4-enoate (11 )

[0191] TBSO OTES

[0192]

[0193] The terminal alkene 10 (268 mg, 0.461 mmol, 1.0 eq.) is dissolved in methanol (6.1 ml, 99.5 % laboratory grade). After sequential addition of triethylamine (0.07 ml, 0.461 mmol, 1.0 eq.) and Grubbs II M204 (40.9 mg, 0.048 mmol, 0.1 eq.) the flask is closed by a glass cap and heated to 55 °C (oil bath temperature). The yellow / brown solution is stirred for 18 h after which TLC monitoring (n-pentane / Et2O, 9:1) indi¬ cates full conversion. The solvent is removed under reduced pressure and the brown residue is purified by column chromatography (n-pentane / Et2O, 99:1 — 98:2) to afford the final product 11 (242 mg, 0.417 mmol, 90 %, E / Z ratio 12:1) as a colorless oil.

[0194] 1H NMR (CDCI3, 400 MHz): 57.29 - 7.21 (m, 2H), 6.91 - 6.84 (m, 2H), 5.67 (ddq, 1H, J = 15.5, 9.1, 1.6 Hz), 5.46 (dq, 1H, J = 15.5, 6.4 Hz), 4.47 - 4.35 (m, 2H), 4.11 (dd, 1H, J = 9.4, 1.5 Hz), 3.84 (d, 1H, J = 9.0 Hz), 3.80 (s, 3H), 3.62 (s, 3H), 3.49 (td, 1H, J = 8.9, 4.4 Hz), 3.37 (dt, 1H, J = 8.8, 7.7 Hz), 1.79 (dddd, 1H, J = 13.7, 9.0, 7.5, 1.5 Hz), 1.61 (dd, 3H, J = 6.4, 1.6 Hz), 1.53 (ddd, 1H, J = 9.4, 8.0, 4.2 Hz), 1.22 (s, 3H), 0.89 (t, 9H, J = 7.9 Hz), 0.83 (s, 9H), 0.56 - 0.42 (m, 6H), -0.01 (s, 3H), -0.08 (s, 3H).

[0195] 13C{1H} NMR (CDCI3, 101 MHz): 5 174.80, 159.29, 130.89, 130.77, 129.41, 128.87, 113.86, 79.58, 72.73, 72.30, 67.80, 58.44, 55.43, 51.22, 32.85, 25.82, 18.14, 17.74, 14.06, 7.07, 5.55, -3.40, -4.91.

[0196] P9641PC00Preparation of the analog fragment

[0197] (3R,4S)-4-((tert-butyl(dimethyl)silyl)oxy)-3-((1 S,2E)-1 -hydroxybut-2-en-1 -y I) -3 -methyloxan-2-one (12)

[0198] O OH

[0199]

[0200] ^^OTBS

[0201] 12

[0202] To a solution of lactone 5 (488 mg, 2.0 mmol, 1.0 eq.) in dry dichloromethane (9.2 ml) 1 M Bu2BOTf in dichloromethane (2.2 ml, 2.2 mmol, 1.1 eq.) is injected at -78 °C under nitrogen atmosphere. After stirring of the yellow solution for 20 min at -78 °C triethylamine (0.36 ml, 2.6 mmol, 1.3 eq.) is added slowly which leads to a colorless solution. The reaction mixture is allowed to stir 1 h at this temperature. Then a mix¬ ture of crotonaldehyde (0.33 ml, 4.0 mmol, 2.0 eq.) with anhydrous dichloromethane (0.67 ml) is added slowly dropwise within 20 min. After stirring of the colorless solu¬ tion for 3 h at -78 °C the cold reaction mixture is quenched sequentially with 20 mM aqueous phosphate buffer (8 ml), methanol (4 ml) and 30 % H2O2 (2 ml) at -78 °C. The cooling bath is removed and vigorous stirring is continued for additional 1.5 h. Dichloromethane (10 ml) is added and the organic layer is separated. The aqueous layer is extracted with dichloromethane (3x20 ml) and the combined organic layers are washed once with an aqueous saturated Na2SO3 solution (11 ml). After drying over Mg2SO4, filtration and removing of the solvent the residue is purified by column chromatography (n-pentane / Et2O, 3:2) to afford product 12 (501 mg, 1.6 mmol, 80 %) as colorless crystals.

[0203] 1H NMR (CDCh, 400 MHz): 55.82 - 5.74 (m, 1 H), 5.67 (dq, 1 H, J = 15.3, 6.2 Hz), 4.43 (ddd, 1 H, J = 11.1 , 6.5, 4.6 Hz), 4.27 - 4.22 (m, 1 H), 4.20 (dd, 1 H, J = 8.5, 3.3 Hz), 4.12 (d, 1H, J = 7.9 Hz), 2.29 (s, 1H), 2.03 (dddd, 1H, J = 14.2, 6.5, 4.5, 3.4 Hz), 1.90 (dtd, 1H, J = 14.0, 8.2, 4.6 Hz), 1.72 (dd, 3H, J = 6.0, 1.2 Hz), 1.17 (s, 3H), 0.88 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H).

[0204] 13C{1H} NMR (CDCh, 101 MHz): 5 175.48, 130.45, 129.71, 76.27, 68.82, 65.38, 53.72, 28.97, 25.80, 18.07, 17.87, 16.67, -4.09, -4.91.

[0205] P9641PC00(3R,4S)-4-((tert-butyl(dimethyl)silyl)oxy)-3-((1S,2E)-1-(methoxymethoxy)but-2-en-1-yl)-3-methyloxan-2-one (13)

[0206] O OMOM

[0207]

[0208]

[0209] To a solution of compound 12 (430 mg, 1.37 mmol, 1.0 eq.) in anhydrous dichloro¬ methane (3.8 ml) DIPEA (0.93 ml, 5.47 mmol, 4.0 eq.) and MOMCI (0.31 ml, 4.11 mmol, 3.0 eq.) are added sequentially at 0 °C. After warming to room temperature over 2 h the solution is stirred for 3.5 h at 30 °C, then 18 h at room temperature and after that again 4h at 30 °C. The orange mixture is quenched with water (6 ml) and the organic phase is separated. The aqueous layer is extracted with diethyl ether (3x10 ml) and the combined organic layers are dried over MgSC . After filtration and concentration under reduced pressure the residue is purified by column chromatog¬ raphy (npentane / Et?© 2:1) to yield the product 13 (433 mg, 1.21 mmol, 88.3 %) as a colorless oil.

[0210] 1H NMR (CDCI3, 400 MHz): 55.74 - 5.60 (m, 2H), 4.69 (d, 1H, J = 6.8 Hz), 4.46 (d, 1H, J = 6.8 Hz), 4.40 (ddd, 1H, J = 10.8, 6.2, 4.4 Hz), 4.25 (ddd, 1H, J = 11.1, 8.4, 4.0 Hz), 4.18 (dd, 1H, J = 8.6, 3.5 Hz), 4.07 (d, 1H, J = 8.0 Hz), 3.36 (s, 3H), 2.06 (ddt, 1H, J = 13.8, 6.2, 3.8 Hz), 1.88 (dtd, 1H, J = 13.9, 8.5, 4.4 Hz), 1.76 (d, 3H, J = 4.9 Hz), 1.17 (s, 3H), 0.90 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H).

[0211] 13C{1H} NMR (CDCI3, 101 MHz): 5 174.52, 132.62, 127.32, 93.49, 81.17, 69.95, 65.15, 56.09, 53.73, 29.81, 25.84, 18.16, 17.90, 17.77, -4.16, -4.80.

[0212] Methyl (2R,3S,4E)-2-((1 S)-1 -((tert-butyl(dimethyl)silyl)oxy)-3-hydroxypropyl)-3-(methoxymethoxy)-2 -methylhex -4-enoate (14)

[0213] MOMO OTBS

[0214]

[0215] In a 10 ml pointed flask lactone 13 (75 mg, 0.21 mmol, 1.0 eq.) is dissolved in anhy¬ drous THF (0.13 ml) and anhydrous methanol (0.04 ml). A 1.02 M KOH solution in anhydrous methanol (0.30 ml, 0.306 mmol, 1.457 eq.) is added slowly at 0 °C and

[0216] P9641PC00the reaction solution is allowed to stir for further 50 min at 0 °C. After stirring for 41 h at room temperature the clear bright yellow solution is monitored by TLC (n-pen-tane / Et2O, 1 :1 ), which shows full conversion. A solution of camphorsulfonic acid (70.9 mg, 0.305 mmol, 1.453 eq.) in anhydrous methanol (1.4 ml) is added slowly dropwise at 0 °C. After stirring for 15 min at 0 °C diethyl ether (3.3 ml) is added. Then the reaction mixture is allowed to stir further 15 min at 0 °C before it is warmed to room temperature. The reaction solution becomes a white suspension which must be stirred more vigorous to keep it homogeneous. TMSCH2N2solution in hexane is injected in several portions until TLC shows full conversion of the carbox¬ ylic acid intermediate (Rf: 0.1 (n-pentane / Et2O, 1:1)). Excess of TMSCH2N2repre¬ sented by a yellow suspension must be destroyed by slowly dropwise addition of acetic acid glacial at 0 °C. To the resulting white suspension triethylamine (1.0 ml) is added immediately. After warm up the suspension, dichloromethane (10 ml) and a saturated aqueous NaHCOa solution (1 ml) are poured in a separating funnel. After shaking water (1 ml) is added and the organic layer is separated. The aqueous layer is extracted with dichloromethane (3x10 ml) and the combined organic layers are dried over MgSC , filtered and concentrated under reduced pressure. The residue is purified by column chromatography (n-pentane / Et2O, 2:1) to afford the final product 14 (63 mg, 0.162 mmol, 77.2 %) as a colorless oil.

[0217] 1H NMR (C6D6, 400 MHz): 55.65 (dqd, 1H, J = 15.4, 6.5, 0.7 Hz), 5.37 (ddq, 1H, J = 15.4, 8.8, 1.6 Hz), 4.73 (d, 1H, J = 6.6 Hz), 4.68 (d, 1H, J = 8.9 Hz), 4.45 (d, 1H, J = 6.6 Hz), 4.00 (dd, 1H, J = 7.8, 2.9 Hz), 3.62 - 3.51 (m, 2H), 3.47 (s, 3H), 3.25 (s, 3H), 2.06 - 1.87 (m, 2H), 1.52 (s, 3H), 1.50 (dd, 3H, J = 6.5, 1.7 Hz), 1.01 (s, 9H), 0.14 (s, 3H), 0.11 (s, 3H).

[0218] 13C{1H} NMR (C6D6, 101 MHz): 5 174.20, 132.76, 126.67, 93.42, 78.93, 73.74, 59.60, 56.40, 55.67, 51.19, 36.91, 26.38, 18.74, 17.89, 15.78, -3.45, -3.85.

[0219] 3-((4-methoxybenzyl)oxy)propan-1 -ol (15)

[0220] H

[0221]

[0222] O^^^OPMB

[0223] 15

[0224] 1,3-propandiol (1.65 g, 21.7 mmol, 1.0 eq.) is dissolved in anhydrous dichloro¬ methane (13 ml). Then p-methoxybenzyl alcohol (3.0 ml, 24.06 mmol, 1.1 eq.) and Amberlyst 15 (0.165 g, 0.1 eq. (w / w)) are added sequentially. After refluxing for

[0225] P9641PC006.5 h the reaction mixture is stirred over night at room temperature. Then the solu¬ tion is refluxed again for 6.5 h. After that MgSC is added at room temperature. MgSC and Amberlyst 15 are removed by filtration. The residue in the filter is washed with dichloromethane. After concentration under reduced pressure the crude is purified by column chromatography (n-pentane / Et?©, 1 :2) to afford product 15 (3.131 g, 15.95 mmol, 73.5 %) as a colorless oil.

[0226] 1H NMR (CDCI3, 400 MHz): 57.27 - 7.20 (m, 2H), 6.90 - 6.83 (m, 2H), 4.43 (s, 2H), 3.78 (s, 3H), 3.73 (t, 2H, J = 5.8 Hz), 3.60 (t, 2H, J = 5.9 Hz), 2.64 (s, 1H), 1.83 (p, 2H, J = 5.9 Hz).

[0227] 13C{1H} NMR (CDCI3, 101 MHz): 5 159.24, 130.24, 129.31, 113.84, 72.87, 68.83, 61.53, 55.27, 32.17.

[0228] 3-((4-methoxybenzyl)oxy)propanal (16)

[0229]

[0230] 16

[0231] Compound 15 (3.04 g, 15.5 mmol, 1.0 eq) is dissolved in anhydrous dichloro¬ methane (59 ml). After sequential addition of DMSO (12 ml) and triethylamine (11 ml, 77.5 mmol, 5.0 eq) the solution is cooled to 0 °C. Then sulphur trioxide pyri¬ dine complex (9.87 g, 62 mmol, 4.0 eq.) is slowly added in portions. The solution is stirred for 3 h at 0 °C. After that diethyl ether (250 ml) is added and the organic phase is washed at first with water (2x70 ml) and then with an aqueous saturated CUSO4 solution (70 ml). After washing several times with more water to remove the CUSO4 solution residues, the organic layer is treated with brine (70 ml), dried over MgSC and filtered. The solvent is removed under reduced pressure and the resi¬ due is purified by column chromatography (npentane / Et2O, 2:1) to obtain the pro¬ tected aldehyde 16 (2.22 g, 11.43 mmol, 74 %) as a colorless oil.

[0232] 1H NMR (CDCI3, 400 MHz): 59.76 (t, 1H, J = 1.8 Hz), 7.28 - 7.20 (m, 2H), 6.91 - 6.83 (m, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 3.76 (t, 2H, J = 6.1 Hz), 2.66 (td, 2H, J = 6.1, 1.9 Hz).

[0233] 13C{1H} NMR (CDCI3, 101 MHz): 5201.29, 159.35, 130.00, 129.39, 113.88, 72.92, 63.56, 55.29, 43.90.

[0234] P9641PC003-((tert-butyl(dimethyl)silyl)oxy)propan-1 -ol (17)

[0235] HO^^^OTBS

[0236] 17

[0237] To a solution of 1 ,3-propandiol (7.61 g, 100 mmol, 1.0 eq.) in anhydrous THF (90 ml) at 0 °C, sodium hydride (4 g, 100 mmol, 1.0 eq) is added in portions. After stir¬ ring for 5 min at 0 °C the reaction mixture continues stirring at room temperature for 45 min. It becomes a grey suspension. To this suspension, TBSCI (15 g, 100 mmol, 1.0 eq.) dissolved in anhydrous THF (42 ml) is added dropwise. After stirring for 3 h at room temperature the white suspension is quenched with water (25 ml). In a sep¬ arating funnel the organic phase is extracted with diethyl ether (3x50 ml). The combined organic layers are washed with an aqueous saturated NaHCOs solution (50 ml), treated with brine (60 ml), dried over MgSC and filtered. The solvent is re¬ moved under reduced pressure. After column chromatography (n-pentane / ethyl ace¬ tate, 9:1 — 4:1) the TBS protected alcohol 17 (14.5 g, 76.17 mmol, 76.17 %) is ob¬ tained as a colorless oil.

[0238] Rf = 0.46 (n-pentane / ethyl acetate, 4:1).

[0239] 3-((tert-butyl(dimethyl)silyl)oxy)propanal (18)

[0240]

[0241] 18

[0242] Compound 17 (8.16 g, 43 mmol, 1.0 eq) is dissolved in anhydrous dichloromethane (162 ml). After sequential addition of DMSO (33 ml) and triethylamine (30 ml, 215 mmol, 5.0 eq) the solution is cooled to 0 °C. Then sulphur trioxide pyridine complex (27 g, 172 mmol, 4.0 eq.) is slowly added in portions. The solution is stirred for 2 h at 0 °C. After that diethyl ether (600 ml) is added and the organic phase is washed sequentially with water (3x110 ml), 1 M HCI solution (100 ml), aqueous saturated NaHCOa solution (100 ml) and brine (100 ml). The organic layer is dried over MgSC and filtered. The solvent is removed under reduced pressure and the resi¬ due is purified by column chromatography (n-pentane / Et?©, 9:1) to obtain the TBS protected aldehyde 18 (6.66 g, 35.4 mmol, 82 %) as a colorless oil.

[0243] 1H NMR (CDCI3, 400 MHz): 59.78 (t, 1H, J = 2.1 Hz), 3.97 (t, 2H, J = 6.0 Hz), 2.58 (td, 2H, J = 6.0, 2.1 Hz), 0.86 (s, 9H), 0.05 (s, 6H).

[0244] 13C{1H} NMR (CDCI3, 101 MHz): 5202.14, 57.52, 46.68, 25.92, 18.33, -5.33.

[0245] P9641PC00

[0246]

[0247] Methyl (2R,3S,4E)-3-((tert-butyl(dimethyl)silyl)oxy)-2-methyl- 2-((1S)-3-oxo-1-((triethylsilyl)oxy)propyl)hex-4-enoate (19)

[0248]

[0249] Compound 11 (160 mg, 0.2754 mmol, 1.0 eq.) is dissolved in dichloromethane (5.80 ml). Then an aqueous pH 7 phosphate buffer solution (0.58 ml) is poured in. After that DDQ (93.8 mg, 0.4131 mmol, 1.5 eq.) is added and the mixture is stirred for 1 h at room temperature. The reaction is quenched with saturated aqueous Na-HCO3 solution (15 ml) and diluted with dichloromethane (15 ml). The organic layer is separated and the aqueous layer is extracted with dichloromethane (3x15 ml). The combined organic layers are dried over MgSC , filtered and concentrated under re¬ duced pressure. This crude mixture, which is directly used for the next step, is dis¬ solved in anhydrous dichloromethane (2.8 ml) and cooled to 0 °C. After sequential addition of NaHCCh (60.15 mg, 0.716 mmol, 2.6 eq.) and DMP (151.85 mg, 0.358 mmol, 1.3 eq.) the resulting suspension is stirred for 15 min at 0 °C. Then stir¬ ring is continued for 3 h at room temperature. The reaction is quenched by addition of saturated aqueous Na2S2O3 solution (3 ml) and saturated aqueous NaHCCh solu¬ tion (3 ml) and the mixture is diluted with diethyl ether (15 ml). The organic layer is separated and the aqueous layer is extracted with diethyl ether (3x20 ml). The com¬ bined organic layers are dried over MgSC , filtered, concentrated under reduced pressure and purified by column chromatography (n-pentane / Et2O, 95:5) to obtain the aldehyde 19 (87 mg, 0.19 mmol, 69 %) as a colorless oil.

[0250] 13C{1H} NMR (C6D6, 151 MHz): 5 199.77, 173.95, 131.28, 129.25, 79.80, 70.32, 58.01, 51.15, 48.29, 26.29, 26.00, 18.30, 17.52, 14.34, 7.21, 7.20, 5.64, 5.61, -3.25, -4.72.

[0251] P9641PC00Methyl (2R,3S,4E)-3-((tert-butyl(dimethyl)silyl)oxy)-2-((1 S,3Z)-1 -hydroxy-4-io-dobut-3-en-1 -yl)-2-methylhex-4-enoate (20)

[0252] TBSO OH

[0253]

[0254] 20 (lodomethyl)triphenylphosphonium iodide (115.6 mg, 0.218 mmol, 1.25 eq.) is suspended in anhydrous THF (1.25 ml). Then 2M NaHMDS solution in THF (0.12 ml, 0.227 mmol, 1.3 eq.) is added to the suspension at room temperature and the mixture is stirred for 10 min. The resulting orange solution is cooled to -78 °C. Aldehyde 19 (80 mg, 0.1744 mmol, 1.0 eq.) is dissolved in anhydrous THF (2.5 ml) and the mixture is injected drop wise. After stirring for 25 min at -78 °C the reaction mixture is allowed to warm up to room temperature over 1h. Then stirring is continued for additional 3.5 h. The reaction is stopped by filtration through a pad of silica, which is flushed with diethyl ether (200 ml). After evaporation of the solvent the crude mixture is purified by column chromatography (n-pentane / Et2O, 99:1) to obtain an inseparable mixture (57 mg) of product (Rt: 0.9 (n-pentane / Et2O 9:1)) and an unknown compound.

[0255] This mixture (57 mg, 0.098 mmol, 1.0 eq.) is used for the next step as if it would be the pure substance. It is dissolved in anhydrous methanol (1.0 ml) and pyridinium p-toluenesulfonate (27.6 mg, 0.11 mmol, 1.1 eq.) is added. The solution is stirred for 20 h at 0 °C. After evaporation of the solvent, the residue is purified by column chromatography (n-pentane / Et2O, 9:1) to obtain the vinyl iodide 20 (16 mg, 0.034 mmol, 20 %) as a colorless oil.

[0256] 13C{1H} NMR (C6D6, 101 MHz): 5 175.34, 139.31, 131.06, 128.73, 83.73, 75.86, 73.40, 56.81, 51.30, 39.18, 26.14, 18.42, 17.66, 14.13, -3.75, -4.81.

[0257] P9641PC00Experimental procedure

[0258] Propargyl Alchol n-Buli (Ph3P)2PdCI2Iodine > Cui, i-Pr2NH^ - - THE -78°C THF

[0259] / ?-Bu3SnH CuCN «-BuLi DCM,rt THF

[0260]

[0261] (lodoethynyl)trimethylsilane (SA-1 )

[0262] TMS - = - 1

[0263] SA-1

[0264] To a solution of trimethylsilylacetylene (10 g, 87.55 mmol, 1.0 equiv) in dry THF (150 mL), n-BuLi (2.5 M in hexane, 70 mL, 175.10 mmol, 2.0 equiv) was added dropwise at -78 °C, and the mixture was stirred for 30 minutes. A solution of l2(26.5 g, 105.06 mmol, 1.2 equiv) in dry THF (80 mL) was then added dropwise. The reaction mixture was stirred for an additional 15 minutes at -78 °C before quenching with saturated aqueous Na2S2O3(200 mL).Et2O (100 mL) and water (100 mL) were added, and the layers were separated. The aqueous layer was extracted with Et2O (3 * 100

[0265] mL). The combined organic layers were dried over MgSO4, filtered, and concen¬ trated under reduced pressure (100 mbar, 40 °C) to afford SA-1 (13.34 g, 59.53

[0266] P9641PC00mmol, 68%) as a pale-yellow liquid with a fruity odor. The product was used directly in the next reaction without purification.

[0267] General data: C5H9ISi; FW: 224.114 ; TLC: Rf=0.54 (Pentane); UV (+); KMNO41H-NMR (400 MHz, CDCI3) 60.18 (s,9H) .

[0268] 13C-NMR (151 MHz, CDCI3) 6 104.37, 30.47, 0.04.

[0269] 5-(T rimethylsilyl)penta-2,4-diyn-1 -ol (SA-2)

[0270]

[0271] SA-2

[0272] A degassed solution of dry THF (300 mL) and DIPA (30 mL, 22.52 g, 150.0 mmol, 3.0 equiv) was prepared by three freeze-pump-thaw cycles. To this solution, Pd(PPh3)2CI2(676 mg, 1.17 mmol, 2 mol%) and Cui (223 mg, 1.17 mmol, 2 mol%) were added, followed by SA-1 (13.00 g, 58.03 mmol, 1.0 equiv) and propargyl alco¬ hol (3.90 g, 43.44 mmol, 1.2 equiv). The resulting yellow-brown solution was stirred at room temperature for 2 h 30 min. The reaction mixture was filtered over a pad of Celite and washed with Et2O. The filtrate was concentrated under reduced pressure to obtain a brown liquid, which was purified by flash chromatography (pentane / Et2O, 3:1) to afford SA-2 (5.74 g, 37.70 mmol, 65%) as a pale yellow oil.

[0273] General data: C8Hi2OSi; FW: 152.265; TLC: Rf=0.5 (3:1 Pen / Et2O); UV (+); Vanillin: dark blue

[0274] 1H-NMR (400 MHz, CDCI3) 64.33 (d, J = 4.8 Hz, 2H), 1.73 (s, 1H), 0.20 (s, 9H).13C-NMR (151 MHz, CDCI3) 687.87, 87.24, 75.87, 70.74, 51.46, -0.39.

[0275] (E)-pent-2-en-4-yn-1-ol (SA-3)

[0276]

[0277] SA-3

[0278] To a solution of SA-2 (5.50 g, 36.13 mmol, 1.0 equiv) in dry Et2O (85 mL), LIAIH4(4.10 g, 108.31 mmol, 3.0 equiv) was added portion-wise at 0 °C. The resulting gray suspension was warmed to room temperature and stirred for 2 hours. The reaction was then cooled to 0 °C, and water (20 mL) was added dropwise, followed by 15% aqueous NaOH (10 mL). The suspension was allowed to warm to room temperature

[0279] P9641PC00and stirred overnight. The mixture was filtered over a pad of Celite and washed with Et2O. The filtrate was dried over MgSO4, filtered, and concentrated under reduced pressure (rt, 400 mbar) to yield an orange solution. The crude residue was purified by flash chromatography (pure pentane to pentane / Et2O, 1:1), affording SA-3 (2.78 g, 22.40 mmol, 62%) as a yellow liquid. Due to the high volatility of SA-3, the solvent was not completely removed after flash chromatography, and the yield was deter¬ mined by1H NMR.

[0280] General data: C5H6O; FW: 82.1; TLC: Rf=0.4 (3:1 Pen / Et2O); UV (+); Vanillin: dark blue

[0281] 1H-NMR (600 MHz, CDCI3) 56.35 (dt, J = 16.0, 5.0 Hz, 1H), 5.74 (dq, J= 16.1, 2.1 Hz, 1 H), 4.22 (ddd, J = 4.3, 1.4, 0.7 Hz, 2H), 2.89 (dd, J = 1.5, 0.7 Hz, 1 H).

[0282] 13C-NMR (151 MHz, CDCI3) 5 143.98, 109.17, 81.72, 77.96, 62.74.

[0283] (2E,5E)-6-(tributylstannyl)hexa-2,5-dien-1 -ol (SA-4)

[0284]

[0285] B u3S

[0286] SA-4

[0287] To a solution of CuCN (2.68 g, 29.90 mmol, 1.15 equiv) in dry THF (60 mL), n-BuLi (2.5 M in hexane, 24 mL, 60.06 mmol, 2.30 equiv) was added dropwise at -78 °C. The resulting suspension was allowed to warm slowly to room temperature. The re¬ action mixture was then cooled again to -78 °C, and n-Bu3SnH (16.2 mL, 60.06 mmol, 2.30 equiv) was added dropwise. The resulting deep yellow solution was stirred at -78 °C for 10 min. Then, a solution of SA-3 (2.60 g, 20.96 mmol, 1.0 equiv) in hexane (50 mL) was added dropwise. The resulting deep orange solution was stirred at -78 °C. After 90 min, aq. sat. NH4CI (540 mL) and 25% aq. NH4OH (60 mL) were added, followed by Et2O (100 mL), and the layers were separated. The aqueous layer was extracted with Et2O (3 * 50 mL). The combined organic layers, dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a yellow liquid. The crude residue was purified by flash chromatography (Hex / Et2O / NEt3, 90:10:1 to 50:50:1) to afford SA-4 (2.31 g, 16.75 mmol, 80%) as a pale-yellow oil.

[0288] General data: Ci7H34OSn; FW: 373.1617; TLC: Rf=0.37 (3:1 Pen / Et2O); UV (+); Vanillin: dark blue

[0289] P9641PC001H-NMR (600 MHz, C6D6) 56.76 - 6.68 (m, 1 H), 6.35 - 6.21 (m, 2H), 5.67 - 5.60 (m, 1 H), 3.91 (s, 2H), 1.62 - 1.54 (m, 6H), 1.41 - 1.34 (m, 6H), 0.99 - 0.89 (m, 15H).

[0290] 13C-NMR (151 MHz, C6D6) 5 147.19, 133.93, 133.42, 132.38, 62.77, 29.60, 27.73, 13.98, 9.82.

[0291] (2E,4E)-5-(tributylstannyl)penta-2,4-dienoic acid (SA-5)

[0292]

[0293] To a solution of SA-4 (2 g, 11.61 mmol, 1.0 equiv) in dry DCM (60 mL) was added NMO (2 g, 16.07 mmol, 3.0 equiv), followed by TPAP (307 mg, 1.16 mmol, 8 mol%) in a single addition. The resulting dark brown suspension was stirred at room tem¬ perature for 3 h. The reaction mixture was then filtered through a pad of silica and washed with Et2O. The yellow filtrate was concentrated under reduced pressure to give 2.05 g of crude SA-4-1 as a brown oil.

[0294] General data: Ci7H32OSn; FW: 371.145;

[0295] TLC: Rf=0.84 (3:1 Pen / Et2O); Rf=0.53 (15:1 Pen / Et2O) UV (+); Vanillin: purple.

[0296] 1H NMR (600 MHz, C6D6) 59.41 (d, J = 7.8 Hz, 1H), 6.78 - 6.57 (m, 2H), 6.44 (dd, J = 15.4, 9.9 Hz, 1H), 5.90 (dd, J = 15.3, 7.8 Hz, 1H), 1.61 - 1.48 (m, 5H), 1.44 - 1.25 (m, 8H), 0.93 (td, J = 7.8, 5.8 Hz, 15H).

[0297] 13C NMR (151 MHz, C6D6) 5 192.90, 152.02, 148.78, 145.00, 29.45, 27.64, 13.91, 9.88.

[0298] The crude aldehyde SA-4-1 was dissolved in t-BuOH (25 mL) and 2-methyl-2-bu- tene (14 ml, 108.44 mmol, 20 equiv). The resulting light brown solution was cooled to 0 °C, and a suspension of NaCIO2(4.00 g, 44.22 mmol, 8 equiv) in water (5.5 mL) and a suspension of NaH2PO4-2H2O (3.45 g, 22.11 mmol, 4 equiv) in water (5.5 mL) were added. The resulting yellow solution was stirred at 0 °C for 1.5 h, then diluted with aq. sat. NH4CI (50 mL). Et2O (50 mL) was added, and the layers were sepa¬ rated. The aqueous layer was extracted with Et2O (2 * 50 mL). The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pres¬ sure to give 2.4 g of crude SA-5.

[0299] P9641PC00General data: Ci7H32O2Sn; FW: 387.144; TLC: Rf=0.47 (3:1 Pen / Et2O); UV (+); KMNO4.1H-NMR (600 MHz, C6D6) 57.42 (dd, J = 15.3, 9.4 Hz, 1H), 6.71 - 6.53 (m, 2H), 5.77 (d, J = 15.2 Hz, 1 H), 1.63 - 1.42 (m, 6H), 1.39 - 1.25 (m, 6H), 0.92 (td, J = 7.7, 3.6 Hz, 15H).13C-NMR (151 MHz, C6D6) 5 179.81, 148.53, 148.34, 144.63, 119.80, 29.43, 27.64, 13.91, 9.81.

[0300] (S)-methyl 3-hydroxy-2-((2E,4E)-5-(tributylstannyl)penta-2,4-dienamido)pro-panoate (SA-6)

[0301] BuaSn

[0302]

[0303] SA-6

[0304] To the solution of the crude carboxylic acid SA-5 (1 g, 2.58 mmol, 1.0 equiv) and L-serine methyl ester hydrochloride (475 mg, 3.10 mmol, 1.2 equiv) in dry THF (20 mL), was added DIPEA (1.1 mL, 6.19 mmol, 2.4 equiv). the resulting solution was cooled to 0 °C, and HBTU (1.18 g, 3.10 mmol, 1.2 equiv) was added to the solution in one portion. The resulting yellow suspension was stirred at 0 °C for 30 min and then allowed to warm to rt. After 4 h, the reaction mixture was diluted with aq. sat. NH4CI (60 mL). Et2O (60 mL) was added, and the layers were separated. The aque¬ ous layer was extracted with Et2O (3 * 50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to give a crude residue. The crude product was purified by flash chromatography (pen- tane / Et2O / TEA, 1 :1 :0.1 ) to afford SA-6 (705 mg, 1.44mmol, 55% over 3 steps) as a pale-yellow oil.

[0305] General data: C^HsgNC Sn; FW: 488.248; TLC: Rf=0.33 (Pen / Et2O 1:1); UV (+); KMNO4,.

[0306] 1H-NMR (600 MHz, C6D6) 57.49 (dd, J = 15.0, 9.9 Hz, 1H), 6.73 - 6.54 (m, 2H), 6.22 (d, J = 7.2 Hz, 1 H), 4.74 (dd, J = 7.2, 3.6 Hz, 1 H), 3.74 (t, J = 3.6 Hz, 2H), 3.24 (s, 3H), 1.61 - 1.54 (m, 6H), 1.38 - 1.35 (m, 6H), 0.99 - 0.91 (m, 15H).

[0307] 13C-NMR (151 MHz, C6D6) 5 171.11, 166.05, 145.15, 144.49, 143.57, 122.92, 63.84, 55.41, 52.03, 30.23, 29.51, 27.72, 13.96, 9.85.

[0308] MS(ESI+): m / z: 490.1974 [M+H]+, 512.1837 [2M+Na]+.

[0309] P9641PC00Methyl 2-((1 E,3E)-4-(tributylstannyl)buta-1 ,3-dien-1 -yl)oxazole-4-carboxylate (SA-7)

[0310]

[0311] To a solution of SA-6 (600 mg, 1.23 mmol, 1.0 equiv) in dry DCM (10 mL) was added DAST (178 pL, 1.35 mmol, 1.1 equiv) dropwise at -78 °C. The resulting pale¬ yellow solution was stirred at -78 °C for 4 h. The reaction was quenched with K2CO3(340 mg, 2.45 mmol, 2.0 equiv), allowed to warm to room temperature over 1 h, and then poured into sat. aq. NaHCO3(40 mL) and DCM (40 mL). The aqueous layer was extracted with DCM (3 * 30 mL). The combined organic layers, dried over MgSO4, filtered, and concentrated under reduced pressure to give 594 mg crude ox¬ azoline as a pale yellow-brown oil.

[0312] General data: C2iH37NO3Sn; FW: 470.23; TLC: Rf=0.33 (Pen / Et2O 1:1); UV (+);

[0313] KMNO4

[0314] 1H NMR (400 MHz, CD3CN) 56.96 - 6.89 (m, 1H), 6.76 (s, 2H), 6.84 - 6.66 (m, 2H), 6.02 (d, J = 15.5 Hz, 1H), 4.77 (dd, J = 10.1, 7.7 Hz, 1H), 4.46 - 4.40 (m, 2H), 3.71 (s, 4H).

[0315] 13C NMR (101 MHz, CD3CN) 5 172.70, 166.42, 145.90, 145.63, 143.83, 118.30, 117.34, 70.20, 69.47, 52.99, 52.97, 52.87, 29.79, 27.93, 13.96, 10.17, 1.94, 1.73, 1.53, 1.32, 1.11, 0.91, 0.70.

[0316] MS(ESI+): m / z 472.1926 [M+H]+.

[0317] The crude oxazoline (594 mg, 1.26 mmol, 1.0 equiv) was dissolved in dry DCM (10 mL). The resulting pale yellow solution was cooled to 0 °C, and BrCCI3(504 pL, 5.05 mmol, 4.0 equiv) was added dropwise, followed by the addition of DBU (622 pL, 4.17 mmol, 3.3 equiv). The reaction mixture was stirred at 0 °C for 17 h, then di¬ luted with hexane (10 mL) and directly purified by flash chromatography (Hex / EtOAc / NEt3, 50:1:1 to 20:1:1) to afford SA-7 (507 mg, 1.08 mmol 86% over 2 steps) as a pale yellow oil.

[0318] General data: C2iH35NO3Sn; FW: 468.217; TLC: Rf=0.61 (Pen / Et2O 1:1); UV (+); KMNO4.

[0319] P9641PC001H-NMR (400 MHz, C6D6) 57.58 (s,1H), 7.23-7.19 (m, 1H), 6.70 - 6.51 (m, 2H), 6.20 (d, J = 16 Hz, 1H), 3.44 (s, 3H), 1.65 - 1.49 (m, 6H), 1.39 - 1.28 (m, 6H), 0.98 - 0.83 (m, 15H).

[0320] 13C-NMR (101 MHz, C6D6) 5 162.42, 161.80, 145.79, 143.72, 143.47, 140.44, 135.64, 128.54, 128.30, 128.06, 115.96, 51.54, 29.74, 27.92, 27.90, 14.18, 14.16, 10.12.

[0321] MS(ESI+): m / z: 470.1759 [M+H]+, 492.1567 [M+Na]+, 961.2868 [2M+Na]+.

[0322] 2-((1 E,3E)-4-(tributylstannyl)buta-1 ,3-dien-1 -yl)oxazole-4-carboxylic acid (SA-8)

[0323] Bu- iSn'

[0324]

[0325] SA-8

[0326] To a solution of SA-7 (500 mg, 1.07 mmol, 1.0 equiv) in 1,2-dichloroethane (10 mL), trimethyltin hydroxide (385 mg, 2.12 mmol, 2.0 equiv) was added. The mixture was heated to 80 °C and stirred for 8 h. After completion, the reaction mixture was di¬ luted with 0.2 M NaH2PO4-2H2O (60 mL) and Et2O (70 mL). The aqueous layer was extracted with Et2O (2 * 50 mL). The organic layers were combined, dried over MgSO4, fi Itered, and concentrated under reduced pressure to give 510 mg crude SA-8 as a yellow wax.

[0327] General data: C2oH33N03Sn; FW: 454.199; UV (+); KMNO4

[0328] 1H-NMR (400 MHz, CDCI3) 58.28 (s, 1H), 7.21 - 7.15 (m, 1 H), 6.82 - 6.66 (m, 2H), 6.37 (d, J = 16 Hz, 1H), 1.64 - 1.43 (m, 6H), 1.42 - 1.25 (m, 6H), 1.04 - 0.83 (m,15H).

[0329] MS (ESP): m / z 456.1620 [M+H]+.

[0330] P9641PC00(1Z,4S,5R,6S,7E)-6-((tert-butyldimethylsilyl)oxy)-1-iodo-5-(methoxycarbonyl)-5-methylnona-1,7-dien-4-yl 2-((1E,3E)-4-(tributylstannyl)buta-1,3-dien-1-yl)oxa-zole-4-carboxylate (SA-9)

[0331]

[0332] To the solution of alcohol (27 mg, 0.0576 mmol, 1.0 equiv) and acid (40 mg, 0.0864 mmol, 1.5 equiv) in dry CH2CI2 (5 mL) under exclusion of light and an argon atmos¬ phere was add NEta (15 pl, 0.1056 mmol, 3.3 equiv), followed by DMAP (4.50 mg, 0.0346 mmol, 60 mol%) and MNBA (36.0 mg, 0.1037 mmol, 1.8 equiv) were added to the solution. The resulting yellow solution was stirred at rtfor 18 h. aq. sat. Na-HCO3 (10 mL) and CH2CI2 (10 mL) were added, and the layers were separated. The aq. layer was extracted with CH2CI2 (2x10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The reaction yielded 25 mg (0.0276 mmol, 72% yield) of the SA-9 pale yellow oil. Additionally, 9 mg of the starting alcohol was recovered.

[0333] General data: CssH^NIOeSISn; FW: 904.617; TLC: Rf=0.62 (Hex / EA 8:1); UV (+); Vanillin: blue ,

[0334]

[0335] [a]p5= +15.00 (c = 0.6 in MeOH)

[0336] 1H-NMR (400 MHz, C6D6) 57.84 (s, 1 H), 7.25 - 7.17 (m, 1 H), 6.65 - 6.54 (m, 2H), 6.23 - 6.13 (m, 2H), 5.98 (dd, J = 9.7, 3.2 Hz, 1H), 5.92 - 5.78 (m, 1H), 5.45 (dq, J = 15.4, 6.4 Hz, 1H), 4.38 (d, J = 8.9 Hz, 1H), 3.37 (s, 2H), 2.84 - 2.72 (m, 1H), 2.72 (dd, J = 6.7, 3.1 Hz, 1 H), 2.71 (s, 1 H), 1.61 - 1.42 (m, 11 H), 1.40 - 1.28 (m, 9H), 1.01 - 0.89 (m, 15H), 0.08 (s, 3H), 0.06 (s, 3H).

[0337] 13C-NMR (101 MHz, C6D6) 5173.01, 162.40, 160.16, 145.52, 143.90, 143.29, 140.27, 138.05, 135.26, 131.21, 129.74, 128.30, 128.06, 127.82, 115.67, 84.90, 78.70, 74.81, 65.92, 56.38, 51.46, 37.65, 29.50, 27.67, 26.18, 18.36, 17.67, 15.60, 13.94, 9.87, 9.83, -3.23, -4.72.

[0338] P9641PC00(2R,2'R,3S,3'S,4E,4'E)-dimethyl 2,2'-((4S,6Z,8E,10E,18S,20Z,22E,24E)-2,16-di-oxo-3,13,17,27-tetraoxa-29,30-diazatricyclo[24.2.1.112,15]triaconta-1(28),6,8,10,12(30),14,20,22,24,26(29)-decaene-4,18-diyl)bis(3-((tert-butyldime-thylsilyl)oxy)-2-methylhex-4-enoate) (SA-10)

[0339]

[0340] A stock solution of Pd(PPh3)4(10.8 mg, 9.24 pmol, 60 mol%), CuTC (17.5 mg, 92.4 pmol, 6.0 equiv), and [Ph2PO2][NBu4] (44.6 mg, 97.02 pmol, 6.3 equiv) was pre¬ pared in dry DMF (2 mL) under an argon atmosphere. An aliquot (0.85 mL) of this red-brown solution was transferred to another flask, diluted with dry DMF (8 mL), thoroughly degassed, cooled to 0 °C, and kept in the dark. A degassed solution of SA-9 (15 mg, 15.4 pmol, 2.0 equiv) in dry DMF (2 mL) was added dropwise via sy¬ ringe pump to the stirring reaction mixture at 0 °C. The reaction was stirred for 3 hours and 30 minutes at 0 °C, then filtered through a silica plug and eluted with di¬ ethyl ether (2 * 80 mL). The two pale yellow filtrates were washed with water (80 mL), and the aqueous layer was extracted with diethyl ether (80 mL). The combined organic layers were washed again with water (2 * 80 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to yield an orange oil. The crude residue was purified by flash chromatography (Hexane / EtOAc / TEA, 3:1:0.1) to afford the desired product SA-10 (6 mg, 6.15 pmol, 39%).

[0341] General data: Cs^NzO^; FW: 975.3212; TLC: Rf=0.58 (Hex / EA 3:1); UV (+); Vaniline: green ,

[0342]

[0343] [a]p5= +115.111 (c = 0.45 in MeOH)

[0344] 1H-NMR (600 MHz, MeOD) 58.49 (s, 2H), 6.80 (br s, 2H), 6.69 (t, J = 13.2 Hz, 2H), 6.17 - 6.09 (m, 6H), 5.78 - 5.70 (m, 4H), 5.64 (td, J = 10.3, 7.2 Hz, 2H), 5.44 (d, J = 10.1 Hz, 2H), 4.36 - 4.30 (m, 2H), 3.59 (s, 6H), 2.72 (d, J = 11.6 Hz, 1 H), 2.68 (d, J = 10.5 Hz, 1 H), 2.54 - 2.47 (m, 2H), 1.80 - 1.75 (m, 6H), 1.41 (d, J = 13.7 Hz, 8H), 0.88 (s, 18H), 0.07 (s, 6H), 0.01 (s, 6H).

[0345] P9641PC00(2S,2'S,3S,3'S,4E,4'E)-dimethyl 2,2'-((4S,6Z,8E,10E,18S,20Z,22E,24E)-2,16-di-oxo-3,13,17,27-tetraoxa-29,30-diazatricyclo[24.2.1.112,15]triaconta-1(28),6,8,10,12(30),14,20,22,24,26(29)-decaene-4,18-diyl)bis(3-hydroxy-2-methylhex-4-enoate)) SA-11

[0346] OH O ^0 OH

[0347] O=f ’

[0348] O

[0349]

[0350] To a solution of SA-10 (6 mg, 6.15 pmol, 1.0 equiv) in dry THF (1200 pL) and dry pyridine (396 pL, 4.92 mmol, 800 equiv) at 0 °C in a 10 mL plastic centrifuge tube, HF-py (70% HF, 383 pL, 14.76 mmol, 2400 equiv) was added dropwise under light exclusion. After 40 minutes from the completion of the addition, the reaction mixture was allowed to warm to room temperature and stirred for a total of 20 hours before stopping. The resulting pale yellow solution was diluted with EtOAc (40 mL) and sat¬ urated aqueous NaHCO3(40 mL). The aqueous layer was extracted with EtOAc (2 x 40 mL), and the combined organic layers were washed with saturated aqueous NaHCO3(40 mL), dried over MgSO4, filtered, and concentrated under reduced pres¬ sure. The crude residue was purified by flash chromatography (Hexane / EtOAc, 5:1 to 1:1) to afford the desired product, disorazole Z1 (SA-11) (2 mg, 2.67 pmol, 43%), along with recovered SA-10 (1 mg, 1.03 pmol, 16%) and mono-TBS-protected SA- 10 (2 mg, 2.32 pmol, 37%).

[0351] General data: C40H46N2O12; FW: 746.7994; TLC: Rf=0.25 (Hex / EA 1:1); UV (+); Vaniline: green

[0352] 1H-NMR (600 MHz, MeOD) 58.50 (s, 2H), 6.77 (br s, 2H), 6.66 (t, J = 12 Hz, 2H), 6.15 -6.11 (m, 6H), 5.77 - 5.67 (m, 4H), 5.65 - 5.61 (m, 2H), 5.44 (d, J = 10.3 Hz, 2H), 4.33 (d, J = 7.7 Hz, 2H), 3.62 (s, 6H), 2.70 - 2.57 (m, 4H), 2.01 (s, 3H), 1.74 (dd, J = 6.4, 1.6 Hz, 6H), 1.39 (s, 6H), 1.24 (t, J = 7.1 Hz, 6H).

[0353] Preparation of disorazole Z1 linker conjugates

[0354] The solvents used in this synthesis were purchased in dry form and used as re¬ ceived without further purification. All reactions were conducted under a nitrogen at¬ mosphere unless otherwise specified. Products were purified using flash

[0355] P9641PC00chromatography on Merck silica gel 60 (40-63 pm). Electrospray ionization (ESI) and electron ionization (El) mass spectra were recorded on Finnigan MAT 95 and Waters Xevo G2-TOF spectrometers.

[0356] Synthesis of a mono(methylaminoethyl carbamate) derivative of Disorazole Z1

[0357] (S4)

[0358]

[0359] (2R,3S,E)-2-((4S,6Z,8E,10E,18S,20Z,22E,24E)-18-((2S,3S,E)-3-hydroxy-1-meth-oxy-2-methyl-1-oxohex-4-en-2-yl)-2,16-dioxo-3,13,17,27-tetraoxa-29,30-diazatri- cyclo[24.2.1.112,15]triaconta-1(28),6,8,10,12(30),14,20,22,24,26(29)-decaen-4- yl) -1 -methoxy-2-methyl-1 -oxohex-4-en-3-yl 1 H-imidazole-1 -carboxylate (S3)

[0360]

[0361] S3

[0362] To a solution of Disorazole Z1 (50 mg, 0.068 mmol, 1.0 equiv) in anhydrous MeCN under nitrogen at 0 °C was added CDI (11.6 mg, 0.072 mmol, 1.05 equiv) portion¬ wise. The reaction mixture was stirred at 0 °C to room temperature and monitored carefully by LC-MS. Upon formation of the mono-activated product, the reaction mix¬ ture was diluted with DCM and washed with water. The organic layer was dried over

[0363] P9641PC00Na2SO4, filtered, and concentrated under reduced pressure. Purification by flash chromatography afforded the mono-activated intermediate. MS (ESI) m / z = 840.38 [M+H]+.

[0364] (2R,2'R,3S,3'S,4E,4'E)-dimethyl 2,2'-((4S,6Z,8E,10E,18S,20Z,22E,24E)-2,16-di- oxo-3,13,17,27-tetraoxa-29,30-diazatricyclo[24.2.1.112,15]triaconta- 1(28),6,8,10,12(30),14,20,22,24,26(29)-decaene-4,18-diyl)bis(2-methyl-3-((me- thyl(2-(methylamino)ethyl)carbamoyl)oxy)hex-4-enoate) (S4)

[0365]

[0366] To a solution of the mono-activated intermediate S3 (20 mg, 0.0238 mmol, 1.0 equiv) in anhydrous DMF under nitrogen at 0 °C was added N,N'-dimethylethylenediamine (2.6 pL, 0.026 mmol, 1.1 equiv) dropwise. The reaction mixture was stirred at 0 °C to room temperature and monitored by TLC and LC-MS. Upon completion, the reaction mixture was quenched with saturated aqueous NH4CI, diluted with water, and extracted with DCM. The combined organic layers were washed with brine, dried over MgSC , filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography to afford the mono-aminated product as a yellow oil (13 mg, 65%).MS (ESI) m / z = 861.47 [M + H]+.

[0367] P9641PC00Synthesis of Bis(methylaminoethyl carbamate)-modified Disorazole Z1 (S2)

[0368]

[0369] S2 (2R,2'R,3S,3'S,4E,4'E)-((4S,6Z,8E,10E,18S,20Z,22E,24E)-2,16-dioxo-3,13,17,27-tetraoxa-29,30-diazatricyclo[24.2.1.112,15]triaconta- 1(28),6,8,10,12(30),14,20,22,24,26(29)-decaene-4,18-diyl)bis(1-methoxy-2-me-thyl-1 -oxohex-4-ene-3,2-diyl) bis(1 H-imidazole-1 -carboxylate) (S 1 )

[0370]

[0371] To a solution of Disorazole Z1 (100 mg, 0.134 mmol, 1.0 eq) in anhydrous MeCN (5 mL) at room temperature under a nitrogen atmosphere, CDI (108.5 mg, 0.67 mmol, 5.0 eq) was added in one portion. The reaction mixture was stirred at room tempera¬ ture for 8 hours, and progress was monitored by TLC. Upon completion, the reaction mixture was diluted with DCM (10 mL) and washed with water (5 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using ethyl acetate: methanol (5:0.1) as the eluent, yielding the title compound S1 as a yellow viscous oil (110 mg, 87%).

[0372] MS (ESI) m / z = 935.5412 (M + H+)

[0373] P9641PC00(2R,2'R,3S,3'S,4E,4'E)-dimethyl 2,2'-((4S,6Z,8E,10E,18S,20Z,22E,24E)-2,16-di-oxo-3,13,17,27-tetraoxa-29,30-diazatricyclo[24.2.1.112,15]triaconta- 1(28),6,8,10,12(30),14,20,22,24,26(29)-decaene-4,18-diyl)bis(2-methyl-3-((me-thyl(2-(methylamino)ethyl)carbamoyl)oxy)hex-4-enoate) (S2)

[0374]

[0375] S2

[0376] To solution of S1 (110 mg, 0.117 mmol, 1.0 eq) in anhydrous DMF (5 mL), DMAP (3 mg, 0.024 mmol, 0.2 eq) was added. The mixture was stirred for 5 minutes at room temperature, followed by the dropwise addition of DMEDA (22 pL, 0.23 mmol, 2.0 eq). Subsequently, DIPEA (82 pL, 0.46 mmol, 4.0 eq) was added. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was quenched by adding 10 mL of cold saturated NaCI solution. The mixture was ex¬ tracted with ethyl acetate (3 * 10 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using ethyl acetate: methanol: TEA = 3:2:0.1 as the eluent to afford the title compound S2 as a yellow viscous oil (85 mg, 75%).

[0377] MS (ESI) m / z = 975.4731 (M + H+)

[0378] P9641PC00Synthesis of the Di-Conjugated Bis-MAEC Disorazole Z1 -Silyl Ether Linker (L1)

[0379] TBSCI, Imidazole OTBS DCM,rt,ON, 99% HO Ll-2

[0380] 2-(2-aminoethoxy)ethan-l-ol NaHCO3, H2O, OC to room Ih, 57%

[0381] Ll-4

[0382]

[0383] 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (L1 -2)

[0384]

[0385] 4-(hydroxylmethyl)phenol (1.00 g, 8.06 mmol, 1 eq) was dissolved in dry DCM, then cooled in an ice-water bath. Imidazole (0.658 g, 9.67 mmol, 1.2 eq) and tert-

[0386] P9641PC00butyldimethylsilyl chloride (1.46 g, 9.67 mmol 1.2 eq) were added and the reaction warmed to room temperature. After 3 hours, the reaction was diluted with ether (50 ml) and washed with saturated NH4CI (50 ml) and brine (25 ml). The organic layer was dried over Na2SO4 and concentrated in vacuo as a yellow oil. The crude product was purified by column chromatography to obtain compound L1-2 as a pale-yellow oil (1.82 g, 95% yield). HRMS (ESP) m / z: calcd for Ci3H26O2Si [M+NH4]+: 256.1728, found: 256.1733.

[0387] 1 -(2-(2-hydroxyethoxy)ethyl)-1 H-pyrrole-2, 5-dione (L1 -1 )

[0388]

[0389] Maleimide (2.0 g, 20.6 mmol) was dissolved in ethyl acetate (80 mL) and the solution was cooled to 0 °C using an ice bath. N-Methylmorpholine (1.85 g, 20.5 mmol) was then added dropwise. Subsequently, a solution of methyl chloroformate (2.46 g, 20 mmol) in ethyl acetate (5 mL) was added dropwise, and the reaction mixture was stirred at room temperature for 1 h. Upon completion of the reaction, the resulting insoluble salt was removed by filtration. The filtrate was concentrated under reduced pressure to afford crude methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1 -carboxylate. Due to its instability, the crude product was used directly in the next step without further purification.

[0390] 2-(2-Aminoethoxy)ethan-1-ol (2.0 g, 20.6 mmol) was dissolved in saturated aqueous sodium bicarbonate solution (60 mL) and cooled to 0 °C. Once the desired tempera¬ ture was reached, this solution was added to the crude intermediate (3.28 g, crude). The reaction mixture was stirred at 0 °C for 30 min and then gradually allowed to warm to room temperature, where stirring was continued for an additional 1 h. After completion of the reaction, the mixture was extracted with ethyl acetate. The com¬ bined organic extracts were concentrated, and the crude product was purified by col¬ umn chromatography to afford compound L1-1 as a white solid (2.04 g, 53% yield over two steps). HRMS (ESI+) m / z: calcd for CsH-^NC 186.076; found 186.076. ESI m / z

[0391] P9641PC001-(2-(2-(((4-(((tert-butyldimethylsilyl)oxy)methyl)phenoxy)diisopropylsi- lyl)oxy)ethoxy)ethyl)-1 H-pyrrole-2, 5-dione (L1 -3)

[0392]

[0393] Imidazole (150 mg, 2.16 mmol, 2.0 equiv) was added to a solution of dichlorodiiso¬ propylsilane (200 mg, 1.08 mmol, 1.0 equiv) in anhydrous DCM (20 mL). The reaction mixture was cooled to approximately -5 °C. A solution of L1-2 (0.26 g, 1.08 mmol, 1.0 equiv) in dichloromethane (5 mL) was added dropwise, and the reaction mixture was stirred for 10 min at the same temperature. A solution of L1-1 (200 mg, 1.08 mmol, 1.0 equiv) in dichloromethane (10 mL) was then added dropwise. The reaction mixture was gradually allowed to warm to room temperature and stirred until completion of the reaction. Water was added, and the mixture was extracted with dichloromethane (3 x 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by col¬ umn chromatography to afford L1-3 (0.40 g, 70% yield). HRMS (ESI+) calcd for C27H49N2O6Si2[M+NH4]+: 553.3038, found: 553.3132.

[0394] 1-(2-(2-(((4-(hydroxymethyl)phenoxy)diisopropylsilyl)oxy)ethoxy)ethyl)-1H-pyr- role-2, 5-dione (L1-4)

[0395]

[0396] To the solution of L1-3 (150 mg, 0.28 mmol, 1.0 equiv) in ethanol (5 mL) was treated with pyridinium p-toluenesulfonate (PPTS) (7 mg, 0.028 mmol, 0.1 equiv). The reac¬ tion mixture was stirred at room temperature overnight. Upon completion of the reac¬ tion, triethylamine (0.14 mL, 1.0 mmol, 3.6 equiv) was added to quench the reaction mixture. The solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography to afford L1-4 as a colorless oil (98 mg, 80% yield). HRMS (ESP) calcd for C2iH35N2O6Si [M+NH4]+: 439.2259, found: 439.2344.

[0397] P9641PC004-(((2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethoxy)diisopropylsi- lyl)oxy)benzyl (4-nitrophenyl) carbonate (L1 )

[0398]

[0399] L1-4 (80 mg, 0.18 mmol, 1.0 equiv) was dissolved in anhydrous DMF (10 mL). Bis(p-nitrophenyl) carbonate (113 mg, 0.36 mmol, 2.0 equiv) and N,N-diisopropylethylamine (DIPEA) (47 pL, 0.27 mmol, 1.5 equiv) were added, and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction, the solvent was removed under reduced pressure. The resulting residue was dispersed in diethyl ether and the precipitate was collected by filtration to afford a crude product. The crude material was further purified by column chromatography to yield L1 -5 as a white solid powder (78 mg, 70% yield). HRMS (ESI+) calcd for C28H38N3OioSi [M+NH4]+: 604.3; found 604.5.

[0400] P9641PC00Synthesis of a Bis(Silyl Ether Linker-MAEC) Disorazole Z1 Conjugate (1)

[0401] HOBT DIPEA DMF, r.t.

[0402]

[0403] 1

[0404] To the solution of L1 (30 mg, 0.062 mmol, 3 eq) in 2 mL of anhydrous DMF, HOBt (5.5 mg ,0.041 mmol, 2 eq) was added at room temperature under a nitrogen atmos¬ phere. Subsequently, S2 (20 mg, 0.021 mmol, 1.0 eq) and DIPEA (14.3 pL, 0.082 mmol, 4 eq) were added. The reaction mixture was stirred at room temperature for 18 hours, and the progress was monitored by TLC. After completion, the solvent was removed under reduced pressure to yield the crude product. MS (ESI) m / z = 1886.7853 [M + NH4]+.

[0405] P9641PC00Synthesis the Mc-Val-Cit-PABOH linker (L2)

[0406]

[0407] P9641PC00(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanoic acid (L2-1)

[0408]

[0409] L-Citrulline (3.00 g, 17.13 mmol, 1.1 equiv) was dissolved in water (75 mL), and so¬ dium bicarbonate (2.88 g, 34.40 mmol, 2.2 equiv) was added. The resulting solution was stirred at room temperature for 1 h. A solution of Fmoc-CI (4.00 g, 15.57 mmol, 1.0 equiv) in 1,2-dimethoxyethane (DME) (80 mL) was then added to the reaction mixture. The reaction was stirred at room temperature for 24 h. After completion of the reaction, the solvent was removed under reduced pressure. The residue was ex¬ tracted with ethyl acetate (3*). The organic layers were discarded and the aqueous phase was acidified with 2 M HCI, resulting in the formation of a white precipitate partially soluble in water. A mixture of isopropanol- ethyl acetate (10% v / v iPrOH) was added and the biphasic mixture was stirred until clear phase separation was achieved. The organic layer was collected, and the aqueous phase was re-extracted twice with the same solvent system. The combined organic extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford a clear viscous residue. The crude product was sonicated in diethyl ether and triturated twice, decanting the solvent each time, to afford the product as a white solid. Drying under reduced pressure afforded compound L2-1 as a white solid (5.30 g, 14.78 mmol, 95%) without further purification. HRMS (ESI+) m / z calcd for C2iH23N3NaO5[M+Na]+: 420.1535; found: 420.1531.

[0410] P9641PC00(S)-(9H-fluoren-9-yl)methyl (1 -((4-(hydroxymethyl)phenyl)amino)-1 -oxo-5- ureidopentan-2-yl)carbamate (L2-2)

[0411]

[0412] A solution of L2-1 (2.00 g, 5.03 mmol, 1.0 equiv) and 4-aminobenzyl alcohol (1.86 g, 15.1 mmol, 3.0 equiv) in DMF (50 mL) was treated with DIPEA (1.30 mL, 7.32 mmol, 1.5 equiv) and stirred at room temperature for 15 min. HATU (3.1 g, 8.05 mmol, 1.6 equiv) was then added, and the reaction mixture was stirred at room temperature for 48 h under exclusion of light. After completion of the reaction, the solvent was re¬ moved under reduced pressure. The residue was purified by flash column chroma¬ tography to afford L2-2 as a white solid (71% yield). HRMS (ESP) m / z calcd for C28H30N4NaO5[M+Na]+: 525.2114; found: 525.2116.

[0413] (9H-fluoren-9-yl)methyl ((S)-1 -(((S)-1 -((4-(hydroxymethyl)phenyl)amino)-1 -oxo- 5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (L2-3)

[0414]

[0415] A solution of L2-2 (2 g, 3.98 mmol, 1.0 equiv) in DMF (20 mL) was treated with tri¬ ethylamine (11.1 mL, 79.6 mmol, 20 equiv) and the reaction mixture was stirred at room temperature for 24 h. The solvent and excess triethylamine were removed under reduced pressure. The resulting residue was dissolved in DMF (40 mL), and Fmoc-Val-OSu (1.91 g, 4.38 mmol, 1.1 equiv) was added. The reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, DMF was removed under reduced pressure and the residue was purified by flash column chromatog¬ raphy to afford the product as a white solid (78% yield). HRMS (ESP) m / z calcd for C33H39N5NaO6[M+Na]+: 624.2798; found: 624.2794.

[0416] P9641PC006-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y l)-N -((S)-1 -<«S)-1 -((4-(hydroxyme- thyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2- yl)hexanamide (L2-4)

[0417]

[0418] A solution of L2-3 (1.00 g, 1.67 mmol, 1.0 equiv) in DMF (10 mL) was treated with triethylamine (4.63 mL, 33.0 mmol, 19.8 equiv), and the reaction mixture was stirred at room temperature for 24 h. The DMF and excess triethylamine were removed under reduced pressure. The resulting residue was dissolved in DMF (20 mL), and Mc-OSu (0.56 g, 1.85 mmol, 1.1 equiv) was added. The reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, DMF was removed under re¬ duced pressure, and the residue was purified by flash column chromatography to af¬ ford the product as a white solid (86% yield). HRMS (ESI+) m / z calcd for C28H40N6NaO7[M+Na]+: 595.2856; found: 595.2854.

[0419] 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3 -methyl-butanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (L2)

[0420]

[0421] L2-4 (200 mg, 0.349 mmol, 1.0 equiv) was dissolved in anhydrous pyridine (6 mL) under an argon atmosphere at room temperature. The reaction mixture was cooled to 0 °C, and a solution of p-nitrophenyl chloroformate (212 mg, 1.05 mmol, 3.0 equiv) in CH2CI2(4 mL) was added in one portion. The mixture was maintained at 0 °C for 10 min and then allowed to warm to room temperature, where it was stirred for an

[0422] P9641PC00additional 2 h. After completion of the reaction, ethyl acetate (90 mL) and 15% aque¬ ous citric acid (130 mL) were added. The organic layer was separated and washed sequentially with additional 15% citric acid solution, water, and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under re¬ duced pressure to afford a pale-yellow residue. The crude material was purified by flash column chromatography to afford L2 as a white solid (77 mg, 30% yield). HRMS (ESP) m / z calcd for C35H45N7OH [M+H]+: 737.3021 ; found: 737.3017.

[0423] Synthesis of the Di-Conjugated Bis-MAEC Disorazole Z1- dipeptide (Val-Cit) Linker (2)

[0424]

[0425] To the dipeptide linker L2 (30 mg, 0.062 mmol, 3 eq) in anhydrous DMF (2 mL), HOBt (5.5 mg, 0.041 mmol, 2 eq) was added under a nitrogen atmosphere and stirred at room temperature for 10 minutes. Subsequently, S2 (20 mg, 0.021 mmol, 1 eq) in DMF (1 ml) was added dropwise to the reaction mixture, followed by addition of DIPEA

[0426] P9641PC00(14.2 pL, 0.081 mmol, 4 eq). The reaction mixture was stirred at room temperature for 12 hours, and the progress of the reaction was monitored by TLC. After the disappearance of S2 on TLC, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography. MS (ESI) m / z = 2143.9864 [M + H]+.

[0427] Mono-Conjugated Mono-MAEC Disorazole Z1 -Dipeptide (Val-Cit) Linker Conju-

[0428]

[0429] To a solution of dipeptide linker L2 (17 mg, 0.035 mmol, 1.5 equiv) in anhydrous DMF (2 mL), HOBt (6.3 mg, 0.046 mmol, 2.0 equiv) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 10 min. Subsequently, S4 (20 mg, 0.023 mmol, 1.0 equiv) dissolved in DMF (1 mL) was added dropwise, followed by DIPEA (8.1 pL, 0.047 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature, and the progress of the reaction was monitored by TLC. After disappearance of the starting material, the reaction mixture was concentrated under reduced pressure. The residue was purified by column

[0430] P9641PC00chromatography to afford the desired mono-conjugated product. MS (ESI) m / z = 1445.6517 [M + H]+.

[0431] Preparation of MC-Gly-Gly-Phe-Gly-OH (L3)

[0432] HOSu, EDCI, CH3CN L3-1 L-phenylalanine HOSu, EDCI NaHCO3, DME / H2O CH3CN L3-2

[0433] Gly-OH NaHCO3, DME / H2O L3-3

[0434] Piperidine DMF, 20min MC-OSu, DI PEA, DMF, 10 min, 80%

[0435]

[0436] L3 P9641PC002,5-dioxopyrrolidin-1-yl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acet- amido)acetate (L3-1)

[0437] Fmocx

[0438]

[0439] Fmoc-Gly-Gly (1.80 g, 5.14 mmol) was dissolved in acetonitrile (18 mL) in a reaction flask and stirred at room temperature until a clear solution was obtained. EDC-HCI (1.00 g, 5.22 mmol) and N-hydroxysuccinimide (NHS) (0.64 g, 5.54 mmol) were added to the reaction mixture, and stirring was continued at room temperature for 12 h. TLC analysis indicated complete conversion of the starting material. The reaction mixture was filtered and the collected solid was dried under reduced pressure to afford L3-1 as a white solid (2.0 g, 86% yield). HRMS (ESI): m / z calculated for C23H22N3O7 [M + H]+452.145; found 452.147.

[0440] (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-oic acid (L3-2)

[0441] 0r Fmoc .Ph

[0442] N n N n

[0443]

[0444] HAHA

[0445] L-Phenylalanine (1.50 g, 9.09 mmol) and sodium bicarbonate (1.50 g, 17.86 mmol) were dissolved in water (40 mL), and the mixture was stirred at room temperature until a clear solution was obtained. L3-1 (4.1 g, 9.15 mmol) was dissolved in DME (10 mL), and this solution was added dropwise to the above aqueous solution with stirring. The reaction mixture was stirred at room temperature for 12 h, after which TLC anal¬ ysis indicated complete consumption of the starting material. The organic solvent was removed under reduced pressure, and the remaining aqueous reaction mixture was slowly added to 0.5 M aqueous HCI (94 mL), resulting in the formation of a precipitate. The suspension was filtered and the collected solid was dried under reduced pressure to afford L3-2 as a solid (2.81 g, 61% yield). HRMS (ESI): m / z calcd for C28H28N3O6[M+H]+502.197; found 502.198.

[0446] P9641PC00(S)-(9H-fluoren-9-yl)methyl (2-((2-((1 -(2,5-dioxopyrrolidin-1 -y l)-1 -oxo-3-phe- nylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate (L3-3)

[0447]

[0448] L3-2 (1.00 g, 2.00 mmol) was dissolved in acetonitrile (13 mL) in a reaction flask. EDC-HCI (0.40 g, 2.09 mmol) and N-hydroxysuccinimide (NHS) (0.27 g, 2.32 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 12 h, after which TLC analysis indicated complete conversion. The reaction mixture was filtered and the collected solid was dried under reduced pressure to afford L3-3 as a solid (870 mg, 73% yield). HRMS (ESI): m / z calcd for C32H31N4O8[M+H]+583.211; found 583.215.

[0449] (S)-1 -(9H-fluoren-9-yl)-3,6,9, 12-tetraoxo-11 -phenyl-2-oxa-4,7,10, 13-tetraazapen- tadecan-15-oic acid (L3-4)

[0450]

[0451] Glycine (0.60 g, 8.0 mmol) and sodium bicarbonate (1.34 g, 16.0 mmol) were dis¬ solved in water (30 mL), and the mixture was stirred at room temperature until a clear solution was obtained. L3-3 (2.60 g, 4.35 mmol) was dissolved in DME (8 mL), and this solution was added dropwise to the above aqueous solution with stirring. The reaction mixture was stirred at room temperature for 12 h, after which TLC analysis indicated complete consumption of the starting material. The organic solvent was re¬ moved under reduced pressure, and the remaining aqueous reaction mixture was slowly added to 0.5 M aqueous HCI (60 mL), resulting in the formation of a large amount of precipitate. The suspension was filtered and the collected solid was dried under reduced pressure to afford L3-4 acid as a solid (2.0 g, 83% yield). HRMS (ESI): m / z calcd for C29H29N4O7 [M+H]+544.195; found 544.198.

[0452] P9641PC00(S)-2-(2-(2-(2-aminoacetamido)acetamido)-2-phenylacetamido)acetic acid (L3-5)

[0453]

[0454] L3-4 (1.00 g, 1.79 mmol) was dissolved in 20% piperidine in DMF (10 mL) and stirred at room temperature for 10 min. The reaction mixture was concentrated in vacuo to afford crude L3-5, which was used directly in the next step without further purification. MS (ESI) m / z = 337.2 [M + H]+.

[0455] (S)-5-benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-4,7,10,13-tetraoxo- 3,6,9, 12-tetraazaoctadecan-1-oic acid (L3)

[0456]

[0457] The crude L3-4 obtained from the previous step (1.00 g, 1.79 mmol, 1.0 equiv) was dissolved in DMF (18 mL), and Mc-OSu (591 mg, 1.97 mmol, 1.1 equiv) was added. The reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, DMF was removed under reduced pressure, and the resulting residue was purified by column chromatography to L3 as a solid (815 mg, 86% yield). HRMS (ESI): m / z calcd for C25H32N5O8[M+H]+530.224; found 530.226.

[0458] P9641PC00Synthesis of the Di-Conjugated Bis-MAEC Disorazole Z1 -Tetrapeptide (GGFG)

[0459]

[0460] To a solution of tetrapeptide linker L3 (40 mg, 0.062 mmol, 3 eq) in anhydrous DMF (2 mL) under a nitrogen atmosphere, DMTMM (26 mg, 0.086 mmol, 4.2 eq) was added. The reaction mixture was stirred at room temperature for 5 minutes to activate the linker. Subsequently, S2 (20 mg, 0.021 mmol, 1 eq) in 1 ml DMF was added drop¬ wise, followed by the addition of DIPEA (27.7 pL, 0.154 mmol, 7.5 eq). The reaction was monitored by TLC and LC-MS. After completion, the solvent was removed under reduced pressure to yield the crude product. MS (ESI) m / z = 1997.8877 [M + H]+.

[0461] P9641PC00mono-conjugated mono-MAEC Disorazole Z1 -tetrapeptide (GGFG) linker conjugate (5)

[0462]

[0463] To a solution of tetrapeptide linker L3 (23 mg, 0.035 mmol, 1.5 equiv) in anhydrous DMF (2 mL) under a nitrogen atmosphere, DMTMM (15.0 mg, 0.049 mmol, 2.1 eq) was added. The reaction mixture was stirred at room temperature for 5 min to activate the linker. Subsequently, the S4 (20 mg, 0.0232 mmol, 1.0 equiv) in DMF (1 mL) was added dropwise, followed by the addition of DIPEA 15.2 pL (0.087 mmol, 3.75 eq) . The reaction was monitored by TLC and LC-MS. After completion, the solvent was removed under reduced pressure to yield the crude product. MS (ESI) m / z = 1372.5936 [M + H]+.

[0464] P9641PC00MC-GGFG-Hemiaminal-Glycolic acid Synthesis (L4)

[0465] Pb(OAc)4, pyridine. Benzyl glycolate, PPTS, FmocHN THF, Toluene 3h, 65% DCM, refluxed, Over night, 99%

[0466] piperidine, DMF, 20% Frnoc-Phe-Osu DMF, DIPEA DMF,30min, 68%

[0467] Fmoc-Gly-Gly-OH DIPEA, HATU, DMF,30min, 55%

[0468] MC-OSu, DIPEA, DMF, 80%

[0469] (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methyl acetate (L4-1) FmocHN

[0470]

[0471] Fmoc-Gly-Gly-OH (2.00 g, 5.66 mmol) was suspended in a mixture of THF (51 mL), toluene (17 mL), and pyridine (0.85 mL). The suspension was stirred until partially dissolved, after which lead tetraacetate (3.13 g, 7.08 mmol) was added. Upon

[0472] P9641PC00addition, the reaction mixture developed an orange color. The mixture was heated to reflux and maintained under stirring. After approximately 1 h, the solution became colorless and a white precipitate formed. The reaction was continued at reflux for a total of 3 h. The mixture was then filtered through a pad of Celite, washing the pad with ethyl acetate. The combined filtrates were concentrated under reduced pressure to afford a crude residue. Purification by flash column chromatography provided the desired product as a colorless solid (1.2 g, 65% yield). MS (ESI) m / z: [M+H]+calcd. for CI8H17N2O3309.12; found 309.13. Only the iminium ion was observed due to fragmentation of the corresponding hemiaminal during MS analysis.

[0473] benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oate (L4-2)

[0474] H1

[0475] FmocHN'z^X|| / ^ / i3Bn

[0476]

[0477] L4-1 (1.00 g, 2.71 mmol) was dissolved in anhydrous DCM (15 mL). Benzyl glycolate (3.85 mL, 27.1 mmol) was added, followed by PPTS (68 mg, 0.27 mmol). The reaction mixture was heated to reflux and stirred overnight. Reaction progress was monitored by LC-MS, which indicated near complete conversion. The mixture was diluted with ethyl acetate (60 mL) and washed with water (3 * 60 mL). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography to afford the desired product as a white solid (1.25 g, 99% yield). MS (ESI) m / z: [M+Na]+calcd for C27H26N2NaO6497.17; found 497.09.

[0478] benzyl 2-((2-aminoacetamido)methoxy)acetate (L4-3)

[0479]

[0480] L4-2 (1.00 g, 2.11 mmol) was dissolved in 20% piperidine in DMF (10 mL) and the reaction mixture was stirred at room temperature for 20 min. The solvent was removed under reduced pressure to afford the crude amine, which was used directly in the subsequent step without further purification. MS (ESI) m / z = 253.111 [M + H]+.

[0481] P9641PC00(S)-benzyl 5-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2,12-dioxa-4,7,10- triazatetradecan-14-oate (L4-4)

[0482] FmocHN

[0483]

[0484] The crude amine obtained from the previous step (2.00 mmol, 1.0 equiv) was dis¬ solved in DMF (2 mL). DIPEA (0.69 mL, 4.00 mmol, 2.0 equiv) was added, followed by Fmoc-Phe-OSu (1.62 g, 3.00 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 30 min. After completion of the reaction, the solvent was re¬ moved under reduced pressure and the crude residue was purified by flash column chromatography to afford the desired product as a white solid (869 mg, 1.40 mmol, 68% yield). MS (ESI) m / z [M+Na]+calcd for C36H35N3NaO7644.24; found 644.04.

[0485] (S)-benzyl 2-((2-(2-amino-3-phenylpropanamido)acetamido)methoxy)acetate (L4-5)

[0486]

[0487] L4-4 (869 mg, 1.40 mmol) was dissolved in 20% piperidine in DMF and stirred for 20 minutes. The reaction was concentrated in vacuo and used in next step without further purification. MS (ESI) m / z = 340.179 [M + H]+.

[0488] (S)-benzyl 11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa- 4,7,10,13,16-pentaazaicosan-20-oate (L4-6)

[0489] FmocHI

[0490] N •Bn

[0491]

[0492] The crude product obtained from the previous step (1.40 mmol, 1.0 equiv) was dis¬ solved in anhydrous DMF (2 mL). DIPEA (0.96 mL, 5.52 mmol, 3.9 equiv) and Fmoc- Gly-Gly-OH (1.03 g, 2.90 mmol, 2.1 equiv) were added, followed by HATU (1.05 g, 2.76 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 30 min. The reaction was quenched with acetic acid and the solvent was removed under reduced pressure. The crude residue was chromatographed to afford the desired

[0493] P9641PC00product as a white solid (567 mg, 0.77 mmol, 55% yield). MS (ESI) m / z [M+Na]+calcd for C40H41N5NaO9758.28; found 758.0.

[0494] (R)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10,13,16- pentaazaicosan-20-oic acid (L4-7)

[0495]

[0496] L4-6 (567 mg, 0.77 mmol, 1.0 equiv) was suspended in EtOH / EtOAc (2:1, 10.5 mL) and placed under a nitrogen atmosphere. 10% Pd / C (115 mg) was added, and hydrogen was bubbled through the reaction mixture at 1 atm for 1 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was filtered through a pad of Celite, rinsing with methanol, and the filtrate was concentrated under reduced pressure to afford the crude product, which was used directly in the subsequent step without further purification. MS (ESI) m / z = 646.257 [M + H]+.

[0497] (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-

[0498]

[0499] The substrate (0.77 mmol) was dissolved in 20% piperidine in DMF (8 mL) and stirred at room temperature for 20 min. The reaction mixture was concentrated in vacuo, and the resulting crude product was used directly in the next step without further purification. MS (ESI) m / z = 424.273 [M + H]+.

[0500] (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-6,9,12, 15, 18-pentaoxo- 3-oxa-5,8,11,14,17-pentaazatricosan-1-oic acid (L4)

[0501]

[0502] The crude product obtained from the previous step (0.77 mmol, 1.0 equiv) was dis¬ solved in DMF (8.8 mL). DIPEA (0.88 mL, 5.0 mmol, 6.5 equiv) was added, followed

[0503] P9641PC00by MC-OSu (356 mg, 1.16 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 10 min. Complete conversion was observed by LC-MS. The reaction was quenched by addition of AcOH (0.88 mL). The crude residue was chromato¬ graphed to afford the desired product as a white solid (380 mg, 0.62 mmol, 80% yield). MS (ESI) m / z [M+H]+calcd for C28H37N6O10617.26; found 617.07.

[0504] Synthesis of the Di-Conjugated Bis-MAEC Disorazole Z1-MC-GGFG-Glycolic Linker (6)

[0505] DIPEA, COMU, DMF, rt, 20%

[0506]

[0507] L4 (46 mg, 0.075 mmol, 2.0 equiv) was dissolved in DMF (0.5 mL). DIPEA (26 pL, 0.149 mmol, 4.0 equiv) was added, followed by COMU (32 mg, 0.075 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 30 min to form the activated acid. The resulting solution was added directly to S2 (36.6 mg, 0.0375 mmol, 1.0 equiv). Complete conversion was observed by LC-MS . The reaction was quenched with acetic acid and purified to afford the desired product 17 mg, 0.0079 mmol, 21 % yield. HRMS (ES ) m / z calcd for C106H135N18O31[M+H]+2171.941 ; found 2171.982

[0508] P9641PC00Synthesis of the Mono-Conjugated Mono-MAEC Disorazole Z1-MC-GGFG- Glycolic Linker (7)

[0509]

[0510] L4 (16 mg, 0.028 mmol, 1.2 equiv) was dissolved in DMF (0.5 mL). DIPEA (7 pL, 0.041 mmol, 2.0 equiv) was added, followed by COMU (9 mg, 0.023 mmol, 1.0 equiv). The reaction mixture was stirred at room temperature for 30 min to form the activated acid. The resulting solution was added directly to S4 (20 mg, 0.023 mmol, 1.0 equiv). Reaction progress was monitored by LC-MS. The reaction was quenched with acetic acid and purified to afford the desired product. MS (ESI) m / z = 1445. 61 [M + H]+.

[0511] P9641PC00MC-GGFG-PAB-PNP Synthesis (L5)

[0512] 4-aminobenzyl alcohol, HATU, DPEA DMF, 48 h, 65%

[0513] L5-1 L3-4 1. TEA, DMF 2. MC-OSu, 4-Nitrophenylchlorformat DMF, 48h, 80% dry Pyridine, 0oCto RT , 2 h.

[0514]

[0515] (S)-(9H-fluoren-9-yl)methyl (2-((2-((2-((2-((4-(hydroxymethyl)phenyl)amino)-2- oxoethyl)amino)-2 -oxo-1 -phenylethyl)amino)-2-oxoethyl)amino)-2-ox- oethyl)carbamate (L5-1)

[0516]

[0517] A solution of compound L3-3 (1.00 g, 2.52 mmol, 1.0 equiv) and 4-aminobenzyl al¬ cohol (929 mg, 7.55 mmol, 3.0 equiv) in DMF (25 mL) was treated with DIPEA (0.44 mL, 2.52 mmol, 1.0 equiv) and stirred at room temperature for 15 min. HATU (1.00 g, 2.77 mmol, 1.1 equiv) was then added, and the reaction mixture was stirred at room temperature for 48 h in the dark. After completion of the reaction, DMF was re¬ moved under reduced pressure and the residue was purified by flash column chro¬ matography to afford the product (1.06 g, 1.64 mmol, 65% yield). HRMS (ESI): m / z calcd for CayHayNsOyNa [M+Na]+; found 686.259.

[0518] P9641PC00(S)-6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y l)-N -(2 -((2 -(( 1 -((2-((4-(hydroxyme-thyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-ox- oethyl)amino)-2-oxoethyl)hexanamide (L5-2)

[0519]

[0520] A solution of compound L5-1 (200 mg, 0.333 mmol, 1.0 equiv) in DMF (1.7 mL, 0.2 M) was treated with triethylamine (0.93 mL, 6.59 mmol, 19.8 equiv), and the reaction mixture was stirred at room temperature for 24 h. The solvent and excess triethyla¬ mine were removed under reduced pressure. The resulting residue was dissolved in DMF (3.3 mL, 0.1 M), and Mc-OSu (111 mg, 0.366 mmol, 1.1 equiv) was added. The reaction mixture was stirred at room temperature for 20 h. After completion of the reaction, DMF was removed under reduced pressure, and the residue was purified by flash column chromatography to afford the desired product (137 mg, 0.216 mmol, 65% yield). HRMS (ESI): m / z calcd for C32H39N6O8[M+H]+635.282; found 635.286

[0521] (S)-4-(5-benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-4,7, 10,13-tetraoxo- 3,6,9, 12-tetraazaoctadecanamido)benzyl (4-nitrophenyl) carbonate (L5)

[0522]

[0523] L5-2 (50 mg, 0.079 mmol, 1.0 equiv) was dissolved in anhydrous DMF (5 mL). Bis(p-nitrophenyl) carbonate (50 mg, 0.158 mmol, 2.0 equiv) and N,N-diisopropylethyla-mine (DIPEA) (21 pL, 0.119 mmol, 1.5 equiv) were added, and the reaction mixture was stirred at room temperature for 1 h. Reaction progress was monitored by LC-MS. After completion of the reaction, the solvent was removed under reduced pressure. The residue was dispersed in diethyl ether, and the precipitate was collected by filtra¬ tion to afford a crude product. The crude material was further purified by manual col¬ umn chromatography to yield the product as a white solid. HRMS (ESI): m / z calcd for C39H42N7O12[M+H]+800.286 ; found 800.288

[0524] P9641PC00Synthesis of the Di-Conjugated Bis-MAEC Disorazole Z1-MC-GGFG-PAB Linker (8)

[0525] o

[0526]

[0527] To a solution of L5 (73 mg, 0.092 mmol, 3.0 equiv) in anhydrous DMF (2 mL), HOBt (8.4 mg, 0.0616 mmol, 2.0 equiv) was added at room temperature under a nitrogen atmosphere. Subsequently, S2 (30 mg, 0.0308 mmol, 1.0 equiv) and DIPEA (21 pL, 0.123 mmol, 4.0 equiv) were added. The reaction mixture was stirred at room tem¬ perature and the progress was monitored by LC-MS. After completion, the solvent was removed under reduced pressure to yield the crude product. MS (ESI) m / z = 2281.96 [M+H]+.

[0528] P9641PC00Synthesis of the Mono-Conjugated Mono-MAEC Disorazole Z1-MC-GGFG-PAB Linker (9)

[0529]

[0530] 9

[0531] To a solution of L5 (27 mg, 0.035 mmol, 1.5 equiv) in anhydrous DMF (2 mL), HOBt (3.2 mg, 0.0232 mmol, 1.0 equiv) was added at room temperature under a nitrogen atmosphere. Subsequently, S4 (20 mg, 0.0232 mmol, 1.0 equiv) and DIPEA (8.10 pL, 0.047 mmol, 2.0 equiv) were added. The reaction mixture was stirred at room tem¬ perature and the progress was monitored by LC-MS. After completion, the solvent was removed under reduced pressure to yield the crude product. MS (ESI) m / z = 1507.64 [M+H]+.

[0532] P9641PC00

Claims

Claims1. Compound according to formula I,Iwherein Ri is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-alkyl, cyclo-alkenyl, aryl, hetero-aryl;R2is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-al- kyl, cyclo-alkenyl, aryl, hetero-aryl;R3 is independently selected from H, alkyl, iso-alkyl, alkenyl, alkinyl, cyclo-al- kyl, cyclo-alkenyl, aryl, hetero-aryl;R4 is independently selected from the group comprising H, carbonyl-N-imidaz- olyl, carbonyl-N,N-dimethylaminoethan, silyl ether linkers, dipeptide linkers and tetrapeptide linkers,X is independently selected from O, S and N,and the E / Z-isomers, diastereomers and enantiomers of the compound ac¬ cording to formula I,under the provision that the compound according to formula I is not disorazole Z1.

2. The compound according to claim 1 wherein R4 is independently selected from silyl ether linkers, dipeptide linkers and tetrapeptide linkers of the group com¬ prising 4-(((2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)diiso- propylsilyl)oxy)benzyl hydrogen carbonate (L1), 4-((R)-2-((R)-2-(6-(2,5-dioxo- 2, 5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3-methylbutanamido)-5-ureidopentan- amido)benzyl hydrogen carbonate— methane (1 / 1) (L2), (6-(2,5-dioxo-2,5-P9641PC00dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-D-phenylalanylglycine (L3), (S)- 10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -y I )-6 , 9 , 12,15,18-pentaoxo-3- oxa-5, 8,11,14, 17-pentaazatricosanoic acid (L4), (R)-4-(5-benzyl-18-(2,5-dioxo- 2,5-dihydro-1 H-pyrrol-1 -y I )-4 , 7 , 10, 13-tetraoxo-3,6,9, 12-tetraazaoctade- canamido)benzyl hydrogen carbonate (L5).

3. Compound according to claim 1 or 2, wherein Ri, R2and R3are methyl, and X is O.

4. Compound according to any of the preceding claims that is:P9641PC00P9641PC00P9641PC00P9641PC005. Method for the preparation of a compound according to any of claims 1 to 4, P9641PC00wherein in a first part a)(+)-Disorazole Z1(+)-disorazole Z1 is prepared by reacting a compound of formula SA-8SA-8with a compound of formula 20to yield a compound of formula SA-9P9641PC00that is reacted to a compound of formula SA-10SA- 10that is reacted to (+)-disorazole Z1 ,and wherein in a part b1) after part a) (+)-disorazole Z1 is reacted to com¬ pound S1that is converted to yield compound S2P9641PC00S2or wherein in a part b2) after part a) (+)-Disorazole Z1 is reacted to compound S3that is converted to yield compound S4and wherein in a third part c) of the method after part b1 ) or after part b2) com¬ pound S2 or compound S4 are reacted with silyl ether linkers, dipeptide linkers and tetrapeptide linkers of the group comprisingP9641PC004-(((2-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)ethoxy)ethoxy)diisopropylsi- lyl)oxy)benzyl hydrogen carbonate (L1 ),4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido)-3-methyl- butanamido)-5-ureidopentanamido)benzyl hydrogen carbonate-methane (1 / 1) (L2),(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl)glycylglycyl-D-phenylalanyl- glycine (L3), (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)- 6,9,12,15,18-pentaoxo-3-oxa-5,8,11 ,14,17-pentaazatricosanoic acid (L4), (R)-4-(5-benzyl-18-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-4,7, 10,13-tetraoxo- 3,6,9, 12-tetraazaoctadecanamido)benzyl hydrogen carbonate (L5), to yield a compound according to claim 4 that is compound 1 , 2, 3, 4, 5, 6, 7, 8, or 9.

6. Use of a compound according to any of claims 1 to 4 in the preparation of pay¬ loads for Antibody-Drug-Conjugates (ADCs).

7. Use of a compound according to any of claims 1 to 4 as payloads for Anti- body-Drug-Conjugates (ADCs).P9641PC00

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