RU2798981C2 - Antibody conjugate with non-natural amatoxin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions including a conjugate of a non-natural amatoxin with an antibody having antitumor or anticancer activity, and the use of the conjugate for the production of an antitumor drug or an anticancer drug are described. In one embodiment, the conjugate has the structural formula (I).

EFFECT: invention expands the arsenal of agents with antitumor or anticancer activity.

13 cl, 4 tbl, 8 ex

Description

The field of technology to which the invention belongs

The present invention relates to a bicyclic octapeptide derivative which can be conjugated to an appropriate target-binding group in a specific chemical structure. This structure is stable in blood plasma and degrades to the active pharmaceutical ingredient in a specific biological environment, thus maximizing the killing effects on target cells and minimizing toxic side effects on non-target cells, which can be used to treat various malignancies.

Prerequisites for the creation of the invention

Amanitin is one of the peptide amanitatoxins isolated from deadly poisonous mushrooms and is a bicyclic peptide consisting of eight amino acids. There are nine natural amanitins that have been isolated and purified, which are respectively α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, β-amanitin are the main toxins causing death. Amanitin is a class of slowly acting toxins and can inhibit the transcription of eukaryotic RNA polymerase II and RNA polymerase III, leading to protein deficiency and cell death. This class of toxins has an extremely high inhibitory effect on RNA polymerase II and its KD can be as high as 3 nM. Toxins are periodically absorbed into the body as a result of enterohepatic circulation in the gastrointestinal tract and can cause permanent serious damage to organs such as the liver, kidneys, heart and lungs.

Upon conjugation of amanitin to a large biomolecular carrier (such as an antibody molecule), the toxicity of amanitin is substantially reduced and amanitin becomes even relatively non-toxic; and the cytotoxicity of amanitin can only be observed after removal of the biomolecular carrier under specific physiological conditions.

According to studies by Theodor Wieland's group, when the sulfoxide structure in natural amanitin is replaced with elemental sulfur, the cellular toxicity of the non-natural amanitin thus formed does not change much. The natural amanitin molecule was conjugated with a monoclonal antibody by Heidelberg Pharmaceutical Co., Ltd., Germany, resulting in a drug molecule having antitumor activities.

In the present invention, non-natural amanitin, similar in toxicity to natural amanitin, is conjugated to a biomolecule capable of binding to a target through a biopharmaceutically acceptable binding structure, to thereby obtain a compound that is stable in plasma and can effectively kill tumor cells in cells.

Definitions

, используемый в формулах и таблицах, представляет собой связь, использующуюся как точка для присоединения фрагмента или заместителя к ядру структуры соединения.In accordance with the standards and practice in the art, the symbol

, used in formulas and tables, is a bond used as a point for attaching a fragment or substituent to the core of the compound structure.

In accordance with the standards and practice in the art, hetero (atom, alkyl, aryl, cyclo) refers to the corresponding chemical structure containing an atom (or atoms) other than carbon.

The essence of the invention

The present invention provides a conjugate of a bicyclic octapeptide amanitin derivative and a biomacromolecule, which is stable in the bloodstream and is cleaved after endocytosis by target cells, while releasing the amanitin derivative as an RNA polymerase inhibitor, and exhibits strong toxicity in cells by specifically inhibiting mRNA synthesis in eukaryotes, and the conjugate is in particular a toxin conjugate of structural formula (I):

Where:

R ¹ is H, -OH or -OLA;

R ² is H and -OH;

R ³ is OC _1-6 alkyl;

R ⁴ is H or -LA;

R ⁵ is -NH ₂ , -OH, -NH-LA or -OLA;

where O is an oxygen atom, N is a nitrogen atom, and H is a hydrogen atom.

A is a biomacromolecular fragment that binds to a target site;

as a preferred embodiment, L includes the following structure:

-L ₁ -[L ₂ ] _m -[AA] _n -L ₃ -

where: L ₁ is a linker for binding to biomacromolecule A; L ₂ is a spacer, and L ₂ is a fragment that binds L ₁ to AA; m is an integer 1-6; L ₃ binds to the toxin shown in the structural formula (I); AA is a fragment of 1-6 amino acids and n is 0 or 1.

As a preferred embodiment, A is a biomacromolecular fragment that binds to a target site and includes an antibody or antigen-binding fragments thereof, an antibody-like protein, a nucleic acid aptamer, etc.

As a preferred embodiment, the target site-binding biomacromolecule is an antibody, or an antigen-binding fragment thereof, which is selected from a chimeric antibody, a deimmunized antibody, a humanized antibody, a human antibody, a diabody, a triabody, and a nanobody.

Even more preferably, the antigen-binding moiety is selected from the group consisting of Fab, F(ab'), Fd, Fv, single chain Fv and disulfide-linked Fv(dsFv).

As a preferred embodiment, one and only one of R ¹ , R ⁴ and R ⁵ contains the -LA structure.

As a preferred embodiment, L ₁ is a linker for binding to biomacromolecule A, and is selected from:

etc.; where the wavy line connects with biomacromolecule A.

As a preferred embodiment, L ₂ is a spacer selected from one or more of the same or different combinations of substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heterocycloalkyl, substituted or unsubstituted C5-C20 aryl, substituted or unsubstituted C5-C20 heteroaryl, and -(CH ₂ CH ₂ O) _a - (where a is an integer from 1 to 20), and the spacers are connected to each other through an appropriate chemical bond.

Even more preferably, the substituent is selected from one or more of the same or different combinations of hydroxyl group, sulfhydryl group, halogen, carboxyl group, amino group, phosphate group, nitro group, cyano group, sulfo group, substituted or unsubstituted C1-C6 alkyl, etc. .

Even more preferably, the substituent is selected from one or more of the same or different combinations of hydroxyl group, sulfhydryl group, halogen, carboxyl group, amino group, phosphate group, nitro group, cyano group, sulfonic group, etc.

As a preferred embodiment, AA is a fragment consisting of 1-6 amino acids, which are L-amino acids selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine , asparagine, glutamine, threonine, aspartate, glutamate, lysine, arginine, histidine, etc.,

and even more preferably phenylalanine, citrulline, valine, lysine, serine, glutamate, aspartate and glycine.

As a preferred embodiment, L ₃ is selected from

any linking group that binds to a toxin, etc.,

where the wavy line binds to AA and the asterisk binds to the toxin shown in structural formula (I).

Even more preferably, L4 is selected from a carbonyl group or a single bond.

Even more preferably, R6 and R7 are each independently selected from hydrogen, C1-C6 alkyl, etc.

Even more preferably, L5 is C2-C12 alkyl.

As a preferred embodiment, the drug contains a toxin conjugate of structural formula (I) described above, or a salt thereof.

As a preferred embodiment, the use of a toxin conjugate of structural formula (I) described above, or a salt thereof, for the preparation of an antitumor drug or an anticancer drug is disclosed.

As a preferred embodiment, the anticancer drug or anticancer drug is lung cancer drug, kidney cancer drug, urethral cancer drug, colorectal cancer drug, prostate cancer drug, glioblastoma drug , ovarian cancer drug, pancreatic cancer drug, breast cancer drug, melanoma drug, liver cancer drug, bladder cancer drug, malignant lymphoma drug, leukemia drug, medicines for stomach cancer or a medicine for cancer of the esophagus.

Common abbreviations and symbols: G: gram mg: milligram min: minutes ml: milliliter they say: molar °C: degrees Celsius boc: tert-butoxycarbonyl Pybop: 1H-benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Citation: citrulline _CO2 : carbon dioxide DCM: dichloromethane DIPEA: diisopropylethylamine DMF: dimethylformamide DMSO: dimethyl sulfoxide DPBS: Dulbecco's phosphate buffer solution DTPA: diethylenetriaminepentaacetic acid DTT: dithiothreitol EA: ethyl acetate EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum HATU: O-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate _H2O : water HOBt: 1-hydroxybenzotriazole mAbs: monoclonal antibody MEM: Minimum culture medium MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium internal salt MTT: 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide PAB: para-aminobenzyloxy PBS: phosphate buffer solution Sodium pyruvate: Na pyruvate THF: tetrahydrofuran TLC: thin layer chromatography tris: trishydroxymethylaminomethane Val: valine Trt-Cl: triphenylmethyl chloride TBTU: O-(1H-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate HOBT: 1-hydroxybenzotriazole TFA: trifluoroacetic acid TBS-Cl: tert-butyldimethylsilyl chloride HOSu: N-hydroxysuccinimide _{Na2CO3 :} sodium carbonate EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride CuBr: copper bromide

Detailed description of embodiments

The present invention is further illustrated by combining the specific examples below. These examples are used only to describe the present invention and not to limit the scope of the present invention. Unless otherwise indicated, all professional and scientific terms used have the same meanings as commonly known to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described in this application, all can be used in the method of the present invention. The preferred embodiments and materials described are for illustrative purposes only.

Example 1 Synthesis of low molecular load ama-0301

1) Solid phase synthesis of intermediate 08

The N-fluorenylmethoxycarbonyl-O-tert-butyl-L-hydroxyproline-preloaded resin used as starting material was treated with 20% piperidine solution (adding 20 ml of 20% piperidine solution to 1 g of resin) to remove the Fmoc protecting group and washed at with DMF 5 times until a neutral pH is reached. Then, to DMF, which was used as a solution (20 ml/g), Fmoc-N-propynyl-L-asparagine (Fmoc-Asn(Trt)-OH) (3 eq.), TBTU (2.5 eq. ), HOBT (1.8 eq.) and DIPEA (6 eq.), the mixture was allowed to react for 2 h at room temperature (28°C) and then washed 3 times with DMF (each time adding 20 ml of DMF to 1 g resin), followed by binding with amino acids in accordance with the previous procedure. After completion of the final binding, the resulting compound was cleaved from the resin using a 1% TFA solution in dichloromethane (each time adding 20 ml to 1 g of resin; 1% TFA for 5 min; repeated three times); this solution was removed on a rotary evaporator; and stirring was carried out with methyl t -butyl ether to crystallize, thereby obtaining compound 08 with an overall yield of about 43% and a high purity of 81.3%. MS: [M+H] ^+1244.6521 .

2) Synthesis of compound 09

8 g of compound 08, crude product, was dissolved using TFA (10 ml/g) and then stirred and reacted for 5 h at room temperature; TFA was removed under reduced pressure at 50°C; and carried out the purification using preparative liquid chromatography to obtain about 3.8 g of pure product compound 09 with a yield of 71% and a purity of 96.4%. MS: [M+H] ^+828.3241 .

3) Synthesis of compound 14

2.94 g of (4S)-hydroxylysoleucine, 40 ml of 1,4-dioxane and 40 ml of saturated sodium carbonate solution were added to a 250 ml one-necked flask and stirred homogeneously, followed by addition of Fmoc-OSu portionwise. After 10 minutes, stirring was continued at room temperature for 12 h until the interaction of the starting materials was completed. 50 ml of water was added to the reaction liquid, and a 5% citric acid solution was used to adjust the pH to about 4. Ethyl acetate was used for extraction, 3 times (50 ml each time). The organic layer was collected, washed once with 50 ml brine, dried over anhydrous sodium sulfate and concentrated to give a pale yellow oil which was directly used in the next step without purification in >100% yield.

4) Synthesis of compound 15

The crude product of compound 13 above was dissolved using 40 ml of DMF and then 2.68 g (2 eq.) of imidazole was added followed by portionwise addition of TBS-Cl; after that, stirring was carried out at room temperature for 12 h until the interaction of the initial substances was completed. 50 ml of water and 50 ml of ethyl acetate were added and stirred. The organic layer was separated and the aqueous layer was extracted twice with ethyl acetate (each time 50 ml). The organic layer was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow oil, which was subjected to silica gel column chromatography (elution: PE:EA=5:1) to give 4.5 g of an oil with a yield of about 46, 6% for two stages.

5) Synthesis of compound 10

Compound 14, HOSu (1.23 g, 1.15 eq.), DCC (2.23 g, 1.15 eq.) and 50 ml THF were added sequentially to a 250 ml one-necked flask and stirred at room temperature for 6 h in a nitrogen atmosphere. After completion of the reaction, 50 ml of water and 50 ml of ethyl acetate were added and stirred for 10 minutes, and then the organic layer was separated. The aqueous layer was then extracted twice with ethyl acetate (50 ml each) and the organic layer was combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a pale yellow oil which was purified by preparative liquid chromatography to give about 3.24 g of a white foamy solid. substances with a yield of 60%. 1H-NMR (400 MHz, DMSO- d ⁶ ): 0.08 (s, 6H), 0.86 (s, 9H), 0.98 (d, 3H, J=8.0 Hz), 1.06 (d, 3H, J=5.6), 1.95 (t, J=10.8), 2.83 (s, 4H), 4.21 (dd, 1H, J=16.8 Hz, 8 .0 Hz), 4.34 (dd, 1H, J=12 Hz, 4 Hz), 4.67-4.73 (m, 1H), 7.31 (d, 2H, J=8.0 Hz) , 7.34-7.46 (m, 2H), 7.70-7.76 (m, 2H), 7.89 (t, 2H, J=12.0 Hz), 8.24 (d, 1H , J=8.8 Hz); MS: 581.34[M+H].

6) Synthesis of compound 11

0.5 g of compound 09 was dissolved using 1.5 ml of anhydrous DMF and then compound 10 (701 mg, 2 eq.) was added. The pH was adjusted to 8-9 with DIPEA; the reaction was carried out under nitrogen atmosphere for 5 hours at room temperature and monitored by HPLC until the reaction of the starting substance 09 was essentially complete. The product was directly used in the next step without further processing.

7) Synthesis of compound 12

0.3 ml (20%) of piperidine was added to the above reaction liquid; the reaction mixture was stirred for 2 hours at room temperature and the stirring was stopped when the reaction of the starting materials was completed (monitored by HPLC). For purification, preparative liquid chromatography (neutral conditions, acetonitrile/pure aqueous system) was used to collect the fraction corresponding to the target peak; after removal of the acetonitrile under reduced pressure, 277 mg of a white powdery solid was obtained by lyophilization in a yield of about 42.8% over two steps. MS: [M+H] ⁺ 1071.5120.

8) Synthesis of compound 13

270 mg of compound 12 was dissolved using anhydrous DMF. EDCI (96.6 mg, 2 eq) was added; HOBT (170 mg, 5 eq.) and DIPEA (0.22 ml, 5 eq.) and stirred for 4 h at room temperature until the reaction was complete (monitored by HPLC). For purification, preparative liquid chromatography (neutral conditions, acetonitrile/pure aqueous system) was used to collect the fraction corresponding to the target peak; after removal of the acetonitrile under reduced pressure, about 136.4 mg of a white powdery solid was obtained by lyophilization in a yield of 51.4% and MS: [M+H] ⁺ 1053.4908.

9) Synthesis of compound 16

1 g of N-Succinimidyl-6-maleimidohexanoate was dissolved using 10 ml of tetrahydrofuran and then 0.678 g of 2-[2-(2-azidoethoxy)ethoxy]ethylamine (1.2 eq.) was added; stirred at room temperature for 4 hours under nitrogen until the reaction was complete (monitored by TLC); then water was added and EA was used for extraction 3 times (each time 10 ml). The organic layer was combined, dried, concentrated and purified by column chromatography to give about 0.8 g of the desired product in 84% yield.

10) Synthesis of compound 17

80 mg of compound 16 was dissolved using 5 ml of DMSO, compound 13 (114.7 mg, 0.5 eq.), 44.7 mg (1.5 eq.) of copper bromide and 0.2 ml of purified water was added; stirring was carried out for 3 h at room temperature in a nitrogen atmosphere until the reaction of compound 13 was completed (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak, and the organic solvent was removed on a rotary evaporator; received about 50.5 mg of a white solid by lyophilization with a yield of 32.6%. MS: [M+H] ^+1420.7031 .

11) Synthesis of compound ama-0301

To 45 mg of compound 17 was added 1 ml of 5% TFA/MeOH to achieve clarification upon dissolution. The reaction was carried out at room temperature for 1 hour under a nitrogen atmosphere. After completion of the reaction of the starting material 17 (monitored by HPLC) and removal of the solvent by purging with nitrogen, purification was carried out using preparative liquid chromatography and the organic solvent was concentrated and removed to obtain 15.4 mg of a white solid by lyophilization with a yield of 37.2%. MS: [M+H] ⁺ 1306.6013.

Example 2 Synthesis of low molecular load ama-0302

1) Solid phase synthesis of key intermediate 24

Prepared with reference to the synthesis of compound 08. After crystallization with methyl tert -butyl ether, about 10.6 g of a tan solid was obtained with a high purity of about 79.8%.

2) Synthesis of compound 25

The synthesis was carried out with reference to the synthesis of compound 09. The crude product of compound 24 was dissolved using 50 ml of TFA and stirred at room temperature for 5 h under nitrogen until the reaction of the starting materials was essentially complete, according to HPLC; purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak. After the organic solvent was removed under reduced pressure, 2.38 g of a white solid was obtained by lyophilization. MS: [M+H] ^+831.4251 .

3) Synthesis of compound 26

The synthesis was carried out with reference to the synthesis of compound 11. 1.0 g of starting material 25 was added and about 820 mg of a white solid was obtained by lyophilization in a yield of 52.5%. MS: [M+H] ^+1296.6431 .

4) Synthesis of compound 27

The synthesis was carried out with reference to the synthesis of compound 12. 800 mg of starting material 26 was added and about 308.8 mg of a white solid was obtained by lyophilization in a yield of about 46.6%. MS: [M+H] ^+1074.5184 .

5) Synthesis of compound 28

The synthesis was carried out with reference to the synthesis of compound 13. 300 mg of starting material 27 was added and about 208.4 mg of a white solid was obtained by lyophilization in a yield of about 70.6%. MS: [M+H] ^+1056.5243 .

6) Synthesis of compound 29

100 mg of compound 28 was dissolved using 1 ml of 5% TFA solution in methanol; stirring was carried out for 2 hours at room temperature under nitrogen until the reaction of the starting materials was complete (monitored by HPLC); Purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak, and received about 69.5 mg of a pale yellow solid by lyophilization with a yield of 77.9%. MS: [M+H]+ 942.4571.

7) Synthesis of compound 30

50 mg of compound 29 was added to a 10 ml reaction flask and 67.5 mg (1.1 eq.) tetrakis(triphenylphosphine palladium) was added under nitrogen atmosphere. The air was replaced with nitrogen, and 5 ml of anhydrous tetrahydrofuran and 0.1 ml of anhydrous morpholine were added to the reaction flask via a syringe. The starting materials were dissolved and stirred for 12 hours at room temperature until the reaction of the starting materials was complete (monitored by HPLC). Purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak, and received about 35.2 mg of a pale yellow solid by lyophilization with a yield of 73.5%. MS: [M+H] ^+902.4123 .

8) Synthesis of compound 31

Fmoc-L-valine (20 g) and HoSu (7.46 g, 1.1 eq.) were dissolved using 200 ml THF; then the reaction flask was placed in a bath of ice salt and cooled to 0°C. The DCC condensing agent (14.6 g, 1.1 eq.) was added slowly and the reaction temperature was controlled at 0-5° C., the addition being completed within 3 hours. The resulting material was removed from the ice bath and stirred and reacted for 12 hours. The reaction was not stopped until the reaction of Fmoc-L-valine was completed (monitored by TLC). Suction filtration was carried out under reduced pressure and the filter cake was washed with 100 ml of THF. The filtrate was dried on a rotary evaporator. 100 ml of DCM was added to the residue, dissolved with stirring at 35°C, filtered to remove a small amount of insoluble matter using an organic membrane filter, and then placed in an oil bath at 35°C. Added 100 ml of petroleum ether with stirring; crystallization was carried out by natural cooling for 1 hour and then cooling in an ice-salt bath for 2 hours. Suction filtration was carried out and the solid was washed with petroleum ether and dried in a vacuum oven at 40° C. to give 21.86 g of a white powdery solid in about 85% yield.

9) Synthesis of compound 32

20 g of compound 31 was dissolved in 200 ml of THF as solvent and 9.64 g (1.2 eq.) of L-citrulline was added; added 1 M sodium carbonate to bring the pH to 8-9, and clarification of the soluble reaction mixture was not achieved. Stirring was carried out for 48 hours at room temperature until the reaction was complete (monitored by TLC). While stirring in an ice bath, the reaction liquid was adjusted to pH 3-4 using an aqueous citric acid solution and extracted 3 times with isopropanol:EA=1:5 (40 ml isopropanol+200 ml EA). The organic phase was combined, dried over anhydrous sodium sulfate, filtered and subjected to drying on a rotary evaporator, followed by the addition of 200 ml of methyl t -butyl ether and stirring for 2 hours; suction filtration was then carried out to collect the filter cake, which was dried in a vacuum oven at 45° C., to give 18.6 g of a white solid in about 81.7% yield.

10) Synthesis of compound 33

18 g of compound 32 was added to a 500 ml reaction flask and dissolved with 200 ml of DMF; then 8 g (1.0 eq.) of 4-[(N-tert-butoxycarbonyl)aminomethyl]aniline, 20 g (1.5 eq.) of HATU and DIPEA (18 ml, 3 eq.) were added successively and stirred for 24 h at room temperature. After completion of the reaction of the starting materials (monitored by TLC), 200 ml of water and 200 ml of dichloromethane were added and stirred for 10 minutes. Then the organic layer was separated and washed twice with water (50 ml each time). The organic layer was collected, dried over anhydrous sodium sulfate and then filtered and concentrated to give a brown oil. To this oil was added 200 ml of methyl tert -butyl ether and stirred for 30 minutes; then the solid was washed and filtered and the filter cake was washed twice with methyl t -butyl ether (50 ml each time). The filter cake was collected and dried in a vacuum oven at 50° C. to give 18.9 g of a brown solid in 74.4% yield.

11) Synthesis of compound 34

15 g of compound 33 was added to a 250 ml reaction flask, to which was added 75 ml of a 20% solution of piperidine in DMF; stirring was carried out for 1 h at room temperature until the reaction of the starting materials was completed (monitored by TLC). The solvent was distilled off under reduced pressure using an oil pump, and then 200 ml of methyl t -butyl ether was added and stirred for 2 hours at room temperature. A brown solid precipitated and filtered. The filter cake was washed twice with methyl tert -butyl ether (50 ml each), collected and dried in a vacuum oven at 50° C. to give 6.8 g of a brown solid in 66.4% yield.

12) Synthesis of compound 35

1 g of compound 34 was dissolved using 10 ml of DMF and then 773 mg of N-succinimidyl-6-maleimidohexanoate (1.2 eq.) was added; stirring was carried out for 5 hours at room temperature until the reaction of the starting materials was completed (monitored by TLC). 10 ml of water and 20 ml of ethyl acetate were added to the reaction liquid, and stirred for 10 minutes. The organic layer was then separated, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography (petroleum ether:ethyl acetate=50:1) to give 956 mg of a pale yellow foamy solid in 68.3% yield.

13) Synthesis of compound 36

30 mg of compound 35 was dissolved using a 20% TFA solution in dichloromethane and then stirred for 2 h at room temperature until the reaction was complete (monitored by TLC). The solvent was removed on a rotary evaporator under reduced pressure to give a crude product, compound 36, which was used directly in the next step without purification.

14) Synthesis ama-0302

The crude product of compound 36 (1.2 eq.) obtained in the previous step was dissolved using 1 ml of DMF; then 32.5 mg of compound 30 and 20.5 mg of HATU (1.5 eq.) were added and the pH was adjusted to 8-9 with DIPEA; stirring was carried out for 6 hours at room temperature under nitrogen until the reaction of starting material 30 was complete (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak. After lyophilization, 15.3 mg of a pale yellow solid was obtained with a yield of 29.1%. MS: [M+H]+ 1455.7011.

Example 3 Synthesis of low molecular load ama-0303

1) Synthesis of compound 37

The synthesis was carried out with reference to the synthesis method described in J. Am. Chem. soc. 2018, 140, 6513-6517.

2) Synthesis of compound 38

The synthesis was carried out with reference to the synthesis of compound 11. After lyophilization, about 751.5 mg of the target compound were obtained with a yield of 56.8%. MS: [M+H]+ 1426.6751.

3) Synthesis of compound 39

The synthesis was carried out with reference to the synthesis of compound 12. 700 mg of compound 38 were added and about 482.3 mg of a white solid was obtained by lyophilization in a yield of 81.6%. MS: [M+H]+ 1205.6124.

4) Synthesis of compound 40

The synthesis was carried out with reference to the synthesis of compound 13. 450 mg of compound 39 was added. After purification using preparative liquid chromatography, about 384.2 mg of a white solid was obtained by lyophilization in a yield of 86.7%. [M+H]+ 1186.6012.

5) Synthesis of compound 41

The synthesis was carried out with reference to the synthesis of compound 29. 100 mg of compound 40 was added; after purification using preparative liquid chromatography, about 54.2 mg of a white solid was obtained by lyophilization in a yield of about 67.2%. [M+H]+ 958.4250.

6) Synthesis of compound 42

The synthesis was carried out with reference to the synthesis of compound 30. 50 mg of compound 41 was added; after purification using preparative liquid chromatography, about 28.6 mg of a white solid was obtained by lyophilization of the target peak, with a yield of 59.7%. [M+H]+ 918.4413.

7) Synthesis of compound ama-0303

The synthesis was carried out with reference to the synthesis of compound ama-0302. 25 mg of compound 42 was added; after purification using preparative liquid chromatography, about 15.2 mg of a pale yellow solid was obtained by lyophilization in a yield of 38%. [M+H]+ 1471.6820.

Example 4 Synthesis of low molecular load ama-0304

1) Synthesis of compound 49

As for the synthesis method, it was carried out with reference to the synthesis of compound 08; obtained about 2.4 g of the crude product of compound 49 with a purity of about 86.2%, which was directly used in the next stage without purification.

2) Synthesis of compound 50

The synthesis was carried out with reference to the synthesis of compound 09; after purification using preparative liquid chromatography, about 1.1 g of the title compound was obtained. [M+H]+ 790.4126.

3) Synthesis of compound 51

The synthesis was carried out with reference to the synthesis of compound 11. 500 mg of compound 50 was added; after purification using preparative liquid chromatography, 341.5 mg of the title compound were obtained in a yield of 43%. [M+H]+ 1255.6195.

4) Synthesis of compound 52

The synthesis was carried out with reference to the synthesis of compound 12. 300 mg of compound 51 was added; after purification using preparative liquid chromatography, about 186.4 mg of a white solid was obtained by lyophilization of the fraction corresponding to the target peak in a yield of 75.5%. [M+H]+ 1033.5013.

5) Synthesis of compound 53

The synthesis was carried out with reference to the synthesis of compound 13. 150 mg of compound 52 was added; after purification using preparative liquid chromatography, about 86.5 mg of a white solid was obtained by lyophilization of the fraction corresponding to the target peak in a yield of 58.7%. [M+H]+ 1015.5121.

6) Synthesis of compound 54

80 mg of compound 53 was dissolved using anhydrous DMSO; then 159 mg of tert-butyl-N-(4-bromobutyl)carbamic acid (8 eq.) and 88 mg of potassium tert-butoxide (10 eq.) were added; the reaction mixture was stirred for 12 h at room temperature and then 159 mg of tert-butyl-N-(4-bromobutyl)carbamic acid (8 eq.) and 88 mg of potassium tert-butoxide (10 eq.) were added. The reaction mixture was stirred for another 24 h at room temperature until the disappearance of the starting materials (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak; and received 18.2 mg with a yield of 19.5%. [M+H]+ 1186.6137.

7) Synthesis of compound 55

18.2 mg of compound 54 obtained as described above was dissolved using 0.1 ml of trifluoroacetic acid and stirred for 30 minutes at room temperature. 2 ml dichloromethane was added; after stirring until homogeneity, the solvent was removed on a rotary evaporator under reduced pressure and the resulting material was stored for later use.

8) Synthesis of compound ama-0304

The crude product of compound 55, obtained as described above, was dissolved using 1 ml of DMF and then 9.5 mg of N-succinimidyl-6-maleimidohexanoate (2 eq.) was added. The pH was adjusted to 8-9 with DIPEA; stirring was carried out under nitrogen atmosphere for 5 h at room temperature until the reaction of the starting materials was completed (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak. After removal of the organic solvent on a rotary evaporator, 10.4 mg of an off-white solid was obtained by lyophilization in 53% yield. [M+H] ⁺ 1279.6537.

Example 5 Synthesis of low molecular load ama-0305

1) Synthesis of compound 56

The synthesis was carried out with reference to the synthesis of compound 11. 200 mg of starting material 50 was added and about 211.3 mg of a white solid was obtained by lyophilization in a yield of 60.2%. [M+H] ⁺ 1385.6713.

2) Synthesis of compound 57

The synthesis was carried out with reference to the synthesis of compound 12. 200 mg of starting material 56 was added and about 114.8 mg of a white solid was obtained by lyophilization in a yield of 68.3%. [M+H] ⁺ 1163.5793.

3) Synthesis of compound 58

The synthesis was carried out with reference to the synthesis of compound 13. 110 mg of starting material 57 was added and about 72.5 mg of an off-white solid was obtained by lyophilization in a yield of 66.9%. [M+H] ⁺ 1145.5901.

4) Synthesis of compound 59

About 70 mg of compound 58, obtained as described above, was dissolved using 0.5 ml of a solution of TFA in methanol; then stirring was carried out for 1 hour at room temperature until the reaction of the starting materials was completed (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak. After removal of the organic solvent on a rotary evaporator, 45.7 mg of an off-white solid was obtained by lyophilization in a yield of 81.6%. [M+H] ⁺ 917.4013.

5) Synthesis of compound 60

The synthesis was carried out with reference to the method described in patent WO2012041504

6) Synthesis of compound 61

About 42 mg of starting material 59 was dissolved using 1 ml of anhydrous DMF; then 22 mg of compound 60 (2 eq.) and 58 mg of dibutyltin dilaurate were added; followed by stirring for 24 hours at room temperature under a nitrogen atmosphere. Again 22 mg of compound 60 (2 eq) was added and stirring was continued for 52 h at room temperature until the starting material 59 disappeared (monitored by HPLC). 0.2 ml of methanol was added to the above reaction liquid to stop the reaction; purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak. The organic solvent was removed on a rotary evaporator and about 9.6 mg of a pale yellow solid was obtained by lyophilization in a yield of 18.1%. [M+H] ⁺ 1159.5703.

7) Synthesis of compound 62

9.6 mg of compound 61 obtained as described above was dissolved using 0.1 ml of trifluoroacetic acid and stirred for 30 minutes at room temperature. 2 ml dichloromethane was added; after stirring until homogeneity, the solvent was removed on a rotary evaporator under reduced pressure and the resulting material was used directly in the next step without purification.

8) Synthesis of compound ama-0305

Compound 62, obtained as described above, was dissolved using 1 ml of anhydrous DMF, and then 5.1 mg of N-succinimidyl-6-maleimidohexanoate (2 eq.) was added; stirring was carried out under nitrogen atmosphere for 5 h at room temperature until the reaction of the starting materials was completed (monitored by HPLC); purification was carried out using preparative liquid chromatography to collect the fraction corresponding to the target peak; after drying with nitrogen gas, 4.6 mg of an off-white solid was obtained in a yield of 44.4%. [M+H] ⁺ 1252.6091.

Example 6 Preparation of an Antibody-Drug Conjugate

1) General conjugation method : After preliminary purification, antibody molecules with a monomer yield of more than 95% were subjected to medium exchange and added to a phosphate buffer solution containing EDTA through an ultrafiltration centrifuge tube at a concentration of 10 mg/ml. TCEP was added at 10 times the molar amount of antibody molecules and reacted at room temperature for 2 hours. The resulting substance was subjected to a medium change procedure and added to a phosphate buffer solution at pH 6.5 through an ultrafiltration centrifuge tube; then DHAA was added in 10 times the molar amount of antibody molecules and the reaction was carried out at room temperature for 2 hours. Then the payload was added in 3 times the molar number of antibody molecules and the interaction was carried out at room temperature for 4 hours. completion of the reaction, the resulting substance was subjected to a medium exchange procedure and added to PBS through an ultrafiltration centrifuge tube having a molecular weight cutoff of 30 kDa, and the unbound load was removed.

2) Determination of the DAR of the antibody-drug

Detection conditions for monomer release:

Samples were centrifuged at 14,000 rpm for 5 minutes and the supernatant was used to analyze the injection.

Device: Waters e2695 (2489 UV/Visible)

Chromatographic column: TSKgel G3000SWXL (7.8 × 300 mm, 5 µm)

Mobile phase: A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5

isocratic elution with mobile phase A for 30 min; flow rate: 0.714 ml/min; column temperature: 25°C; and detection wavelength: 280 nm.

Conditions for determining DAR:

Samples were centrifuged at 14,000 rpm for 5 minutes and the supernatant was used to analyze the injection.

Device: Waters H-class (TUV)

Chromatography column: Proteomix HIC Butyl-NP5 (4.6 x 35 mm, 5 µm)

Mobile phase: A: 1.5 M ammonium sulfate, 0.025 M anhydrous sodium phosphate, pH 7.0

B: 0.025 M anhydrous sodium phosphate, 25% IPA, pH 7.0

equilibrating the chromatographic column with mobile phase A; gradient elution with mobile phase A and B; flow rate: 0.8 ml/min; column temperature: 25°C; and detection wavelength: 214 nm.

3) Results

Antibody drug conjugate Antibody Payload Monomer yield DAR (Theory 2.0) Tras-0301 Trastuzumab ama-0301 96.1 1.81 Tras-0302 Trastuzumab ama-0302 96.4 1.80 Tras-0303 Trastuzumab ama-0303 96.8 1.84 Tras-0304 Trastuzumab ama-0304 97.1 1.78 Tras-0305 Trastuzumab ama-0305 96.7 1.76

4) Conclusion

After binding ama-0301/ama-0302/ama-0303/ama-0304/ama-0305 to trastuzumab, the binding efficiency was higher and the monomer yield was higher.

Example 7 Plasma Stability

1) Procedures

A certain amount of ADC sample was added to human plasma from which human IgG had been removed. Each ADC was repeated three times in three parallel assays and placed in a 37° C. water bath for incubation. After incubation for 72 h and 144 h, respectively, the ADC sample was removed and 100 µl of ProteinA resin (MabSelect SuReTM LX Lot: # 10221479 GE, washed with PBS) was added to each tube, which was shaken using a vertical adsorption mixer for 2 h; after washing and elution steps, ADCs were obtained after incubation, and ADC samples incubated for a certain time were detected by RP-HPLC.

2) Results

Antibody drug conjugate DAR (Theory 2.0) 72 h 144 h Tras-0301 1.78 1.76 Tras-0302 1.80 1.78 Tras-0303 1.83 1.82 Tras-0304 1.76 1.76 Tras-0305 1.74 1.72

3) Conclusion

In human plasma, each antibody drug conjugate showed almost no degradation after 3 and 6 days and had good stability.

Example 8 in vitro activity test

1) Materials used in the experiment

Cells: from Cell Bank of Chinese Academy of Sciences

Tumor cell culture medium: Gibco

FBS: BIOWEST

2) Obtaining the culture medium

Culture medium (with 10% FBS, Penicillin/Streptomycin (100 U/mL))

Detection medium (with 1% FBS, Penicillin/Streptomycin (100 U/mL))

3) Procedures

The ultraviolet lamp of the biological safety cabinet was turned on 30 minutes in advance for irradiation and then ventilated for 3 minutes. The culture medium, detection medium, D-PBS, and trypsin were preheated in a water bath with a 37°C thermostat, the surface was disinfected with alcohol, and placed in a biological safety box. Cells with a confluence of about 80% were selected and placed in a biological safety box; after the old medium was removed, the cells were washed with D-PBS, which was then aspirated; and washed cells were digested with trypsin for 2-3 minutes, nutrient medium was added to neutralize and centrifuged at 1200 rpm for 3 minutes. The supernatant obtained after centrifugation was removed, and 4 ml of detection medium was used for homogeneous mixing. 100 µl was used for counting, while 50 µl of cell fluid was taken and 50 µl of trypan blue dye was added for homogeneous mixing and then counting. According to the previously determined number, cells were seeded at a density of 80 μl/well in a 96-well plate, where only 80 μl of detection medium were added to wells E11, F11 and G11 only, and the outer wells were filled with 150 μl of DPBS. The dilution of the antibody solution includes: using the detection medium to obtain 300 μl of a test sample solution with an initial concentration of 5 μM in the first column of a 96-well plate (V-type); adding 210 µl of detection medium to the second to tenth column from the end; adding 30 μl of homogeneously mixed solution from the first column to the second column; mixing this until homogeneous 10 times with a pipette up and down; and removing the pipette tip and repeating the procedure sequentially for the next 7 concentrations; 24 hours after seeding, adding diluted antibodies at 20 µl per well and establishing controls by adding only 20 µl of detection medium to column 11; repetition of each concentration in 2 wells; and after the addition, performing homogeneous mixing using a vortex cell shaker at 550 rpm for 3 minutes.

4) Detection

After 4 days, the MTS reagent was removed, thawed at room temperature, protected from light, and homogeneously mixed in a vortex mixer. In a biological safety cabinet, 20 µl of CellTiter 96® One Solution Reagen MTS reagent was added along the side wall of the wells for every 100 µl of cell culture volume. The MTS solution was mixed homogeneously by gently patting the surface of the plate and placed in a cell incubator protected from light for 2 hours for continuous incubation. After completion of the reaction, the 96-well plate was removed and the absorbance value at OD 490 nm was measured using a microplate reader; and carried out the registration, sorting and storage of data.

5) Results

Number SKBR3
IC50 (nM) SKOV3
IC50 (nM) NCI-N87
IC50 (nM) Tras-0303 0.37 1.43 7.92 trastuzumab > 1000 > 1000 200-1000

Claims (40)

1. A conjugate of a non-natural amatoxin with an antibody having antitumor or anticancer activity, containing a toxin fragment and a biomacromolecular fragment A, where the conjugate has the structural formula (I), or a pharmaceutically acceptable salt thereof,

Where: R ¹ is H, -OH or -OLA; R ² is H or -OH; R ³ is OC _1-6 alkyl; R ⁴ is H or -LA; R ⁵ is -NH ₂ , -OH, -NH-LA or -OLA; A is a biomacromolecular fragment that binds to a target site; where O is an oxygen atom, N is a nitrogen atom, and H is a hydrogen atom; where L includes the following structure: -L ₁ -[L ₂ ] _m -[AA] _n -L ₃ -, Where: L ₁ or L ₃ are each independently selected from one or more of succinimide and its ring-opening form, -C(=O)-, -C(=O)NH-, C1-C6 alkyl, C1-C6 alkoxy, triazole , aminobenzyl alcohol and their derivatives; L ₂ is a spacer selected from one or more of substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C3-C20 heterocycloalkyl, substituted or unsubstituted C5-C20 aryl, substituted or unsubstituted C5-C20 heteroaryl and -(CH ₂ CH ₂ O)a-, where a is an integer selected from 1-20, and where the substituent is selected from one or more of hydroxyl group, sulfhydryl group, halogen, carboxyl group, amino group, phosphate group , nitro, cyano, sulfo, or C1-C6 alkyl which is unsubstituted or substituted by one or more of a hydroxyl group, a sulfhydryl group, a halogen, a carboxyl group, an amino group, a phosphate group, a nitro group, a cyano group, or a sulfo group; L ₁ binds to biomacromolecular fragment A; L ₂ binds L ₁ to AA; L ₃ binds to a fragment of the toxin; m is an integer selected from 1-6; AA is a structural fragment consisting of 1-6 amino acids and n is 0 or 1. 2. A non-natural amatoxin conjugate with an antibody according to claim 1 or a pharmaceutically acceptable salt thereof, where A is a biomacromolecular fragment that binds to the target site and includes an antibody, its antigen-binding fragment, an antibody-like protein. 3. A non-natural amatoxin conjugate with an antibody according to claim 2 or a pharmaceutically acceptable salt thereof, where the biomacromolecule that binds to the target site is an antibody or its antigen-binding fragment, which is selected from a chimeric antibody, a deimmunized antibody, a humanized antibody, a human antibody , diabody, triabody, tetrabody, or nanobody. 4. A non-natural amatoxin-antibody conjugate of claim 2 or 3, or a pharmaceutically acceptable salt thereof, wherein the antigen-binding moiety is selected from the group consisting of Fab, F(ab'), Fd, Fv, single chain Fv, or disulfide-linked Fv( dsFv). 5. The non-natural amatoxin antibody conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein one and only one of R ¹ , R ⁴ and R ⁵ contains the -LA structure. 6. A non-natural amatoxin conjugate with an antibody according to claim 1 or a pharmaceutically acceptable salt thereof, where L ₁ is a linker for binding to biomacromolecule A and is selected from:

where the wavy line links with biomacromolecule A. 7. A non-natural amatoxin conjugate with an antibody according to claim 1 or a pharmaceutically acceptable salt thereof, where AA is a fragment consisting of 1-6 amino acids selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine , tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartate, glutamate, lysine, arginine or histidine. 8. The non-natural amatoxin-antibody conjugate of claim 7, or a pharmaceutically acceptable salt thereof, wherein the amino acids are L-amino acids selected from phenylalanine, citrulline, valine, lysine, serine, glutamate, aspartate, or glycine. 9. A non-natural amatoxin conjugate with an antibody according to claim 1 or a pharmaceutically acceptable salt thereof, where L ₃ is selected from

,

or

or any linking group that binds to the toxin; Where: L ₄ or L ₅ are each independently selected from one or more of succinimide and its ring-opening form, -C(=O)-, -C(=O)NH-, C1-C6 alkyl, C1-C6 alkoxy, triazole , aminobenzyl alcohol and their derivatives; R ₆ and R ₇ are each independently selected from a hydrogen atom, a deuterium atom, a C1-C6 alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or a heteroaryl group; where the wavy line is associated with AA and the asterisk is associated with the toxin, as shown in the structural formula of formula (I). 10. An unnatural amatoxin-antibody conjugate of claim 9, or a pharmaceutically acceptable salt thereof, wherein L4 is a carbonyl group or a single bond. 11. The non-natural amatoxin antibody conjugate of claim 9, or a pharmaceutically acceptable salt thereof, wherein R6 and R7 are each independently selected from hydrogen or C1-C6 alkyl. 12. A non-natural amatoxin-antibody conjugate of claim 9, or a pharmaceutically acceptable salt thereof, wherein L ₅ is C2-C12 alkyl. 13. The use of a non-natural amatoxin conjugate with an antibody having antitumor or anticancer activity, according to any one of paragraphs. 1-12 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable ingredient for producing an antitumor drug or an anticancer drug.