Multispecific bioconjugate linker and synthesis method thereof

Abstract

The present application relates to a multi-specific bioconjugate linker and a method for synthesizing the same, and specifically to a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof. The present application also relates to a method for preparing the compound and its use in tumor treatment.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to a multi-specific biological coupling linker and a method for synthesizing the same. Background Art

[0002] Multispecific drug molecules are a hot topic in cancer treatment today. Compared with the traditional drug design that relies on one drug for one target, multispecific drugs can target multiple targets at the same time, so they can attack cancer cells more accurately and efficiently, and reduce the toxic side effects caused by off-target effects.

[0003] Although bispecific antibodies and multispecific antibodies have shown good effects in tumor treatment, there are still many challenges in the development of multispecific antibody drugs. One of the most important problems is the preparation of multispecific antibodies. Traditional multispecific antibody preparation platforms mostly use antibody fusion expression for production. This method has the problem of antibody mismatch, which makes the downstream purification process extremely difficult, and therefore difficult to expand production. There are some multifunctional compounds, but these compounds do not have specificity and bioorthogonality, are prone to mutual interference, and cannot truly achieve precise coupling of multiple compounds. Summary of the invention

[0004] The present application provides a compound, which may be a multispecific bioconjugate linker or a multispecific conjugate. The present application may have one or more of the following effects: (1) The compound of the present application may be a multispecific compound, and the multispecific conjugate may exhibit good anti-tumor activity, IC 50 The value can be lower than 10 nanomoles; (2) In the preparation method of the compound of the present application, the reaction can achieve fixed-point, quantitative, and modular connection, and ultimately a multispecific conjugate with clear structure, uniformity, and controllable quality can be obtained.

[0005] The present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which has a structure as shown in Formula Ia or Formula Ib:

[0006]

[0007] Said Wa is a trivalent group, and Wb is a tetravalent group;

[0008] The A, B and C are each independently selected from the following groups:

[0009]

[0010] Where R 12 , R 13 and R 14are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0011] Among them, A, B and C, Cannot exist at the same time;

[0012] Among them, A, B and C, Cannot exist at the same time;

[0013] Among them, A, B and C, Cannot exist at the same time;

[0014] When A, B and C, and When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0015] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0016] A, B and C are not the same

[0017] In one embodiment, the compound wherein:

[0018] (1) A is B is C is

[0019] (2) A is B is C is

[0020] (3) A is B is C is

[0021] (4) A is B is C is

[0022] (5) A is B is C is

[0023] (6) A is B is C is or

[0024] (7) A is B is C is or

[0025] (8) A is B is C is

[0026] (9) A is B is C is

[0027] (10) A is B is C is

[0028] (11) A is B is C is or

[0029] (12) A is B is C is

[0030] (13) A is B is C is

[0031] (14) A is B is C is

[0032] (15) A is B is C is

[0033] Where R 12 , R 13 and R 14are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0034] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0035] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0036] In one embodiment, the compound, wherein:

[0037] (1) A is B is C is

[0038] (2) A is B is C is

[0039] (3) A is B is C is

[0040] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0041] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0042] In one embodiment, the compound, wherein:

[0043] (1) A is B is C is

[0044] (2) A is B is C is

[0045] (3) A is B is C is

[0046] Where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0047] When A, B and C, When both exist, the R 12 Not amino.

[0048] In one embodiment, the compound, wherein:

[0049] (1) A is B is C is

[0050] (2) A is B is C is

[0051] (3) A is B is C is

[0052] Where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site.

[0053] In one embodiment, the compound wherein: A is B is C is

[0054] Where R 12 a group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, Represents the junction site.

[0055] In one embodiment, the compound, wherein:

[0056] for

[0057] for

[0058] The W is a trivalent group or a tetravalent group, and the W is optionally substituted by one or more R,

[0059] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0060] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0061] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0062] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0063] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1-, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0064] R, R 1 , R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0065] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0.

[0066] In one embodiment, the compound, wherein J 1 , J 2 , and J 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0067] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0068] In one embodiment, the compound wherein X 1 , X 2 , and X 3 Each is independently selected from the group consisting of -NH- and -O-.

[0069] In one embodiment, the compound, wherein K 1 , K 2 , and K 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0070] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0071] In one embodiment, the compound, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0072] In one embodiment, the compound, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0.

[0073] In one embodiment, the compound, wherein Y 1 , Y 2 , and Y 3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-;

[0074] R 1 Selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanol;

[0075] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0076] In one embodiment, the compound, wherein (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0.

[0077] In one embodiment, the compound, wherein L 1 , L 2 , and L 3 Each is independently selected from the following group: chain alkyl, and aryl, or any combination of the foregoing; wherein, q1, q2, and q3 are each independently selected from the following group: 0, 1, 2 and 3.

[0078] In one embodiment, the compound, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0079] In one embodiment, the compound, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0080] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0081] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0082] In one embodiment, the compound, wherein:

[0083] W is selected from the following group:

[0084] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0085] In one embodiment, the compound, wherein:

[0086] W is selected from the following group:

[0087]

[0088] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S and P;

[0089] The --- represents a double bond or a single bond.

[0090] In one embodiment, the compound, wherein:

[0091] W is selected from the following group:

[0092]

[0093] In one embodiment, the compound is selected from the following group:

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] In one embodiment, the compound is selected from the following group:

[0100]

[0101] A compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula IIa or Formula IIb, wherein:

[0102]

[0103] The Wc is a trivalent group, and Wd is a tetravalent group;

[0104] The A 2 , B 2 and C 2 Each independently selected from the following group:

[0105] and covalent bonds;

[0106] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0107] represents the junction site,

[0108] The A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect;

[0109] Where P1 , P 2 and P 3 Each is independently selected from the following group: lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0110] In one embodiment, the compound, wherein:

[0111] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0112] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0113] (3)A 2 for B 2 for C 2 for or covalent bond; or

[0114] (4)A 2 for B 2 for C 2 for or covalent bond; or

[0115] (5)A 2 for B 2 for or C 2 for or covalent bond; or

[0116] (6)A 2 for B 2 for C 2 for or covalent bond; or

[0117] (7)A 2 for B 2 for C 2 for or covalent bond; or

[0118] (8)A 2 for B 2 for C 2 for or a covalent bond; or

[0119] (9)A 2 for B 2 for C 2 for or covalent bond; or

[0120] (10)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or

[0121] (11)A 2 for B 2 for C 2 for or a covalent bond; or

[0122] (12)A 2 for B 2 for C 2 for or a covalent bond; or

[0123] (13)A 2 for B 2 for C 2 for or covalent bonds;

[0124] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0125] represents the junction site,

[0126] The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0127] In one embodiment, the compound, wherein:

[0128] (1)A 2 for B 2 for C 2 for or a covalent bond; or

[0129] (2)A 2 for B 2 for C 2 for or a covalent bond; or

[0130] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0131] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0132] represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0133] In one embodiment, the compound, wherein:

[0134] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0135] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0136] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0137] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0138] represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0139] In one embodiment, the compound, wherein:

[0140] (1)A2 for B 2 for C 2 for or covalent bond; or

[0141] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0142] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0143] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl,

[0144] represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0145] In one embodiment, the compound wherein: A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2The two binding sites of Wc and P are randomly connected 3 connect.

[0146] In one embodiment, the compound wherein: A 2 for B 2 for, C 2 for or covalent bond; where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0147] In one embodiment, the compound, wherein:

[0148] for

[0149] for

[0150] The W is a trivalent group or a tetravalent group, and the W is optionally substituted by one or more R,

[0151] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0152] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0153] The Lc is -(J 3 ) m3 -C(=O)-X 3-(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0154] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0155] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0156] R, R 1 , R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0157] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0.

[0158] In one embodiment, the compound, wherein J 1 , J 2 , and J 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0159] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0160] In one embodiment, the compound wherein X 1 , X 2 , and X 3 Each is independently selected from the group consisting of -NH- and -O-.

[0161] In one embodiment, the compound, wherein K 1 , K 2 , and K 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination of the foregoing; wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2 and 3.

[0162] In one embodiment, the compound, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0163] In one embodiment, the compound, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0.

[0164] In one embodiment, the compound, wherein Y 1 , Y 2 , and Y 3Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 Selected from the following group: hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2 and 3.

[0165] In one embodiment, the compound, wherein (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0.

[0166] In one embodiment, the compound, wherein L 1 , L 2 , and L 3 Each is independently selected from the following group: chain alkyl, and aryl, or any combination of the foregoing; wherein, q1, q2, and q3 are each independently selected from the following group: 0, 1, 2 and 3.

[0167] In one embodiment, the compound, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0168] In one embodiment, the compound, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0169] In one embodiment, the compound, wherein:

[0170] W is selected from the following group:

[0171] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0172] In one embodiment, the compound, wherein:

[0173] W is selected from the following group:

[0174]

[0175] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S and P;

[0176] The --- represents a double bond or a single bond.

[0177] In one embodiment, the compound, wherein:

[0178] W is selected from the following group:

[0179]

[0180]

[0181] In one embodiment, the compound wherein P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

[0182] In one embodiment, the compound, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

[0183] In one embodiment, the compound, wherein the antibody targets a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0184] In one embodiment, the compound, wherein the amino acid sequence of the antibody is selected from the following group: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0185] In one embodiment, the compound is selected from the following group:

[0186]

[0187] Among them, P 1 , P 2 and P 3 Each is independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0188] In one embodiment, the compound is:

[0189]

[0190] Among them, P 1 , P 2 and P 3 The amino acid sequences can be:

[0191] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0192] SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or

[0193] SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or

[0194] SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 3, or

[0195] SEQ ID NO: 10, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0196] SEQ ID NO: 11, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0197] SEQ ID NO: 12, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0198] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10, or

[0199] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, or

[0200] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, or

[0201] SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0202] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula Ia or Formula Ib:

[0203]

[0204] in:

[0205] for

[0206] for

[0207] in:

[0208] (1) A is B is C is

[0209] (2) A is B is C is

[0210] (3) A is B is C is

[0211] Where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the junction site;

[0212] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L1 ) q1 -;

[0213] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0214] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0215] Among them, J 1 , J 2 , and J 3 Each is independently selected from the following group: alkyl, and polyethylene glycol, or any combination thereof; m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0216] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0217] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkanol-polyethylene glycol-alkanol-, alkanol and polyethylene glycol, or n1, n2 or n3 is each independently 0;

[0218] Y 1 , Y 2 , and Y 3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; n1, n2, and n3 are each independently selected from the group consisting of 0, 1, 2, and 3;

[0219] (L1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 are each independently 0;

[0220] W is selected from the following group:

[0221]

[0222] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0223] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0224] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula Ia or Formula Ib:

[0225]

[0226] in:

[0227] for

[0228] for

[0229] Where: A is B is C is Where R 12 a group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, represents the junction site;

[0230] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0231] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0232] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0233] J 1 , J 2 , and J 3 Each is independently selected from the following group: alkyl, and polyethylene glycol, or any combination of the foregoing, m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0234] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0235] (K 1 )n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0;

[0236] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0;

[0237] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0;

[0238] W is

[0239]

[0240] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0241] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0242] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula IIa or Formula IIb:

[0243]

[0244] in,

[0245] for

[0246] for

[0247] in,

[0248] A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect;

[0249] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0250] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0251] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0252] Among them, J 1 , J 2 , and J 3 Each is independently selected from the following group: alkyl, and polyethylene glycol, or any combination thereof; m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0253] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0254] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkanol-polyethylene glycol-alkanol-, alkanol and polyethylene glycol, or n1, n2 or n3 is each independently 0;

[0255] Y 1 , Y 2 , and Y 3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; n1, n2, and n3 are each independently selected from the group consisting of 0, 1, 2, and 3;

[0256] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -alkanyl-aryl-, alkanyl and polyethylene glycol, or q1, q2 and q3 are each independently 0;

[0257] W is selected from the following group:

[0258]

[0259]

[0260] P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

[0261] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0262] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0263] In one embodiment, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which may have a structure as shown in Formula IIa or Formula IIb:

[0264]

[0265] in,

[0266] for

[0267] for

[0268] in,

[0269] A 2 for B 2 for, C 2 for or covalent bond; where R12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect;

[0270] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0271] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0272] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0273] J 1 , J 2 , and J 3 Each is independently selected from the following group: alkyl, and polyethylene glycol, or any combination of the foregoing, m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3;

[0274] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-;

[0275] (K 1 ) n1 , (K2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0;

[0276] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0;

[0277] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0;

[0278] W is

[0279]

[0280] P 1 , P 2 and P 3 Each is independently selected from the following group: an antibody targeting 4-1BB, an antibody targeting EGFR, an antibody targeting CD3, an antibody targeting Her2, an antibody targeting CD47, an antibody targeting CD20, a branched peptide containing CMV, neo2 protein, IFNα protein, fluorescein isothiocyanate (FITC), and CPG nucleic acid.

[0281] For example, La, Lb and Lc can be each independently selected from the following group: -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)8-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)8-NH-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-NH-C(=O)-CH2-, - C(=O)-NH-(CH2)2-NH-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0282] For example, La, Lb and Lc can each be independently selected from the following groups: -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-phenyl-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH-C(=O)-CH2-, -C(=O)-NH-(CH2)2-(CH2-O-CH2)3-(CH2)2-NH- and -C(=O)-O-.

[0283] In one aspect, the present application provides a method for preparing the compound described in the present application, comprising:

[0284] reacting the compound represented by formula (Ia-I) with reactant 1, reactant 2 and reactant 3;

[0285] or reacting the compound represented by formula (Ia-I) with reactant 1 and reactant 2;

[0286] or reacting the compound represented by formula (Ia-I) with reactant 1;

[0287]

[0288] The reactant 1 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A,

[0289] The reactant 2 is HX1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B,

[0290] The reactant 3 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C,

[0291] Said W is a trivalent group, said W is optionally substituted by one or more R,

[0292] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0293] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0294] R, R 1, R 1a and R 1b each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0295] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0;

[0296] A, B and C are each independently selected from the following group:

[0297]

[0298] Where R 12 , R 13 and R 14 each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0299] Among them, A, B and C, Cannot exist at the same time;

[0300] Among them, A, B and C, Cannot exist at the same time;

[0301] Among them, A, B and C, Cannot exist at the same time;

[0302] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0303] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0304] A, B and C are not the same

[0305] Among them, when A, B or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid is added to remove Boc.

[0306] In one embodiment of the preparation method, the order of adding reactant 1, reactant 2 and reactant 3 is determined according to the order of introducing A, B and C. The order of introducing A, B and C is as follows from first to last:

[0307] Where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0308] In one aspect, the present application provides a method for preparing the compound described in the present application, comprising:

[0309] Reacting the compound of formula Ia described in any one of the present application with reactant 4, reactant 5 and reactant 6;

[0310] The reactant 4 is A 1 -P 1 ,

[0311] The reactant 5 is B 1 -P 2 ,

[0312] The reactant 6 is C 1 -P 3 ,

[0313] Among them, A of reactant 4 1 React with A of the compound described in Formula Ia, B of the reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia;

[0314] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0315] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0316] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0317] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0318] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0319] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0320] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0321] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0322] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0323] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0324] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0325] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0326] Where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0327] In one embodiment, the preparation method described,

[0328] in, Cu was added to the reaction + As a catalyst.

[0329] In one embodiment, the preparation method described,

[0330] in, 1-100% equivalent of SrtA ligase was added to the reaction as a catalyst.

[0331] A pharmaceutical composition comprising the compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0332] Use of the compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition described in the present application in the preparation of a medicament for treating and / or preventing tumors.

[0333] In one embodiment of the use, the tumor is selected from tumors associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0334] In one embodiment of the use, the tumor is selected from the group consisting of solid tumors and hematological cancers.

[0335] In one embodiment of the use, the tumor is selected from the group consisting of lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0336] A method for treating and / or preventing tumors, comprising administering to a subject in need thereof the compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same.

[0337] According to the method for treating and / or preventing tumors described in the present application, the tumor may be a tumor associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0338] According to the method for treating and / or preventing tumors described in the present application, the tumor is selected from the following group: solid tumors and blood cancers.

[0339] According to the method for treating and / or preventing tumors described in the present application, the tumor can be selected from the following group: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0340] A compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same, for use in treating and / or preventing tumors.

[0341] A compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same, for use in treating and / or preventing a tumor selected from the following group: a solid tumor and a blood cancer.

[0342] A compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same, for treating and / or preventing a tumor, wherein the tumor may be selected from a tumor associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

[0343] A compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same, for use in treating and / or preventing tumors, wherein the tumor may be selected from the following group: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0344] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0345] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

[0346] Figure 1 Shown is the H NMR spectrum of the Scaffold 1 of a multi-specific bioconjugate described in the present application.

[0347] Figure 2 Shown is a mass spectrum of a multi-specific bioconjugate Scaffold 1 described in the present application

[0348] Figure 3 The NMR spectrum of Scaffold 2 of a multi-specific bioconjugate described in this application is shown.

[0349] Figure 4 Shown is a mass spectrum of a multi-specific bioconjugate Scaffold 2 described in the present application

[0350] Figure 5 Shown is the N3-NHS-NHS H NMR spectrum of the intermediate N3-NHS-NHS of the synthetic multi-specific bioconjugate scaffold 1 (Scaffold 1) or scaffold 2 (Scaffold 2) described in the present application.

[0351] Figure 6 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-FITC described in the present application.

[0352] Figure 7 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-FITC described in the present application.

[0353] Figure 8 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-CPG described in the present application.

[0354] Fig. 9 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-CPG described in the present application.

[0355] Fig.10 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-CMV described in the present application.

[0356] Fig.11 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-CMV described in the present application.

[0357] Fig.12 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-neo2 described in the present application.

[0358] Fig.13 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-neo2 described in the present application.

[0359] Fig.14 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-IFNα described in the present application.

[0360] Fig.15 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-IFNα described in the present application.

[0361] Fig.16 Shown is the SDS-PAGE image of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0362] Fig.17 Shown is the mass spectrum of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0363] Fig.18 Shown are the tumor cell growth inhibition results of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0364] Fig.19 Shown is the reaction route of a multispecific conjugate EGFR-CD3-CMV described in the present application.

[0365] Fig. 20 Shown is the reaction route of a multispecific conjugate EGFR-CD3-4-1BB described in the present application.

[0366] Fig.21 Shown are the reaction routes of the control group EGFR-TZ-4-1BB and EGFR-CD3-Gly described in the present application. DETAILED DESCRIPTION

[0367] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0368] Definition of terms

[0369] In this application, the terms "protein" or "polypeptide" are used interchangeably and generally refer to a chain of at least two (2) or more amino acids linked together by peptide bonds or amide bonds, and independent of post-translational modifications (e.g., glycosylation, acylation, phosphorylation, etc.). Antibodies are specifically intended to be included within the scope of this definition. The polypeptides of the present invention may comprise more than one subunit, wherein each subunit is encoded by a separate DNA sequence. Proteins may also comprise more than one polypeptide unit, including dimers, trimers, tetramers or a variety of higher order structures.

[0370] In this application, the term "equivalent" or "equivalent percentage" can be used interchangeably and generally refers to the number of moles of "X" relative to the number of moles of "Y." For example, 5 molar equivalents of X relative to Y means that if 1 mole of Y is used, 5 moles of X can be used.

[0371] In this application, the term "corresponding" of the selection of the X group to the Y group generally means that when the X group selects a certain group, the Y group selects another specific group accordingly. For example, the selection of A1 of the reactant 4 corresponding to A of the compound described in Formula Ia can be when A is When, accordingly, A1 can choose Corresponding to it.

[0372] In this application, the term "sequence of introduction" generally refers to the order in which a plurality of different specific groups are introduced into a starting compound. The order of introducing the starting compounds may refer to first adding a The reactant 1 is then added with Reactant 2, finally add The reactant 3 is first introduced Reintroduction Last introduction For example, introducing Before that, you can Replace with Acid was then added to remove Boc.

[0373] In the present application, the term "group X can be connected to group Y and group Z in either orientation" generally means that when group X is used to connect group Y and group Z, the two or more connection sites of group X can be connected to group Y or group Z at will. For example, group The connection to the group W and the group P can be in any orientation and can be a group The C atom connects the group W, the N atom connects the group P, or the group The N atom connects the group W, and the C atom connects the group P.

[0374] In this application, the term "enantiomers" generally refers to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0375] In the present application, the term "polysaccharide" generally refers to a molecule composed of a chain of monosaccharide units connected by glycosidic bonds. For example, the term "polysaccharide" may include molecules composed of two or more monosaccharide units, for example, molecules in which the longest monosaccharide chain is 3 to 9 monosaccharide units. The term "polysaccharide" may include linear and branched molecules, isolated molecules and molecules associated with polypeptides, sialylated and non-sialylated molecules.

[0376] In this application, the term "diastereomer" generally refers to stereoisomers with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers may have different physical properties, for example, melting points, boiling points, spectral properties and reactivity.

[0377] In this application, the terms "tautomer" or "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerizations and imine-enamine isomerizations. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0378] In this application, the term "mesomeric compound" generally refers to a molecule containing an asymmetric atom, but having symmetric factors such that the total optical rotation in the molecule is zero. The term "racemic compound" or "racemic mixture" refers to a composition consisting of equimolar amounts of two enantiomeric substances.

[0379] In this application, the term "lipid" generally refers to a straight-chain, branched, saturated or unsaturated aliphatic carboxylic acid, a phospholipid or a sterol. Examples of aliphatic carboxylic acids are lauric acid, palmitic acid, stearic acid, oleic acid and (CH3(CH2) n )2CHCOOH, wherein n is a number of at least 1. The phospholipid may be phosphatidylethanolamine, such as dioleoylphosphatidylethanolamine.

[0380] In this application, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" generally refer to polymers of nucleotides (e.g., ribonucleotides or deoxyribonucleotides), and include naturally occurring (adenine, guanine, cytosine, uracil and thymine), non-naturally occurring and modified nucleic acids. The term is not limited by the length of the polymer (e.g., the number of monomers). Nucleic acids can be single-stranded or double-stranded, and generally contain a 5'-3' phosphodiester bond, although in some cases, nucleotide analogs can have other connections. Monomers are generally referred to as nucleotides. The term "non-natural nucleotide" or "modified nucleotide" refers to a nucleotide containing a modified nitrogenous base, sugar or phosphate group, or a nucleotide incorporating a non-natural part into its structure. Examples of non-natural nucleotides include dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated and fluorescently labeled nucleotides.

[0381] In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1000, a compound with a molecular weight of more than 1000, or a compound with a molecular weight of more than 10000 or more than 100000. In the present application, a compound may also refer to a compound connected by chemical bonds, for example, a compound in which one or more molecules with a molecular weight of less than 1000 are connected to a biomacromolecule by chemical bonds, and the biomacromolecule may be a high polysaccharide, protein, nucleic acid, polypeptide, etc. For example, the compound of the present application may include a compound in which a protein is connected to one or more molecules with a molecular weight of less than 1000, a compound in which a protein is connected to one or more molecules with a molecular weight of less than 10000, or a compound in which a protein is connected to one or more molecules with a molecular weight of less than 100000.

[0382] In this application, the term "mixture" generally refers to a blend of two or more individual compounds.

[0383] In the present application, the term "antibody" or "antibody or its antigen-binding fragment" generally refers to immunological binding agents extending to all antibodies from all species, including dimers, trimers and multimer antibodies, bispecific antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, recombinant and remodeled antibodies, nanobodies and their fragments. The term "antibody or its fragment" may refer to any antibody-like molecule with an antigen-binding region, and the term includes antigen-binding active substance fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), linear antibodies, double antibodies, nanobodies, etc. The term "antigen-binding fragment" may refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. For example, a fragment of a full-length antibody may be used to perform the antigen-binding function of an antibody. The preparation and use of various antibody-based constructs and fragments are well known in the art. In one embodiment, it may refer to one or more of an anti-EGFR antibody, an anti-CD3 antibody, and an anti-41-BB antibody.

[0384] In the present application, the term "Nanobody" generally refers to those antibodies as defined in WO 2008 / 020079 or WO 2009 / 138519, and thus in certain aspects generally refers to a VHH, a humanized VHH or a camelized VH (e.g. a camelized human VH) or generally a sequence-optimized VHH (e.g. optimized for chemical stability and / or solubility, maximum overlap with known human framework regions and maximum expression).

[0385] In the present application, the term "any combination" generally means that the group can be a group selected from any number of groups in the group in any order. 1 is selected from the following group: chain alkyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof, and in one embodiment may refer to L 1 It can be an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, or a polyethylene glycol group. In one embodiment, it can refer to L 1 It may be a combination of an alkyl group and an aryl group or a combination of an alkyl group and polyethylene glycol. In one embodiment, it may refer to -alkyl group-aryl group or -alkyl group-polyethylene glycol-alkyl group.

[0386] In this application, the term "trivalent" group or "trivalent group" generally refers to a group having three bonding positions in the group. For example, trivalent groups include, but are not limited to, trivalent alkanes, trivalent cycloalkyls, trivalent heterocycloalkyls, trivalent alkenyls, trivalent alkynyls, trivalent aryls, and trivalent heteroaryls. For example, in one embodiment, it may include, but is not limited to

[0387] In the present application, the term "quaternary group" generally refers to a group having 4 bonding positions in the group.

[0388] In this application, the term "polyethylene glycol" or "PEG" generally refers to a polyethylene glycol of the general formula -(OCH2CH2) n -represents a polymer group derived from ethylene oxide in branched or linear form, wherein n is an integer of 3, 4, 5, 6, 7, 8, 9 or greater. The polyethylene glycol chain includes polymers of ethylene glycol having an average total molecular weight selected from the range of about 500 to about 40,000 daltons. The average molecular weight of the PEG chain is indicated by a number, for example, PEG-5,000 refers to a polyethylene glycol chain having an average total molecular weight of about 5,000. The polyethylene glycol can be substituted or unsubstituted.

[0389] In this application, the term "halogen" is generally meant to include fluorine, chlorine, bromine and iodine.

[0390] In this application, the term "urea" generally refers to -(HN-CO-)2N-.

[0391] In the present application, the term "alkanyl" generally refers to a residue derived from an alkanyl group by removing a hydrogen atom. Alkanyl groups can be substituted or non-substituted, substituted or non-substituted. The term "alkanyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group, which has a residue derived from removing a hydrogen atom from the same carbon atom or two different carbon atoms of a parent alkane, which can be a straight or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, for example, an alkanyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkanyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl radical may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic group.

[0392] In the present application, the term "cycloalkyl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of a carbocyclic ring by removing hydrogen atoms. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, and the carbocyclic ring contains 3 to 20 carbon atoms, can contain 3 to 12 carbon atoms, can contain 3 to 10 carbon atoms, and can contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocyclic rings include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, etc.; polycyclic carbocyclic rings can include spirocyclic, condensed ring and bridged ring carbocyclic rings. Cycloalkyl can be substituted or unsubstituted.

[0393] In the present application, the term "heterocycloalkyl" generally refers to a stable 3-7-membered monocyclic structure without aromaticity, a fused 7-10-membered bicyclic heterocyclic structure or a bridged 6-10-membered bicyclic heterocyclic structure, which can be saturated or partially saturated. In addition to carbon atoms, these cyclic structures also contain one or more heteroatoms, wherein the heteroatoms can be selected from the following groups: oxygen, sulfur and nitrogen. For example, it contains 1-4 heteroatoms as defined above. When used to represent atoms on a heterocyclic ring structure, the term "nitrogen" can include nitrogen that has undergone a substitution reaction. Heterocycloalkyl can be substituted or unsubstituted.

[0394] In this application, the term "alkenyl" generally refers to a straight or branched hydrocarbon group containing one or more double bonds. Illustrative examples of alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. C-1-4-yl having more than one double bond is preferably a cyclopentyl or a dicyclopentyl. 2-6 Illustrative examples of alkenyl include butadienyl, pentadienyl, hexadienyl and hexatrienyl and their branched forms. The position of the unsaturated bond (double bond) can be at any position of the carbon chain. The alkenyl group can be substituted or unsubstituted.

[0395] In the present application, the term "alkynyl" generally refers to an unsaturated straight or branched chain alkynyl group, for example, ethynyl, 1-propynyl, propargyl, butynyl, etc. The alkynyl group may be substituted or unsubstituted.

[0396] In the present application, the term "aryl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of an aromatic ring by removing a hydrogen atom. The term "aromatic ring" may refer to a 6 to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., a ring sharing adjacent carbon atom pairs) with a conjugated π electron system, which may be 6 to 10 members, such as benzene and naphthalene. The aromatic ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring. The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups, independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio and heterocycloalkylthio.

[0397] In the present application, the term "heteroaryl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of a heteroaromatic ring by removing hydrogen atoms. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms can be selected from the following groups: oxygen, sulfur and nitrogen. The heteroaryl group can be 5 to 10 yuan, and can be 5 yuan or 6 yuan, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio and heterocycloalkylthio.

[0398] In the present application, the term "alkoxy" generally refers to an alkyl group to which an oxygen group is attached. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0399] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group may but need not be present, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.

[0400] In the present application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are replaced independently of one another by a corresponding number of substituents. The substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.

[0401] Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are consistent with the structures of the present application except that a hydrogen atom is replaced by deuterium or tritium, or a carbon atom is replaced by carbon 13 or carbon 14, are within the scope of the present application.

[0402] In this application, the term "small molecule" generally refers to any chemical moiety or other moiety having a molecular weight less than about 5000 Daltons (Da). In some embodiments, the molecular weight of the small molecule is less than about 2500 Daltons, about 1000 Daltons, or about 500 Daltons. In some embodiments, the small molecule may not be a polymer. In some embodiments, the small molecule may not be a protein. In some embodiments, the small molecule may not be a nucleic acid. In some embodiments, the small molecule may not be a polysaccharide. In some embodiments, the small molecule may have biological activity and / or play an effect that affects a biological process.

[0403] In some embodiments, the small molecule can be a natural product or first prepared by chemical synthesis. For example, in one embodiment the small molecule can be a therapeutic drug, a chemical toxin, or a detection substance, such as the dye molecule FITC.

[0404] In the present application, the term "antigen" generally refers to peptides, polypeptides and proteins encoded by the gene construct of the present invention, which are used as targets for inducing an immune response. The target protein can be an immunogenic protein for which immunity is induced, and it can have at least one identical epitope with a protein from a pathogen or an undesirable cell type, and the undesirable cell type can be, for example, a cancer cell or a cell involved in an autoimmune disease. The immune response to the target protein can protect an individual from a specific infection or disease associated with the target protein, or can treat the above-mentioned infection or disease of an individual.

[0405] In this application, the term "cytokine" generally includes, for example, interleukins, interferons, chemokines, hematopoietic growth factors, tumor necrosis factors and transforming growth factors. Generally speaking, they can be low molecular weight proteins that can regulate the maturation, activation, proliferation and differentiation of cells of the immune system.

[0406] In the present application, the term "dye molecule" generally refers to a substance that dyes a target substance. For example, the "dye molecule" may include a fluorescent marker, and may also include a near-infrared fluorescent marker. Fluorescent labeling is achieved using chemically reactive derivatives of fluorophores. Common reactive groups may include amine-reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine-reactive succinimidyl esters such as NHS-fluorescein, and sulfhydryl-reactive maleimide-activated fluorescein (fluor) such as fluorescein-5-maleimide. Any of these reactive dyes may react with another molecule to produce a stable covalent bond between fluorophores and labeled molecules. Other suitable fluorescent markers included in the present application are Alexa Fluor, DylightFluor, and ATTO dyes. For example, fluorescent proteins included in the present application are such as GFP, YFP, or CFP. For example, activatable dye molecules activated by pH changes or voltage and temperature included in the present application.

[0407] In the present application, the term "pharmaceutically acceptable salt", "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" generally refers to salts of the compounds of the present application. Such salts may be safe and / or effective when used in mammals and may have the desired biological activity. The compounds of the present application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0408] In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. For example, the carrier may be suitable for parenteral (e.g., intravenous, intramuscular, subcutaneous, intrathecal) administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0409] In the present application, the term "Boc" or "t-Butyloxy carbonyl" generally refers to an amino protecting group commonly used in organic synthesis.

[0410] In the present application, the term "Cu+" or "monovalent copper" generally refers to a monovalent copper catalyst. For example, Cu+ can be CuCN, CuSCN, CuI or CuBr.

[0411] In the present application, the term "SrtA ligase" generally refers to a ligase that mediates a coupling reaction. The principle of the coupling reaction mediated by SrtA ligase (Chen, Long, et al. "Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency." Scientific reports 6.1 (2016): 1-12.) is that SrtA ligase can specifically recognize the LPETG sequence shown in SEQ ID NO: 7, and connect the substrate molecule with naked glycine at the N-terminus to the target protein.

[0412] In the present application, the term "specific" generally refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, a natural antibody is monospecific. As in the present application, the term "multispecific" refers to the selectivity of having two or more antigen binding sites, at least two of which bind to different antigens or different epitopes of the same antigen. For example, it can be multispecific for at least two different antigens (i.e., EGFR as a first antigen and 4-1BB as a second antigen). In one embodiment of the present invention, the multispecific antibody according to the present invention can be bispecific. In another embodiment of the present invention, the multispecific antibody according to the present invention can be trispecific.

[0413] In the present application, the term "coupling" generally refers to connecting two compounds by a covalent bond or by a strong non-covalent interaction, such as a connection by a covalent bond. For example, the present application provides a connection in which N3 of one compound can react with an alkynyl group of another compound to form a covalent bond, and a conjugate of the two compounds can be formed.

[0414] In the present application, the term "tether" generally refers to a central compound to which different other compounds can be connected. The central compound can be connected to one or more other compounds to form another larger compound by covalent bonds or by strong non-covalent interactions. For example, the tether of the present application can be connected to one or more compounds having lipids, proteins, nucleic acids, small molecules or polysaccharides, or any combination thereof to form a conjugate.

[0415] In the present application, the term "comprising" generally refers to including the features explicitly specified, but not excluding other elements. The terms "above" and "below" generally refer to the case where the number is inclusive.

[0416] In this application, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below a specified value. DETAILED DESCRIPTION OF THE INVENTION

[0418] In one aspect, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which has a structure as shown in Formula Ia or Formula Ib:

[0419]

[0420] Said Wa may be a trivalent group, and Wb may be a tetravalent group;

[0421] The A, B and C can be independently selected from the following groups:

[0422]

[0423] Where R 12 , R 13 and R 14 may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0424] Among them, A, B and C, Cannot exist at the same time;

[0425] Among them, A, B and C, Cannot exist at the same time;

[0426] Among them, A, B and C, Cannot Exist at the same time; when A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0427] When A, B and C, When both exist, the R 12 , R 13 and R14 Not amino,

[0428] A, B and C are not the same

[0429] In another embodiment, A, B and C can be independently selected from the following groups:

[0430] Where R, R 12 each X may be independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, each X may be independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen;

[0431] Among them, when R is C5H 11 , X is H, or R is CH2OH, X is H, or R is CH2CH3, X is F, in A, B and C, Cannot exist at the same time;

[0432] Wherein, when R is CH3 and X is F, or R is CH2CH3 and X is F, among A, B and C, Cannot At the same time,

[0433] Among them, A, B and C, Cannot At the same time,

[0434] Among them, A, B and C, Cannot Exist at the same time.

[0435] In one embodiment, the selection of A, B and C groups can be the following group:

[0436] (1) A is B is C is

[0437] (2) A is B is C is

[0438] (3) A is B is C is

[0439] (4) A is B is C is

[0440] (5) A is B is C is

[0441] (6) A is B is C is

[0442] (7) A is B is C is

[0443] (8) A is B is C is

[0444] (9) A is B is C is

[0445] (10) A is B is C is

[0446] (11) A is B is C is

[0447] (12) A is B is C is

[0448] (13) A is B is C is

[0449] (14) A is B is C is

[0450] Where R 12 , R 13 and R 14may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0451] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0452] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0453] In another embodiment, the selection of A, B and C groups can be the following group:

[0454] (1) A is B is C is

[0455] (2) A is B is C is

[0456] (3) A is B is C is

[0457] Where R 12 , R 13 and R 14 may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0458] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0459] In another embodiment, the selection of A, B and C groups can be the following group:

[0460] (1) A is B is C is

[0461] (2) A is B is C is

[0462] (3) A is B is C is

[0463] Where R 12 may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0464] When A, B and C, When both exist, the R 12 Not amino.

[0465] For example, where R 12 may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site.

[0466] For example, where A can be B can be C can be Where R 12 A group may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, Represents the junction site.

[0467] In the present application, Wa may be a trivalent group, and Wb may be a tetravalent group; for example:

[0468] Can be

[0469] or Can be

[0470] The W may be a trivalent group or a tetravalent group, and the W may be optionally substituted by one or more R,

[0471] The La can be -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 )q1 -;

[0472] The Lb can be -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0473] The Lc can be -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0474] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each may be independently selected from the following group: an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and polyethylene glycol, or any combination thereof;

[0475] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be independently selected from the following group: -NR 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR1a -C(=O)-NR 1b -;

[0476] R, R 1 , R 1a and R 1b may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0477] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0.

[0478] In another embodiment, wherein J 1 , J 2 , and J 3 Each of them can be independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; wherein, m1, m2, and m3 can be independently selected from the following group: 0, 1, 2, and 3.

[0479] In another embodiment, wherein X 1 , X 2 , and X 3 may each be independently selected from the group consisting of -NH- and -O-.

[0480] In another embodiment, wherein K 1 , K 2 , and K 3 Each of them can be independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; wherein, n1, n2, and n3 can be independently selected from the following group: 0, 1, 2, and 3.

[0481] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each may be independently selected from the group consisting of -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 may be independently 0.

[0482] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may be each independently 0.

[0483] In another embodiment, wherein Y 1 , Y 2 , and Y 3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 Selected from the following group: hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 can be each independently selected from the following group: 0, 1, 2 and 3.

[0484] For example, where (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 may each independently be 0.

[0485] In another embodiment, wherein L 1 , L 2 , and L 3 can be independently selected from the following group: alkyl, and aryl, or any combination thereof; wherein, q1, q2, and q3 can be independently selected from the following group: 0, 1, 2, and 3.

[0486] For example, where (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 may be each independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 may be each independently 0.

[0487] For example, where (L 1 ) q1 , (L 2 ) q2 , and (L 3 )q3 may be each independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 may be each independently 0.

[0488] In another embodiment, wherein:

[0489] W can be selected from the following group:

[0490] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0491] For example, where:

[0492] W can be selected from the following group:

[0493]

[0494] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 may be each independently selected from the following group: C, N, O, S and P;

[0495] The --- represents a double bond or a single bond.

[0496] For example, where:

[0497] W can be selected from the following group:

[0498]

[0499]

[0500] In another embodiment, the compound can be

[0501]

[0502] The La is -(J 1 ) m1 -C(=O)-X 1-(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0503] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0504] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0505] Among them, J 1 , J 2 , and J 3 can be independently selected from the following groups: chain alkyl, and polyethylene glycol, or any combination thereof; wherein m1, m2, and m3 can be independently selected from the following groups: 0, 1, 2, and 3;

[0506] Among them, X 1 , X 2 , and X 3 may each be independently selected from the following group: -NH- and -O-;

[0507] Among them, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may each independently be 0;

[0508] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 may each independently be 0;

[0509] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 may be independently selected from the following group: -CH2-aryl-, -(CH2)2-, -CH2-, alkyl and polyethylene glycol, or q1, q2 and q3 may be independently 0;

[0510] W can be selected from the following group:

[0511]

[0512] A can be B can be C can be Where R 12 A group may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, Represents the junction site.

[0513] For example, W can be selected from the following group:

[0514]

[0515] In another embodiment, the La, Lb and Lc can be each independently selected from the following group: a linear hydrocarbon chain, a linear heterohydrocarbon chain containing 1 or more heteroatoms, a branched hydrocarbon chain containing 1 or more branches, and a linear heterohydrocarbon chain containing 1 or more heteroatoms and containing 1 or more branches.

[0516] In another embodiment, the La, Lb and Lc may be each independently selected from the following group: a linear alkyl chain, and a linear heteroalkyl chain containing 1 or more heteroatoms.

[0517] In another embodiment, the La, Lb and Lc can be each selected from -C nx H 2nx -, where nx is a number greater than 0. For example, nx is a number greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, or greater than 9. For example, nx is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0518] In another embodiment, the La, Lb and Lc may each be independently selected from a polymer.

[0519] In another embodiment, the La, Lb and Lc can be each independently selected from the following group: polysaccharide, polyamino acid, polynucleotide, and polylactic acid.

[0520] In another embodiment, the La, Lb and Lc can be polysaccharides independently of each other. Polysaccharides can be monomers or polymers of sugar residues, and can be straight or branched. Polysaccharides can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo N-acetylglucosamine, etc.). In another embodiment, the La, Lb and Lc can be homopolymers and heteropolymers of sugar residues independently of each other.

[0521] In another embodiment, the La, Lb and Lc can each independently be a polynucleotide. Polynucleotides can include single-stranded and double-stranded nucleotide polymers. The nucleotides that make up the polynucleotide can be ribonucleotides or deoxyribonucleotides, or a modified form of either type of nucleotide. In another embodiment, the La, Lb and Lc can each independently be a base-modified polynucleotide,

[0522] In another embodiment, the La, Lb and Lc may each independently be polyglycine, for example, polyglycine polymerized from more than one glycine. In certain embodiments, polyglycine polymerized from more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, or more than nine glycine. In certain embodiments, polyglycine polymerized from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine.

[0523] In another embodiment, each of the groups may be independently selected from the following groups, which may mean that each group is selected from one or more of the following groups, or it may mean that some of the groups are selected from one or more of the following groups, and another part of the groups are selected from one or more of the following groups in any definition of the group in this application.

[0524] In another embodiment, the compound can be selected from the following group:

[0525]

[0526]

[0527]

[0528]

[0529] In another aspect, the present application provides a compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, which has a structure as shown in Formula IIa or Formula IIb, wherein:

[0530]

[0531] The Wc is a trivalent group, and Wd is a tetravalent group;

[0532] The A 2 , B 2 and C 2 Each independently selected from the following group:

[0533]

[0534] and covalent bonds;

[0535] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0536] represents the attachment site, and P 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect;

[0537] Where P 1 , P 2 and P 3 Each is independently selected from the following group: lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0538] The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two attachment sites of are arbitrarily connected to Wc in one embodiment, wherein A 2 , B 2 and C 2 Can be independently selected from the following groups:

[0539] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0540] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0541] (3)A 2 for B 2 for C 2 for or covalent bond; or

[0542] (4)A 2 for B 2 for C 2 for or covalent bond; or

[0543] (5)A 2 for B 2 for C 2 for or covalent bond; or

[0544] (6)A 2 for B 2 for C 2 for or covalent bond; or

[0545] (7)A 2 for B 2 for C 2 for or covalent bond; or

[0546] (8)A 2 for B 2 for C 2 for or a covalent bond; or

[0547] (9)A 2 for B 2 for C 2 for or a covalent bond; or

[0548] (10)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or

[0549] (11)A 2 for B 2 for C 2 for or a covalent bond; or

[0550] (12)A 2 for B 2 for C 2 for or a covalent bond; or

[0551] (13)A 2 for B 2 for C 2 for or covalent bonds;

[0552] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0553] represents the junction site,

[0554] The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0555] For example, where A 2 , B 2 and C 2 Can be independently selected from the following groups:

[0556] (1)A 2 for B 2 for C 2 for or a covalent bond; or

[0557] (2)A 2 for B 2 for C 2 for or a covalent bond; or

[0558] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0559] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0560] represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2The two binding sites of Wc and P are randomly connected 3 connect.

[0561] For example, where A 2 , B 2 and C 2 Can be independently selected from the following groups:

[0562] (1)A 2 for B 2 for C 2 for or a covalent bond; or

[0563] (2)A 2 for B 2 for C 2 for or a covalent bond; or

[0564] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0565] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0566] represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0567] For example, R 12 , R 13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl,

[0568] represents the connection site, the A 2The two connection sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two connection sites can be arbitrarily connected with Wc and P 2 connection, the C 2 The two connection sites can be arbitrarily connected with Wc and P 3 connect.

[0569] For example, where A 2 , B 2 and C 2 Can be: A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 may be each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, represents the connection site, the A 2 The two connection sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two connection sites can be arbitrarily connected with Wc and P 2 connection, the C 2 The two connection sites can be arbitrarily connected with Wc and P 3 connect.

[0570] For example, A 2 Can be B 2 Can be, C 2 Can be or covalent bond; where R 12 may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the connection site, the A 2 The two connection sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two connection sites can be arbitrarily connected with Wc and P 2 connection, the C 2 The two connection sites can be arbitrarily connected with Wc and P 3 connect.

[0571] In another embodiment, Can be Can be

[0572] The W may be a trivalent group, and the W may be optionally substituted by one or more R,

[0573] The La can be -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0574] The Lb can be -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0575] The Lc can be -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0576] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each may be independently selected from the following group: an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and polyethylene glycol, or any combination thereof;

[0577] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be independently selected from the following group: -NR 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0578] R, R 1 , R 1a and R 1b may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0579] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0.

[0580] In another embodiment, wherein J 1 , J 2 , and J 3 Each of them can be independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; wherein, m1, m2, and m3 can be independently selected from the following group: 0, 1, 2, and 3.

[0581] In another embodiment, wherein X 1 , X 2 , and X 3 may each be independently selected from the group consisting of -NH- and -O-.

[0582] In another embodiment, wherein K 1 , K 2 , and K 3 Each of them can be independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof; wherein, n1, n2, and n3 can be independently selected from the following group: 0, 1, 2, and 3.

[0583] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 )n3 Each may be independently selected from the group consisting of -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 may be independently 0.

[0584] For example, where (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may be each independently 0.

[0585] In one embodiment, wherein Y 1 , Y 2 , and Y 3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 Selected from the following group: hydrogen, protium, deuterium, tritium, and chain alkyl; wherein n1, n2, and n3 can be each independently selected from the following group: 0, 1, 2 and 3.

[0586] For example, where (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 may each independently be 0.

[0587] In one embodiment, wherein L 1 , L 2 , and L 3 can be independently selected from the following group: alkyl, and aryl, or any combination thereof; wherein, q1, q2, and q3 can be independently selected from the following group: 0, 1, 2, and 3.

[0588] For example, where (L 1 ) q1 , (L 2 ) q2 , and (L3 ) q3 may be each independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 may be each independently 0.

[0589] For example, where (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 may be each independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 may be each independently 0.

[0590] In one embodiment, W can be selected from the following group:

[0591] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0592] For example, W can be selected from the following group:

[0593]

[0594] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 may be each independently selected from the following group: C, N, O, S and P;

[0595] The --- represents a double bond or a single bond.

[0596] For example, W can be selected from the following group:

[0597]

[0598] In another embodiment, wherein P 1 , P 2 and P 3Each may be independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, a fusion protein, and an antibody or an antigen-binding fragment thereof.

[0599] For example, the antibody can be selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

[0600] For example, the antibody targeting target can be selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0601] For example, the amino acid sequence of the antibody can be selected from the following group: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0602] In another embodiment, the compound can be

[0603]

[0604] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0605] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0606] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0607] Among them, J 1 , J 2 , and J 3can be independently selected from the following groups: chain alkyl, and polyethylene glycol, or any combination thereof; wherein m1, m2, and m3 can be independently selected from the following groups: 0, 1, 2, and 3;

[0608] Among them, X 1 , X 2 , and X 3 may each be independently selected from the following group: -NH- and -O-;

[0609] Among them, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 may be each independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 may each independently be 0;

[0610] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 can be independently selected from the following groups: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 may each independently be 0;

[0611] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 may be independently selected from the following group: -CH2-aryl-, -(CH2)2-, -CH2-, alkyl and polyethylene glycol, or q1, q2 and q3 may be independently 0;

[0612] W can be selected from the following group:

[0613]

[0614] A 2 Can be B 2 Can be, C 2 Can be or covalent bond; where R 12 may be selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the junction site,

[0615] Where P 1 , P 2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof;

[0616] The A 2 The two connection sites can be arbitrarily connected with Wc and P 1 Connect the B 2 The two connection sites can be arbitrarily connected with Wc and P 2 connection, the C 2 The two connection sites can be arbitrarily connected with Wc and P 3 connect.

[0617] For example, A 2 The a-linked site and P 1 Connection, b connection site and Wc in (L 1 ) q1 Connect, B 2 The b-linking site is connected to P 2 Connection, a connection site and Wc in (L 2 ) q2 Connect, C 2

[0618] The a-linked site and P 3 Connection, a connection site and Wc in (L 3 ) q3 connect.

[0619] For example, W can be selected from the following group:

[0620]

[0621] In another embodiment, the one compound can be selected from the following group:

[0622]

[0623] Among them, P 1 , P 2 and P 3 Each may be independently selected from the following group: lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0624] In one embodiment, P1 , P 2 and P 3 Can be independently selected from lipids. 1 , P 2 and P 3 Can be independently selected from the following group: aliphatic carboxylic acid, phospholipid and sterol. In one embodiment, P 1 , P 2 and P 3 Each may be independently selected from a linear, branched, saturated or unsaturated aliphatic carboxylic acid.

[0625] In one embodiment, P 1 , P 2 and P 3 Can be independently selected from proteins. In one embodiment, P 1 , P 2 and P 3 Each can be independently selected from a polypeptide. The protein or polypeptide may include post-expression modifications, such as glycosylation, acetylation, phosphorylation, etc. The protein or polypeptide may include one or more amino acid analogs, polypeptides with substituted bonds, and modified polypeptides known in the art, including both naturally occurring and non-naturally occurring modifications.

[0626] In one embodiment, P 1 , P 2 and P 3 Can be independently selected from nucleic acid. The nucleic acid can refer to a polymer of nucleotides (e.g., ribonucleotides or deoxyribonucleotides), and includes naturally occurring (adenine, guanine, cytosine, uracil and thymine), non-naturally occurring and modified nucleic acids. The nucleic acid may not be limited by the length of the polymer (e.g., the number of monomers). The nucleic acid may be single-stranded or double-stranded, and generally contains a 5'-3' phosphodiester bond, and nucleotide analogs may also have other connections. The monomers of the nucleic acid are generally referred to as nucleotides. The non-natural nucleotides or the modified nucleotides refer to nucleotides containing modified nitrogenous bases, sugars or phosphate groups, or nucleotides incorporating non-natural parts into their structures. The non-natural nucleotides may include dideoxynucleotides, biotinylated, aminized, deaminated, alkylated, benzylated and fluorescently labeled nucleotides.

[0627] In one embodiment, P 1 , P 2 and P 3Each of the small molecules can be independently selected from small molecules. The small molecules can be mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemoprotectants, hormones, anti-hormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors and radiosensitizers.

[0628] In one embodiment, P 1 , P 2 and P 3 Can be independently selected from polysaccharides. The polysaccharide can include molecules composed of two or more monosaccharide units, for example, can include molecules in which the longest monosaccharide chain is 3 to 9 monosaccharide units. The polysaccharide can include straight chain and branched chain molecules. Any combination of the polysaccharide and lipid, protein, nucleic acid, or small molecule can include isolated and polypeptide-bound molecules, sialylated and non-sialylated molecules.

[0629] Among them, P 1 , P 2 and P 3 Each may be independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0630] For example, P 1 , P 2 and P 3 The amino acid sequences can be:

[0631] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0632] SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or

[0633] SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or

[0634] SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 3, or

[0635] SEQ ID NO: 10, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0636] SEQ ID NO: 11, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0637] SEQ ID NO: 12, SEQ ID NO: 2, and SEQ ID NO: 3, or

[0638] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10, or

[0639] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11, or

[0640] SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12, or

[0641] SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0642] On the other hand, P in this application 1 , P 2 and P 3 Can be each independently a protein having a ligase recognizable sequence, and the ligase can connect the substrate molecule to the protein. On the other hand, P in this application 1 , P 2 and P 3 Each of them can be independently a protein having a LPETG sequence, and the LPETG sequence as shown in SEQ ID NO: 7 can be specifically recognized by a ligase, such as SrtA ligase.

[0643] On the other hand, P in this application 1 , P 2 and P 3 Each can be independently selected from a VHH nanobody targeting EGFR, a VHH nanobody targeting CD3, a VHH nanobody targeting 4-1BB, a VHH nanobody targeting Her2, a VHH nanobody targeting CD47, a VHH nanobody targeting CD20, a branched peptide containing a CMV antigen, a fusion protein of cytokines IL2 / IL5, a cytokine IFNα, a dye molecule FITC and a CPG nucleic acid molecule.

[0644] Preparation method of compound

[0645] In one aspect, the present application provides a method for preparing a compound, comprising reacting a compound represented by formula (Ia-I) with reactant 1, reactant 2, and reactant 3;

[0646] Alternatively, the compound represented by formula (Ia-I) may be reacted with reactant 1 and reactant 2;

[0647] Alternatively, the compound represented by formula (Ia-I) may be reacted with reactant 1;

[0648]

[0649] The reactant 1 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A,

[0650] The reactant 2 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B,

[0651] The reactant 3 can be HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C,

[0652] The W may be a trivalent group, and the W may be optionally substituted by one or more R,

[0653] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each may be independently selected from the following group: an alkyl group, a cycloalkyl group, a heterocycloalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, and polyethylene glycol, or any combination thereof;

[0654] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Can be independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1-C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0655] R, R 1 , R 1a and R 1b may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0656] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 can each be independently selected from a number greater than 0;

[0657] A, B and C can each be independently selected from the following groups:

[0658]

[0659] Where R 12 , R 13 and R 14 may be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0660] Among them, A, B and C, Cannot exist at the same time;

[0661] Among them, A, B and C, Cannot exist at the same time;

[0662] Among them, A, B and C, Cannot exist at the same time;

[0663] When A, B and C, When both exist, the R 12 , R 13and R 14 is not an alkynyl group;

[0664] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0665] A, B and C are not the same

[0666] Among them, when A, B or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid may be added to remove Boc.

[0667] For example, acid (0%-300% hydrochloric acid or trifluoroacetic acid) can be added to remove Boc. In another embodiment, the order of adding reactant 1, reactant 2 and reactant 3 can be determined according to the order of introducing A, B and C. The order of introducing A, B and C from first to last can be:

[0668] Where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0669] For example, when you need to introduce When you import the Reintroduction Last introduction To decide to add N3-Amine first, then TZ-Amine, and finally Boc-Gly-Amine.

[0670] For example, in the above steps, the compound represented by formula (Ia-I) reacts with reactant 1, reactant 2 and reactant 3 in an equivalent ratio ranging from 20 / 1 to 1 / 20, and 1%-100% molar equivalent of triethylamine or ethylenediamine or dimethylaminopyridine or pyridine or N,N-diisopropylethylamine is added as a catalyst. The reaction conditions are 4-100° C., and the reaction solvent can be water, other water-based solutions, DMF, DMSO, methanol, acetonitrile, tetrahydrofuran, dichloromethane or their corresponding mixed solvents, etc. The reaction concentration can be in the range of 1 nanomolar to 10 molar, and the reaction time can be 0-24 hours.

[0671] For example, step 1: the starting material NHS reagent can be reacted with N3-Amine in a ratio of 1 / 1.2, and the reaction conditions can be DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and reacting at 25°C for 2 hours. After the reaction is completed, the solvent can be removed by rotary evaporation, and then it can be purified by HPLC (high performance liquid chromatography) to obtain the first intermediate N3-NHS-NHS.

[0672] Step 2: N3-NHS-NHS and TZ-Amine can be reacted in a ratio of 1 / 1.2. The reaction conditions can be DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and react at 25°C for 2 hours. After the reaction, the solvent can be removed by rotary evaporation, and then it can be purified by HPLC (high performance liquid chromatography) to obtain a second intermediate, which is the multispecific bioconjugated scaffold 2.

[0673] Step 3: The second intermediate, i.e., the multi-specific bioconjugated scaffold 2, can be reacted with Boc-Gly-Amine in a ratio of 1 / 1.2. The reaction conditions can be DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and a reaction at 25°C for 2 hours. After the reaction, the solvent can be removed by rotary evaporation, and then it can be purified by HPLC (high performance liquid chromatography). The purified product can be dissolved in DCM (dichloromethane), and then 20% TEA (triethylamine) can be added by volume. The Boc (tert-butyloxycarbonyl) group can be removed by reacting at room temperature for 5 minutes. Then, HPLC (high performance liquid chromatography) can be used to purify the final product, i.e., the multi-specific bioconjugated scaffold 1.

[0674] On the other hand, the present application also provides a method for preparing a compound, which can react the compound described in any one of Formula Ia in the present application with reactant 4, reactant 5 and reactant 6;

[0675] The reactant 4 is A 1 -P 1 ,

[0676] The reactant 5 is B 1 -P 2 ,

[0677] The reactant 6 is C 1 -P 3 ,

[0678] Among them, A of reactant 4 1 React with A of the compound described in Formula Ia, B of the reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia;

[0679] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0680] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0681] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0682] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0683] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0684] When at least one of A, B or C is When A 1 , B1 or C 1 At least one

[0685] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0686] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0687] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0688] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0689] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0690] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0691] Where R 12 , R 13 and R 14 Each may be independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0692] In some embodiments, when at least one of A, B or C is When A 1 , B1 or C 1 At least one

[0693] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0694] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0695] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0696] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0697] Where R, R 12 can be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, and each X can be each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen.

[0698] For example, the groups in A, B, or C are 1 , B 1 or C 1 The group A in can be B is C is When A 1 for B 1 for C 1 for Where R 12 It may be hydrogen.

[0699] For example, Cu can be added to the reaction +As a catalyst. For example, adding a catalytic amount of Cu+ to generate Cu+ in other forms as a catalyst.

[0700] For example, 1-100% equivalent of SrtA ligase can be added to the reaction as a catalyst.

[0701] For example, the reaction of the compound described in Formula Ia with reactant 4, reactant 5 and reactant 6 can be carried out in a molar ratio range of 20 / 1 to 1 / 20, the reaction solvent can be water, other water-based solutions, DMF, DMSO, methanol, acetonitrile, tetrahydrofuran, dichloromethane or their corresponding mixed solvents, etc., the reaction concentration can be in the range of 1 nanomole to 100 moles, and the reaction time can be 0-24 hours.

[0702] Treatments and uses

[0703] In one aspect, the present application provides a pharmaceutical composition comprising any compound of the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0704] On the other hand, the present application provides a compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the preparation of a drug for treating and / or preventing a tumor. For example, the tumor may be selected from tumors associated with the expression of the following groups: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the tumor may be selected from the following groups: solid tumors and hematological cancers. For example, the tumor may be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0705] On the other hand, the present application provides a method for treating and / or preventing a tumor, comprising administering to a subject in need thereof a compound described herein or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition comprising the same. For example, the tumor may be selected from tumors associated with the following group of expressions: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the tumor may be selected from the following groups: solid tumors and hematological cancers. For example, the tumor may be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0706] On the other hand, the present application provides a compound described in the present application or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or pharmaceutically acceptable salt thereof, and / or pharmaceutical composition comprising the same, for treating and / or preventing tumors. For example, the tumor may be selected from tumors associated with the following group of expressions: 4-1BB, EGFR, CD3, Her2, CD47, and CD20. For example, the tumor may be selected from the following groups: solid tumors and hematological cancers. For example, the tumor may be selected from the following groups: lung cancer, renal cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, and esophageal cancer.

[0707] Technical Solution

[0708] 1. A compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula Ia or Formula Ib:

[0709]

[0710] Said Wa is a trivalent group, and Wb is a tetravalent group;

[0711] The A, B and C are each independently selected from the following groups:

[0712]

[0713] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0714] Among them, A, B and C, Cannot exist at the same time;

[0715] Among them, A, B and C, Cannot exist at the same time;

[0716] Among them, A, B and C, Cannot exist at the same time;

[0717] Among them, A, B and C, Cannot exist at the same time;

[0718] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0719] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0720] A, B and C are not the same

[0721] 2. The compound according to technical solution 1, wherein:

[0722] (1) A is B is C is

[0723] (2) A is B is C is

[0724] (3) A is B is C is

[0725] (4) A is B is C is

[0726] (5) A is B is C is

[0727] (6) A is B is C is

[0728] (7) A is B is C is

[0729] (8) A is B is C is

[0730] (9) A is B is C is

[0731] (10) A is B is C is

[0732] (11) A is B is C is

[0733] (12) A is B is C is

[0734] (13) A is B is C is

[0735] (14) A is B is C is

[0736] (15) A is B is C is

[0737] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0738] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0739] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0740] 3. A compound according to any one of technical solutions 1-2, wherein:

[0741] (1) A is B is C is

[0742] (2) A is B is C is

[0743] (3) A is B is C is

[0744] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0745] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino.

[0746] 4. A compound according to any one of technical solutions 1-3, wherein:

[0747] (1) A is B is C is

[0748] (2) A is B is C is

[0749] (3) A is B is C is

[0750] Where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0751] When A, B and C, When both exist, the R12 Not amino.

[0752] 5. A compound according to any one of technical solutions 1 to 4, wherein:

[0753] (1) A is B is C is

[0754] (2) A is B is C is

[0755] (3) A is B is C is

[0756] Where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, Represents the junction site.

[0757] 6. A compound according to any one of technical solutions 1-5, wherein: A is B is C is

[0758] Where R 12 a group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, Represents the junction site.

[0759] 7. A compound according to any one of technical solutions 1-6, wherein:

[0760] for

[0761] or for

[0762] The W is a trivalent group or a tetravalent group, and the W is optionally substituted by one or more R,

[0763] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0764] The Lb is -(J 2 )m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0765] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0766] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0767] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0768] R, R 1, R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0769] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0.

[0770] 8. The compound according to technical solution 7, wherein J 1 , J 2 , and J 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0771] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0772] 9. A compound according to any one of technical solutions 7-8, wherein X 1 , X 2 , and X 3 Each is independently selected from the group consisting of -NH- and -O-.

[0773] 10. A compound according to any one of technical solutions 7-9, wherein K 1 , K 2 , and K 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0774] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0775] 11. A compound according to any one of technical solutions 7-10, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0776] 12. A compound according to any one of technical solutions 7-11, wherein (K 1 ) n1 , (K 2 ) n2 , and (K3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0.

[0777] 13. A compound according to any one of technical solutions 7-12, wherein Y 1 , Y 2 , and Y 3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-;

[0778] R 1 Selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkanol;

[0779] wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0780] 14. A compound according to any one of technical solutions 7-13, wherein (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0.

[0781] 15. A compound according to any one of technical solutions 7-14, wherein L 1 , L 2 , and L 3 Each is independently selected from the following group: chain alkyl, and aryl, or any combination of the foregoing; wherein, q1, q2, and q3 are each independently selected from the following group: 0, 1, 2 and 3.

[0782] 16. A compound according to any one of technical solutions 7-15, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0783] 17. A compound according to any one of technical solutions 7-16, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0784] 18. A compound according to any one of technical solutions 7-17, wherein:

[0785] W is selected from the following group:

[0786] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0787] 19. A compound according to any one of technical solutions 7-18, wherein:

[0788] W is selected from the following group:

[0789]

[0790] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S and P;

[0791] The --- represents a double bond or a single bond.

[0792] 20. A compound according to any one of technical solutions 7-19, wherein:

[0793] W is selected from the following group:

[0794]

[0795]

[0796] 21. A compound according to any one of technical solutions 1-20, which is selected from the following group:

[0797]

[0798]

[0799]

[0800] 22. A compound according to any one of technical solutions 1-21, which is selected from the following group:

[0801]

[0802] 23. A compound or its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula IIa or Formula IIb, wherein:

[0803]

[0804] The Wc is a trivalent group, and Wd is a tetravalent group;

[0805] The A 2 , B 2 and C 2 Each independently selected from the following group:

[0806] and covalent bonds;

[0807] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0808] represents the junction site,

[0809] The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2The two binding sites of Wc and P 3 connect;

[0810] Where P 1 , P 2 and P 3 Each is independently selected from the following group: lipids, proteins, nucleic acids, small molecules and polysaccharides, or any combination thereof.

[0811] 24. The compound according to technical solution 23, wherein:

[0812] (1)A 2 for B 2 for C 2 for

[0813]

[0814] or covalent bond; or

[0815] (2)A 2 for B 2 for C 2 for

[0816] or covalent bond; or

[0817] (3)A 2 for B 2 for C 2 for or covalent bond; or

[0818] (4)A 2 for B 2 for C 2 for

[0819] or covalent bond; or

[0820] (5)A 2 for B 2 for C 2 for or covalent bond; or

[0821] (6)A 2 for B 2 for C 2 for or covalent bond; or

[0822] (7)A 2 for B 2 for C 2 for or covalent bond; or

[0823] (8)A 2 for B 2 for C 2 for or covalent bond; or

[0824] (9)A 2 for B 2 for C 2 for or covalent bond; or

[0825] (10)A 2 for or covalent bond, B 2 is a covalent bond, C 2 is a covalent bond; or

[0826] (11)A 2 for B 2 for C 2 for or covalent bond; or

[0827] (12)A 2 for B 2 for C2 for or a covalent bond; or

[0828] (13)A 2 for B 2 for C 2 for or covalent bonds;

[0829] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0830] represents the junction site,

[0831] The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0832] 25. A compound according to any one of technical solutions 23-24, wherein:

[0833] (1)A 2 for B 2 for C 2 for or a covalent bond; or

[0834] (2)A 2 for B 2 for C 2 for or a covalent bond; or

[0835] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0836] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0837] represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0838] 26. A compound according to any one of technical solutions 23-25, wherein:

[0839] (1)A 2 for B 2 for C 2 for or covalent bond; or

[0840] (2)A 2 for B 2 for C 2 for or covalent bond; or

[0841] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0842] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl,

[0843] represents the connection site, the A 2The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0844] 27. A compound according to any one of technical solutions 23-26, wherein:

[0845] (1)A 2 for B 2 for C 2 for or a covalent bond; or

[0846] (2)A 2 for B 2 for C 2 for or a covalent bond; or

[0847] (3)A 2 for B 2 for C 2 for or covalent bonds;

[0848] Where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl,

[0849] represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

[0850] 28. A compound according to any one of technical solutions 23-27, wherein: A 2 for B 2 for C 2 for or covalent bond; where R 12 , R 13 and R 14 are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkyl, represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0851] 29. A compound according to any one of technical solutions 23-28, wherein: A 2 for B 2 for, C 2 for or covalent bond; where R 12 is selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and alkanol, represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

[0852] 30. A compound according to any one of technical solutions 23-29, wherein:

[0853] for

[0854] or for

[0855] The W is a trivalent group or a tetravalent group, and the W is optionally substituted by one or more R,

[0856] The La is -(J 1 ) m1 -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -;

[0857] The Lb is -(J 2 ) m2 -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -;

[0858] The Lc is -(J 3 ) m3 -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -;

[0859] The J 1 , J 2 , J 3 , K 1 , K 2 , K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0860] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0861] R, R 1 , R 1a and R 1b are each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl, represents the junction site;

[0862] Among them, m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0.

[0863] 31. The compound according to technical solution 30, wherein J 1 , J 2 , and J 3 Each independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination thereof;

[0864] wherein m1, m2, and m3 are each independently selected from the following group: 0, 1, 2, and 3.

[0865] 32. A compound according to any one of technical solutions 30-31, wherein X 1 , X 2 , and X 3 Each is independently selected from the group consisting of -NH- and -O-.

[0866] 33. A compound according to any one of technical solutions 30-32, wherein K 1 , K 2 , and K 3 Each is independently selected from the following group: chain alkyl, and polyethylene glycol, or any combination of the foregoing; wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2 and 3.

[0867] 34. A compound according to any one of technical solutions 30-33, wherein (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -alkane-polyethylene glycol-alkane-, alkane and polyethylene glycol, or n1, n2 or n3 is each independently 0.

[0868] 35. A compound according to any one of technical solutions 30-34, wherein (K 1 ) n1 , (K 2 )n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)-, -(CH2)2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-, and -(CH2)8-, or n1, n2 or n3 is each independently 0.

[0869] 36. A compound according to any one of technical solutions 30-35, wherein Y 1 , Y 2 , and Y 3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-; R 1 , R 1a and R 1b Each independently selected from the following group:

[0870] Hydrogen, protium, deuterium, tritium, and alkyl; wherein n1, n2, and n3 are each independently selected from the following group: 0, 1, 2, and 3.

[0871] 37. A compound according to any one of technical solutions 30-36, wherein (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each is independently selected from the following group: -C(=O)-, -NR 1 -C(=O)-, -NR 1 -, and -O-, R 1 is selected from the group consisting of hydrogen, protium, deuterium, tritium, and alkyl; or p1, p2 or p3 are each independently 0.

[0872] 38. A compound according to any one of technical solutions 30-37, wherein L 1 , L 2 , and L 3 Each is independently selected from the following group: chain alkyl, and aryl, or any combination of the foregoing; wherein, q1, q2, and q3 are each independently selected from the following group: 0, 1, 2 and 3.

[0873] 39. A compound according to any one of technical solutions 30-38, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3Each is independently selected from the group consisting of -alkyl-aryl-, alkyl and polyethylene glycol, or q1, q2 and q3 are each independently 0.

[0874] 40. A compound according to any one of technical solutions 30-39, wherein (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the following group: -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2 and q3 are each independently 0.

[0875] 41. A compound according to any one of technical solutions 30-40, wherein:

[0876] W is selected from the following group:

[0877] a trivalent alkanyl group, a trivalent cycloalkyl group, a trivalent heterocycloalkyl group, a trivalent alkenyl group, a trivalent alkynyl group, a trivalent aryl group, and a trivalent heteroaryl group.

[0878] 42. A compound according to any one of technical solutions 30-41, wherein:

[0879] W is selected from the following group:

[0880]

[0881] The X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 and X 18 Each independently selected from the group consisting of C, N, O, S and P;

[0882] The --- represents a double bond or a single bond.

[0883] 43. A compound according to any one of technical solutions 30-42, wherein:

[0884] W is selected from the following group:

[0885]

[0886]

[0887] 44. A compound according to any one of technical solutions 23-43, wherein P 1 , P 2 and P 3 Each is independently selected from the following group: a nucleic acid molecule, a dye molecule, a cytokine, an antigen, and an antibody or an antigen-binding fragment thereof, or any combination thereof.

[0888] 45. A compound according to technical solution 44, wherein the antibody is selected from the following groups: monoclonal antibodies, single-chain antibodies, chimeric antibodies, humanized antibodies, fully human antibodies and nanobodies.

[0889] 46. ​​A compound according to any one of technical solutions 44-45, wherein the antibody targets a target selected from the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0890] 47. A compound according to any one of technical solutions 44-46, wherein the amino acid sequence of the antibody is selected from the following group: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0891] 48. A compound according to any one of technical solutions 23-47, which is selected from the following group:

[0892]

[0893] Among them, P 1 , P 2 and P 3 Each is independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0894] 49. A compound according to any one of technical solutions 23-48, which is:

[0895]

[0896] Among them, P 1 , P 2 and P 3The amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO: 3.

[0897] 50. A method for preparing the compound according to any one of technical solutions 1 to 22, comprising:

[0898] reacting the compound represented by formula (Ia-I) with reactant 1, reactant 2 and reactant 3;

[0899] or reacting the compound represented by formula (Ia-I) with reactant 1 and reactant 2;

[0900] or reacting the compound represented by formula (Ia-I) with reactant 1;

[0901]

[0902] The reactant 1 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A,

[0903] The reactant 2 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B,

[0904] The reactant 3 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C,

[0905] Said W is a trivalent group, said W is optionally substituted by one or more R,

[0906] The J 1 , J 2 , J 3 , K 1 , K 2, K 3 , L 1 , L 2 , and L 3 Each is independently selected from the following group: alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, and polyethylene glycol, or any combination thereof;

[0907] The X 1 , X 2 , X 3 , Y 1 , Y 2 , and Y 3 Each independently selected from the following group: 1 -, -O-, -S-, -C(=O)-, -C(=S)-, -C(R 1a )(R 1b )-,-NR 1 -C(=O)-, -C(=O)-NR 1 -,-NR 1 -C(=S)-, -C(=S)-NR 1 -, -OC(=O)-, -C(=O)-O-, -OC(=S)-, -C(=S)-O-, -OC(=O)-O-, -OC(=O)-NR 1 -,-NR 1 -C(=O)-O-, and -NR 1a -C(=O)-NR 1b -;

[0908] R, R 1 , R 1a and R 1b each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0909] wherein m1, m2, m3, n1, n2, n3, p1, p2, p3, q1, q2, and q3 are each independently selected from a number greater than 0;

[0910] A, B and C are each independently selected from the following group:

[0911]

[0912] Where R 12 , R 13 and R 14each independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl;

[0913] Among them, A, B and C, Cannot exist at the same time;

[0914] Among them, A, B and C, Cannot exist at the same time;

[0915] Among them, A, B and C, Cannot exist at the same time;

[0916] When A, B and C, When both exist, the R 12 , R 13 and R 14 is not an alkynyl group;

[0917] When A, B and C, When both exist, the R 12 , R 13 and R 14 Not amino,

[0918] A, B and C are not the same

[0919] Among them, when A, B, or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid is added to remove Boc.

[0920] 51. According to the preparation method of technical solution 50, the order of adding reactant 1, reactant 2 and reactant 3 is determined according to the introduction order of A, B and C, and the introduction order of A, B and C is from first to last:

[0921] Where R 12 , R 13 and R 14 Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0922] 52. A method for preparing the compound according to any one of technical solutions 23-49, comprising:

[0923] reacting the compound of formula Ia in any one of technical solutions 1 to 22 with reactant 4, reactant 5 and reactant 6;

[0924] The reactant 4 is A 1 -P 1 ,

[0925] The reactant 5 is B 1 -P 2 ,

[0926] The reactant 6 is C 1 -P 3 ,

[0927] Among them, A of reactant 4 1 React with A of the compound described in Formula Ia, B of the reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia;

[0928] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0929] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0930] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0931] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0932] When at least one of A, B or C is When A 1, B 1 or C 1 At least one

[0933] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0934] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0935] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0936] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0937] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0938] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0939] When at least one of A, B or C is When A 1 , B 1 or C 1 At least one

[0940] Where R 12 , R 13 and R 14Each is independently selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, alkanyl, cycloalkyl, heterocycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl.

[0941] 53. The preparation method according to technical solution 52,

[0942] in, Cu was added to the reaction + As a catalyst.

[0943] 54. The preparation method according to any one of technical solutions 52-53,

[0944] in, 1-100% equivalent of SrtA ligase was added to the reaction as a catalyst.

[0945] 55. A pharmaceutical composition comprising a compound according to any one of technical solutions 1 to 49 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0946] 56. Use of a compound according to any one of technical schemes 1 to 49 or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition according to technical scheme 55 in the preparation of a medicament for treating and / or preventing tumors.

[0947] 57. According to the use of technical solution 56, the tumor is selected from tumors associated with the expression of the following group: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

[0948] 58. According to the use described in any one of technical solutions 56-57, the tumor is selected from the following group: solid tumors and blood cancers.

[0949] 59. According to the use described in any one of technical solutions 56-58, the tumor is selected from the following group: lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

[0950] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method and use of the present application, and are not intended to limit the scope of the present invention.

[0951] Example

[0952] The materials and equipment used in this application include:

[0953] N3-Amine, TZ-Amine, Boc-Gly-Amine, FITC-Amine, DBCO-Gly, and BCN-Gly were purchased from Xi'an Dianhua Biotechnology Co., Ltd. or Click Chemistry Tools. Other compounds and solvents were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. or Beijing Bailingwei Technology Co., Ltd. unless otherwise specified.

[0954] CPG-Amine nucleic acid was purchased from Suzhou Jinweizhi Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO: 4, specifically: 5`-TCC ATG ACG TTC CTG ACG TT-3`, wherein the 5` thereof contains a C12 amino modification.

[0955] The branched peptide containing CMV antigen was purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd., and its structure is shown in the formula CMV:

[0956]

[0957] The amino acids of the three branches are shown in SEQ ID NO: 5, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0958] All VHH nanobodies were expressed in E. coli.

[0959] The amino acid sequence of the VHH nanobody targeting EGFR is shown in SEQ ID NO: 2, specifically:

[0960] MDEVQLVESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSGGGGSLPETGGHHHHHH;

[0961] The amino acid sequence of the VHH nanobody targeting CD3 is shown in SEQ ID NO: 3, specifically:

[0962] MDEVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMGWFRQAPGKEREFVAAIVWSGGNTYYEDSVKGRFTISRDNAKNTMYLQMTSLKPEDSATYYCAAKIRPYIFKIAGQYDYWGQGTQVTVSSGGGGSGGGGSLPETGGHHHHHH;

[0963] The amino acid sequence of the VHH nanobody targeting 4-1BB is shown in SEQ ID NO: 1, specifically:

[0964] MQVQLQESGGGLVQPGGSLRLSCAASGYTYSSNCMGWFRQAPGKGLEGVAVICTGGGSSPYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADLLRAGTPLSSYEFNYWGQGTLVTVSSGGGGSLPETGGAAALEHHHHHH.

[0965] SrtA ligase is expressed by E. coli. The principle of SrtA ligase-mediated coupling reaction (Chen, Long, et al. Scientific reports 6.1 (2016): 1-12.) is that SrtA ligase can specifically recognize the LPETG sequence shown in SEQ ID NO: 7 and transfer the substrate molecule with naked glycine at the N-terminus to the target protein.

[0966] The neo2 protein of the present application is a fusion protein of cytokines IL2 / IL5, as shown in SEQ ID NO: 8, specifically:

[0967] PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFSGGGGSGGGGSLPETGGHHHHHH.

[0968] The IFNα protein of the present application, as shown in SEQ ID NO: 9, is specifically:

[0969] CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKELPETGHHHHHH. The instruments used included: Waters preparative high performance liquid chromatograph (HPLC), Waters liquid chromatography-mass spectrometry (LC-MS), using a C18 column; size exclusion gel-filtration system, using a Superdex 75HR 16 / 600 column; Bio-red gel imaging system; Bruker400M nuclear magnetic resonance.

[0970] Module preparation method:

[0971] (1) Preparation of EGFR-DBCO

[0972] The preparation of EGFR-DBCO adopts a coupling reaction mediated by SrtA ligase. There is an LPETG sequence on the VHH nanobody targeting EGFR, and the SrtA ligase can connect the substrate molecule DBCO-Gly with a naked glycine at the N-terminus to the EGFR nanobody. Specifically, 1 millimole of DBCO-Gly and 5 micromoles of SrtA ligase are added to 100 micromoles of VHH nanobody PBS solution, and EGFR-DBCO can be obtained after reacting at room temperature for 2 hours. The yield after size exclusion purification is about 80%.

[0973] (2) Preparation of CD3-BCN

[0974] The preparation of CD3-BCN adopts SrtA ligase-mediated coupling reaction. There is LPETG sequence on the VHH nanobody targeting CD3, and SrtA ligase can connect the substrate molecule BCN-Gly with naked glycine at the N-terminus to the CD3 nanobody. 1 mmol of BCN-Gly and 5 mmol of SrtA ligase are added to 100 micromoles of VHH nanobody PBS solution targeting CD3. After reacting at room temperature for 2 hours, CD3-BCN can be obtained. The yield is about 90% after size exclusion purification.

[0975] Example 1 Preparation of multispecific bioconjugate linker

[0976] The method for preparing a multi-specific bioconjugate tether provided in the present application synthesizes tether 1 (Scaffold 1) and tether 2 (Scaffold 2), and the specific reaction formula is as follows:

[0977]

[0978] Step 1: The starting material NHS reagent is reacted with N3-Amine in a ratio of 1 / 1.2. The reaction conditions are DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and the reaction is carried out at 25°C for 2 hours. After the reaction is completed, the solvent is removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography) to obtain the first intermediate N3-NHS-NHS with a yield of 74%.

[0979] Step 2: N3-NHS-NHS and TZ-Amine are reacted in a ratio of 1 / 1.2. The reaction conditions are DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and the reaction is carried out at 25°C for 2 hours. After the reaction, the solvent is removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography) to obtain a second intermediate, which is a multispecific bioconjugated scaffold 2, with a yield of 65%.

[0980] Step 3: The second intermediate, i.e., the multi-specific bioconjugated scaffold 2, is reacted with Boc-Gly-Amine in a ratio of 1 / 1.2. The reaction conditions are DMF (N,N-dimethylformamide) as a solvent, a catalytic amount of TEA (triethylamine) as a catalyst, and a reaction at 25°C for 2 hours. After the reaction, the solvent is removed by rotary evaporation, and then purified by HPLC (high performance liquid chromatography). The purified product is dissolved in DCM (dichloromethane), and then 20% TEA (triethylamine) is added by volume, and the Boc (tert-butyloxycarbonyl) group is removed by reaction at room temperature for 5 minutes. Then, the final product, i.e., the multi-specific bioconjugated scaffold 1 (Scaffold 1), is purified by HPLC (high performance liquid chromatography) with a yield of 48%.

[0981] use 1 The prepared Scaffold 1 and Scaffold 2 were characterized by H-NMR and liquid chromatography-mass spectrometry (ESI-MS). Figure 1-4 The results shown are analyzed as follows:

[0982] Scaffold1 1 The H-NMR characterization results are: 1H NMR(CD3OD,400MHz)δ(ppm):10.28(s,1H),8.45-8.43(d,2H),8.38-8.36 (m,4H),7.89-7.87(d,1H),7.62-7.60(d,1H),7.51-7.49(d,2H),7.43-7. 40(d,1H),7.35-7.33(d,1H),5.45(s,1H),3.84(s,4H),3.64(s,2H),3.6 0-3.38(m,18H),3.26-3.22(m,6H),1.89-1.84(m,6H),1.72-1.66(m,6H). ESI-MS characterization results: C53H82N14O15, 1154.3 (m / z); [M+H] + :1155.5,[M+Na] + :1177.5(m / z).

[0983] Scaffold2 1 The H-NMR characterization results are: 1 H NMR (CD3OD, 400MHz)δ(ppm):10.43(s,1H),8.69(d,1H),8.66(m,2H),8.47-8.45(d,2H),8.35-8.31(m,2H),7.59-7.57(d,2H),7.53-7.47(m,2H),3.88(s,2H),3.67(s,2H),3.61-3.43(m,12H),3.31-3.26(m,4H),1.91-1.84(m,4H),1.74-1.67(m,4H).ESI-MS characterization results:C45H60N12O14,992.4(m / z);[M+H] + :993.4,[M+Na] + :1015.3(m / z).

[0984] use 1 The intermediate N3-NHS-NHS in the synthesis of Scaffold 1 and Scaffold 2 was characterized by H-NMR. Figure 5 The results shown are analyzed as follows: 1H NMR(CD2Cl2,400MHz)δ(ppm):8.97-8.96(t,1H),8.875-8.871(d,2H),7.79-7.77(t,1H),6.92(s,1H),3.93(s,2H),3.69- 3.60(m,8H),3.56-3.54(m,2H),3.46-3.4(m,4H),3.37-3.32(m,2H),2.17(s,8H),1.96-1.90(dd,2H),1.69-1.75(dd,2H).

[0985] Example 2 Preparation of multispecific conjugate EGFR-CD3-FITC

[0986] The multispecific conjugate is synthesized by the method for preparing a multispecific conjugate using a multispecific bioconjugate linker provided in the present application, for example, a multispecific conjugate EGFR-CD3-FITC, and the specific reaction formula is as follows:

[0987]

[0988] The preparation method is as follows: first, the above-mentioned multi-specific bioconjugated scaffold 2 and FITC-Amine are reacted. FITC-Amine (final concentration 10 mmol) is added to a DMF (N, N-dimethylformamide) solution of 1 mmol of scaffold 2, and N3-TZ-FITC is purified by HPLC after reacting at room temperature for 2 hours. Then, N3-TZ-FITC reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromolar, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 1 hour, N3-CD3-FITC is purified by size exclusion chromatography. Finally, N3-CD3-FITC reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration is 100 micromolar, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 2 hours, EGFR-CD3-FITC is purified by size exclusion chromatography.

[0989] EGFR-CD3-FITC was characterized by SDS-PAGE and Coomassie blue staining. Figure 6 As shown, its purity is >90%.

[0990] The results of EGFR-CD3-FITC characterization by LC-MS are as follows Figure 7 shown.

[0991] Example 3 Preparation of multispecific conjugate EGFR-CD3-CPG

[0992] The multispecific conjugate is synthesized by the method for preparing a multispecific conjugate using a multispecific bioconjugate linker provided in the present application, for example, a multispecific conjugate EGFR-CD3-CPG, and the specific reaction formula is as follows:

[0993]

[0994] The preparation method is as follows: first, the above-mentioned multi-specific bioconjugated scaffold 2 and CPG-Amine are reacted. 1 mmol of scaffold 2 is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of CPG-Amine, and N3-TZ-CPG is obtained by HPLC purification after reacting for 2 hours at room temperature. Then, N3-TZ-CPG reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and N3-CD3-CPG is purified by size exclusion chromatography after reacting for 1 hour at room temperature. Finally, N3-CD3-CPG reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and EGFR-CD3-CPG is purified by size exclusion chromatography after reacting for 2 hours at room temperature.

[0995] EGFR-CD3-CPG was characterized by SDS-PAGE and Coomassie blue staining. Figure 8 As shown, its purity is >90%.

[0996] The results of LC-MS characterization of EGFR-CD3-CPG are as follows Fig. 9 shown.

[0997] Example 4 Preparation of multispecific conjugates EGFR-CD3-CMV, EGFR-CD3-neo2 and EGFR-CD3-IFNα

[0998] Multispecific conjugates, such as the multispecific conjugates EGFR-CD3-CMV, EGFR-CD3-neo2 and EGFR-CD3-IFNα, were synthesized by the method for preparing multispecific conjugates using multispecific bioconjugate linkers provided in the present application.

[0999] The preparation method of EGFR-CD3-CMV is to first react the above-mentioned multi-specific bioconjugated linker 1 (Scaffold1) with a branched peptide containing a CMV antigen. 1 millimole of linker 1 (Scaffold1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of branched peptide, and then the above-mentioned 5 micromoles of SrtA ligase is added. After reacting at room temperature for 2 hours, size exclusion purification is used to obtain N3-TZ-CMV. Then, N3-TZ-CMV reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 1 hour, size exclusion chromatography is used to purify N3-CD3-CMV. Finally, N3-CD3-CMV reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 2 hours, size exclusion chromatography is used to purify EGFR-CD3-CMV. The specific reaction formula is as follows: Fig.19 shown.

[1000] The preparation method of EGFR-CD3-neo2 is as follows: first, the above-mentioned multi-specific bioconjugated scaffold 1 is reacted with neo2 having a sequence as shown in SEQ ID NO: 8 of the present application. 1 millimole of scaffold 1 is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of branched peptide, and then the above-mentioned 5 micromoles of SrtA ligase are added. After reacting at room temperature for 2 hours, size exclusion purification is used to obtain N3-TZ-neo2. Then, N3-TZ-neo2 is reacted with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 1 hour, size exclusion chromatography is used to purify N3-CD3-neo2. Finally, N3-CD3-neo2 is reacted with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as the solvent, and after reacting for 2 hours at room temperature, EGFR-CD3-neo2 was purified by size exclusion chromatography.

[1001] The preparation method of EGFR-CD3-IFNα is to first react the above-mentioned multi-specific bioconjugated linker 1 (Scaffold1) with the IFNα sequence shown in SEQ ID NO: 9 of the present application. 1 millimole of linker 1 (Scaffold1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of branched peptide, and then the above-mentioned 5 micromoles of SrtA ligase are added. After reacting at room temperature for 2 hours, size exclusion purification is used to obtain N3-TZ-IFNα. Then, N3-TZ-IFNα and CD3-BCN are reacted at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and after reacting at room temperature for 1 hour, size exclusion chromatography is used to purify N3-CD3-IFNα. Finally, N3-CD3-IFNα and EGFR-DBCO are reacted at a molar ratio of 1 / 1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as the solvent, and after reacting for 2 hours at room temperature, EGFR-CD3-IFNα was purified by size exclusion chromatography.

[1002] EGFR-CD3-CMV was characterized by SDS-PAGE and Coomassie blue staining. Fig.10 As shown, its purity is >90%.

[1003] The results of LC-MS characterization of EGFR-CD3-CMV are as follows Fig.11 shown.

[1004] EGFR-CD3-neo2 was characterized by SDS-PAGE and Coomassie blue staining. Fig.12 As shown, its purity is >90%.

[1005] The results of LC-MS characterization of EGFR-CD3-neo2 are as follows Fig.13 shown.

[1006] EGFR-CD3-IFNα was characterized by SDS-PAGE and Coomassie blue staining. Fig.14 As shown, its purity is >90%.

[1007] The results of EGFR-CD3-IFNα characterization by LC-MS are as follows Fig.15 shown.

[1008] Example 5 Preparation of multispecific conjugate EGFR-CD3-4-1BB

[1009] The multispecific conjugate is synthesized by the method for preparing a multispecific conjugate using a multispecific bioconjugate linker provided in the present application, for example, a trispecific VHH nanobody EGFR-CD3-4-1BB, and the specific reaction formula is as follows: Fig. 20 shown.

[1010] The preparation method is to first react the above-mentioned multi-specific bioconjugated linker 1 (Scaffold1) with the VHH nanobody targeting 4-1BB. 1 millimole of linker 1 (Scaffold1) is added to a PBS (phosphate buffered saline) solution containing 100 micromoles of VHH nanobody, and then the above-mentioned 5 micromoles of SrtA ligase are added. After reacting for 2 hours at room temperature, size exclusion purification is used to obtain N3-TZ-4-1BB. Then, N3-TZ-4-1BB reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromoles, PBS (phosphate buffered saline) is used as a solvent, and N3-CD3-4-1BB is purified by size exclusion chromatography after reacting for 1 hour at room temperature. Finally, N3-CD3-4-1BB reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration was 100 micromolar, PBS (phosphate buffered saline) was used as the solvent, and after reacting for 2 hours at room temperature, EGFR-CD3-4-1BB was purified by size exclusion chromatography.

[1011] EGFR-CD3-4-1BB was characterized by SDS-PAGE and Coomassie blue staining. Fig.16 As shown, its purity is >90%.

[1012] The results of LC-MS characterization of EGFR-CD3-4-1BB are as follows Fig.17 shown.

[1013] Example 6 Multispecific conjugate EGFR-CD3-4-1BB inhibits tumor cell growth

[1014] The application of the EGFR-CD3-4-1BB prepared above in anti-tumor is specifically the killing of tumor cells by PBMCs mediated by EGFR-CD3-4-1BB. PBMCs are from Shanghai Ausail Biotechnology Co., Ltd., and the tumor cells used are A431 cells from ATCC. The specific experimental details are as follows:

[1015] On day 1, A431 cells were seeded in a 96-well plate at a cell seeding density of 10,000 cells per well in a volume of 100 μl. The culture medium used was DMEM containing 20% ​​FBS (fetal bovine serum) and 10% double antibody (penicillin-streptomycin mixture). The culture conditions were a 37°C incubator containing 5% carbon dioxide.

[1016] On the second day, the old culture medium in the 96-well plate was aspirated and 100 microliters of new culture medium was added. Then, 100 microliters of 1640 culture medium (containing 20% ​​FBS (fetal bovine serum), 10% double antibody (penicillin-streptomycin mixture)) containing 150,000 PBMCs was added to each well. Then, different concentrations of EGFR-CD3-4-1BB were added to the experimental group, corresponding concentrations of EGFR-TZ-4-1BB or EGFR-CD3-Gly were added to the control group, and corresponding volumes of PBS (phosphate buffered saline) were added to the blank group. The preparation methods of the control group EGFR-TZ-4-1BB and EGFR-CD3-Gly refer to the above examples and as shown in the following example. Fig.21 shown.

[1017] Specifically, 1 millimole of the connecting arm (Scaffold1) was added to a PBS (phosphate buffered saline) solution containing 100 micromoles of VHH nanoantibodies targeting 4-1BB, and then the above-mentioned 5 micromoles of SrtA ligase were added, and N3-TZ-4-1BB was obtained by size exclusion purification after reacting for 2 hours at room temperature. Then, N3-TZ-4-1BB and EGFR-DBCO were reacted at a molar ratio of 1 / 1. The reaction concentration was 100 micromoles, PBS (phosphate buffered saline) was used as a solvent, and EGFR-TZ-4-1BB was obtained by size exclusion chromatography purification after reacting for 2 hours at room temperature.

[1018] Specifically, the connecting arm (Scaffold1) reacts with CD3-BCN at a molar ratio of 1 / 1. The reaction concentration is 100 micromolar, PBS (phosphate buffered saline) is used as a solvent, and N3-CD3-Gly is purified by size exclusion chromatography after reacting for 1 hour at room temperature. Then, N3-CD3-Gly reacts with EGFR-DBCO at a molar ratio of 1 / 1. The reaction concentration is 100 micromolar, PBS (phosphate buffered saline) is used as a solvent, and EGFR-CD3-Gly is purified by size exclusion chromatography after reacting for 2 hours at room temperature.

[1019] On the third day, the LDH lactate dehydrogenase cytotoxicity assay was used to test the killing effect of the above experimental groups on tumor cells. The kit used was KTA1030 LDH Cytotoxicity Assay Kit LDH Cytotoxicity Assay Kit (Abbkine), and the specific operation steps were carried out according to the instructions provided by the kit. The results are as follows Fig.18 shown.

[1020] The present application uses the above-mentioned embodiments to illustrate the multi-specific bioconjugate linker provided by the present application and its synthesis, the multi-specific conjugate prepared by using the bioconjugate linker, and their preparation methods and uses. However, the present application is not limited to the above-mentioned process steps, that is, it does not mean that the present application must rely on the above-mentioned process steps to be implemented. The technicians in the relevant technical field should understand that any improvement to the present application, the equivalent replacement of the raw materials selected by the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application. Sequence Listing <110> Beijing University <120> Multispecific bioconjugate linker and synthesis method thereof <130> 0198-PA-001 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> 4-1BB VHH <400> 1 Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser 20 25 30 Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly 35 40 45 Val Ala Val Ile Cys Thr Gly Gly Gly Ser Pro Ser Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Asp Leu Leu Arg Ala Gly Thr Pro Leu Ser Ser Tyr Glu 100 105 110 Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Leu Pro Glu Thr Gly Gly Ala Ala Ala Leu Glu His His 130 135 140 His His His His 145 <210> 2 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> EGFR VHH <400> 2 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr 1 5 10 15 Gly Gly Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg 20 25 30 Ser Tyr Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Asp Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Ala Ala Ala Gly Ser Ala Trp Tyr Gly Thr Leu Tyr Glu 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His 130 135 140 His His His His 145 <210> 3 <211> 148 <212> PRT <213> Artificial Sequence <220> <223> CD3 VHH <400> 3 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Pro Val Gln Ala 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Tyr Arg 20 25 30 Gly Tyr Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Val Trp Ser Gly Gly Asn Thr Tyr Tyr Glu Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Met Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr Cys Ala Ala Lys Ile Arg Pro Tyr Ile Phe Lys Ile Ala Gly Gln 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His 130 135 140 His His His His 145 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CPG-Amine <220> <221> misc_feature <222> (1)..(1) <223> C12 amino modification <400> 4 tccatgacgt tcctgacgtt 20 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CMV1 / 2 <400> 5 Asn Leu Val Pro Met Val Ala Thr Val Pro Arg Gly Leu Arg Lys 1 5 10 15 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CMV3 <400> 6 Lys Gly Gly Asn Gln Leu Pro Glu Thr Gly Gly 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> SrtA target <400> 7 Leu Pro Glu Thr Gly 1 5 <210> 8 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> neo2 <400> 8 Pro Lys Lys Lys Ile Gln Leu His Ala Glu His Ala Leu Tyr Asp Ala 1 5 10 15 Leu Met Ile Leu Asn Ile Val Lys Thr Asn Ser Pro Pro Ala Glu Glu 20 25 30 Lys Leu Glu Asp Tyr Ala Phe Asn Phe Glu Leu Ile Leu Glu Glu Ile 35 40 45 Ala Arg Leu Phe Glu Ser Gly Asp Gln Lys Asp Glu Ala Glu Lys Ala 50 55 60 Lys Arg Met Lys Glu Trp Met Lys Arg Ile Lys Thr Thr Ala Ser Glu 65 70 75 80 Asp Glu Gln Glu Glu Met Ala Asn Ala Ile Ile Thr Ile Leu Gln Ser 85 90 95 Trp Ile Phe Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Pro 100 105 110 Glu Thr Gly Gly His His His His His His 115 120 <210> 9 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> IFN alpha <400> 9 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu Leu Pro Glu Thr Gly His His His His His His 165 170 175 <210> 10 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> Her2 Ab <400> 10 Met Val Asp Asn Lys Phe Asn Lys Glu Met Arg Asn Ala Tyr Trp Glu 1 5 10 15 Ile Ala Leu Leu Pro Asn Leu Asn Asn Gln Gln Lys Arg Ala Phe Ile 20 25 30 Arg Ser Leu Tyr Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu 35 40 45 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Gly Leu Pro Glu Thr 50 55 60 Gly Gly Leu Glu His His His His His His 65 70 <210> 11 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> CD47 Ab <400> 11 Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val Ala 1 5 10 15 Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe Pro 20 25 30 Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val Leu 35 40 45 Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val Ser 50 55 60 Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn 65 70 75 80 Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg Lys 85 90 95 Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser 100 105 110 Val Arg Ala Lys Pro Ser Gly Gly Gly Ser Gly Gly Gly Ser Leu Pro 115 120 125 Glu Thr Gly Gly His His His His His His 130 135 <210> 12 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> CD20 Ab <400> 12 Met Asp Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser 20 25 30 Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser 50 55 60 Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly His His His His His His 130 135 140

Claims

1. A compound, or a mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula Ia: in for The W is The La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -; The Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -; The Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -; in, X 1 , X 2 , and X 3 are each independently selected from the group consisting of -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 are each independently selected from the following group: -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl is a 6- to 14-membered aryl group, The A, B and C are each independently selected from the following groups: Among them, A, B and C, Cannot exist at the same time; Among them, A, B and C, Cannot exist at the same time; Among them, A, B and C, Cannot exist at the same time; When A, B and C, and When both exist, the R 12 is not an alkynyl group; When A, B and C, and When both exist, the R 12 Not amino, A, B and C are not the same Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, Represents the junction site.

2. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is or (3) A is B is C is or (4) A is B is C is or (5) A is B is C is or (6) A is B is C is or Where R 12 are each independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, represents the junction site; When A, B and C, When both exist, the R 12 is not an alkynyl group; When A, B and C, When both exist, the R 12 Not amino.

3. The compound according to claim 1, wherein: (1) A is B is C is or (2) A is B is C is Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, represents the junction site; When A, B and C, When both exist, the R 12 Not amino.

4. The compound according to claim 1, wherein: (1) A is B is C is or (2) or A is B is C is Where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, Represents the junction site.

5. The compound according to claim 1, wherein: A is B is C is Where R 12 A group selected from the group consisting of hydrogen, protium, deuterium, tritium, halogen and C1-C 12 Alkyl, Represents the junction site.

6. The compound according to claim 1, which is selected from the following group:

7. The compound according to claim 1, which is selected from the following group:

8. A compound or a mixture thereof, or a pharmaceutically acceptable salt thereof, having a structure as shown in Formula IIa, wherein: in, for The W is The La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -; The Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -; The Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -; in, X 1 , X 2 , and X 3 are each independently selected from the group consisting of -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 are each independently selected from the following group: -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl is a 6- to 14-membered aryl group, The A 2 , B 2 and C 2 Each independently selected from the following group: and covalent bonds; Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect, P 1 , P 2 and P 3 Each is independently selected from the group consisting of lipids, proteins, nucleic acids, small molecules and polysaccharides, or a combination thereof.

9. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or a covalent bond; or (2)A 2 for B 2 for C 2 for or a covalent bond; or (3)A 2 for B 2 for C 2 for or a covalent bond; or (4)A 2 for B 2 for C 2 is a covalent bond; or (5)A 2 for B 2 is a covalent bond, C 2 is a covalent bond; or (6)A 2 for B 2 for C 2 for or a covalent bond; or Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, represents the junction site, The A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P are randomly connected 2 connection, the C 2 The two binding sites of Wc and P are randomly connected 3 connect.

10. The compound according to claim 8, wherein: (1)A 2 for B 2 for C 2 for or a covalent bond; or (2)A 2 for B 2 for C 2 for or covalent bonds; Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl, represents the connection site, the A 2 The two binding sites of Wc and P are randomly connected 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

11. The compound according to claim 8, wherein: A 2 for B 2 for, C 2 for or covalent bond; where R 12 Selected from the group consisting of hydrogen, protium, deuterium, tritium, halogens and C1-C 12 Alkyl, represents the connection site, the A 2 The two binding sites of Wc and P 1 Connect the B 2 The two binding sites of Wc and P 2 connection, the C 2 The two binding sites of Wc and P 3 connect.

12. The compound according to claim 8, wherein P 1 , P 2 and P 3 Each is independently selected from the group consisting of a protein, a polypeptide, a nucleic acid and a small molecule.

13. The compound according to claim 8, wherein P 1 , P 2 and P 3 Each is independently selected from the group consisting of a dye molecule, a cytokine, an antigen and an antibody.

14. The compound of claim 13, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and a nanobody.

15. The compound of claim 13, wherein the antibody targets a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47, and CD20.

16. The compound of claim 13, wherein the amino acid sequence of the antibody is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO:

12.

17. The compound according to claim 8, which is selected from the following group: in, P 1 , P 2 and P 3 Each is independently selected from antibodies targeting a target selected from the group consisting of 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

18. The compound according to claim 8, which is: in, P 1 , P 2 and P 3 The amino acid sequences are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 3, or SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NO:

3.

19. A method for preparing the compound according to any one of claims 1 to 7, comprising: reacting the compound represented by formula (Ia-I) with reactant 1, reactant 2 and reactant 3; or reacting the compound represented by formula (Ia-I) with reactant 1 and reactant 2; or reacting the compound represented by formula (Ia-I) with reactant 1; The reactant 1 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -A, The reactant 2 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -B, The reactant 3 is HX 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -C, in, X 1 , X 2 , and X 3 are each independently selected from the group consisting of -NH- and -O-, (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each is independently selected from the following group: -(CH2)-(CH2-O-CH2)3-(CH2)- and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2 or n3 are each independently 0, (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0, (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 are each independently selected from the following group: -CH2-aryl-, -(CH2)2- and -CH2-, or q1, q2 and q3 are each independently 0, wherein the aryl is a 6- to 14-membered aryl group, The W is A, B and C are each independently selected from the following group: Among them, A, B and C, Cannot exist at the same time; Among them, A, B and C, Cannot exist at the same time; Among them, A, B and C, Cannot exist at the same time; When A, B and C, When both exist, the R 12 is not an alkynyl group; When A, B and C, When both exist, the R 12 Not amino, A, B and C are not the same Among them, when A, B, or C is When the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, Replace with After the compound represented by the formula (Ia-I) reacts with reactant 1, reactant 2 or reactant 3, an acid is added to remove Boc.

20. The preparation method according to claim 19, wherein the order of adding reactant 1, reactant 2 and reactant 3 is determined according to the order of introducing A, B and C, and the order of introducing A, B and C from first to last is: Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl.

21. A method for preparing the compound according to any one of claims 8 to 18, comprising: reacting the compound of formula Ia according to any one of claims 1 to 7 with reactant 4, reactant 5 and reactant 6; The reactant 4 is A 1 -P 1 , The reactant 5 is B 1 -P 2 , The reactant 6 is C 1 -P 3 , Among them, A of reactant 4 1 React with A of the compound described in Formula Ia, B of the reactant 5 1 With the compound B of formula Ia, C of reactant 6 1 Reaction with C of the compound described in Formula Ia; When at least one of A, B or C is When A 1 , B 1 or C 1 At least one When at least one of A, B or C is When A 1 , B 1 or C 1 At least one When at least one of A, B or C is When A 1 , B 1 or C 1 At least one When at least one of A, B or C is When A 1 , B 1 or C 1 At least one When at least one of A, B or C is When A 1 , B 1 or C 1 At least one When at least one of A, B or C is When A 1 , B 1 or C 1 At least one Where R 12 Selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, C3-C 12 Cycloalkyl, 3- to 7-membered heterocycloalkyl, C2-C6 alkenyl, ethynyl, 1-propynyl, propargyl, butynyl, 6- to 14-membered aryl, and 5- to 10-membered heteroaryl.

22. The preparation method according to claim 21, in, 1-100% equivalent of SrtA ligase was added to the reaction as a catalyst.

23. A pharmaceutical composition comprising a compound according to any one of claims 8 to 18 or a mixture thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

24. Use of a compound according to any one of claims 8 to 18 or a mixture thereof, or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition according to claim 23 in the preparation of a medicament for treating and / or preventing tumors.

25. The use according to claim 24, wherein the tumor is selected from tumors associated with the following group of expressions: 4-1BB, EGFR, CD3, Her2, CD47 and CD20.

26. The use according to claim 24, wherein the tumor is selected from the group consisting of solid tumors and blood cancers.

27. The use according to claim 24, wherein the tumor is selected from the group consisting of lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer and esophageal cancer.

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