Compounds for complex formation of rare earth elements and / or s-, p-, d-block metals, their coordination compounds, peptide conjugates, methods for preparing them, and their uses.

Abstract

Title: Compounds for complexing rare earth elements and / or s-, p-, d-block metals, their coordination compounds, peptide conjugates, methods for their preparation and their uses The present invention relates to compounds of general formula (I), [Formula 1] JPEG2024533430000465.jpg31169 where Y is nitrogen or N-oxide and R 1 is hydrogen; halogen; hydroxyl; azide; amine; trifluoromethyl; -COO-alkyl; -CH2CH(OMe)2; -SH; -SO3H; -SO2Ar (where Ar is phenyl); NO2; [Case 2] JPEG2024533430000466.jpg7169 ;C6~C 10 Aryl (optionally substituted with -NH2, -NO2, -N, -COOH, -CH2Cl and / or -CH2COOH); C7-C 10 arylalkyl (optionally substituted with -NH2, -NO2, -COOH, -CH2Cl, and / or -CH2COOH); R 2 is an azide group; or [C3] JPEG2024533430000467.jpg9169 (wherein n is an integer ranging from 1 to 3); A is H;-(CH2) n COOH; -CH(CH3)COOH; -CH2P(=O)(OR)2 (where R is H or alkyl); -CH((CH2) n COOH)COOH;-CH((CH2) n -NH2)COOH, where n is an integer from 1 to 3; -CH2C(=O)(NH2); -CH2C(=O)(NH)-CH2COOH; -CH2P(=O)(OH)(Ar), where Ar is phenyl optionally substituted with C1-C6 alkyl; Z is [C4] JPEG2024533430000468.jpg60169 selected from the group consisting of; R 3 teeth, [C5] JPEG2024533430000469.jpg6169 And R 4 is H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; C1-C6 alkyl; C3-C6 cycloalkyl; -CF3; -(CH2) n NH2;-(CH2) n NHFmoc;-(CH2) n NHBoc;adamantyl; [C6] JPEG2024533430000470.jpg7169 R 5 is R 1 ,or, [C7] JPEG2024533430000471.jpg6169 and / or R 2 and R 3 Both, forming a 1,2,3-triazole group; with the proviso that at most one A is H. The invention further relates to coordination compounds thereof, peptide conjugates thereof, methods for their preparation and their use.

Description

Detailed description of the invention

[0001] [Current state of technology] The present invention relates to novel macrocyclic compounds suitable as crosslinking chelating agents for complexing rare earth elements and / or s-, p-, d-block metals to form extremely stable coordination compounds. These coordination compounds are suitable for use as labels for the quantitative detection of peptide conjugates and / or as contrast agents for magnetic resonance imaging. The present invention further relates to a method for preparing the chelating agents and a method for tracking peptide / protein-containing pharmaceuticals.

[0002] [Background technology] Metallic elements are used in biomedical applications such as imaging contrast agents, radiotherapy agents, or labels for bioanalysis. Most of these applications require the metals to be bound to stable chelates, allowing them to covalently bond with other molecules, such as peptide or antibody-based targeting vectors. The most common example of a chelating agent applicable to most metallic elements is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and its derivatives. In addition, a wide range of chelating agents specific to particular metal elements have been developed. [Price EW, Orvig C. (2014), Chem. Soc. Rev. 43(1), 260 - 290].

[0003] Rare earth elements (scandium-Sc, yttrium-Y, lanthanum-La, cerium-Ce, praseodymium-Pr, neodymium-Nd, promethium-Pm, samarium-Sm, europium-Eu, gadolinium-Gd, terbium-Tb, dysprosium-Dy, holmium-Ho, erbium-Er, thulium-Tm, ytterbium-Yb, and lutetium-Lu) are a group of metals that offer a wide range of medical applications. 90 Y, 153 Sm and 177 Lu-based radiopharmaceuticals have received FDA approval. 166Clinical trials are underway for Ho, and there are also others that exhibit favorable characteristics for positron emission tomography (PET), single photon emission computed tomography (SPECT), or therapy ( 44 Sc, 47 Sc, 86 Y, 149 Pm, 159 Gd, 149 Tb, 161 Tb, 165 Dy, 161 Ho, 169 Er and 175 Yb). Stable non-radioactive Gd chelates are clinically used as contrast agents in magnetic resonance imaging (MRI). Stable isotopes of rare earth elements are also used as labels for analytical purposes. Compounds of interest labeled in this way can be visualized, traced, and quantified based on the intrinsic luminescence or isotope mass of the element with zero background in biological systems. The intrinsic isotope mass is particularly useful, and typically, with an inductively coupled plasma mass spectrometer (ICP-MS), many compounds can be quantified simultaneously in one analysis (multiplexing) [Bodenmiller B. et al. (2012), Nat. Biotechnol. 30(9), 858-867]. Other metal elements belonging to the s-, p-, d-blocks of the periodic table, such as 89 Sr, 223 Ra, 117m Sn, 212 Pb, 213 Bi, 64 Cu, 225 Ac are used in radiopharmaceutical compounds for imaging or therapy.

[0004] For practical applications, metal chelates must be extremely stable so that metal ions do not easily escape from the chelating agent or the support molecule. In this respect, thermodynamic stability constants are insufficient information, as most applications are carried out under conditions entirely different from thermodynamic equilibrium (e.g., in vivo). Instead, kinetic inertness, which characterizes the rate at which the metal ions desorb from the chelate under given conditions, must be considered. Kinetic inertness is strongly correlated with the rigidity of the chelating agent. DTPA-type (diethylenetriaminepentaacetic acid) acyclic chelating agents have a more flexible structure, lower kinetic inertness, and are more rigid than DOTA-type macrocyclic chelating agents. In particular, chelates with high kinetic inertness are desirable for in vivo applications where the metal chelate is exposed to excessive competing biochelating agents and metal ions. For this reason, rigid, kinetically inert macrocyclic chelating agents are preferred over flexible acyclic chelating agents. For example, approved or under-development radionuclides 177 Substantially all radiopharmaceuticals based on Lu (half-life 6.7 days) utilize the macrocyclic chelating agent DOTA. This is because, in order to achieve the desired therapeutic effect, the metal must remain bound to the target molecule in vivo for several weeks. Similarly, in MRI contrast agents, macrocyclic chelates of gadolinium(III) are preferred over acyclic DTPA types. Recently, it has been found that large amounts of free gadolinium are released in vivo from acyclic agents and deposit in the human brain for long periods [Fur ML, Caravan P. (2019), Metallomics 11(2), 240-254]. In response to this finding, and to prevent potential harm to patients, the use of acyclic MRI contrast agents is strictly restricted worldwide by drug regulatory agencies. Therefore, the importance of the kinetic inertness of metal chelates is increasing in medical and other applications, and there is a need for chelating agents that provide higher inertness.

[0005] A method to further enhance the kinetic inertness is to reinforce the macrocyclic chelating agent with another ring and harden it. For example, the crosslinked macrocyclic chelating agent cb-TE2A (4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane) provides a chelate with copper(II) ions that is far more inert than its non-crosslinked analogue [Boswell CA et al. (2004), J. Med. Chem. 47(6), 1465-1474]. Another similar chelating agent, cb-TEDPA (6,6'-((1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)bis(methylene))dipicolinic acid), has been shown to provide an exceptionally inert lanthanide(III) chelate [Rodriguez-Rodriguez A. et al. (2016), Inorg. Chem. 55(5), 2227-2239]. Furthermore, another cyclene-based crosslinking chelating agent provided an extremely inert copper(II) chelate [Esteves, CV et al. (2013), Inorg. Chem. 52(9), 5138-5153]. A common feature of these chelating agents is that the crosslinks are independent of the pendant arms to which they coordinate and are oriented opposite to the pendant arms in the chelate. Although chelating agents have been developed in which the aforementioned crosslinking connects the pendant arm itself, the effect on the stability of the chelate was strongly negative [Vipond, J. et al. (2007), Inorg. Chem. 46(7), 2584-2595].

[0006] Furthermore, excessively high kinetic inertness can be counterproductive, and there are practical limitations. This is because the energy barrier that the metal ion must overcome to exit the chelate is similar to the barrier that the metal ion must overcome to enter the chelate during chelate formation. Generally, the more inert the chelate, the more difficult its formation becomes. Increasing the reaction time at high temperatures accelerates the formation kinetics. However, such reaction conditions are often incompatible with the half-lives of metal nuclides in the preparation of highly sensitive targeting vectors (e.g., antibodies) and / or radiopharmaceuticals.

[0007] There is still a strong demand for a new type of chelating agent that can rapidly form kinetically inert metal complexes under mild synthetic conditions.

[0008] [Disclosure of the Invention] We have overcome the problems of the background art with a novel type of chelating agent that forms a crosslink after the complex formation of the metal ion, thereby hardening the structure and increasing kinetic inertness. The crosslink is formed between two coordination pendant arms based on a cycloaddition reaction between the alkyne and the azide substituent on the opposite pendant arm. This reaction does not require catalysis by Cu(I) ions as is usually the case and occurs spontaneously after the formation of the non-crosslinked chelate. Thus, the chelating agent acts as a unidirectional trap of the metal ion. The initial formation of the non-crosslinked chelate is relatively fast. Subsequently, the crosslink is formed and the metal is trapped within the chelating agent. In the case of rare earth elements, the resulting crosslinked chelate exhibits extremely high kinetic inertness, up to six orders of magnitude greater than similar chelates using DOTA. Such extremely high inertness allows it to withstand harsh hydrolysis conditions in concentrated acid, enabling a new use of the chelate as an analytical label, where the chelate in the solution can be directly quantified using liquid chromatography-mass spectrometry (LC-MS). The use of LC-MS is advantageous because it is a much more common instrument than ICP-MS. Because free metals are not released in vivo from the coordination compound, and therefore no metal deposition occurs in the human or animal body, the extremely high inertness is advantageous even when the chelate is to be used in vivo in the future, such as in MRI contrast agents or radiopharmaceuticals.

[0009] The compound according to the present invention can act as an extremely efficient molecular trap, allowing for extremely stable, rigid, and clear coordination of metal ions. The ligand can be prepared in a relatively small number of steps in organic synthesis. After the metal ion is coordinated, a click reaction occurs between the azide group and the triple bond present in the opposing pendant arm of the macrocyclic compound, forming a triazole bridge and encapsulating the metal ion within the cage (forming a so-called click-zip complex). The formation of the triazole is irreversible and is illustrated in Scheme 1 below.

[0010] Scheme 1. Schematic diagram of the click-zip principle as shown in the example of ligand TD647.

[0011] [ka]

[0012] In a first embodiment, the subject matter of the present invention relates to compounds of general formula (I).

[0013] [ka]

[0014] Here, Y is nitrogen (N); N-oxide (N + -O - Selected from the group consisting of; R 1 H; halogen; -OH; -N3; ​​-(CH2) n N3 (where n is an integer in the range of 1 to 3);-NR2 (where R is independently selected from H or C1-C6 alkyl groups that may be branched or linear);-(CH2) n NR2 (where n and R are as defined above); C6~C 10 Aryl(arbitrary, -NH2, -NO2, -N3,

[0015] [ka]

[0016] , can be substituted with -COOH, -CH2Cl and / or -CH2COOH); C7~C 10 Arylalkyl (optionally, -NH2, -NO2, -N3,

[0017] [ka]

[0018] , can be substituted with -COOH, -CH2Cl, and / or -CH2COOH); -CF3; -COOR (where R is as defined above); -(CH2) n COOR(where n and R are as defined above);

[0019] [ka]

[0020] ;-CH2CH(OMe)2;

[0021] [ka]

[0022] Selected from the group consisting of ;-SH;-SO3H;-SO2Ar (where Ar is phenyl);NO2; R 2 teeth,

[0023] [ka]

[0024] ;or

[0025] [ka]

[0026] (where n is an integer in the range of 1 to 3); and preferably, R 2 teeth

[0027] [ka]

[0028] or

[0029] [ka]

[0030] And; more preferably, R 2 teeth

[0031] [ka]

[0032] and; A is H;-(CH2) n COOH(where n is an integer from 1 to 3);-CH(CH3)COOH;-CH((CH2) n CH3)COOH(where n is as defined above);-CH2P(=O)(OR)2(where R is as defined above);-CH((CH2) n COOH)COOH(where n is an integer from 1 to 3);-CH((CH2) n Independently selected from the group consisting of NH2)COOH (where n is an integer from 1 to 3);-CH2C(=O)(NH2);-CH2C(=O)(NH)-CH2COOH;-CH2P(=O)(OH)(Ar) (where Ar is phenyl which can be optionally substituted with C1-C6 alkyl); Z is

[0033] [ka]

[0034] Selected from the group consisting of, Here, R 3 teeth,

[0035] [ka]

[0036] And, R 4H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; C1-C6 alkyl (may be branched or linear); C3-C6 cycloalkyl; -CF3; -(CH2) n NHR 6 (Here, n is an integer in the range of 1 to 3, and R 6 (Selected from H, fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl);-C(CH3)2(NHR) 6 )(Here, R 6 (As defined above); Adamantine;

[0037] [ka]

[0038] ;-(CH2) n COOH (where n is as defined above); C6~C 10 Aryl(arbitrary, -NH2, -NO2, -N3,

[0039] [ka]

[0040] Selected from the group consisting of: -COOH, -CH2Cl, and / or -CH2COOH (which may be substituted); R 5 R 1 ,or,

[0041] [ka]

[0042] (Here, R 4 (is as defined above); preferably, R 5 H;-CF3;halogen;-OH;C7~C 10Arylalkyl (which may optionally be substituted with -NH2, -NO2, -COOH, -CH2Cl, and / or -CH2COOH); or

[0043] [ka]

[0044] ; and; and / or, R 2 and R 3 Both are, formula

[0045] [ka]

[0046] , or formula

[0047] [ka]

[0048] The 1,2,3-triazole group (where R 4 It is as defined above); forming; However, at most one A is H.

[0049] If the compound of the present invention contains a chiral center, all enantiomers, mixtures of enantiomers, and racemates fall within the scope of the present invention. The present invention further includes the compound of general formula (I) in the form of a salt with an alkali metal, ammonium, or amine, as well as the compound of general formula (I) in the form of an addition salt with an acid.

[0050] The term "halogen" refers to any isotope (iozotop) of F, Cl, Br, and I. In particular, this term means: 19 F, 35 Cl, 37 Cl, 79 Br, 81 Br, 127Non-radioactive isotopes such as I; and 18 F, 36 Cl, 77 Br, 83 Br, 123 I, 124 I, 125 I, 131 It contains radioactive isotopes such as I.

[0051] In one embodiment, A is H;-(CH2) n COOH(where n is an integer from 1 to 3);-CH(CH3)COOH;-CH((CH2) n A molecule is independently selected from the group consisting of CH3)COOH (where n is an integer from 1 to 3);-CH2P(=O)(OR)2 (where R is as defined above);-CH2C(=O)(NH2);-CH2C(=O)(NH)-CH2COOH;-CH2P(=O)(OH)(Ar) (where Ar is phenyl which can be optionally substituted with C1-C6 alkyl groups).

[0052] In one embodiment, R 1 H;halogen;-OH;-N3;-CH2N3;-NR2 (where R is independently selected from H or a C1-C6 alkyl group that may be branched or linear);-CH2NR2 (where R is as defined above);C6-C 10 Aryl (can optionally be substituted with -NH2, -NO2, -COOH and / or -CH2COOH); C7~C 10 Arylalkyl (which can optionally be substituted with -NH2, -NO2, -COOH, -CH2Cl and / or -CH2COOH; -CF3; -COOR (where R is as defined above); -(CH2) n COOR(where n and R are as defined above);

[0053] [ka]

[0054] ;-CH2CH(OMe)2;

[0055] [ka]

[0056] Selected from the group consisting of ;-SH;-SO3H;-SO2Ar (where Ar is phenyl);NO2; Preferably, R 1 H; halogen; -OH; -N3; ​​-CH2N3; -N(CH3)2; phenyl; carboxyphenyl; -CF3; -COOR (where R is as defined above); and

[0057] [ka]

[0058] Selected from.

[0059] In one embodiment, R 4 H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; branched or linear C1-C6 alkyl; C3-C6 cycloalkyl; -CF3; -CH2NHR 6 (Here, R 6 (Selected from H, fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl);-C(CH3)2(NHR) 6 )(Here, R 6 (As defined above); Adamantine;

[0060] [ka]

[0061] Selected from the group consisting of, Preferably, R 4is selected from the group consisting of H; trifluoromethyl; 4-piperidinyl; -CH2-NH2; phenyl; cyclopropyl; adamantyl; terc-butyl; trimethylsilyl (TMS); triisopropylsilyl (TIPS); halogen (F, Cl, Br, I); -C(CH3)2NH2; and -C(CH3)2NHBoc. More preferably, R 4 It is hydrogen.

[0062] In one embodiment of the present invention, the compound of general formula (I) comprises the following substituents: Y is nitrogen (N); R 1 H; halogen; -OH; -N3; ​​-CH2N3; -NR2 (where R is independently selected from H or C1-C6 alkyl groups that may be branched or linear); C6-C 10 Aryl (can optionally be substituted with -NH2, -NO2, -COOH and / or -CH2COOH); C7~C 10 Arylalkyl (which can optionally be substituted with -NH2, -NO2, -COOH, -CH2Cl and / or -CH2COOH); -CF3; -COOR (where R is as defined above);

[0063] [ka]

[0064] ;-CH2CH(OMe)2;

[0065] [ka]

[0066] Selected from the group consisting of; R 2 teeth

[0067] [ka]

[0068] and; A is independently selected from the group consisting of: H; -(CH2) n COOH (where n is an integer from 1 to 3); -CH(CH3)COOH; -CH((CH2) n CH3)COOH (where n is as defined above); -CH2P(=O)(OR)2 (where R is as defined above); -CH2C(=O)(NH2); -CH2C(=O)(NH)-CH2COOH; -CH2P(=O)(OH)(Ar) (where Ar is phenyl optionally substituted with C1-C6 alkyl); Z is

[0069]

Chemical formula

[0070] selected from the group consisting of where R 3 is

[0071]

Chemical formula

[0072] and; R 4 is H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; C1-C6 alkyl (which may be branched or straight-chain); C3-C6 cycloalkyl; -CF3; -CH2NHR 6 (where R 6 is selected from H, fluorenylmethyloxycarbonyl and benzyloxycarbonyl); -C(CH3)2(NHR 6 )(where R 6 is as defined above); adamantyl;

[0073]

Chemical formula

[0074] selected from the group consisting of; R 5 is H; -CF3; halogen; -OH; C7-C 10 arylalkyl (optionally substituted with -NH2, -NO2, -COOH, -CH2Cl and / or -CH2COOH); or

[0075]

Chemical formula

[0076] (where R 4 is as defined above); and and / or, R 2 and R 3 together are of the formula

[0077]

Chemical formula

[0078] , or of the formula

[0079]

Chemical formula

[0080] a 1,2,3-triazole group (where R 4 is as defined above); to form provided that at most one A is H.

[0081] In a preferred embodiment, Y is nitrogen, and thus R 1 and R 2 form a pendant arm containing a pyridyl moiety substituted with.

[0082] In one embodiment, R 1 is in the meta position from Y and R 2 is present in the para position from -CH2-.

[0083] In one embodiment, R 1 It is in the meta position from Y, R 2 It is located in the ortho position from -CH2-.

[0084] In another embodiment, R 1 It exists at the para position from Y.

[0085] In one preferred embodiment, R 2 teeth

[0086] [ka]

[0087] That is the case.

[0088] Preferably, R 2 teeth

[0089] [ka]

[0090] And R 1 is selected from the group consisting of H, phenyl, carboxyphenyl, halogen (preferably Cl), trifluoromethyl, carboxyl, carboxylic acid ester, or amine, more preferably R 1 is H or carboxyphenyl.

[0091] In one embodiment, Z is

[0092] [ka]

[0093] That is the case.

[0094] In one preferred embodiment, Z is

[0095] [ka]

[0096] And R 3 and R 5 is as defined above. Preferably, Z is

[0097] [ka]

[0098] And moreover, Z is

[0099] [ka]

[0100] That is the case.

[0101] In the compound of general formula (I), A may be the same or different. Preferably, A is the same.

[0102] In one embodiment, Z is

[0103] [ka]

[0104] That is the case.

[0105] In one embodiment, Z is

[0106] [ka]

[0107] That is the case.

[0108] In one embodiment, A is -(CH2)COOH;-(CH2)2COOH;-CH2P(=O)(OEt)2;-CH2P(=O)(OH)2;-CH2P(=O)(OH)(OEt);-CH2P(=O)(Ph)(OH);-(CH2)COONH2;-(CH2)C(=O)NH-CH2COOH;-(CH2)COO t Selected from the group containing Bu;-CH(CH2COOH)COOH.

[0109] Preferably, A is selected from the group including -(CH2)COOH;-CH2P(=O)(OEt)2;-CH2P(=O)(OH)2;-CH2P(=O)(OH)(OEt); and -CH2P(=O)(Ph)(OH). Most preferably, A is -(CH2)COOH.

[0110] In one embodiment, Y is nitrogen; R 1 H;Cl;-N(CH3)2;phenyl (can optionally be substituted with -COOH or -CH2COOH);benzyl (can optionally be substituted with -COOH or -CH2COOH);-CF3;-COOCH3;-COOCH(CH3)2;-COOtBu;

[0111] [ka]

[0112] Selected from the group consisting of ;-SH;; A is H;-(CH2) n Independently selected from the group consisting of COOH (where n is 1 or 2);-CH2P(=O)(OH)2;-CH2P(=O)(OH)(OEt);-CH2P(=O)(OEt)2;-CH2C(=O)(NH2);-CH2C(=O)(NH)-CH2COOH;-CH2P(=O)(OH)(Ph); Z is

[0113] [ka]

[0114] Selected from the group consisting of, Here, R 3 teeth

[0115] [ka]

[0116] and; R 4 It is selected from the group consisting of H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; cyclopropyl, terc-butyl; -CF3; -CH2NH2; -CH2N(H)(Fmoc); -C(CH3)2(NH2); -C(CH3)2(NHBoc); adamantyl; R 5 is H, or,

[0117] [ka]

[0118] (Here, R 4 (as defined above); However, it is a compound in which at most one A is replaced by H.

[0119] In one preferred embodiment, Z is

[0120] [ka]

[0121] And; R 3 teeth

[0122] [ka]

[0123] And; R 4 and R 5This is as defined above, and preferably R 4 and R 5 H is H.

[0124] In another embodiment, R 2 and R 3 Both are, formula

[0125] [ka]

[0126] , or formula

[0127] [ka]

[0128] The 1,2,3-triazole group (where R 4 This is as defined above, and preferably R 4 is hydrogen); forms. In such embodiments, A bridge is formed between two opposing nitrogen atoms of the cyclic portion, and the compound of general formula (I) thus forms a cage-type structure. The bridge is of general formula (IIa) or general formula (IIb)

[0129] [ka]

[0130] It is the entity of; Here L is

[0131] [ka]

[0132] A linker selected from the group consisting of; R 1 , R 4 and R 5The above definition is correct. Linker L is derived from the Z group as defined above. Preferably, L is

[0133] [ka]

[0134] And more preferably, L is

[0135] [ka]

[0136] That is the case.

[0137] In one preferred embodiment, the compound of general formula (I) is selected from the group including compounds having the following combinations of substituents: Y is N + -O - And; R 1 ,R 4 and R 5 H is:

[0138] [Table 1]

[0139] Y is N; R 1 , R 4 and R 5 H is:

[0140] [Table 2-1]

[0141] [Table 2-2]

[0142] Y is N; Z is

[0143] [ka]

[0144] And; R is

[0145] [ka]

[0146] is:

[0147] [Table 3]

[0148] Y is N; R 2 and R 3 is, formula

[0149] [ka]

[0150] They form the 1,2,3-triazole groups together:

[0151] [Table 4]

[0152] Y is N; R 2 and R 3 is, formula

[0153] [ka]

[0154] They form the 1,2,3-triazole groups together:

[0155] [Table 5]

[0156] Y is N; R 1 is phenyl; R 2 teeth

[0157] [ka]

[0158] And:R 3 teeth

[0159] [ka]

[0160] is:

[0161] [Table 6]

[0162] Y is N; A is -(CH2)COOH; R 1 H is R 2 teeth

[0163] [ka]

[0164] is:

[0165] [Table 7-1]

[0166] [Table 7-2]

[0167] Y is N; A is -(CH2)COOH; R 2 teeth

[0168] [ka]

[0169] Z is

[0170] [ka]

[0171] is:

[0172] [Table 8]

[0173] Y is N; A is -CH(CH3)COOH; R 2 teeth

[0174] [ka]

[0175] Z is

[0176] [ka]

[0177] is:

[0178] [Table 9]

[0179] In the most preferred embodiment, the compound of general formula (I) is such that Y is nitrogen and Z is

[0180] [ka]

[0181] The compounds are selected from the group consisting of compounds in which the remaining substituents are present in the following combinations:

[0182] [Table 10-1]

[0183] [Table 10-2]

[0184] [Table 10-3]

[0185] .

[0186] In another embodiment, the subject of the present invention is a method for synthesizing the compound of general formula (I), comprising the following steps: i) Provides an alkyne intermediate of the general formula Z-Cl (where Z is as defined above); ii) Azide intermediate of general formula (III)

[0187] [ka]

[0188] To provide; Here, Y, R 1 and R 2 This is as defined above; iii) Cyclene derivatives of general formula (IV)

[0189] [ka]

[0190] To provide Here, pA is -(CH2) n COO tBu(where n is an integer from 1 to 3); benzyloxycarbonyl; -CH(CH3)COO t Bu;-CH(CH3)COOR(where R is tert-butyl, methyl, or ethyl);-CH((CH2) n CH3)COOR(where n and R are as defined above;-CH((CH2) n COOR)COOR(where n is as defined above, and R is independently selected from tert-butyl, methyl, or ethyl);-CH2P(=O)(OR)2(where R is a C1-C6 alkyl which may be branched or linear);-CH2C(=O)(NH2);-CH2C(=O)(NH)-CH2COOH;-CH2P(=O)(OH)(Ar)(where Ar is a phenyl which may optionally be substituted with a C1-C6 alkyl); selected from the group comprising -CH2COOtBu;-CH2P(O)(OEt)2;-(CH2)2COOtBu; iv-A) The cyclene derivative of general formula (IV) is reacted with the alkyne intermediate Z-Cl to obtain the intermediate of general formula (V).

[0191] [ka]

[0192] To obtain Here, Z and pA are as defined above; or iv-B) The cyclene derivative of general formula (IV) is reacted with the azide intermediate of general formula (III) to obtain the intermediate of general formula (VI).

[0193] [ka]

[0194] To obtain Here, Y, R 1 , R 2 And pA are as defined above; vA) The intermediate of general formula (V) is reacted with the azide intermediate of general formula (III) to obtain the intermediate of general formula (VII).

[0195] [ka]

[0196] To obtain Here, Y, R 1 , R 2 pA and Z are as defined above; or vB) The intermediate of general formula (VI) is reacted with the alkyne intermediate Z-Cl to obtain the intermediate of general formula (VII); vi) Optionally, hydrolysis of the protecting group is performed to obtain the compound of general formula (I).

[0197] Generally, compounds of the general formula Z-Cl are commercially available or can be synthesized using a Pd(0)-catalyzed Sonogashira cross-coupling reaction from the corresponding 2-halopyridine (preferably Br,I) and a terminally alkyne or silyl-protected acetylene.

[0198] Compounds of general formula (III) can be obtained from 2,6-bis(halomethyl)pyridine (preferably Cl) by demethrization using sodium azide as the source of the azide moiety.

[0199] Cyclene derivatives of general formula (IV) are commercially available or can be prepared by reacting a cyclene or a trans-N-bis protected cyclene (preferably carbamate protected), alkyl acrylate (Michael addition), or trialkyl phosphite having an alkyl haloacetate (alkylation) in the presence of (para)formaldehyde (Kabachnik-Fields reaction).

[0200] Steps iv-A), iv-B), vA), and vB) are carried out in a solvent selected from the group including MeCN (preferably), DMSO, or DMF. The reaction is carried out at room temperature, usually for several hours to up to several days.

[0201] Step vi) of deprotecting the pA group is optional; for example, if pA is -CH2P(O)(OEt)2, and no deprotection is performed, then A in the resulting general formula (I) is equal to pA. The specific conditions for deprotecting the protecting group depend on the chemical properties of the protecting group and are known to those skilled in the art. Typically, terc-butyl ester protecting groups and Boc protecting groups are hydrolyzed under acidic conditions (TFA) or thermally, methyl protecting groups or isopropyl protecting groups are hydrolyzed under alkaline conditions, and ethylphosphonic acid esters are converted to the corresponding phosphonic acid by transesterification with trimethylsilyl bromide in the presence of a pyridine base.

[0202] Another subject of the present invention is the compound of the general formula (I) defined above, and a lanthanide (III) cation, Na + Ba 2+ Pb 2+ Sr 2+ Ca 2+ , Cd 2+ Zn 2+ Mn 2+ Pt 2+ Cu 2+ Ni 2+ , Sc 3+ , Y 3+ , Bi 3+ In 3+ , Ru 3+ , Ir 3+ , Ga 3+ , Tl 3+ , Pd 2+ A coordination compound with a metal cation selected from the group consisting of; preferably, the metal cation is La 3+ Ce 3+ , Pr 3+ , Nd 3+ Sm 3+ ,EU 3+ , Gd 3+ , Tb 3+ Dy3+ Ho 3+ Er 3+ , Tm 3+ Yb 3+ Lu 3+ , Y 3+ , Sc 3+ , Bi 3+ In 3+ , Tl 3+ Pb 2+ Ca 2+ , Cd 2+ Zn 2+ Cu 2+ Ni 2+ Mn 2+ , Pd 2+ na + It is selected from the group consisting of the following.

[0203] The aforementioned metal ions are 40 Ca, 42 Ca, 43 Ca, 44 Ca, 46 Ca, 48 Ca, 45 Sc, 89 Y, 138 La, 139 La, 136 Ce, 138 Ce, 140 Ce, 142 Ce, 141 Pr, 142 Nd, 143 Nd, 144 Nd, 145 Nd, 146 Nd, 148 Nd, 150 Nd, 144 Sm, 147 Sm, 148 Sm, 149 Sm, 150 Sm, 152 Sm, 154 Sm, 151 EU, 153 EU, 152 Gd, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 160 Gd, 159 Tb,156 Dy, 158 Dy, 160 Dy, 161 Dy, 162 Dy, 163 Dy, 164 Dy, 165 Ho, 162 Er, 164 Er, 166 Er, 167 Er, 168 Er, 170 Er, 169 Tm, 168 Yb, 170 Yb, 171 Yb, 172 Yb, 173 Yb, 174 Yb, 176 Y, 175 Lu, 176 Stable isotopes such as Lu, and 44 Sc, 47 Sc, 64 Cu, 67 Cu, 86 Y, 90 Y, 140 Nd, 149 PM, 151 PM, 153 Sm, 159 Gd, 149 Tb, 161 Tb, 165 Dy, 161 Ho, 166 Ho, 169 Er, 167 Tm, 175 Yb, 177 It should be understood as containing all isotopes of a particular metal, including radioactive isotopes such as Lu.

[0204] Lanthanides (Ln) are defined as chemical elements that include 15 metallic chemical elements with atomic numbers ranging from 57 to 71 (from lanthanum to lutetium).

[0205] Preferably, the coordination compound is of general formula (VIII)

[0206] [ka]

[0207] It holds.

[0208] Here, M is the aforementioned metal cation, and R 1 , R 4 , R 5 , and A are as defined above, and n is selected from 1, 2, 3, and 4; More preferably, the coordination compound is of general formula (VIIIa)

[0209] [ka]

[0210] It holds.

[0211] The coordination compounds according to the present invention also include solvates and salts of pharmaceutically acceptable acids. The positive charge of the metal cation is offset by two negative charges arising from the A pendant arm (e.g., acetate). Thus, the overall charge of the coordination compound is -1 (for monovalent metal ions), 0 (for divalent metal ions), or +1 (for trivalent metal ions). The counterion that compensates for the total charge of the coordination compound is selected from the group including anions derived from the following pharmaceutically acceptable acids: 1-Hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid; 2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor-10-sulfonic acid (+); capric acid (decanoic acid); caproic acid (hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecyl sulfate; ethane-1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); Glucuronic acid (D); Glutamic acid; Glutaric acid; Glycerophosphate; Glycolic acid; Hypric acid; Hydrobromic acid; Hydrochloric acid; Isobutyric acid; Lactic acid (DL); Lactobionic acid; Lauric acid; Maleic acid; Malic acid (-L); Malonic acid; Mandelic acid (DL); Methanesulfonic acid; Naphthalene-1,5-disulfonic acid; Naphthalene-2-sulfonic acid; Nicotinic acid; Nitric acid; Oleic acid; Oxalic acid; Palmitic acid; Pamoic acid; Phosphoric acid; Propionic acid; Pyroglutamic acid (-L); Salicylic acid; Sebacic acid; Stearic acid; Succinic acid; Sulfuric acid; Tartaric acid (+L); Thiocyanic acid; Toluenesulfonic acid (p); Undecylenic acid; Trifluoroacetic acid.

[0212] The coordination compound can be prepared from a free ligand of general formula (I) (preferably non-cage type, and therefore preferably without trazole crosslinking) and a metal salt (typically a water-soluble metal salt, preferably selected from the group including chloride, nitrate, acetate, formate, trifluoroacetate, and trifluoromethylsulfonate, more preferably chloride or nitrate) according to Scheme 1 above. By stirring these reactants in an aqueous environment (pH 5-7) at a temperature in the range of 25°C to 100°C, the metal cation coordinates to the compound of general formula (I), and then the R 2 and R 3 The substituents form the triazole crosslinks, causing the metal cation to "click-zip" into the cage (the reaction temperature and time are highly dependent on the choice of ligand and metal cation).

[0213] However, suitable ligands are not limited to the non-cage type compounds of general formula (I), but a wider range of metal cations (such as lanthanides) are preferred because they can form cage type coordination compounds. Crosslinked (cage type) compounds of general formula (I) having 1,5-triazole crosslinks are typically Tl 3+ Pb 2+ , Bi 3+ In 3+ , Cd 2+ Ca 2+ Cu 2+ Ni 2+ Mn 2+ , Pd 2+ na + It can form a coordination compound with a metal cation selected from the group including the following. The crosslinked (cage-type) compound of general formula (I) having a 1,4-triazole crosslink is typically La 3+ Ce 3+ , Pr 3+ , Nd 3+ Sm 3+ ,EU 3+ , Gd 3+ , Tb 3+ Dy 3+ Ho 3+ Er 3+ , Tm 3+ Yb 3+ Lu 3+ , Y 3+ , Tl 3+ Pb 2+ , Bi 3+ In 3+ , Cd 2+ Ca 2+ Cu 2+ Ni 2+ Mn 2+ , Pd 2+ na + A coordination compound can be formed with a metal cation selected from the group including the following:

[0214] The resulting coordination compounds are highly inert and stable, making them suitable for a variety of applications, including MRI contrast agents, diagnostic or therapeutic radiology, and drug tracking involving peptides or proteins.

[0215] The coordination of the compound of general formula (I) to the metal cation occurs via the macrocyclic nitrogen atom of the cyclone moiety, the oxygen atoms present in the A group (pendant arm) and the Y group (when Y is an N-oxide), and via the nitrogen atom of the Y group (when Y is nitrogen) and the nitrogen atoms present in the Z group.

[0216] In one embodiment, the coordination compound according to the present invention can undergo a post-click-zip transformation. This means that after the metal cation has coordinated into the cage of the compound of general formula (I), either by following scheme 1 or by coordinating to a cage-type compound of general formula (I), it may undergo further reactions if several functional groups are present. Typically, such functional groups are substituents R of the coordination compound. 1 or R 5 The halogen present inside is preferably Cl, NO2, or SH, but the post-click-zip conversion is R 1 , R 4 , R 5 and / or may include reactions of the A group (such as amide coupling reactions of uncoordinated carboxyl groups or amino groups).

[0217] For example, R 1 is a halogen, preferably Cl; or R 1 C6~C substituted with -CH2Cl 10 If it is an aryl, the coordination compound of general formula (VIII) is phenylboronic acid (3-borono-5-nitrobenzoic acid, or 2-(4-(4,4,5 , The compound can undergo a post-click transformation using a Pd(0)-catalyzed Suzuki cross-coupling reaction with 5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetic acid or 2-(4-boronophenyl)acetic acid, during which the halogen is substituted, so the resulting coordination compound of general formula (VIII) is R 1 As -COOH, or -COOH or -CH 2 COOH-substituted C 6 ~C 10 ArielThis results in the presence of the carboxylic acid moiety, which can be used in amide coupling reactions for the synthesis of peptide conjugates. The conjugate with oligoarginine can pass through the cell membrane, allowing the metal cage to be taken up by the cell.

[0218] Another possible post-click transformation involves the reaction with NaN3, which substituted the -N3 group with R. 1 , R 4 and / or R 5 This involves the substitution of halogens present within the substituents. The resulting azide can be used to couple the metal cage with another alkyne-containing moiety using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

[0219] Another possible post-click transform is R 1 , R 4 and / or R 5 The method involves hydrolyzing the TIPS protecting group present in the substituent and protecting the triple bond (R 1 , R 3 and / or R 5 teeth

[0220] [ka]

[0221] The hydrolysis was carried out using aqueous K2CO3, and as a result, R 1 , R 3 and / or R 5 teeth

[0222] [ka]

[0223] The resulting deprotected triple bond is suitable for further conjugation via reactions with alkynes, such as click reactions with azides and the formation of triazole bridges (e.g., CuAAC reactions).

[0224] In another embodiment, the post-click transform is R 1 , R 4 and / or R 5 This could also be an addition reaction of methanol to R, where R 1 , R 3 and / or R 5 teeth

[0225] [ka]

[0226] Therefore, the triple bond is converted to a dimethyl acetal (a protected form of the aldehyde). This represents a conversion from one functional group (alkyne) to another (aldehyde). Aldehydes can be used in conjugation reactions based on the formation of Schiff bases with amines, hydrazines, and acid hydrazides.

[0227] In yet another embodiment, the post-click conversion may be a deuteration reaction of the CH2 group of the pendant arm in D2O in the presence of DBU converted to a CD2 group. This conversion does not change the physicochemical properties of the molecule (in terms of stability or reactivity), but changes the overall mass of the molecule so that it can be completely distinguished from the undeuterated parent molecule using mass spectrometry. In other words, the metal ion cage having a particular metal can be used as two different mass tags.

[0228] In yet another embodiment, the post-click transformation may be a selective reduction of the pyridyl cycle of the Z substituent, where Z is

[0229] [ka]

[0230] And R 3 and R 5As defined above, NaBH4 or NaBD4 is used as the reducing agent, thereby removing the Z group, respectively.

[0231] [ka]

[0232] ,or,

[0233] [ka]

[0234] This conversion alters the mass of the molecule, similar to the deuteration of the CH2 group, for the purpose of a metal tag.

[0235] In yet another embodiment, the post-click transform is R 1 and / or R 4 and / or R 5 and / or NH2 groups of A (if present) (where R 1 and / or R 4 and / or R 5 is -NH2 or -(CH2) n The reaction may also involve a reaction between NH2 (containing NH2) and FmocCl, resulting in the conversion of the -NH2 group to an -NHFmoc group. The Fmoc group is the most commonly used amine protecting group in solid-phase peptide synthesis (SPPS). Therefore, introducing the Fmoc group facilitates the use of the aforementioned metal cage in SPPS.

[0236] In yet another embodiment, the post-click transform is R 1 and / or R 4 and / or R 5 The reaction may also be between the NH2 group of A (if present) and the carboxyl group of another molecule of general formula (I) as defined above or a metal complex thereof, resulting in a conjugate in which two or more of the compounds of general formula (I) or their metal complexes are linked via peptide (amide) bonds.

[0237] In yet another embodiment, the post-click transform is R 1 and / or R 4 and / or R 5 And / or, if present in A, the reaction may also be between the uncoordinated COOH group and the amino group of an amino acid or peptide, thus forming a peptide bond for conjugation purposes.

[0238] In yet another embodiment, the post-click transform is R 1 , R 4 , and / or R 5 The reaction may also involve an S-alkylation reaction between a substituted halogen, preferably Cl, and a thiol, resulting in the conversion of the halogen to a sulfide. The thiol may be, for example, Na2S, NaSH, N-Boc-cysteine, or a cysteine-containing peptide, and the reaction may be carried out in the presence of DIPEA. Incorporating cysteine ​​(an amino acid) into the structure of the coordination compound according to the present invention further expands the possibilities of amide bonds (e.g., peptide bonds).

[0239] In yet another embodiment, the post-click transform is R 1 , R 3 , and / or R 5 of

[0240] [ka]

[0241] Base (here, R 1 , R 3 , and / or R 5 teeth

[0242] [ka]

[0243] The reaction may also involve a reaction between (including) and an azide, preferably an alkyl- or aryl azide, thereby combining the alkyl- or aryl- and R 1 , R 3 , and / or R 5 A triazole crosslink is formed connecting the groups. The aryl azide is preferably a C6-C10 aryl azide, such as benzyl azide, and is optionally substituted with -COOH or -CH2COOH. The alkyl azide is preferably a C1-C7 alkyl azide, where the alkyl can be linear or branched.

[0244] In yet another embodiment, the post-click transform is R 1 or R 5 -N3 group (where R 1 or R 5 The reaction may also involve a reaction between a compound (containing a -N3 group) and a substituent containing a carbon-carbon triple bond, thereby forming a triazole bridge that connects the substituent and the coordination compound. The substituent containing the triple bond may be, for example, a C7-C10 arylalkynyl optionally substituted with -COOH or -CH2COOH (e.g., phenylacetylene), where the phenyl portion may optionally be substituted with -COOH or -CH2COOH.

[0245] In yet another embodiment, the post-click zip transformation is R 1 and / or R 5 The reaction may involve the -SH group of and a substituent containing maleimide, thereby forming a thiosuccinimide linkage that connects the substituent and the coordination compound; or R 1 and / or R 5 The reaction of the -SH group with another substituent containing a -SH group forms a disulfide bridge linking the substituent and the coordination compound; or R 1 and / or R 5 The reaction of the -SH group with an alkyl- or aryl halogenide forms a thioether linkage that connects the alkyl- or aryl with the coordination compound.

[0246] In yet another embodiment, the post-click zip transformation is R 1 and / or R 5 The reaction may also involve the reaction between the -NO2 group and a substituent containing an -SH group, thereby forming a thioether bond that links the substituent and the coordination compound.

[0247] In one embodiment, the post-click conversion may involve a reaction with another coordination compound of the present invention, thereby forming a chain or dimer. The chain or dimer of the coordination compound may contain the same metal cation in both click-zip cages, or each chelate may contain a different metal cation. The advantage of the chain and dimer lies in the possibility of combining the properties of the two cage-type chelates, for example, their inherent weights, to produce mass tags with large weight variability.

[0248] Typically, the coordination compound is the R of the first coordination compound. 1 or R 5 The group and the R of the following coordination compound 1 or R 5 The chain is bonded by forming a triazole crosslink between the group and the (R 5 The use of the base is Z

[0249] [ka]

[0250] ,or,

[0251] [ka]

[0252] (Only possible in this case).

[0253] The general basic structure of the aforementioned coordination compound chain is, for example, (IXa) or (IXb).

[0254] [ka]

[0255] [ka]

[0256] .

[0257] In one embodiment, the coordination compound is an amine group (R 1 and / or R 4 and / or R 5 A substituent of a coordination compound containing and / or A), and a carboxyl group (R 1 and / or R 4 and / or R 5 It is attached to the chain by an amide bond formed between it and a substituent of another coordination compound containing and / or A).

[0258] The aforementioned chain typically contains two coordination compounds according to the present invention, but may contain three, four or more coordination compounds.

[0259] In yet another embodiment, the two types of linkage described above are possible in a single chain, for example, two adjacent coordination compounds are linked by a single triazole crosslink, R 5 or R 1 Another coordination compound according to the present invention can be linked via a triazole crosslink, or via an amide bond if an amino group or carboxyl group is present, while leaving the substituent intact.

[0260] In one embodiment, the coordination compound is a substituent R of a coordination compound containing an -SH group. 1 or R 5 And substituent R of another coordination compound containing an -SH group 1 or R 5 It is bonded to the chain by a disulfide bond (-SS-) formed between it and the other element.

[0261] The coordination compound dimer is the R of the first coordination compound. 1 The group and the R of the second coordination compound 5 By forming a triazole crosslink between the group and the R of the first coordination compound, 5 The group and the R of the second coordination compound 1 The present invention comprises two coordination compounds bonded together by forming a triazole crosslink between them.

[0262] The general structure of the aforementioned coordination compound dimer is, for example, (X).

[0263] [ka]

[0264] .

[0265] The synthesis of the coordination compound chain or dimer is based on a click reaction between the azide group of the first coordination compound and the triple bond of the second coordination compound, thereby forming a triazole crosslink and linking the two coordination compounds to a dimer. The triazole crosslink is selected from the group including: First-coordination compound R 1 or R 4 or R 5 The -N3 group of the second coordination compound and the R 1 or R 4 or R 5 A formula is formed between the aforementioned triple bond and the triple bond.

[0266] [ka]

[0267] , or formula

[0268] [ka]

[0269] The 1,2,3-triazole group.

[0270] Preferably, the metal cation is different, for example, the metal cation is Tb 3+ and 176 Yb 3+ Selected from.

[0271] Another objective of the present invention is to provide conjugates suitable for use as markers for drug tracking. Many drugs are peptide or protein-based, and their biodistribution can be tracked by attaching fluorescent or radioactive markers to the structure of the peptide or protein. The coordination compounds and dimers of the present invention are suitable for conjugation to the peptide or protein of the drug to be tracked. Therefore, isolated cell cultures or tissues can be analyzed for the presence of the metal complex using conventional methods such as LC-MS, which are commonly used and relatively inexpensive instruments. The cells or tissues can be completely hydrolyzed with a strong acid without degrading the coordination compound according to the present invention. The coordination compound can then be detected from the hydrolyzed sample. Such methods are inexpensive and reliable due to the extremely high stability of the coordination compound. Furthermore, LC-MS allows for the use of multiple coordination compounds of different metal isotopes (multiplexing), so all coordination compounds can be analyzed in a single LC-MS analysis.

[0272] The conjugate according to the present invention comprises the coordination compound according to the present invention as defined above, or a dimer or chain of the coordination compound as defined above, conjugated to a peptide or protein. The conjugation occurs via an amide bond formed between the amino group of the peptide or protein and the carboxyl group of the coordination compound or chain or dimer, or vice versa. Therefore, the coordination compound or chain or dimer must contain either an -NH2 or -COOH group prior to the conjugation of the peptide or protein. Preferably, the -NH2 or -COOH group is R1 and / or R 4 and / or R 5 It is part of the base, and therefore the conjugate is -NH2;-(CH2) n NH2;-COOH;-(CH2) n COOH; -NH2, -COOH, -(CH2) n NH2, or -(CH2) n COOH-substituted C6~C 10 R selected from the group containing aryls (where n is an integer in the range of 1 to 3) 1 and / or R 4 and / or R 5 It is formed from a coordination compound having a group or a coordination compound dimer.

[0273] The aforementioned conjugation reaction conditions are known to those skilled in the art, and the reaction is typically carried out in DMSO at 25°C in the presence of an organic base (triethylamine, ethyldiisopropylamine) and a commonly used peptide coupling agent (preferably HATU, PyAOP). The reaction is usually completed in a few minutes.

[0274] The peptide is preferably selected from the group comprising oligopeptides of 3 to 20 amino acids; The protein is preferably selected from the group including antibodies, preferably monoclonal antibodies.

[0275] Another object of the present invention is a method for tracking a drug, preferably a peptide-based drug or a protein-based drug, comprising the following steps: i) Supply a cell culture or tissue to be analyzed, comprising at least one conjugate as defined above, wherein the peptide or protein is the peptide-based drug or protein-based drug to be tracked; ii) Hydrolyze the cell culture or tissue from step i) using a strong acid, preferably a non-oxidizing aq.HCl, to obtain a hydrolysate; iii) Preferably, the presence of the coordination compound according to the present invention or the coordination compound dimer is qualitatively and / or quantitatively analyzed using LC-MS for the hydrolysate from step ii).

[0276] A further object of the present invention is the in vitro use of the coordination compound, or the coordination compound dimer, or the conjugate according to the present invention, in pharmaceuticals, preferably for the development and testing of new drugs, and more preferably for drug marking and tracking.

[0277] Another object of the present invention is to use the coordination compound or the coordination compound dimer according to the present invention as a medical diagnostic, preferably an MRI contrast agent. Preferably, since Gd has a very high relaxation rate, Gd 3+ The coordination compound or dimer is used as an MRI contrast agent. Due to the extremely high kinetic inertness of the coordination compound (approximately 100 times higher than that of GdDOTA, a commonly used MRI contrast agent), even higher doses can be used, and free gadolinium, which is itself toxic to humans or animals, is not released from the coordination compound according to the present invention. Therefore, the claimed coordination compound is expected to be safe for use in humans or animals.

[0278] Another object of the present invention is the use of the coordination compound, or the coordination compound chain, or the coordination compound dimer according to the present invention, where M is a radionuclide, preferably 44 Sc, 47 Sc, 64 Cu, 67 Cu, 86 Y, 90 Y, 140 Nd, 149 PM, 151 PM, 153 Sm, 159 Gd, 149 Tb, 161 Tb, 165 Dy, 161 Ho, 166 Ho, 169 Er, 167Tm, 175 Yb, 177 Selected from the group including Lu, and used in medicine as a radiodiagnostic agent and / or radiopharmaceutical. Alternatively, the radionuclide may be present in the side chain of the coordination compound, such as a radioisotope of a halogen, for example, R 1 and / or R 5 teeth, 18 F is also acceptable.

[0279] The present invention is further demonstrated by the following embodiments.

[0280] [Examples] [Example 1: Synthesis of an intermediate for an alkyne pendant arm] Synthesis of TD701:

[0281] [ka]

[0282] (6-bromopyridine-2-yl)methanol (2.47 g; 13.1 mmol; 1.0 equivalent) and a magnetic stirrer were placed in a pear-shaped glass flask (50 mL) and protected with argon three times. Then, solid CuI (149 mg; 782 μmol; 6.0 mol%) and [Pd(PPh3)2Cl2] (225 mg; 321 μmol; 2.4 mol%) were added and the mixture was further protected with argon (three times). Next, dry THF (25 mL) was added through the septum under a constant flow of argon, followed by the addition of ethynyltrimethylsilane (2.0 mL; 14.5 mmol; 1.1 equivalent) and TEA (5.5 mL; 39.5 mmol; 3.0 equivalent). The mixture then turned dark brown. The flask was left stirred at room temperature for 3 hours under the septum (without external argon). Subsequently, the mixture was transferred to a separatory funnel containing Depositphotos (150 mL) and H₂O (100 mL). After shaking, the dark (not completely homogeneous) organic layer was separated, and the aqueous layer was further extracted with Depositphotos (5 × 50 mL). The combined organic layers were filtered through a cotton stopper, the filtrate was further dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting dark brown oily residue was purified by flash chromatography (330 g SiO₂, 100% DCM to 10% Depositphotos in DCM). The combined fraction containing the product was evaporated to dryness and then evaporated once more together with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a dark yellow oil. Yield: 2.16 g (80%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 0.24(CH3,s,9H);4.52(CH2,d,2H,J 3 HH =6);5.48(OH,t,1H, 3 J HH =6);7.39(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.46(arom.,dd,1H, 3 J HH =8; 4 J HH=1);7.80(arom.,t,1H, 3 J HH =8). 13 C{ 1 H}δ C -0.3(CH3,s); 63.9(CH2,s); 93.4 and 104.5(C≡C;2×s); 120.2(arom.,s); 125.4(arom.,s); 137.2(arom.,s); 140.8(arom.,s); 162.7(arom.,s). ESI-HRMS:206.0995[M+H] + (Theoretical value [C 11 H 16 N1O1Si1] + (=206.0996).

[0283] Synthesis of TD557:

[0284] [ka]

[0285] In a round-bottom glass flask (100 mL), TD701 (2.16 g; 10.5 mmol; 1.0 equivalent) was dissolved in DCM (40 mL). Next, a freshly prepared solution of SOCl2 (1.50 mL; 20.7 mmol; 2.0 equivalent) in DCM (10 mL) was added, and the open flask was stirred at room temperature for 1 hour. Next, a diluted aqueous solution of NaHCO3 (50 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for another 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. A brown residue ( 1The product (already pure according to 1HNMR) was purified by flash chromatography (330 g SiO2, 60% PE in DCM to 20% PE in DCM) to remove heavily colored impurities. The fraction combined with the product was evaporated to dryness and then evaporated once more with the DCM. The remaining nearly colorless oil was left in a freezer to solidify. The resulting solid was pulverized and dried overnight under high vacuum to obtain the product in the form of free base as a fine white powder. Yield: 2.14 g (87%; 1 step; TD701 standard). NMR (DMSO-d6): 1 H δ H 0.25(CH3,s,9H);4.76(CH2,s,2H);7.50(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.55(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.86(arom.,t,1H, 3 J HH =8). 13 C{ 1 H}δ C -0.4(CH3,s);46.3(CH2,s);94.3 and 103.8(C≡C;2×s);123.3(arom.,s);126.7(arom.,s);138.1(arom.,s);141.5(arom.,s);157.0(arom.,s). ESI-HRMS:224.0657[M+H] + (Theoretical value [C 11 H 15 N1Cl1Si1] + (=224.0657).

[0286] Synthesis of TD558:

[0287] [ka]

[0288] In a pear-shaped glass flask (100 mL), TD557 (1.05 g; 4.69 mmol; 1.0 equivalent) was dissolved in MeCN (40 mL). Next, a freshly prepared solution of KF (406 mg; 7.0 mmol; 1.5 equivalent) in H2O (7 mL) was added, and the flask was vigorously stirred at room temperature for 2 hours. The mixture was concentrated to ~10 mL and diluted with DCM (50 mL) and diluted aqueous solution of NaHCO3 (50 mL). The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free base as a pale yellow oil. Yield: 702 mg (99%; 1 step; based on TD557). NMR (DMSO-d6): 1 H δ H 4.37(C≡CH,s,1H);4.76(CH2,s,2H);7.53(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.57(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.87(arom.,t,1H, 3 J HH =8). 13 C{ 1 H}46.3(CH2,s);80.6 and 82.6(C≡CH;2×s);123.4(arom.,s);126.8(arom.,s);138.1(arom.,s);141.3(arom.,s);157.0(arom.,s). ESI-HRMS:152.0262[M+H] + (Theoretical value [C8H7N1Cl1]) + (=152.0262).

[0289] Synthesis of TD712:

[0290] [ka]

[0291] (6-bromopridin-2-yl)methanol (284 mg; 1.51 mmol; 1.0 equivalent) and a magnetic stirrer were placed in a pear-shaped glass flask (10 mL) and protected with argon three times. Subsequently, solid CuI (14.5 mg; 76 μmol; 5.0 mol%) and [Pd(PPh3)2Cl2] (21 mg; 30 μmol; 2.0 mol%) were added and the flask was further protected with argon (three times). Then, dry THF (4 mL) was added through the septum under a constant flow of argon, followed by the addition of ethinyltriisopropylsilane (370 μL; 1.65 mmol; 1.1 equivalent) and TEA (630 μL; 4.52 mmol; 3.0 equivalent), after which the mixture turned dark brown. The flask was left stirred under the septum (without external argon) at room temperature for 16 hours. The mixture was then transferred to a separatory funnel containing Depositphotos (30 mL) and H₂O (30 mL). After shaking, the dark (not perfectly homogeneous) organic layer was separated, and the aqueous layer was further extracted with Depositphotos (5 × 25 mL). The combined organic layers were filtered through a cotton stopper, the filtrate was further dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting dark brown oily residue was purified by flash chromatography (120 g SiO₂, 100% DCM to 10% Depositphotos in DCM). The combined fraction containing the product was evaporated to dryness and then evaporated once more with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 387 mg (89%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 0.92-1.26(i-Pr,m,21H);4.53(CH2,d,2H, 3 J HH =6);5.48(OH,t,1H, 3 J HH =6);7.40(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.47(arom.,dd,1H, 3 J HH =8; 4 JHH =1);7.80(arom.,t,1H, 3 J HH =8). 13 C{ 1 H} δ C 10.7(i-Pr,s);18.5(i-Pr,s);64.0(CH2,s);89.4 and 106.6(C≡C;2×s);120.2(ar om.,s);125.8(arom.,s);137.2(arom.,s);140.9(arom.,s);162.7(arom.,s). ESI-HRMS:290.1932[M+H] + (Theoretical value [C 17 H 28 N1O1Si1] + (=290.1935).

[0292] Synthesis of TD723:

[0293] [ka]

[0294] In a round-bottom glass flask (25 mL), TD712 (384 g; 1.33 mmol; 1.0 equivalent) was dissolved in DCM (5 mL). Next, a freshly prepared solution of SOCl2 (200 μL; 2.75 mmol; 2.1 equivalents) in DCM (1 mL) was added, and the open flask was stirred at room temperature for 1 hour. Next, a diluted aqueous solution of NaHCO3 (10 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for another 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. Brown residue ( 1The product (already pure according to 1HNMR) was purified by flash chromatography (120 g SiO2, 20% DCM in PE to 30% DCM in PE) to remove strongly colored impurities. The fraction combined with the product was evaporated to dryness and then evaporated once more with the DCM. The remaining colorless oil was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless solid. Yield: 356 mg (87%; 1 step; TD712 standard). NMR (DMSO-d6): 1 H δ H 0.91-1.28(i-Pr,m,21H);4.78(CH2,s);7.52(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.57(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.86(arom.,t,1H, 3 J HH =8). ESI-HRMS:308.1593[M+H] + (Theoretical value [C 17 H 27 N1Cl1Si1] + (=308.1596).

[0295] Synthesis of TD530:

[0296] [ka]

[0297] In a 50 mL pear-shaped glass flask, (6-bromopyridine-2-yl)methanol (1.00 g; 5.35 mmol; 1.0 equivalent), N-Boc-propargylamine (1.00 g; 6.44 mmol; 1.2 equivalents), and a magnetic stirrer were placed and protected with argon three times. Subsequently, solid CuI (102 mg; 536 μmol; 10.0 mol%) and [Pd(PPh3)2Cl2] (187 mg; 266 μmol; 5.0 mol%) were added, and the flask was protected with argon three more times. Dry THF (27 mL) was added through the septum under a constant flow of argon, followed by the addition of TEA (2.24 mL; 16.1 mmol; 3.0 equivalents), after which the mixture turned dark brown. The flask was left stirred at room temperature for 18 hours under the septum (without external argon). The mixture was then transferred to a separatory funnel containing Depositphotos (30 mL) and H₂O (50 mL). After shaking, the dark (not perfectly homogeneous) organic layer was separated, and the aqueous layer was further extracted with Depositphotos (3 × 30 mL). The combined organic layers were filtered through a cotton stopper, the filtrate was further dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting dark brown oily residue was purified by flash chromatography (220 g SiO₂, 100% DCM to 100% Depositphotos). The combined fraction containing the product was evaporated to dryness and then evaporated once more with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 610 mg (43%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 1.40(CH3,s,9H);3.99(CH2,d,2H, 3 J HH =6);4.51(CH2,d,2H, 3 J HH =6);5.47(OH,t,1H, 3 J HH =6);7.32(arom,dd,1H, 3 J HH =8; 4 J HH =1);7.40(NH,t,1H, 3 J HH=6);7.44(arom.,dd,1H,J 3 HH =8; 4 J HH =1);7.79(arom.,t,1H, 3 J HH =8). 13 C{ 1 H} δ C 28.2(CH3,s);29.9(CH2,s);64.0(CH2,s);78.4(C-CH3,s);81.3 and 86.9(C≡C;2×s);119.8(arom.,s);125. 0(arom.,s);137.2(arom.,s);141.1(arom.,s);155.3(CO,s);162.5(arom.,s).ESI-HRMS:263.1390[M+H] + (Theoretical value [C 14 H 19 N2O3] + (=263.1390).

[0298] Synthesis of TD538:

[0299] [ka]

[0300] In a round-bottom glass flask (250 mL), TD530 (568 mg; 2.17 mmol; 1.0 equivalent) was dissolved in DCM (20 mL), followed by the addition of TEA (900 μL; 6.46 mmol; 3.0 equivalents). Next, a freshly prepared solution of MsCl (355 μL; 4.33 mmol; 2.0 equivalents) dissolved in DCM (5 mL) was added. The resulting solution was stirred at room temperature for 20 minutes. The mixture was then diluted with DCM (50 mL), followed by the addition of a diluted aqueous solution of NaHCO3 (50 mL). The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 341.1 [M + H] + (Theoretical value [C 15 H 21N2O5S1] + (=341.1). All of the TD538 obtained in this way was used directly in TD539 without further purification or characterization.

[0301] Synthesis of TD1045:

[0302] [ka]

[0303] In a 25 mL pear-shaped glass flask, (6-bromopyridine-2-i)methanol (500 mg; 2.67 mmol; 1.0 equivalent), 1,1-dimethylpropagylamine (420 μL; 3.99 mmol; 1.5 equivalents), and a magnetic stirrer were placed and the flask was protected with argon three times. Subsequently, solid CuI (21 mg; 110 μmol; 4.0 mol%) and [Pd(PPh3)2Cl2] (19 mg; 27 μmol; 1.0 mol%) were added, and the flask was protected with argon three more times. After adding dry THF (10 mL) through a septum under a constant flow of argon, TEA (1.10 mL; 7.89 mmol; 3.0 equivalents) was added, and the mixture subsequently turned dark brown. The flask was left with the septum closed (but without external argon) and stirred at room temperature for 18 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid phase was further washed with MeCN. The filtrate was evaporated to dryness, and the resulting dark brown solid residue was purified by flash chromatography (100% Â to 40% Â in 120 g SiO2, MeOH). The combined fraction containing the product was evaporated to dryness and then evaporated once together with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow solidified oil. Yield: 283 mg (56%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 1.38(CH3,s,6H);3.17(NH2,s,2H);4.51(CH2,s,2H);5.44(OH,bs,1H);7.26(arom.,dd,1H, 3 J HH =8;4 J HH =1);7.40(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.76(arom.,t,1H, 3 J HH =8). ESI-HRMS:191.1180[M+H] + (Theoretical value [C 11 H 15 N2O1] + (=191.1180).

[0304] Synthesis of TD1048:

[0305] [ka]

[0306] TD1045 (280 mg; 1.47 mmol; 1.0 equivalent) was placed in a pear-shaped glass flask (25 mL), followed by the addition of MeCN (10 mL) and a solution of Boc2O (2.0 m in dry THF; 1.0 mL; 2.00 mmol; 1.4 equivalents). The resulting mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the resulting yellow residue was purified by flash chromatography (120 g SiO2, 100% DCM to 100% siRNA). The combined fraction containing the product was evaporated to dryness and then evaporated once more with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless solid. Yield: 242 mg (57%; 1 step; based on TD1045). NMR (DMSO-d6): 1 H δ H 1.40(CH3,s,9H),1.53(CH3,s,6H);4.51(CH2,d,2H, 3 J HH =4);5.44(OH,t,1H, 3 J HH =4);7.12(NH,bs,1H);7.25(arom,dd,1H, 3 J HH =8; 4 J HH=1);7.41(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.77(arom.,t,1H, 3 J HH =8). ESI-HRMS:291.1704[M+H] + (Theoretical value [C 16 H 23 N2O3] + (=291.1703).

[0307] Synthesis of TD1050:

[0308] [ka]

[0309] In a 20 mL glass vial, TD1048 (19 mg; 65 μmol; 1.0 equivalent) was dissolved in 3 mL of DCM, and TEA (28 μL; 201 μmol; 3.1 equivalents) was added. Next, a freshly prepared solution of MsCl (10 μL; 129 μmol; 2.0 equivalents) in 1 mL of DCM was added. The resulting solution was stirred at room temperature for 20 minutes. The mixture was then diluted with 15 mL of DCM, and then 15 mL of diluted aqueous solution of NaHCO3 was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 3 × 20 mL of DCM. The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. ESI-MS (LC-MS): 369.1 [M + H] + (Theoretical value [C 17 H 25 N2O5S1] + (=369.1). All of the TD1050 obtained in this way was used directly in TD1054 (and TD1105 in a similar manner) without further purification or characterization.

[0310] Synthesis of TD966:

[0311] [ka]

[0312] In a 100 mL pear-shaped glass flask, (6-bromopyridine-2-yl)methanol (1.00 g; 5.35 mmol; 1.0 equivalent), N-Boc-4-ethynylpiperidine (1.23 g; 5.88 mmol; 1.1 equivalent), and a magnetic stirrer were placed and protected with argon three times. Subsequently, solid CuI (41 mg; 215 μmol; 4.0 mol%) and [Pd(PPh3)2Cl2] (75 mg; 107 μmol; 2.0 mol%) were added, and the flask was protected with argon three more times. Dry THF (40 mL) was added through a septum under a constant flow of argon, followed by TEA (2.20 mL; 15.7 mmol; 3.0 equivalent). The mixture turned dark brown. The flask was left with stirring under the septum (but without external argon) at room temperature for 2 days. The mixture was then transferred to a separatory funnel containing Depositphotos (30 mL) and H₂O (50 mL). After shaking, the dark (not perfectly homogeneous) organic layer was separated, and the aqueous layer was further extracted with Depositphotos (3 × 30 mL). The combined organic layers were filtered through a cotton stopper, the filtrate was further dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting dark brown oily residue was purified by flash chromatography (100% Depositphotos from 220 g SiO₂, 100% DCM). The combined fraction containing the product was evaporated to dryness and then evaporated once more with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 1.47 g (87%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 1.40(CH3,s,9H);1.44-1.57(CH2,m,2H);1.75-1.89(CH2,m,2H);2.87(CH-C≡Ctt,1H, 3 J HH =8, 3 J HH =4);3.01-3.17(CH2,m,2H);3.59-3.73(CH2,m,2H);4.51(CH2-O,d,2H, 3 J HH=6);5.44(OH,t,1H, 3 J HH =6);7.32(arom,dd,1H, 3 J HH =8; 4 J HH =1);7.41(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.76(arom.,t,1H, 3 J HH =8). ESI-HRMS:317.1860[M+H] + (Theoretical value [C 18 H 25 N2O3] + (=317.1860).

[0313] Synthesis of TD1117:

[0314] [ka]

[0315] In a 20 mL glass vial, TD952 (50.0 mg; 158 μmol; 1.0 equiv) was dissolved in 6 mL of DCM, and TEA (66 μL; 473 μmol; 3.0 equiv) and neat MsCl (25 μL; 323 μmol; 2.0 equiv) were added. The resulting solution was stirred at room temperature for 20 minutes, and then a diluted aqueous solution of DCM (10 mL) and NaHCO3 (10 mL) was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 395.2 [M + H] + (Theoretical value [C 19 H 27 N2O5S1] + (=395.2). All of the TD1117 obtained in this way was used directly in TD1118 without further purification or characterization.

[0316] Synthesis of TD936:

[0317] [ka]

[0318] In a 4 mL glass vial, (6-bromopyridine-2-yl)methanol (135 mg; 722 μmol; 1.0 equivalent), CuI (5.5 mg; 29 μmol; 4.0 mol%), and [Pd(PPh3)2Cl2] (10.0 mg; 14 μmol; 2.0 mol%) were placed with a magnetic stirrer and protected with argon three times. Then, dry THF (3 mL) was added through a septum under a constant flow of argon, followed by phenylacetylene (95 μL; 865 μmol; 1.2 equivalents) and TEA (300 μL; 2.15 mmol; 3.0 equivalents). The mixture turned dark brown. The vial was left to stand with stirring under the septum (but without external argon) at room temperature for 16 hours. The mixture was filtered through a syringe microfilter (PTFE) and washed with MeCN. The filtrate was evaporated to dryness, redissolved in MeCN (3 mL), and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fraction containing the product was neutralized with a diluted aqueous solution of NaHCO3 and evaporated to dryness. The residue was dissolved in a mixed solvent of DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After a short shaking, the bottom phase was separated, and the aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residual solid was dried briefly under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 141 mg (93%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 4.57(CH2,d,2H, 3 J HH =6);5.50(OH,t,1H, 3 J HH =6);7.42-7.54(arom.,Ph,m,2+3H);7.57-7.64(Ph,m,2H);7.85(arom.,t,1H, 3 JHH =8). ESI-HRMS:210.0913[M+H] + (Theoretical value [C 14 H 12 N1O1] + (=210.0913).

[0319] Synthesis of TD939:

[0320] [ka]

[0321] In a 20 mL glass vial, TD936 (22 mg; 105 μmol; 1.0 equivalent) was dissolved in 6 mL of DCM, and TEA (44 μL; 316 μmol; 3.0 equivalents) and neat MsCl (16 μL; 207 μmol; 2.0 equivalents) were added. The resulting solution was stirred at room temperature for 20 minutes, and a dilute aqueous solution of NaHCO3 (5 mL) was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 4 × 25 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 288.1 [M + H] + (Theoretical value [C 15 H 14 N1O3S1] + (=288.1). All of the TD939 obtained in this way was used directly in TD944 without further purification or characterization.

[0322] Synthesis of TD937:

[0323] [ka]

[0324] In a 4 mL glass vial, (6-bromopyridine-2-yl)methanol (135 mg; 722 μmol; 1.0 equivalent), CuI (5.5 mg; 29 μmol; 4.0 mol%), and [Pd(PPh3)2Cl2] (10.0 mg; 14 μmol; 2.0 mol%) were placed with a magnetic stirrer and protected with argon three times. Then, dry THF (3 mL) was added through a septum under a constant flow of argon, followed by cyclopropylacetylene (75 μL; 886 μmol; 1.2 equivalents) and TEA (300 μL; 2.15 mmol; 3.0 equivalents), after which the mixture turned dark brown. The vial was left to stand with stirring under the septum (but without external argon) at room temperature for 16 hours. The mixture was filtered through a syringe microfilter (PTFE) and washed with MeCN. The filtrate was evaporated to dryness, redissolved in MeCN (3 mL), and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fraction containing the product was neutralized with a diluted aqueous solution of NaHCO3 and evaporated to dryness. The residue was dissolved in a mixed solvent of DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After a short shaking, the bottom phase was separated, and the aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residual solid was dried briefly under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 98 mg (78%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 0.69-0.83(CH2-CH,m,2H);0.83-0.99(CH2-CH,m,2H);1.56(CH,tt, 3 J HH =8, 3 J HH =5);4.49(CH2,d,2H, 3 J HH =6);5.42(OH,t,1H, 3 J HH =6);7.27(arom,dd,1H, 3 J HH =8, 4 J HH =1);7.38(arom.,dd,1H,3 J HH =8, 4 J HH =1);7.74(arom.,t,1H, 3 J HH =8). ESI-HRMS:174.0913[M+H] + (Theoretical value [C 11 H 12 N1O1] + (=174.0913).

[0325] Synthesis of TD940:

[0326] [ka]

[0327] In a 20 mL glass vial, TD937 (26 mg; 150 μmol; 1.0 equivalent) was dissolved in DCM (9 mL), and TEA (63 μL; 452 μmol; 3.0 equivalents) and neat MsCl (23 μL; 297 μmol; 2.0 equivalents) were added. The resulting solution was stirred at room temperature for 20 minutes, and a diluted aqueous solution of NaHCO3 (5 mL) was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 252.0 [M + H] + (Theoretical value [C 12 H 14 N1O3S1] + (=252.1). All of the TD940 obtained in this way was used directly in TD943 without further purification or characterization.

[0328] Synthesis of TD952:

[0329] [ka]

[0330] In a 4 mL glass vial, (6-bromopyridine-2-yl)methanol (150 mg; 802 μmol; 1.0 equivalent), CuI (6.1 mg; 32 μmol; 4.0 mol%), and [Pd(PPh3)2Cl2] (11.5 mg; 16 μmol; 2.0 mol%) were placed with a magnetic stirrer and protected with argon three times. Then, dry THF (3 mL) was added through a septum under a constant flow of argon, followed by the addition of tert-butylacetylene (200 μL; 1.62 mmol; 2.0 equivalent) and TEA (340 μL; 2.44 mmol; 3.0 equivalent), after which the mixture turned dark brown. The vial was left with the septum closed (but without external argon) and stirred at room temperature for 24 hours. The mixture was filtered through a syringe microfilter (PTFE) and washed with MeCN. The filtrate was evaporated to dryness, redissolved in MeCN (3 mL), and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fraction containing the product was neutralized with diluted aqueous NaHCO3 solution and evaporated to dryness. The residue was dissolved in a mixed solvent of DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After a short shake, the bottom phase was separated, and the aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residual solid was dried briefly under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 146 mg (96%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 1.30(CH3,s,9H);4.51(CH2,d,2H, 3 J HH =6);5.43(OH,t,1H, 3 J HH =6);7.26(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.39(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.74(arom.,t,1H, 3 JHH =8). ESI-HRMS:190.1227[M+H] + (Theoretical value [C 12 H 16 N1O1] + (=190.1226).

[0331] Synthesis of TD956:

[0332] [ka]

[0333] In a 20 mL glass vial, TD952 (8 mg; 42 μmol; 1.0 equivalent) was dissolved in 3 mL of DCM, and TEA (18 μL; 129 μmol; 3.0 equivalents) and neat MsCl (6.5 μL; 84 μmol; 2.0 equivalents) were added. The resulting solution was stirred at room temperature for 20 minutes, and then 5 mL of diluted aqueous solution of DCM and NaHCO3 was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 4 × 10 mL of DCM. The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. ESI-MS (LC-MS): 268.1 [M + H] + (Theoretical value [C 13 H 18 N1O3S1] + (=268.1). All of the TD956 obtained in this way was used directly in TD959 without further purification or characterization.

[0334] Synthesis of TD965:

[0335] [ka]

[0336] A glass vial (4 mL) contained (6-bromopyridine-2-yl)methanol (53 mg; 283 μmol; 1.0 equivalent), 1-ethinyladamantane (50 mg; 312 μmol; 1.1 equivalent), CuI (2.7 mg; 14 μmol; 5.0 mol%), [Pd(PPh3)2Cl2] (5.0 mg; 7 μmol; 2.5 mol%), and a magnetic stirrer, and was protected with argon three times. Dry THF (1.5 mL) was added through a septum under a constant flow of argon, followed by TEA (120 μL; 861 μmol; 3.0 equivalents). The vial was left to stand with stirring under the septum (but without external argon) for 2 days. The mixture was filtered through a syringe microfilter (PTFE) and washed with MeCN. The filtrate was evaporated to dryness, redissolved in MeCN (3 mL), and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fraction containing the product was neutralized with diluted aqueous NaHCO3 solution and evaporated to dryness. The residue was dissolved in a mixed solvent of DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After a short shaking, the bottom phase was separated, and the aqueous phase was further extracted with DCM (4 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residual solid was dried briefly under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 67 mg (89%; 1 step; based on (6-bromopyridine-2-yl)methanol). NMR (DMSO-d6): 1 H δ H 1.61-1.76(Adm.,m,6H);1.82-1.93(Adm.,m,6H);1.94-2.00(Adm.,m,3H);4.50(CH2,d,2H, 3 J HH =6);5.43(OH,t,1H, 3 J HH =6);7.25(arom,dd,1H, 3 J HH =8, 4 J HH =1);7.38(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.74(arom.,t,1H,J3 HH =8). ESI-HRMS:268.1694[M+H] + (Theoretical value [C 18 H 22 N1O1] + (=268.1696).

[0337] Synthesis of TD990:

[0338] [ka]

[0339] In a 20 mL glass vial, TD965 (21 mg; 79 μmol; 1.0 equivalent) was dissolved in 3 mL of DCM, and TEA (33 μL; 237 μmol; 3.0 equivalents) and neat MsCl (12 μL; 155 μmol; 2.0 equivalents) were added. The resulting solution was stirred at room temperature for 20 minutes, and then 10 mL of diluted aqueous solution of DCM and NaHCO3 was added. The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 3 × 15 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 346.1 [M+H] + (Theoretical value [C 19 H 24 N1O3S1] + (=346.1). All of the TD990 obtained in this way was used directly in TD992 without further purification or characterization.

[0340] Synthesis of TD1194:

[0341] [ka]

[0342] In a glass vial (4 mL), TD558 (110 mg; 726 μmol; 1.0 equivalent) was dissolved in MeCN (3.3 mL), and NIS (180 mg; 800 μmol; 1.1 equivalent) and AcOH (50 μL; 875 μmol; 1.2 equivalents) were added. The resulting mixture was stirred at 80°C for 3 hours. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and diluted with DCM (50 mL). The resulting two-phase mixture was transferred to a separatory funnel, and diluted aqueous solution of NaHCO3 and aqueous solution of Na2S3O3 were added. After a short shaking, the bottom phase was separated, and the aqueous layer was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was dried under high vacuum for a short time to obtain the product in the form of free base as a pale red solid. Yield: 61.5 mg (31%; 1 step; TD558 standard). NMR (DMSO-d6): 1 H δ H 4.74(CH2,s,2H);7.48(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.54(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.85(arom.,t,1H, 3 J HH =8). ESI-HRMS:277.9230[M+H] + (Theoretical value [C8H6N1I1Cl1]) + (=277.9228).

[0343] Synthesis of TD1178:

[0344] [ka]

[0345] A glass vial (20 mL) was filled with CuI (60.0 mg; 315 μmol; 40 mol%), Phen (114 mg; 633 μmol; 80.0 mol%), KHCO3 (159 mg; 1.59 mmol; 2.0 equivalents), and a magnetic stirrer, and protected with argon three times. Next, under a constant flow of argon, a solution of TD558 (120 mg; 792 μmol; 1.0 equivalent) and Togni I (288 mg; 872 μmol; 1.1 equivalents) in dry DCM (8 mL) was added through a septum, and the resulting mixture was stirred at room temperature for 2 days. The mixture was filtered through a syringe microfilter (PTFE), and the solid phase was further washed with DCM. The filtrate was evaporated to dryness, and the residue was resuspended in MeCN (3 mL). The mixture was filtered again through a syringe microfilter (PTFE), and the filtrate was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and diluted with DCM (50 mL). The resulting two-phase mixture was transferred to a separatory funnel, and a diluted aqueous solution of NaHCO3 was added. After a short shaking, the bottom phase was separated, and the aqueous layer was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The resulting yellow oil was dried briefly under high vacuum (until the oil crystallized), and the product in the form of free bases was obtained as a pale yellow solid (part of the product sublimated as colorless crystals at the top of the flask). Yield: 48.5 mg (28%; 1 step; TD558 standard). NMR (DMSO-d6): 1 H δ H 4.83(CH2,s,2H);7.78(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.88(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.04(arom.,t,1H, 3 J HH =8). 19 F{ 1 H}δ F -49.2(s).ESI-HRMS:220.0140[M+H] +(Theoretical value [C9H6N1F3Cl1]) + (=220.0135).

[0346] Synthesis of TD797:

[0347] [ka]

[0348] In a round-bottom glass flask (50 mL), methyl 4,6-dibromopicolinate (200 mg; 678 μmol; 1.0 equivalent) was dissolved in a mixed solvent of THF (4 mL) and MeOH (2 mL). To the resulting slightly yellowish solution, solid NaBH4 (205 mg; 5.42 mmol; 8.0 equivalents) was added partially (over 10 minutes without stoppering the flask). During this time, hydrogen gas was vigorously generated, and the mixture became almost colorless. The mixture was transferred to a separatory funnel and diluted with DCM (100 mL) and H2O (100 mL). After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a white solid. Yield: 179 mg (99%; 1 stage; based on methyl 4,6-dibromopicolinate). NMR (DMSO-d6): 1 H δ H 4.53(CH2,s,2H);5.68(OH,bs,1H);7.67(arom.,m,1H);7.89(arom.,m,1H). ESI-HRMS:265.8809[M+H] + (Theoretical value [C6H6N1O1Br2]) + (=265.8811).

[0349] Synthesis of TD804:

[0350] [ka]

[0351] TD797 (100 mg; 375 μmol; 1.0 equivalent) and a magnetic stirrer were placed in a glass vial (4 mL) and protected with argon three times. Then, solid CuI (4.3 mg; 23 μmol; 6.0 mol%) and [Pd(PPh3)2Cl2] (8.0 mg; 11 μmol; 3.0 mol%) were added, and the vial was protected with argon three more times. Next, dry THF (1.5 mL) was added through the septum under a constant flow of argon, followed by the addition of ethynyltrimethylsilane (160 μL; 1.16 mmol; 3.1 equivalents) and TEA (160 μL; 1.15 mmol; 3.1 equivalents), after which the mixture turned dark brown. The flask was left stirred at room temperature for 16 hours under the septum (without external argon). The mixture was then transferred to a separatory funnel containing Depositphotos (50 mL) and H2O (50 mL). After shaking, the dark organic layer was separated, and the aqueous layer was further extracted with Depositphotos (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The resulting dark brown oily residue was purified by flash chromatography (120 g SiO2, 100% DCM to 20% Depositphotos in DCM). The combined fraction containing the product was evaporated to dryness and then co-evaporated once more over DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a dark brown oil. Yield: 99 mg (88%; 1 step; TD797 standard). NMR (DMSO-d6): 1 H δ H 0.24(CH3,s,9H);0.25(CH3,s,9H);4.51(CH2,d,2H, 3 J HH =6);5.53(OH,t,1H, 3 J HH =6);7.40(arom.,d,1H, 4 J HH =2);7.42(arom.,d,1H, 4 J HH =2). ESI-HRMS:302.1389[M+H] + (Theoretical value [C 16 H 24 N1O1Si2] + (=302.1391).

[0352] Synthesis of TD806:

[0353] [ka]

[0354] In a pear-shaped glass flask (50 mL), TD804 (98 mg; 325 μmol; 1.0 equivalent) was dissolved in DCM (3 mL). Next, a freshly prepared solution of SOCl2 (47 μL; 647 μmol; 2.0 equivalent) in DCM (1 mL) was added, and the open flask was stirred at room temperature for 1 hour. The reaction mixture was diluted with DCM (16 mL), and a diluted aqueous solution of NaHCO3 (20 mL) was added. The resulting two-phase mixture was vigorously stirred at room temperature for another 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 20 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as brown oil. Yield: 102 mg (98%; 1 stage; based on TD804). NMR (DMSO-d6): 1 H δ H 0.25(CH3,s,18H);4.74(CH2,s,2H);7.53(arom.,d,1H, 4 J HH =2);7.60(arom.,d,1H, 4 J HH =2). ESI-HRMS:320.1051[M+H] + (Theoretical value [C 16 H 23 [N1O1Cl1Si2] + (=320.1052).

[0355] Synthesis of TD807:

[0356] [ka]

[0357] In a round-bottom glass flask (50 mL), TD806 (101 mg; 316 μmol; 1.0 equivalent) was dissolved in MeCN (5 mL). Next, a freshly prepared solution of KF (46.4 mg; 800 μmol; 2.5 equivalents) dissolved in H2O (800 μL) was added, and the flask was stirred at room temperature for 5 hours. The mixture was concentrated to less than 1 ml and diluted with DCM (50 mL) and a diluted aqueous solution of NaHCO3 (50 mL). The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a brown solid. Yield: 48 mg (87%; 1 step; based on TD806). NMR (DMSO-d6): 1 H δ H 4.48(C≡CH,s,1H);4.75(C≡CH,s,1H);4.76(CH2,s,2H);7.61(arom.,d,1H, 4 J HH =1);7.65(arom.,d,1H, 4 J HH =1). ESI-HRMS:176.0267[M+H] + (Theoretical value [C 10 H7N1Cl1] + (=176.0267).

[0358] Synthesis of TD662:

[0359] [ka]

[0360] In a glass vial (4 mL), dipropargylamine (116 μL; 1.12 mmol; 1.0 equivalent) was dissolved in MeCN (2.5 mL), followed by the addition of neat 1,2-dibromoethane (965 μL; 11.2 mmol; 10 equivalents) and NaHCO3 (113 mg; 1.35 mmol; 1.2 equivalents). The resulting mixture was stirred at 80°C for 2 days. Next, the mixture was diluted with DCM (50 mL), followed by the addition of a diluted aqueous solution of NaHCO3 (50 mL). The resulting two-phase mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further purified by column chromatography (SiO2; 25 g; DCM). The combined fraction containing the product was evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 69 mg (31%; 1 step; based on dipropargylamine). NMR (DMSO-d6): 1 H δ H 2.86(CH2,t,2H, 3 J HH =7);3.21(C≡CH,t,2H, 4 J HH =2);3.21(CH2,t,4H, 4 J HH =2);3.55(CH2,t,2H, 3 J HH =7). 13 C{ 1 H} δ C 30.3(CH2,s); 41.6(CH2,s); 53.9(CH2,s); 76.4 and 78.5(C≡CH,2×s). ESI-HRMS: 200.0070[M+H] + (Theoretical value [C8H 11 N1Br1] + (=200.0070).

[0361] [Example 2: Synthesis of an azid pendant arm intermediate] Synthesis of TD406:

[0362] [ka]

[0363] In a round-bottom glass flask (250 mL), 2,6-bis(chloromethyl)pyridine (6.17 g; 35.0 mmol; 1.2 equivalents) was dissolved in MeCN (80 mL), followed by the addition of solid NaN3 (1.90 g; 29.2 mmol; 1.0 equivalent) and anhydrous K2CO3 (4.04 g; 29.2 mmol; 1.0 equivalent). The resulting suspension was stirred at 50°C for 5 days. The mixture was filtered through glass frit S3, and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the orange oily residue was purified by column chromatography (SiO2, DCM, 30% PE to 100% DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a faint yellow oil. Yield: 2.62g (49%; 1 stage; based on NaN3). Recovery: 2,6-bis(chloromethyl)pyridine 2.21g (36% of initial usage). NMR (DMSO-d6): 1 H δ H 4.53(CH2,s,2H);4.78(CH2,s,2H);7.40(arom.,d,1H, 3 J HH =8; 4 J HH =1);7.52(arom.,d,1H, 3 J HH =8; 4 J HH =1);7.89(arom.,t,1H, 3 J HH =8). 13 C{ 1 H} δ C 46.6(CH2,s);54.2(CH2,s);121.9(arom.,s);122.6(arom.,s);138.5(arom.,s);155.7(arom.,s);156.3(arom.,s). ESI-HRMS:183.0433[M+H] + (Theoretical value [C7H8N4Cl1]) + (=183.0432).

[0364] Synthesis of TD595:

[0365] [ka]

[0366] In a 20 mL glass vial, TD406 (30 mg; 162 μmol; 1.0 equivalent) was dissolved in 5 mL of distilled coffee (DCM), followed by the addition of a freshly prepared solution of MCPBA (77%; 72 mg; 320 μmol; 2.0 equivalents) in 1 mL of DCM. The resulting solution was stirred at room temperature for 3 hours. The mixture was diluted with 20 mL of DCM and 25 mL of diluted aqueous solution of NaHCO3, and transferred to a separatory funnel. After shaking, the bottom layer was separated, and the aqueous layer was further extracted with 3 × 20 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. ESI-MS (LC-MS): 199.0 [M+H] + (Theoretical value [C7H8N4O1Cl1]) + (=199.0). All of the TD595 obtained in this way was used directly in TD596 without further purification or characterization.

[0367] Synthesis of TD726:

[0368] [ka]

[0369] In a round-bottom glass flask (1000 mL), dimethyl 4-chloropyridine-2,6-dicarboxylate (15.0 g; 65.3 mmol; 1.0 equivalent) was gradually dissolved in a mixed solvent of THF (540 mL) and MeOH (120 mL). The resulting slightly yellowish solution was cooled in an ice bath (~5°C), and then solid NaBH4 (12.4 g; 328 mmol; 5.0 equivalent) was added partially (over 1 hour without stoppering the flask). During this time, hydrogen gas was vigorously generated, and the color of the mixture changed from red to orange and finally to yellow. After the addition, the flask was warmed to room temperature and then stirred at room temperature for 16 hours. The resulting slightly yellowish milky white solution was filtered through glass frit (S3), the filtrate was evaporated to dryness, and then co-evaporated with DCM (as a suspension). The residue was dissolved in boiling H2O (less than 600 mL), and the resulting highly alkaline solution was continuously extracted with DCM overnight. The organic layer (containing partially crystallized product) was evaporated to dryness. The residue was mechanically ground and further dried under high vacuum (until a certain mass was obtained) to obtain the product in the form of free base as a nearly colorless microcrystalline powder. Yield: 10.84 g (96%; 1 step; based on dimethyl 4-chloropyridine-2,6-dicarboxylate). NMR (DMSO-d6): 1 H δ H 4.53(CH2,d,4H, 3 J HH =6);5.54(OH,t,2H, 3 J HH =6);7.36(arom.,s,2H). 13 C{ 1 H} δ C 63.7(CH2,s);118.0(arom.,s);144.0(arom.,s);163.5(arom.,s). ESI-HRMS:174.0317[M+H] + (Theoretical value [C7H9N1O2Cl1]) + =174.0316). EA(C7H8N1O2Cl1,M R =173.6):C48.4(48.5);H4.7(4.5);N7.7(8.1);Cl20.4(21.2).

[0370] Synthesis of TD759:

[0371] [ka]

[0372] In a round-bottom glass flask (100 mL), TD726 (626 mg; 3.61 mmol; 1.0 equivalent) was suspended in DCM (20 mL). Next, a freshly prepared solution of SOCl2 (780 μL; 10.7 mmol; 3.0 equivalent) in DCM (10 mL) was added, and the open flask was stirred at room temperature for 90 minutes to produce a clear solution. Next, a diluted aqueous solution of NaHCO3 (40 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The resulting crystallized residue was pulverized and further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless microcrystalline powder. Yield: 711 mg (94%; 1 stage; TD726 standard). NMR (DMSO-d6): 1 H δ H 4.79(CH2,s,4H);7.70(arom.,s,2H). 13 C{ 1 H} δ C 45.7(CH2,s);122.8(arom.,s);144.4(arom.,s);158.2(arom.,s). ESI-HRMS:209.9638[M+H] + (Theoretical value [C7H7N1Cl3]) + (=209.9639).

[0373] Synthesis of TD760:

[0374] [ka]

[0375] In a glass vial (20 mL), TD759 (708 mg; 3.36 mmol; 1.3 equivalents) was dissolved in MeCN (20 mL), followed by the addition of solid NaN3 (168 mg; 2.58 mmol; 1.0 equivalent) and dried K2CO3 (360 mg; 2.61 mmol; 1.0 equivalent). The resulting suspension was stirred at 80°C for 24 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the yellow oily residue was purified by column chromatography (SiO2, PE 40% to PE 10% in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum for 1 hour to obtain the product in the form of free base as a faint yellow oil. Yield: 323 mg (58%; 1 step; based on NaN3). Recovery amount: 218 mg of TD759 (31% of the initial dose). NMR (DMSO-d6): 1 H δ H 4.56(CH2,s,2H);4.78(CH2,s,2H);7.58(arom.,d,1H, 4 J HH =2);7.69(arom.,d,1H, 4 J HH =8). 13 C{ 1 H} δ C 45.8(CH2,s);53.6(CH2,s);121.9(arom.,s);122.5(arom.,s);144.4(arom.,s);157.9(arom.,s);158.2(arom.,s). ESI-HRMS:217.0040[M+H] + (Theoretical value [C7H7N4Cl]2) + (=217.0042).

[0376] Synthesis of TD1146:

[0377] [ka]

[0378] In a glass vial (20 mL), TD726 (495 mg; 2.85 mmol; 1.0 equivalent) was dissolved in DMF (11.0 mL; 143 mmol; 50 equivalents), and then freshly powdered KOH (1.6 g; 28.5 mmol; 10 equivalents) was added. The resulting suspension was stirred at 100 °C for 3 days in the presence of air (two thick needles were used to pierce holes in the septum of the vial). Thereafter, the mixture was filtered through a syringe microfilter (PTFE; the solid was washed with DMF). The filtrate was evaporated to dryness and purified by preparative HPLC (C18; H2O-MeCN gradient, with addition of TFA). The fractions containing the product were combined and directly freeze-dried to obtain the product as a white foam in the form of trifluoroacetate. Yield: 187 mg (22%; one-step; based on TD726). NMR (DMSO-d6): 1 H δ H 3.18 (CH3, s, 6H); 4.59 (CH2, d, 4H, 3 J HH = 6); 5.91 (OH, t, 2H, 3 J HH = 6); 6.84 (arom., s, 2H). ESI-HRMS: 183.1128 [M+H] + (theoretical value [C9H 15 N2O2] + = 183.1128). EA (C9H 14 N2O2-1.0TFA, M R = 296.2): C 44.6 (44.1); H 5.1 (4.9); N 9.5 (9.1); F 19.2 (18.2).

[0379]

Chemical Structure

[0380] Synthesis of TD1154: In a round-bottom glass flask (100 mL), TD1146 1.0 TFA (173.3 mg; 585 μmol; 1.0 equivalent) was suspended in DCM (17 mL), and neat SOCl2 (780 μL; 2.34 mmol; 4.0 equivalents) was added. The resulting mixture was stirred at room temperature for 90 minutes to obtain a clear solution. Next, a diluted aqueous solution of NaHCO3 (20 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a white solid. Yield: 105.6 mg (82%; 1 step; based on TD726). NMR (DMSO-d6): 1 H δ H 2.98(CH3,s,6H);4.60(CH2,s,4H);6.73(arom.,s,2H).ESI-HRMS:219.0450[M+H] + (Theoretical value [C9H 13 N2Cl2] + (=219.0450).

[0381] Synthesis of TD1163:

[0382] [ka]

[0383] In a glass vial (20 mL), TD1154 (103.9 mg; 474 μmol; 1.3 equivalents) was dissolved in MeCN (7 mL), followed by the addition of solid NaN3 (23.7 mg; 365 μmol; 1.0 equivalent) and dry K2CO3 (50.3 mg; 364 μmol; 1.0 equivalent). The resulting suspension was stirred at 70 °C for 24 h. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the residue was purified by column chromatography (SiO2, 5% EtOAc in DCM to 10% EtOAc in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum for 1 h to afford the product in the form of a free base as a colorless oil. Yield: 33.8 mg (41%; one-step; based on NaN3). Recovered: 11.0 mg of TD1154 (11% of the initial amount used). NMR (DMSO-d6): 1 H δ H 2.99 (CH3, s, 6H); 4.32 (CH2, s, 2H); 4.60 (CH2, s, 2H); 6.60 (arom., d, 1H, 4 J HH = 2); 6.73 (arom., d, 1H, 4 J HH = 2). ESI-HRMS: 226.0853 [M + H] + (calcd for [C9H 13 N5Cl1] + = 226.0854).

[0384] Synthesis of TD1024:

[0385]

Chem.

[0386] A glass vial (100 mL) was filled with CuI (70 mg; 0.37 mmol; 4.0 mol%), [Pd(PPh3)2Cl2] (125 mg; 0.18 mmol; 2.0 mol%), and a magnetic stirrer, and protected with argon three times. Subsequently, a solution of dimethyl 4-iodopyridine-2,6-dicarboxylate (2.93 g; 9.13 mmol; 1.0 equivalent) in a mixture of dry THF (40 mL), dry toluene (40 mL), and dry DMF (2 mL) was added through the septum. After adding ethinyltriisopropylsilane (2.25 mL; 10.0 mmol; 1.1 equivalent) and TEA (3.80 mL; 27.3 mmol; 3.0 equivalent), the mixture turned pale red. The flask was left under the septum (without external argon) and stirred at room temperature for 20 hours. The resulting dark-colored mixture was filtered through a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was dissolved in DCM (250 mL), and the resulting red solution was washed once with an aqueous solution of Na2S2O3. The organic layer was separated, dried over Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by flash chromatography (20% siRNA in DCM from 220 g SiO2, 100% DCM). The combined fraction containing the product was evaporated to dryness and then evaporated once more with DCM. The residue was further dried overnight under high vacuum to obtain the pre-purification product in the form of free base as an orange oil. Yield: 3.16 g (92%; 1 step; based on dimethyl 4-iodopyridine-2,6-dicarboxylate). NMR (DMSO-d6): 1 H δ H 0.92-1.27(i-Pr,m,21H);3.92(CH3,s,6H);8.16(arom,s,2H). 13 C{ 1 H} δ C 10.6(i-Pr,s);18.4(i-Pr,s);52.9(CH3,s);99.0(C≡C-Si,s);102.5(C≡C-a rom.;s);129.3(arom.,s);132.7(arom.,s);148.4(arom.,s);164.0(CO,s). ESI-HRMS:376.1939[M+H] + (Theoretical value [C 20H 30 N1O4Si1] + (=376.1939).

[0387] Synthesis of TD808:

[0388] [ka]

[0389] In a round-bottom glass flask (250 mL), TD1024 (3.15 g; 8.39 mmol; 1.0 equivalent) was dissolved in a mixed solvent of THF (60 mL) and MeOH (30 mL). Next, solid NaBH4 (2.55 g; 67.4 mmol; 8 equivalents) was partially added over 1 hour (during which hydrogen gas was vigorously generated), and the color of the mixture changed to dark red. After the addition, the flask was stirred at room temperature for another 1 hour. The mixture was evaporated to dryness. The residue was suspended in DCM (200 mL) and H2O (200 mL) and transferred to a separatory funnel. After shaking, the bottom phase (as a suspension) was separated. The aqueous phase was further extracted with DCM (3 × 100 mL). The combined organic layers were diluted with ELISA (500 mL). The resulting brown solution was dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a light brown solid. Yield: 2.61 g (97%; 1 step; TD1024 standard). NMR (DMSO-d6): 1 H δ H 1.04-1.17(i-Pr,m,21H);4.51(CH2,d,4H, 3 J HH =6);5.48(OH,t,2H, 3 J HH =6); 7.31(arom, s, 2H). 13 C{ 1 H} δ C 10.6(i-Pr,s);18.5(i-Pr,s);63.9(CH2,s);94.6(C≡C-Si,s);105.1(C≡C-arom.;s);119.8(arom.,s);130.8(arom.,s);161.9(arom.,s). ESI-HRMS:320.2036[M+H]+ (Theoretical value [C 18 H 30 [N1O2] + (=320.2040).

[0390] Synthesis of TD815:

[0391] [ka]

[0392] In a round-bottom glass flask (250 mL), pre-purified TD808 (2.61 g; 8.17 mmol; 1.0 equivalent) was suspended in DCM (100 mL). Next, a freshly prepared solution of SOCl2 (1.80 mL; 24.8 mmol; 3.0 equivalent) in DCM (10 mL) was added, and the open flask was stirred at room temperature for 1 hour. Then, a diluted aqueous solution of NaHCO3 (100 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 75 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 2.87g (99%; 1 stage; TD808 standard). NMR (DMSO-d6): 1 H δ H 0.97-1.20(i-Pr,m,21H);4.78(CH2,s,4H);7.59(arom,s,2H). 13 C{ 1 H} δ C 10.6(i-Pr,s);18.4(i-Pr,s);45.9(CH2,s);96.6(C≡C-Si,s);103.5(C≡C-arom.;s);124.6(arom.,s);132.0arom.,s);157.1(arom.,s). ESI-HRMS:356.1368[M+H] + (Theoretical value [C 18 H 28 N1Cl2Si1] + (=356.1368).

[0393] Synthesis of TD817:

[0394] [ka]

[0395] In a round-bottom glass flask (250 mL), TD815 (2.86 g; 8.02 mmol; 1.2 equivalents) was dissolved in MeCN (130 mL), followed by the addition of solid NaN3 (435 mg; 6.69 mmol; 1.0 equivalent) and dried K2CO3 (920 mg; 6.67 mmol; 1.0 equivalent). The resulting suspension was stirred at 80°C for 16 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the orange oily residue was purified by column chromatography (220 g SiO2, 30% PE in DCM to 10% PE in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum for 1 hour to obtain the product in the form of free base as a slightly yellow oil. Yield: 1.23 g (51%; 1 step; based on NaN3). Recovery amount: 709 mg of TD815 (25% of the initial dose). NMR (DMSO-d6): 1 H δ H 1.01-1.21(i-Pr,m,21H);4.55(CH2,s,2H);4.78(CH2,s,2H);7.47(arom.,d,1H, 4 J HH =1);7.58(arom, d,1H, 4 J HH =1). 13 C{ 1 H} δ C 10.6(i-Pr,s);18.4(i-Pr,s);46.0(CH2,s);53.7(CH2,s);96.5(C≡C-Si,s);103.7( C≡C-arom,s);124.3(arom.,s);131.8(arom.,s);156.6(arom.,s);157.1(arom.,s). ESI-HRMS:363.1763[M+H] + (Theoretical value [C 18 H 28N4Cl1Si1] + (=363.1766).

[0396] Synthesis of TD1052:

[0397] [ka]

[0398] In a glass vial (20 mL), dimethyl 4-iodopyridine-2,6-dicarboxylate (500 mg; 1.56 mmol; 1.0 equivalent), CuI (600 mg; 3.15 mmol, 2.0 equivalents), and [(dppf)PdCl2] (7.0 mg, 10 μmol; 0.6 mol%) were dissolved in dry DMF (8 mL), followed by the addition of methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (600 mg; 3.12 mmol; 2.0 equivalents) to dry DMF (2 mL). The resulting dark mixture was stirred at 100 °C for 16 hours. After cooling, the mixture was diluted with DCM (10 mL) and filtered through a syringe microfilter (PTFE). The solid was further washed with DCM. The filtrate was further diluted with DCM (50 mL) and washed with a diluted aqueous solution of NaHCO3 (5 × 25 mL). The organic layer was dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by column chromatography (120 g SiO2, 100% DCM to 15% siRNA in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless solid. Yield: 366 mg (89%; 1 step; based on dimethyl 4-iodopyridine-2,6-dicarboxylate). NMR (DMSO-d6): 1 H δ H 3.97(CH3,s,6H);8.49(arom.,q,2H, 4 J HF =1). 19 F { 1 H} δ F -63.4(s). ESI-HRMS:264.0480[M+H] + (Theoretical value [C 10 H9N1O4F3] +=264.0478).

[0399] Synthesis of TD1053:

[0400] [Chemical formula]

[0401] In a round-bottom glass flask (100 mL), TD1052 (365 mg; 1.39 mmol; 1.0 equivalent) was dissolved in a mixed solvent of MeOH (5 mL) and THF (10 mL). The resulting colorless solution was cooled in an ice bath (to 5 °C or lower), and then solid NaBH4 (420 mg, 11.1 mmol, 8.0 equivalents) was added partially (without plugging the flask, over 30 minutes). During this process, hydrogen gas evolved vigorously, and the color of the mixture changed to red and orange. After the addition, the flask was warmed to room temperature and further stirred at room temperature for 30 minutes. The resulting yellow solution was evaporated to dryness. The residue was purified by column chromatography (120 g SiO2, solid loading method, from 100% EtOAc to 15% MeOH in EtOAc). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum overnight to obtain the product in the form of a yellow oil as a free base. Yield: 171 mg (60%; one-step; based on TD1052). NMR (DMSO-d6): 1 H δ H 4.62 (CH2, s, 4H); 5.65 (OH, s, 2H); 7.60 (arom., q, 2H, 4 J HF = 1). 19 F{ 1 H} δ F -63.6 (s). ESI-HRMS: 208.0580 [M + H] + (theoretical value [C8H9N1O2F3] + = 208.0580).

[0402] Synthesis of TD1055:

[0403] [Chemical formula]

[0404] In a round-bottom glass flask (100 mL), TD1053 (169 mg; 816 μmol; 1.0 equivalent) was dissolved in DCM (20 mL). Next, a freshly prepared solution of SOCl2 (237 μL; 3.26 mmol; 4.0 equivalents) in DCM (5 mL) was added, and the resulting mixture was stirred at room temperature for 16 hours. Then, a diluted aqueous solution of NaHCO3 (25 mL) was added, and the resulting two-phase mixture was vigorously stirred at room temperature for a further 10 minutes, during which gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 168 mg (84%; 1 step; based on TD1053). NMR (DMSO-d6): 1 H δ H 4.90(CH2,s,4H);7.94(arom.,s,2H). 19 F{ 1 H} δ F -63.4s).APCI-HRMS:243.9902[M+H] + (Theoretical value [C8H7N1F3Cl2]) + (=243.9902).

[0405] Synthesis of TD1057:

[0406] [ka]

[0407] In a glass vial (4 mL), TD1055 (143 mg; 586 μmol; 1.8 equivalents) was dissolved in MeCN (3.5 mL), followed by the addition of solid NaN3 (21.5 mg; 331 μmol; 1.0 equivalent) and dried K2CO3 (46 mg; 333 μmol; 1.0 equivalent). The resulting suspension was stirred at 70°C for 3 days. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the orange oily residue was purified by column chromatography (80 g SiO2, 30% PE in DCM to 40% DCM in PE). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum for 1 hour to obtain the product in the form of free base as a colorless oil. Yield: 43.9 g (53%; 1 step; NaN3). Recovery amount: 32.1 mg of TD1055 (22% of the initial usage). NMR (DMSO-d6): 1 H δ H 4.69(CH2,s,2H);4.90(CH2,s,2H);7.81(arom.,d,1H, 4 J HH =1);7.93(arom.,d,1H, 4 J HH =1). 19 F{ 1 H} δ F -63.4(s). APCI-HRMS:251.0306[M+H] + (Theoretical value [C8H7N4F3Cl1]) + (=251.0306).

[0408] Synthesis of TD549:

[0409] [ka]

[0410] Dimethyl 4-chloropyridine-2,6-dicarboxylate (5.00 g; 21.8 mmol; 1.0 equivalent), phenylboronic acid (3.20 g; 26.2 mmol; 1.2 equivalents), and XPhos Pd G2 (510 mg; 648 μmol; 3.0 mol%) were placed in a pear-shaped glass flask (250 mL) and protected with argon three times. Next, dry DMF (110 mL) was added through the septum under a constant flow of argon, followed by freshly dried Cs2CO3 (15.6 g; 47.9 mmol; 2.2 equivalents, the flask was briefly opened for addition). The mixture was then stirred under the septum (without external argon) at 80°C for 20 hours. The resulting dark mixture was filtered through glass frit (S3), and the filtrate was poured into a stirring beaker containing H2O (500 mL). The precipitate was collected on glass frit (S2), washed with H2O, and dried overnight under high vacuum to obtain the product in the form of a free base as an off-white solid. Yield: 3.01 g (51%; 1 step; based on dimethyl 4-chloropyridine-2,6-dicarboxylate). NMR (DMSO-d6): 1 H δ H 3.95(CH3,s,6H);7.45-7.57(Ph,m,3H);7.82-8.00(Ph,m,2H);8.48(arom.,s,2H); 13 C{ 1 H} δ C 52.7(CH3,s);125.0(arom,s);127.2(Ph.,s);129.5(Ph.,s);130.2(Ph.,s);135.5(Ph.,s);148.6(arom.,s);150.0(arom.,s);164.7(CO.,s). ESI-HRMS:272.0916[M+H] + (Theoretical value [C 15 H 14 N1O4] + (=272.0917).

[0411] Synthesis of TD563:

[0412] [ka]

[0413] In a round-bottom glass flask (500 mL), TD549 (2.98 g; 11.0 mmol; 1.0 equivalent) was dissolved in a mixed solvent of MeOH (100 mL) and THF (100 mL). The resulting slightly yellow solution was cooled in an ice bath (below 5°C), and then solid NaBH4 (2.90 g, 76.7 mmol, 9.0 equivalent) was added partially (over 30 minutes without stoppering the flask). During this time, hydrogen gas was vigorously generated, and the color of the mixture changed to red and orange. After the addition, the flask was warmed to room temperature and stirred for another 30 minutes at room temperature. The resulting slightly yellow, opaque solution was filtered through a syringe microfilter (PTFE). The filtrate was evaporated to dryness and then co-evaporated with DCM (as a suspension). The residue was dissolved in a mixed solvent of H2O (300 mL) and DCM (300 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (8 × 75 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting crystallized residue was pulverized and further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless solid. Yield: 2.29 g (97%; 1 step; TD549 standard). NMR (DMSO-d6): 1 H δ H 4.59(CH2,s,4H);5.45(OH,s,2H);7.45-7.57(Ph,m,3H);7.60(arom.,s,2H);7.68-7.82(Ph,m,2H). 13 C{ 1 H} δ C 64.2(CH2,s);115.7(arom.,s);126.7(Ph,s);129.1(Ph,s);129.3(Ph,s);138.1(Ph,s);148.2(arom.,s);161.7(arom.,s). ESI-HRMS:216.1022[M+H] + (Theoretical value [C 13 H 14 [N1O2] + (=216.1019).

[0414] Synthesis of TD564:

[0415] [ka]

[0416] In a round-bottom glass flask (500 mL), TD563 (2.25 g; 10.5 mmol; 1.0 equivalent) was dissolved in DCM (250 mL) (with gentle heating). Next, a freshly prepared solution of SOCl2 (2.27 mL; 31.3 mmol; 3.0 equivalent) in DCM (10 mL) was added. Subsequently, a precipitate began to form. After stirring at room temperature for 1 hour, all of the previously formed precipitate dissolved again. Next, a diluted aqueous solution of NaHCO3 (150 mL) was added to the clear yellow solution, and the resulting two-phase mixture was vigorously stirred at room temperature for another 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellowish solid. Yield: 2.59 g (98%; 1 step; TD563 standard). NMR (DMSO-d6): 1 H δ H 4.88(CH2,s,4H);7.47-7.58(Ph,m,3H);7.77-7.85(Ph,m,2H);7.86(arom.,s,2H). 13 C{ 1 H} δ C 46.6(CH2,s);120.3(arom.,s);126.9(Ph,s);129.3(Ph,s);129.7(Ph,s);136.5(P h,s);149.5(arom,s);157.1(arom.,s);157.1(arom.,s);ESI-HRMS:252.0342[M+H] + (Theoretical value [C 13 H 12 N1Cl2] + (=252.0341).

[0417] Synthesis of TD566:

[0418] [ka]

[0419] In a pear-shaped glass flask (100 mL), TD564 (1.60 g; 6.35 mmol; 1.3 equivalents) was dissolved in MeCN (60 mL) (gently heated), followed by the addition of solid NaN3 (320 mg; 4.92 mmol; 1.0 equivalent) and dried K2CO3 (680 mg; 4.93 mmol; 1.0 equivalent). The resulting suspension was stirred at 80°C for 24 hours. The mixture was filtered through glass frit S3, and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the orange oily residue was purified by column chromatography (SiO2, DCM, 15% PE to 100% DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried under high vacuum for 1 hour to obtain the product in the form of free base as a white solid. Yield: 691 mg (54%; 1 step; based on NaN3). Recovery amount: 613 mg of TD564 (38% of the initial dose). NMR (DMSO-d6): 1 H δ H 4.59(CH2,s,2H);4.84(CH2,s,2H);7.46-7.60(Ph,m,3H);7.74(arom.,d,1H, 4 J HH =2);7.77-7.85(Ph,m,2H);7.86(arom.,d,1H, 4 J HH =2). 13 C{ 1 H} δ C 46.7(CH2,s);54.2(CH2,s);119.4(arom.,s);120.0(arom.,s);126.9(Ph,s);129.3(P h,s);129.7(Ph,s);136.6(Ph,s);149.3(arom.,s);156.6(arom.,s);157.1(arom.,s). ESI-HRMS:259.0747[M+H] + (Theoretical value [C 13 H 12 N4Cl1] + (=259.0745).

[0420] Synthesis of TD1083:

[0421] [ka]

[0422] In a round-bottom glass flask (100 mL), methyl isonicotinate (250 mg; 1.83 mmol; 1.0 equivalent) was dissolved in MeOH (10 mL), and concentrated H2SO4 (25 μL) was added. The resulting mixture was stirred at 55°C for 30 minutes. After cooling, a solution of (NH4)2S2O8 (4.16 g; 18.2 mmol; 10 equivalents) in H2O (10 mL) was added dropwise, and the resulting mixture was stirred further at 55°C for 16 hours. The reaction was then carefully quenched with aqueous NaHCO3 solution. The mixture was transferred to a separatory funnel and extracted with ELISA (5 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were coupled and directly freeze-dried to obtain the product in the form of free base as a white, fluffy solid. Yield: 99.5 mg (28%; 1 step; based on methyl isonicotinate). NMR (DMSO-d6): 1 H δ H 3.92(CH3,s,3H);4.60(CH2,d,4H, 3 J HH =6); 5.58(OH,t,2H, 3 J HH =6);7.80(arom.,s,2H).ESI-HRMS:198.0759[M+H] + (Theoretical value [C9H 12 N1O4] + =198.0761). EA(C9H 11 N1O4,M R =197.2):C54.8(54.8);H5.6(5.5);N7.1(7.0).

[0423] Synthesis of TD1087:

[0424] [ka]

[0425] In a round-bottom glass flask (50 mL), TD1083 (96.2 mg; 488 μmol; 1.0 equivalent) was dissolved in DCM (20 mL), followed by the addition of SOCl2 (106 μL; 1.46 mmol; 3.0 equivalents). The mixture was stirred at room temperature for 1 hour. Then, a diluted aqueous solution of NaHCO3 (20 mL) was added to quench the mixture. The resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum (where the product slowly crystallized), yielding the product in the form of free base as a bright yellow solid. Yield: 112.8 mg (99%; 1 step; based on TD1083). NMR (DMSO-d6): 1 H δ H 3.92(CH3,s,3H);4.90(CH2,s,4H);7.98(arom.,s,2H).ESI-HRMS:234.0085[M+H] + (Theoretical value [C9H 10 [N1O2Cl2] + (=234.0083).

[0426] Synthesis of TD1089:

[0427] [ka]

[0428] In a glass vial (4 mL), TD1087 (112.0 mg; 478 μmol; 1.2 equivalents) was dissolved in MeCN (2 mL), followed by the addition of solid NaN3 (26.0 mg; 400 μmol; 1.0 equivalent) and dried K2CO3 (55.0 mg; 400 μmol; 1.0 equivalent). The resulting suspension was stirred at 70°C for 2 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the oily residue was purified by column chromatography (SiO2, DCM, 10% ethyl acetate in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the pre-purification product in the form of free base (containing less than 20% bisazide by-products) as a yellow oil. Yield: 49.6 mg. ESI-HRMS: 241.0490[M+H] + (Theoretical value [C9H 10 N4O2Cl1] + (=241.0487). A portion of the TD1089 obtained in this way was used directly in TD1092 without further purification or characterization.

[0429] Synthesis of TD1101:

[0430] [ka]

[0431] In a round-bottom glass flask (100 mL), isopropyl isonicotinate (600 mg; 3.63 mmol; 1.0 equivalent) was dissolved in MeOH (20 mL), and concentrated H2SO4 (50 μL) was added. Next, a solution of (NH4)2S2O8 (8.32 g; 36.5 mmol; 10 equivalents) in H2O (20 mL) was added dropwise, and the resulting mixture was stirred at 70°C for 16 hours. The reaction products were then carefully neutralized with aqueous NaHCO3 solution. The mixture was transferred to a separatory funnel and extracted with RINKAN (5 × 50 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were joined and directly freeze-dried to obtain the product in the form of free bases as a white, fluffy solid. Yield: 284 mg (35%; 1 level; based on isopropyl isonicotinate). NMR (DMSO-d6): 1 H δ H 1.34(CH3,d,6H, 3 J HH =6);4.59(CH2,d,4H, 3 H J HH =6);5.19(CH,hept,1H, 3 J HH =6);5.57(OH,t,2H, 3 J HH =6);7.78(arom.,s,2H).ESI-HRMS:226.1076[M+H] + (Theoretical value [C 11 H 16 N1O4] + (=226.1074).

[0432] Synthesis of TD1109:

[0433] [ka]

[0434] In a round-bottom glass flask (100 mL), TD1101 (283 mg; 1.26 mmol; 1.0 equivalent) was dissolved in DCM (40 mL), followed by the addition of SOCl2 (275 μL; 3.79 mmol; 3.0 equivalent). The mixture was stirred at room temperature for 1 hour. Then, a diluted aqueous solution of NaHCO3 (40 mL) was added to quench the mixture. The resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum (where the product slowly crystallized), yielding the product in the form of free base as a pale yellow solid. Yield: 322 mg (98%; 1 step; based on TD1101). NMR (DMSO-d6): 1 H δ H 1.35(CH3,d,6H, 3 J HH =6);4.90(CH2,s,4H);5.20(CH,hept,1H, 3 J HH =6);7.96(arom.,s,2H).ESI-HRMS:262.0399[M+H] + (Theoretical value [C 11 H 14 [N1O2Cl2] + (=262.0396).

[0435] Synthesis of TD1112:

[0436] [ka]

[0437] In a glass vial (20 mL), TD1109 (321 mg; 1.23 mmol; 1.2 equivalents) was dissolved in MeCN (6 mL), followed by the addition of solid NaN3 (66.5 mg; 1.02 mmol; 1.0 equivalent) and dried K2CO3 (141 mg; 1.02 mmol; 1.0 equivalent). The resulting suspension was stirred at 70°C for 16 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the oily residue was purified by column chromatography (SiO2, DCM to 3% Â in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 116 mg (42%; 1 step; based on NaN3). Recovery: 124 mg of TD1109 (39% of the amount initially used). NMR (DMSO-d6): 1 H δ H 1.35(CH3,d,6H, 3 J HH =6);4.67(CH2,s,2H);4.89(CH2,s,4H);5.19(CH,hept,1H, 3 J HH =6);7.83(arom.,d,1H, 4 J HH =1).7.95(arom.,d,1H, 4 J HH =1). ESI-HRMS:269.0800[M+H] + (Theoretical value [C 11 H 14 N4O2Cl1] + (=269.0800).

[0438] Synthesis of TD1119:

[0439] [ka]

[0440] In a glass vial (40 mL), tert-butyl isonicotinate (486 mg; 2.71 mmol; 1.0 equivalent) was dissolved in MeOH (15 mL), and then concentrated H2SO4 (40 μL) was added. Next, a solution of (NH4)2S2O8 (6.20 g; 27.2 mmol; 10 equivalents) in H2O (15 mL) was added dropwise, and the resulting mixture was further stirred at 80°C for 30 minutes. The reaction was then carefully neutralized with NaHCO3. The mixture was transferred to a separatory funnel and extracted with RINKAN (5 × 30 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product in the form of free base as a white, fluffy solid. Yield: 161 mg (25%; 1 level; based on tert-butyl isonicotinate). NMR (DMSO-d6): 1 H δ H 1.57(CH3,s,9H);4.58(CH2,d,4H, 3 J HH =4);5.54(OH,t,2H, 3 J HH =4);7.73(arom.,s,2H).ESI-HRMS:240.1230[M+H] + (Theoretical value [C 12 H 18 N1O4] + (=240.1230).

[0441] Synthesis of TD1129:

[0442] [ka]

[0443] In a round-bottom glass flask (100 mL), TD1119 (159 mg; 664 μmol; 1.0 equiv) was dissolved in DCM (20 mL), followed by the addition of SOCl2 (145 μL; 2.00 mmol; 3.0 equiv). The mixture was stirred at room temperature for 1 hour. Then, a diluted aqueous solution of NaHCO3 (20 mL) was added to quench the mixture. The resulting two-phase mixture was vigorously stirred at room temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum (where the product slowly crystallized), yielding the product in the form of free base as a faint yellow solid. Yield: 175 mg (95%; 1 step; based on TD1119). NMR (DMSO-d6): 1 H δ H 1.57(CH3,s,9H);4.89(CH2,s,4H);7.91(arom.,s,2H).ESI-HRMS:276.0552[M+H] + (Theoretical value [C 12 H 16 [N1O2Cl2] + (=276.0553).

[0444] Synthesis of TD1130:

[0445] [ka]

[0446] In a glass vial (4 mL), TD1129 (174.0 mg; 630 μmol; 1.2 equivalents) was dissolved in MeCN (3 mL), followed by the addition of solid NaN3 (34.0 mg; 523 μmol; 1.0 equivalent) and dried K2CO3 (72.0 mg; 522 μmol; 1.0 equivalent). The resulting suspension was stirred at 70°C for 6 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the oily residue was purified by column chromatography (SiO2, DCM to 3% Â in DCM). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 58.2 mg (39%; 1 step; based on NaN3). Recovery: 90.5 mg of TD1129 (52% of the initial amount used). NMR (DMSO-d6): 1 H δ H 1.57(CH3,s,9H);4.66(CH2,s,2H);4.89(CH2,s,2H);7.79(arom.,d,1H, 4 J HH =2).7.91(arom.,d,1H, 4 J HH =2). ESI-HRMS:305.0772[M+Na] + (Theoretical value [C 12 H 15 [N4O2Cl1Na1] + (=305.0776).

[0447] Synthesis of TD725:

[0448] [ka]

[0449] A glass vial (40 mL) was filled with TD726 (628 mg; 3.62 mmol; 1.0 equivalent), (4-(tert-butoxycarbonyl)phenyl)boronic acid (880 mg; 3.96 mmol; 1.1 equivalent), and XPhos Pd G2 (57 mg; 7.2 μmol; 2.0 mol%), and protected with argon three times. Next, dry 1,4-dioxane (16 mL) was added through the septum under a constant flow of argon, followed by the addition of a freshly prepared solution of K3PO4-H2O (920 mg; 4.00 mmol; 1.1 equivalent) in H2O (8 mL) (which had been briefly washed with argon before addition). This mixture was stirred under the septum (but without external argon) at 80°C for 16 hours. The resulting dark mixture was transferred to a separatory funnel and diluted with siRNA (40 mL) and H₂O (50 mL). After shaking, the upper layer was separated, and the aqueous layer was further extracted with siRNA (5 × 30 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was purified by flash chromatography (120 g SiO₂, 100% siRNA to 30% MeOH in siRNA). The combined fraction containing the product was evaporated to dryness and then co-evaporated once using DCM. The residue was further dried overnight under high vacuum to obtain the pre-purification product in the form of free base as an off-white powder. Yield: 1.04 g, NMR (DMSO-d6): 1 H δ H 1.57(CH3,s,9H);4.61(CH2,d,4H, 3 J HH =6);5.48(OH,t,2H, 3 J HH =6);7.64(arom.,s,2H);7.88(arom.,dm,2H, 3 J HH =9);8.04(arom, dm,2H, 3 J HH =9). 13 C{ 1 H} δ C27.8(CH3,s);64.2(CH2,s);81.0(C-CH3,s);115.8(arom.,s);127.0(arom,s);129.9(arom .,s);131.6(arom.,s);142.2(arom.,s);147.0(arom.,s);162.0(arom.,s);164.6(CO,s). ESI-HRMS:316.1539[M+H] + (Theoretical value [C 18 H 22 N1O4] + (=316.1543).

[0450] Synthesis of TD730:

[0451] [ka]

[0452] In a round-bottom glass flask (250 mL), pre-purified TD725 (1.04 g; ≤3.30 mmol; 1.0 equivalent) was suspended in DCM (35 mL). Next, a freshly prepared solution of SOCl2 (721 μL; 9.93 mmol; ≥3.0 equivalent) in DCM (5 mL) was added. A clear solution was immediately formed, and the open flask was stirred at room temperature for 1 hour. The mixture was quenched by adding a diluted aqueous solution of NaHCO3 (60 mL). The resulting two-phase mixture was vigorously stirred at constant temperature for a further 1 hour, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (5 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by flash chromatography (120 g SiO2, 100% DCM to 20% siRNA in DCM). The combined fraction containing the product was evaporated to dryness and then evaporated once more in DCM. The remaining slightly yellowish oil slowly crystallized. The solid was pulverized and dried overnight under high vacuum to obtain the product in the form of free base as an off-white powder. Yield: 839 mg (66%; 2 steps; TD726 standard). NMR (DMSO-d6): 1 H δ H1.58(CH3,s,9H);4.86(CH2,s,4H);7.92(arom.,s,2H);7.94(arom.,dm,2H, 3 J HH =9);8.05(arom.,dm,2H, 3 J HH =9). 13 C{ 1 H} δ C 27.8(CH3,s);46.5(CH2,s);81.2(C-CH3,s);120.6(arom,s);127.2(arom.,s);129.9(arom .,s);132.1(arom.,s);140.6(arom.,s);148.3(arom.,s);157.3(arom.,s);164.5(CO,s). ESI-HRMS:352.0866[M+H] + (Theoretical value[C 18 H 20 [N1O2Cl2] + =352.0866).

[0453] Synthesis of TD733:

[0454]

change

[0455] In a 20 mL glass vial, TD730 (837 mg; 2.38 mmol; 1.3 equivalents) was dissolved in MeCN (18 mL), followed by the addition of solid NaN3 (119 mg; 1.83 mmol; 1.0 equivalent) and dried K2CO3 (253 mg; 1.83 mmol; 1.0 equivalent). The resulting suspension was stirred at 80°C for 24 hours. The mixture was filtered through a syringe microfilter (PTFE), and the solid residue was washed with MeCN. The filtrate was evaporated to dryness, and the oily residue was purified by column chromatography (SiO2, 20% pentane in DCM to 1% Â in DCM). The fractions containing the product were combined and evaporated to dryness. The resulting nearly colorless oil was further dried overnight under high vacuum (where the product slowly crystallized), and the product in the form of free base was obtained as a colorless solid. Yield: 230 mg (35%; 1 step; based on NaN3). Recovery amount: 442 mg of TD730 (53% of the initial amount used). NMR (DMSO-d6): 1 H δ H 1.57(CH3,s,9H);4.62(CH2,s,2H);4.86(CH2,s,2H);7.80(arom.,d,1H, 4 J HH =2);7.91(arom.,d,1H, 4 J HH =2);7.94(arom, dm,2H, 3 J HH =9);8.05(arom.,dm,2H, 3 J HH =9). 13 C{ 1 H}δ C 27.8(CH3,s);46.6(CH2,s);54.2(CH2,s);81.2(C-CH3,s);119.6(arom,s);120.3(arom.,s);127.2(arom.,s);129. 9(arom.,s);132.1(arom.,s);140.7(arom.,s);148.1(arom.,s);156.8(arom.,s);157.3(arom.,s);164.5(CO,s). ESI-HRMS:359.1270[M+H] + (Theoretical value [C 18 H 20 N4O2Cl1]+ (=359.1269).

[0456] Synthesis of TD1425:

[0457] [ka]

[0458] A glass vial (40 mL) was filled with methyl 2-bromo-6-methylisonicotinate (1.00 g; 4.35 mmol; 1.0 equivalent), NBS (recrystallized from boiling H2O; 770 mg; 4.33 mmol; 1.0 equivalent), (BnO)2 (53 mg; 219 μmol; 5 mol%), and a magnetic stirrer, and protected with argon three times. Under a constant flow of argon, CCl4 (21 mL) was added through the septum. The vial was left to stand with stirring under the septum (but without external argon) at 75°C for 24 hours. After cooling, the mixture was filtered through a syringe microfilter (PTFE), and the solid was washed with DCM. The filtrate was evaporated to dryness, resuspended in c-Hex (25 ml), and filtered through a syringe microfilter (PTFE). The filtrate was evaporated to dryness, and the residue was purified by flash chromatography (120 g SiO2, 100% c-Hex to 80% DCM in c-Hex). The combined fraction containing the product was evaporated to dryness. The residual oil was further dried overnight under high vacuum (where the product crystallized), yielding the product in the form of free base as a slightly yellow solid. Yield: 571 mg (43%; 1 step; based on methyl 2-bromo-6-methylisonicotinate). Recovery: 314 mg of methyl 2-bromo-6-methylisonicotinate (31% of the initial amount used). NMR (DMSO-d6): 1 H δ H 3.91(CH3,s,3H);4.79(CH2,s,2H);7.94(arom.,d,1H, 4 J HH =1);8.05(arom.,d,1H, 4 J HH =1). ESI-HRMS:307.8916[M+H] + (Theoretical value [C8H8N1O2Br2]) +(=307.8916).

[0459] Synthesis of TD1428:

[0460] [ka]

[0461] TD1425 (523 mg; 1.69 mmol; 1.0 equivalent), N-Boc-1,1-dimethylpyropagylamine (310 mg; 1.69 mmol; 1.0 equivalent), and a magnetic stirrer were placed in a pear-shaped glass flask (25 mL) and protected with argon three times. Then, solid CuI (13.0 mg; 68 μmol; 4.0 mol%) and [Pd(PPh3)2Cl2] (24 mg; 34 μmol; 2.0 mol%) were added and protected with argon again (three times). Next, dry THF (7.5 mL) was added through a septum under a constant flow of argon, followed by the addition of DIPEA (885 μL; 5.08 mmol; 3.0 equivalent). The flask was left at room temperature for two days with stirring under the septum (but without external argon). The mixture was evaporated to dryness, resuspended in DCM (15 mL), filtered through a syringe microfilter (PTFE), and the solid phase was further washed with DCM. The filtrate was purified by flash chromatography (120 g SiO2, 10% Â to 50% Â in c-Hex). The combined fraction containing the product was evaporated to dryness and re-purified once by flash chromatography (80 g SiO2, 100% Â to 5% Â in DCM). The combined fraction containing the product was evaporated to dryness and further evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellow solidified oil. Yield: 175 mg (25%; 1 step; TD1425 standard). NMR (DMSO-d6): 1 H δ H 1.42(CH3-CO,s,9H);1.56(CH3-CN,s,6H);3.91(CH3-O,s,3H);4.79(CH2,s,2H);7.23(NH,bs,1H);7.71(arom.,d,1H, 4 JHH =1);7.97(arom.,d,1H, 4 J HH =1). ESI-HRMS:411.0913[M+H] + (Theoretical value [C 18 H 24 N2O4Br1] + (=411.0914).

[0462] Synthesis of TD1386:

[0463] [ka]

[0464] In a round-bottom glass flask (100 mL), pyridine-2,6-diyldimethanol (2.00 g; 14.4 mmol; 1.0 equivalent) and imidazole (0.98 g; 14.4 mmol; 1.0 equivalent) were dissolved in DMF (20 mL), and TBDMSCl (2.17 g; 14.4 mmol; 1.0 equivalent) was added. The resulting solution was stirred at room temperature for 2 hours. The mixture was evaporated to dryness, and the residue was dissolved in a mixed solvent of DCM (50 mL) and diluted aqueous NaHCO3 (50 mL) and transferred to a separatory funnel. After shaking briefly, the bottom phase was separated, and the aqueous layer was further extracted with DCM (5 × 30 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was purified by column chromatography (100 g SiO2, DCM to 50% ethyl phosphate in DCM). The fractions containing the product were combined and evaporated to dryness, then co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 1.54 g (42%; 1 step; based on pyridine-2,6-diyldimethanol). NMR (DMSO-d6): 1 H δ H 0.09(CH3,s,6H);0.92(CH3,s,9H);4.51(CH2,d,2H, 3 J HH =5);4.71(CH2,s,2H);5.37(OH,t,1H, 3 J HH=5);7.28(arom,dd,1H, 3 J HH =8; 4 J HH =1);7.34(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.81(arom.,t,1H, 3 J HH =8). ESI-HRMS:254.1569[M+H] + (Theoretical value [C 13 H 24 N1O2Si1] + (=254.1571).

[0465] Synthesis of TD1389:

[0466] [ka]

[0467] In a round-bottom glass flask (250 mL), TD1386 (1.54 g; 6.08 mmol; 1.0 equivalent) was dissolved in DCM (60 mL), followed by the addition of SOCl2 (885 μL; 12.0 mmol; 2.0 equivalents). The mixture was stirred at room temperature for 1 hour. Then, a diluted solution of NaHCO3 (60 mL) was added to quench the mixture. The resulting two-phase mixture was vigorously stirred at room temperature for a further 30 minutes, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 1.63 g (99%; 1 step; based on TD1386). NMR (DMSO-d6): 1 H δ H 0.10(CH3,s,6H);0.92(CH3,s,9H);4.74(CH2,s,2H);4.75(CH2,s,2H);7.40(arom.,dd,1H, 3 J HH =8;4 J HH =1);7.42(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.87(arom.,t,1H, 3 J HH =8). ESI-HRMS:272.1231[M+H] + (Theoretical value [C 13 H 23 N1O1Cl1Si1] + (=272.1232).

[0468] Synthesis of TD1390:

[0469] [ka]

[0470] In a glass flask (20 mL), KCN (390 mg; 5.99 mmol; 1.05 equivalents) was dissolved in H2O (2 mL), followed by the addition of TD1389 (1.55 g; 5.70 mmol; 1.0 equivalent) in DMF (6 mL). The resulting mixture was vigorously stirred at 100 °C for 70 minutes. After cooling to room temperature, the mixture was diluted with H2O (4 mL) and MeCN (8 mL) and filtered through a syringe microfilter (PTFE). The filtrate was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The remaining pale yellow oil was solidified in a freezer. The resulting solid was pulverized and dried overnight under high vacuum to obtain the product in the form of a free base as a faint yellow powder. Yield: 1.23 g (82%; 1 step; TD1389 standard). NMR (DMSO-d6): 1 H δ H0.10(CH3,s,6H);0.92(CH3,s,9H);4.17(CH2,s,2H);4.75(CH2,s,2H);7.30(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.40(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.86(arom.,t,1H, 3 J HH =8). ESI-HRMS: 263.1573 [M+H] + (Theoretical value[C 14 H 23 N2O1Si1] + =263.1574).

[0471] Synthesis of TD1393:

[0472]

change

[0473] In a pear-shaped glass flask (100 mL), TD1390 (700 mg; 2.67 mmol; 1.00 equivalent) was dissolved in MeOH (2.70 mL; 66.7 mmol; 25 equivalents), followed by the addition of TMSCl (2.37 mL; 18.7 mmol; 7.0 equivalents). The resulting mixture was vigorously stirred at 60 °C for 2 hours. After cooling to room temperature, the mixture was quenched with H₂O (4 mL) and partially neutralized by slowly adding saturated aqueous solution of NaHCO₃ (5 mL) (to pH 3–4). The resulting two-phase mixture was carefully evaporated to dryness, and H₂O (10 mL) was added to the residue. The mixture was filtered through a syringe microfilter (RC), and the filtrate was directly purified by preparative HPLC (C18; H₂O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous solution of NaHCO₃, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free base as a slightly yellow oil. Yield: 404 mg (84%; 1 step; TD1390 standard). NMR (DMSO-d6): 1 H δ H 3.61(CH3,s,3H);3.81(CH2,s,2H);4.51(CH2,d,2H, 3 J HH =5);5.39(OH,t,1H, 3 J HH =5);7.20(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.36(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.76(arom.,t,1H, 3 J HH =8). ESI-HRMS:182.0811[M+H] + (Theoretical value [C9H 12 N1O3] + (=182.0812).

[0474] Synthesis of TD1395:

[0475] [ka]

[0476] In a 50 mL round-bottom glass flask, TD1393 (340 mg; 1.88 mmol; 1.0 equivalent) and imidazole (190 mg; 2.79 mmol; 1.5 equivalents) were dissolved in 6 mL of DMF, and then TBDMSCl (425 mg; 2.82 mmol; 1.5 equivalents) was added. The resulting solution was stirred at room temperature for 16 hours. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with NaHCO3 and diluted aqueous solution, and evaporated to dryness. The residue was dissolved in 50 mL of DCM and 50 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 4 × 25 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a faint yellow oil. Yield: 537 mg (97%; 1 step; TD1393 standard). NMR (DMSO-d6): 1 H δ H 0.09(CH3,s,6H);0.91(CH3,s,9H);3.61(CH3,s,3H);3.81(CH2,s,2H);4.70(CH2,s,2H);7.23(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.32(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.79(arom.,t,1H, 3 J HH =8). ESI-HRMS:296.1676[M+H] + (Theoretical value [C 15 H 26 N1O3Si1] +(=296.1677).

[0477] Synthesis of TD1396:

[0478] [ka]

[0479] In a round-bottom glass flask (100 mL), TD1395 (537 mg; 1.82 mmol; 1.0 equivalent) was dissolved in a mixed solvent of THF (9 mL) and MeOH (9 mL). To the resulting colorless solution, solid NaBH4 (2.06 g; 54.5 mmol; 30 equivalents) was added partially (over 10 minutes without stoppering the flask). During this time, hydrogen gas was vigorously generated. The mixture was further stirred at room temperature for 1 hour, during which time it was diluted twice with MeOH (9 mL each) to ensure stirring. The mixture was diluted with DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 483 mg (99%; 1 step; TD1395 standard). NMR (DMSO-d6): 1 H δ H 0.09(CH3,s,6H);0.92(CH3,s,9H);2.84(CH2,t,2H, 3 J HH =7);3.71(CH2,td,2H, 3 J HH =7,J 3 HH =5);4.62(OH,t,1H, 3 J HH =5);4.71(CH2,s,2H);7.14(arom,dd,1H, 3 J HH =8; 4 J HH =1);7.24(arom.,dd,1H, 3 J HH =8; 4 J HH=1);7.70(arom.,t,1H, 3 J HH =8). ESI-HRMS:268.1726[M+H] + (Theoretical value [C 14 H 26 N1O2Si1] + (=268.1727).

[0480] Synthesis of TD1401:

[0481] [ka]

[0482] In a round-bottom glass flask (50 mL), TD1396 (94 mg; 352 μmol; 1.0 equivalent) was dissolved in DCM (3.5 mL), followed by the addition of SOCl2 (51 μL; 702 μmol; 2.0 equivalent). The mixture was stirred at room temperature for 3 hours. The mixture was diluted with DCM (20 mL) and quenched with a diluted aqueous solution of NaHCO3 (10 mL). The resulting two-phase mixture was vigorously stirred at room temperature for a further 30 minutes, during which time gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 15 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was dissolved in DMF (1.7 mL), followed by the addition of solid NaN3 (46 mg; 708 μmol; 2.0 equivalent). The resulting mixture was stirred overnight at 80°C. After cooling, the mixture was directly purified by preparative HPLC (C18; TD1390 standard). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 17.6 mg (28%; 2 steps; TD1396 standard). NMR (DMSO-d6): 1 H δH 2.98(CH2,t,2H, 3 J HH =7);3.69(CH2,td,2H, 3 J HH =7, 3 J HH =5);4.53(CH2,d,2H, 3 J HH =6);5.37(OH,t,1H, 3 J HH =6);7.18(arom、dd,1H, 3 J HH =8; 4 J HH =1);7.32(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.73(arom.,t,1H, 3 J HH =8). ESI-HRMS:201.0746[M+Na] + (Theoretical value[C8H 10 N4O1Na1] + =201.0747).

[0483] Synthesis of TD1406:

[0484]

change

[0485] In a round-bottom glass flask (25 mL), TD1401 (16.5 mg; 92.6 μmol; 1.0 equiv) was dissolved in DCM (1.85 mL), followed by the addition of SOCl2 (13.5 μL; 186 μmol; 2.0 equiv). The mixture was stirred at room temperature for 1 hour. The mixture was diluted with DCM (10 mL) and quenched with diluted aqueous solution of NaHCO3 (10 mL). The resulting two-phase mixture was vigorously stirred at room temperature for a further 30 minutes, during which gas bubbles slowly formed. The mixture was transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless solid. Yield: 17.7 mg (97%; 1 stage; TD1401 standard). NMR (DMSO-d6): 1 H δ H 3.02(CH2,t,2H, 3 J HH =7);3.71(CH2,td,2H, 3 J HH =7, 3 J HH =5);4.75(CH2,s,2H);7.32(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.41(arom.,dd,1H, 3 J HH =8; 4 J HH =1);7.79(arom.,t,1H, 3 J HH =8). ESI-HRMS:197.0589[M+H] + (Theoretical value [C8H 10 N4Cl1] + (=197.0589).

[0486] Synthesis of TD1339:

[0487] [ka]

[0488] 3.54 g of ethyl l-lactate (30.0 mmol; 1.00 equivalent) and a magnetic stirrer were placed in a 250 mL pear-shaped glass flask and briefly protected with argon three times. Next, 100 mL of dry DCM was added through a septum under a constant flow of argon, and the mixture was cooled in an ice bath (5°C). Subsequently, anhydrous triflick (5.3 mL; 31.5 mmol; 1.05 equivalent) was added dropwise, followed immediately by the addition of dry pyridine (2.54 mL; 31.5 mmol; 1.05 equivalent). The resulting mixture was stirred at 5°C for 30 minutes. The resulting suspension was directly purified by column chromatography (140 g SiO2, DCM). The combined fraction containing the product was evaporated to dryness and briefly dried under high vacuum to obtain the product as a slightly pinkish oil. Yield: 5.90 g (79%; 1 step; based on ethyl l-lactate). NMR (DMSO-d6): 1 H δ H 1.26(CH3-CH2,t,3H, 3 J HH =7);1.51(CH3-CH,d,3H, 3 J HH =7);4.19-4.31(CH2,m,2H);5.27(CH,q,1H, 3 J HH =7). 19 F{ 1 H}δ F -77.7(s). CI-HRMS:251.0193[M+H] + (Theoretical value [C6H 10 [O5S1F3] + (=251.0196).

[0489] Synthesis of TD1488:

[0490] [ka]

[0491] Dimethyl l-malate (200 mg; 1.23 mmol; 1.00 equivalent) and a magnetic stirrer were placed in a pear-shaped glass flask (25 mL) and protected with argon for three short periods. Next, dry DCM (4 mL) was added through a septum under a constant flow of argon. The mixture was cooled in an ice bath (5°C), followed by the dropwise addition of anhydrous triflick (220 μL; 1.31 mmol; 1.06 equivalent), and then immediately followed by the dropwise addition of dry pyridine (105 μL; 1.30 mmol; 1.06 equivalent). The resulting mixture was stirred at room temperature for 30 minutes. The resulting suspension was directly purified by column chromatography (30 g SiO2, DCM). The combined fraction of the product was evaporated to dryness and dried briefly under high vacuum to obtain the product as a colorless oil. Yield: 258 mg (77%; 1 step; based on dimethyl l-malate). NMR (DMSO-d6): 1 H δ H 3.08(CH2,dd,1H, 2 J HH =17, 3 J HH =5);3.13(CH2,dd,1H, 2 J HH =17, 3 J HH =6);4.65(CH3,s,3H);3.78(CH3,s,3H);5.48(CH,dd,1H, 3 J HH =6, 3 J HH =5). 19 F{ 1 H}δ F -77.7(s). ESI-HRMS:316.9910[M+Na] + (Theoretical value [C7H9O7F3S1Na1]) + (=316.9913).

[0492] [Example 3: Synthesis of a protected macrocyclic intermediate] Synthesis of TD680:

[0493] [ka]

[0494] In a pear-shaped glass flask (250 mL), Cbz2 cyclene (free base; 1.54 g; 3.50 mmol; 1.6 equivalents) was dissolved in MeCN (100 mL), followed by the addition of dry K2CO3 (300 mg; 2.17 mmol; 1.0 equivalent). To the vigorously stirred reaction mixture, a solution of tert-butyl bromoacetate (427 mg; 2.19 mmol; 1.0 equivalent) in dry MeCN (25 mL) was added dropwise over 2 hours. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (150 mL) and H2O (150 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 75 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 624 mg (51%; 1 step; based on tert-butyl bromoacetate). NMR (CD₃CN): 1 H δ H 1.32-1.49(CH3,m,9H);2.57-2.76(mc,m,4H);2.81-2.98(mc,m,4H);3.23-3.64(mc,CH2-CO,m,8+2H);5.00-5.15 CH2-Ph,m,4H);7.19-7.48(Ph,m,10H).ESI-HRMS:555.3171[M+H] + (Theoretical value [C 30 H 43 N4O6] + (=555.3177).

[0495] Synthesis of TD686:

[0496] [ka]

[0497] In a glass vial (4 mL), TD680 (150 mg; 270 μmol; 1.0 equiv) was dissolved in MeCN (3 mL), and Boc2O (2.0 M solution in THF; 200 μL; 400 μmol; 1.5 equiv) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, immediately neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 170 mg (96%; 1 stage; TD680 standard). NMR (CD3CN): 1 H δ H 1.17-1.56(CH3,m,18H);2.62-2.99(mc,m,4H);3.10-3.53(mc,CH2-CO,m,12+2H );4.96-5.16(CH2-Ph,m,4H);7.20-7.47(Ph,m,10H).ESI-HRMS:655.3691[M+H] + (Theoretical value [C 35 H 51 N4O8] + (=655.3701).

[0498] Synthesis of TD691:

[0499] [ka]

[0500] TD686 (170 mg; 260 μmol) and a magnetic stirrer were placed in a pear-shaped glass flask (25 mL) and protected with argon three times. Then, solid palladium carbon (17 mg) was added and protected with argon three more times. MeOH (10 mL) was added through a septum under a constant flow of argon. Then, the argon inflow was removed, and H2 gas (from a balloon) was bubbling into the mixture, and it was left at room temperature for 30 minutes. The catalyst was then filtered using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 100 mg (≥99%; 1 step; TD686 reference). ESI-MS (LC-MS): 387.3 [M+H] + (Theoretical value [C 19 H 39 N4O4] + (=387.3). The entire amount of TD691 was used directly in TD692 without any further characterization.

[0501] Synthesis of TD1106:

[0502] [ka]

[0503] In a 50 mL pear-shaped glass flask, Cbz2 cyclene (free base, 500 mg; 1.14 mmol; 1.0 equivalent) was dissolved in 10 mL of MeCN, followed by the addition of dried K2CO3 (870 mg; 5.69 mmol; 5.0 equivalents) and methyl bromoacetate (235 μL; 2.51 mmol; 2.2 equivalents) in 5 mL of MeCN. The resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in 50 mL of DCM and 50 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 422 mg (64%; 1 step; based on Cbz₂ cycline). NMR (CD₃CN): 1 H δ H 2.63-2.88(mc,m,8H);3.19-3.49(mc,CH2-CO,m,8+4H);3.59(CH3,s,6H);5.07(CH2-Ph,s,4H);7.21-7.41(Ph,m,10H).ESI-LCMS:585.3[M+H] + (Theoretical value [C 30 H 41 N4O8] + =585.3).

[0504] Synthesis of TD1116:

[0505] [ka]

[0506] Palladium carbon (42 mg) and a magnetic stirrer were placed in a pear-shaped glass flask (50 mL) and protected with argon three times. Next, a solution of TD1106 (418 mg; 715 μmol) in MeOH (25 mL) was added through the septum under a constant flow of argon. The argon inflow was then removed, and H2 gas (from a balloon) was bubbling into the mixture. The mixture was left at room temperature for 1 hour. The catalyst was then filtered using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. This crystallized upon standing. Yield: 223 mg (99%; 1 step; TD1106 reference). NMR (CD3CN): 1 H δ H 2.48-2.57(mc,m,8H);2.63-2.74(mc,m,8H);3.36(CH2-CO,s,4H);3.64(CH3,s,6H).ESI-MS LC-MS):317.2[M+H] + (Theoretical value [C 14 H 29 N4O4] + =317.2).

[0507] Synthesis of TD687:

[0508] [ka]

[0509] Mix TD680 (150 mg; 270 μmol; 1.0 equivalent) and P(OEt)3 (232 μL; 1.35 mmol; 5.0 equivalents) in a glass vial (2 mL), followed by solid (CH2O) n(12 mg; 400 μmol; 1.5 equivalents) was added. The resulting suspension was stirred at room temperature for 3 days. The reaction mixture was diluted with MeCN (700 μL) and filtered through a syringe microfilter (PTFE). The filter was further washed with MeCN. The filtrate was purified directly by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, immediately neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a faint yellow oil. Yield: 167 mg (87%; 1 step; TD680 basis). ESI-MS (LC-MS): 705.4 [M+H] + (Theoretical value [C 35 H 54 N4O9P1] + (=705.4). The entire amount of TD687 was used directly in TD695 without any further characterization.

[0510] Synthesis of TD695:

[0511] [ka]

[0512] TD687 (167 mg; 237 μmol) and a magnetic stirrer were placed in a pear-shaped glass flask (25 mL) and protected with argon three times. Next, solid palladium carbon (17 mg) was added and protected with argon three more times. MeOH (10 mL) was added through the septum under a constant flow of argon. Then, the argon inflow was removed, and H2 gas (from a balloon) was bubbled into the mixture and left at room temperature for 30 minutes. The reaction mixture was further stirred at room temperature under a hydrogen atmosphere (from a balloon) for 16 hours. The catalyst was then filtered off using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellowish oil. Yield: 100 mg (97%; 1 step; TD686 reference). ESI-MS (LC-MS): 437.2 [M+H] + (Theoretical value [C 19 H 41 N4O5P1] + (=37.3). The entire amount of TD695 was used directly in the TD700 without any further characterization.

[0513] Synthesis of TD913:

[0514] [ka]

[0515] In a glass vial (20 mL), dissolve Cbz2 cyclene (free base; 242 mg; 549 μmol; 1.0 equivalent) in MeCN (12 mL), and then solid (CH2O) n(50 mg; 1.67 mmol; 3.0 equivalents) and PhP(OMe)2 (350 μL; 2.2 mmol; 4.0 equivalents) were added. The resulting suspension was stirred at 80°C for 24 hours. The reaction mixture was filtered through a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (75 mL) and H2O (75 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 259 mg (61%; 1 level; based on Cbz2 cycline). NMR (CD3CN): 1 H δ H 2.30-3.38(mc,CH2-P,m,16+4H);3.47(CH3,d,6H, 3 J HP =11);5.00-5.12(CH2-Ph,m,4H);7.27-7.40(Ph,m,10H);7.40-7.51(Ph,m,4H);7.51-7.60(Ph,m,2H);7.60-7.82(Ph,m,4H). 31 P δ P 42.8(m). ESI-HRMS:777.3178[M+H] + (Theoretical value [C 40 H 51 N4O8P2] + (=777.3177).

[0516] Synthesis of TD910:

[0517] [ka]

[0518] TD913 (250 mg; 322 μmol) and a magnetic stirrer were placed in a pear-shaped glass flask (50 mL) and protected with argon three times. Next, solid palladium carbon (50 mg) was added and protected with argon three more times. MeOH (25 mL) was added through a septum under a constant flow of argon. Then, the argon inflow was removed, and H2 gas (from a balloon) was bubbling into the mixture, and the mixture was left at room temperature for 2 hours. The catalyst was then filtered using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 161 mg (98%; 1 step; TD913 reference). NMR (CD3CN): 1 H δ H 2.28-3.14(mc,CH2-P,m,16+4H);3.53(CH3,d,3H, 3 J HP =11);3.54(CH3,d,3H, 3 J HP =11);7.46-7.63(Ph,m,6H);7.71-7.83(Ph,m,4H). 31 P δ P 43.3(m). ESI-HRMS:509.2438[M+H] + (Theoretical value [C 24 H 39 N4O4P2 + ]=509.2441).

[0519] Synthesis of TD571:

[0520] [ka]

[0521] In a glass vial (4 mL), Cbz2 cyclene (free base; 1.00 g; 2.27 mmol; 1.0 equivalent) and P(OEt)3 (2.00 mL; 11.7 mmol; 5.1 equivalents) were mixed, followed by solid (CH2) n(164 mg; 5.47 mmol; 2.4 equivalents) was added. The resulting suspension was stirred at room temperature for 24 hours. The reaction mixture was evaporated to dryness, and the residue was purified by column chromatography (SiO2 80 g; DCM-MeOH-NH3 aqueous solution 150:10:1). The fractions containing the product were combined and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellowish oil. Yield: 1.29 g (77%; 1 step; Cbz2 cycline basis). ESI-MS (LC-MS): 741.3 [M+H] + Theoretical value [C 34 H 55 N4O 10 P2] + (=741.3). The entire amount of TD571 was used directly in TD573 without any further characterization.

[0522] Synthesis of TD573

[0523] [ka]

[0524] TD571 (1.29 g, 1.74 mmol) and a magnetic stirrer were placed in a pear-shaped glass flask (100 mL) and protected with argon three times. Then, solid palladium carbon (129 mg) was added and protected with argon three more times. EtOH (96%, 70 mL) was added through the septum under a constant flow of argon. Then, the argon inflow was removed and H2 gas (from a balloon) was bubbled into the mixture and left at room temperature for 30 minutes. The reaction mixture was further stirred at room temperature under a hydrogen atmosphere (from a balloon) for 16 hours. The catalyst was then filtered off using a syringe microfilter (PTFE; the filter was further washed with EtOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellow oil. Yield: 776 mg (95%; 1 step; based on TD571). ESI-MS (LC-MS): 473.2 [M+H] + (Theoretical value [C 18 H 43 N4O4P2]+ (=473.3). The majority of the TD573 was used directly in the TD579 without further characterization.

[0525] Synthesis of TD423:

[0526] [ka]

[0527] In a 25 mL pear-shaped glass flask, tBuDO2A (free base; 410 mg; 1.03 mmol; 1.4 equivalents) was dissolved in 8 mL of dry MeCN, followed by the addition of Cs2CO3 (840 mg; 2.58 mmol; 3.4 equivalents) and KI (172 mg; 1.04 mmol; 1.4 equivalents). A solution of 2-chloro-N-(prop-2-in-1-yl)acetamide (100 mg; 760 μmol; 1.0 equivalent) in 2 mL of dry MeCN was added, and the resulting mixture was stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness to obtain the pre-purification product in the form of free base as yellow oil. Yield: 180 mg (mixture of ~75% TD423 and 25% bis(substituted) by-products). ESI-MS (LC-MS): 496.4 [M + H] + (Theoretical value [C 25 H 46 N5O5] + (=496.3). TD423 was used directly in TD425 without any purification or characterization.

[0528] Synthesis of TD635:

[0529] [ka]

[0530] In a pear-shaped glass flask (250 mL), tBuDO2A (free base; 1.21 g; 3.02 mmol; 2.3 equivalents) was dissolved in MeCN (150 mL). To the vigorously stirred reaction mixture, a solution of TD558 (1202 mg; 1.33 mmol; 1.0 equivalent) in MeCN (100 mL) was added dropwise (over 2 hours). The resulting mixture was further stirred at room temperature for 2 days and then evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a faint yellow oil. This crystallized upon standing. Yield: 453 mg (66%; 1 step; TD558 standard). NMR (CD3CN): 1 Hδ H 1.39(CH3,s,18H);2.53(mc,m,4H);2.61(mc,m,4H);2.73mc,m,4H);2.77(mc,m,4H); 3.01(CH2-CO,s,4H);3.44(C≡CH,s,1H);3.66(CH2-arom,s,2H);7.38(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.70(arom.,t,1H, 3 J HH =8);7.76(arom.,dd,1H,J 3 HH =8, 4 J HH =1). 13 C{ 1 H}δ C28.4(CH3,s);48.2(mc,s);51.8(mc,s);52.6(mc,s);54.9(mc,s);57.6(CH2-CO,s);59.3(CH2-arom,s);78.0(C≡CH,s);81.3(C -CH3,s);84.0(C≡CH,s);124.9(arom.,s);126.5(arom.,s);137.8(arom.,s);141.6(arom.,s);162.8(arom.,s);172.0(CO,s). ESI-HRMS:516.3542[M+H] + (Theoretical value [C 28 H 46 N5O4] + (=516.3544).

[0531] Synthesis of TD539:

[0532] [ka]

[0533] In a pear-shaped glass flask (100 mL), tBuDO2A (free base; 1.73 g; 4.32 mmol; ≥2.0 equivalents) was dissolved in dry MeCN (15 mL), followed by the addition of dry K2CO3 (900 mg; 6.52 mmol; ≥3.0 equivalents). To the vigorously stirred reaction mixture, a solution of freshly prepared and isolated TD538 (≤2.17 mmol; 1.0 equivalent) in dry MeCN (10 mL) was added dropwise over 15 minutes. The resulting mixture was further stirred at room temperature for 20 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (150 mL) and H2O (150 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow solidified oil. Yield: 653 mg (47%; 2 steps; TD530 standard). NMR (CD3CN): 1 H δ H 1.39(CH3,s,18H);1.43(CH3,s,9H);2.51-2.84(mc,m,16H);3.01(CH2-CO,s,4H);3.64(CH2-arom,s,2H);4.05(CH2-C≡C,bd,2H, 3 J HH =5);5.97(NH-CO,bt,1H, 3 J HH =5);7.27(arom.,dd,1H, 3 J HH =7, 4 J HH =2); 7.66-7.75 (arom, m, 2H). 13 C{ 1 H} δ C28.4(CH3,s);28.6(CH3,s);31.2(CH2-C≡C,bs);48.1(mc,s);51.8(mc,s);52.8(mc,s);55.1(mc,s);57.6(CH2-CO,s);59.6(CH2-arom,s);8 1.3(C-CH3, 2×s); 82.6 and 87.0(C≡C, 2×s); 124.3(arom, s); 126.0(arom, s); 137.7(arom, s); 142.3(arom, s); 162.7(arom, s); 172.0(CO, s). ESI-HRMS:645.4331[M+H] + (Theoretical value [C 34 H 57 N6O6] + (=645.4334).

[0534] Synthesis of TD1118:

[0535] [ka]

[0536] In a pear-shaped glass flask (50 mL), tBuDO2A (free base; 160 mg; 399 μmol; ≥2.5 equivalents) was dissolved in MeCN (10 mL), followed by the addition of dried K2CO3 (65 mg; 470 μmol; ≥3 equivalents). To the vigorously stirred reaction mixture, a solution of freshly prepared isolated TD1117 (≤470 mmol; 1.0 equivalent) in MeCN (10 mL) was added dropwise over 5 minutes. The resulting mixture was further stirred at room temperature for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 30 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless solidified oil. Yield: 62.2 mg (57%; 2 steps; TD966 standard). NMR (CD₃CN): 1 H δ H 1.40(CH3,s,18H);1.43(CH3,s,9H);1.53-1.64(CH2,m,2H);1.82-1.91(CH2,m,2H);2.47-2.79(mc,m,16H);2.79-2.88(CH- C≡C,m,1H);3.01(CH2-CO,s,4H);3.06-3.18(CH2,m,2H);3.63(CH2-arom,s,2H);3.71-3.82(CH2,m,2H);7.27(arom.,dd,1H, 3 J HH =7, 4 J HH =2);7.62-7.72(arom.,m,2H).ESI-HRMS:699.4801[M+H] + (Theoretical value [C 38 H 63 N6O6] + (=699.4804).

[0537] Synthesis of TD692:

[0538] [ka]

[0539] In a pear-shaped glass flask (250 mL), TD681 (100 mg; 259 μmol; 1.5 equivalents) was dissolved in MeCN (25 mL). To the vigorously stirred reaction mixture, a solution of TD558 (26 mg; 172 μmol; 1.0 equivalent) in MeCN (25 mL) was added dropwise (over 1 hour). The resulting mixture was further stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the product were combined, immediately neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 38 mg (44%; 1 step; TD558 standard). ESI-MS (LC-MS): 502.4 [M+H] + (Theoretical value [C 27 H 44 N5O4 + ] + (=502.3). The entire amount of TD692 was used directly in TD703 without any further characterization.

[0540] Synthesis of TD700:

[0541] [ka]

[0542] In a pear-shaped glass flask (100 mL), TD695 (100 mg; 229 μmol; 1.5 equivalents) was dissolved in MeCN (25 mL), followed by the addition of dry K2CO3 (21 mg; 152 μmol; 1.0 equivalent). To the vigorously stirred reaction mixture, a solution of TD558 (23 mg; 152 μmol; 1.0 equivalent) in MeCN (25 mL) was added dropwise over 1 hour. The resulting mixture was further stirred at room temperature for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (75 mL) and H2O (75 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted using DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 20 mg (24%; 1 step; TD558 as reference). ESI-MS (LC-MS): 552.3 [M + H] + (Theoretical value [C 27 H 47 N5O5P1] + (=552.3). The entire amount of TD700 was used directly in TD705 without any further characteristic analysis.

[0543] Synthesis of TD579:

[0544] [ka]

[0545] In a pear-shaped glass flask (25 mL), TD573 (600 mg; 1.27 mmol; 2.0 equivalents) was dissolved in MeCN (10 mL). To the vigorously stirred reaction mixture, a solution of TD558 (30 mg; 488 μmol; 1.0 equivalent) in MeCN (10 mL) was added dropwise (over 30 minutes). The resulting mixture was further stirred at room temperature for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (125 mL), H2O (125 mL), and aqueous NaOH (1%; 20 mL) solutions and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted using DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a yellow oil. Yield: 137 mg (48%; 1 step; TD558 standard). ESI-MS (LC-MS): 588.3 [M + H] + (Theoretical value [C 26 H 48 N5O6P2] + (=588.3). The TD579 was used directly in the TD575 and TD580 without any further characterization.

[0546] Synthesis of TD799:

[0547] [ka]

[0548] In a pear-shaped glass flask (100 mL), tBuDO2Prop (free base; 165 mg; 385 μmol; 2.0 equivalents) was dissolved in MeCN (30 mL). To the vigorously stirred reaction mixture, a solution of TD558 (29 mg; 191 μmol; 1.0 equivalent) in MeCN (30 mL) was added dropwise (over 6 hours). The resulting mixture was further stirred at room temperature for 10 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a slightly yellowish oil. Yield: 47 mg (45%; 1 step; TD558 as reference). NMR (CD3CN): 1 H δ H 1.38(CH3,s,18H);2.19-2.25(CH2-CO,m,4H);2.44-2.64(mc,CH2-CH2-CO,m,16+4H);3.41(C≡CH,s,1H);3.66(CH2-arom,s,2H);7.36(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.70(arom.,t,1H, 3 J HH =8);7.77(arom.,bd,1H, 3 J HH =8). ESI-HRMS:544.3854[M+H] + (Theoretical value [C 30 H 50 N5O4] + (=544.3857).

[0549] Synthesis of TD663:

[0550] [ka]

[0551] In a 50 mL pear-shaped glass flask, tBuDO2A (free base; 210 mg; 524 μmol; 2.7 equivalents) was dissolved in 15 mL of MeCN, followed by the addition of dried K2CO3 (27 mg; 195 μmol; 1.0 equivalent). To the vigorously stirred reaction mixture, a solution of TD566 (50 mg; 193 μmol; 1.0 equivalent) in 15 mL of MeCN was added dropwise over 15 minutes. The resulting mixture was further stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was separated and subjected to preparative HPLC (C18; H2O-Me The product was purified by a CN gradient (with FA addition). The fractions containing the product were combined, neutralized with a diluted aqueous solution of NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 83 mg (69%; 1 step; TD566 as reference). NMR (CD3CN): 1 H δ H 1.33(CH3,s,18H);2.62(mc,m,4H);2.66(mc,m,4H);2.74(mc,m,4H);2.80(mc,m,4H);2.82(CH2-CO,s,4H);3.74(CH2-arom,s,2 H);4.46(CH2-N3,s,2H);2.46-7.58(Ph,arom.,m,3+1H);7.78-7.87(Ph,m,2H);8.18(arom.,bs,1H).ESI-HRMS:623.4022[M+H] + (Theoretical value [C 33 H 51 N8O4] + (=623.4028).

[0552] Synthesis of TD711:

[0553] [ka]

[0554] In a pear-shaped glass flask (100 mL), tBuDO2A (free base; 564 mg; 1.41 mmol; 2.5 equivalents) was dissolved in MeCN (40 mL). To the vigorously stirred reaction mixture, a solution of TD406 (103 mg; 564 μmol; 1.0 equivalent) in MeCN (40 mL) was added dropwise (over 2 hours). The resulting mixture was further stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 217 mg (70%; 1 step; TD406 as reference). NMR (CD3CN): 1 H δ H 1.39(CH3,s,18H);2.55(mc,m,4H);2.60(mc,m,4H);2.76(mc,m,8H);3.03(CH 2-CO,s,4H);3.70(CH2-arom,s,2H);4.39(CH2-N3,s,2H);7.21(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.67(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.73(arom.,t,1H, 3 J HH =8). 13 C{ 1 H} δ C28.4(CH3,s);48.3(mc,s);51.8(mc,s);52.7(mc,s);54.7(mc,s);56.1(CH2-N3,s);57.7(CH2-CO,s);59.0(CH2-arom,s) ;81.2(C-CH3,s);121.2(arom.,s);124.1(arom.,s);138.2(arom.,s);155.6(arom.,s);161.9(arom.,s);172.0(CO,s). ESI-HRMS:547.3716[M+H] + (Theoretical value [C 27 H 47 N8O4] + (=547.3715).

[0555] Synthesis of TD596:

[0556] [ka]

[0557] In a 25 mL pear-shaped glass flask, tBuDO2A (free base; 162 mg; 405 μmol; ≥2.5 equivalents) was dissolved in 10 mL of MeCN. To the vigorously stirred reaction mixture, a solution of crude TD633 (≤162 μmol; 1.0 equivalent) in 10 mL of MeCN was added dropwise over 15 minutes. The resulting mixture was further stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in 50 mL of DCM and 50 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated, and the aqueous phase was further extracted with 3 × 25 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 44 mg (48%; 2 steps; TD406 as baseline). ESI-MS (LC-MS): 563.4 [M+H] + (Theoretical value [C 27H 47 N8O5] + (=563.4). The entire amount of TD596 was used directly in TD604 without any further characterization.

[0558] Synthesis of TD1340:

[0559] [ka]

[0560] In a 50 mL pear-shaped glass flask, Cbz2 cyclene (free base; 1.32 g; 3.0 mmol; 1.0 equivalent) was dissolved in 15 mL of dry MeCN, followed by the addition of 1.65 g of dry K2CO3 (1.65 g; 12.0 mmol; 4.0 equivalents). Next, a solution of TD1339 (1.50 g; 6.0 mmol; 2.0 equivalents) in 5 mL of dry MeCN was added, and the resulting suspension was stirred at room temperature for 8 hours. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in 150 mL of DCM and 150 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 1.57 g (82%; 1 step; Cbz₂ cycline as reference). NMR (CD₃CN): 1 H δ H 1.09(CH3-CH,d,6H, 3 J HH =7);1.19(CH3-CH2,t,6H, 3 J HH =7);2.58-2.70(mc,m,4H);2.76-2.98(mc,m,4H);3.23-3.52(mc,m,8H);3.52-3.79(CH,m,2H);4.06(CH2-CH3,q,4H, 3 JHH =7);5.00-5.13(CH2-Ph,m,4H);7.24-7.41(Ph,m,10H).ESI-HRMS:641.3539[M+H] + (Theoretical value [C 34 H 49 N4O8] + (=641.3545).

[0561] Synthesis of TD1341:

[0562] [ka]

[0563] Palladium carbon (127 mg) and a magnetic stirrer were placed in a pear-shaped glass flask (100 mL) and protected with argon three times. Next, a solution of TD1340 (1.27 g; 1.98 mmol) in MeOH (50 mL) was added through the septum. Then, the argon was removed, and H2 gas (from a balloon) was bubbling through the mixture at room temperature for 1 hour. The catalyst was then filtered using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 723 mg (98%; 1 step; TD1340 reference). NMR (CD3CN): 1 H δ H 1.16-1.33(CH3,m,12H);2.35-2.45(mc,m,4H);2.56-2.65(mc,m,4H);2.65-2.76(mc,m,4H);2.76-2.85(mc,m,4H);3.54(CH,q,2H, 3 J HH =7);4.00-4.24(CH2-CH3,q,4H, 3 J HH =7). ESI-HRMS:372.2808[M+H] + (Theoretical value [C 18 H 37 N4O4 + ] + (=373.2809).

[0564] Synthesis of TD1343:

[0565] [ka]

[0566] In a pear-shaped glass flask (50 mL), TD1341 (143 mg; 384 μmol; 2.0 equivalents) was dissolved in MeCN (15 mL), and dried K2CO3 (26.3 mg; 191 μmol; 1.0 equivalent) was added. To the vigorously stirred reaction mixture, a solution of TD558 (29.0 mg; 191 μmol; 1.0 equivalent) in MeCN (15 mL) was added dropwise (over 15 minutes). The resulting mixture was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free base as a nearly colorless oil. Yield: 46.0 mg (49%; 1 step; TD558 standard). NMR (CD₃CN): 1 H δ H 1.14(CH3-CH,d,6H, 3 J HH =7);1.19(CH3-CH2,t,6H, 3 J HH =7);2.28-2.38(mc,m,4H);2.45-2.59(mc,m,4H);2.59-2.94(mc,m,8H);3.23(CH,q,2H, 3 J HH =7);3.41(CH2-arom, d,1H, 2 J HH =15);3.45(C≡CH,s,1H);3.88(CH2-arom.,d,1H, 2 JHH =15);4.06(CH2-CH3,q,4H, 3 J HH =7);7.35-7.42(arom.,m,1H);7.66-7.79(arom.,m,2H).ESI-HRMS:488.3231[M+H] + (Theoretical value [C 26 H 42 N5O4] + (=488.3231).

[0567] Synthesis of TD1345:

[0568] [ka]

[0569] In a 25 mL pear-shaped glass flask, TD1343 (45.0 mg; 92 μmol; 1.0 equivalent) was dissolved in 2 mL of MeCN, followed by the addition of dry K2CO3 (51 mg; 196 μmol; 4.0 equivalents). Next, a solution of TD406 (20.0 mg; 110 μmol; 1.2 equivalents) in 2 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 7 days. The solid was filtered through a syringe microfilter (PTFE), and the filtrate was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in 25 mL of DCM and 25 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with 4 × 10 mL of DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free bases as a nearly colorless oil. Yield: 52.9 mg (90%; 1 step; TD1343 standard). ESI-HRMS: 634.3825 [M+H] + (Theoretical value [C 33 H 48 N9O4] + (=634.3824). The entire amount of TD1345 was used directly in TD1346 without any further characterization.

[0570] Synthesis of TD1447:

[0571] [ka]

[0572] In a pear-shaped glass flask (100 mL), TD1341 (722 mg; 1.94 mmol; 2.0 equivalents) was dissolved in MeCN (40 mL), followed by the addition of dry K2CO3 (133 mg; 964 μmol; 1.0 equivalent). To the vigorously stirred reaction mixture, a solution of TD1057 (241 mg; 962 μmol; 1.0 equivalent) in MeCN (40 mL) was added dropwise over 2 hours. The resulting mixture was further stirred at room temperature for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (100 mL) and H2O (100 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of formate as a faint yellow oil. Yield: 367 mg (60% assuming M-1.0FA; M R =632.7; 1 step; TD1057 reference). Recovery amount: 352 mg of TD1341 (49% of initial usage). NMR (CD3CN): 1 H δ H 1.18(CH3-CH,d,6H, 3 J HH =7);1.21(CH3-CH2,t,6H, 3 J HH =7);2.52-2.62(mc,m,4H);2.64-2.76(mc,m,4H);2.81-2.94(mc,m,4H);2.95-3.06(mc,m,4H);3.39(CH-CH3,q,2H, 3 J HH=7);3.81(CH2-arom、d,1H, 2 J HH =15);3.90(CH2-arom.,d,1H, 2 J HH =15);4.09(CH2-CH3,q,4H, 3 J HH =7);4.62(CH2-N3,s,2H);7.58(arom.,d,1H, 4 J HH =2);7.66(arom.,d,1H, 4 J HH =2). 19 F δ F -65.10(s). ESI-HRMS:587.3266[M+H] + (Theoretical value[C 26 H 42 N8O4F3] + =587.3276).

[0573] Synthesis of TD1449:

[0574]

change

[0575] In a pear-shaped glass flask (25 mL), TD1447 (assuming M-FA; 263.0 mg; 418 μmol; 1.0 equivalent) was dissolved in MeCN (5 mL), followed by the addition of dried K2CO3 (231 mg; 1.67 μmol; 4.0 equivalent). Next, a solution of TD1428 (173.0 mg; 421 μmol; 1.0 equivalent) in MeCN (10 mL) was added, and the resulting suspension was stirred at 40°C for 1 hour. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free bases as a yellow waxy oil. Yield: 309 mg (81%; 1 step; TD1447 standard). NMR (CD₃CN): 1 H δ H 1.10(CH3-CH,d,6H, 3 J HH =7);1.13(CH3-CH2,t,6H, 3 J HH =7);1.43(CH3-CO,s,9H);1.61(CH3-CN,s,6H);2.53-2.80(mc,m,12H);2.89-3.03(mc,m,4H);3.32(CH-CH3,q,2H, 3 J HH =7);3.68(CH2-arom, s,2H);3.71(CH2-arom, d,1H, 2 J HH =15);3.77(CH2-arom.,d,1H, 2 J HH =15);3.89(CH3-O,s,3H);3.99(CH2-CH3,q,4H,J 3 HH =7);4.52(CH2-N3,s,2H);5.56(NH,bs,1H);7.50(arom,d,1H, 4 JHH =2);7.71(arom.,d,1H, 4 J HH =2);8.00(arom.,d,1H, 4 J HH =2);8.16(arom.,d,1H, 4 J HH =2). 19 F δ F -64.98(s). ESI-HRMS:917.4851[M+H] + (Theoretical value[C 44 H 64 N 10 O8F3] + =917.4855).

[0576] Synthesis of rac-TD1489:

[0577]

change

[0578] In a glass vial (20 mL), Cbz2 cyclene (free base; 178 mg; 404 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (225 mg; 1.63 mmol; 4.0 equivalents). Next, a solution of TD1488 (250 mg; 850 μmol; 2.1 equivalents) in MeCN (2 mL) was added, and the resulting suspension was stirred at 40°C for 4 days (racemization occurred during the reaction). The solid was filtered using a syringe microfilter (PTFE) and then washed with MeCN. The filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and concentrated using a rotary evaporator to remove most of the MeCN. The mixture was diluted with DCM (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 172 mg (73%; 1 step; based on Cbz₂ cycline). ESI-MS (LC-MS): 585.3 [M + H] + (Theoretical value [C 30 H 41 N4O8] + =585.3).

[0579] Synthesis of TD1493:

[0580] [ka]

[0581] In a glass vial (20 mL), rac-TD1489 (free base; 172 mg; 294 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (162 mg; 1.17 mmol; 4.0 equivalents). Next, a solution of TD1399 (148 mg; 592 μmol; 2.0 equivalents) in MeCN (2 mL) was added, and the resulting suspension was stirred at 40°C for 70 minutes. The solid was filtered using a syringe microfilter (PTFE) and further washed with MeCN. The filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the pure product (separation of the more hydrophobic diastereomer with different malic acid arm chirality was successful) were combined, neutralized with diluted aqueous NaHCO3, concentrated using a rotary evaporator, and most of the MeCN was removed. The mixture was diluted with DCM (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 88.1 mg (44%; 1 step; relative to rac-TD1489). NMR (CD₃CN): 1 H δ H 1.09(CH3-CH,d,3H, 3 J HH =7);1.19(CH3-CH2,t,3H, 3 J HH =7);2.44(CH2-CH,dd,1H, 2 J HH =17, 3 J HH =6);2.55-2.70(mc,CH2-CH,m,3+2H);2.73-7.43(Ph,m,10H).ESI-HRMS:685.3439[M+H] + (Theoretical value [C 35 H 49 O 10 N4] + (=685.3443).

[0582] Synthesis of TD1495:

[0583] [ka]

[0584] Palladium carbon (9 mg) and a magnetic stirrer were placed in a pear-shaped glass flask (25 mL) and protected with argon three times. Next, a solution of TD1493 (88.0 mg; 129 μmol) in MeOH (6 mL) was added through the septum. Then, the argon inflow was removed, and H2 gas (from a balloon) was bubbling into the mixture, and it was left at room temperature for 1 hour. The catalyst was then filtered using a syringe microfilter (PTFE; the filter was further washed with MeOH). The filtrate was evaporated to dryness and co-evaporated once with DCM. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 52.6 mg (98%; 1 step; based on TD1493). ESI-HRMS: 417.2706[M+H] + (Theoretical value [C 19 H 37 O6N4] + (=417.2708).

[0585] Synthesis of TD1497:

[0586] [ka]

[0587] In a pear-shaped glass flask (25 mL), TD1495 (55 mg; 132 μmol; 1.7 equivalents) was dissolved in MeCN (5 mL), followed by the addition of dry K2CO3 (11.0 mg; 80 μmol; 1.0 equivalent). To the vigorously stirred reaction mixture, a solution of TD558 (12.0 mg; 79 μmol; 1.0 equivalent) in MeCN (5 mL) was added dropwise over 15 minutes. The resulting mixture was stirred at room temperature for 4 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted using DCM (3 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free base as a nearly colorless oil. Yield: 12.1 mg (29%; 1 step; TD558 standard). ESI-MS (LC-MS): 532.3 [M + H] + (Theoretical value [C 27 H 42 N5O6] + =532.3).

[0588] Synthesis of TD1500:

[0589] [ka]

[0590] In a glass vial (4 mL), TD1497 (12.1 mg; 23 μmol; 1.0 equivalent) was dissolved in a solution of TD406 (5.8 mg; 32 μmol; 1.4 equivalents) in MeCN (2 mL), and then dried K2CO3 (17.5 mg; 127 μmol; 4.0 equivalents) was added. The resulting suspension was stirred at 40°C for 1 day. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was diluted with H2O (1 mL). The resulting solution was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3 solution, and evaporated to dryness. The residue was dissolved in DCM (25 mL) and H2O (25 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (4 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried under high vacuum for a short time to obtain the product in the form of free bases as a faint yellow oil. Yield: 11.9 mg (77%; 1 step; TD1497 standard). ESI-MS (LC-MS): 678.3 [M+H] + (Theoretical value [C 34 H 48 N9O6] + =678.4).

[0591] [Example 4: Synthesis of uncaged macrocyclic ligands] Synthesis of TD425:

[0592] [ka]

[0593] In a pear-shaped glass flask (50 mL), crude TD423 (less than 75% of the mixture with less than 25% bis(substituted) byproducts; 180 mg; ≥1.0 equivalent) was dissolved in dry MeCN (10 mL), followed by the addition of Cs2CO3 (360 mg; 1.11 mmol; 3.8 equivalents). Next, a solution of TD566 (75 mg; 290 μmol; 1.0 equivalent) in MeCN (5 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation with MeOH twice. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 129 mg (27%; 3 stages; based on 2-chloro-N-(prop-2-in-1-yl)acetamide). NMR (D2O, pD~5): 1 H δ H 2.62(C≡CH,t,1H, 4 J HH =3);2.89-3.74(mc,CH2-CO,m,16+6H);3.79(CH2-C≡CH,t,2H, 4 J HH =3);4.03(CH2-arom, bs,2H);4.68(CH2-N3,s,2H);7.55-7.67(Ph,arom.,m,3+1H);7.78(arom.,d,1H, 4 J HH =2);7.85-7.92(Ph,m,2H);8.17(arom.,d,1H, 4 J HH =2). ESI-HRMS:606.3151[M+H] + (Theoretical value [C 30 H 40 N9O5] + =606.3147). EA(C30 H 39 N9O5-0.1FA-1.3H2O,M R =633.7):C57.0(56.8);H6.6(6.3);N19.9(19.5). Synthesis of TD669:

[0594] [ka]

[0595] In a glass vial (4 mL), TD663 (30 mg; 48 μmol, 1.0 equivalent) was dissolved in dry MeCN (500 μL), followed by the addition of K2CO3 (20 mg; 145 μmol; 3 equivalents). Next, a solution of TD662 (10 mg; 50 μmol; 1.0 equivalent) in MeCN (500 μL) was added, and the resulting suspension was stirred at room temperature for 2 days. Then, another portion of TD662 (6 mg; 30 μmol; 0.6 equivalents) in MeCN (200 μL) was added, and the mixture was stirred at room temperature for another 2 days. The solid was filtered through a syringe microfilter (PTFE), and the filtrate was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 27 mg (≤88%) M-0.1FA-xH2O;M R =634.4; 2 steps; assumed to be based on TD663). NMR (D2O, pD~5): 1 H δ H2.59-3.82(mc,CH2-CO,CH2-CH2-N(CH2-C≡CH)2,m,16+4+10H);4.00(CH2-arom,bs,2H);4.64(CH2-N3,s,2H);7.55-7.65(Ph,m,3H);7.74(arom.,d,1H, 4 J HH =2);7.83-7.91(Ph,m,2H);8.17(arom.,d,1H, 4 J HH =2). ESI-HRMS: 630.3510 [M+H] + (Theoretical value[C 33 H 44 N9O4] + =630.3511).

[0596] Synthesis of TD604:

[0597]

change

[0598] In a glass vial (4 mL), TD596 (44 mg; 78 μmol; 1.0 equivalent) was dissolved in MeCN (3 mL), and dried K2CO3 (33 mg; 239 μmol; 3.0 equivalents) was added. Next, a solution of TD558 (13 mg; 86 μmol; 1.1 equivalents) in MeCN (600 μL) was added, and the resulting suspension was stirred at room temperature for 16 days. Next, an additional portion of TD558 (2 mg; 13 μmol; 0.2 equivalents) in MeCN (400 μL) was added. The reaction mixture was stirred further at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 38 mg (81%; 2 steps; TD596 standard). NMR (D2O, pD~4): 1 H δ H 2.80-3.68(mc,CH2-CO,m,16+4H);3.72(C≡CH,s,1H);3.93(CH2-arom.,bs,2H);4.17(CH2 -arom.,s,2H);4.74(CH2-N3,s,2H);7.48-7.67(arom.,m,3H);7.74-7.95(arom.,m,3H). 13 C{ 1 H}δ C48.3(mc,s);48.9(mc,s);50.6(CH2-N3,s);51.4(CH2-arom.,s);51.5(mc,s);51.7 (mc,s);56.7(CH2-CO,s);58.8(CH2-arom.,s);80.7 and 82.2(C≡CH;2×s);126.0(aro m, s);126.5(arom, s);127.9(arom, s);128.5(arom, s);131.5(arom, s);140.3(arom s);141.2(arom, s);148.4(arom, s);148.7(arom, s);157.3(arom, s);169.5(CO, s). ESI-HRMS: 566.2836 [M+H] + (Theoretical value [C 27 H 36 N9O5] + =566.2834). EA(C 27 H 35 N9O5-0.1FA-1.6H2O,M R =599.1):C54.3(54.4);H6.5(6.1);N21.0(20.7).

[0599] Synthesis of TD556:

[0600] [ka]

[0601] In a 25 mL pear-shaped glass flask, TD635 (216 mg; 419 μmol; 1.0 equivalent) was dissolved in 12 mL of MeCN, followed by the addition of dried K2CO3 (174 mg; 1.26 mmol; 3.0 equivalents). Next, a solution of TD406 (92 mg; 504 μmol; 1.2 equivalents) in 3 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in 3 mL of TFA, and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fraction containing the product was coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 161 mg (66%; 2 steps; TD635 standard). NMR (D2O, pD~4): 1 H δ H 2.80-3.60(mc,CH2-CO,m,16+4H);3.74(C≡CH,s,1H);3.86(CH2-arom.,bs,2H);3.94(CH2-arom.,bs,2H);4.72(CH2-N3,s,2H);7.65(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.67(arom, dd,1H, 3 J HH =8, 4 J HH =1);7.95-8.03(arom.,m,3H);8.12(arom.,t,1H, 3 J HH =8); 13 C{ 1 H} δ C48.3(mc,s);48.4(mc,s);51.4(mc,s);51.5(mc,s);54.3(CH2-N3,s);56.6(CH2-CO, s);58.0(CH2-arom, s);58.9(CH2-arom.,s);80.9 and 82.0(C≡CH;2×s);124.5(arom., s);126.2(arom.,s);126.3(arom.,s);128.5(arom, s);140.5(arom.s);141.2(arom .s);142.7(arom.s);155.0(arom.s);155.5(arom.s);157.2(arom.s);169.2(CO, s). ESI-HRMS: 550.2880[M+H] + (Theoretical value [C 27 H 36 N9O4] + =550.2885). EA(C 27 H 35 N9O4-0.1FA-1.6H2O,M R =583.1):C55.8(55.5);H6.6(6.1);N21.6(21.2).

[0602] Synthesis of TD810:

[0603] [ka]

[0604] In a 20 mL glass vial, TD711 (103 mg; 188 μmol; 1.0 equivalent) was dissolved in 5 mL of MeCN, and dried K2CO3 (130 mg; 942 μmol; 5.0 equivalents) was added. Next, a solution of TD807 (33 mg; 188 μmol; 1.0 equivalent) in 2 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in 3 mL of TFA, and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as an off-white, fluffy solid in the form of a mixed salt of trifluoroacetate / formate. Yield: 55 mg (43%; 2 steps; TD711 standard). NMR (D2O, pD~4): 1 H δ H 2.79-3.63(mc,CH2-CO,m,16+4H);3.74(C≡CH,s,1H);3.82(CH2-arom.,bs,2H);3. 87(CH2-arom.,bs,2H);3.92(C≡CH,s,1H);4.63(CH2-N3,s,2H);7.55(arom, dd,1H, 3 J HH =8, 4 J HH =1);7.72(arom.,d,1H, 4 J HH =1);8.10(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.16(arom.,t,1H, 3 J HH =8);8.26(arom.,d,1H, 4 J HH =1). ESI-HRMS:574.2882[M+H] +(Theoretical value [C 29 H 36 N9O4] + =574.2885). EA(C 29 H 35 N9O4-0.5TFA-0.1FA-2.1H2O,M R =673.1):C53.7(53.8);H6.0(6.2);N18.7(18.7).

[0605] Synthesis of TD827:

[0606] [ka]

[0607] In a glass vial (20 mL), TD635 (157 mg; 304 μmol; 1.0 equivalent) was dissolved in dry MeCN (7 mL), and dry K2CO3 (210 mg; 1.52 mmol; 5.0 equivalents) was added. Next, a solution of TD817 (110 mg; 303 μmol; 1.0 equivalent) in dry MeCN (3 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the tert-butyl-protected product were combined, concentrated to approximately half volume (to remove most of the MeCN), and diluted with DCM (125 mL). The resulting two-phase mixture was transferred to a separatory funnel. The aqueous phase was neutralized with diluted aqueous NaHCO3. After shaking, the bottom phase was separated, and the aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were filtered through glass frit (S3) with anhydrous Na2SO4 and evaporated to dryness. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and most of the TFA was removed by co-evaporation with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as an off-white, fluffy solid in the form of formate. Yield: 149 mg (62%; 2 steps; TD711 standard). NMR (D2O + FA, pD~3):1 H δ H 0.86 - 1.33 (i-Pr, m, 21H); 2.58 - 3.78 (mc, CH2-CO, C≡CH, bm, 16 + 4 + 1H); 3.93 (CH2-arom., bs, 2H); 4.06 (CH2-arom, bs, 2H); 4.56 (CH2-N3, s, 2H); 7.37 (arom., s, 1H); 7.53 (arom., d, 1H, 3 J HH = 8); 7.74 (arom., s, 1H); 7.80 (arom., d, 1H, 3 J HH = 8); 7.96 (arom., t, 1H, 3 J HH = 8). ESI-HRMS: 730.4216 [M + H] + (theoretical value [C 38 H 56 N9O4Si1] + = 730.4219). EA (C 38 H 55 N9O4Si1-0.8FA-1.6H2O, M R = 795.6): C 58.6 (58.7); H 7.6 (7.7); N 15.8 (15.7); Si 3.5 (3.2).

[0608] Synthesis of TD718:

[0609]

Chemical formula

[0610] In a 10 mL pear-shaped glass flask, TD711 (31 mg; 57 μmol; 1.0 equivalent) was dissolved in 2 mL of dry MeCN, followed by the addition of dry K2CO3 (31 mg; 225 μmol; 4.0 equivalents). Next, a solution of TD706 (13 mg; 58 μmol; 1.0 equivalent) in 2 mL of dry MeCN was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the tert-butyl-protected product were combined, diluted with DCM (100 mL), and transferred to a separatory funnel. The aqueous phase was neutralized with a diluted aqueous solution of NaHCO3. After shaking, the bottom phase was separated, dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and most of the TFA was removed by co-evaporation with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain the amphoteric product as a white, fluffy solid. Yield: 23 mg (61%; 2 steps; TD706 standard). NMR (D2O, pD~5): 1 H δ H 0.28(CH3,s,9H);2.28-3.67(mc,CH2-CO,m,16+4H);3.89(CH2-arom.,bs,4H);4.57(CH2-N3,s,2H);7.49(arom.,dd,1H, 3 J HH =7, 4 J HH =2);7.58-7.64(arom.,m,1H);7.78-7.98(arom.,m,4H).ESI-HRMS:622.6277[M+H] + (Theoretical value [C 30 H 44 N9O4Si1] + =622.3280). EA(C 30 H 43 N9O4Si1-2.6H2O,M R=668.7):C53.9(53.9);H7.3(7.0);N18.9(18.7). Synthesis of TD728:

[0611] [ka]

[0612] In a 10 mL pear-shaped glass flask, TD711 (66 mg; 121 μmol; 1.0 equivalent) was dissolved in 2 mL of MeCN, followed by the addition of dry K2CO3 (67 mg; 486 μmol; 4.0 equivalent). Next, a solution of TD723 (37 mg; 120 μmol; 1.0 equivalent) in 2 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the tert-butyl-protected product were combined and diluted with DCM (150 mL), and the resulting two-phase mixture was transferred to a separatory funnel. The aqueous phase was neutralized with a diluted aqueous solution of NaHCO3. After shaking, the bottom phase was separated, and the aqueous phase was further extracted with DCM (2 × 50 mL). The combined organic layers were filtered through anhydrous Na2SO4 and glass frit (S3), and evaporated to dryness. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and most of the TFA was removed by co-evaporation with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain the amphoteric product as a white, fluffy solid. Yield: 64 mg (71%; 2 steps; TD723 standard). NMR (D2O, pD~5): 1 H δ H 0.84-1.34(i-Pr,m,21H);2.41-3.78(mc,CH2-CO,m,16+4H);3.89(CH2-arom.,bs,2H);3.95(CH2-arom.,bs,2H);4.52(CH2-N3,s,2H);7.42(arom,d,1H, 3 J HH=8);7.46(arom.,d,1H, 3 J HH =8);7.74(arom.,d,1H, 3 J HH =8);7.78(arom.,d,1H, 3 J HH =8);7.85(arom.,t,1H, 3 J HH =8);7.87(arom.,t,1H, 3 J HH =8). ESI-HRMS: 706.4221 [M+H] + (Theoretical value[C 36 H 56 N9O4Si1] + =706.4219). EA(C 36 H 55 N9O4Si1-2.6H2O,M R =752.8):C57.4(57.7);H8.1(8.0);N16.7(16.5);Si3.7(3.8).

[0613] Synthesis of TD1204:

[0614]

change

[0615] In a glass vial (20 mL), TD711 (35.0 mg; 64.0 μmol; 1.0 equivalent) was dissolved in MeCN (1 mL), and dried K2CO3 (44.2 mg; 320 μmol; 5.0 equivalents) was added. Next, a solution of TD1194 (21.4 mg; 77.1 μmol; 1.2 equivalents) in MeCN (2 mL) was added, and the resulting suspension was stirred at room temperature for 2 hours. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a white, fluffy solid in the form of formate. Yield: 23.3 mg (49%; 2 steps; TD711 as reference). NMR (D2O, pD~7): 1 H δ H 2.83-3.67(mc,CH2-CO,m,16+4H);3.88(CH2-arom.,bs,2H);3.92(CH2-arom.,bs,2H);4.56(CH2-N3,s,2H);7.49(arom.,d,1H, 3 J HH =8);7.54(arom.,d,1H, 3 J HH =8);7.87-8.00(arom.,m,4H).ESI-HRMS:676.1855[M+H] + (Theoretical value [C 27 H 35 N9O4I1 + ]=676.1851). EA(C 27 H 34 N9O4I1-0.3FA-2.7H2O,M R =738.0):C44.4(44.6);H5.5(5.1);N17.1(16.7).

[0616] Synthesis of TD944:

[0617] [ka]

[0618] In a pear-shaped glass flask (10 mL), TD711 (58 mg; 106 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (87 mg; 629 μmol; 6.0 equivalents). Next, a solution of freshly prepared and isolated TD939 (≤105 μmol; ≤1.0 equivalent) in MeCN (2 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the amphoteric product as a faint yellow solid. Yield: 38 mg (55%; 3 stages; TD936 standard). NMR (D2O, pD~7): 1 H δ H 2.82-3.67(mc,CH2-CO,m,16+4H);3.91(CH2-arom.,bs,2H);3.95(CH2-arom.,bs,2 H);4.58(CH2-N3,s,2H);7.40-8.07(Ph,arom.,m,5+6H).ESI-HRMS:626.3197[M+H] + (Theoretical value [C 33 H 40 N9O4] + =626.3198). EA(C 30 H 39 N9O4-1.5H2O,M R =652.8):C60.7(61.0);H6.5(6.3);N19.3(18.8).

[0619] Synthesis of TD943:

[0620] [ka]

[0621] In a pear-shaped glass flask (10 mL), TD711 (82 mg; 150 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (125 mg; 904 μmol; 6.0 equivalents). Next, a solution of freshly prepared and isolated TD940 (≤150 μmol; ≤1.0 equivalent) in MeCN (2 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the amphoteric product as a faint yellow solid. Yield: 66 mg (71%; 3 stages; TD937 standard). NMR (D2O, pD~7): 1 H δ H 0.74-1.06(CH2-CH,m,4H);1.35-1.66(CH2-CH,m,1H);2.69-3.65(mc,CH2-CO,m,16+4H);3.86(CH2-arom,bs,2H);3.92( CH2-arom.,bs,2H);4.57(CH2-N3,s,2H);7.42-7.53(arom.,m,2H);7.83-8.01(arom.,m,4H).ESI-HRMS:590.3196[M+H] + (Theoretical value [C 30 H 40 N9O4] + =590.3198). EA(C 30H 39 N9O4-1.5H2O,M R =616.7):C58.4(58.6);H6.9(6.9);N20.4(20.3).

[0622] Synthesis of TD959:

[0623] [ka]

[0624] In a pear-shaped glass flask (10 mL), TD711 (23 mg; 42 μmol; 1.0 equivalent) was dissolved in MeCN (1 mL), followed by the addition of dried K2CO3 (35 mg; 253 μmol; 6.0 equivalents). Next, a solution of freshly prepared and isolated TD956 (≤42 μmol; ≤1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were coupled and directly freeze-dried to obtain the amphoteric product as a nearly colorless solid. Yield: 18 mg (67%; 3 stages; TD952 as reference). NMR (D2O, pD~6): 1 H δ H 1.34(CH3,s,9H);2.72-3.69(mc,CH2-CO,m,16+4H);3.88(CH2-arom.,bs,2H);3.92(CH2-arom.,bs,2H );4.57(CH2-N3,s,2H);7.46-7.55(arom.,m,2H);7.84-8.03(arom.,m,4H).ESI-HRMS:606.3513[M+H] + (Theoretical value [C31 H 44 N9O4] + =606.3511). EA(C 31 H 43 N9O4-1.8H2O,M R =638.2):C58.3(58.7);H7.4(7.0);N19.8(19.4) Synthesis of TD992:

[0625] [ka]

[0626] In a glass vial (4 mL), TD711 (43 mg; 79 μmol; 1.0 equivalent) was dissolved in MeCN (1 mL), followed by the addition of dried K2CO3 (44 mg; 318 μmol; 4.1 equivalents). Next, a solution of freshly prepared and isolated TD990 (≤79 μmol; ≤1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at 40°C for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and evaporated twice with MeOH to remove most of the TFA. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as an off-white, fluffy solid in the form of formate. Yield: 42.0 mg (73%; 3 stages; TD965 standard). NMR (D2O, pD~5): 1 H δ H1.62 - 1.83 (Adm., m, 6H); 1.86 - 2.04 (Adm., m, 9H); 2.77 - 3.70 (mc, CH2-CO, m, 16 + 4H); 3.90 (CH2-arom, bs, 2H); 3.94 (CH2-arom., bs, 2H); 4.57 (CH2-N3, s, 2H); 7.44 - 7.56 (arom., m, 2H); 7.82 - 8.00 (arom., m, 4H). ESI-HRMS: 684.3982 [M+H] + (Theoretical value [C 37 H 50 N9O4] + = 684.3980). EA (C 37 H 49 N9O4-0.3FA-1.9H2O, M R = 731.9): C 61.2 (61.2); H 7.1 (7.1); N 17.2 (17.0). Synthesis of TD703:

[0627]

Chem.

[0628] In a 20 mL glass vial, TD692 (38 mg; 76 μmol; 1.0 equivalent) was dissolved in 3 mL of MeCN, followed by the addition of dried K2CO3 (31 mg; 217 μmol; 3.0 equivalents). Next, a solution of TD406 (14 mg; 77 μmol; 1.0 equivalent) in 2 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 16 hours. Then, dried K2CO3 (31 mg; 217 μmol; 3.0 equivalents) and another portion of TD406 (10 mg; 55 μmol; 0.7 equivalents) in 1 mL of MeCN were added, and the resulting suspension was further stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the protected product were combined, evaporated to dryness, and then evaporated twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and then evaporated three times with MeOH to remove most of the TFA. The residue was purified directly by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a slightly yellowish viscous oil in the form of a mixed salt of trifluoroacetate / formate. Yield: 16 mg (34%; 2 steps; TD692 standard). NMR (D2O, pD~5): 1 H δ H 2.59-3.73(mc,CH2-CO,m,16+2H);3.75(C≡CH,s,1H);3.84(CH2-arom.,bs,2H);4.29-4.62(CH2-arom.,CH2-N3,m,2+2H);7.37(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.45(arom, dd,1H, 3 J HH =8, 4 J HH =1);7.52(arom.,t,1H, 3 J HH =8);7.56(arom.,dd,1H, 3 J HH =8, 4 J HH=1);7.59(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.89(arom.,t,1H, 3 J HH =8).ESI-HRMS:492.2829[M+H] + (Theoretical value [C 25 H 34 N9O2] + =492.2830). EA(C 25 H 33 N9O2-0.3TFA-0.7FA-3.5H2O,M R =621.1):C50.9(51.1);H6.8(6.8);N20.3(20.0).

[0629] Synthesis of TD705:

[0630] [ka]

[0631] In a 20 mL glass vial, TD700 (20 mg; 36 μmol; 1.0 equivalent) was dissolved in MeCN (3 mL), followed by the addition of dried K2CO3 (20 mg; 145 μmol; 4.0 equivalents). Next, a solution of TD406 (9 mg; 49 μmol; 1.4 equivalents) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 22 mg (87%; 1 step; TD406 standard). ESI-MS (LC-MS): 698.4 [M+H]+ (Theoretical value [C 34 H 53 N9O5P1] + (=698.4). The entire amount of TD705 was used directly in TD714 without any further characterization.

[0632] Synthesis of TD714:

[0633] [ka]

[0634] In a glass vial (4 mL), TD705 (22 mg; 32 μmol; 1.0 equivalent) was dissolved in dry pyridine (1 mL), and neat TMSBr (50 μL; 379 μmol; 12.0 equivalents) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with 50% MeOH (1 mL) aqueous solution, and the mixture was stirred further at room temperature for 1 hour. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and then evaporated twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and then evaporated twice with MeOH to remove most of the TFA. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 12 mg (≤64%) M-0.1FA-xH2O;M R =590.2; 2 steps; assumed to be based on TD705). NMR (D2O, pD~5): 1 H δ H 2.73-3.71(mc,CH2-CO,CH2-P,m,16+2+1H);3.74(C≡CH,s,1H);3.84(CH2-arom,bs,1H);3.99(CH2-arom,bs,1H);4.17(CH2-arom.,bs,2H);4.47(CH2-P;bd, 2 J H=13);4.67(CH2-N3,s,1H);4.68(CH2-N3,s,1H);7.54(arom,d,1H, 3 J HH =8);7.61(arom.,d,1H, 3 J HH =8);7.62(arom.,d,1H, 3 J HH =8);7.64(arom.,d,1H, 3 J HH =8);7.90(arom.,t,1H, 3 J HH =8);7.93(arom.,t,1H, 3 J HH =8). ESI-HRMS: 586.2649 [M+H] + (Theoretical value[C 26 H 37 N9O5P1] + =586.2650).

[0635] Synthesis of TD921:

[0636]

change

[0637] In a 25 mL pear-shaped glass flask, TD910 (160 mg; 315 μmol; 2.0 equivalents) was dissolved in 10 mL of MeCN, followed by the addition of a solution of dry K2CO3 (22 mg; 160 μmol; 1.0 equivalent). Next, a solution of TD558 (24 mg; 158 μmol; 1.0 equivalent) in 5 mL of MeCN was added dropwise over 15 minutes, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in 50 mL of DCM and 50 mL of H2O and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered through glass frit (S₃), and evaporated to dryness. The resulting alkyne-containing intermediate (along with a partially N-methylated / demethylated by-product as a result of ongoing automethylation; 49 mg; ≤78 μmol; ≤1.0 equivalent) was dissolved in MeCN (7 mL), followed by the addition of TD406 (18 mg; 119 μmol; ≥1.5 equivalents) and K₂CO₃ (54 mg; 391 μmol; ≥5.0 equivalents). The resulting suspension was stirred at room temperature for 16 hours. The solid was filtered through a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C₁₈; H₂O-MeCN gradient, with TFA). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO₃, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The methyl ester of the obtained product (along with a partially N-methylated / demethylated by-product as a result of ongoing automethylation; 37 mg; ≤48 μmol; ≤1.0 equivalent) was dissolved in dry pyridine (2 mL), followed by the addition of neat TMSBr (65 μL; 492 μmol; ≥12.0 equivalents), and the resulting mixture was stirred at room temperature for 16 hours.The reaction mixture was quenched with 50% MeOH (1 mL) aqueous solution, and the mixture was further stirred at room temperature for 1 hour. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were coupled and directly freeze-dried to obtain the amphoteric product as a white, fluffy solid. Yield: 25.6 mg (20%; 3 stages; TD558 standard). NMR (D2O, pD~4): 1 H δ H 2.56-3.75(mc,CH2-P,C≡CH,m,16+4+1H);4.20(CH2-arom.,bs,2H);4.29(CH2-arom. , bs,2H);4.45(CH2-N3,s,2H);7.23-7.77(Ph,arom.,m,10+4H);7.96(arom.,t,1H, 3 J HH =8);7.97(arom.,t,1H, 3 J HH =8). 31 P{ 1 H} δ P 25.1(bs).ESI-HRMS:796.2334[M+H] + (Theoretical value [C 44 H 34 N 10 [O2P2] + =796.2336). EA(C 37 H 45 N9O4P2-4.6H2O,M R =824.6):C53.9(54.5);H6.6(6.3);N15.3(14.7).

[0638] Synthesis of TD580:

[0639] [ka]

[0640] In a 20 mL glass vial, TD579 (137 mg; 233 μmol; 1.0 equivalent) was dissolved in MeCN (7 mL), and dried K2CO3 (141 mg; 1.02 mmol; 4.4 equivalents) was added. Next, a solution of TD406 (50 mg; 274 μmol; 1.2 equivalents) in MeCN (3 mL) was added, and the resulting suspension was stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a colorless oil. Yield: 125 mg (73%; 1 step; TD579 standard). NMR (CD3CN): 1 H δ H 1.21(CH3,t,12H, 3 J HH =7);2.90(mc,m,8H);2.97(CH2-P,d,4H, 2 J HP =10);2.99(mc,m,4H);3.24(mc,m,8H);3.55(C≡CH,s,1H);4.01(CH2-OP,m,8H);4.21 (CH2-arom, s,2H);4.48(CH2-N3,s,2H);4.55(CH2-arom,s,2H);7.37(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.49(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.51(arom,dd,1H,J 3 HH =8, 4 J HH =1);7.54(arom.,dd,1H, 3 J HH=8, 4 J HH =1);7.80(arom.,t,1H, 3 J HH =8);7.84(arom.,t,1H, 3 J HH =8). 31 P{ 1 H} δ P 24.8(s). ESI-MS(LC-MS):734.5[M+H] + (Theoretical value [C 33 H 54 N9O6P2] + =734.4).

[0641] Synthesis of TD582:

[0642] [ka]

[0643] In a glass vial (4 mL), TD580 (25 mg; 34 μmol; 1.0 equivalent) was dissolved in a mixture of NaOH (10%; 1 mL) aqueous solution and EtOH (300 μL), and the resulting solution was stirred at room temperature for 2 days. The reaction mixture was quenched with AcOH (200 μL), and the mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were joined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 9 mg (≤39% M-0.1FA-xH2O; M R =682.3; 1 stage; assuming TD580 standard). ESI-MS(LC-MS):678.4[M+H] + (Theoretical value [C 29 H 46 N9O6P2] + =678.3).

[0644] Synthesis of TD581:

[0645] [ka]

[0646] In a glass vial (4 mL), TD580 (25 mg; 34 μmol; 1.0 equivalent) was dissolved in dried pyridine (1 mL), and neat TMSBr (110 μL; 833 μmol; ≤25 equivalents) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with 50% MeOH (1 mL) aqueous solution, and the mixture was stirred further at room temperature for 1 hour. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were joined and directly freeze-dried to obtain a nearly amphoteric product as a slightly yellow, fluffy solid. Yield: 6 mg (≤28% M-0.1FA-xH2O; M R =626.2; 1 stage; assuming TD580 standard). ESI-MS(LC-MS):622.3[M+H] + (Theoretical value [C 25 H 38 N9O6P2] + =622.2).

[0647] Synthesis of TD575:

[0648] [ka]

[0649] In a glass vial (20 mL), TD579 (51 mg; 87 μmol; 1.2 equivalents) was dissolved in MeCN (3 mL), followed by the addition of dried K2CO3 (30 mg; 217 μmol; 3.0 equivalents). Next, a solution of TD566 (20 mg; 73 μmol; 1.0 equivalent) in MeCN (2 mL) was added, and the resulting suspension was stirred at room temperature for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined, neutralized with diluted aqueous NaHCO3, and evaporated to dryness. The residue was dissolved in DCM (50 mL) and H2O (50 mL) and transferred to a separatory funnel. After shaking, the bottom phase was separated. The aqueous phase was further extracted with DCM (3 × 25 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered through glass frit (S3), and evaporated to dryness. The residue was further dried overnight under high vacuum to obtain the product in the form of free base as a nearly colorless oil. Yield: 46 mg (90%; 1 step; TD566 standard). ESI-MS (LC-MS): 810.4 [M+H] + (Theoretical value [C 39 H 58 N9O6P2] + (=810.4). The TD575 was used directly in the TD576 without any further characteristic analysis.

[0650] Synthesis of TD576:

[0651] [ka]

[0652] In a glass vial (4 mL), TD575 (23 mg; 28 μmol; 1.0 equivalent) was dissolved in dried pyridine (1 mL), and neat TMSBr (220 μL; 1.67 mmol; ≤60 equivalents) was added. The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with 50% MeOH (1 mL) aqueous solution, and the mixture was stirred further at room temperature for 1 hour. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 15 mg (≤75% M-0.1FA-xH2O; M R =702.3; 1 step; assumed to be based on TD575). NMR (D2O + NaOD, pD ~ 8): 1 H δ H 2.54-3.76(mc,CH2-P,m,16+4H);3.93(CH2-arom.,bs,2H);4.10(CH2-arom.,bs,2H);4.60(CH2-N3,s,2H);7.44(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.46-7.51(Ph,m,3H);7.53(arom, dd,1H, 3 J HH =8, 4 J HH =1);7.66-7.70(Ph,m,2H);7.71arom.,d,1H, 4 J HH =2);7.77(arom.,d,1H, 4 J HH =2);7.81(arom.,t,1H, 3 J HH =8). 31 P{ 1 H} δ P 8.2 (bs).ESI-HRMS: 696.2575 [MH] - (Theoretical value [C 31 H 40 N9O6P2] - (=696.2582).

[0653] Synthesis of TD801:

[0654] [ka]

[0655] In a 20 mL glass vial, TD799 (46 mg; 85 μmol; 1.0 equivalent) was dissolved in 5 mL of MeCN, and dried K2CO3 (48 mg; 348 μmol; 4.1 equivalents) was added. Next, a solution of TD406 (20 mg; 110 μmol; 1.3 equivalents) in 1 mL of MeCN was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in 3 mL of TFA, and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 33 mg (48%; 2 steps; TD799 standard). NMR (D2O, pD~3): 1 H δ H 2.41-3.78(mc,CH2-CO,CH2-P,m,16+2+2H);3.89(CH2-arom.,bs,2H);3.95(CH2-arom.,bs,2H);4.52(CH2-N3,s,2H);7.42(arom,d,1H, 3 J HH =8);7.46(arom.,d,1H, 3 J HH =8);7.74(arom.,d,1H, 3 J HH =8);7.78(arom.,d,1H, 3 J HH =8);7.85(arom.,t,1H, 3 J HH =8);7.87(arom.,t,1H,3 J HH =8). ESI-HRMS:578.3208[M+H] + (Theoretical value [C 29 H 40 N9O4] + =578.3203). EA(C 29 H 39 N9O4-2.0TFA,M R =805.7):C49.2(49.3);H5.1(5.4);N15.6(15.7). Synthesis of TD764:

[0656] [ka]

[0657] In a 20 mL glass vial, TD635 (304 mg; 589 μmol; 1.1 equivalents) was dissolved in MeCN (7 mL), and dried K2CO3 (305 mg; 2.21 mmol; 4.0 equivalents) was added. Next, a solution of TD760 (120 mg; 553 μmol; 1.0 equivalent) in MeCN (3 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 196 mg (51%; 2 steps; TD635 standard). NMR (D2O, pD~3): 1 H δ H2.86-3.60(mc,CH2-CO,m,16+4H);3.77(C≡CH,s,1H);3.79-4.06(CH2-arom.,bm,4H);4.62(CH2-N3,s,2H);7.62(arom,d,1H, 4 J HH =2);7.67-7.73(arom.,m,1H);8.07-8.14(arom.,m,2H);8.17(arom.,d,1H, 4 J HH =2). ESI-HRMS: 582.2344 [MH] - (Theoretical value[C 27 H 33 [N9O4Cl1]=582.2350). EA(C 27 H 34 N9O4Cl1-0.7TFA-1.7H2O,M R =694.5):C49.1(49.0);H5.5(5.4);N18.2(18.4);Cl5.1(5.3).

[0658] Synthesis of TD1063:

[0659]

change

[0660] In a glass vial (4 mL), TD635 (50 mg; 97 μmol; 1.0 equivalent) was dissolved in MeCN (1 mL), and dried K2CO3 (67 mg; 485 μmol; 5.0 equivalents) was added. Next, a solution of TD1057 (24 mg; 96 μmol; 1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 33.4 mg (55%; 2 steps; TD635 standard). NMR (D2O, pD~7): 1 H δ H 2.81-3.68(mc,CH2-CO,m,16+4H);3.72(C≡CH,s,1H);3.82(CH2-arom.,bs,2H);4.05(CH2-arom,bs,2H);4.70 (CH2-N3,s,2H);7.62-7.69(arom.,m,1H);7.82(arom.,s,1H);7.92-7.99(arom.,m,2H);8.38(arom.,s,1H). 19 F{ 1 H} δ F -64.2(s).ESI-HRMS:618.2759[M+H] + (Theoretical value [C 28 H 35 N9O4F3] + =618.2759). EA(C 28 H 34 N9O4F3-0.1FA-1.2H2O,M R =643.8):C52.4(52.8);H5.7(5.4);N19.6(19.4);F8.8(7.8).

[0661] Synthesis of TD1092:

[0662] [ka]

[0663] In a glass vial (4 mL), TD635 (35 mg; 68 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (44 mg; 318 μmol; 4.7 equivalents). Next, a solution of pre-purified TD1089 (containing less than 20% bisazide by-products; 20 mg; <83 μmol; <1.2 equivalents) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation with MeOH twice. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 31.9 mg (74%; 2 steps; TD635 standard). NMR (D2O, pD~6): 1 H δ H 2.76-3.62(mc,CH2-CO,m,16+4H);3.73(C≡CH,s,1H);3.83(CH2-arom.,bs,2H);3. 99(CH3,s,3H);4.05(CH2-arom.,bs,2H);4.69(CH2-N3,s,2H);7.70(arom,dd,1H, 3 J HH =8, 4 J HH =1);8.02(arom.,d,1H, 4 J HH =1);8.05(arom.,dd,1H,3 J HH =8, 4 J HH =1);8.15(arom.,t,1H, 3 J HH =8);8.58(arom.,d,1H, 4 J HH =1). ESI-HRMS: 608.2939 [M+H] + (Theoretical value[C 29 H 38 N9O6] + =608.2940). EA(C 29 H 37 N9O6-0.1FA-1.1H2O,M R =632.1):C55.3(55.1);H6.3(6.0);N19.9(19.6).

[0664] Synthesis of TD1148:

[0665]

change

[0666] In a glass vial (4 mL), TD635 (44 mg; 85 μmol; 1.0 equivalent) was dissolved in MeCN (2.5 mL), followed by the addition of dried K2CO3 (47 mg; 340 μmol; 4.0 equivalents). Next, a solution of TD1112 (23 mg; 86 μmol; 1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 3 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 30.7 mg (55%; 2 steps; TD635 standard). NMR (D2O, pD~6): 1 H δ H 1.40(CH3,d,6H, 3 J HH =6);2.77-3.63(mc,CH2-CO,m,16+4H);3.70(C≡CH,s,H);3.80(CH2-arom.,bs,2H);4.06(CH2-arom,bs,2H);4.65(CH2-N3,s,2H);5.26(CH,hept,1H, 3 J HH =6);7.65(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.93-8.00(arom.,m,2H);8.12(arom.,t,1H, 3 J HH =8);8.45(arom.,d,1H, 4 J HH =2).ESI-HRMS:636.3247[M+H] + (Theoretical value [C 31 H 42 N9O6]+ =636.3253). EA(C 31 H 41 N9O6-0.1FA-1.0H2O,M R =658.3):C56.7(56.4);H6.6(6.4);N19.1(18.8). Synthesis of TD1160:

[0667] [ka]

[0668] In a pear-shaped glass flask (100 mL), TD1116 (50 mg; 158 μmol; 2.0 equivalents) was dissolved in MeCN (20 mL), followed by the addition of dried K2CO3 (11 mg; 80 μmol; 1.0 equivalent). Next, a solution of TD558 (12 mg; 79 μmol; 1.0 equivalent) in MeCN (20 mL) was added dropwise over 2 hours, and the resulting suspension was stirred at room temperature for 7 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the monoalkylation intermediate were combined and directly freeze-dried. The resulting solid (15.4 mg; ≤36 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), and dried K2CO3 (25 mg; 181 μmol; ≥5.0 equivalents) was added. Next, a solution of TD1130 (15 mg; 53 μmol; ≥ 1.5 equivalents) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 7 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fraction containing the methyl-protected product was joined and directly lyophilized. The residue was redissolved in a mixture of MeOH (2 mL) and H2O (1 mL), followed by the addition of solid LiOH-H2O (15 mg; 360 μmol; > 10 equivalents). The resulting mixture was stirred at room temperature for 15 minutes, quenched with FA (15 μL), and directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fraction containing the product was joined and directly lyophilized to obtain the product as a white solid. Yield: 1.7 mg (<5%; 3 stages; TD558 standard). ESI-HRMS:650.3407[M+H] + (Theoretical value [C 32 H 44 N9O6] + (=650.3409).

[0669] Synthesis of TD1176:

[0670] [ka]

[0671] In a glass vial (4 mL), TD635 (43.5 mg; 84.4 μmol; 1.1 equivalents) was dissolved in MeCN (2 mL), and dried K2CO3 (42.5 mg; 308 μmol; 4.0 equivalents) was added. Next, a solution of TD1163 (17.3 mg; 76.7 μmol; 1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 2 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a white, fluffy solid in the form of formate. Yield: 43.6 mg (80%; 2 steps; TD1163 standard). NMR (D2O, pD~7): 1 H δ H 2.74-3.62(mc,CH2-CO,CH3,m,16+4+6H);3.70(C≡CH,s,1H);3.76(CH2-arom.,bs,2H);3.84(CH2-arom.,bs,2H);~4.7-4.8(CH2-N3,HOD signal obscured);6.85(arom,bs,1H);7.02(arom.,bs,1H);7.64(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.88(arom.,bt,1H, 3 J HH =8);7.94(arom.,bd,1H, 3 J HH =8). ESI-HRMS:593.3310[M+H] + (Theoretical value [C 29 H 41 N 10 O4] + =593.3307). EA(C29 H 40 N 10 O4-0.8FA-4.4H2O,M R =708.8):C50.5(50.8);H7.2(7.1);N19.8(19.5).

[0672] Synthesis of TD647

[0673] [ka]

[0674] In a pear-shaped glass flask (50 mL), TD635 (375 mg; 727 μmol; 1.0 equivalent) was dissolved in dry MeCN (20 mL), followed by the addition of dry K2CO3 (300 mg; 2.17 mmol; 3.0 equivalents). Next, a solution of TD566 (189 mg; 731 μmol; 1.0 equivalent) in dry MeCN (5 mL) was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 342 mg (72%; 2 steps; TD635 standard). NMR (D2O, pD~4): 1 H δ H 2.70-3.59(mc,CH2-CO,m,16+4H);3.66(CH2-arom.,bs,2H);3.69(C≡CH,s,1H);3.90(CH2 -arom.,bs,2H);4.64(CH2-N3,s,2H);7.46-7.60(Ph,arom,m,3+1H);7.64(arom.,dd,1H, 3J HH = 8, 4 J HH = 2); 7.68 (arom., t, 1H, 3 J HH = 8); 7.69 (arom., d, 1H, 4 J HH = 2); 7.79 - 7.88 (Ph, m, 2H); 8.28 (arom., d, 1H, 4 J HH = 2). 13 C{ 1 H} δ C 48.2 (mc, s); 48.3 (mc, s); 51.2 (mc, s); 51.3 (mc, s); 54.7 (CH2 - N3, s); 56.4 (CH2 - CO, s); 57.8 (CH2 - arom., s); 59.1 (CH2 - arom., s); 80.3 and 82.4 (C≡CH; 2×s); 121.9 (arom., s); 124.4 (arom, s); 125.6 (arom, s); 128.2 (Ph, s); 128.5 (Ph, s); 130.0 (Ph, s); 130.8 (arom, s); 137.7 (Ph, s); 140.3 (arom, s); 141.2 (arom, s); 153.6 (arom, s); 155.4 (arom, bs); 156.2 (arom, s); 157.6 (arom, s); 168.7 (CO, s). ESI - HRMS: 626.3195 [M + H] + (Theoretical value [C 33 H 40 N9O4] + = 626.3198). EA (C 33 H 39 N9O4 - 0.2FA - 0.9H2O, M R = 651.2): C 61.2 (61.6); H 6.4 (6.1); N 19.4 (19.1).

[0675] Synthesis of TD722:

[0676]

Chem.

[0677] In a glass vial (20 mL), TD539 (350 mg; 543 μmol; 1.0 equivalent) was dissolved in MeCN (15 mL), followed by the addition of dried K2CO3 (375 mg; 2.72 mmol; 5.0 equivalent). Next, a solution of TD566 (140 mg; 541 μmol; 1.0 equivalent) in MeCN (3 mL) was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl / Boc-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (5 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a slightly yellow solid in the form of trifluoroacetate. Yield: 295 mg (69%; 2 steps; TD566 standard). NMR (D2O, pD~7): 1 H δ H 2.74-3.64(mc,CH2-CO,m,16+4H);3.72(CH2-arom.,bs,2H);3.98(CH2-arom.,bs,2H);4.1 0(CH2-C≡C,s,2H);4.65(CH2-N3,s,2H);7.47-7.69(Ph,arom,m,3+3H);7.72(arom.,d,1H, 4 J HH =2);7.85-7.96(Ph,m,2H);8.41(arom.,d,1H, 4 J HH =2). ESI-HRMS:655.3460[M+H] + (Theoretical value C) 34 H 43 N 10 O4] + =655.3463). EA(C 34 H 42 N 10 O4-0.7TFA-2.0H2O,M R=770.6):C55.2(54.9);H6.1(5.8);N18.2(18.3);F5.2(5.3).

[0678] Synthesis of TD1054:

[0679] [ka]

[0680] In a glass vial (4 mL), TD663 (30.0 mg; 45.3 μmol; 1.0 equivalent) was dissolved in MeCN (1 mL), and dried K2CO3 (37.5 mg; 271 μmol; 5.0 equivalents) was added. Next, a solution of freshly prepared and isolated TD1050 (≤65.3 μmol; ≤1.5 equivalents) in MeCN (1 mL) was added, and the resulting suspension was stirred at room temperature for 3 days. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl / Boc-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a colorless solid in the form of a mixed salt of trifluoroacetate / formate. Yield: 23.0 mg (65%; 2 steps; TD663 standard). NMR (D2O, pD~8): 1 H δ H 1.72(CH3,s,6H);2.67-3.61(mc,CH2-CO,m,16+4H);3.70(CH2-arom.,bs,2H);3.97(CH2 -arom, bs,2H);4.64(CH2-N3,s,2H);7.45-7.67(Ph,arom.,m,3+3H);7.72(arom.,d,1H, 4 J HH=2);7.86-7.93(Ph,m,2H);8.40(arom.,d,1H, 4 J HH =2). ESI-HRMS: 683.3775 [M+H] + (Theoretical value[C 36 H 47 N 10 O4] + =683.3776). EA(C 36 H 46 N 10 O4-0.3TFA-0.8FA-2.3H2O,M R =777.3):C57.8(58.0);H6.5(6.5);N18.0(17.7).

[0681] Synthesis of TD1105:

[0682]

change

[0683] In a glass vial (4 mL), TD711 (101 mg; 185 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (102 mg; 738 μmol; 4.0 equivalents). Next, a solution of freshly prepared and isolated TD1050 (≤222 μmol; ≤1.2 equivalents) in MeCN (1 mL) was added, and the resulting suspension was stirred at 40°C for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl / Boc-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the completely deprotected intermediate were combined and directly lyophilized. The resulting solid was dissolved in a mixed solvent of H2O (6 mL), MeCN (6 mL), and THF (2 mL), followed by the addition of Boc2O (2.0 M in dry THF; 315 μL; 630 μmol; ≥3.4 equivalents) and NaHCO3 (210 mg; 2.50 mmol; ≥13.5 equivalents). The resulting mixture was stirred at room temperature for 2 days, after which another portion of Boc2O (2.0 M in dry THF; 315 μL; 630 μmol; ≥3.4 equivalents) was added. The mixture was further stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined to obtain a nearly zwitterionic product as a white, fluffy solid. Yield: 36.8 mg (27%; 3 stages; TD711 standard). NMR (D2O, pD~7): 1 H δ H 1.47(CH3,s,9H);1.65(CH3,s,6H);2.85-3.65(mc,CH2-CO,m,16+4H);3.91(CH2- arom.,bs,2H);3.95(CH2-arom,bs,2H);4.59(CH2-N3,s,2H);7.51(arom.,dd,1H, 3 JHH =7, 4 J HH =2);7.59(arom.,dd,1H,J 3 HH =7,J 4 HH =2);7.89-8.05(arom.,m,4H).ESI-HRMS:707.3988[M+H] + (Theoretical value[C 35 H 51 N 10 O6] + =707.3988). EA(C 35 H 50 N 10 O6-0.1FA-2.0H2O,M R =747.5):C56.4(56.7);H7.3(7.0);N18.7(18.5).

[0684] Synthesis of TD1127:

[0685]

change

[0686] In a glass vial (20 mL), TD1118 (56.9 mg; 81.4 μmol; 1.0 equivalent) was dissolved in MeCN (2 mL), followed by the addition of dried K2CO3 (45.0 mg; 326 μmol; 4.0 equivalents) and TD406 (16.0 mg; 87.6 μmol; 1.1 equivalents) in MeCN (1 mL). The resulting suspension was stirred at room temperature for 16 hours. The solid was filtered through a syringe microfilter (PTFE), and the filtrate was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the tert-butyl / Boc-protected product were combined and evaporated to dry, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as a colorless solid in the form of a mixed salt of trifluoroacetate / formate. Yield: 49.0 mg (71%; 2 steps; TD1118 standard). NMR (D2O, pD~7): 1 H δ H 1.96-2.11(CH2,m,2H);2.14-2.29(CH2,m,2H);2.86-3.63(mc,CH2-CO,CH2,CH,m,16+4+4+1H);3.90(CH2-a rom.,bs,2H);3.94(CH2-arom,bs,2H);4.59(CH2-N3,s,2H);7.49-7.55(arom.,m,1H);7.60(arom.,dd,1H, 3 J HH =8, 4 J HH =2);7.92-8.04(arom.,m,4H).ESI-HRMS:633.3612[M+H] + (Theoretical value [C 32 H 45 N 10 O4] + =633.3620). EA(C 32 H 44 N 10 O4-1.8TFA-0.1FA,M R=878.6):C50.9(50.7);H5.5(5.9);N16.6(16.9).

[0687] Synthesis of TD742:

[0688] [ka]

[0689] In a glass vial (20 mL), TD635 (90 mg; 163 μmol; 1.1 equivalents) was dissolved in MeCN (6 mL), followed by the addition of dried K2CO3 (85 mg; 616 μmol; 4.0 equivalents). Next, a solution of TD733 (55 mg; 153 μmol; 1.0 equivalent) in MeCN (2 mL) was added, and the resulting suspension was stirred at room temperature for 18 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the tert-butyl-protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fraction containing the product was coupled and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 87 mg (80%; 2 steps; TD733 standard). NMR (D2O, pD~4): 1 H δ H 2.76-3.64(mc,CH2-CO,m,16+4H);3.69(C≡CH,s,1H);3.72(CH2-arom.,bs,2H);3.99( CH2-arom.,bs,2H);4.77(CH2-N3,s,2H);7.54-7.70(arom,m,3H);7.85(arom.,d,1H, 4 J HH =2);7.96(arom.,d,2H, 3 J HH =8);8.11(arom.,d,2H,3 J HH =8);8.38(arom.,d,1H, 4 J HH =2). ESI-HRMS:670.3094[M+H] + (Theoretical value [C 34 H 40 N9O6] + =670.3096). EA(C 34 H 40 N9O6-0.2FA-1.6H2O,M R =707.8):C58.0(58.4);H6.1(5.9);N17.8(17.5).

[0690] Synthesis of TD744:

[0691] [ka]

[0692] In a 20 mL glass vial, TD539 (80 mg; 124 μmol; 1.1 equivalents) was dissolved in MeCN (3 mL), followed by the addition of dried K2CO3 (65 mg; 471 μmol; 4.0 equivalents). Next, a solution of TD733 (42 mg; 117 μmol; 1.0 equivalent) in MeCN (3 mL) was added, and the resulting suspension was stirred at room temperature for 24 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA addition). The fractions containing the protected product were combined and evaporated to dryness, and the majority of the MeCN was removed by evaporation twice with MeOH. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice with MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain the product as an off-white, fluffy solid in the form of a mixed salt of trifluoroacetate / formate. Yield: 44 mg (45%; 2 steps; TD733 standard). NMR (D2O, pD~4): 1 H δ H2.71-3.64(mc,CH2-CO,m,16+4H);3.70(CH2-arom.,bs,2H);3.98(CH2-arom.,bs,2H);4 .12(CH2-C≡C,s,2H);4.70(CH2-N3,s,2H);7.50-7.59(arom, m,2H);7.63(arom.,dd,1H,J 3 HH =7,J 4 HH =2);7.78(arom.,d,1H, 4 J HH =2);7.95(arom.,d,2H, 3 J HH =9);8.07(arom.,d,2H, 3 J HH =9);8.42(arom.,d,1H, 4 J HH =2). ESI-HRMS:699.3358[M+H] + (Theoretical value [C 35 H 43 N 10 O6] + =699.3362). EA(C 35 H 42 N 10 O6-1.0TFA-1.3H2O,M R =836.2):C53.1(52.8);H5.5(5.8);N16.8(16.9).

[0693] Synthesis of TD750: In a glass vial (2 mL), TD744-1.0TFA-1.3H2O (1.5 mg; ~1.8 μmol; 1.0 equivalent) was dissolved in an aqueous solution of boric acid / NaOH buffer (200 ml; pH 9.0; 900 μL; 180 mmol; ~100 equivalents). Then, a freshly prepared solution of FmocCl (0.6 mg; ~2.3 μmol; 1.3 equivalents) in MeCN (900 μL) was added. The resulting clear solution was stirred at room temperature for 2 hours. The mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 0.8 mg (≤48%) M-0.2FA-xH2O; M R=930.2; 1 step; (assuming TD744-1.0TFA-1.3H2O reference). ESI-HRMS: 921.4044[M+H] + (Theoretical value [C 50 H 53 N 10 O8] + (=921.4042).

[0694] Synthesis of TD779:

[0695] [ka]

[0696] In a glass vial (4 mL), TD647-0.2FA-0.9H2O (10 mg; 15 μmol; 1.0 equivalent) and ammonium chloride (2 mg; 38 μmol; 2.5 equivalents) were dissolved in DMSO (2 mL), and DIPEA (22 μL; 126 μmol; 8.2 equivalents) and solid HATU (18 mg; 47 μmol; 3.1 equivalents) were added. The resulting yellow solution was stirred at room temperature for 15 minutes. The mixture was quenched with FA (6 μL; 169 μmol; 10 equivalents) and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly lyophilized to obtain a nearly amphoteric product as a white, fluffy solid. Yield: 7 mg (≤73%) M-0.1FA-xH2O;M R =628.4; 1 step; (assuming TD647-0.2FA-0.9H2O reference). NMR (D2O+FA, pD~3): 1 H δ H 2.83-3.74(mc,CH2-CO,bm,16+4H);3.65(C≡CH,s,1H);4.13-4.68(CH2-arom., bm4H);4.69(CH2-N3,s,2H);7.52-7.66(Ph,arom,m,3+1H);7.71(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.80-7.88(Ph,m,2H);7.90(arom.,d,1H,J 4 HH=2);7.91(arom.,t,1H, + J HH =8);8.00(arom.,bs,1H).ESI-HRMS:624.3503[M+H] + (Theoretical value [C 33 H 42 N 11 O2] + (=624.3504).

[0697] [ka]

[0698] Synthesis of TD778: In a glass vial (4 mL), TD647-0.2FA-0.9H2O (12 mg; 18 μmol; 1.0 equivalent) and glycine tert-butyl hydrochloride (8 mg; 48 μmol; 2.6 equivalents) were dissolved in DMSO (2 mL), followed by the addition of DIPEA (26 μL; 149 μmol; 8.1 equivalents) and solid HATU (21 mg; 55 μmol; 3.0 equivalents). The resulting yellow solution was stirred at room temperature for 20 minutes. The mixture was then quenched with FA (7 μL; 186 μmol; 10 equivalents) and purified directly by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the tert-butyl protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by evaporation twice over MeOH. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain a nearly zwitterionic product as a white, fluffy solid. Yield: 9 mg (62%; 2 steps; based on TD647-0.2FA-0.9H2O). NMR (D2O, pD~5): 1 Hδ H 2.83-3.93-(mc,CH2-CO,C≡CH,bm,16+8+1H);4.35-4.69(CH2-arom.,CH2-N3.,bm,4+2H);7.49(arom.,d,1H,3 J HH =8);7.56-7.73(Ph,arom.,m,3+1H);7.75-7.92(Ph,arom.,m,2+2H);8.06(arom.,s,1H). ESI-HRMS:738.3471[MH] - (theor.[C 37 H 44 N 11 O6] - =738.3482). EA(C 37 H 45 N 11 O6-0.1FA-2.4H2O,M R =787.7):C56.6(55.9);H6.4(5.8);N19.6(20.3).

[0699] [ka]

[0700] Synthesis of TD1408: In a glass vial (25 mL), TD635 (51 mg; 99 μmol; 1.1 equivalent) was dissolved in MeCN (1 mL), followed by the addition of dried K2CO3 (54 mg; 391 μmol; 4.4 equivalents). Next, a solution of TD1406 (17.5 mg; 89 μmol; 1.0 equivalent) in MeCN (1 mL) was added, and the resulting suspension was stirred at 40°C for 16 hours. The solid was filtered using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness, and the majority of the TFA was removed by two evaporations with MeOH. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with added FA). The fractions containing the product were combined and directly freeze-dried to obtain a nearly zwitterionic product as a white, fluffy solid. Yield: 41.7 mg (77%; 2 steps; based on TD1406). NMR (D2O, pD~7):1 Hδ H 2.74 - 4.16 (mc, CH2 - CO, CH2 - arom., CH2 - CH2 - N3, C≡CH, m, 16 + 4 + 4 + 4 + 1H); 7.37 - 7.45 (arom., m, 1H); 7.65 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1); 7.85 - 7.93 (arom., m, 2H); 7.96 (arom., t, 1H, 3 J HH = 8); 8.06 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1). ESI - HRMS: 564.3044 [M + H] + (theor. [C 28 H 38 N9O4] + = 564.3041). EA (C 28 H 37 N9O4 - 0.1FA - 2.4H2O, M R = 611.5): C 55.2 (56.0); H 6.9 (6.7); N 20.6 (19.8).

[0701]

Chem.

[0702] Synthesis of TD1346: In a pear-shaped glass flask (25 mL), TD1345 (52.7 mg; 83 μmol; 1.0 equivalent) was dissolved in 60% aq. MeCN (2.7 mL), followed by the addition of freshly prepared aq. LiOH (1.0 M; 830 μL; 830 μmol; 10 equivalents). The resulting solution was stirred at room temperature for 5 hours. The mixture was quenched by the addition of FA (31 μL; 822 μmol; 10 equivalents). The resulting solution was briefly concentrated to remove most of the MeCN, and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly lyophilized to obtain the product in the form of free base as a white, fluffy solid. Yield: 35.4 mg (71%; 1 step; based on TD1345). NMR (D2O, pD~7): 1 Hδ H 1.24-1.42(CH3,m,6H);2.44-3.81(mc,CH2-arom.,C≡CH,m,16+4+1H);4.41-4.53(CH-CH3,m,2H);4.56(CH2-N3,s,2H);7.49(arom.,d,1H, 3 J HH =8);7.63(arom.,d,1H, 3 J HH =8);7.93(arom.,t,1H, 3 J HH =8);7.96(arom.,t,1H, 3 J HH =8);8.05(arom.,d,1H, 3 J HH =8);8.10(arom.,d,1H, 3 J HH =8). ESI-HRMS:578.3197[M+H] + (theor.[C 29 H 40 N9O4] + =578.3198). EA(C 29 H 39 N9O4-1.2H2O,M R =599.3):C58.1(58.0);H7.0(6.5);N21.0(20.6).

[0703] [ka]

[0704] Synthesis of TD1451: In a pear-shaped glass flask (25 mL), TD1449 (302 mg; 330 μmol; 1.0 equivalent) was dissolved in 60% aq. MeCN (17 mL), followed by the addition of freshly prepared aq. LiOH (1.0 M; 3.3 mL; 3.3 mmol; 10 equivalents). The resulting solution was stirred at room temperature for 2 hours. The mixture was briefly concentrated to remove most of the MeCN, and the mixture was directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain a nearly zwitterionic product as a white, fluffy solid. Yield: 257 mg (87%; 1 step; based on TD1449). NMR (D2O, pD~3): 1 Hδ H 1.37-1.60(CH3-CO,CH3-CH,m,9+6H);1.67(CH3-CN,s,6H);2.66-4.30(mc,CH2-arom.,CH-CH3,m,16+4+2 H);4.68(CH2-N3,s,2H);7.80(arom.,s,1H);7.92(arom.,s,1H);7.98(arom.,s,1H);8.02(arom.,s,1H). 19 Fδ F -64.44(s). ESI-HRMS:847.4070[M+H] + (theor.[C 39 H 54 N 10 O8F3] + =634.3824). EA(C 39 H 53 N 10 O8F3-0.1FA-2.7H2O,M R =900.1):C52.2(53.1);H6.6(6.2);N15.6(15.5);6.3(5.4).

[0705] [ka]

[0706] Synthesis of TD1504: In a glass vial (25 mL), TD1500 (11.9 mg; 18 μmol; 1.0 equivalent) was dissolved in MeCN (800 μL) and H2O (550 μL), followed by the addition of freshly prepared aq.LiOH (1.0 M; 263 μL; 263 μmol; 15 equivalents). The resulting solution was stirred at room temperature for 3 hours. The mixture was quenched by the addition of FA (8 μL; 212 μmol; 12 equivalents). The resulting solution was diluted with H2O (2 mL) and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly lyophilized to obtain the product in the form of free base as a white, fluffy solid. Yield: 8.9 mg (~77% M-2H2O; M R =657.7; 2 steps; assumed to be based on TD1500). NMR (D2O, pD~3): 1 Hδ H 1.28-1.45(CH3,m,3H);2.49-3.70(mc,CH-CH3,CH-CH2,CH2-arom.,m,16+1+3+4H);3.76(C≡CH,s,1H);4.35-4.51(CH2-N3,m,2H);7.69(arom.,dd,2H, 3 J HH =8, 4 J HH =1);7.98(arom.,t,1H, 3 J HH =8);8.05-8.21(arom.,m,3H). ESI-MS(LC-MS):622.3[M+H] + (theor.[C 30 H 40 N9O6] + =622.3).

[0707] [Example 5: Synthesis of a cage-type macrocyclic ligand (crosslinked with a triazole group)]

[0708] [ka]

[0709] Synthesis of cz-TD425: It was obtained as a byproduct during the synthesis of TD425 as a white, fluffy solid in a nearly zwitterionic form. Yield: 3 mg (≤1%) M-0.1FA-xH2O;M R =609.9; 3 steps; assumed to be based on 2-chloro-N-(prop-2-in-1-yl)acetamide). NMR (D2O, pD~3): 1 Hδ H 2.52-5.20(mc,CH2-CO,CH2-NH-CO-CH2,CH2-arom.,bm,16+4+4+2H);6.24(CH2-N3,bs,2 H);7.56-7.72(Ph,m,3H);7.88(CH-N3,s,1H);7.88-7.94(Ph,m,2H);8.33(arom.,d,1H, 4 J HH =2);8.73(arom.,d,1H, 4 J HH =2). ESI-HRMS:606.3143[M+H] + (theor.[C 30 H 40 N9O5] + (=606.3147).

[0710] [ka]

[0711] Synthesis of cz-TD556: It was obtained as a by-product during the synthesis of TD556 in the form of trifluoroacetate, as a white, fluffy solid. Yield: 9 mg (~2%; 2 steps; based on TD635). NMR (D2O, pD~3): 1 Hδ H 2.54-4.06(mc,CH2-CO,bm,16+4H);4.59(CH2-arom.,bs,2H);5.02(CH2-arom.,bs,2H);5.86(CH2-N3,bs,2H);7.67(arom.,bd,1H, 3 J HH =8);7.70(arom.,bd,1H, 3 J HH =8);7.83(arom.,bd,1H, 3 JHH =8);7.87(arom.,bd,1H, 3 J HH =8);8.14(arom.,bt,1H, 3 J HH =8);8.16(CH-N3,bs,1H);8.17(arom.,bt,1H, 3 J HH =8). ESI-HRMS:550.2889[M+H] + (theor.[C 27 H 36 N9O4] + =550.2885). EA(C 27 H 35 N9O4-2.7TFA-0.5H2O,M R =866.4):C44.9(44.8);H4.5(4.8);N14.5(14.8).

[0712] [ka]

[0713] Synthesis of cz-TD764: It was obtained as a by-product during the synthesis of TD764 in the form of trifluoroacetate, as a white, fluffy solid. Yield: 9 mg (~2%; 2 steps; based on TD635). NMR (D2O, pD~3): 1 Hδ H 2.48-3.94(mc,CH2-CO,bm,16+4H);4.57(CH2-arom.,bs,2H);5.00(CH2-arom.,bs,2H);5.94(CH2-N3,bs,2H);7.64(arom.,d,1H, 3 J HH =8);7.79(arom.,s,1H);7.84(arom.,d,1H, 3 J HH =8);7.92(arom.,s,1H);8.14(arom.,t,1H, 3 J HH =8);8.15(CH-N3,s,1H). ESI-HRMS:582.2344[MH] - (theor.[C 27 H 33N9O4Cl1] - =582.2350). EA(C 27 H 34 N9O4Cl1-1.8TFA-3.3H2O,M R =848.7):C43.3(43.3);H5.0(4.5);N14.9(14.5);Cl4.2(4.2);F12.1(12.0).

[0714] [ka]

[0715] Synthesis of cz-TD1063: It was obtained as a byproduct during the synthesis of TD1113 in the form of trifluoroacetate, as a white, fluffy solid. Yield: 1.7 mg. NMR (D2O, pD~3): 1 Hδ H 2.57-3.84(mc,CH2-CO,m,16+4H);4.70(CH2-arom.,s,2H);4.99(CH2-arom.,bs,2H);6.03(CH2-N3,bs,2H);7.65(arom.,d,1H, 3 J HH =8);7.86(arom.,d,1H, 3 J HH =8);8.05(arom.,s,1H);8.12-8.18(arom.,CH-N3,m,1+1H);8.19(arom.,s,1H). 19 Fδ F -65.82(s). NMR (at 10% H2O, pH 7.0, ~50 mM MOPS / NaOH buffer, ~0.5 mM cz-TD1063): 19 Fδ F -65.66(s;ΔH 1 / 2 =4.6Hz). ESI-HRMS:618.2754[M+H] + (theor.[C 29 H 35 N9O4F3] + (=618.2759).

[0716] [ka]

[0717] Synthesis of TD1188: In a glass vial (4 mL), TD711 (70.0 mg; 128 μmol; 1.0 equivalent) was dissolved in MeCN (1.5 mL), followed by the addition of dried K2CO3 (71 mg; 514 μmol; 4.0 equivalent). Next, a solution of TD1178 (31 mg; 141 μmol; 1.1 equivalent) was added to MeCN (1.5 mL), and the resulting suspension was stirred at room temperature for 7 days. The solid was filtered off using a syringe microfilter (PTFE), and the filtrate was evaporated to dryness. The residue was purified by preparative HPLC (C18; H2O-MeCN gradient, with FA). The fractions containing the tert-butyl-protected product were combined, evaporated to dryness, and evaporated twice with MeOH to remove most of the MeCN. The residue was dissolved in TFA (3 mL), and the resulting clear mixture was stirred at room temperature for 16 hours. The mixture was evaporated to dryness and then evaporated twice with MeOH to remove most of the TFA. The residue was directly purified by preparative HPLC (C18; H2O-MeCN gradient, with FA added). The fractions containing the product were combined and directly freeze-dried to obtain the zwitterionic product as a white, fluffy solid. Yield: 9.5 mg (10%; 2 steps; based on TD711). NMR (D2O, pD~5): 1 Hδ H 2.11-4.00(mc,CH2-CO,bm,16+4H);4.52(CH2-arom.,bs,2H);4.88-6.58(CH2-arom.,CH2-N3,bm,4H);7.66(arom.,bd,1H, 3 J HH =8);7.68(arom.,d,1H, 3 J HH =8);7.75(arom.,bd,1H, 3 J HH =8);7.85(arom.,d,1H, 3 J HH =8);8.08(arom.,bt,1H, 3 J HH =8);8.16(arom.,t,1H, 3 J HH =8). 19 FδF -59.35(s). ESI-HRMS:618.2756[M+H] + (theor.[C 28 H 35 N9O4F3] + =618.2759). EA(C 28 H 34 N9O4F3-0.2TFA-0.4FA-4.3H2O,M R =736.3):C47.0(46.6);H6.0(5.6);N17.1(16.8);F9.3(10.1).

[0718] [ka]

[0719] Synthesis of TD650: In a glass vial (40 mL), TD647-0.2FA-0.9H2O (130 mg; 200 μmol; 1.0 equivalent) was dissolved in H2O (20 mL), followed by the addition of aq. citric acid (100 mM; 20 mL; 2.0 mmol; 10 equivalents) to adjust the pH to 2.4. The resulting mixture was then stirred at 80°C for 2 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 109 mg (77%; 1 step; based on TD647-0.2FA-0.9H2O). NMR (D2O, pD ~ 5, 95℃): 1 Hδ H 2.69-3.63(mc,CH2-CO,m,16+4H);4.22(CH2-arom.,s,2H);4.58(CH2-arom.,s,2H);6.22(CH2-N3,s,2H);7.61(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.60-7.67(Ph,m,3H);7.83(arom.,s,1H);7.85(arom.,dd,1H, 3 JHH =8, 4 J HH =1); 7.86 - 7.92 (Ph, m, 2H); 8.00 (arom., s, 1H); 8.11 (arom., t, 1H, 3 J HH =8); 8.21 (CH - N3, s, 1H). 13 C{ 1 H}δ C 48.5 (mc, s); 49.9 (mc, s); 50.2 (mc, s); 50.4 (mc, s); 53.4 (CH2 - N3, s); 55.6 (CH2 - CO, s); 57.8 (CH2 - arom., s); 59.2 (CH2 - arom., s); 122.6 (arom., s); 124.2 (arom., s); 125.4 (arom., s); 125.9 (arom., s); 127.8 (Ph, s); 129.9 (Ph, s); 130.7 (Ph, s); 134.5 (CH - N3, s); 137.0 (Ph, s); 138.0 (arom., s); 139.9 (arom., s); 147.1 (arom., s); 152.3 (arom., bs); 153.6 (arom., bs); 153.8 (arom., bs); 156.8 (arom., s); 172.6 (CO, s). ESI - HRMS: 626.3200 [M + H] + (theor. [C 33 H 40 N9O4] + = 环戊二烯 33 H 39 N9O4 - 0.9FA - 2.0H2O, M R = 703.2): C 57.9 (58.3); H 6.4 (5.9); N 17.9 (17.5).

[0720]

Chem.

[0721] It should be noted that the chemical structure and some chemical terms may not be accurately translated without more context. The above translation is mainly based on literal conversion while trying to maintain the integrity of the chemical formula and related notations.Synthesis of TD871: In a glass vial (40 mL), TD647-1.3TFA (78.0 mg; 103 μmol; 1.0 equivalent) was dissolved in H2O (34 mL), followed by the addition of aq. Borate / CsOH buffer (200 mM; pH 9.0; 6.0 mL; 1.2 mmol; ~12 equivalents). The resulting mixture was then stirred at 80°C for 6 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 49.3 mg (70%; 1 step; based on TD647-1.3TFA). NMR (D2O, pD ~5, 95°C): 1 Hδ H 2.93-3.56(mc,CH2-CO,m,16+4H);4.41(CH2-arom.,s,2H);4.49(CH2-arom.,s,2H);6.00(CH2-N3,s,2H);7.54(arom.,dd,1H, 3 J HH =7, 4 J HH =2);7.56-7.66(Ph,m,3H);7.77(arom.,d,1H, 4 J HH =1);7.79-7.87(Ph,m,2H);7.94(arom.,d,1H, 4 J HH =1);8.05(arom.,dd,1H, 3 J HH =8, 4 J HH =2);8.07(arom.,dd,1H, 3 J HH =8, 3 J HH =7);8.93(CH-N3,s,1H). 13 C{ 1 H}δ C50.0(mc,s);50.7(mc,bs);52.0(mc,s);52.3(mc,s);54.8(CH2-N3,s);56.6(CH2-CO,s);59.9(CH2-arom .,s);60.2(CH2-arom.,bs);121.2(arom.,s);121.7(arom.,s);123.3(arom.,s);124.5(arom.,s);127. 0(CH-N3,s);127.7(Ph,s);129.9(Ph,s);130.5(Ph,s);137.1(Ph,s);140.2(arom.,s);148.0(arom.,s) ;150.3(arom.,s);151.7(arom.,s);152.5(arom.,bs);154.9(arom.,s);155.0(arom.,s);174.4(CO,s). ESI-HRMS:626.3199[M+H] + (theor.[C 33 H 40 N9O4] + =626.3198). EA(C 33 H 39 N9O4-0.6FA-2.3H2O,M R =696.8):C58.1(57.9);H6.5(6.2);N18.1(18.2).

[0722] [Example 6: Synthesis of coordination compounds]

[0723]

change

[0724] Synthesis of TD734: In a glass vial (20 mL), TD714-0.1FA-xH2O (6 mg; ~10 μmol; 1.0 equivalent) was dissolved in H2O (4 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 100 mL; 500 μmol; ~50 equivalents) and aq. LuCl3 (100 mM; 110 μL; 11 μmol; ~1.1 equivalents). The mixture was stirred at 80°C for 16 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then purified directly by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate, as a mixture of two diastereoisomers (~11:1). Yield: 5 mg (≤61% [M]) + [FA] - -xH2O;M R =803.6; 1 step; assumed to be based on TD714-0.1FA-xH2O). NMR (D2O, pD~4, signals of major isomers): 1 Hδ H 2.50-3.82(mc,CH2-CO,CH2-P,m,16+2+1H);3.85-4.00(CH2-arom.,CH2-P,m,1+1H);4.10(CH2-arom.,d,1H, 2 J HH =16);4.23(CH2-arom.,d,1H, 2 J HH =15);4.99(CH2-arom.,d,1H, 2 J HH =15);5.53(CH2-N3,d,1H, 2 J HH =15);7.56(CH2-N3,d,1H, 2 J HH =15);7.67(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.73(arom.,dd,1H, 3 J HH =8, 4 J HH= 1); 7.81 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 2); 7.83 (CH - N3, s, 1H); 7.98 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 2); 7.18 (arom., t, 1H, 3 J HH = 8); 8.20 (arom., t, 1H, 3 J HH = 8); 8.35 (FA, s, 1H). 13 C{ 1 H}δ C 48.9 (mc, s); 51.3 (mc, s); 51.6 (CH2 - P, d, 1 J CP = 73); 52.0 (mc, s); 52.3 (mc, s); 53.4 (mc, s); 53.5 (CH2 - N3, s); 53.6 (mc, s); 55.0 (mc, s); 56.3 (mc, s); 59.7 (CH2 - arom., s); 60.0 (CH2 - arom., s); 61.0 (CH2 - CO, s); 125.9 (arom., s); 127.6 (arom., s); 128.9 (arom., s); 130.4 (arom., s); 136.4 (CH - N3, s); 138.7 (arom., s); 142.0 (arom., s); 142.9 (arom., s); 146.2 (arom., s); 156.8 (arom., s); 157.6 (arom., s); 158.6 (arom., s); 169.5 (FA, s); 178.2 (CO, s). 31 P{ 1 H}δ P 17.8 (s). ESI - HRMS: 758.1822 [M] + (theor. [C 26 H 34 N9O5P1Lu1] + = 758.1823).

[0725]

Chem.

[0726] Synthesis of TD925: In a glass vial (20 mL), TD921-4.6H2O (6.1 mg; 7.4 μmol; 1.0 equivalent) was dissolved in H2O (18 mL) and i-PrOH (1 mL). Subsequently, aq. MOPS / NaOH buffer (500 mM; pH 7.0; 400 μL; 200 μmol; 27 equivalents) and aq. GdCl3 (100 mM; 95 μL; 9.5 μmol; 1.3 equivalents) were added, and the mixture was stirred at 80°C for 7 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, TFA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 2.5 mg (≤33% [M]) + [TFA] - -xH2O;M R =1010.0; 1 step; (assuming it is based on TD921-4.6H2O). ESI-HRMS: 897.2158[M] + (theor.[C 37 H 43 N9O4P2Gd1] + (=897.2149).

[0727] [ka]

[0728] Synthesis of TD560: In a glass vial (20 mL), TD556-0.1FA-1.6H2O (39 mg; 69 μmol; 1.0 equivalent) was dissolved in H2O (15 mL), then aq. MOPS / NaOH buffer (500 mM; pH 7.0; 3.40 mL; 1.70 mmol; 25 equivalents) and aq. EuCl3 (100 mM; 745 μL; 75 μmol; 1.1 equivalents) were added, and the mixture was stirred at 80°C for 4 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 24 mg (43%; 1 stage; based on TD556-0.1FA-1.6H2O). ESI-HRMS: 700.1867[M] + (theor.[C 27 H 33 N9O4Eu1] + =700.1862). EA([C 27 H 33 N9O4Eu1] + [FA] - -4.5H2O,M R =825.7):C40.7(40.7);H5.2(4.8);N15.3(15.0);Eu18.4(17.7).

[0729] [ka]

[0730] Synthesis of TD561: In a glass vial (20 mL), TD556-0.1FA-1.6H2O (40 mg; 69 μmol; 1.0 equivalent) was dissolved in H2O (15 mL), followed by the addition of aq.MOPS / NaOH buffer (500 mM; pH 7.0; 3.40 mL; 1.70 mmol; 25 equivalents) and aq.GdCl3 (100 mM; 745 μL; 75 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 4 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 24 mg (42%; 1 step; based on TD556-0.1FA-1.6H2O). ESI-HRMS: 705.1893[M] + (theor.[C 27 H 33 N9O4Gd1] + =705.1891). EA([C 27 H 33 N9O4Gd1] + [FA] - -4.6H2O,M R =832.8):C40.4(40.8);H5.2(4.9);N15.1(15.5);Gd18.9(18.1).

[0731] [ka]

[0732] Synthesis of TD562: In a glass vial (20 mL), TD556-0.1FA-1.6H2O (40 mg; 69 μmol; 1.0 equivalent) was dissolved in H2O (15 mL), followed by the addition of aq.MOPS / NaOH buffer (500 mM; pH 7.0; 3.40 mL; 1.70 mmol; 25 equivalents) and aq.TbCl3 (100 mM; 745 μL; 75 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 4 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 27 mg (47%; 1 stage; based on TD556-0.1FA-1.6H2O). ESI-HRMS: 706.1906[M] + (theor.[C 27 H 33 N9O4Tb1] + =706.1903). EA([C 27 H 33 N9O4Tb1] + [FA] - -4.7H2O,M R =836.2):C40.2(40.5);H5.2(4.9);N15.1(14.7);Tb19.0(18.7).

[0733] [ka]

[0734] Synthesis of TD1069: In a glass vial (20 mL), TD556-0.1FA-1.6H2O (20.8 mg; 35.7 μmol; 1.0 equivalent) was dissolved in H2O (17 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 1.80 mL; 900 μmol; 25 equivalents) and aq. LuCl3 (100 mM; 400 μL; 40.0 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 16 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 26.6 mg (82%; 1 step; based on TD556-0.1FA-1.6H2O). NMR (D2O, pD~3): 1 Hδ H 2.64-3.82(mc,CH2-CO,m,16+4H);3.95(CH2-arom.,d,1H, 2 J HH =15);4.15(CH2-arom.,d,1H, 2 J HH =16);4.29(CH2-arom.,d,1H, 2 J HH =15);4.85(CH2-arom.,d,1H, 2 J HH =16);5.60(CH2-N3,d,1H, 2 J HH =15);7.10(CH2-N3,d,1H, 2 J HH =15);7.73(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.80(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.85(arom.,dd,1H, 3 J HH =8, 4 J HH=1);7.88(CH-N3,s,1H);8.02(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.25(arom.,t,1+1H, 3 J HH =8). ESI-HRMS:722.2060[M] + (theor.[C 27 H 33 N9O4Lu1] + =722.2058). EA([C 27 H 33 N9O4Lu1] + [TFA] - -0.3TFA-2.4H2O,M R =913.0):C38.9(38.7);H4.2(3.8);N13.8(13.7);Lu19.2(16.5).

[0735] [ka]

[0736] Synthesis of TD751: In a glass vial (20 mL), TD718-2.6H2O (14 mg; 21 μmol; 1.0 equivalent) was dissolved in H2O (18 mL), followed by the addition of aq.MES / NaOH buffer (500 mM; pH 5.2; 1.30 mL; 650 μmol; 31 equivalents) and aq.LuCl3 (100 mM; 236 μL; 24 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 3 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then purified directly by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 13 mg (65%; 1 step; based on TD718-2.6H2O). NMR (D2O, pD~6): 1 Hδ H0.10(CH3,s,9H);2.61-3.83(mc,CH2-CO,m,16+4H);3.94(CH2-arom.,d,1H, 2 J HH =15);4.24(CH2-arom.,d,1H, 2 J HH =16);4.24(CH2-arom.,d,1H, 2 J HH =15);4.84(CH2-arom.,d,1H, 2 J HH =16);5.54(CH2-N3,d,1H, 2 J HH =15);6.92(CH2-N3,d,1H, 2 J HH =15);7.70(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.75(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.85(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.98(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.22(arom.,t,1H, 3 J HH =8);8.26(arom.,t,1H, 3 J HH =8);8.45(FA,s,1H)。 13 C{ 1 H}δ C-1.85 (CH3, s); 48.4 (mc, s); 48.7 (mc, s); 50.7 (mc, s); 51.9 (mc, s); 52.0 (CH2-N3, s); 53.2 (mc, s); 53.8 (mc, s); 54.4 (mc, s); 55.5 (mc, s); 58.4 (CH2-CO, s); 59.0 (CH2-arom., s); 59.1 (CH2-arom., s); 60.9 (CH2-CO, s); 125.9 (arom., s); 126.7 (arom., s); 128.3 (arom., s); 130.4 (arom., s); 141.4 (arom., s); 142.3 (arom., s); 142.6 (arom., s); 146.3 (arom., s); 147.5 (arom., s); 155.1 (arom., s); 157.7 (arom., s); 158.7 (arom., s); 17​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Synthesis of TD737: In a glass vial (20 mL), TD728-2.6H2O (15 mg; 20 μmol; 1.0 equivalent) was dissolved in H2O (17 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 2.00 mL; 1.00 mmol; 50 equivalents) and aq. LuCl3 (100 mM; 220 μL; 22 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 5 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, FA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of formate. Yield: 12 mg (58%; 1 step; based on TD728-2.6H2O). NMR (D2O, pD~5): δ H 0.96(i-Pr,d,9H, 3 J HH =8);0.99(i-Pr,d,9H, 3 J HH =8);1.26(i-Pr,hept,3H, 3 J HH =8);2.62-3.84(mc,CH2-CO,m,16+4H);4.05(CH2-arom.,s,2H);4.29(CH2-arom.,d,1H, 2 J HH =17);4.88(CH2-arom.,d,1H, 2 J HH =17);5.43(CH2-N3,d,1H, 2 J HH =15);6.74(CH2-N3,d,1H, 2 J HH =15);7.67(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.82(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.83(arom.,dd,1H, 3 J HH =8, 4 JHH = 1); 7.94 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1); 8.20 (arom., t, 1H, 3 J HH = 8); 8.22 (arom., t, 1H, 3 J HH = 8); 8.43 (FA, s, 1H). 13 C{ 1 H}δ C 12.1 (i-Pr, s); 18.8 (i-Pr, 2×s); 47.9 (mc, bs); 50.0 (mc, bs); 50.4 (mc, bs); 52.2 (mc, bs); 53.0 (CH2-N3, s); 54.0 (mc, s); 55.2 (mc, s); 57.2 (mc, s); 57.6 (mc, s); 59.3 (CH2-CO, s); 60.0 (CH2-arom., s); 62.3 (CH2-arom., bs); 64.0 (CH2-CO, s); 126.1 (arom., s); 127.4 (arom., s); 128.9 (arom., s); 130.8 (arom., s); 141.6 (arom., s); 143.3 (arom., s); 143.8 (arom., s); 144.6 (arom., s); 147.1 (arom., s); 156.1 (arom., s); 158.4 (arom., s); 159.6 (arom., s); 171.5 (FA, s); 178.0 (CO, s); 179.1 (CO, s). ESI-HRMS: 878.3396 [M] + (theor. [C 36 H 53 N9O4Si1Lu1] + = 878.3392). EA ([C 36 H 53 N9O4Si1Lu1] + [FA] - -5.9H2O, M R = 1030.2): C 43.1 (43.4); H 12.2 (11.8); N 12.2 (11.8); Si 2.7 (2.9); Lu 17.0 (16.9).

[0739] [Chem.]

[0740] Synthesis of TD946: In a glass vial (20 mL), TD943-1.5H2O (28.1 mg; 45.6 μmol; 1.0 equivalent) was dissolved in H2O (16 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 3.00 mL; 1.50 mmol; 33 equivalents) and aq. LuCl3 (100 mM; 540 μL; 54 μmol; 1.2 equivalents). The mixture was stirred at 80°C for 3 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then purified directly by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 32.6 mg (77%; 1 step; based on TD943-1.5H2O). NMR (D2O, pD~5): δ H 0.69-0.94(CH2-CH,m,2H);0.94-1.07(CH2-CH,m,2H);1.65(CH2-CH,tt,1H, 3 J HH =8, 3 J HH =5);2.58-3.86(mc,CH2-CO,m,16+4H);3.97(CH2-arom.,d,1H, 2 J HH =15);4.19(CH2-arom.,d,1H, 2 J HH =16);4.29(CH2-arom.,d,1H, 2 J HH =15);4.87(CH2-arom.,d,1H, 2 J HH =16);5.53(CH2-N3,d,1H, 2 J HH =15);6.99(CH2-N3,d,1H, 2 J HH =15);7.73(arom.,dd,1H, 3 J HH =8, 4 J HH= 1); 7.86 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1); 7.93 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1); 7.99 (arom., dd, 1H, 3 J HH = 8, 4 J HH = 1); 8.24 (arom., t, 1H, 3 J HH = 8); 8.29 (arom., t, 1H, 3 J HH = 8). ESI-HRMS: 762.2375 [M] + (theor. [C 30 H 37 N9O4Lu1] + = 762.2371). EA ([C 30 H 37 N9O4Lu1] + [TFA] - -0.2TFA - 1.8H2O, M R = 930.8): C 41.8 (42.2); H 4.4 (4.2); N 13.5 (13.1); F 7.3 (7.0).

[0741]

Chem.

[0742] Synthesis of TD961: In a glass vial (20 mL), TD959-1.5H2O (14.7 mg; 23.0 μmol; 1.0 equivalent) was dissolved in H2O (16 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 1.50 mL; 0.75 mmol; 33 equivalents) and aq. LuCl3 (100 mM; 280 μL; 28 μmol; 1.2 equivalents). The mixture was stirred at 80°C for 3 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 20.1 mg (90%; 1 step; based on TD959-1.8H2O). NMR (D2O, pD~4): δ H 1.18(CH3,s,9H);2.64-3.86(mc,CH2-CO,m,16+4H);4.01(CH2-arom.,d,1H, 2 J HH =15);4.16(CH2-arom.,d,1H, 2 J HH =15);4.35(CH2-arom.,d,1H, 2 J HH =17);4.87(CH2-arom.,d,1H, 2 J HH =17);5.48(CH2-N3,d,1H, 2 J HH =15);6.66(CH2-N3,d,1H, 2 J HH =15);7.70(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.84(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.87(arom.,dd,1H, 3 J HH =8, 4 J HH=1);7.93(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.22(arom.,t,1H, 3 J HH =8);8.26(arom.,t,1H, 3 J HH =8). ESI-HRMS:778.2685[M] + (theor.[C 31 H 41 N9O4Lu1] + =778.2684). EA([C 31 H 41 N9O4Lu1] + [TFA] - -0.2TFA-3.0H2O,M R =968.5):C41.4(41.5);H4.9(4.6);N13.0(12.7);F7.1(7.4).

[0743] [ka]

[0744] Synthesis of TD951: In a glass vial (20 mL), TD944-1.5H2O (14.4 mg; 22.1 μmol; 1.0 equivalent) was dissolved in H2O (18 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 1.20 mL; 600 μmol; 27 equivalents) and aq. LuCl3 (100 mM; 265 μL; 26.5 μmol; 1.2 equivalents). The mixture was stirred at 80°C for 4 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 17.4 mg (80%; 1 step; based on TD944-1.5H2O). NMR (D2O, pD~2): δ H2.69-3.88(mc,CH2-CO,m,16+4H);3.97(CH2-arom.,d,1H, 2 J HH =15);4.16-4.36(CH2-arom.,m,2H);4.88(CH2-arom.,d,1H, 2 J HH =17);5.51(CH2-N3,d,1H, 2 J HH =15);6.97(CH2-N3,d,1H, 2 J HH =15);7.16-7.25(Ph,m,2H);7.28-7.41(Ph,m,3H);7.49(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.73(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.83(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.03(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.06(arom.,t,1H, 3 J HH =8);8.25(arom.,t,1H, 3 J HH =8)。ESI-HRMS:798.2379[M] + (theor.[C 33 H 37 N9O4Lu1] + =798.2371)。EA([C 33 H 37 N9O4Lu1] + [TFA] - -0.2TFA-3.0H2O,M R =988.5):C43.0(43.0);H4.4(4.3);N12.8(12.6);F6.9(6.9)。

[0745] [ka]

[0746] Synthesis of TD994: In a glass vial (20 mL), TD992-0.3FA-1.9H2O (41.6 mg; 56.8 μmol; 1.0 equivalent) was dissolved in H2O (16 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 2.85 mL; 1.43 mmol; 25 equivalents) and aq. LuCl3 (100 mM; 625 μL; 62.5 μmol; 1.1 equivalents). The mixture was stirred at 80°C for 8 days. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, TFA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 52.3 mg (86%; 1 step; based on TD992-0.3FA-1.9H2O). NMR (D2O, pD~4): δ H 1.52-1.78(Adm.,m,9H);1.80-1.95(Adm.,m,6H);2.60-3.83(mc,CH2-CO,m,16+4H);3.99(CH2-arom.,d,1H, 2 J HH =15);4.11(CH2-arom.,d,1H, 2 J HH =15);4.33(CH2-arom.,d,1H, 2 J HH =17);4.84(CH2-arom.,d,1H, 2 J HH =17);5.43(CH2-N3,d,1H, 2 J HH =15);6.58(CH2-N3,d,1H, 2 J HH =15);7.66(arom.,d,1H, 3 J HH =8);7.80(arom.,d,1H, 3 J HH=8);7.88-7.92(arom.,m,2H);8.18(arom.,t,1H, 3 J HH =8);8.22(arom.,t,1H, 3 J HH =8). ESI-HRMS:856.3154[M] + (theor.[C 37 H 47 N9O4Lu1] + =856.3153). EA([C 37 H 47 N9O4Lu1] + [TFA] - -0.4TFA-3.0H2O,M R =1069.4):C44.7(44.3);H5.0(4.6);N11.8(11.5);F7.5(7.4);Lu16.4(14.0).

[0747] [ka]

[0748] Synthesis of TD1221: In a glass vial (20 mL), TD1204-0.3FA-2.7H2O (22.5 mg; 30.5 μmol; 1.0 equivalent) was dissolved in H2O (18 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 1.57 mL; 785 μmol; 26 equivalents) and aq. LuCl3 (100 mM; 375 μL; 37.5 μmol; 1.2 equivalents). The mixture was stirred at 80°C for 24 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, TFA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 24.5 mg (74%; 1 step; based on TD1204-0.3FA-2.7H2O). NMR (D2O, pD~2): 1 Hδ H2.59-3.83(mc,CH2-CO,m,16+4H);3.95(CH2-arom.,d,1H, 2 J HH =15);4.23(CH2-arom.,d,1H, 2 J HH =17);4.27(CH2-arom.,d,1H, 2 J HH =15);4.85(CH2-arom.,d,1H, 2 J HH =17);5.62(CH2-N3,d,1H, 2 J HH =15);7.01(CH2-N3,d,1H, 2 J HH =15);7.72(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.87(arom.,d,1H, 4 J HH =2);7.90(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.97(arom.,dd,1H, 3 J HH =8, 4 J HH =1);8.23(arom.,t,1H, 3 J HH =8);8.29(arom.,t,1H, 3 J HH =8)。ESI-HRMS:848.1020[M] + (theor.[C 27 H 32 N9O4I1Lu1] + =848.1024)。EA([C 27 H 32 N9O4I1Lu1] + [TFA] - -0.5TFA-4.1H2O,M R =1092.3):C33.0(33.8);H3.8(3.6);N11.5(11.8);I11.6(10.9);F7.8(7.0)。

[0749] [ka]

[0750] Synthesis of TD782: In a glass vial (40 mL), TD764-0.7TFA-1.7H2O (190 mg; 274 μmol; 1.0 equivalent) was dissolved in H2O (21 mL), followed by the addition of aq. MOPS / NaOH buffer (500 mM; pH 7.0; 15.4 mL; 7.70 mmol; 28 equivalents) and aq. LuCl3 (100 mM; 3.30 mL; 330 μmol; 1.2 equivalents). The mixture was stirred at 80°C for 16 hours. The mixture was then filtered through a syringe microfilter (RC). The filtrate was concentrated using a rotary evaporator and then directly purified by preparative HPLC (C18; H2O-MeCN gradient, with TFA added). The fractions containing the product were combined and directly lyophilized to obtain the product as a white, fluffy solid in the form of trifluoroacetate. Yield: 224 mg (85%; 1 step; based on TD764-0.7TFA-1.7H2O). NMR (D2O, pD~3): 1 Hδ H 2.61-3.84(mc,CH2-CO,m,16+4H);3.94(CH2-arom.,d,1H, 2 J HH =15);4.15(CH2-arom.,d,1H, 2 J HH =16);4.29(CH2-arom.,d,1H, 2 J HH =15);4.84(CH2-arom.,d,1H, 2 J HH =16);5.58(CH2-N3,d,1H, 2 J HH =15);7.10(CH2-N3,d,1H, 2 J HH =15);7.80(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.83(arom.,d,1H, 4 JHH =2);7.85(arom.,dd,1H, 3 J HH =8, 4 J HH =1);7.88(CH-N3,s,1H);8.13(arom.,d,1H, 4 J HH =2);8.24(arom.,t,1H, 3 J HH =8). ESI-HRMS:756.1664[M] + (theor.[C 27 H 32 N9O4Cl1Lu1] + =756.1668). EA([C 27 H 32 N9O4Cl1Lu1] + [TFA] - -5.1H2O,M R =961.9):C36.2(36.4);H4.4(4.0);N13.1(12.9);Cl3.7(3.7);F5.9(6.4);Lu18.2(16.8).

[0751] [ka]

[0752] Synthesis of TD819: In a glass vial (4 mL), [TD782] + [TFA] --5.1H2O (10.0 mg; 10.4 μmol; 1.0 equivalent), 3-Borono-5-nitrobenzoic acid (4.4 mg; 21 μmol; 2.0 equivalent), and XPhosPdG2 (0.5 mg; ~0.6 μmol; 6 mol%) were packed into the container and fixed with argon three times. Next, dry DMF (420 μL) was added through the septum under a constant flow of argon, followed by the addition of a freshly prepared (and briefly washed with argon before addition) solution of K3PO4-H2O (326 mM; 160 μL; 52 μmol; 5.0 equivalent). The mixture was then stirred under the septum (without external argon) at 80°C for 16 hours. The resulting heterogeneous mixture was diluted with H2O (2 mL), and the resulting slightly opaque pale y...

Claims

1. Compounds of general formula (I) 【Chemistry 1】 Here, Y is selected from the group consisting of nitrogen; N-oxide; R １ is H; halogen; -OH; -N ３ ; -(CH ２ ) ｎ N ３ (where n is an integer in the range of 1 to 3); -NR ２ (where R is H, or C １ to C ６ alkyl which can be branched or linear, and is independently selected); -(CH ２ ) ｎ NR ２ (where n and R are as defined above); C ６ to C １０ aryl (optionally, -NH ２ , -NO ２ , -N ３ , 【Chemistry 2】 , -COOH, -CH ２ Cl and / or -CH ２ (Can be substituted with COOH); C ７ ~C １０ Arylalkyl (optionally, -NH ２ , -NO ２ , -N ３ , 【Transformation 3】 , -COOH, -CH ２ Cl, and / or -CH ２ (May be substituted with COOH); -CF ３ ;-COOR(where R is as defined above);-(CH ２ ) ｎ COOR (where n and R are as defined above); 【Chemistry 4】 ;-CH ２ CH(OMe) ２ ; 【Transformation 5】 ;-SH;-SO ３ H; -SO ２ Ar (where Ar is phenyl); NO ２ Selected from the group consisting of; R ２ teeth, 【Transformation 6】 ； or 【Transformation 7】 (where n is an integer in the range of 1 to 3); and; A is H; -(CH ２ ) ｎ COOH (where n is an integer from 1 to 3); -CH (CH ３ )COOH;-CH((CH ２ ) ｎ CH ３ )COOH (where n is as defined above); -CH ２ P (= O) (OR) ２ (Here, R is as defined above); -CH((CH ２ ) ｎ COOH)COOH(where n is an integer from 1 to 3); -CH((CH ２ ) ｎ NH ２ )COOH (where n is an integer from 1 to 3); -CH ２ C(=O)(NH ２ ); -CH ２ C(=O)(NH)-CH ２ COOH; -CH ２ P(=O)(OH)(Ar) (where Ar is arbitrarily C) １ ~C ６ Independently selected from the group consisting of; (phenyl which can be substituted with alkyl); Z is, 【Transformation 8】 Selected from the group consisting of, Here, R ３ teeth, 【Chemistry 9】 And, R ４ H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; C １ ~C ６ Alkyl (can be branched or linear); C ３ ~C ６ Cycloalkyl; -CF ３ ;-(CH ２ ) ｎ NHR ６ (Here, n is an integer in the range of 1 to 3, and R ６ (is selected from H, fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl and benzyloxycarbonyl); C (CH ３ ) ２ (NHR ６ ) (Here, R ６ (as defined above); adamantyl; 【Chemistry 10】 ;-(CH ２ ) ｎ COOH (where n is as defined above); C ６ ~C １０ Aryl (optional, -NH) ２ , -NO ２ , -N ３ 【Chemistry 11】 , -COOH, -CH ２ Cl, and / or -CH ２ Selected from the group consisting of (which can be substituted with COOH); R ５ R １ ,or, 【Chemistry 12】 (Here, R ４ (is as defined above); and / or, R ２ and R ３ Both are, formula 【Chemistry 13】 , or formula 【Chemistry 14】 The 1,2,3-triazole group (where R ４ (as defined above); form; However, at most one A is H.

2. A compound of general formula (I) as described in claim 1: Y is nitrogen; R １ is H; Cl; -N(CH ３ ) ２ ; phenyl (optionally substituted with -COOH or -CH ２ COOH); benzyl (optionally substituted with -COOH or -CH ２ COOH); -CF ３ ; -COOCH ３ ; -COOCH(CH ３ ) ２ ; -COOtBu; 【Chemistry 15】 Selected from the group consisting of; A is H; -(CH ２ ), where n is 1 or 2; -CH ｎ COOH; -CH ２ P(=O)(OH) ２ ; -CH ２ P(=O)(OH)(OEt); -CH ２ P(=O)(OEt) ２ ; -CH ２ C(=O)(NH ２ ); -CH ２ C(=O)(NH)-CH ２ COOH; -CH ２ P(=O)(OH)(Ph); and is independently selected from the group consisting of; Z is, 【Chemistry 16】 Selected from the group consisting of, Here, R ３ teeth 【Chemistry 17】 And; R ４ H; halogen; piperidinyl; trimethylsilyl; triisopropylsilyl; phenyl; cyclopropyl; tert-butyl; -CF ３ ;-CH ２ NH ２ ;-CH ２ N(H)(Fmoc);-C(CH ３ ) ２ (NH ２ ); -C(CH ３ ) ２ Selected from the group consisting of (NHBoc); adamantyl; R ５ H, or, [Chemistry 18] (Here, R ４ (as defined above); However, it is a compound in which at most one A is H.

3. The compound according to claim 1, Z is, 【Chemistry 19】 And; R ３ teeth 【Chemistry 20】 And; R ４ and R ５ A compound as defined in claim 1.

4. The compound according to claim 1, R ２ and R ３ Both are such that the bridge between two opposing nitrogen atoms in the cyclone portion is an entity of general formula (IIa) or (IIb), 【Chemistry 21】 ,formula 【Chemistry 22】 , or formula 【Chemistry 23】 Forms a 1,2,3-triazole group; Here, L is 【Chemistry 24】 Selected from the group consisting of; R １ , R ４ and R ５ A compound as defined in claim 1.

5. Y is nitrogen, Z 【Chemistry 25】 And, A compound of general formula (I) according to claim 1, selected from the group consisting of compounds in which the remaining substituents are present in the following combinations: Table 1-1 Table 1-2 Table 1-3 Table 1-4 。

6. A method for preparing a compound of general formula (I) according to any one of claims 1 to 5, comprising the following steps: i) Provide an alkyne intermediate of general formula Z-Cl, Here, Z is as defined in claim 1; ii) Azide intermediate of general formula (III) 【Chemistry 26】 To provide Here, Y, R １ and R ２ This is as defined in claim 1; iii) Cyclene derivatives of general formula (IV) 【Chemistry 27】 To provide Here, pA is -(CH ２ ) ｎ COO ｔ Bu (where n is an integer between 1 and 3); benzyloxycarbonyl; -CH(CH ３ )COOR(where R is tert-butyl, methyl, or ethyl);-CH((CH ２ ) ｎ CH ３ )COOR(where n is an integer from 1 to 3 and R is tert-butyl, methyl, or ethyl);-CH((CH ２ ) ｎ COOR)COOR(where n is an integer from 1 to 3, and R is independently selected from tert-butyl, methyl, or ethyl);-CH ２ P (= O) (OR) ２ (Here, R can be branched or linear C) １ ~C ６ (It is alkyl); -CH ２ C(=O)(NH ２ ); -CH ２ C(=O)(NH)-CH ２ COOH; -CH ２ P(=O)(OH)(Ar) (where Ar is arbitrarily C) １ ~C ６ Selected from the group consisting of: (phenyl that can be substituted with alkyl); iv-A) The cyclone derivative of general formula (IV) is reacted with the alkyne intermediate Z-Cl to obtain the intermediate of general formula (V). 【Chemistry 28】 To obtain Here, Z is as defined in claim 1, and pA is as defined above; or iv-B) The cyclone derivative of general formula (IV) is reacted with the azide intermediate of general formula (III) to obtain the intermediate of general formula (VI). 【Chemistry 29】 To obtain Here, Y, R １ , R ２ And pA are as defined above; v-A) The intermediate of general formula (V) is reacted with the azide intermediate of general formula (III) to obtain the intermediate of general formula (VII). 【Transformation 30】 To obtain Here, Y, R １ , R ２ , pA and Z are as defined above; or v-B) The intermediate of general formula (VI) is reacted with the alkyne intermediate Z-Cl to obtain the intermediate of general formula (VII); vi) Optionally, hydrolysis of the protecting group is performed to obtain the compound of general formula (I).

7. A compound of general formula (I) as described in claim 1, a lanthanide (III) cation, and Na ＋ Ba ２＋ Pb ２＋ , Sr ２＋ Ca ２＋ , Cd ２＋ , Zn ２＋ Mn ２＋ , Pt ２＋ ,Cd ２＋ Ni ２＋ , Sc ３＋ , Y ３＋ , Bi ３＋ In ３＋ Ru ３＋ , Ir ３＋ Ga ３＋ , Tl ３＋ , Pd ２＋ A coordination compound with a metal cation selected from the group consisting of the following.

8. A method for preparing a coordination compound according to claim 7, characterized by comprising the following steps: i) Synthesize the compound of general formula (I) described in claim 1; ii) Inorganic acids, lanthanide (III) cations, Na ＋ Ba ２＋ Pb ２＋ , Sr ２＋ Ca ２＋ , Cd ２＋ , Zn ２＋ Mn ２＋ , Pt ２＋ ,Cd ２＋ Ni ２＋ , Sc ３＋ , Y ３＋ , Bi ３＋ In ３＋ Ru ３＋ , Ir ３＋ Ga ３＋ , Tl ３＋ , Pd ２＋ To provide a salt with a metal cation selected from the group consisting of; iii) The compound of general formula (I) derived from step i) and the metal salt derived from step ii) are mixed in an aqueous solution, resulting in the chelation of the metal cation by the compound of general formula (I), and the coordination compound described in claim 7 is formed; iv) Optionally, convert the coordination compound derived from step iii), the conversion being selected from at least one of the following reactions: -R １ and / or R ４ and / or R ５ A Pd(0)-catalyzed Suzuki cross-coupling reaction between a halogen group present and phenylboronic acid, thereby introducing -COOH into -R1 and / or R4 and / or R5; -NaN ３ R １ and / or R ４ and / or R ５ The halogen group present and -N ３ Substitution reaction with a group; -R １ and / or R ４ and / or R ５ Hydrolysis of the TIPS protecting group present in the substituent, where R １ and / or R ３ and / or R ５ teeth, 【Chemistry 31】 And as a result, R １ and / or R ３ and / or R ５ teeth, 【Chemistry 32】 This will result in; - Methanol and R １ and / or R ４ and / or R ５ (Here, R １ and / or R ３ and / or R ５ teeth, 【Transformation 33】 The addition reaction with (which converts the triple bond into a dimethyl acetal; - D in which DBU exists ２ CH of group A in O ２ Deuteration of the base, CH ２ The base is CD ２ Convert to base; - Selective reduction of the pyridyl ring of the Z substituent, where Z is 【Transformation 34】 And R ３ and R ５ This is as defined in claim 1; -Z substituent (where Z is 【Chemistry 35】 And R ３ and R ５ The pyridyl ring (as defined in claim 1), with NaBH as the reducing agent. ４ or NaBD ４ Selective reduction using the following method, thereby removing the Z groups, 【Transformation 36】 or 【Chemistry 37】 Convert to; -R １ and / or R ４ and / or R ５ and / or NH of A ２ Base (R here) １ and / or R ４ and / or R ５ and / or A is -NH ２ or - (CH ２ ) ｎ NH ２ (including) and the reaction with FmocCl, resulting in -NH ２ Convert the group to the -NHFmoc group; -R １ and / or R ４ and / or R ５ and / or the reaction of the -COOH group present in A with the amino group of an amino acid or peptide, thereby forming a peptide bond; -R １ , R ４ , R ５ S-alkylation reaction of substituted halogens with thiols, resulting in the conversion of halogens to sulfides; -R １ , R ３ , and / or R ５ of 【Transformation 38】 Base (R here) １ , R ３ , and / or R ５ teeth, 【Chemistry 39】 The reaction of (including) with azide, thereby the alkyl or aryl and R １ , R ３ , and / or R ５ Forms triazole crosslinks that bridge the groups; -R １ and / or R ５ -N ３ Base (here, R １ and / or R ５ is, -N ３ A reaction between a group (including a carbon-carbon triple bond) and a substituent containing a carbon-carbon triple bond, thereby forming a triazole crosslink that bridges the substituent and the coordination compound; -R １ and / or R ５ The reaction of the -SH group with a substituent containing maleimide, thereby forming a thiosuccinimide bond that bridges the substituent and the coordination compound; -R １ and / or R ５ The reaction of the -SH group with another substituent containing a -SH group, thereby forming a disulfide bridge that bridges the substituent with the coordination compound; -R １ and / or R ５ The reaction of the -SH group with an alkylhalogenide or arylhalogenide, thereby forming a thioether bond between the alkyl or aryl and the coordination compound; -R １ and / or R ５ NO - ２ The reaction between the group and the substituent containing the -SH group forms a thioether bond that connects the substituent to the coordination compound.

9. A coordination compound chain comprising at least two coordination compounds according to claim 7, R of the first coordination compound １ or R ５ The group and the R of the following coordination compound １ or R ５ Binding via triazole crosslinks formed between the group and / or, A coordination compound substituent R containing a certain amine group １ or R ４ or R ５ Alternatively, A and substituent R of another coordination compound containing a carboxyl group. １ or R ４ or R ５ or bonded via an amide bond formed between A and, and / or, A coordination compound substituent R containing a certain -SH group １ or R ５ And substituent R of another coordination compound containing an -SH group １ or R ５ A coordination compound chain, linked via a disulfide bond formed between and .

10. Coordination compound dimer of general formula (X) 【Chemistry 40】 Here, M １ and M ２ This is a metal cation as defined in claim 7, R １ , R ４ and R ５ This is as defined in claim 1.

11. A conjugate for drug tracking comprising the coordination compound described in claim 7 and conjugated to a peptide or protein; Here, the coordination compound described in claim 7 is -NH ２ or R containing a -COOH group １ and / or R ４ and / or R ５ Includes the base; R １ and / or R ４ and / or R ５ The peptide or protein is bound to the -COOH group present in the peptide or protein via an amide bond formed between the -COOH group present in the peptide and the amino group of the peptide or protein, or R １ and / or R ４ and / or R ５ -NH ２ The peptide or protein is bound via an amide bond formed between the group and the carboxyl group of the peptide or protein; further, The peptide is selected from the group comprising oligopeptides of 3 to 20 amino acids; further, The aforementioned protein is a conjugate selected from the group including antibodies.

12. A conjugate for drug tracking comprising the coordination compound chain described in claim 9 and conjugated to a peptide or protein; Here, the coordination compound chain described in claim 9 is -NH ２ or R containing a -COOH group １ and / or R ４ and / or R ５ Includes the base; R １ and / or R ４ and / or R ５ The peptide or protein is bound to the -COOH group present in the peptide or protein via an amide bond formed between the -COOH group present in the peptide and the amino group of the peptide or protein, or R １ and / or R ４ and / or R ５ -NH ２ The peptide or protein is bound via an amide bond formed between the group and the carboxyl group of the peptide or protein; further, The peptide is selected from the group comprising oligopeptides of 3 to 20 amino acids; further, The aforementioned protein is a conjugate selected from the group including antibodies.

13. A method for tracking drugs, characterized by comprising the following steps: i) supplying a cell culture or tissue to be analyzed, comprising at least one conjugate according to claim 11, wherein the peptide or protein is a peptide-based or protein-based drug to be tracked; ii) Hydrolyze the cell culture or tissue from step i) using a strong acid to obtain a hydrolysate; iii) The presence of the coordination compound of claim 7 is qualitatively and / or quantitatively analyzed by performing a hydrolysis of the hydrolysate from step ii).

14. A method for tracking drugs, characterized by comprising the following steps: i) supplying a cell culture or tissue to be analyzed, comprising at least one conjugate according to claim 12, wherein the peptide or protein is a peptide-based or protein-based drug to be tracked; ii) Hydrolyze the cell culture or tissue from step i) using a strong acid to obtain a hydrolysate; iii) The presence of the coordination compound chain of claim 9 is qualitatively and / or quantitatively analyzed by the hydrolysate of step ii).

15. In vitro use of the coordination compound according to claim 7, or the coordination compound chain according to claim 9, or the conjugate according to claim 11 or 12, in pharmaceuticals, for the development and testing of new drugs.

16. In vitro use of the coordination compound dimer according to claim 10 for the development and testing of new drugs in pharmaceuticals.

17. A coordination compound according to claim 7, or a coordination compound chain according to claim 9, for use in medical diagnosis.

18. A coordination compound dimer according to claim 10 for use in medical diagnosis.

19. A coordination compound according to claim 7, or a coordination compound chain according to claim 9, Here, the metal is ４４ Sc, ４７ Sc, ６４ Cd, ６７ Cd, ８６ Y, ９０ Y, １４０ Nd, １４９ Pm, １５１ Pm, １５３ Sm, １５９ Gd, １４９ Tb, １６１ Tb, １６５ Dy, １６１ Ho, １６６ Ho, １６９ Er, １６７ Tm, １７５ Yb, １７７ Selected from the group including Lu, and / or R １ , R ４ , and / or R ５ It contains radioactive halogens, A coordination compound according to claim 7, or a coordination compound chain according to claim 9, for use in medicine as a radiodiagnostic agent and / or radiopharmaceutical.

20. A coordination compound dimer according to claim 10, Here, M １ and M ２ teeth, ４４ Sc, ４７ Sc, ６４ Cd, ６７ Cd, ８６ Y, ９０ Y, １４０ Nd, １４９ Pm, １５１ Pm, １５３ Sm, １５９ Gd, １４９ Tb, １６１ Tb, １６５ Dy, １６１ Ho, １６６ Ho, １６９ Er, １６７ Tm, １７５ Yb, １７７ Selected from the group including Lu, and / or R １ , R ４ , and / or R ５ It contains radioactive halogens, The coordination compound dimer according to claim 10, for use in medicine as a radiodiagnostic agent and / or radiopharmaceutical.

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