RU2840926C2 - Conjugate of antibody with amatoxin of non-natural type - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to biotechnology and can be used in medicine. Described is use of a non-natural amatoxin conjugate with an antibody as an anti-tumour or anti-cancer drug with high activity and improved stability in blood plasma.

EFFECT: invention widens the range of agents possessing pronounced cytotoxic activity, and can be used in cancer therapy.

2 cl, 4 tbl, 8 ex

Description

Field of technology to which the invention relates

The present invention relates to a bicyclic octapeptide derivative which can be conjugated with a corresponding target-binding group in a special chemical structure. This structure is stable in blood plasma and decomposes into an active pharmaceutical ingredient in a certain biological environment, thereby maximizing the killing effects on target cells and minimizing toxic side effects on non-target cells, which can be used to treat various malignant tumors.

Prerequisites for the creation of an 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, amanullin, amanullic acid and proamanullin, with α-amanitin and β-amanitin being the major toxins that cause death. Amanitin is a class of slow-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 reach 3 nM. Toxins are periodically absorbed into the body through enterohepatic circulation in the gastrointestinal tract and can cause permanent, severe damage to organs such as the liver, kidneys, heart, and lungs.

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

According to the research conducted by Theodor Wieland's group, when the sulfoxide structure in natural amanitin is replaced by elemental sulfur, the cell toxicity of the non-natural amanitin formed in this way changes only slightly. The natural amanitin molecule was conjugated with a monoclonal antibody by Heidelberg Pharmaceutical Co., Ltd., Germany, resulting in a drug molecule with antitumor activities.

In the present invention, non-natural amanitin, similar in toxicity to natural amanitin, is conjugated with 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

In accordance with standards and practices in the art, the symbol used in formulas and tables, represents a bond used as a point for attaching a moiety or substituent to the core of a compound structure.

In accordance with 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, thereby 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 represents 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 represents an oxygen atom, N represents a nitrogen atom and H represents a hydrogen atom.

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

as a preferred embodiment, L comprises 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 moiety linking L ₁ to AA; m is an integer of 1-6; L ₃ binds to the toxin shown in structural formula (I); AA is a moiety consisting 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 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 fragment 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 comprises the structure -LA.

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

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

As a preferred embodiment, L ₂ is a spacer selected from one or more, 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 linked to each other via an appropriate chemical bond.

Even more preferably, the substituent is selected from one or more identical 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 identical or different combinations of hydroxyl group, sulfhydryl group, halogen, carboxyl group, amino group, phosphate group, nitro group, cyano group, sulfo 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 from phenylalanine, citrulline, valine, lysine, serine, glutamate, aspartate and glycine.

As a preferred embodiment, L ₃ is selected from

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

where the wavy line represents AA and the asterisk represents 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 medicament comprises 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 production of an antitumor drug or an anticancer drug is disclosed.

As a preferred embodiment, the antitumor drug or anticancer drug is a drug for lung cancer, a drug for kidney cancer, a drug for urethral cancer, a drug for colorectal cancer, a drug for prostate cancer, a drug for glioblastoma, a drug for ovarian cancer, a drug for pancreatic cancer, a drug for breast cancer, a drug for melanoma, a drug for liver cancer, a drug for bladder cancer, a drug for malignant lymphoma, a drug for leukemia, a drug for gastric cancer, or a drug for esophageal cancer.

Common abbreviations and symbols: G: gram mg: milligram min: minutes ml: milliliter mol.: molar °C: degrees Celsius Boc: tert-butoxycarbonyl PyBOP: 1H-benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Cit: citrulline _CO2 : carbon dioxide DCM: dichloromethane DIPEA: diisopropylethylamine DMF: dimethylformamide DMSO: dimethyl sulfoxide DPBS: Dulbecco's phosphate buffered saline 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 mAb: monoclonal antibody MEM: Minimum Nutrient 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 specific examples presented below. These examples are used only to describe the present invention, but not to limit the scope of the present invention. Unless otherwise specified, all professional and scientific terms used have the same meanings as are commonly known to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described in this application can all be used in the method of the present invention. The described preferred embodiments and materials are intended for illustrative purposes only.

Example 1 Synthesis of low molecular weight load ama-0301

1) Solid-phase synthesis of intermediate compound 08

The N-fluorenylmethoxycarbonyl-O-tert-butyl-L-hydroxyproline preloaded resin, which was used as a 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 with DMF 5 times until neutral pH was reached. Then, Fmoc-N-propynyl-L-asparagine (Fmoc-Asn(Trt)-OH) (3 equiv), TBTU (2.5 equiv), HOBT (1.8 equiv) and DIPEA (6 equiv) were added successively to DMF, which was used as a solution (20 ml/g), reacted for 2 h at room temperature (28 °C) and then washed 3 times with DMF (each time adding 20 ml DMF to 1 g resin), followed by coupling with amino acids according to the previous procedure. After completion of the final coupling, the obtained compound was cleaved from the resin using 1% TFA solution in dichloromethane (each time adding 20 ml to 1 g resin; 1% TFA for 5 min; repeated three times); this solution was removed on a rotary evaporator; and stirred with methyl tert -butyl ether for crystallization, thereby obtaining compound 08 in 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, a 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 purified 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 single-neck flask and mixed homogeneously, followed by the addition of Fmoc-OSu portionwise. After 10 min, stirring was continued at room temperature for 12 h until the reaction of the starting materials was completed. 50 ml of water was added to the reaction liquid, and 5% citric acid solution was used to adjust the pH to about 4. Ethyl acetate was used for extraction 3 times (50 ml each). The organic layer was collected, washed once with 50 ml of saturated 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 the above compound 13 was dissolved with 40 ml of DMF, and then 2.68 g (2 equiv.) of imidazole was added, followed by portionwise addition of TBS-Cl, and then stirred at room temperature for 12 h until the reaction of the starting materials 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 (50 ml each). 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 (eluting with PE:EA=5:1) to give 4.5 g of oil with a yield of about 46.6% for two steps.

5) Synthesis of compound 10

Compound 14, HOSu (1.23 g, 1.15 equiv), DCC (2.23 g, 1.15 equiv) and 50 mL THF were successively added to a 250 mL single neck flask and stirred at room temperature for 6 h under nitrogen atmosphere. After completion of the reaction, 50 mL of water and 50 mL of ethyl acetate were added and stirred for 10 min 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 white foamy solid in 60% yield. 1H-NMR (400 MHz, DMSO -d6 ): 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 with 1.5 mL of anhydrous DMF and then compound 10 (701 mg, 2 equiv) was added. The pH was adjusted to 8-9 with DIPEA; the reaction was carried out under nitrogen atmosphere for 5 h at room temperature and monitored by HPLC until the reaction of starting material 09 was essentially complete. The product was used directly in the next step without further work-up.

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 stirring was stopped when the reaction of the starting materials was completed (monitored by HPLC). For purification, preparative liquid chromatography (neutral condition, acetonitrile/pure water system) was used to collect the fraction corresponding to the target peak; after removing acetonitrile under reduced pressure, 277 mg of white powdery solid was obtained by lyophilization in a yield of about 42.8% for 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 equiv); HOBT (170 mg, 5 equiv) and DIPEA (0.22 mL, 5 equiv) were added 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 water system) was used to collect the fraction corresponding to the target peak; after removing acetonitrile under reduced pressure, about 136.4 mg of white powdery solid was obtained by lyophilization with 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 with 10 ml of tetrahydrofuran and then 0.678 g of 2-[2-(2-azidoethoxy)ethoxy]ethylamine (1.2 equiv) was added; stirring was carried out at room temperature for 4 h under nitrogen atmosphere until the reaction was complete (monitored by TLC); then water was added and EA was used for extraction 3 times (10 ml each). The organic layer was combined, dried, concentrated and purified by column chromatography to obtain about 0.8 g of the desired product in 84% yield.

10) Synthesis of compound 17

80 mg of compound 16 was dissolved with 5 ml of DMSO, compound 13 (114.7 mg, 0.5 equiv), 44.7 mg (1.5 equiv) of copper bromide and 0.2 ml of purified water were added, stirring was carried out for 3 h at room temperature under 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 by rotary evaporator; about 50.5 mg of white solid was obtained by lyophilization in 32.6% yield. 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 h under nitrogen atmosphere. After completion of the reaction of starting material 17 (monitored by HPLC) and removal of the solvent by purging with nitrogen, purification was performed using preparative liquid chromatography and the organic solvent was concentrated and removed to obtain 15.4 mg of white solid by lyophilization in 37.2% yield. MS: [M+H] ⁺ 1306.6013.

Example 2 Synthesis of low molecular weight 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 yellow-brown solid with a high purity of about 79.8% was obtained.

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 with 50 ml of TFA and stirred at room temperature for 5 h under nitrogen atmosphere 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 white solid was obtained by lyophilization with 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 white solid was obtained by lyophilization with 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 white solid was obtained by lyophilization with 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 h at room temperature under nitrogen atmosphere 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 about 69.5 mg of pale yellow solid was obtained by lyophilization in 77.9% yield. 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 equiv) of 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 syringe. The starting materials were dissolved and stirred for 12 h at room temperature until the reaction of the starting materials was complete (monitored by HPLC). Purification was performed using preparative liquid chromatography to collect the fraction corresponding to the target peak, and about 35.2 mg of pale yellow solid was obtained by lyophilization in 73.5% yield. MS: [M+H] ⁺ 902.4123.

8) Synthesis of compound 31

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

9) Synthesis of compound 32

20 g of compound 31 were dissolved in 200 ml of THF as a solvent and 9.64 g (1.2 equiv.) of L-citrulline were added; 1 M sodium carbonate was added to adjust the pH to 8-9, and the dissolving reaction mixture did not become clear. Stirring was carried out for 48 h 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 aqueous citric acid solution and extracted 3 times with isopropanol:EA=1:5 mixture (40 ml of isopropanol + 200 ml of EA). The organic phase was combined, dried over anhydrous sodium sulfate, filtered and dried on a rotary evaporator, followed by the addition of 200 ml of methyl tert -butyl ether and stirring for 2 h; then suction filtration was performed to collect the filter cake, which was dried in a vacuum oven at 45°C to obtain 18.6 g of a white solid product with a yield of about 81.7%.

10) Synthesis of compound 33

18 g of compound 32 were added to a 500 ml reaction flask and dissolved with 200 ml of DMF; then 8 g (1.0 equiv) of 4-[(N-tert-butoxycarbonyl)aminomethyl]aniline, 20 g (1.5 equiv) of HATU and DIPEA (18 ml, 3 equiv) were added successively and stirred for 24 h at room temperature. After the 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 min. 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 min; the solid was then washed and filtered and the filter cake was washed twice with methyl tert -butyl ether (50 ml each). 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 75 ml of 20% piperidine in DMF solution was added; stirring was carried out for 1 h at room temperature until the reaction of the starting materials was complete (monitored by TLC). The solvent was distilled off under reduced pressure using an oil pump and then 200 ml of methyl tert -butyl ether were added and stirred for 2 h at room temperature. A brown solid precipitated and was 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 obtain 6.8 g of a brown solid in 66.4% yield.

12) Synthesis of compound 35

1 g of compound 34 was dissolved with 10 ml of DMF, and then 773 mg of N-succinimidyl 6-maleimidohexanoate (1.2 equiv) was added; stirring was carried out for 5 h 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 obtain 956 mg of pale yellow foamy solid in 68.3% yield.

13) Synthesis of compound 36

30 mg of compound 35 was dissolved using 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 the crude product, compound 36, which was used directly in the next step without purification.

14) Synthesis of ama-0302

The crude product 36 (1.2 equiv.) obtained in the previous step was dissolved with 1 mL of DMF; then 32.5 mg of 30 and 20.5 mg of HATU (1.5 equiv.) were added and the pH was adjusted to 8-9 with DIPEA; stirring was carried out for 6 h at room temperature under nitrogen atmosphere 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 pale yellow solid was obtained in 29.1% yield. MS: [M+H]+ 1455.7011.

Example 3 Synthesis of low molecular weight 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 was 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 was added and about 482.3 mg of white solid was obtained by lyophilization with 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 pale yellow solid was obtained by lyophilization in 38% yield. [M+H]+ 1471.6820.

Example 4 Synthesis of low molecular weight ama-0304

1) Synthesis of compound 49

As for the synthesis method, it was carried out with reference to the synthesis of compound 08; about 2.4 g of crude product of compound 49 with a purity of about 86.2% was obtained, which was directly used in the next step 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 target 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 target compound was obtained in 43% yield. [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, with 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, with 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 equiv.) and 88 mg of potassium tert-butoxide (10 equiv.) 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 equiv.) and 88 mg of potassium tert-butoxide (10 equiv.) were added. The reaction mixture was stirred for another 24 h at room temperature until the starting materials disappeared (monitored by HPLC); purification was performed using preparative liquid chromatography to collect the fraction corresponding to the target peak; and 18.2 mg was obtained in 19.5% yield. [M+H]+ 1186.6137.

7) Synthesis of compound 55

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

8) Synthesis of compound ama-0304

The crude product of compound 55, prepared as described above, was dissolved with 1 mL of DMF and then 9.5 mg of N-succinimidyl 6-maleimidohexanoate (2 equiv.) 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 complete (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 weight 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 white solid was obtained by lyophilization with 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 white solid was obtained by lyophilization with 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 were added and about 72.5 mg of off-white solid was obtained by lyophilization in 66.9% yield. [M+H] ⁺ 1145.5901.

4) Synthesis of compound 59

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

5) Synthesis of compound 60

The synthesis was carried out with reference to the method described in patent WO 2012041504.

6) Synthesis of compound 61

About 42 mg of starting material 59 was dissolved with 1 ml of anhydrous DMF; then 22 mg of compound 60 (2 equiv.) and 58 mg of dibutyltin dilaurate were added; then stirring was carried out for 24 h at room temperature under nitrogen atmosphere. Again 22 mg of compound 60 (2 equiv.) was added and stirring was continued for 52 h at room temperature until the starting material 59 disappeared (monitored by HPLC). To the above reaction liquid, 0.2 ml of methanol was added 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 by rotary evaporation and about 9.6 mg of pale yellow solid was obtained by lyophilization in 18.1% yield. [M+H] ⁺ 1159.5703.

7) Synthesis of compound 62

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

8) Synthesis of compound ama-0305

Compound 62, prepared as described above, was dissolved with 1 mL of anhydrous DMF and then 5.1 mg of N-succinimidyl 6-maleimidohexanoate (2 equiv) was added; stirring was carried out under nitrogen atmosphere for 5 h 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; after drying with nitrogen gas, 4.6 mg of off-white solid was obtained in 44.4% yield. [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 a medium exchange procedure and added to a phosphate buffer solution containing EDTA through an ultrafiltration centrifuge tube at a concentration of 10 mg/mL. TCEP was added in an amount 10 times the molar amount of antibody molecules and the reaction was carried out at room temperature for 2 h. The resulting substance was subjected to a medium exchange procedure and added to a phosphate buffer solution at pH 6.5 through an ultrafiltration centrifuge tube; then DHAA was added in an amount equal to 10 times the molar amount of antibody molecules and the reaction was carried out at room temperature for 2 h. Then the payload was added in an amount equal to 3 times the molar amount of antibody molecules and the reaction was carried out at room temperature for 4 h. After 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 DAR of antibody-drug

Detection conditions for monomer release:

Samples were centrifuged at 14,000 rpm for 5 min and the supernatant was used for feed analysis.

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 min and the supernatant was used for feed analysis.

Device: Waters H-class (TUV).

Chromatography column: Proteomix HIC Butyl-NP5 (4.6×35 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,

equilibration of 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 of 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 Stability in plasma

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 triplicate 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 an overhead mixer for adsorption for 2 h; after washing and elution steps, the incubation ADC was obtained and the 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 the Cell Bank of Chinese Academy of Sciences

Tumor cell culture medium: Gibco

FBS: BIOWEST

2) Obtaining culture medium

Nutrient 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 in advance for 30 minutes 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 thermostat at 37°C, the surface was disinfected with alcohol and placed in the biological safety cabinet. The cells with a confluency of about 80% were collected and placed in the biological safety cabinet; after the old medium was removed, the cells were washed with D-PBS, which was then aspirated; and the washed cells were digested with trypsin for 2-3 minutes, the culture medium was added for neutralization, and centrifuged at 1200 rpm for 3 minutes. The supernatant obtained after centrifugation was discarded, and 4 ml of the detection medium was used for homogeneous mixing. 100 μl was used for counting, where 50 μl of the cell fluid was taken out and 50 μl of trypan blue dye was added for homogeneous mixing and then counting. According to the previously determined amount, the cells were seeded at a density of 80 μl/well in a 96-well plate, where only 80 μl of the detection medium was added to the well of E11, F11 and G11, 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 the test sample solution with an initial concentration of 5 μM in the first column of the 96-well plate (V type); adding 210 μl of the detection medium to the second to tenth column from the end; adding 30 μl of the homogeneously mixed solution from the first column to the second column; mixing this to homogeneity 10 times by pipetting up and down; and removing the pipette tip and repeating the procedure sequentially for the next 7 concentrations; 24 h after plating, adding diluted antibodies at 20 µl per well and establishing controls by adding only 20 µl of detection medium to column 11; repeating each concentration in 2 wells; and after addition, performing homogeneous mixing using a vortex cell shaker at 550 rpm for 3 min.

4) Detection

After 4 days, the MTS reagent was removed, thawed at room temperature, protected from light, and mixed homogeneously 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 tapping the surface of the plate and placed in a cell incubator, protected from light, for 2 hours of continuous incubation. After completion of the reaction, the 96-well plate was removed and the optical density value at OD 490 nm was measured using a microplate reader; and the data were recorded, sorted, and stored.

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

This application covers the following aspects.

1. A conjugate of a non-natural amatoxin with an antibody, comprising a fragment of the toxin having the structural formula (I), or a pharmaceutically acceptable salt thereof, and a biomacromolecular fragment A having binding affinity for a target,

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;

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

2. A conjugate of a non-natural amatoxin with an antibody according to claim 1, wherein L comprises the following structure:

-L ₁ -[L ₂ ] _m -[AA] _n -L ₃ -

where: L ₁ is a linker that binds to the biomacromolecular fragment A; L ₂ is a spacer that binds L ₁ to AA; m is an integer selected from 1-6; L ₃ is a structural fragment that binds to the toxin fragment; AA is a structural fragment consisting of 1-6 amino acids, and n is 0 or 1.

3. A conjugate of a non-natural amatoxin with an antibody according to claim 1, wherein A comprises an antibody or an antigen-binding fragment thereof, an antibody-like protein or a nucleic acid aptamer.

4. A conjugate of a non-natural amatoxin with an antibody according to claim 3, wherein the antibody or its antigen-binding fragment comprises a chimeric antibody, a deimmunized antibody, a humanized antibody, a human antibody, a bispecific antibody, a tetraspecific antibody, a nanobody or their antigen-binding fragments.

5. A conjugate of a non-natural amatoxin with an antibody according to claim 3, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab’), Fd, Fv, single-chain Fv or disulfide-linked Fv(dsFv).

6. A conjugate of a non-natural amatoxin with an antibody according to claim 1, wherein only one of R ¹ , R ⁴ and R ⁵ comprises the -LA structure.

7. A conjugate of a non-natural amatoxin with an antibody according to claim 2, wherein L ₁ is a linker that binds to A, and wherein L ₁ is selected from:

where the wavy line represents the connection with A.

8. A conjugate of a non-natural amatoxin with an antibody according to claim 2, wherein L ₂ is 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, or -(CH ₂ CH ₂ O)a-, where a is an integer selected from 1-20.

9. A conjugate of a non-natural amatoxin with an antibody according to claim 8, wherein substituted includes substitution with a hydroxyl group, a sulfhydryl group, a halogen, a carboxyl group, an amino group, a phosphate group, a nitro group, a cyano group, a sulfo group, or an additionally substituted or unsubstituted C1-C6 alkyl.

10. A conjugate of a non-natural amatoxin with an antibody according to claim 9, wherein additionally substituted includes substitution with 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.

11. A conjugate of a non-natural amatoxin with an antibody according to claim 2, wherein the amino acids 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 or histidine.

12. A conjugate of a non-natural amatoxin with an antibody according to claim 11, wherein the amino acids are selected from phenylalanine, citrulline, valine, lysine, serine, glutamate, aspartate or glycine.

13. A conjugate of non-natural amatoxin with an antibody according to claim 2, wherein L ₃ comprises

or any linking group that binds to a toxin moiety;

where the wavy line represents binding to AA and the asterisk represents binding to the toxin fragment.

14. A conjugate of a non-natural amatoxin with an antibody according to claim 13, wherein L4 is a carbonyl group or a single bond.

15. A conjugate of a non-natural amatoxin with an antibody according to claim 13, wherein R6 and R7 are each independently selected from hydrogen or C1-C6 alkyl.

16. A conjugate of a non-natural amatoxin with an antibody according to claim 13, wherein L5 is C2-C12 alkyl.

17. A pharmaceutical composition comprising a conjugate of an antibody with a non-natural type amatoxin according to claim 1 and a pharmaceutically acceptable carrier.

18. Use of a conjugate of an antibody with a non-natural type amatoxin according to claim 1 for obtaining an antitumor drug or an anticancer drug.

19. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is a drug for lung cancer, a drug for kidney cancer, a drug for urethral cancer, a drug for colorectal cancer, a drug for prostate cancer, a drug for glioblastoma, a drug for ovarian cancer, a drug for pancreatic cancer, a drug for breast cancer, a drug for melanoma, a drug for liver cancer, a drug for bladder cancer, a drug for malignant lymphoma, a drug for leukemia, drugs for gastric cancer, or a drug for esophageal cancer.

Claims (23)

1. The use of a conjugate of a non-natural amatoxin with an antibody having antitumor or anticancer activity, or a pharmaceutically acceptable salt thereof, as an antitumor or anticancer drug, where the conjugate of non-natural amatoxin with an antibody contains a toxin fragment and a biomacromolecular fragment A and has the structural formula (I): 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 represents an oxygen atom, N represents a nitrogen atom and H represents 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 derivatives thereof; 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 wherein the substituent is selected from 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, a sulfo group, or a C1-C6 alkyl that 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 the biomacromolecular fragment A; L ₂ links L ₁ to AA; L ₃ binds to the toxin fragment; m is an integer chosen from 1-6; AA is a structural fragment consisting of 1-6 amino acids, and n has a value of 0 or 1. 2. The use according to claim 1, wherein the anticancer drug is a drug for lung cancer, a drug for kidney cancer, a drug for urethral cancer, a drug for colorectal cancer, a drug for prostate cancer, a drug for glioblastoma, a drug for ovarian cancer, a drug for pancreatic cancer, a drug for breast cancer, a drug for melanoma, a drug for liver cancer, a drug for bladder cancer, a drug for malignant lymphoma, a drug for leukemia, drugs for gastric cancer or a drug for esophageal cancer.