Conjugates comprising cleavable linkers

Abstract

The specification relates to conjugates comprising one or more tubulin inhibiting moieties DM and pharmaceutically acceptable salts thereof. The specification also relates the use of the conjugates for the treatment of diseases such as cancer, and intermediates useful for the synthesis of the conjugates.

Description

[0001] MTI-106-PCT01-NP

[0002] CONJUGATES COMPRISING CLEAVABLE LINKERS

[0003] Cross-Reference to a Related Patent Application

[0004] This specification claims the benefit of priority to United States Provisional Application US 63 / 752,068 (filed 31 January 2025). The entire text of the above-referenced patent application is incorporated by reference into this specification.

[0005] Field

[0006] This specification relates to certain conjugates comprising microtubule inhibitor (MTI) payloads, and to pharmaceutical compositions containing them. This specification also relates to the use of the conjugates in methods of treating diseases such as cancer. This specification further relates to processes and intermediate compounds involved in the preparation of the conjugates.

[0007] Background

[0008] Microtubules are dynamic filaments of tubulin important for key survival steps in mammalian dividing cells. Targeting microtubule with a microtubule inhibitor (MTI) has been used widely in the treatment of several solid cancer and haematological malignancies, however MTI therapy is often associated with non-favourable side effects (Dumontet etal.; Nature Reviews, 2010, Volume 9, Pages 790-803). In an attempt to address this, MTIs have been converted into Antibody-Drug Conjugates (ADC). A number of approved ADCs have an MTI mode of action (Yaghoubi et al.; Journal of Cellular Physiology, 2020, Volume 235, Pages 31-64). Several binding sites can be targeted with ligands of interest, including colchicine, maytansine and vinca binding sites (Chen et al.; Molecules, 2017, Volume 2, Issue 1281).

[0009] There remains a need for conjugates comprising an MTI payload. The conjugates of the disclosure may be used for the treatment of diseases such as cancer.

[0010] General Description

[0011] In a first aspect there is provided a conjugate of Formula (I)

[0012] Ab – (GA–JA–DM)k(I)

[0013] or a pharmaceutically acceptable salt thereof, wherein

[0014] Ab is an antibody or antigen-binding fragment thereof,

[0015] k is an integer from 1 to 10,

[0016] each GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof,MTI-106-PCT01-NP

[0017] each DMis independently a group of Formula (ID)

[0018] (ID)

[0019]

[0020] wherein

[0021] either (i) R1and R2are independently selected from Ci-g alkyl, C3-6 cycloalkyl and H, or (ii) R1and R2, together with the carbon atom to which they are attached, form a cyclopropane or cyclobutane ring, R3and R10are independently C1-3 alkyl,

[0022] R4and R5are independently C1-6alkyl or C3-6cycloalkyl,

[0023] R6, R7, R8and R9are independently H, C1-6alkyl or C3-6cycloalkyl,

[0024] R11is H, C1-6 alkyl, C3-6 cycloalkyl or phenyl, and

[0025] RX1is C1-6 alkyl or C3-6 cycloalkyl, Rx2is H or F, and Rx3is H or F,

[0026] each JAis independently either

[0027] (i) a group of Formula (IA)

[0028]

[0029] wherein

[0030] E is (CH2)n1, wherein n1 is 0, 1, 2 or 3,

[0031] R12is C1-4 alkyl or H,

[0032] OH HQ' J^ JDH

[0033] O^XO'Z'^R14A

[0034]

[0035] R13Ais, wherein R14Ais CO2H or CH2OH,

[0036] X1is (CH2)n2, wherein n2 is 0, 1, 2 or 3,

[0037] Y is O or NRB, wherein RBis H, C1-4 alkyl or C3-4 cycloalkyl,

[0038] Z1is (CH2)n3, wherein n3 is 0, 1, 2, 3, 4 or 5,

[0039] m is an integer from 2 to 17,

[0040] p is 1 or 0, and

[0041] q is 1 or 0, or

[0042] (ii) a group of Formula (IB)MTI-106-PCT01-NP

[0043]

[0044] wherein

[0045] Z2is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5,

[0046] X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2, 3, 4 or 5,

[0047] j is an integer from 0 to 16,

[0048] r is 1 or 0, and

[0049]

[0050] R13Bis, wherein R14Bis CO2H or CH2OH,

[0051] and wherein

[0052] (GA) indicates the point of attachment to GA, and

[0053] (DM) indicates the point of attachment to DM.

[0054] In a further aspect there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0055] In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0056] In a further aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0057] In a further aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament.

[0058] In a further aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer.

[0059] In a further aspect there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.MTI-106-PCT01-NP

[0060] In a further aspect there is provided a compound of Formula (II)

[0061] GB–JB–DM(II)

[0062] or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,

[0063] each DMis independently a group of Formula (ID)

[0064] (ID)

[0065]

[0066] wherein

[0067] either (i) R1and R2are independently selected from Ci-g alkyl, C3-6 cycloalkyl and H, or (ii) R1and R2, together with the carbon atom to which they are attached, form a cyclopropane or cyclobutane ring, R3and R10are independently C1-3 alkyl,

[0068] R4and R5are independently C1-6alkyl or C3-6cycloalkyl,

[0069] R6, R7, R8and R9are independently H, C1-6alkyl or C3-6cycloalkyl,

[0070] R11is H, C1-6 alkyl, C3-6 cycloalkyl or phenyl, and

[0071] RX1is C1-6 alkyl or C3-6 cycloalkyl, Rx2is H or F, and Rx3is H or F,

[0072] each JBis independently either

[0073] (i) a group of Formula (IIA)

[0074] O O O R13A

[0075]

[0076] wherein

[0077] E is (CH2)ni, wherein nl is 0, 1, 2 or 3,

[0078] R12is C1-4 alkyl or H,

[0079]

[0080] 14Ais CO2H or CH2OH,

[0081] X1is (CH2)n2, wherein n2 is 0, 1, 2 or 3,

[0082] Y is O or NRB, wherein RBis H, C1-4 alkyl or C3-4 cycloalkyl,MTI-106-PCT01-NP

[0083] Z1is (CH2)n3, wherein n3 is 0, 1, 2, 3, 4 or 5,

[0084] m is an integer from 2 to 17,

[0085] p is 1 or 0, and

[0086] q is 1 or 0, or

[0087] (ii) a group of Formula (I I B)

[0088]

[0089] wherein

[0090] Z2is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5,

[0091] X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2, 3, 4 or 5,

[0092] j is an integer from 0 to 16,

[0093] r is 1 or 0, and

[0094] OH

[0095]

[0096] R13Bis, wherein R14Bis CO2H or CH2OH,

[0097] and wherein (GB) indicates the point of attachment to GB, and (DM) indicates the point of attachment to DM.

[0098] In a further aspect there is provided a method of making a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, using a compound of Formula (II), or a salt thereof.

[0099] In a further aspect there is provided a compound of Formula (III)

[0100]

[0101] or a salt thereof, wherein

[0102] either (i) R1and R2are independently selected from C1-6 alkyl, C3-6 cycloalkyl and H, or (ii) R1and R2, together with the carbon atom to which they are attached, form a cyclopropane or cyclobutane ring, R3and R10are independently C1-3 alkyl,MTI-106-PCT01-NP

[0103] R4and R5are independently C1-6alkyl or C3-6cycloalkyl,

[0104] R6, R7, R8and R9are independently H, C1-6alkyl or C3-6cycloalkyl,

[0105] R11is H, C1-6 alkyl, C3-6 cycloalkyl or phenyl, and

[0106] RX1is C1-6 alkyl or C3-6 cycloalkyl, Rx2is H or F, and Rx3is H or F.

[0107] In a further aspect there is provided a method of making a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a salt thereof, using a compound of Formula (III), or a salt thereof.

[0108] A conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may undergo enzymatic cleavage to release the compound of Formula (III). The compound of Formula (III) provides an anticancer effect by, as a minimum, acting as a microtubule inhibitor. The compound of Formula (III) may also exhibit advantageous physical properties (for example, lower lipophilicity, higher aqueous solubility, higher permeability and / or lower plasma protein binding), and / or favourable toxicity profiles (for example a decreased activity at hERG), and / or favourable metabolic or pharmacokinetic profiles, in comparison with other microtubule inhibitors. Further, Conjugates of Formula (I) may exhibit improved efficacy and / or advantageous physical properties (for example, higher colloidal stability, higher chemical stability, lower lipophilicity, higher aqueous solubility, higher permeability and / or lower plasma protein binding), and / or favourable toxicity profiles (for example reduced off target toxicity), and / or favourable metabolic or pharmacokinetic profiles, in comparison with other conjugates. As such, conjugates of Formula (I) and the compound of Formula (III) may be especially suitable as therapeutic agents, such as for the treatment of cancer. A compound of Formula (III), or a salt thereof, may also be useful in the synthesis of a compound of Formula (II), or a salt thereof. Definitions

[0109] So that the present specification may be more readily understood, certain terms are explicitly defined below. In addition, definitions are set forth as appropriate throughout the detailed description. Where examples are provided for a definition, they are not limiting.

[0110] The prefix Cx-y, where x and y are integers, indicates the numerical range of carbon atoms that are present in a group.

[0111] As used herein the term "alkyl" refers to a saturated, linear or branched hydrocarbon radical having the specified number of carbon atoms. Examples of C1.4 alkyl groups include methyl (Me), ethyl (Et), n-propyl (”Pr), i-propyl ('Pr), n-butyl (”Bu), i-butyl ('Bu), s-butyl (sBu), and t-butyl (tBu). Examples of C1-6alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl and n-hexyl.MTI-106-PCT01-NP

[0112] As used herein the term "cycloalkyl" refers to a saturated, cyclic hydrocarbon radical having the specified number of carbon atoms. Examples of C3-6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0113] As used herein the term "conjugation group for conjugation to an antibody, or antigen-binding fragment thereof" refers to an atom or group of atoms capable of forming at least one covalent bond to an antibody, or antigen-binding fragment thereof, through a chemical reaction.

[0114] The use of " " in formulas of this specification indicates the point of attachment to the antibody

[0115] or antigen-binding fragment thereof. By way of illustration

[0116]

[0117] indicates that that there is a covalent bond connecting the antibody, or antigen-binding fragment thereof, to the carbon atom marked 1.

[0118] For the avoidance of doubt, the use of " — I— " in formulas of this specification denotes the point of covalent attachment to a group, where the group is other than the antibody or antigen-binding fragment thereof.

[0119] Units, prefixes, and symbols are denoted in their International System of Units (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.

[0120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0121] Description of Figures

[0122] Embodiments and experiments illustrating the principles of the disclosure will now be discussed with reference to the accompanying figures in which:

[0123] Figure 1 shows cytotoxicity data for ADC-2, ADC-3, ADC-4, ADC-5 and ADC-6 in a Her2+++ NCI-N87 cell line.MTI-106-PCT01-NP

[0124] Figure 2 shows cytotoxicity data for ADC-2, ADC-3, ADC-4, ADC-5 and ADC-6 in a Her2- MDAMB468 cell line.

[0125] Figure 3 shows cytotoxicity data for ADC-2, ADC-3, ADC-4, ADC-5 and ADC-6 in a 3D Her2+++ SKOV3 cell line.

[0126] Figure 4 shows cytotoxicity data for ADC-7 (Control), ADC-8 (Control) and ADC-9 (Control) in a Her2+++ NCI-N87 cell line.

[0127] Detailed Description

[0128] In one aspect, this specification provides a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as defined above.

[0129] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In further embodiments k is an integer from 2 to 10. In further embodiments k is an integer from 2 to 8. In further embodiments k is 4. In further embodiments k is 8.

[0130] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis independently selected from

[0131]

[0132] wherein RKis H or CH3, RLis C1-6alkyl,

[0133]

[0134] and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.MTI-106-PCT01-NP

[0135] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis independently selected from

[0136]

[0137] and

[0138] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein GAis

[0139] O

[0140]

[0141] In embodiments there is provided a conjugate of Formula, (I), or a pharmaceutically acceptable salt thereof, wherein each JAis independently a group of Formula ( I A)

[0142]

[0143] In embodiments there is provided a conjugate of Formula, (I), or a pharmaceutically acceptable salt thereof, wherein E is (CH2)n1, wherein n1 is 0, 1, 2 or 3. In further embodiments E is a covalent bond. In further embodiments E is CH2. In further embodiments E is (CH2)2. In further embodiments E is (CH2)3.

[0144] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. In further embodiments m is an integer from 6 to 16. In further embodiments m is an integer from 7 to 15. In furtherMTI-106-PCT01-NP

[0145] embodiments m is an integer from 8 to 14. In further embodiments m is an integer from 9 to 13. In further embodiments m is an integer from 10 to 12. In further embodiments m is 11.

[0146] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein p is 1.

[0147] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein q is 1.

[0148] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein R12is C1-4 alkyl or H. In further embodiments R12is C1-4 alkyl. In further embodiments R12is CH3.

[0149] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0150] thereof, wherein R 14A

[0151]

[0152] is CO2H or CH2OH. In further embodiments, R14Ais CO2H.

[0153] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt OH

[0154] thereof, wherein

[0155]

[0156] R13Ais wherein R14Ais CO2H or CH2OH. In further embodiments, R14Ais CO2H.

[0157] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0158] thereof, wherein each

[0159]

[0160] R13Ais

[0161] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt OH

[0162] thereof, wherein

[0163]

[0164] R13AisMTI-106-PCT01-NP

[0165] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein X1is (CH2)n2, wherein n2 is 0, 1, 2 or 3. In further embodiments X1is a covalent bond. In further embodiments X1is CH2. In further embodiments X1is (CH2)2. In further embodiments X1is (CH2)3.

[0166] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is O.

[0167] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Y is NRB, wherein RBis H, Ci.4alkyl or C3-4 cycloalkyl. In further embodiments, Y is NH.

[0168] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Z1is (CH2)n3, wherein n3 is 1, 2, 3, 4 or 5. In further embodiments Z1is CH2. In further embodiments Z1is (CH2)2. In further embodiments Z1is (CH2)3. In further embodiments Z1is (CH2)4. In further embodiments Z1is (CH2)5.

[0169] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula ( I Al)

[0170] (IA1)

[0171]

[0172] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IA2)MTI-106-PCT01-NP

[0173] OH

[0174]

[0175] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA-JA-DM) is

[0176]

[0177] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA-JA-DM) is

[0178]

[0179] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA-JA-DM) isMTI-106-PCT01-NP

[0180] OH

[0181]

[0182] In embodiments there is provided a conjugate of Formula, (I), or a pharmaceutically acceptable salt thereof, wherein each JAis independently a group of Formula (IB)

[0183]

[0184] wherein

[0185] Z2is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5,

[0186] X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2, 3, 4 or 5,

[0187] j is an integer from 0 to 16,

[0188] r is 1 or 0, and

[0189] OH

[0190]

[0191] R13Bis, wherein R14Bis CO2H or CH2OH.

[0192] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein j is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In further embodiments j is an integer from 0 to 12. In further embodiments j is an integer from 0 to 8. In further embodiments j is 0. In further embodiments j is 8.MTI-106-PCT01-NP

[0193] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0194] thereof, wherein R

[0195]

[0196] is CO2H or CH2OH. In further embodiments, R14Bis CO2H.

[0197] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0198] thereof, wherein R

[0199]

[0200] is CO2H or CH2OH. In further embodiments, R14Bis CO2H.

[0201] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt

[0202] thereof, wherein each

[0203]

[0204] R13Bis

[0205] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt OH

[0206] thereof, wherein

[0207]

[0208] R13Bis

[0209] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein r is 1.

[0210] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2, 3, 4 or 5. In further embodiments, X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2 or 3. In embodiments, X2and X3are both CH2.

[0211] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein Z2is (CH2)n4, wherein n4 is 1, 2, 3, 4 or 5. In further embodiments Z2is CH2. In further embodiments Z2is (CH2)2. In further embodiments Z2is (CH2)3. In further embodiments Z2is (CH2)4. In further embodiments Z2is (CH2)5.MTI-106-PCT01-NP

[0212] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB1)

[0213] (IB1)

[0214]

[0215] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each JAis a group of Formula (IB2)

[0216] O

[0217] (IB2)

[0218]

[0219] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA-JA-DM) is

[0220]

[0221] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein each (GA-JA-DM) isMTI-106-PCT01-NP

[0222] OH

[0223]

[0224] In a further aspect there is provided a compound of Formula (II)

[0225] GB_JB_DM (II)

[0226] or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,

[0227] DMis as defined in any embodiment of a conjugate of Formula (I) disclosed herein,

[0228] each JBis independently either

[0229] (i) a group of Formula (IIA)

[0230]

[0231] wherein E, R12, R13A, X1, Y, Z1, m, p and q are as defined in any embodiment of a conjugate of Formula (I) disclosed herein, or

[0232] (ii) a group of Formula (I I B)

[0233]

[0234] wherein j, r, R13B, X2, X3, and Z2are as defined in any embodiment of a conjugate of Formula (I) disclosed herein,MTI-106-PCT01-NP

[0235] and wherein (GB) indicates the point of attachment to GB, and (DM) indicates the point of attachment to DM.

[0236] In embodiments there is provided a compound of Formula, (II), or a salt thereof, wherein GBis selected from

[0237]

[0238] wherein X1is CH or N, h is 0 or 1, Hal is Cl, Br or I, RKis H or CH3, and RLis C1-6alkyl.

[0239] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis selected from

[0240] O

[0241]

[0242] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein GBis

[0243] O

[0244]

[0245] MTI-106-PCT01-NP

[0246] In embodiments there is provided a compound of Formula (ll)7or a salt thereof, wherein JBis a group of Formula (IIA1)

[0247] (IIA1)

[0248]

[0249] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein JBis a group of Formula (IIA2)

[0250] OH

[0251] (IIA2)

[0252]

[0253] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein JBis a group of Formula (IIB1)

[0254] (IIB1)

[0255]

[0256] In embodiments there is provided a compound of Formula (II), or a salt thereof, wherein JBis a group of Formula (IIB2)MTI-106-PCT01-NP

[0257] o o

[0258] N

[0259] (GB) H (IIB2)

[0260]

[0261] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-1-(((S)-1-(((S,E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0262] OH

[0263]

[0264] salt thereof.

[0265] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanamido) methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) amino)-2-fluoro-3-methylphenyl) propan-2-yl) amino) propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acidMTI-106-PCT01-NP

[0266] OH

[0267]

[0268] or a salt thereof.

[0269] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-1-(((S)-1-(((S,E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)propan-2-yl)-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0270] OH

[0271]

[0272] salt thereof.

[0273] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-1-(((S)-1-(((S,E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-(33-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,31-dioxo-6,9,12,15,18,21,24,27-octaoxa-2,30-diazatritriacontyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidMTI-106-PCT01-NP

[0274] OH

[0275]

[0276] or a salt thereof.

[0277] In embodiments there is provided a compound of Formula (II), or a salt thereof, that is (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid

[0278] OH

[0279]

[0280] or a salt thereof. In a further aspect there is provided a method of making a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, using a compound of Formula (II), or a salt thereof, as disclosed in any of the embodiments herein.

[0281] In a further aspect there is provided a compound of Formula (III)

[0282] Rx1

[0283]

[0284] MTI-106-PCT01-NP

[0285] or a salt thereof, wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, Rxl, Rx2and Rx3are as defined in any embodiment of a conjugate of Formula (I) disclosed herein. In further embodiments, the salt thereof is a pharmaceutically acceptable salt thereof. In further embodiments, there is provided a compound of Formula (III).

[0286] In embodiments, the compound of Formula (III), or a salt thereof, is a compound of Formula (IIIA)

[0287] (IIIA)

[0288]

[0289] ,or a salt thereof. In further embodiments, the salt thereof is a pharmaceutically acceptable salt thereof. In further embodiments, there is provided a compound of Formula (IIIA).

[0290] In embodiments, the compound of Formula (III), or a salt thereof, is a compound of Formula (IIIB) Rx1

[0291] (IIIB)

[0292]

[0293] ,or a salt thereof. In further embodiments, the salt thereof is a pharmaceutically acceptable salt thereof. In further embodiments, there is provided a compound of Formula (IIIB).

[0294] In embodiments, the compound of Formula (III), or a salt thereof, is

[0295] (S, E)-4-((S)-2-((R)-2-((2-(3-amino-5-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,

[0296] (S, E)-4-((S)-2-((R)-2-((2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)arnino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,

[0297] (S, E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((R)-2-((2-(3-methyl-5-(methylamino)phenyl)propan-2-yl)amino)propanamido)butanamido)hex-2-enoic acid,

[0298] (S, E)-4-((S)-2-((R)-2-((2-(3-amino-5-ethylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,

[0299] (S, E)-4-((S)-2-((R)-2-((2-(5-amino-3-ethyl-2-fluorophenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,

[0300] (S, E)-4-((S)-2-((R)-2-((2-(2-fluoro-3-methyl-5-(methylamino)phenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid, orMTI-106-PCT01-NP

[0301] (S, E)-4-((S)-2-((R)-2-((2-(3-amino-4-fluoro-5-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,

[0302] or a salt thereof.

[0303] In further embodiments, the salt thereof is a pharmaceutically acceptable salt thereof.

[0304] In embodiments, the compound of Formula (III), or a salt thereof, is (S, E)-4-((S)-2-((R)-2-((2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-

[0305] F. HN. J Si N dimethy1hex-2-enoic acid O

[0306]

[0307] ,or a salt thereof. In further embodiments, the salt thereof is a pharmaceutically acceptable salt thereof.

[0308] In embodiments, there is provided (S, E)-4-((S)-2-((R)-2-((2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-

[0309] F. HN. J Si N

[0310] eno c ac d

[0311]

[0312] O

[0313] i i

[0314] In a further aspect there is provided a method of making a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, or a compound of Formula (II), or a salt thereof, using a compound of Formula (III), or a salt thereof, as disclosed in any of the embodiments herein.

[0315] The present specification is intended to include all isotopes of atoms occurring in the present compounds and conjugates. Isotopes will be understood to include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include13C and14C. Isotopes of nitrogen include15N.

[0316] The compounds disclosed herein may contain one or more chiral centers. Accordingly, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e. as individual enantiomers, diastereoisomers, or as a stereoisomerically enriched mixture. All such stereoisomer (and enriched) mixtures are included within the scope of the embodiments, unless otherwise stated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of suchMTI-106-PCT01-NP

[0317] compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0318] Unless stereochemistry is explicitly indicated in a chemical structure or chemical name, the chemical structure or chemical name is intended to embrace all possible stereoisomers, diastereoisomers, conformers, rotamers and tautomers of the compound depicted. For example, a compound containing a chiral carbon atom is intended to embrace both the (R) enantiomer and the (S) enantiomer, as well as mixtures of the enantiomers, including racemic mixtures; and a compound containing two chiral carbons is intended to embrace all enantiomers and diastereoisomers including (R, R), (S, S), (R, S) and (S, R).

[0319] The compound of Formula (II), and salts thereof, may be prepared, used or supplied in amorphous form, crystalline form, or semicrystalline form and any given compound of Formula (II), or salt thereof, may be capable of being formed into more than one crystalline / polymorphic form, including hydrated and / or solvated forms. It is to be understood that the present specification encompasses any and all such solid forms of the compound of Formula (II), and salts thereof.

[0320] The compound of Formula (III), and salts thereof (e.g. pharmaceutically acceptable salts thereof), may be prepared, used or supplied in amorphous form, crystalline form, or semicrystalline form and any given compound of Formula (III), and salts thereof (e.g. pharmaceutically acceptable salts thereof) may be capable of being formed into more than one crystalline / polymorphic form, including hydrated and / or solvated forms. It is to be understood that the present specification encompasses any and all such solid forms of the compound of Formula (III), and salts thereof (e.g. pharmaceutically acceptable salts thereof).

[0321] The term "pharmaceutically acceptable salt" is used to specify that a salt is suitable for use in patients. An example list of pharmaceutically acceptable salts and preparation methods can be found in the Handbook of Pharmaceutical Salts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth, editors, Weinheim / Zurich: Wiley-VCH / VHCA, 2002.

[0322] A suitable pharmaceutically acceptable salt of a conjugate of Formula (I) or compound of Formula (III) is, for example, an acid addition salt. In general, an acid addition salt can be prepared using various inorganic or organic acids. Such salts can typically be formed by, for example, mixing the compound with an acid (e.g., a stoichiometric amount of acid) using various methods known in the art.

[0323] A further suitable pharmaceutically acceptable salt of a conjugate of Formula (I) is, for example, a salt formed within a patient's body after administration of a conjugate of Formula (I) to the patient.MTI-106-PCT01-NP

[0324] A further suitable pharmaceutically acceptable salt of a compound of Formula (III) is, for example, a salt formed within a patient's body after administration of a compound of Formula (III) to the patient.

[0325] The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient, and which contains no additional components which are unacceptably toxic to a patient to which the composition would be administered. Such compositions can be sterile. A pharmaceutical composition according to the present specification will comprise a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0326] In embodiments there is provided a pharmaceutical composition comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable, non-toxic, sterile carrier. In further embodiments the carrier is a physiological saline, non-toxic buffer, or preservative. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012), the contents of which are incorporated by reference.

[0327] Pharmaceutical compositions comprising a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, may be comprised within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a nasal aerosol, or a combination thereof.

[0328] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0329] In one aspect there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0330] Where "cancer" is mentioned, this includes both non-metastatic cancer and also metastatic cancer, such that treating cancer involves treatment of both primary tumours and also tumour metastases. In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, for use in the treatment of HER2 positive cancer.

[0331] In one aspect there is provided the use of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.MTI-106-PCT01-NP

[0332] In one aspect there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof.

[0333] As used herein, the term "treating" or "treatment" refers to alleviating the specified condition, eliminating or reducing one or more symptoms of the condition, slowing or eliminating the progression of the condition, and delaying the reoccurrence of the condition in a previously afflicted or diagnosed patient or subject.

[0334] As used herein, the term "effective amount" means that amount of a conjugate of Formula (I) that will elicit the biological or medical response of a tissue, system, animal or human this is being sought, for instance, by a researcher or clinician.

[0335] The term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope the amounts effective to enhance normal physiological function.

[0336] The term "patient" refers to any animal (e.g., a mammal), including, but not limited to humans, nonhuman primates, rodents, and the like, which is to be the recipient of a particular treatment. In embodiments the term "patient" refers to a human subject.

[0337] In embodiments there is provided a method of treating cancer in a patient in need thereof, comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the cancer is a HER2 positive cancer.

[0338] In embodiments there is provided a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and an additional anti-tumour substance for the conjoint treatment of cancer.

[0339] In embodiments there is provided a combination for use in the treatment of cancer comprising a conjugate of the Formula (I), or a pharmaceutically acceptable salt thereof and an additional antitumour agent.

[0340] In embodiments there is provided a conjugate of the Formula (I), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent.

[0341] Herein, where the term "conjoint treatment" is used in reference to a combination treatment, it is to be understood that this may refer to simultaneous, separate or sequential administration. In one aspect, "conjoint treatment" refers to simultaneous administration. In another aspect, "conjointMTI-106-PCT01-NP

[0342] treatment" refers to separate administration. In a further aspect, "conjoint treatment" refers to sequential administration.

[0343] In embodiments there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, and the additional antitumour substance are jointly effective in producing an anti-cancer effect.

[0344] In one aspect there is provided a compound of Formula (III), or a pharmaceutically acceptable salt thereof, for use in therapy.

[0345] In one aspect there is provided a compound of Formula (III), or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

[0346] In one aspect there is provided the use of a compound of Formula (III), or a pharmaceutically acceptable salt thereof, as described herein, in the manufacture of a medicament, such as a medicament for the treatment of cancer.

[0347] In one aspect there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a compound of Formula (III), or a pharmaceutically acceptable salt thereof.

[0348] In embodiments there is provided a combination for use in the treatment of cancer comprising a compound of Formula (III), or a pharmaceutically acceptable salt thereof and an additional antitumour agent.

[0349] In embodiments there is provided a compound of Formula (III), or a pharmaceutically acceptable salt thereof, in combination with an additional anti-tumour agent.

[0350] In embodiments there is provided a method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a compound of Formula (III), or a pharmaceutically acceptable salt thereof, and simultaneously, separately or sequentially administering at least one additional anti-tumour substance to said patient, where the amounts of the compound of Formula (III) or a pharmaceutically acceptable salt thereof, and the additional antitumour substance are jointly effective in producing an anti-cancer effect.

[0351] Conjugation

[0352] T1MTI-106-PCT01-NP

[0353] Examples of GAand GBinclude, but are not limited to, the following, wherein X1is CH or N, h is 0 or 1, RKis H or CH3, Hal is Cl, Br or I, RLis C1-6alkyl, and indicates the point of attachment to the antibody, or antigen-binding fragment thereof.

[0354] GAGB

[0355] 0 0

[0356] AisA

[0357] ^ X) ^ X)

[0358] . NH2

[0359] 01

[0360] V N^ y

[0361] ^ X)

[0362] 0 0

[0363] AA

[0364] 0 0 rj 0

[0365] Z

[0366] 0 0

[0367] o V

[0368] ■A V 6

[0369] vA 0 A w ”

[0370] 0 0 /

[0371] tn

[0372] *

[0373] RK70

[0374] s 7

[0375] sx

[0376] (O2N)h-“-

[0377] 0 0

[0378] A

[0379] NxHal^NA

[0380] H H

[0381] 0 0

[0382] AA Hal^cA

[0383] 0 0

[0384] AV VA 0

[0385] 0

[0386]

[0387] MTI-106-PCT01-NP

[0388] OH

[0389] H '

[0390] *

[0391] *

[0392] ^z z ZNX- / "

[0393] ZZ- N

[0394] / v A

[0395] V= / $

[0396] ^^ z *—>

[0397] ==r "N

[0398] a:

[0399] C= / '=c

[0400] *

[0401] HN^J L

[0402] RL'

[0403] R i^hN

[0404] JQL

[0405] * z *

[0406] Hal^\

[0407] N3^

[0408] H \

[0409] A H2N.0A

[0410] * 0x

[0411] A J HsC^V

[0412] 2. / 'A

[0413] O O

[0414]

[0415] Antibody or antigen-binding fragment thereof

[0416] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen.MTI-106-PCT01-NP

[0417] In embodiments the antibody is isolated or recombinant. " Isolated", when used herein refers to a polypeptide, e.g., an antibody, that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated antibody will be prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody which is substantially free of other antibodies having different antigenic specificities.

[0418] In embodiments the antibody comprises at least two "light chains" (LC) and two "heavy chains" (HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as " VH") and a heavy chain constant region (abbreviated herein as " CH"). The heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain (abbreviated herein as " VL") and a light chain constant domain (abbreviated herein as " CL").

[0419] In embodiments the antibody is a full-length antibody. An "intact" or "full-length" antibody, as used herein, refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0420] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) (also known as hypervariable regions), interspersed with regions that are more conserved, termed framework regions (FRs). In embodiments each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region.

[0421] Binding between an antibody and its target antigen or epitope is mediated by the CDRs. The term "epitope" refers to a target protein region (e.g. polypeptide) capable of binding to (e.g. being bound by) an antibody or antigen-binding fragment of the disclosure. The CDRs are the main determinants of antigen specificity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes ( Al-lazikani et al. (1997) J. Molec. Biol.

[0422] 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.MTI-106-PCT01-NP

[0423] The "constant domains" (or "constant regions") of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0424] There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE and IgM, each with characteristic effector functions designated by isotype. Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and Fey Rl 11. Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.

[0425] In embodiments the antibody or antigen-binding fragment thereof is an IgG isotype. The antibody or antigen-binding fragment thereof can be any IgG subclass, for example IgGl, lgG2, lgG3, or lgG4 isotype. In embodiments the antibody or antigen-binding fragment thereof is based on an IgGl isotype.

[0426] The terms " Fc region", " Fc part" and " Fc" are used interchangeably herein and refer to the portion of a native immunoglobulin that is formed by two Fc chains. Each " Fc chain" comprises a constant domain CH2 and a constant domain CH3. Each Fc chain may also comprise a hinge region. A native Fc region is homodimeric. In embodiments the Fc region may be heterodimeric because it may contain modifications to enforce Fc heterodimerisation. The Fc region contains the carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor), and has no antigen binding activity. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been found in a number of Fc domain sites, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, resulting in minor variations between the sequences described in the instant application and sequences known in the art. As a result, every naturally occurring IgG Fc region is referred to as a "wild type IgG Fc domain" or " WT IgG Fc domain" (i.e., any allele). Human IgGl, lgG2, lgG3, and lgG4 heavy chain sequences can be obtained in a variety of sequence databases, including the UniProt databaseMTI-106-PCT01-NP

[0427] (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG4_HUMAN) respectively.

[0428] In embodiments the antibody of the disclosure is a monoclonal antibody. A "monoclonal antibody" (mAb) refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" can encompass both full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. In embodiments the antibody of the disclosure is an isolated monoclonal antibody. In further embodiments the antibody is a fully human monoclonal antibody.

[0429] In embodiments the antibody of the disclosure is a full-length antibody described above.

[0430] Alternatively, the antibody can be an antigen-binding fragment. The term "antigen-binding fragment" as used herein incudes any naturally-occurring or artificially-constructed configuration of an antigen-binding polypeptide comprising one, two or three light chain CDRs, and / or one, two or three heavy chain CDRs, wherein the polypeptide is capable of binding to the antigen.

[0431] In embodiments the antigen-binding fragment of the disclosure is a Fab fragment. The antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BITE), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-lg, an (scFv)2, a mAb2 or a DARPin.

[0432] The terms " Fab fragment" and " Fab" are used interchangeably herein and contain a single light chain (e.g. a constant domain CL and a VL) and a single heavy chain (e.g. a constant domain CHI and a VH). The heavy chain of a Fab fragment is not capable of forming a disulfide bond with another heavy chain.

[0433] A "Fab' fragment" contains a single light chain and a single heavy chain but in addition to the CH1 and the VH, a "Fab' fragment" contains the region of the heavy chain between the CH1 and CH2 domains that is required for the formation of an inter-chain disulfide bond. Thus, two "Fab' fragments" can associate via the formation of a disulfide bond to form a F(ab')2 molecule.MTI-106-PCT01-NP

[0434] A " F(ab')2 fragment" contains two light chains and two heavy chains. Each chain includes a portion of the constant region necessary for the formation of an inter-chain disulfide bond between two heavy chains.

[0435] An " Fv fragment" contains only the variable regions of the heavy and light chain. It contains no constant regions.

[0436] A "single-domain antibody" is an antibody fragment containing a single antibody domain unit (e.g., VH or VL).

[0437] A "single-chain Fv" ("scFv") is antibody fragment containing the VH and VL domain of an antibody, linked together to form a single chain. A polypeptide linker is commonly used to connect the VH and VL domains of the scFv.

[0438] A "tandem scFv", also known as a TandAb, is a single-chain Fv molecule formed by covalent bonding of two scFvs in a tandem orientation with a flexible peptide linker.

[0439] A "bi-specificT cell engager" (BiTE) is a fusion protein consisting of two single-chain variable fragments (scFvs) on a single peptide chain. One of the scFvs binds to T cells via the CD3 receptor, and the other to a tumour cell antigen.

[0440] A "diabody" is a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.

[0441] A " DARPin" is a bispecific ankyrin repeat molecule. DARPins are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell-free and is described in He M. and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.MTI-106-PCT01-NP

[0442] In embodiments the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatised moieties can improve the solubility, the biological half-life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0443] Examples

[0444] The specification will now be illustrated by the following non-limiting Examples.

[0445] General Information

[0446] Reagents and solvents (all anhydrous HPLC -grade) were obtained from commercial suppliers and used without any further purification unless otherwise stated. All reagents were weighed and handled in air unless otherwise stated. Brine refers to a saturated solution of NaCI. Concentration under reduced pressure refers to the use of a rotary evaporator. Pegylated reagents were obtained from PUREPEG. MC-Val-Cit-PAB-MMAE was purchased from Med Chem Express (CAS: 646502-53-6) and is referred to as LP1 (reference)

[0447] List of abbreviations used

[0448] Alloc: Allyloxycarbonyl; TFA: trifluoroacetic acid; FA: Formic acid; FC: flash chromatography; FMOC: fluorenylmethyloxycarbonyl; DCM: dichloromethane; DBA: Dibenzylideneacetone; DMF: N, N-dimethylformamide; DMSO: dimethylsulfoxide; dppf: l,l'-Bis(diphenylphosphino)ferrocene; HFIP: l,l,l,3,3,3-Hexafluoro-2-propanol; HATU: l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; Tf: triflate.

[0449] Flash Column chromatography

[0450] Method A

[0451] Flash chromatography was performed using a BIOTAGE ISOLERA and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed using MERCK KIESELGEL 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light, chromatography solvents were bought and used without further purification from VWR U. K. This method was applied to all intermediates, unless stated differently.

[0452] Method BMTI-106-PCT01-NP

[0453] Chromatographic purification of products was accomplished with a CHEETAH MP200 system with integrated UV detection, using column chromatography on (A) silica gels (40-60 pm), eluted with petroleum ether and ethyl acetate or dichloromethane and methanol, (B) C18 spherical (20-35 pm), eluted with water and acetonitrile with formic acid (0.1%) or trifluoracetic acid (0.05%) or ammonium formate (10 mmol) modifier. This method was applied to all other A and B intermediates not stated in FC method A, unless stated differently

[0454] LC / MS conditions

[0455] Method A

[0456] Positive mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2. Mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds' period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. Flow rate was 0.8 mL / minute. Detection was at 254 nm. Columns: WATERS ACQUITY UPLC BEH SHIELD RP18 1.7pm 2.1 x 50 mm at 50 °C fitted with WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD Pre-column, 130A, 1.7pm, 2.1 mm x 5 mm. This method was applied to intermediates A2-A6, A39, A72 and A73, unless stated differently.

[0457] Method B

[0458] Samples were analysed using a QEXACTIVE ORBITRAP (THERMO) coupled to a DIONEX ULTIMATE 3000 UPLC (THERMO) equipped with an ACQUITY UPLC CSH C18 RP column (2.1 mm x 50 mm, 1.7 pm particle (Waters)). Autosampler and column temperatures were set to 50 °C and 10 °C respectively. Five pL of 200uM sample dissolved in 1:1 v / v acetonitrile / water were injected on column and separated over 9 minutes (reduced samples) or 10 minutes (intact samples) using a gradient of 5-100% mobile phase B (0.1% FA in acetonitrile) in mobile phase A (0.1% TFA in water) at 0.5 mL / min. Settings for heated electrospray source were as follows: sheath gas flow rate 50, auxiliary gas flow rate 15 and spray voltage 3.5 kV. RF level was set to 50. Capillary temperature and auxiliary gas heater temperature were set to 350°C and 400°C respectively. UV trace was recorded at 223 nm and 330 nm. Full scan mass spectra were acquired in positive mode with resolution of 70000, AGC target set to 3E6, maximum injection time set to 200 ms and mass range of 150 - 2000 m / z. Fragmentation spectra were obtained using all ion fragmentation mode for 133-2000 m / z with stepped normalized collision energy of 15, 28 and 45. The acquired data was analysed with THERMO XCALIBUR QUAL BROWSER (version 3.1.66.10).MTI-106-PCT01-NP

[0459] Method C

[0460] Samples were analysed using a QEXACTIVE ORBITRAP (THERMO) coupled to a DIONEX ULTIMATE 3000 UPLC (THERMO) equipped with an ACQUITY UPLC CSH C18 RP column (2.1 mm x 50 mm, 1.7 pm particle (WATERS)). Five pl of 200uM sample dissolved in 1:1 v / v acetonitrile / water were injected on column and separated over 9 minutes (reduced samples) or 10 minutes (intact samples) using a gradient of 20-60% mobile phase B (0.1% FA in acetonitrile) in mobile phase A (0.1% TFA in water) at 0.5 mL / min. Settings for heated electrospray source were as follows: sheath gas flow rate 50, auxiliary gas flow rate 15 and spray voltage 3.5 kV. RF level was set to 50. Capillary temperature and auxiliary gas heater temperature were set to 350°C and 400°C respectively. UV trace was recorded at 223 nm and 330 nm Full scan mass spectra were acquired in positive mode with resolution of 70000, AGC target set to 3E6, maximum injection time set to 200 ms and mass range of 150 - 2000 m / z. Fragmentation spectra were obtained using all ion fragmentation mode for 133-2000 m / z with stepped normalized collision energy of 15, 28 and 45. The acquired data was analysed with THERMO XCALIBUR QUAL BROWSER (version 3.1.66.10).

[0461] Method D

[0462] Samples were analysed using a SHIMADZU LCMS-2020 with electrospray ionization in positive ion detection mode with 20ADXR pump, SIL-20ACXR autosampler, CTO-20AC column oven, M20A PDA Detector and LCMS 2020 MS detector. LC was run in two set ups: 1) Halo C18 column (2.0 pm 3.0x30 mm) in combination with a gradient (5-100% B in 1.2 minutes) of water and FA (0.1%) (A) and CH3CN and FA (0.1%) (B)at a flow rate of 1.5 mL / min; 2) POROSHELL HPH C18 column (2.7 pm 3.0x50 mm) in combination with a gradient (5-95% B in 2 minutes) of aqueous 46 mM ammonium carbonate / ammonia buffer at pH 10 (A) and MeCN (B) at a flow rate of 1.2 mL / min; 3) Halo C18 column (2.0 pm 3.0x30 mm) in combination with a gradient (5-95% B in 2 minutes) of water and TFA (0.05%) (A) and CH3CN and TFA (0.05%) at a flow rate of 1.5 mL / min (B). The Column Oven (CTO-20AC) temperature was 40.0°C. The injection volume was 1 pl. PDA (SPD-M20A) detection was in the range 190-400 nm. The MS detector, which was configured with electrospray ionization as ionizable source; Acquisition mode: Scan; Nebulizing Gas Flow:1.5 L / min; Drying Gas Flow:15 L / min; Detector Voltage: Tuning Voltage ± 0.2 kv; DL Temperature: 250 °C; Heat Block Temperature: 250 °C; Scan Range: 90.00 - 900.00 m / z. This method was applied to all other A and B intermediates not stated in LCMS method A, unless stated differently.

[0463] HPLC conditions

[0464] Method AMTI-106-PCT01-NP

[0465] Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a SHIMADZU PROMINENCE machine using a PHENOMENEX GEMINI NX 5p C18 column (at 50 °C) dimensions: 150 x 21.2 mm. Eluents used were solvent A (H2O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid) or solvent A (H2O with 0.1% trifluoroacetic acid acid) and solvent B (CH3CN with 0.1% trifluoroacetic acid), unless stated differently.

[0466] Method B

[0467] Preparative HPLC was performed with a WATERS MASSLYNX system with integrated MS detection and equipped with Prep C18 OBD 5pm 30 x 150 mm columns from XBRIDGE or XSELECT CSH.

[0468] Alternatively, GILSON GX-281 with integrated UV detection was used, equipped with either XBRIDGE or SUNFIRE C18 10pm, 19x150 ID or 19x250mm. As eluent (acidic) gradients of water / MeCN / FA acid (95 / 5 / 0.1) or water / 0.05% TFA (A) and MeCN / MeOH (B) or 0.05% ammonia in water / 10 mmol NH4HCO3 (A) and MeCN / MeOH (B) were applied.

[0469] Method C

[0470] Preparative CHIRAL HPLC was performed with a GILSON GX-281 system with integrated UV detection and equipped with one of CHIRALPAK AS, AD, CHIRALCEL OD, OJ CHIRALPAK I A, I B, IC, ID, I E, I F, IG, IH columns (DAICEL CHEMICAL INDUSTRIES, LTD.) (R, R)-Whelk-Ol, (S, S)-Whelk-Ol columns (REGIS TECHNOLOGIES, INC. ) CHIRAL Cellulose-SB, SC, SA columns (YMC CO., LTD.) at different column size (250x20mm, 250x30mm) with noted percentage of either ethanol in hexane (%Et / Hex) or isopropanol in hexane (%IPA / Hex) as isocratic solvent systems.

[0471] NMR Method

[0472] Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a BRUKER AV400. All1H and13C NMR are quoted in ppm for measurement against TMS or residual solvent peaks as internal standards. Unless otherwise stated all experiments were carried out using DMSO-d6 as solvent.1H NMR chemical shifts (δ) are given in ppm ± 0.01, and coupling constants (J) are given in Hz ± 0.1 Hz. The1H NMR spectra are reported as follows: <5 / ppm (multiplicity, coupling constant(s) J / Hz, number of protons). Multiplicity is abbreviated as follows: s = singlet, br s = broad singlet, d = doublet, br d = broad doublet, dd = doublet of doublets, t = triplet, dt = doublet of triplet, q = quartet, dq = doublet of quartet, quint = quintet, m = multiplet.13C NMR chemical shifts (5) are given in ppm ± 0.1.

[0473] General procedure I (Amino acid-coupling on resin)MTI-106-PCT01-NP

[0474] A solution of amino acid (0.20 mmol), HATU (76 mg, 0.20 mmol) and N-ethyl-N-isopropylpropan-2-amine (38.8 mg, 0.30 mmol) in DMF (2 mL) was added to the resin loaded with (S, E)-4-((S)-2-amino-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid (Intermediate 4,100 mg, loading 1.13 mmol / g ) and shaken at 25 °C for 2h. Resin was washed with DMF (3x5mL) and DCM (3x5mL).

[0475] General procedure II (Alloc-deprotection on resin)

[0476] A solution of Pd(PPh3)4 (11.56 mg, 10.00 pmol) and phenylsilane (32.5 mg, 0.30 mmol) in DCM (2.000 mL) was added to the resin and shaken at 25 °C for 1h. Resin was washed with DCM (3x5mL) General procedure III (Amide coupling on resin)

[0477] Illa: A solution of 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (53.2 mg, 0.20 mmol) and N-ethyl-N-isopropylpropan-2-amine (38.8 mg, 0.30 mmol) in DMF (2 mL) was added to the resin and shaken at 25 °C for 1h. Resin was washed with DMF (3x5mL) and DCM (3x5mL)

[0478] lllb: A solution of 2,5-dioxopyrrolidin-l-yl l-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oate (138 mg, 0.20 mmol) and N-ethyl-N-isopropylpropan-2-amine (38.8 mg, 0.30 mmol) in DMF (2 mL) was added to the resin and shaken at 25 °C for 1h. Resin was washed with DMF (3x5mL) and DCM (3x5mL)

[0479] General procedure IV (Cleavage from resin)

[0480] IVa: a solution of 20% l,l,l,3,3,3-Hexafluoro-2-propanol in DCM (3 mL) was added to resin and shaken for 1h at 25 °C before filtering the resin off. Resin was washed with DCM and MeOH. Filtrate was concentrated to dryness

[0481] IVb: a solution of 20% TFA in DCM (3 mL) was added to resin and shaken for 1h at 25 °C before filtering the resin off. Resin was washed with DCM and MeOH. Filtrate was concentrated to dryness General procedure V (Grignard)

[0482] Methyl magnesium bromide (3 eq.) was added to correspondent benzonitrile (1 eq.) in ethyl ether at 0°C under nitrogen. The resulting mixture was stirred at 0°C for 2 hours. Titanium isopropoxide (1 eq.) was added to one portion at 0°C. The resulting mixture was stirred at 45°C for 5h. 10% Aqueous sodium hydroxide and ethyl acetate were added to the reaction mixture, and the resulting mixture was stirred at 25 °C for 1h. The reaction mixture was filtered through Celite, and then the layers of the filtrate were separated and concentrated to dryness.

[0483] General procedure VI (Amine Alkylation)MTI-106-PCT01-NP

[0484] Esters of (S)-2-(((trifluoromethyl)sulfonyl)oxy)propanoate (2 eq.) was added to benzylamine analogues (1 eq.) and DIEA (3 eq.) in DCM at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3-16h. In some cases, the reaction mixture was quenched with water, extracted twice with DCM, the organic layer was dried over Na₂SO₄U, filtered and evaporated to afford residue. General procedure VII (Ester hydrolysis)

[0485] Vila: Pd-C (0.527 g, 0.50 mmol) was added to benzyl ester (2 g, 4.96 mmol) in EtOH (20 mL) and water (10.00 mL) at 25°C under hydrogen. The resulting mixture was stirred at 25 °C-100°C for 2-16h hours.

[0486] Vllb: Ester in 4 N HCI (EA) was stirred at 25 °C for 5h. The solvent was removed under reduced pressure.

[0487] Vile: NaOH (5 eq.) was added to (1 eq.) in MeOH / THF 1 / 1 at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 16h. The solvent was removed under reduced pressure. The reaction mixture was acidified with 2M HCI.

[0488] Vlld: H2O2(30% in water) (100 eq.) was added slowly to benzyl ester (1 eq.) and NaOH (2 M in water) (1 eq) in tetrahydrofuran at 0°C over a period of 2 minutes under nitrogen. The resulting mixture was stirred at 30 °C for 12h. The reaction mixture was evaporated and The reaction mixture was acidified with 2M HCI. The reaction mixture was evaporated to afford crude product.

[0489] Vile: Ester was stirred in 4 N HCI (40 mL) at 25°C under hydrogen. The resulting mixture was stirred at 100 °C for 16 hours.

[0490] General procedure VIII (Carbamate formation)

[0491] Villa: allyl ((S)-3-methyl-l-(((S)-l-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2-yl)carbamate (2.181 g, 4.02 mmol) was added to 2-Hydroxypyridine-N-oxide (0.558 g, 5.02 mmol), DIEA (1.755 mL, 10.05 mmol) and alanine derivative (i.e. B intermediates) (1.3 g, 5.02 mmol) in DCM (30 mL) at 25 °C under nitrogen. The resulting mixture was stirred at 25 °C for 6 hours. The solvent was removed under reduced pressure Vlllb A solution of allyl ((S)-3-methyl-l-(((S)-l-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-l-oxopropan-2-yl)amino)-l-oxobutan-2-yl)carbamate (163 mg, 0.30 mmol), 2-hydroxypyridine 1-oxide (33.3 mg, 0.30 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.081 mL, 0.45 mmol) in DMF (2 mL) was added to the resin and shaken at 25 °C for 24h. Resin was washed with DMF (3x5mL) and DCM (3x5mL)

[0492] General procedure IX (Fmoc deprotection on resin)MTI-106-PCT01-NP

[0493] 5% DBU solution in DMF was added to the resin and shaken at room temperature for 30 min, then washed with DCM and DMF.

[0494] Intermediate 1

[0495] f Ik A H

[0496] Fmoc / N

[0497] Fmoc''

[0498]

[0499] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methyl-L-valine (1 g, 2.83 mmol) was dissolved in THF (10 mL), and di(lH-imidazol-l-yl)methanone (0.596 g, 3.68 mmol) was added in one portion. The solution was stirred for 15 min at room temperature and cooled down to 0 °C for 10 min. To this solution, sodium tetrahydroborate (0.214 g, 5.66 mmol) in water (3 mL) was injected fast and uniformly and stirred at 0 °C for 1h. The reaction was quenched by addition of 1 N HCI (5 mL) and 10 mL of water. The reaction mixture was extracted with EtOAc (3 x 30 mL). The combined organic extracts were washed with saturated NaHCO3 and brine ) and dried over MgSO4. The solution was filtered through a thin pad of Celite, and the solvent was evaporated and vacuum-dried to give 9H-fluoren-9-yl)methyl (S)-(l-hydroxy-3-methylbutan-2-yl)(methyl)carbamate which was used in the next step without purification, dissolved in DCM (10.00 mL). 3-oxo-1l5-benzo[d][1,2]iodaoxole-1,1,1(3H)-triyl triacetate (1.440 g, 3.40 mmol) was added and the solution was stirred overnight at 25 °C. The reaction mixture was filtered through celite and the residue was purified by flash silica chromatography (FC method A), elution gradient 5 to 60% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (9H-fluoren-9-yl)methyl (S)-methyl(3-methyl-l-oxobutan-2-yl)carbamate (Intermediate 1, 0.800 g, 84 %) as a colourless oil.1H NMR (CHLOROFORM-d, mixture of rotamers) 6: 9.11-9.75 (m, 1H), 7.76-7.82 (m, 2H), 7.63 (br d, J=7.2 Hz, 1H), 7.56 (br d, J=7.3 Hz, 1H), 7.43 (br t, J=7.4 Hz, 2H), 7.34 (tt, J=7.4, 1.4 Hz, 2H), 4.45-4.76 (m, 2H), 4.24-4.33 (m, 1H), 4.16 (d, J=9.9 Hz, 1H), 2.79-2.88 (m, 3H), 2.22-2.33 (m, 1H), 1.15 (d, J=6.5 Hz, 2H), 0.79-1.02 (m, 3H), 0.62 (br d, J=6.6 Hz, 1H). MS (ESI) [M + H+] 338.3.

[0500] Intermediate 2

[0501] N

[0502] Fmoc^ N

[0503] O

[0504]

[0505] MTI-106-PCT01-NP

[0506] Intermediate 1 (0.8 g, 2.37 mmol) was dissolved in dry DCM (10 mL) in an oven-dried 100 mL round bottom flask under nitrogen, tert-butyl 2-(triphenyl-l5-phosphaneylidene)propanoate (1.852 g, 4.74 mmol) was added and the solution was stirred for 24h. The reaction mixture was evaporated onto silica gel and purified by flash silica chromatography, elution gradient 5 to 60% EtOAc in heptane (FC method A). Pure fractions were evaporated to dryness to afford tert-butyl (S, E)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2,5-dimethy1hex-2-enoate (Intermediate 2, 0.630 g, 59.1 %) as a colourless oil.1H NMR (400 MHz, CHLOROFORM-d, mixture of rotamers) 6 = 7.78 (d, J = 7.6 Hz, 2H), 7.60 (br t, J = 8.9 Hz, 2H), 7.41 (t, J = 7.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 6.65 - 6.35 (m, 1H), 4.76 -4.49 (m, 2H), 4.46 - 4.35 (m, 1H), 4.30 - 4.22 (m, 1H), 3.92 - 3.78 (m, 1H), 2.84 - 2.63 (m, 3H), 2.06 (s, 1H), 1.90 (s, 2H), 1.57 - 1.43 (m, 10H), 1.36 - 1.26 (m, 2H), 0.97 - 0.84 (m, 3H), 0.69 - 0.40 (m, 3H). MS (ESI) for [M + H+] 451.

[0507] Intermediate 3

[0508] Fmoc^

[0509]

[0510] A solution of Intermediate 2 (630 mg, 1.40 mmol) in DCM (5 mL) and TFA (3.00 mL) was stirred at 25 °C for 1h. The reaction was concentrated. To remove traces of TFA, the residue was purified by reversed phase chromatography (BIOTAGE SFAR C18D Duo 100A 30pm 30g, (FC method A)), using decreasingly polar mixtures of pure water (neutral) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (S, E)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-2,5-dimethy1hex-2-enoic acid (Intermediate 3, 506 mg, 92 % as a white powder.1H NMR (CHLOROFORM-d, mixture of rotamers): 6 = 7.85 - 7.74 (m, 2H), 7.60 (br t, J = 8.0 Hz, 2H), 7.45 - 7.37 (m, 2H), 7.36 - 7.30 (m, 2H), 6.87 - 6.50 (m, 1H), 4.83 - 4.39 (m, 2H), 4.34 -4.18 (m, 1H), 3.80 (br t, J = 10.0 Hz, 1H), 2.80 - 2.63 (m, 3H), 2.02 - 1.86 (m, 2H), 1.74 - 1.48 (m, 1H), 1.40 - 1.26 (m, 2H), 0.91 (br t, J = 5.1 Hz, 3H), 0.68 - 0.37 (m, 3H). MS (ESI) for C24H27NO4 [M + H+] calculated 394.3, obtained 394.4.

[0511] Intermediate 4- (Loaded on resin)

[0512] Fmoc^

[0513]

[0514] MTI-106-PCT01-NP

[0515] A solution of Intermediate 3 (0.512 g, 1.3 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.699 mL, 4.00 mmol) in DCM (5 mL) was added to 720 mg of 2 -Chlorotrityl chloride resin (1.5 - 2 mmol / g) (1 mmol) and the resulting suspension was stirred at 25 °C for 3h. Resin was washed with 3x DCM / MeOH / DIPEA (17:2:1), 3xDCM; 2xDMF, 2xDCM and dried in vacuo.5% solution of DBU in DMF was added and the resin was shaken 30 min, then washed with DMF and DCM. (S)-2-[(9H-Fluoren-9-yl)methoxycarbonylamino]-3,3-dimethylbutanoic acid (Intermediate 4, 0.2 mmol) was added following general procedure I, followed by general procedure IX.

[0516] Intermediate 5

[0517] OH

[0518]

[0519] Trifluoromethanesulfonic anhydride (47.8 g, 169.30 mmol) was added to ethyl (S)-2-hydroxypropanoate (10 g, 84.65 mmol) and 2,6-dimethylpyridine (18.14 g, 169.30 mmol) in DCM (100 mL) at 0°C. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with saturated brine (50 mL), and washed sequentially with saturated brine (50 mLX 3), the organic layer was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. Residue reacted following general procedure VI, using 2-phenylpropan-2-amine (17.17 g, 126.98 mmol). The crude product was purified by flash C18-flash chromatography (FC method B), elution gradient 50 to 90% MeCN in water (0.1% NH4HCO3). Pure fractions were evaporated to dryness to afford ethyl (2-phenylpropan-2-yl)-D-alaninate (Intermediate 5, 15.00 g, 75 %) as a brown oil. 1H NMR (300 MHz, DMSO) 6 1.01 - 1.14 (m, 6H), 1.30 (d, J = 4.6 Hz, 6H), 2.40 (s, 1H), 2.90 (q, J = 7.0 Hz, 1H), 3.93 (q, J = 7.1 Hz, 2H), 7.14 - 7.21 (m, 1H), 7.28 (ddd, J = 1.3, 6.8, 7.8 Hz, 2H), 7.39 - 7.50 (m, 2H). m / z (ES+), [M+H]+ = 236; tfa, HPLC tR = 0.630 min.

[0520] Intermediate 6

[0521]

[0522] Intermediate 5 (15 g, 63.74 mmol) was added to HCI (191 ml, 764.89 mmol) at 25°C. The resulting mixture was stirred at 80 °C for 2h. The reaction mixture was poured into saturated brine (20 mL), extracted with 1:4 isopropanol / chloroform (3 x 20 mL), the organic layer was dried over Na₂SO₄,MTI-106-PCT01-NP

[0523] filtered and evaporated to afford yellow oil. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 10% MeCN in water (0.1% FA). Pure fractions were evaporated to dryness to afford (2-phenylpropan-2-yl)-D-alanine (Intermediate 6, 4.26 g, 32.2 %) as a yellow solid. 1H NMR (300 MHz, DMSO) 6 1.09 (d, J = 7.2 Hz, 3H), 1.52 (s, 6H), 2.87 (q, J = 7.1 Hz, 1H), 7.23 - 7.42 (m, 3H), 7.48 - 7.57 (m, 2H). m / z (ES+), [M+H]+ = 208; tfa, HPLC tR = 0.775 min

[0524] Reference compound Pl

[0525]

[0526] Reference compound Pl was synthesized according to general procedure I using Intermediate 6 (48.4 mg, 0.23 mmol), cleaved from resin following procedure IVb. The crude product was purified by preparative HPLC (HPLC method A, SHIMADZU, 15 min, 15-95 % MeCN / Water), using decreasingly polar mixtures of water and MeCN (both containing 0.1% TFA) as eluents. Fractions containing the desired compound were evaporated to dryness in GENEVAC, then freeze dried to afford (S, Ej-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((R)-2-((2-phenylpropan-2-yl)amino)propanamido)butanamido)hex-2-enoic acid (Pl, 15.20 mg, 25.07 %) as a white crystalline solid. HPLC tR (method C)= 5.72 min, m / z (ES+), [M+H]+ =474;1H NMR (400 MHz, DMSO-d6) 6 ppm 0.7 (d, 7=6.5 Hz, 3 H) 0.8 (d, 7=6.6 Hz, 3 H) 0.9 (s, 9 H) 1.0 (d, 7=7.1 Hz, 3 H) 1.3 (d, 7=12.8 Hz, 6 H) 1.8 (d, 7=1.4 Hz, 3 H) 1.9 (dt, 7=10.7, 6.6 Hz, 1 H) 2.8 (q, 7=7.0 Hz, 1 H) 2.9 (s, 4 H) 4.7 (d, 7=9.9 Hz, 1 H) 4.9 (s, 1 H) 6.6 (dd, 7=9.3, 1.5 Hz, 1 H) 7.2 - 7.2 (m, 1 H) 7.3 (t, 7=7.6 Hz, 2 H) 7.5 - 7.5 (m, 2 H) 8.1 (d, 7=9.8 Hz, 1 H) 8.2 (s, 1 H)

[0527] Intermediate 7

[0528]

[0529] Tf₂O (70.3 mL, 416.20 mmol) was added to 2,6-dimethylpyridine (59.5 g, 554.93 mmol) and benzyl (S)-2-hydroxypropanoate (50 g, 277.46 mmol) in DCM (300 mL) at -60°C. The resulting mixture was stirred at -50 °C for 2h.MTI-106-PCT01-NP

[0530] The reaction mixture was quenched with water (500 mL), and washed sequentially with Citric acid (10%), the organic layer was dried over Na₂SO₄, filtered and evaporated to afford benzyl (S)-2-(((trifluoromethyl)sulfonyl)oxy)propanoate (Intermediate 7, 80 g, 92 %) as a yellow oil. 1H NMR (300 MHz, Chloroform-d) 6 1.71 (d, 3H), 5.24 - 5.31 (m, 3H), 7.32 - 7.43 (m, 5H).

[0531] Intermediate 8

[0532]

[0533] Intermediate 8 was synthesized according to procedure V using 3-bromo-5-methylbenzonitrile (10 g, 51.01 mmol) in diethyl ether (80 mL). The crude product was purified by flash silica chromatography, elution gradient 0 to 20% DCM in MeOH. Pure fractions were evaporated to dryness to afford 2-(3-bromo-5-methylphenyl)propan-2-amine (Intermediate 8, 5.00 g, 43.0 %) as a yellow oil. m / z (ES+), [M+H]+ = 228; acid, HPLC tR = 0.691 min.1H NMR (300 MHz, Chloroform-d) 6 1.48 (s, 6H), 1.71 (d, J = 8.2 Hz, 2H), 2.29 - 2.37 (m, 3H), 7.19 - 7.21 (m, 1H), 7.25 (q, J = 0.8 Hz, 1H), 7.46 (s, 1H).

[0534] Intermediate 9

[0535]

[0536] Intermediate 9 was synthesized according to procedure VI using Intermediate 8 (4.8 g, 21.04 mmol) and Intermediate 7 (9.86 g, 31.56 mmol) in DCM (60 mL). The crude product was purified by flash silica chromatography, elution gradient 0 to 60% water in MeCN. Pure fractions were evaporated to dryness to afford benzyl (2-(3-bromo-5-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 9, 6.00 g, 73.1 %) as a colourless oil. m / z (ES+), [M+H]+ = 390; base, HPLC tR = 1.405 min.1H NMR (300 MHz, Chloroform-d) 6 1.24 (d, J = 7.1 Hz, 3H), 1.38 (s, 6H), 2.32 (s, 3H), 3.16 (q, J = 7.0 Hz, 1H), 4.56 (s, 1H), 5.05 (d, J = 1.1 Hz, 2H), 6.55 (s, 1H), 6.93 (d, J = 1.7 Hz, 1H), 7.11 (t, J = 2.0 Hz, 1H), 7.27 - 7.38 (m, 5H).

[0537] Intermediate 10

[0538]

[0539] 10MTI-106-PCT01-NP

[0540] Cs₂CO₃ (2.504 g, 7.69 mmol) was added to tert-Butyl carbamate (0.675 g, 5.76 mmol), Intermediate 9 (1.5 g, 3.84 mmol), Pd₂(dba)₃ (0.352 g, 0.38 mmol) and 2-Dicyclohexylphosphino-2',4',6'-tri-iso-propyl-1,1'-biphenyl (0.366 g, 0.77 mmol) in 1,4-dioxane (1 mL) at 25°C. The resulting mixture was stirred at 90 °C under nitrogen for 1h. The reaction mixture was diluted with DCM (100 mL), filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% petroleum ether in EtOAc. Pure fractions were evaporated to dryness to afford benzyl (, 2-(3-((tert-butoxycarbonyl)amino)-5-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 10, 1.500 g, 92 %) as a yellow oil. m / z (ES+), [M+H]+ = 427; base, HPLC tR = 1.320 min.1H NMR (400 MHz, DMSO-d6) 6 1.13 (d, J = 7.0 Hz, 3H), 1.25 (s, 6H), 1.37 (s, 4H), 1.47 (s, 9H), 5.01 (s, 2H), 6.86 (s, 1H), 7.16 (s, 1H), 7.26 - 7.42 (m, 6H).

[0541] Intermediate 11

[0542]

[0543] 11 Intermediate 11 was synthesized according to procedure Vila using Intermediate 10 (1.45 g, 3.40 mmol) in MeOH (2 mL). The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 50% water in MeCN. Pure fractions were evaporated to dryness to afford (2-(3-((tert-butoxycarbonyl)amino)-5-methylphenyl)propan-2-yl)-D-alanine (Intermediate 11, 0.700 g, 61.2 %) as a white solid, m / z (ES+), [M+Na]+ = 359; acid, HPLC tR = 0.963 min.1H NMR (300 MHz, DMSO-d6) 6 1.13 (d, J = 7.0 Hz, 3H), 1.47 (s, 15H), 2.08 (s, 1H), 2.26 (s, 3H), 2.89 (q, J = 7.1 Hz, 1H), 6.98 (s, 1H), 7.24 (s, 1H), 7.40 (d, J = 2.3 Hz, 1H), 9.30 (s, 1H).

[0544] Synthesis of P2

[0545]

[0546] P2MTI-106-PCT01-NP

[0547] Compound P2 was synthesized according to general procedure I using Intermediate 11 (101 mg, 0.30 mmol), cleaved from resin following procedure IVb. The crude product was purified by flash C18 silica (FC method A, BIOTAGE SFAR BIO C18 D Duo 300 A 20 pm 10 g) chromatography, elution gradient 5 to 30% MeCN in water (both with 0.1% FA). Pure fractions were freeze dried to give (S, E)-4-((S)-2-((R)-2-((2-(3-amino-5-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid (P2, 50.2 mg, 54.2 %) as a white powder. HPLC tR (method B)= 4.93 min, m / z (ES+), [M+H]+ =503; 1H NMR (400 MHz, DMSO, 27°C) 0.76 (3H, d), 0.82 (3H, d), 0.88 (9H, s), 1.30 (3H, d), 1.40 (3H, s), 1.57 (3H, s), 1.78 (3H, d), 1.95 - 2.06 (1H, m), 2.22 (3H, s), 2.98 (3H, s), 3.79 (1H, q), 4.66 (1H, d), 4.93 (1H, dd), 6.57 (1H, s), 6.61 - 6.69 (2H, m), 6.71 (1H, s), 8.66 (1H, d), 8.71 (1H, br s), 9.02 (1H, br s).

[0548] Intermediate 12

[0549]

[0550] 12

[0551] 5-bromo-2-fluoro-3-methylbenzaldehyde (20 g, 92.15 mmol) was added to sodium acetate (12.10 g, 147.44 mmol) and hydroxylammonium chloride solution (9.60 g, 138.23 mmol) in EtOH (200 mL) under air. The resulting mixture was stirred at 20 °C for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-2-fluoro-3-methylbenzaldehyde oxime (Intermediate 12, 20.00 g, 94 %) as a pale yellow oil. m / z (ES-), [M+H]+ = 232; base, HPLC tR = 1.023 min; 1H NMR (400 MHz, DMSO-d6) 6 2.24 (d, J = 2.3 Hz, 3H), 7.51 - 7.56 (m, 1H), 7.64 - 7.68 (m, 1H), 8.16 (s, 1H), 11.77 (s, 1H).

[0552] Intermediate 13

[0553]

[0554] Palladium II acetate (0.223 g, 0.99 mmol) was added to Intermediate 12 (23 g, 99.12 mmol) in MeCN (300 mL) at 20°C under nitrogen. The resulting mixture was stirred at 70 °C for 16 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silicaMTI-106-PCT01-NP

[0555] chromatography, elution gradient 0 to 10% EA in petroleum ether. Pure fractions were evaporated to dryness to afford 5-bromo-2-fluoro-3-methylbenzonitrile (Intermediate 13, 14.00 g, 66.0 %) as a white solid, m / z (ES+), NH4HCO3, HPLC tR = 1.463 min, NO Ms sign. 1H NMR (400 MHz, CDCI3) 6 1.26 (td, J = 7.0, 1.6 Hz, 3H), 7.52 - 7.65 (m, 2H).

[0556] Intermediate 14

[0557]

[0558] Intermediate 14 was synthesized according to general procedure V using Intermediate 13 (12 g, 56.06 mmol). The crude product was purified by flash silica chromatography, elution gradient 0 to 80% EA in petroleum ether. Pure fractions were evaporated to dryness to afford 2-(5-bromo-2-fluoro-3-methylphenyl)propan-2-amine (Intermediate 14, 4.00 g, 29.0 %) as a pale yellow oil. m / z (ES+), [M+H]+ = 246; FA, HPLC tR = 0.777 min;1H NMR (400 MHz, CDCI3) 6 1.56 (s, 6H), 2.26 (d, J = 2.6 Hz, 3H), 7.23 (dd, J = 6.3, 2.5 Hz, 1H), 7.42 (dd, J = 6.9, 2.5 Hz, 1H).

[0559]

[0560] Intermediate 15 was synthesized according to general procedure VI using Intermediate 14 (A78, 3.5 g, 14.22 mmol). The crude product was purified by flash silica chromatography, elution gradient 0 to 25% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(5-bromo-2-fluoro-3-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 15, 5.50 g, 95 %) as a pale yellow oil. m / z (ES+), [M+H]+ = 408; TFA, HPLC tR = 0.827 min; 1H NMR (300 MHz, CDCI3) 6 1.26 (d, J = 7.0 Hz, 3H), 1.46 (s, 6H), 2.22 (d, J = 2.6 Hz, 3H), 3.25 (q, J = 7.0 Hz, 1H), 4.87 - 5.04 (m, 2H), 7.21 (dd, J = 6.6, 2.5 Hz, 1H), 7.26 - 7.42 (m, 8H).

[0561] Intermediate 16

[0562]

[0563] MTI-106-PCT01-NP

[0564] Brettphos Pd G3 (1.243 g, 1.37 mmol) was added to Cs₂CO₃ (13.41 g, 41.15 mmol), Intermediate 15 (5.6 g, 13.72 mmol) and tert-butyl carbamate (1.928 g, 16.46 mmol) in 1,4-dioxane (100 mL) at 25°C under nitrogen. The resulting mixture was stirred at 90 °C for 16 hours. The reaction mixture was poured into water (150 mL), extracted with EtOAc (2 x 150 mL). the combined organic layers was dried over Na₂SO₄, filtered and evaporated to afford brown oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(5-((tert-butoxycarbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 16, 4.60 g, 75 %) as a yellow oil. m / z (ES+), [M+H]+ = 445; TFA, HPLC tR = 0.926 min;lH NMR (400 MHz, CDCI3) 67.47 - 7.22 (m, 7H), 7.22 - 7.19 (m, 1H), 6.97 (dd, J = 2.7, 6.6 Hz, 1H), 6.34 (s, 1H), 5.30 (s, 1H), 4.94 (d, J = 2.7 Hz, 2H), 3.79 - 3.68 (m, 1H), 3.24 (q, J = 7.0 Hz, 1H), 2.20 (d, J = 2.5 Hz, 3H), 1.89 - 1.81 (m, 1H), 1.51 (s, 15H), 1.28 - 1.24 (m, 3H), 0.94- 0.79 (m, 1H).

[0565] Intermediate 17

[0566]

[0567] 4N HCI in 1.4-dioxane (50 mL, 200.00 mmol) was added to Intermediate 16 (4.5 g, 10.12 mmol) at 25°C under nitrogen. The resulting mixture was stirred at 25°C for 16 hours. The solvent was removed under reduced pressure afford crude. Allyl chloroformate (0.739 mL, 12.15 mmol) was added to the mixture with DIEA (5.30 mL, 30.37 mmol) in DCM (50 mL) at 0°C under nitrogen. The resulting mixture was stirred at 25 °C for 4 hours. The reaction mixture was poured into water (100 mL), extracted with DCM (2 x 50 mL), the combined organic layer was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(5-(((allyloxy)carbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 17, 2.90 g, 66.9 %) as a yellow oil. m / z (ES+), [M+H]+ = 429; TFA, HPLC tR = 0.873 min; 1H NMR (300 MHz, CDCI3) 67.42 - 7.24 (m, 6H), 7.10 (dd, J = 2.8, 6.7 Hz, 1H), 6.54 (s, 1H), 5.98 (ddt, J = 5.7, 10.4, 17.2 Hz, 1H), 5.38 (dq, J = 1.5, 17.2 Hz, 1H), 5.33 - 5.21 (m, 2H), 4.96 (d, J = 1.1 Hz, 2H), 4.67 (dt, J = 1.4, 5.7 Hz, 2H), 3.28 (q, J = 7.0 Hz, 1H), 2.22 (d, J = 2.6 Hz, 3H), 1.50 (d, J = 8.5 Hz, 6H), 1.29 (dd, J = 4.0, 7.0 Hz, 3H).MTI-106-PCT01-NP

[0568] Intermediate 18

[0569]

[0570] Intermediate 18 was synthesized according to general procedure VIIC using Intermediate 17 (2.3 g, 5.37 mmol) in THF (30 mL) at 25°C under nitrogen. The crude product was purified by flash silica chromatography, elution gradient 0 to 18% water in MeCN Fractions containing the desired compound were evaporated to dryness to afford (2-(5-(((allyloxy)carbonyl)amino)-2-fluoro-3- methylphenyl)propan-2-yl)-D-alanine (Intermediate 18, 1.100 g, 60.6 %) as a white solid, m / z (ES+), [M+H]+ = 339; FA, HPLC tR = 0.888 min 1H NMR (300 MHz, DMSO-d6) 69.67 (s, 1H), 7.39 (dd, J = 2.7, 7.1 Hz, 1H), 7.31 (dd, J = 2.5, 6.5 Hz, 1H), 5.98 (ddt, J = 5.4, 10.6, 17.2 Hz, 1H), 5.35 (dq, J = 1.7, 17.2 Hz, 1H), 5.24 (dq, J = 1.5, 10.4 Hz, 1H), 4.60 (dt, J = 1.5, 5.4 Hz, 2H), 3.02 (q, J = 7.0 Hz, 1H), 2.19 (d, J = 2.4 Hz, 3H), 1.47 (d, J = 8.3 Hz, 6H), 1.19 (d, J = 7.0 Hz, 3H).

[0571] Synthesis of P3

[0572]

[0573] Compound P3 was synthesized according to general procedure I using Intermediate 18 (67.7 mg, 0.20 mmol), followed by procedure II and cleaved from resin following procedure IVb. The crude product was purified by flash C18 silica chromatography (10g cartridge), elution gradient 5 to 35% MeCN+0.1%TFA in water+0.1%TFA. Pure fractions were evaporated to dryness to afford (S, E)-4-((S)- 2-((R)-2-((2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3- trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid (P3, 36.4 mg, 50.9 %) as a white solid. HPLC tR (method B)= 5.19 min, m / z (ES+), [M+H]+ =521; 1H NMR (400 MHz, DMSO) 68.96 (br s, 1H), 8.70 (d, J = 8.9 Hz, 1H), 6.71 - 6.50 (m, 3H), 4.92 (dd, J = 10.7, 9.5 Hz, 1H), 4.68 (d, J = 8.9 Hz, 1H), 4.06 - 3.96 (m, 1H), 2.98 (s, 3H), 2.16 (d, J = 2.2 Hz, 3H), 2.05 - 1.92 (m, 1H), 1.78 (d, J = 1.5 Hz, 3H), 1.60 (s, 3H), 1.52 (s, 3H), 1.38 (d, J = 6.9 Hz, 3H), 0.90 (s, 9H), 0.82 (d, J = 6.5 Hz, 3H), 0.73 (d, J = 6.5 Hz, 3H).MTI-106-PCT01-NP

[0574]

[0575] 2-Di-t-butylphosphino-2',4',6'-tri-i-propyl-1,1'-biphenyl (0.544 g, 1.28 mmol) and tBuXPhos Pd G3 (1.017 g, 1.28 mmol) was added to Intermediate 9 (5g, 12.81 mmol), Cs₂CO₃ (12.52 g, 38.43 mmol) and tert-butyl methylcarbamate (5.04 g, 38.43 mmol) in toluene (100mL) at 25°C under air. The resulting mixture was stirred at 100 °C for 8 hours. The reaction mixture was quenched with water (200 mL), extracted with EtOAc (3 x 100 mL). The combined organic layers was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(3-((tert-butoxycarbonyl)(methyl)amino)-5-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 19, 7.05 g, 50% LCMS purity) as a pale yellow liquid, m / z (ES+), [M+H]+ = 441; NH4HCO3, HPLC tR = 1.304 min;1H NMR (400 MHz, DMSO) 6 1.37 (d, J = 4.6 Hz, 18H), 1.75 - 2.48 (m, 3H), 4.80 - 5.13 (m, 1H), 6.60 - 7.57 (m, 7H).

[0576] Intermediate 20

[0577]

[0578] Intermediate 19 (7g, 15.89 mmol) (50% pure) was added in HCI-l,4-dioxane (30mL) at 25°C under air. The resulting mixture was stirred at 25 °C for 8 hours. The solvent was removed under reduced pressure to afford benzyl (2-(3-methyl-5-(methylamino)phenyl)propan-2-yl)-D-alaninate (Intermediate 20, 5.60 g) as a yellow oil; m / z (ES+), [M+H]+ = 341; NH4HCO3, HPLC tR = 1.164 min.

[0579] 1H NMR (300 MHz, DMSO) 6 1.29 - 1.88 (m, 8H), 2.30 (d, J = 26.1 Hz, 3H), 2.78 (s, 2H), 3.85 (s, 1H), 5.77 (s, 1H), 6.74 - 7.52 (m, 7H).

[0580] Intermediate 21

[0581]

[0582] MTI-106-PCT01-NP

[0583] allyl carbonochloridate (2.149 g, 17.83 mmol) was added to DIEA (7.78 mL, 44.57 mmol) and Intermediate 2 (5.6 g, 14.86 mmol) in DCM (100mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 5 hours. The reaction mixture was quenched with water (150 mL), extracted with DCM (3 x 100 mL). The combined organic layer was dried over Na₂SO₄, filtered and evaporated to afford pale yellow liquid. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(3-(((allyloxy)carbonyl)(methyl)amino)-5-methylphenyl)propan-2-yl)-D-alaninate (Intermediate 21, 2 g,31.7%) as a pale yellow liquid.m / z (ES+), [M+H]+ = 425; TFA, HPLC tR = 0.857 min;1H NMR (400 MHz, CDCI3) 6 1.21 - 1.43 (m, 9H), 2.34 (s, 3H), 2.82 (d, J = 4.9 Hz, 3H), 3.16 (q, J = 7.1 Hz, 1H), 4.59 -4.61 (m, 2H), 5.28 (d, J = 30.5 Hz, 4H), 5.91 - 5.96 (m, 1H), 6.88 - 7.03 (m, 1H), 7.09 - 7.48 (m, 7H).

[0584] Intermediate 22

[0585]

[0586] Intermediate 22 was synthesized according to general procedure VIIC using (2-(3-(((allyloxy)carbonyl)(methyl)amino)-5-methylphenyl)propan-2-yl)-D-alaninate (A84, 1.9 g, 4.48 mmol) in THF (30 mL) at 25°C under nitrogen. The crude product was purified by flash silica chromatography, elution gradient 0 to 25% water in MeCN Fractions containing the desired compound were evaporated to dryness to afford (2-(3-(((allyloxy)carbonyl)(methyl)amino)-5-methylphenyl)propan-2-yl)-D-alanine (Intermediate 22, 0.605 g, 40.4 %) as a white solid. m / z (ES+), [M+H]+ = 335; FA, HPLC tR = 1.351 min; 1H NMR (400 MHz, DMSO) 6 1.11 (d, J = 7.1 Hz, 3H), 1.49 (s, 6H), 2.32 (s, 3H), 2.85 (t, J = 7.1 Hz, 1H), 3.22 (s, 3H), 4.38 - 4.57 (m, 2H), 5.11 - 5.30 (m, 2H), 5.90 (ddt, J = 17.2, 10.4, 5.1 Hz, 1H), 7.07 (s, 1H), 7.19 (d, J = 2.0 Hz, 1H), 7.27 (t, J = 2.0 Hz, 1H).

[0587] Synthesis of P4

[0588]

[0589] MTI-106-PCT01-NP

[0590] Compound P4 was synthesized according to general procedure I using Intermediate 22 (66.9 mg, 0.20 mmol), followed by procedure II and cleaved from resin following procedure IVa. The crude product was purified by preparative HPLC (20-50 % MeCN / Water, both containing 0.1% TFA).

[0591] Fractions containing the desired compound were combined and freeze-dried to afford (S, E)-2,5-dimethyl-4-((S)-N,3,3-trimethyl-2-((R)-2-((2-(3-methyl-5-(methylamino)phenyl)propan-2-yl)amino)propanamido)butanamido)hex-2-enoic acid (P4, 39.5 mg, 56.5 %) as a white crystalline solid. HPLC tR (method C)= 5.91 min, m / z (ES+), [M+H]+ =517;1H NMR (400 MHz, DMSO-d6) 6 = 9.07 - 8.92 (m, 1H), 8.79 - 8.69 (m, 1H), 8.66 (d, J = 8.7 Hz, 1H), 6.65 (dd, J = 1.5, 9.4 Hz, 1H), 6.56 -6.47 (m, 2H), 6.41 (s, 1H), 5.02 - 4.84 (m, 2H), 4.78 - 4.55 (m, 2H), 3.83 - 3.69 (m, 1H), 3.03 - 2.93 (m, 3H), 2.69 (s, 3H), 2.22 (s, 3H), 2.01 (td, J = 6.6, 10.7 Hz, 1H), 1.78 (d, J = 1.4 Hz, 3H), 1.60 (s, 3H), 1.40 (s, 2H), 1.31 (d, J = 7.0 Hz, 3H), 0.94 - 0.85 (m, 9H), 0.83 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H) Intermediate 23

[0592]

[0593] 23

[0594] butyllithium (37.9 mL, 94.71 mmol) was added to 1,3-dibromo-5-ethylbenzene (25 g, 94.71 mmol) in THF (500 mL) at -78°C under nitrogen. The resulting mixture was stirred at -78 °C for 10 minutes. Then propan-2-one (55.0 g, 947.11 mmol) was added to the mixture warmed to 25°C. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with saturated NH4CI (50 mL) and water(200 mL), extracted with EtOAc (3 x 300 mL), the organic layer was dried over Na₂SO₄, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford 2-(3-bromo-5-ethylphenyl)propan-2-ol (Intermediate 23, 22.00 g, 96 %) as a white solid, m / z (ES+), [M+H-H2O]+ = 225; TFA, HPLC tR = 0.905 min;1H NMR (400 MHz, DMSO) 6 1.17 (t, J = 7.6 Hz, 3H), 2.60 (q, J = 7.6 Hz, 2H), 7.24 (t, J = 2.4, 1.7 Hz, 1H), 7.29 (t, J = 1.7 Hz, 1H), 7.45 (t, J= 1.8 Hz, 1H).

[0595] Intermediate 24MTI-106-PCT01-NP

[0596]

[0597] 23

[0598] H₂SO₄ (13.81 mL, 259.11 mmol) was added to Intermediate 23 (21 g, 86.37 mmol) in MeCN (200 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 1 hour. The reaction mixture was quenched with water (200 mL), extracted with EtOAc (2 x 300 mL). The organic layer was dried over Na2SO4, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EA in petroleum ether. Pure fractions were evaporated to dryness to afford N-(2-(3-bromo-5-ethylphenyl)propan-2-yl)acetamide (Intermediate 24, 8.30 g, 33.8 %) as a white solid, m / z (ES+), [M+H]+ = 284; FA, HPLC tR = 0.835 min;1H NMR (400 MHz, DMSO) 6 1.15 - 1.19 (m, 3H), 1.51 (s, 6H), 1.84 (s, 3H), 2.59 (q, J = 7.6 Hz, 2H), 7.14 (t, J = 1.7 Hz, 1H), 7.23 (dt, J = 12.1, 1.8 Hz, 2H), 8.09 (s, 1H).

[0599] Intermediate 25

[0600]

[0601] 25

[0602] NaOH (11.26 g, 281.49 mmol) was added to Intermediate 24 (8 g, 28.15 mmol) in ethylene glycol (100 mL) at 25°C under nitrogen. The resulting mixture was stirred at 170 °C for 16 hours. The reaction mixture was poured into water (200 mL), extracted with EtOAc (3 x 150 mL), the combined organic layers was dried over Na2SO4, filtered and evaporated to afford 2-(3-bromo-5-ethylphenyl)propan-2-amine (Intermediate 25, 5.00 g, 73.3 %) as a yellow oil.m / z (ES+), [M+H-NH3]+ = 225; FA, HPLC tR = 0.655 min;1H NMR (300 MHz, DMSO) 6 1.11 - 1.19 (m, 6H), 1.31 (s, 3H), 2.57 (q, J = 7.6 Hz, 2H), 7.19 (t, J = 1.7 Hz, 1H), 7.32 (d, J = 1.6 Hz, 1H), 7.50 (t, J = 1.8 Hz, 1H).

[0603] Intermediate 26

[0604]

[0605] 25MTI-106-PCT01-NP

[0606] Intermediate 26 was synthesized according to general procedure VI using Intermediate 25 (5 g, 20.65 mmol) The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 100% MeCN in water. Pure fractions were evaporated to dryness to afford benzyl (2-(3-bromo-5-ethylphenyl)propan-2-yl)-L-alaninate (Intermediate 26, 8.00 g, 96 %) as a yellow oil. m / z (ES+), [M+H]+ = 404; TFA, HPLC tR = 0.808 min;1H NMR (400 MHz, DMSO) 61.11 - 1.14 (m, 6H), 1.29 (d, J = 6.8 Hz, 6H), 2.54 - 2.59 (m, 2H), 3.01 (q, J = 6.9 Hz, 1H), 4.97 (d, J = 3.8 Hz, 2H), 7.23 (t, J = 1.7 Hz, 1H), 7.28 - 7.36 (m, 6H), 7.42 (t, J = 1.8 Hz, 1H).

[0607] Intermediate 27

[0608]

[0609] Brettphos Pd G3 (0.673 g, 0.74 mmol) was added to Intermediate 26 (3 g, 7.42 mmol), tert-butyl carbamate (1.043 g, 8.90 mmol) and Cs₂CO₃ (7.25 g, 22.26 mmol) in 1,4-dioxane (40 mL) at 25°C under nitrogen. The resulting mixture was stirred at 90 °C for 16 hours. The reaction mixture was poured into water (100 mL), extracted with EtOAc (3 x 100 mL), the combined organic layers was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(3-((tert-butoxycarbonyl)amino)-5-ethylphenyl)propan-2-yl)-D-alaninate (Intermediate 27, 1.600 g, 48.9 %) as a yellow oil. m / z (ES+), [M+H]+ = 441; FA, HPLC tR = 0.737 min1H NMR (300 MHz, CDCI3) 61.22 - 1.28 (m, 6H), 1.40 (d, J = 1.5 Hz, 6H), 1.54 (s, 9H), 2.62 (q, J = 7.6 Hz, 2H), 3.16 (q, J = 7.0 Hz, 1H), 5.06 (d, J = 2.5 Hz, 2H), 6.98 (t, J = 1.6 Hz, 1H), 7.13 (t, J = 1.9 Hz, 1H), 7.20 - 7.41 (m, 6H).

[0610] Intermediate 28

[0611]

[0612] Intermediate 27 (2 g, 4.54 mmol) was in HCI in 1,4-dioxane 4N (20 mL) at 25°C under nitrogen. The resulting mixture was stirred at r.t for 16 hours. The solvent was removed under reduced pressure afford benzyl (2-(3-amino-5-ethylphenyl)propan-2-yl)-D-alaninate (Intermediate 28, 1.600 g, 94MTI-106-PCT01-NP

[0613] %)(HCI salt) as brown oil. m / z (ES+), [M+H]+ = 341; TFA, HPLC tR = 0.653 min;1H NMR (400 MHz, DMSO) 6 1.13 - 1.28 (m, 6H), 1.37 (d, J = 6.9 Hz, 3H), 1.73 (s, 3H), 2.60 (q, J = 7.6 Hz, 2H), 3.51 (s, 1H), 3.82 (s, 1H), 4.94 (d, J = 12.3 Hz, 1H), 5.02 (d, J = 12.3 Hz, 1H), 7.15 (d, J = 3.1 Hz, 1H), 7.30 - 7.42 (m, 6H), 7.46 (s, 1H), 9.44 (s, 1H), 10.11 (s, 1H).

[0614] Intermediate 29

[0615]

[0616] Allyl chloroformate (0.767 g, 6.37 mmol) was added Intermediate 29 (2 g, 5.31 mmol) and DIEA (2.78 mL, 15.92 mmol) in DCM (30 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25°C for 5 hours. The reaction mixture was poured into water (50 mL), extracted with DCM (3 x 50 mL). The combined organic layers was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EA in petroleum ether. Pure fractions were evaporated to dryness to afford benzyl (2-(3-(((allyloxy)carbonyl)amino)-5-ethylphenyl)propan-2-yl)-D-alaninate (Intermediate 29, 1.700 g, 75 %) as a yellow oil. m / z (ES+), [M+H]+ = 425; TFA, HPLC tR = 0.894 min1H NMR (400 MHz, DMSO) 6 1.14 (t, J = 7.5 Hz, 9H), 1.27 (s, 3H), 2.54 (d, J = 7.6 Hz, 2H), 2.98 (q, J = 7.0 Hz, 1H), 4.58 - 4.61 (m, 2H), 5.00 (s, 2H), 5.19 - 5.27 (m, 1H), 5.31 - 5.41 (m, 1H), 5.91 - 6.05 (m, 1H), 7.19 (s, 1H), 7.27 - 7.40 (m, 7H), 9.59 (s, 1H).

[0617] Intermediate 30

[0618]

[0619] Intermediate 30 was synthesized according to general procedure VIIC using Intermediate 29 (1.6 g, 3.77 mmol) in THF (20 mL) at 25°C under nitrogen. The precipitate was collected by filtration, washed with water (50 mL) and dried under vacuum to afford (2-(3-(((allyloxy)carbonyl)amino)-5-ethylphenyl)propan-2-yl)-D-alanine (Intermediate 30, 0.750 g, 59.5 %) as a white solid. m / z (ES+), [M+H]+ = 335; FA, HPLC tR = 0.933 min; 1H NMR (300 MHz, DMSO) 69.71 (s, 1H), 7.45 (d, J = 1.9 Hz, 1H), 7.28 (s, 1H), 7.07 (s, 1H), 5.98 (ddt, J = 5.4, 10.6, 17.2 Hz, 1H), 5.36 (dq, J = 1.7, 17.2 Hz, 1H), 5.23MTI-106-PCT01-NP

[0620] (dq, J = 1.5, 10.5 Hz, 1H), 4.60 (dt, J = 1.5, 5.4 Hz, 2H), 2.90 (q, J = 7.1 Hz, 1H), 2.61 - 2.54 (m, 2H), 1.50 (s, 6H), 1.24- 1.10 (m, 6H).

[0621] Synthesis of P5

[0622]

[0623] Compound P5 was synthesized according to general procedure I using Intermediate 30 (66.9 mg, 0.20 mmol), followed by procedure II and cleaved from resin following procedure IVb. The crude product was purified by flash C18 silica chromatography (10g cartridge), elution gradient 5 to 35% MeCN+0.1%TFA in water+0.1%TFA. Pure fractions were evaporated to dryness to afford (S, E)-4-((S)-2-((R)-2-((2-(3-amino-5-ethylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid (P5, 41.0 mg, 58.6 %) as a white solid. HPLC tR (method B)= 5.47 min, m / z (ES+), [M+H]+ =517; 1H NMR (400 MHz, DMSO) 6 9.00 (s, 1H), 8.78 - 8.60 (m, 2H), 6.74 (s, 1H), 6.66 (dq, J = 9.5, 1.5 Hz, 1H), 6.58 (s, 1H), 6.55 (s, 1H), 4.98 - 4.89 (m, 1H), 4.66 (d, J = 8.7 Hz, 1H), 3.83 - 3.72 (m, 1H), 2.99 (s, 3H), 2.56 - 2.51 (m, 2H), 2.09 - 1.93 (m, 1H), 1.78 (d, J = 1.4 Hz, 3H), 1.58 (s, 3H), 1.39 (s, 3H), 1.31 (d, J = 6.9 Hz, 3H), 1.17 (t, J = 7.6 Hz, 3H), 0.89 (s, 9H), 0.83 (d, J = 6.5 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H).

[0624] Compounds P6-P8 were prepared using synthetic methods analogous to those described above.

[0625] Entry Structure and Name Characterisation

[0626] P6 HPLC tR (method B)= 4.72 min, m / z (ES+),

[0627] [M+H]+ = 536; 1H NMR (400 MHz, DMSO) 6 I Hh i

[0628] / \ 1H= 8.95 (br s, 1H), 8.72 (d, J = 8.9 Hz, 1H), 6.67 - 6.62 (m, 2H), 6.57 - 6.51 (m, 1H), 4.92 (dd, J = (S, E)-4-((S)-2-((R)-2-((2-(5-amino-3-ethyl-2- 10.6, 9.5 Hz, 1H), 4.69 (d, J = 8.9 Hz, 1H), 4.06 - fluorophenyl)propan-2- 3.96 (m, 1H), 2.98 (s, 3H), 2.61 - 2.52 (m, 2H), yl)amino)propanamido)-N,3,3- 2.05 - 1.94 (m, 1H), 1.78 (d, J = 1.5 Hz, 3H), 1.60

[0629]

[0630] MTI-106-PCT01-NP

[0631] trimethylbutanamido)-2,5-dimethy1hex-2- (s, 3H), 1.51 (s, 3H), 1.38 (d, J = 6.9 Hz, 3H), 1.15 enoic acid (t, J = 7.5 Hz, 3H), 0.90 (s, 9H), 0.82 (d, J = 6.6 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H).

[0632] P7 HPLC tR (method B)= 5.47 min, m / z (ES+),

[0633] [M+H]+ =; 1H NMR (400 MHz, DMSO, 27°C) 0.77 x A u to.

[0634] hA Ihn i i (7H, dd), 0.89 (9H, s), 1.38 (3H, d), 1.52 (3H, s),

[0635] 1.62 (3H, s), 1.77 (3H, d), 1.94 - 2.05 (1H, m), (S, E)-4-((S)-2-((R)-2-((2-(2-fluoro-3-methyl-5- 2.17 (3H, d), 2.66 (4H, s), 2.96 (3H, s), 3.95 - (methylamino)phenyl)propan-2- 4.05 (1H, m), 4.69 (1H, d), 4.92 (1H, t), 6.32 - yl)amino)propanamido)-N,3,3- 6.39 (1H, m), 6.47 - 6.53 (1H, m), 6.64 (1H, dd), trimethylbutanamido)-2,5-dimethy1hex-2- 8.55 (1H, s), 8.69 (1H, d), 8.98 (1H, s). enoic acid

[0636] P8 HPLC tR (method c)= 5.76 min, m / z (ES+), OX0T 1 fl

[0637] [M+H]+ = 5.21; 1H NMR (400 MHz, DMSO, 27°C) 0.79 (6H, dd), 0.88 (9H, s), 1.29 (3H, d), 1.38 (3H, s), 1.57 (3H, s), 1.78 (3H, d), 1.94 - 2.06 (S, E)-4-((S)-2-((R)-2-((2-(3-amino-4-fluoro-5- (1H, m), 2.18 (3H, d), 2.97 (3H, s), 3.71 - 3.81 methylphenyl)propan-2- (2H, m), 4.64 (1H, d), 4.93 (1H, t), 6.57 - 6.68 yl)amino)propanamido)-N,3,3- (2H, m), 6.69 - 6.78 (1H, m), 8.60 (1H, d), 8.71 trimethylbutanamido)-2,5-dimethy1hex-2- (1H, s), 8.93 (1H, s).

[0638] enoic acid

[0639]

[0640] Intermediate 31

[0641]

[0642] Pd-C (0.670 g, 0.63 mmol) was added to Intermediate 16 (2.8g, 6.30 mmol) in EtOH (20 mL) and water (10.00 mL) at 25°C under hydrogen. The resulting mixture was stirred at 25 °C for 4 hours. The mixture was filtered through a Celite pad. The solvent was removed under reduced pressure to afford (2-(5-((tert-butoxycarbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alanine (Intermediate 31, 2.500 g, 112 %) as a pale yellow liquid, m / z (ES+), [M+H]+ = 355; TFA, HPLC tR =MTI-106-PCT01-NP

[0643] 0.992 min. 1H NMR (300 MHz, DMSO) 6 1.48 (d, J = 7.5 Hz, 18H), 2.17 (d, J = 2.4 Hz, 3H), 3.04 (t, J = 7.0 Hz, 1H), 7.31 (dt, J = 15.5, 8.7 Hz, 2H), 9.31 (s, 1H).

[0644] Intermediate 32

[0645]

[0646] 31 32

[0647] Intermediate 31 (2.5 g, 7.05 mmol) was added in HCI-l,4-dioxane (20 mL) at 25°C under air. The resulting mixture was stirred at 25 °C for 5 hours. The solvent was removed under reduced pressure to afford the (2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)-D-alanine (Intermediate 32, 1.500 g, 84 %) as a pale yellow solid.

[0648] Intermediate 33

[0649] OH

[0650]

[0651] 33

[0652] TBS-CI (20.80 g, 138.02 mmol) in DCM (25 mL) was added dropwise to 1H-imidazole (17.90 g, 262.90 mmol) and 2-hydroxy-5-(hydroxymethyl)benzaldehyde (20 g, 131.45 mmol) in DCM (500 mL) at 0°C over a period of 2 hours under nitrogen. The resulting mixture was stirred at 0 °C for 2 hours. The reaction mixture was quenched with water (500 mL), extracted with DCM (2 x 300 mL) and the organic layer was dried over Na₂SO₄, filtered and evaporated to afford 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (Intermediate 33, 35.0 g, 100 %) as a colourless material, m / z (ES+), [M+Na]+= 289; NH4HCO3, HPLC tR = 1.505 min.

[0653] Intermediate 34MTI-106-PCT01-NP

[0654]

[0655] Silver(l) oxide p.a. (52.2 g, 225.22 mmol) was added to 4A MS (60 g, 0.00 mmol), (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (53.7 g, 135.13 mmol), 5 Intermediate 33 (30 g, 112.61 mmol) in MeCN (300 mL) at 0°C over a period of 2 hours under nitrogen. The resulting mixture was stirred at 60 °C for 16 hours. The mixture was filtered through a Celite pad. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% MeCN in water. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 34, 140 g, 213 %) as a pale yellow oil.

[0656] Intermediate 35

[0657]

[0658] LiOH in H2O (73.8 mL, 73.80 mmol) was added to Intermediate 34 (8.6 g, 14.76 mmol) in THF (75 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was adjusted to pH 6 with IN HCI. Extracted with n-BuOH (2 x 100 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford (2S,3S,4S,5R)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 35, 11.00 g, 168 %) as a white solid.

[0659] Intermediate 36MTI-106-PCT01-NP

[0660]

[0661] 35 36

[0662] 3-bromoprop-l-ene (3.53 g, 29.15 mmol) was added to DIEA (6.79 mL, 38.87 mmol) and Intermediate 35 (8.6 g, 19.43 mmol) in DMA (100 mL) at 25°C under nitrogen. The resulting mixture was stirred at 50 °C for 16 hours. The reaction mixture was quenched with water (200 mL), extracted with EtOAc (2 x 200 mL), washed with brine, the organic layer was dried over Na2SO4, filtered and evaporated to afford allyl (2S,3S,4S,5R)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylate (Intermediate 36, 8.20 g, 87 %) as a pale yellow oil.

[0663] Intermediate 37

[0664]

[0665] 36 37

[0666] allyl carbonochloridate (19.98 g, 165.77 mmol) was added dropwise to pyridine (6.56 g, 82.88 mmol), DMAP (5.06 g, 41.44 mmol) and Intermediate 36 (4 g, 8.29 mmol) in DCM (100 mL) at 0°C under nitrogen. The resulting mixture was stirred at 25 °C for 16 hours. The reaction mixture was quenched with water (100 mL), extracted with DCM (2 x 100 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% THF in petroleum ether. Pure fractions were evaporatedMTI-106-PCT01-NP

[0667] to dryness to afford allyl (2S,3S,4S,5R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-2-carboxylate (Intermediate 37, 9.50 g, 156 %) as a pale yellow solid.

[0668] Intermediate 38

[0669]

[0670] 37

[0671] TFA (2 mL) was added to Et3SiH (8 mL), tert-butyl carbamate (3.03 g, 25.86 mmol) and Intermediate 37 (5 g, 6.80 mmol) in MeCN (50 mL) at 0°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with water (100 mL), extracted with EtOAc (3 x 100 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 70% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford allyl (2S,3S,4S,5R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (Intermediate 38, 8.00 g, 163 %) as a white solid.

[0672] Intermediate 39

[0673]

[0674] MTI-106-PCT01-NP

[0675] Intermediate 38 (5g, 6.93 mmol), bis(4-nitrophenyl) carbonate (2.318 g, 7.62 mmol) and DIEA (1.815 mL, 10.39 mmol) in DMF (50 mL). The resulting mixture was stirred at RT for 3 hours. The reaction mixture was quenched with water (50 mL), extracted with EtOAc (2 x 50 mL), the combined organic layer was dried over Na2SO4, filtered and evaporated to afford residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford allyl (2S,3S,4S,5R)-3,4,5-tris(((allyloxy)carbonyl)oxy)-6-(2-(((tert-butoxycarbonyl)amino)methyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)tetrahydro-2H-pyran-2-carboxylate (Intermediate 39, 6.00 g, 98 %) as a white solid.

[0676] Intermediate 40

[0677]

[0678] HOPO (0.673 g, 6.06 mmol) was added to Intermediate 32 (1.4 g, 5.51 mmol), Intermediate 39 (5.86 g, 6.61 mmol) and DIEA (2.88 mL, 16.52 mmol) in DMF (30 mL) at 25°C under air. The resulting mixture was stirred at 25 °C for 4 hours. The reaction mixture was quenched with water (20 mL), extracted with EtOAc (3 x 20 mL), the organic layer was dried over Na2SO4, filtered and evaporated to afford yellow liquid, he crude product was purified by flash silica chromatography, elution gradient 0 to 100% THF in petroleum ether. Pure fractions were evaporated to dryness to afford (2-(5-((((4-(((3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-(((tert-butoxycarbonyl)amino)methyl)benzyl)oxy)carbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alanine (Intermediate 40, 1.200 g, 21.75 %) as a pale yellow liquid. Intermediate 41MTI-106-PCT01-NP

[0679]

[0680] Intermediate 40 (1.4 g, 1.40 mmol) was added in TFA (lOmL) and DCM (10.00 mL) at 25°C under air. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure to afford the (2-(5-((((4-(((3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3- (aminomethyl)benzyl)oxy)carbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alanine (Intermediate 41, 1.000 g, 70.5 %) as a pale yellow solid.

[0681] Intermediate 42

[0682]

[0683] 2,3,5,6-tetrafluorophenyl 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanoate (0.551 g, 1.20 mmol) was added to Intermediate 41 (1 g, 1.00 mmol) in THF (lOmL) and NaHCO3 (5 mL) at 25°C under air. The resulting mixture was stirred at 25 °C for 4 hours. The solvent was removed under reduced pressure to afford the crude as a pale yellow liquid. The crude product was purified by preparative HPLC (Column: Xselect CSH Prep C18 OBD Colum, 30*150nm, 5pm; Mobile Phase A: Water(0.1%FA), Mobile Phase B: ACN; Flow rate: 60 mL / min mL / min; Gradient: 5% B to 5% B in 2 min, 5% B to 21% B in 2.5 min, 51% B to 56% B in 10 min; Wave Length: 254nm / 220nm nm;

[0684] RTl(min): 10.2) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (2-(5-((((3-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(((2S,3R,4S,5S,6S)-6-((allyloxy)carbonyl)-3,4,5-tris(((allyloxy)carbonyl)oxy)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)-D-alanine (Intermediate 42, 0.605 g, 50.6 %) as a white solid.MTI-106-PCT01-NP

[0685] Synthesis of LP2

[0686]

[0687] Compound LP2 was synthesized according to general procedure I using Intermediate 42 (303 mg, 0.28 mmol), followed by procedure II and procedure Illa, and cleaved from resin following procedure IVb. The crude product was purified by prep HPLC with increasing amount of MeCN in water ( 5-90%). Fractions containing compound were evaporated to dryness to obtain (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LP2, 5.9 mg, 5.12 %) (m / z (ES+), [M+H]+ = 1098; TFA, HPLC tR = 6.33 min.

[0688] Intermediate 43MTI-106-PCT01-NP

[0689]

[0690] TFA (8 mL) was added to Et3SiH (32 mL), tert-butyl carbamate (15.28 g, 130.43 mmol) and Intermediate 34 (20 g, 34.32 mmol) in MeCN (200 mL) at 0°C under nitrogen. The resulting mixture was stirred at 0 °C for 16 hours. The reaction mixture was quenched with saturated NaHCO3 (200 mL), extracted with EtOAc (2 x 300 mL), and the organic layer was dried over Na2SO4, filtered and evaporated to afford a colourless residue. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% THF in petroleum ether. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-(((tert-butoxycarbonyl)amino)methyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 43, 18.80 g, 96 %) as a pale yellow material, m / z (ES+), [M+Na]+ = 592; TFA, HPLC tR = 1.088 min.

[0691] Intermediate 44

[0692]

[0693] Intermediate 43 (10 g, 17.56 mmol) in HCI / l,4-dioxane (40 mL) / l,4-dioxane (40 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 1 hour. The solvent was removed under reduced pressure to afford (2S,3R,4S,5S,6S)-2-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate hydrochloride (Intermediate 44) as a white material, m / z (ES+), [M+H]+ = 470; TFA, HPLC tR = 0.587 min.

[0694] Intermediate 45MTI-106-PCT01-NP

[0695]

[0696] 1-Methylimidazole (0.422 g, 5.14 mmol) was added to Intermediate 44 (1 g, 1.98 mmol) and 2,2-dimethyl-4-oxo-3,8,ll,14,17,20,23,26,29-nonaoxa-5-azadotriacontan-32-oic acid (1.392 g, 2.57 mmol) in dmf (10 mL). Chloro-N, N, N', N'-tetramethylformamidinium hexafluorophosphate (1.442 g, 5.14 mmol) was added after the reaction had been stirred for 20 minutes The resulting mixture was stirred at rt for 16 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 60% MeCN in water. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-(33,33-dimethyl-3,31-dioxo-6,9,12,15,18,21,24,27,32-nonaoxa-2,30-diazatetratriacontyl)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 45, 1.500 g, 76 %) as a colourless oil.

[0697] Intermediate 46

[0698]

[0699] DIEA (0.387 mL, 2.22 mmol) was added to bis(4-nitrophenyl) carbonate (0.371 g, 1.22 mmol) and Intermediate 45 (1.1 g, 1.11 mmol) in DMA (15 mL). The resulting mixture was stirred at rt for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 50% MeCN in water. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-(33,33-dimethyl-3,31-dioxo-6,9,12,15,18,21,24,27,32-nonaoxa-2,30-diazatetratriacontyl)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 46, 0.920 g, 71.7 %) as a colourless oil.

[0700] Synthesis of LP3MTI-106-PCT01-NP

[0701]

[0702] Intermediate 46 (142 mg in 1 mL DCM) added to a solution of P3 ( 312 mg, 0.6 mmol) and HOPO (40 mg) in DMF (1 mL). DIPEA (100 uL) added and mixture allowed to stir at RT. After 2 hrs, product observed with near-complete conversion of the free warhead. Mixture condensed in vacuo and redissolved in 1:1 TFA / DCM before being dried and dissolved into 4:1 MeOH / H2O, neutralised and then K2CO3 (10 eqv added). Mixture purified by reverse phase flash column chromatography and the combined fractions dried to give a clear colourless oil that was re-dissolved in DMF (1 mL) and 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (58.4 mg, 0.22 mmol) added with DIPEA (8 uL). Purified by reverse phase flash column chromatography (5 to 45% ACN+0.1% FA in Water+0.1% FA). Fractions containing compound were purified to dryness giving (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-(33-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-3,31-dioxo-6,9,12,15,18,21,24,27-octaoxa-2,30-diazatritriacontyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as colorless oil (LP3, 16.7 mg, 5.35%) (m / z (ES+), [M+H]+ = 1451; TFA, HPLC tR = 6.83 min.

[0703] Intermediate 47MTI-106-PCT01-NP

[0704] N

[0705] i

[0706] Boc

[0707]

[0708] 47

[0709] tert-butyl 3-(aminomethyl)-3-hydroxyazetidine-l-carboxylate (10 g, 49.44 mmol) was added to N-ethyl-N-isopropylpropan-2-amine (19.17 g, 148.33 mmol) and N-(benzyloxycarbonyloxy)succinimide (14.79 g, 59.33 mmol) in THF (150 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-hydroxyazetidine-l-carboxylate (Intermediate 47, 13.00 g, 78 %) as a yellow oil. m / z (ES+), [M+H]+ = 337; TFA, HPLC tR = 0.903 min

[0710] Intermediate 48

[0711]

[0712] Intermediate 47 (13 g, 38.65 mmol) was added to tert-butyl 2-bromoacetate (9.05 g, 46.38 mmol) and sodium hydride (1.855 g, 77.29 mmol) in THF (150 mL) under nitrogen at 0 °C. The resulting mixture was stirred at 25 °C for 2 hours. The reaction mixture was quenched with water (50 ml), extracted with EtOAc (3 x 150 ml), the top layer was dried over Na₂SO₄, filtered and evaporated to afford yellow oil. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford tert-butyl 3-((((benzyloxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (Intermediate 48, 9.00 g, 51.7 %) as a colourless oil. m / z (ES+), [M+H]+ = 451; TFA, HPLC tR = 1.125 min.

[0713] Intermediate 49

[0714] N

[0715] i

[0716] Boc

[0717]

[0718] MTI-106-PCT01-NP

[0719] Intermediate 48 (9 g, 19.98 mmol) was added to ROYER™ Palladium Catalyst Powder (CAS 7440-05-3, 6.07 g, 19.98 mmol) in THF (120 mL) under hydrogen. The resulting mixture was stirred at 25 °C for 6 hours. The reaction mixture was filtered through silica and washed with THF (3 x 50 mL). The solvent was removed under reduced pressure to afford tert-butyl 3-(aminomethyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (Intermediate 49, 6.00 g, 85 %) as a yellow oil. m / z (ES+), [M+H]+ = 317; TFA, HPLC tR = 0.882 min.

[0720] Intermediate 50

[0721] N

[0722]

[0723] i

[0724] Boc Boc

[0725] 49 50

[0726] Intermediate 49 (5 g, 15.80 mmol) was added to 9-fluorenylmethyl chloroformate (6.13 g, 23.70 mmol) and N, N-diisopropylethylamine (6.13 g, 47.41 mmol) in THF (50 mL) under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in petroleum ether. Pure fractions were evaporated to dryness to afford tert-butyl 3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-3-(2-(tert-butoxy)-2-oxoethoxy)azetidine-l-carboxylate (Intermediate 50, 3.66 g, 43.0 %) as a white solid, m / z (ES+), [M+H]+ = 539; TFA, HPLC tR = 1.230 min.

[0727] Intermediate 51

[0728]

[0729] To Intermediate 50 (615 mg, 1.14 mmol) in DCM (4 mL) was added TFA (4 mL). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure to give a crude product which was used without further purification. A solution of l-[(38-oxo-2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-yl)oxy]-2,5-pyrrolidinedione (708 mg, 1.03 mmol) and DIPEA (0.397 mL, 2.28 mmol) in DMF (5 mL) was added to crude 2-((3-(((((9H-MTI-106-PCT01-NP

[0730] fluoren-9-yl)methoxy)carbonyl)amino)methyl)azetidin-3-yl)oxy)acetic acid (436 mg, 1.14 mmol). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was purified by reverse phase column chromatography (5-60% MeCN in water +0.1% FA) to give 2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetic acid (Intermediate 51, 654 mg, 66.5 %) as a colourless oil. LCMS (15 min): 6.43 min; 954.7 [M+H]+

[0731] Intermediate 52

[0732] OH OAc

[0733] . OAc

[0734] 0 0

[0735]

[0736] 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (26.5 ml, 177.35 mmol) was added dropwise to a 1-L round bottom flask containing (2S,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml) at 21 °C. Next, 3-bromoprop-l-ene (16.72 ml, 193.47 mmol) was added to the reaction mixture dropwise over 10 minutes and the reaction was stirred at 21 °C for 24 hours. Reaction mixture was cooled to 0 °C and treated with pyridine (104 mL, 1289.60 mmol). Acetic anhydride (244 mL, 2579.20 mmol) was next added to the reaction mixture. The reaction was warmed up to room temperature and run for 2 hours at 21 °C. Reaction mixture concentrated under reduced vacuum and the remaining pyridine was azeotropically removed with toluene (1 x 100 mL). Crude material was diluted with DCM (65 mL) and cooled to 0 °C. 30% hydrobromic acid in acetic acid (175 mL, 3226.03 mmol) was next added to the reaction mixture at 0 °C. The reaction was warmed up to room temperature and run for 2 hours 30 minutes at 21 °C. Solvent was evaporated then the compound was purified by normal phase flash column chromatography to afford (2S,3S,4S,5R,6R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 52, 33 g, 48% yield) as a beige translucent material. LCMS (ESI) m / z 445.0 (M + Na)+.

[0737] Intermediate 53MTI-106-PCT01-NP

[0738]

[0739] To a vacuum-dried 500 mL round-bottom flask was added molecular sieves (4 A beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol) and acetonitrile (150 mL), producing a black slurry. To this slurry was added a solution of Intermediate 52 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) over 20 min followed by the addition of Intermediate 33 (13.6 g, 51.1 mmol) in acetonitrile (50 mL) in one portion. The resulting mixture was stirred vigorously at 20 °C for 16 h. After 16 h, the reaction mixture was filtered through a 5-cm pad of Celite and rinsed with dichloromethane (3 x 25 mL). Solvent was evaporated then the compound was purified by normal phase flash column chromatography to afford (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white material (Intermediate 53, 5.2 g, 34% yield). LCMS (ESI) m / z 626.3 (M + NH4)+.

[0740] Intermediate 54

[0741]

[0742] To a solution of Intermediate 53 (5.2 g, 8.6 mmol) in acetonitrile (40 mL) was added tert-butyl carbamate (3.8 g, 32.3 mmol), trifluoroacetic acid (2.0 mL, 25.9 mmol), and triethylsilane (4.1 mL, 25.8 mmol). Stirred for 2 h at 20 °C then solvent was evaporated. To the resulting colorless oil was added 1,4-dioxane (8 mL) and HCI (4.0 M in 1,4-dioxane, 50 mL, 200 mmol). The mixture was stirred at 20 °C for 30 min the solvent was evaporated. The resulting white powder was dissolved in DMSO (3 mL) then passed through cation-exchange resin pre-treated with methanol (WATERS PORAPAK CX). The desired compound was eluted off the resin with methanol to afford (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a white material (Intermediate 54, 2.5 g, 80% over 2 steps). LCMS (ESI) m / z 496.5 (M + H)+.MTI-106-PCT01-NP

[0743] Intermediate 55

[0744] Fmoc^ N

[0745] H

[0746]

[0747] To a stirred reactor containing Intermediate 54 (2.1 kg, 90.5% w / w, 3.57 mol) and acetonitrile (19 L) was added Fmoc-p-alanine (1.11 kg, 3.57 mol). The stirred mixture was cooled to 0°C. To this was added hexafluorophosphate azabenzotriazole tetramethyl uronium (1.36 kg, 3.57 mol) and N, N-diispropylethylamine (0.92 kg, 7.14 mol), and stirred for 4 hours, maintaining the temperature at 0°C. Water (19 L) and ethyl acetate (19 L) was added to the stirred mixture. The organic phase was separated and concentrated to ~19 L under vacuum. Ethyl acetate (28.5 L) was added to the concentrated solution and stirred at 20-25°C for 18 hours. The resulting suspension was filtered, the cake washed with ethyl acetate (3.87 L), and dried under vacuum to (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 55, 1.6 kg, 99% w / w, 56%). LCMS m / z 789 [M+H]+

[0748] Intermediate 56

[0749] H2N

[0750]

[0751] To a stirred reactor containing Intermediate 55 (1 kg, 1.27 mol) and tetrahydrofuran (10 L) at -45°C under nitrogen was added 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (385.98 g, 2.54 mol). The mixture was stirred at -45°C for four hours then diluted with acetonitrile (5 L) and quenched by the addition of hydrogen chloride in tert-butyl methyl ether solution (2.54 L, 2.0 M, 5.07 mol). The mixture was concentrated to ~5 L under vacuum, and diluted with n-heptane (5 L). The acetonitrile layer was collected containing (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-aminopropanamido)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 56, 3.88 kg of MeCN solution, 89.97% area, assumed 100%). LCMS m / z 566.6 [M+H]+MTI-106-PCT01-NP

[0752] Intermediate 57

[0753] H2N

[0754]

[0755] Intermediate 56 (271 mg, 2.10 mmol), 2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (299 mg, 0.79 mmol) and Intermediate 51 (500 mg, 0.52 mmol) in DMF (8 mL) at 25°C under nitrogen. The resulting mixture was stirred at 25 °C for 3 hours. The crude product was purified by flash C18-flash chromatography, elution gradient 0 to 90% MeCN in water. Pure fractions were evaporated to dryness to afford (2S,3R,4S,5S,6S)-2-(2-((3-(2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (Intermediate 57, 340 mg, 43.2 %) as a yellow oil. m / z (ES+), [M+H]+ = 1501

[0756] Intermediate 58MTI-106-PCT01-NP

[0757]

[0758] Charged THF (10.0 V), Intermediate 57 (1.0 eq.) and Bis-PNP (3.5 eq.) to reactor at 25 C. Charged DIEA (2.3 eq.) to reactor. Stirred for 12 hours at 20-25 C. Sampled for IPC. Charged NaCI aq. (20 V) to the reactor. Separated and washed with NaCI aq. (20 V). Collected the organic layer. Concentrated under vacuum at 30-40 C. The crude product was Purified by silica gel column (100% EtOAc and 0%-15% DCM / MeOH). The crude (2S, 3R, 4S, 5S, 6S)-2-(2-((3-(2-((3-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) methyl)-l-(2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenoxy)-6-((allyloxy) carbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate was obtained as yellow oil. (Intermediate 58, 2.4 g, 93.8%). MS ESI: [M / 2+H]+m / z 834

[0759] Intermediate 59MTI-106-PCT01-NP

[0760] OAc

[0761]

[0762] 59

[0763] Charge DMF (20 mL) and Intermediate 58 (2.0 g, 1.20 mmol) to a flask at 5-10 C. Charge HOPO (0.32 g, 2.88 mmol) and DIEA (0.78 g, 6.00 mmol). Charge P3 (1.87 g, 3.60 mmol). Stir for 2 hours at 25 C. Sample for IPC. Quench with 20 w / w% aq. citric acid and purify with prep-HPLC. (H2O in ACN= 0-100%) to give (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-2-fluoro-3-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid as white solid (Intermediate 59, 650 mg, 26.4%). ES+[M / 2+H]+=1024

[0764] Intermediate 60MTI-106-PCT01-NP

[0765] OAc

[0766] 59

[0767]

[0768] Charge MeOH (3.0 mL), Intermediate 59 (300 mg, 0.15 mmol), K2CO3 (101 mg, 0.73 mmol) and H2O (3.0 mL) to a flask at 0 C. Stir for 16 hours at 20-25 C. Sample for IPC. Purify by prep-HPLC (25-40% ACN / 0.1% FA in water). Collect the pure fraction, concentrate and freeze-dry to give (2S,3S,4S,5R,6S)-6-(2-((3-(2-((3-(aminomethyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid was obtained as white solid (Intermediate 60, 145 mg, 59.6%). ES+[M / 2+H=830 Synthesis of LP4MTI-106-PCT01-NP

[0769]

[0770] Charge DMF (2.7 mL ), Intermediate 60 (270 mg, 0.16 mmol) to a flask at 25 C. Charged DIEA (42 mg, 0.33 mmol) and N-Succinimidyl 3-maleimidopropionate (85 mg, 0.33 mmol) to a flask. Stir for 2 h at 25 C. Sample for IPC. Purify by prep-HPLC (25-50% MeCN / 0.1% FA in water). Collect the pure fraction, concentrate and freeze-dry to give (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as white solid. (LP4, 125 mg, 42.4%). ES+ [M / 2+H=906] 1H NMR (500 MHz, DMSO-d6) 6 12.45 (s, 2H), 9.66 (s, 1H), 8.34 (s, 1H), 8.15 (t, J = 6.3 Hz, 1H), 8.02 (s, 1H), 7.86 (t, J = 6.0 Hz, 1H), 7.40 - 7.33 (m, 1H), 7.30 (d, J = 2.2 Hz, 1H), 7.28 (t, J = 3.6 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 6.99 (s, 2H), 6.62 (dd, J = 9.2, 1.6 Hz, 1H), 5.54 (d, J = 4.1 Hz, 1H), 5.24 (d, J = 4.0 Hz, 1H), 5.05 (s, 2H), 4.92 (t, J = 9.9 Hz, 2H), 4.67 (d, J = 9.8 Hz, 1H), 4.31 (t, J = 5.6 Hz, 2H), 4.05 (d, J = 9.4 Hz, 1H), 3.96 - 3.91 (m, 1H), 3.90 - 3.82 (m, 3H), 3.72 (q, J = 10.5 Hz, 3H), 3.63 - 3.56 (m, 4H), 3.56 - 3.45 (m, 43H), 3.43 (dd, J = 5.8, 3.8 Hz, 5H), 3.35 (s, 5H), 3.24 (s, 3H), 2.93 (s, 4H), 2.45 - 2.33 (m, 5H), 2.29 (t, J = 6.5 Hz, 2H), 2.18 (d, J = 2.2 Hz, 3H), 1.93 (ddd, J = 13.5, 10.4, 6.7 Hz, 2H), 1.77 (d, J = 1.4 Hz, 4H), 1.37 (d, J = 14.5 Hz, 6H), 1.03 (d, J = 7.1 Hz, 3H), 0.92 (s, 9H), 0.80 (d, J = 6.5 Hz, 3H), 0.68 (d, J = 6.6 Hz, 3H).MTI-106-PCT01-NP

[0771] Intermediate 61

[0772] OAc

[0773] OAc

[0774]

[0775] Charge toluene (600 mL), Intermediate 33 (60 g, 0.225 mol), (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (138.91 g, 0.338 mol), 4AMS (60 g, 1.0 w / w) and Ag₂O (104.38 g, 0.450 mol) to the flask under N₂, Stir for 16 h at 25±5 C. Sample for IPT. Filter and wash the cake with EtOAc (1.2 L). Concentrate to dryness. Dissolve the residual mixture with EtOAc (50 mL). Charge the silica gel (150 g, 2.5 w / w) and active carbon (12 g, 0.2 w / w). Stir for 16 h at 20 C. Filter and wash the cake with EtOAc (600 mL). Concentrate to dryness. Purify by silica gel column (EtOAc: PE=0-20%) to give the crude (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(4-(((tert-butyldimethylsilyl) oxy) methyl)-2-formylphenoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate as white solid. (Intermediate 61, 60 g, 44.6%). ES+[M+H=597] Intermediate 62

[0776] OAc

[0777]

[0778] Charge MeCN (160 ml), Intermediate 61 (20 g, 0.034 mol) and BocNH₂ (4.71 g, 0.040 mol) to a flask. Cool to 0± 5 C. Charge Et3SiH (11.69 g, 0.101 mol) dropwise to the flask. Charge TFA (3.82 g, 0.034 mol) in MeCN (40 mL) solution at 0 C. Adjust the temperature to 10± 5 C. Stir for 16 h at 10± 5 C. Sample for IPT. Extract the reaction mixture with n-heptane (100 mL) three times to remove the Et3SiH and Et3SiH related impurities. Collect the bottom MeCN phase. Adjust the pH to 6-7 with saturated NaHCOa aqueous solution. Extract the system with MTBE (200 mL) and separation. Wash the organic phase with brine (100 mL). Collect the organic phase and concentrate to 4- 5 V at 45 C. Charge 1,4-dioxane (160 mL) to the system and concentrate dryness to give the crude (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(((tert-butoxycarbonyl) amino)MTI-106-PCT01-NP

[0779] methyl)-4-(((tert-butyldimethylsilyl) oxy) methyl) phenoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate as yellow oil. (Intermediate 62, 21 g, 89.8%). ES+ [M+H=697]

[0780] Intermediate 63

[0781] OAc

[0782]

[0783] Charge the crude Intermediate 62 (20.7 g, 0.030 mol) and 1,4-dioxane (103.5 mL) to a flask. Charge hydrochloric acid (3 M in dioxane) (207 mL) to the flask. Stir for 2 h at 25 C. Sample for I PT. Filter and wash the filter cake with MTBE (41.4 mL). Collect the solid and dry at 30 C under vacuum to give the crude (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(aminomethyl)-4-(hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate as white solid (Intermediate 63, 12 g, 83.7%). ES+[M+H=484]

[0784] Intermediate 64

[0785]

[0786] Charge the 3-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanoic acid (5.92 g, 0.019 mol) and MeCN (90 mL) to a flask. Cool to 0 C. Charge Intermediate 63 (9.0 g, 0.019 mol) and HATU (8.49 g, 0.022 mol) to the flask. Charge the DIEA (7.22 g, 0.056 mol) to a flask. Stir for 2 h at 0-5 C. Sample for IPT. Concentration to dryness. Purify by silica gel column (EtOAc: PE=0-100%) to give (2S,3R,4S,5S,6R)-2-(2-((3-((((9H-fluoren-9-yl) methoxy) carbonyl) amino) propanamido) methyl)-4-(hydroxymethyl) phenoxy)-6-(acetoxymethyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate as yellow solid (Intermediate 64, 4.2 g, 29.0%). ES+ [M+H=777], HPLC purity: 74.5 A%.

[0787] Intermediate 65MTI-106-PCT01-NP

[0788] OAc

[0789]

[0790] Charge the Intermediate 64 (4.0 g, 5.15 mmol) and THF (40 mL) to a flask. Cool to -40 C. Charge DBU (1.57 g, 10.30 mmol) to the flask. Stir for 2 h at -40 C. Sample for IPT. Concentrate to dryness to give the crude (2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2-((3-aminopropanamido) methyl)-4-(hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate as yellow oil (Intermediate 65, 4.7 g). ES+ [M+H=555]

[0791] Intermediate 66

[0792] OAc

[0793]

[0794] 66 Charge the Intermediate 51 (1.33 g, 2.39 mmol) to a flask containing Intermediate 65 (2.39 mmol) in 25 mL DMF. Cool to 0 C. Charge HATU (1.82 g, 4.78 mmol) and DIEA (1.85 g, 14.34 mmol) to the flask. Stir for 2 h at 0 C. Sample for IPT. Purify with reverse prep-HPLC purification (MeCN: 0.1% FA in water =25%-65%, 30min), collect the pure fraction and concentrate to dryness to give (2S,3R,4S,5S,6R)-2-(2-((3-(2-((3-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) methyl) -1-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido)MTI-106-PCT01-NP

[0795] propanamido) methyl) -4-(hydroxymethyl) phenoxy) -6-(acetoxymethyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate as white solid (Intermediate 66, 1.2 g, 16.3%). ES+ [M / 2+H=745],

[0796] Intermediate 67

[0797] OAc

[0798] OAc

[0799]

[0800] Charge the Intermediate 66 (700 mg, 0.470 mmol) and THF (7.0 mL) to a flask. Charge Bis-PNP (500 mg, 1.64 mmol) and DIEA (140 mg, 1.08 mmol) to the flask. Stir for 16 h at 25 C. Sample for IPT. Charge 15% aq. NaCI (using 1 M HCI to adjust the pH to 1-2, 7.0 mL) to quench the reaction at 10 C. Charge 1 M HCI (about 0.42 mL) to adjust the pH to 3-6 at 10 C. Stir for 30 min at 25 C. Separate and collect the upper organic phase. Concentrate to 4-5 V and charge MTBE (10.5 mL) dropwise to the concentrated solution at 25 C. Stir for 1 h at 25 C and stand for 25 min (The crude product precipitates to the bottom layer as oil). Collect the oil to give (2S,3R,4S,5S,6R)-2-(2-((3-(2-((3-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-((((4-nitrophenoxy) carbonyl) oxy) methyl) phenoxy)-6-(acetoxymethyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate as yellow oil (Intermediate 67, 510 mg, 65.6%). ES+ [M / 2+H=827.7], HPLC purity: 96.0 A%.

[0801] Intermediate 68MTI-106-PCT01-NP

[0802]

[0803] Charge the Intermediate 67 (510 mg, 0.308 mmol) and P3 (481 mg, 0.925 mmol) and DMF (5.1 mL) to a flask. Charge HOPO (82.19 mg, 0.740 mmol) and DIEA (159 mg, 1.23 mmol) to the flask. Stir for 2 h at 0 C. Sample for IPT. Purify with reverse prep-HPLC purification (MeCN: 0.1% FA in water= 25%-65%, 30min) to give (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-(((((9H-fluoren-9-yl) methoxy) carbonyl) amino) methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-(((2S,3R,4S,5S,6R)-3,4,5-triacetoxy-6-(acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) amino)-2-fluoro-3-methylphenyl) propan-2-yl) amino) propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid as yellow solid (Intermediate 68, 320 mg, 51.5%). ES+ [M / 2+H=1019],

[0804] Intermediate 69MTI-106-PCT01-NP

[0805] OAc

[0806] 68

[0807] OH

[0808]

[0809] 69

[0810] Charge the Intermediate 68 (320 mg, 0.157 mmol) and MeOH (3.2 mL) to a flask. Charge K2CO3 (217 mg, 1.57 mmol) and H2O (0.64 mL) to the flask. Adjust to 20-25 C. Stir for 2 h at 20-25 C. Sample for I PT. Purify by Prep-HPLC (MeCN: 0.1% FA in water= 20%-35%, 30min) to give (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-(aminomethyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) zetidin-3-yl) oxy) acetamido) ropanamido) ethyl)-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) amino)-2-fluoro-3-methylphenyl) ropan-2-yl) mino) ropanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid as white solid (Intermediate 69, 100 mg, 38.7%). ES+ [M / 2+H=825],

[0811] Synthesis of LP5MTI-106-PCT01-NP

[0812] OH

[0813]

[0814] Charge the Intermediate 69 (165 mg, 0.100 mmol) and DMF (1.7 mL) to a flask. Charge N-Succinimidyl 3-maleimidopropionate (53 mg, 0.200 mmol) and DIEA (26 mg, 0.200 mmol) to the flask. Adjust to 20-25 C. Stir for 2 h at 20-25 C. Sample for IPT. Purify by Prep-HPLC (MeCN: 0.1% FA in water= 20%-55%, 30min) to give (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanamido) methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) amino)-2-fluoro-3-methylphenyl) propan-2-yl) amino) propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid as a white solid (LP5, 63 mg, 35.0%). ES+ [M / 2+H=893], 1H NMR (500 MHz, DMSO-d6) 69.65 (s, 1H), 8.32 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 8.00 (d, J = 9.6 Hz, 1H), 7.87 (s, 1H), 7.45 (s, 1H), 7.36 (s, 1H), 7.27 - 7.17 (m, 4H), 6.99 (s, 1H), 6.66 (s, 1H), 6.59 (d, J = 9.3 Hz, 1H), 5.33 (d, J = 5.1 Hz, 1H), 5.05 (s, 1H), 4.90 (d, J = 9.6 Hz, 1H), 4.73 (d, J = 7.6 Hz, 1H), 4.67 (d, J = 9.8 Hz, 1H), 4.56 (s, 1H), 4.37 (d, J = 6.4 Hz, 1H), 4.21 (s, 1H), 4.05 (d, J = 10.8 Hz, 2H), 3.96 - 3.92 (m, 2H), 3.89 (s, 1H), 3.87 (s, 1H), 3.75 (s, 1H), 3.72 (d, J = 8.0 Hz, 2H), 3.63 (s, 1H), 3.62 (s, 1H), 3.59 (t, J = 6.8 Hz, 4H), 3.51 (d, J = 3.8 Hz, 25H), 3.44 - 3.42 (m, 4H), 3.24 (s, 3H), 2.92 (s, 2H), 2.64 (s, 2H), 2.39 (s, 5H), 2.31 (s, 2H), 2.29 (d, J = 6.8 Hz, 2H), 2.17 (s, 3H), 2.04 - 1.97 (m, 4H), 1.91 (s, 1H), 1.76 (s, 2H), 1.46 (s, 2H), 1.37 (d, J = 14.3 Hz, 4H), 1.24 (s, 9H), 1.02 (d, J = 7.0 Hz, 3H), 0.92 (s, 6H), 0.86 (t, J = 6.7 Hz, 3H), 0.79 (d, J = 6.4 Hz, 3H), 0.68 (d, J = 6.6 Hz, 3H).

[0815] Intermediate 70MTI-106-PCT01-NP

[0816] OAc

[0817]

[0818] 70

[0819] To P2 (96 mg,.19 mmol) was added a solution of Intermediate 58 (312 mg, 0.19 mmol) in DMF (3 mL). DIPEA (0.199 mL, 1.14 mmol) and 2-hydroxypyridine 1-oxide (21.11 mg, 0.19 mmol) were added and the reaction mixture stirred at room temperature for 2.5 h. The reaction mixture was purified by RP chromatography (20-40% MeCN in water +0.1% FA) to give (S, E)-4-((S)-2-((R)-2-((2-(3-((((3-((3-(2-((3-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-(((2S,3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)benzyl)oxy)carbonyl)amino)-5-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid (Intermediate 70, 190 mg, 49.9 %) as a white solid. LCMS (15 min) tR: 6.53 min; 2004.5 [M+H]+

[0820] Intermediate 71MTI-106-PCT01-NP

[0821]

[0822] To Intermediate 70 (190 mg, 0.09 mmol) in MeOH (2 mL) and water (1 mL) was added potassium carbonate (0.108 mL, 1.90 mmol). The reaction mixture was stirred at room temperature for 2 h. acetic acid (0.217 mL, 3.79 mmol) was added and the reaction mixture purified by RP chromatography (5-35% MeCN in water +0.1% FA) to give (2S,3S,4S,5R,6S)-6-(2-((3-(2-((3-(aminomethyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)-4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-5-methylphenyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (Intermediate 71, 109 mg, 70.0 %) as a white solid. LCMS (15 min) tR: 4.67 min; 1642.8 [M+H]+ Synthesis LP6MTI-106-PCT01-NP

[0823] OH

[0824] 71

[0825]

[0826] To Intermediate 71 (109 mg, 0.07 mmol) in DMF (1 mL) was added 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (35.3 mg, 0.13 mmol) and DIPEA (0.069 mL, 0.40 mmol). The reaction mixture was stirred at room temperature for 30 mins. The reaction mixture was purified by RP chromatography (15-30% MeCN in water +0.1% FA) to give (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)methyl)-l- (2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (LP6, 73.7 mg, 61.9 %) as a white solid. LCMS (15 min) tR: 5.23 min; 1793.7 [M+H]+ Cytotoxicity data (i) Pl to P8

[0827] Cells in exponential growth phase were seeded in 96-well culture plates at 2000 cells / well in 80 pl cell culture media and allowed to adhere overnight. Then the cells were treated by adding 20 pl of serial diluted compound with various concentration in duplicate. After 3 (MCF7 and JIMT-l) or 6 days (NCI-N87, N87-Control, N87-Pgp) treatment, the cell viability was determined by CELLTITER-GLO Luminescent Viability Assay (PROMEGA) according to the manufacturer's protocol. Viability was calculated as a percentage of the viable cells cultured in media or treated with DMSO solventMTI-106-PCT01-NP

[0828] control. EC50values were determined by using logistic non-linear regression analysis with PRISM software (GRAPHPAD).

[0829] NCI-N87, MCF7 and JIMT-1 cell lines were obtained from ATCC. N87-Pgp was a P-gp overexpressing NCI-N87 line with transfected MDR1 gene, and N87-Control is a control NCI-N87 line with mocktransfection. Those cells were grown in RPMI1640 media with 10% FBS (NCI-N87, MCF7, N87-Control and N87-Pgp) and DMEM media with 10% FBS (JIMT-1).

[0830] (GraphPad, San Diego, CA).

[0831] Tubulin binding assay

[0832] Tubulin polymerisation assay kit was purchased from CYTOSKELETON INC. ( Cat# BK011P) and performed following the general manufacturer's suggested protocol. Reading were performed using a SPECTRAMAX plate reader with the below instrument settings.

[0833] Settings SPECTRAMAX

[0834] Fluorescence Ex - 340

[0835] Em - 440

[0836] Gain Med

[0837] Reads per well Flashes per read = 6

[0838] Fluor reading from top top

[0839] Integration PMT gain = Medium

[0840] Shaking First read = 5 sec

[0841] Plate type 96 Well Corning Half Area opaque

[0842] Number of Reads 61 read; 1 read per minute

[0843] The assays plate was pre-warmed to 37°C at least 10 minutes prior to initiating the experiment. The final concentration of tubulin reaction mix was 2 mg / mL of tubulin in PIPES pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, 1.0 mM GTP and 15% glycerol. Compounds in a 10 mM DMSO solution were tested at 3mM. The percentages of inhibition of the test articles (final concentration in the well 3mM), were expressed as a percentage of that measured for MMAE by using the terminal fluorescence readings. Monomethylauristatin E (MMAE) was purchased from MED CHEM EXPRESS.

[0844] Table 2: Biological Data for Example PayloadsMTI-106-PCT01-NP

[0845] Example JIMT-1 EC50MCF7 EC50NCI-N87 EC50RI (NCI-N87- Tub inh % wrt Payload (nM) (nM) (nM) PgP EC50 / NCI- MMAE (3μM)

[0846] N87 EC50)

[0847] Pl 12.4 NA 6.42 2.6 67 Reference

[0848] P2 10.41 3.48 3.01 3.9 98

[0849] P3 1.5 0.74 0.45 4.2 88

[0850] P4 4.0 1.3 1.03 6.0 95

[0851] P5 7.4 2.2 1.6 5.3 98

[0852] P6 0.75 0.43 0.58 5.4 100

[0853] P7 1.6 1.0 1.1 3.1 100

[0854] P8 2.9 1.3 1.8 4.0 100

[0855]

[0856] ADC conjugation

[0857] 2.5- equiv. of TCEP (50mM in PBS pH 7.4, 10 prnol, 0.020 mL) was added to solution of antibody in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA). The reduction mixture was allowed to react at RT for 2 hours with gentle shaking. 7 equiv. of Linker payload (lOmM in DMA, 8.15 pmol, 0.08 mL) was added as a DMA solution to the reduced antibody solution (5mg / mL, 1.116 pmol), for a 10% (v / v) final DMA concentration. The resulting solution was mixed for 1 hour at room temperature. The DAR for the conjugate was assessed on a fully reduced reaction sample (DTT IM, sodium borate buffer pH 8.5) using a UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column eluting with a gradient of water and acetonitrile, measured at 214 nm

[0858] A reference ADC (ADC-1) was prepared by conjugating Herceptin-WT to LP1 (Mc-vc-PAB-MMAE). Negative control ADCs (ADC-7, ADC-8, ADC-9) were prepared by conjugating a negative control antibody NIP228 (as disclosed in WO2015127273A1, the contents of which are incorporated by reference) and LP4, LP5 or LP6. NIP228 is an IgGl that has complementary binding to transferrin, a transmembrane protein whose expression is low on the tested cell lines, therefore making it a suitable negative control.MTI-106-PCT01-NP

[0859] Table 3- ADC Conjugation

[0860] ADC mAb TCEP equiv. LP LP equiv. DAR (214nm)

[0861] ADC-1 (Reference) Herceptin-WT 2.6 LP1 6.5 4.0

[0862] ADC-2 Herceptin-WT 2.5 LP2 7 3.7

[0863] ADC-3 Herceptin-WT 2.5 LP3 8 3.5

[0864] ADC-4 Herceptin-WT 2.7 LP4 10.5 4.1

[0865] ADC-5 Herceptin-WT 3.0 LP5 10.5 4.1

[0866] ADC-6 Herceptin-WT 3.2 LP6 9 3.8

[0867] ADC-7 (Control) Nip228-WT 4.5 LP4 5 3.7

[0868] ADC-8 (Control) Nip228-WT 3.3 LP5 5 3.7

[0869] ADC-9 (Control) Nip228-WT 3.3 LP6 9 3.6

[0870]

[0871] Cytotoxicity data (ii) ADCs

[0872] Medium from sub-confluent (80-90% confluency) NCI-N87, MDAMB468, SKOV3 and SKOV3 GUSB KO in a T175 flask was aspirated and the flask rinsed with PBS (about 20 ml) and emptied. TrypLE (4 ml) was added, the flask returned to the 37 °C gassed incubator for up to about 5 minutes, then rapped sharply to dislodge and dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube, diluted with growth medium to a final volume of 15 ml, then centrifuged (400g for 5 min). The supernatant was aspirated, and the pellet re-suspended in 10ml culture medium. Repeated pipetting may be necessary to produce monodisperse cell suspensions. The cell concentration and viability are measured of trypan blue cell-stained cells, using the LUNA II. For the ADCs, a stock solution (300pL) of Antibody Drug Conjugate (ADC) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 9x 5-fold dilutions of the previous ADC solution were made in a ImL deep 96 well plate by serial transfer of 30 pl onto 270 pl of cell culture medium. ADC dilution was dispensed (10 pl / well) into 4 replicate wells of the 384-well plate, containing 30 pl cell suspension seeded the previous day. Control wells received 10 pl cell culture medium. The 384-well plate containing cells and ADCs / payloads was incubated at 37 °C in a CO2-gassed incubator for 6 days. At the end of the incubation period, plates were equilibrated to room temperature for lOminMTI-106-PCT01-NP

[0873] before CellTiter-Glo (Promega) was dispensed (40 pl per well) into each well. IC50was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose response, log(inhibitor) vs. Response - Variable slope (four parameters), constrain at the bottom = 0. Cell growth medium for the cells was RPMI supplemented with 10% Fetal Bovine Serum. The IC50values in each figure were determined by fitting data to a sigmoidal dose-response curve using GraphPad Prism software v9 (GraphPad, San Diego, CA).

[0874] Table 4- SKOV33D, NCI-N87 and MDAMB468 in vitro cytotoxicity data for ADC-1 (Reference), ADC-2 to ADC-6 and ADC-7 to ADC-9 (Controls)

[0875] IC5o NCI-N87(pg / mL) IC50 MDAMB468 (pg / mL) IC50 SKOV33D (Her2- control) (Pg / mL) ADC-1 0.003 >50 0.003 (Reference)

[0876] ADC-2 0.02 >50 0.01 ADC-3 0.014 >50 0.04 ADC-4 0.01 >50 0.03 ADC-5 0.01 >50 0.03 ADC-6 0.02 >50 0.09 ADC-7 (Control) >50 >50 >50 ADC-8 (Control) >50 >50 >50 ADC-9 (Control) >50 >50 >50

[0877]

[0878] The above description of illustrative embodiments is intended only to acquaint others skilled in the art with the Applicant's specification, its principles, and its practical application so that others skilled in the art may readily adapt and apply the specification in its numerous forms, as they may be best suited to the requirements of a particular use. This description and its specific examples, while indicating embodiments of this specification, are intended for purposes of illustration only. This specification, therefore, is not limited to the illustrative embodiments described in this specification, and may be variously modified. In addition, it is to be appreciated that various features of the specification that are, for clarity reasons, described in the context of separate embodiments, also may be combined to form a single embodiment. Conversely, various features of the specification that are, for brevity reasons, described in the context of a single embodiment, also may be combined to form sub-combinations thereof.

Claims

MTI-106-PCT01-NPClaims1. A conjugate of Formula (I)Ab – (GA–JA–DM)k(I)or a pharmaceutically acceptable salt thereof, whereinAb is an antibody or antigen-binding fragment thereof,k is an integer from 1 to 10,each GAis independently a conjugation group conjugated to the antibody or antigen-binding fragment thereof,each DMis independently a group of Formula (ID)whereineither (i) R1and R2are independently selected from Ci-g alkyl, C3-6 cycloalkyl and H, or (ii) R1and R2, together with the carbon atom to which they are attached, form a cyclopropane or cyclobutane ring, R3and R10are independently C1-3 alkyl,R4and R5are independently C1-6alkyl or C3-6cycloalkyl,R6, R7, R8and R9are independently H, C1-6alkyl or C3-6cycloalkyl,R11is H, C1-6 alkyl, C3-6 cycloalkyl or phenyl, andRX1is C1-6 alkyl or C3-6 cycloalkyl, Rx2is H or F, and Rx3is H or F,each JAis independently either(i) a group of Formula (IA)whereinE is (CH2)n1, wherein n1 is 0, 1, 2 or 3,R12is C1-4 alkyl or H,MTI-106-PCT01-NPOH HO^ A JDHO O R14AR13Ais, wherein R14Ais CO2H or CH2OH,X1is (CH2)n2, wherein n2 is 0, 1, 2 or 3,Y is O or NRB, wherein RBis H, C1-4alkyl or C3-4 cycloalkyl,Z1is (CH2)n3, wherein n3 is 0, 1, 2, 3, 4 or 5,m is an integer from 2 to 17,p is 1 or 0, andq is 1 or 0, or(ii) a group of Formula (IB)whereinZ2is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5,X2and X3are independently (CH2)ns, wherein n5 is 0, 1, 2, 3, 4 or 5,j is an integer from 0 to 16,r is 1 or 0, andOH HO^ A JDHand wherein(GA) indicates the point of attachment to GA, and(DM) indicates the point of attachment to DM.

2. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed claim 1, wherein each DMis independently a group of Formula (IDA)MTI-106-PCT01-NP(IDA)3. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 1 or claim 1, wherein each DMis independently a group of Formula (IDB)Rx1(IDB)4. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 3, wherein each DMis a group of Formula (IDC)H (IDC)N N OHH5. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, wherein each JAis independently a group of Formula ( I A)whereinE is (CH2)n1, wherein n1 is 0, 1, 2 or 3,R12is Ci-4 alkyl or H,MTI-106-PCT01-NP14Ais CO2H or CH2OH,X1is (CH2)n2, wherein n2 is 0, 1, 2 or 3,Y is O or NRB, wherein RBis H, C1-4 alkyl or C3-4 cycloalkyl,Z1is (CH2)n3, wherein n3 is 0, 1, 2, 3, 4 or 5,m is an integer from 2 to 17,p is 1 or 0, andq is 1 or 0.

6. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, wherein m is 9, 10, 11, 12 or 13.

7. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, wherein R12is CH3.

8. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in anyone of claims 1 to 7, whereinR9. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 8, wherein E is CH2.

10. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 9, wherein X1is CH2.MTI-106-PCT01-NP11. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 10, wherein Y is O.

12. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 11, wherein Z1is (CH2)2-13. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 12, wherein p is 1.

14. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 13, wherein q is 1.

15. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein each JAis a group of Formula ( I Al)(IA1)16. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 14, wherein each JAis a group of Formula (IA2)MTI-106-PCT01-NP17. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, wherein each JAis independently a group of Formula (IB)whereinZ2is (CH2)n4, wherein n4 is 0, 1, 2, 3, 4 or 5,X2and X3are independently (CH2)n5, wherein n5 is 0, 1, 2, 3, 4 or 5,j is an integer from 0 to 16,r is 1 or 0, andOHR13Bis, wherein R14Bis CO2H or CH2OH.

18. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4 or claim 17, wherein Z2is (CH2)2-19. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4, claim 17 or claim 18, wherein r is 1.MTI-106-PCT01-NP20. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in anyone of claims 1 to 4 or any one of claims 17 to 19, whereinR13Bis21. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4 or any one of claims 17 to 20, wherein X2and X3and both CH?.

22. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 4 or any one of claims 17 to 21, wherein j is an integer from 0 to 8.

23. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 22, wherein GAis independently selected fromwherein RKis H or CH3, RLis C1-6alkyl,and indicates the point of attachment to the antibody or antigen-binding fragment thereof.MTI-106-PCT01-NP24. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 23, wherein GAis25. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 23 or claim 24, wherein each (GA-JA-DM) is26. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in claim 23 or claim 24, wherein each (GA-JA-DM) is1. The conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 26, wherein k is an integer from 2 to 8.MTI-106-PCT01-NP28. A pharmaceutical composition comprising a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 27, and a pharmaceutically acceptable excipient.

29. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 27, or a pharmaceutical composition as claimed in claim 28, for use in therapy.

30. A conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 27, or a pharmaceutical composition as claimed in claim 28, for use in the treatment of cancer.

31. A method of treating cancer in a patient in need thereof comprising administering to the patient an effective amount of a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 27.

32. Use of a conjugate of Formula (I) or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 27, in the manufacture of a medicament for the treatment of cancer.

33. A compound of Formula (II)GB–JB–DM(II)or a salt thereof, wherein GBis a conjugation group for conjugation to an antibody or antigenbinding fragment thereof,DMis as defined for a conjugate of Formula (I) in any one of claims 1 to 4,each JBis independently either(i) a group of Formula (IIA)MTI-106-PCT01-NPwherein E, R12, R13A, X1, Y, Z1, m, p and q are as defined for a conjugate of Formula (I) in claim 1 or any one of claims 5 to 14, or(ii) a group of Formula (I I B)wherein j, r, R13B, X2, X3, and Z2are as defined for a conjugate of Formula (I) in claim 1 or any one of claims 17 to 22,and wherein (GB) indicates the point of attachment to GB, and (DM) indicates the point of attachment to DM.

34. The compound of Formula (II) or a salt thereof, as claimed in claim 33, wherein GBis selected fromMTI-106-PCT01-NPwherein X1is CH or N,h is 0 or 1,Hal is Cl, Br or I,RKis H or CH3, andRLis C1-6 alkyl.

35. The compound of Formula (II) or a salt thereof, as claimed in claim 33 or claim 34, wherein36. The compound of Formula (II) or a salt thereof, as claimed in claim 34, that is (2S,3S,4S,5R,6S)-6-(4-((((3-(2-(((R)-l-(((S)-l-(((S, E)-5-carboxy-2-methy1hex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)amino)-l-oxopropan-2-yl)amino)propan-2-yl)-4-fluoro-5-methylphenyl)carbamoyl)oxy)methyl)-2-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanamido)methyl)-l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-MTI-106-PCT01-NPoyl)azetidin-3-yl)oxy)acetamido)propanamido)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acidOHsalt thereof.

37. The compound of Formula (II) or a salt thereof, as claimed in claim 34, that is (S, E)-4-((S)-2-((R)-2-((2-(5-((((3-((3-(2-((3-((3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl) propanamido) methyl)-1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oyl) azetidin-3-yl) oxy) acetamido) propanamido) methyl)-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) amino)-2-fluoro-3-methylphenyl) propan-2-yl) amino) propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acidOHor a salt thereof.

38. A compound of Formula (III)Rx1MTI-106-PCT01-NPor a salt thereof, whereineither (i) R1and R2are independently selected from C1-6alkyl, C3-6cycloalkyl and H, or (ii) R1and R2, together with the carbon atom to which they are attached, form a cyclopropane or cyclobutane ring, R3and R10are independently C1-3 alkyl,R4and R5are independently C1-6alkyl or C3-6cycloalkyl,R6, R7, R8and R9are independently H, C1-6alkyl or C3-6cycloalkyl,R11is H, C1-6alkyl, C3-6cycloalkyl or phenyl, andRx1is C1-6alkyl or C3-6cycloalkyl, Rx2is H or F, and Rx3is H or F.

39. The compound of Formula (III) or a salt thereof, as claimed in claim 38, that is a compound of Formula (IIIA)(IIIA),or a salt thereof.

40. The compound of Formula (III), or a salt thereof, as claimed in claim 38 or claim 39, that is a compound of Formula (IIIB)Rx1(IIIB),or a salt thereof.

41. The compound of Formula (III), or a salt thereof, as claimed in any one of claims 38 to 40, that is (S, E)-4-((S)-2-((R)-2-((2-(5-amino-2-fluoro-3-methylphenyl)propan-2-yl)amino)propanamido)-N,3,3-trimethylbutanamido)-2,5-dimethy1hex-2-enoic acid,or a salt thereof.

Images