CN110845480A - Difunctional cytotoxin and application thereof - Google Patents

Difunctional cytotoxin and application thereof Download PDF

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CN110845480A
CN110845480A CN201911152237.0A CN201911152237A CN110845480A CN 110845480 A CN110845480 A CN 110845480A CN 201911152237 A CN201911152237 A CN 201911152237A CN 110845480 A CN110845480 A CN 110845480A
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陈虎
罗文婷
叶慧
黄长江
姚雪静
房健民
王惠
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Remegen Co Ltd
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Abstract

The invention provides a bifunctional cytotoxin containing a duocarmycin analogue and a nitrogen mustard compound, and also provides an antibody drug conjugate containing the conjugate and pharmaceutical application thereof. The bifunctional cytotoxin provided by the invention shows good cytotoxic activity on SK-BR-3 (human breast cancer cells), SMMC7721 (human liver cancer cells), SK-OV-3 (human ovarian adenocarcinoma cells), A549 (human non-small cell lung cancer cells), NCI-N87 (human gastric cancer cells), Raji (human lymphoma cells) and Jurkat (acute T cell leukemia cells).

Description

Difunctional cytotoxin and application thereof
Technical Field
The invention relates to a compound, in particular to a duocarmycin cytotoxin, and also relates to a pharmaceutical composition containing the conjugate compound, an Antibody Drug Conjugate (ADC) and pharmaceutical application thereof.
Background
Alkylating agents are chemical highly active compounds, which can form carbonium ions or other compounds with active electrophilic groups in vivo, and further covalently bond with groups (such as amino, sulfhydryl, hydroxyl, carboxyl, phosphate, etc.) which contain abundant electrons in biological macromolecules (such as DNA, RNA, enzyme, etc.) in cells, so that the biological macromolecules lose activity or break DNA molecules, and cause tumor cell death and tumor tissue regression, thus being widely applied to antitumor drugs.
CC-1065 was an alkylating agent isolated from Streptomyces zelesinus (Streptomyces zelensis) in 1978 by Upjohn Inc. [ Hanka, L.J.; dietz, a.; gerpheide, s.a.; kuentzel, s.l.; martin, d.g.j.Antibiot.1978,31,1211-1217.Martin, d.g.; biles, C.; gerpheide, s.a.; hanka, l.j.; krueger, w.c.; McGovren, j.p.; mizsak, s.a.; neil, g.l.; stewart, j.c.; visser, j.j.antiboot.1981, 34,1119-1125 ] which showed highly potent antitumor and antibacterial activity in vitro and in vivo experiments [ Frolova, v.i.; kuzovkov, a.d.; chernyshev, a.i.; taig, m.m.; ircenitskaya, l.p.; terentyeva, T, g.; fomma, I.P. Antibiotiki 1982,27,483-487. CC-1065 binds to specific sequences in the minor groove of DNA, hindering DNA double strand opening, which in turn affects DNA transcription and replication, resulting in loss of cell function and cell death [ Boger, d.l.; johnson, d.s.; yun, w.; tarby, c.m.bioorg.med.chem.1994,2,115-135.Boger, d.l.; ishizaki, t.; zarrimayeh, h.j.am.chem.soc.1991,113,6645-6649.Hurley, l.h.; reynolds, v.l.; swenson, d.h.; petiold, g.l.; scahill, T.A. science 1984,226, 843. cndot. 844. Subsequently, a variety of analogs and prodrugs of CC-1065 were synthesized and reported, such aS CBI ((8bR,9aS) -2-methyl-9, 9 a-dihydro-1H-benzo [ e ] cyclopropyl [ c ] indol-4 (2H) -one), Seco-CBI ((S) -1- (chloromethyl) -3-methyl-2, 3-dihydro-1H-benzo [ e ] indol-5-ol), CPI ((7bR,8aS) -2, 7-dimethyl-1, 2,8,8 a-tetrahydrocyclopropyl [ c ] pyrrolo [3,2-e ] indol-4 (5H) -one), Seco-CPI ((S) -8- (chloromethyl) -1, 6-dimethyl-3, 6,7, 8-tetrahydropyrrolo [3,2-e ] indol-4-ol), these analogs and prodrugs are collectively referred to as the Duocarmycin (Duocarmycin) cytotoxin.
Figure BDA0002283861970000021
The nitrogen-containing compounds are a generic name of β -chloroethyl amine compounds, including mono β -chloroethyl amine and bis β -chloroethyl amine, etc. in the longitudinal view of the structure, nitrogen mustard compounds are generally divided into two parts, namely an alkylation part and a carrier part, wherein the alkylation part is a functional group with anti-tumor activity, and the carrier part can be used for improving the pharmacokinetic properties of the drugs, such as absorption, distribution and the like in vivo, so that the toxicity, selectivity and anti-tumor activity of the drugs are influenced.
The nitrogen mustard compound is simple and convenient to synthesize and low in cost, has more clinical nitrogen mustard medicines, still has the defects of large adverse reaction, low treatment efficiency, poor selectivity and the like, and greatly attracts and encourages countless researchers to be dedicated to research and development of the nitrogen mustard compound so as to obtain the high-efficiency low-toxicity high-selectivity anti-tumor medicine. In particular, in recent years, the demand for anticancer drugs is more urgent, the research on nitrogen mustard compounds is more active at home and abroad, a plurality of nitrogen mustard derivatives with novel structures are reported every year, but the research on the development of bifunctional cytotoxins such as nitrogen mustard drugs and duocarmycin cytotoxin conjugates is less.
Disclosure of Invention
The invention provides bifunctional cytotoxins comprising a duocarmycin analogue and a nitrogen mustard compound, and also provides a pharmaceutical composition, an antibody drug conjugate and pharmaceutical application thereof. The bifunctional cytotoxin provided by the invention has a remarkable inhibiting effect on various tumor cells, has a broad spectrum of tumor cell resistance, and has a remarkable inhibiting or killing effect at an extremely low concentration.
The technical scheme of the invention is as follows:
a compound as described in formula (I),
Q－L－T (I)
wherein:
the structure of Q is
The structure of the T is selected from
Figure BDA0002283861970000032
The structure of the L is selected from
Figure BDA0002283861970000034
Figure BDA0002283861970000035
Further, the compound is selected from the following structures:
Figure BDA0002283861970000041
Figure BDA0002283861970000051
Figure BDA0002283861970000061
the invention also provides a pharmaceutical composition comprising a compound as described in any one of the above.
The invention also provides an antibody drug conjugate which comprises the compound as described in any one of the above as a prodrug of the antibody conjugate.
The invention also provides the use of a compound as defined in any one of the preceding claims in the manufacture of a medicament, wherein the medicament is an antibody drug conjugate.
The invention also provides the use of a compound or pharmaceutical composition or antibody drug conjugate as described in any one of the above in the preparation of a medicament for the treatment of cancer, autoimmune diseases, inflammatory diseases or infectious diseases.
According to the technical scheme, the novel linker structure is adopted to connect the duocarmycin analogue and the nitrogen mustard compound, so that the cytotoxin with double functions and high killing effect is obtained, and the cytotoxin with better killing effect and wider acting cells can be provided for preparing the antibody drug conjugate. And through the connection of the two toxins, namely the two toxins are connected on one connection site of the antibody, so that the drug-loading rate of the antibody drug conjugate is improved in a direction-changing manner.
Drawings
FIG. 1 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 on SK-BR-3 (human breast cancer cells).
FIG. 2 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 on SMMC7721 (human liver cancer cells).
FIG. 3 is a graph showing the growth inhibition rate of SK-OV-3 (human ovarian adenocarcinoma cells) by compounds I-01, I-03, I-05, I-13 and I-15.
FIG. 4 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 against A549 (human non-small cell lung cancer cell).
FIG. 5 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 against NCI-N87 (human gastric cancer cells).
FIG. 6 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 against Raji (human lymphoma cells).
FIG. 7 is a graph showing the growth inhibition rate of compounds I-01, I-03, I-05, I-13 and I-15 against Jurkat (acute T cell leukemia cell).
FIG. 8 is a scatter plot of the IC50 values of compounds I-01, I-03, I-05, I-13, I-15 against SK-BR-3, SMMC7721, SK-OV-3, A549, NCI-N87, Raji, Jurkat cells, respectively.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
[ abbreviations ]
Unless otherwise defined, all abbreviations used herein have the same meaning as understood by one of ordinary skill in the art. As used herein, the abbreviations used and their definitions are as follows:
Figure BDA0002283861970000071
Figure BDA0002283861970000081
[ definitions ]
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Notwithstanding that the numerical ranges and parameter approximations set forth the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference that is said to be "incorporated herein" is to be understood as being incorporated in its entirety.
The term "alkyl" in the present invention denotes a straight or branched chain saturated hydrocarbon (i.e. containing no double or triple bonds). An alkyl group can have 1 to 9 carbon atoms (as it appears in the present invention, a numerical range of "1 to 9" indicates any integer in this range, e.g., "1 to 9 carbon atoms" indicates that the alkyl group can contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 9 carbon atoms. The alkyl group may be a medium size alkyl group containing 1 to 9 carbon atoms. Typical alkyl groups include, but are not limited to: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.
The term "alkenyl" in the present invention denotes a straight or branched chain hydrocarbon containing one or more double bonds. Alkenyl groups may have 2 to 9 carbon atoms and also include alkenyl groups of no specified chain length. The alkenyl group may be a medium size alkenyl group containing 2 to 9 carbon atoms. The alkenyl group may also be a small size alkenyl group containing 2 to 4 carbon atoms. The alkenyl group may be designated as "C2-4 alkenyl" or a similar designation. By way of example only, "C2-4 alkenyl" means 2 to 4 carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the following ranges: vinyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, but-1, 3-dienyl, but-1, 2-dienyl-4-yl. Typical alkenyl groups include, but are not limited to: vinyl, propenyl, butenyl, pentenyl, hexenyl, and the like.
The term "alkynyl" in the present invention denotes a straight or branched chain hydrocarbon containing one or more triple bonds. Alkynyl groups can have from 2 to 9 carbon atoms and also include alkynyl groups of unspecified chain length. The alkynyl group can be a medium size alkynyl group containing 2 to 9 carbon atoms. Alkynyl groups may also be lower alkynyl groups containing 2 to 4 carbon atoms. The alkynyl group may be designed as "C2-4 alkynyl" or a similar design. By way of example only, "C2-4 alkynyl" means 2-4 carbon atoms in the alkynyl chain, i.e., the alkynyl chain may be selected from: ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl and 2-butynyl. Typical alkynyl groups include, but are not limited to: ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
The term "cycloalkyl" in the context of the present invention denotes a fully saturated carbocyclic ring or ring system. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
The term "heterocycloalkyl" in the present invention means that a heterocyclic group as a substituent is bonded to another group through an alkylene group. Examples include, but are not limited to, imidazolinylmethyl and indolinylethyl. The term "heterocyclic group" means that the skeleton of the non-aromatic ring or ring system contains at least one hetero atom. The heterocyclic group may be attached in a fused ring, bridged ring or spiro ring fashion. At least one ring of the heterocyclic ring system is non-aromatic and may have any degree of saturation. The heteroatoms may be located on non-aromatic or aromatic rings of the ring system. The heterocyclic group may have 3 to 20 ring atoms (i.e., the number of atoms constituting the ring backbone, including carbon and heteroatoms), and the present definition also includes heterocyclic groups in ranges not specifying the number of rings. The heterocyclic group may be a medium-sized heterocyclic group containing 3 to 10 ring atoms, and the heterocyclic group may be a medium-sized heterocyclic group containing 3 to 10 ring atoms. The heterocyclic group may also be a small size heterocyclic group containing 3 to 6 ring atoms. Examples of heterocyclyl groups include, but are not limited to: azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepinyl, thietanyl, piperidinyl, piperazinyl, pyrazolinyl, pyrazolidinyl, 1, 3-dioxanyl, 1, 4-dioxanyl, 1, 3-oxathietanyl, 1, 4-oxathietanyl, 2H-1, 3-dioxolanyl, 1, 3-dithiolyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinonyl, thiazolidinyl, 1, 3-oxathiolanyl, 1, 3-oxathietanyl, etc, Indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, tetrahydrothiopyranyl, tetrahydro-1, 4-thiazinyl, thiomorpholinyl, dihydrobenzofuranyl, benzimidazolyl, and tetrahydroquinoline.
The term "aromatic ring" as used herein refers to a monovalent aromatic hydrocarbon radical derived by the removal of a hydrogen atom from one or more carbon atoms of a parent aromatic ring system. Aryl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring or an aromatic carbocyclic ring. Typical aromatic ring groups include, but are not limited to, radicals derived from benzene (phenyl), substituted benzenes, naphthalenes, anthracenes, biphenyls, indenyls, indanyls, 1, 2-dihydronaphthalenes, 1,2,3, 4-tetrahydronaphthylenes, and the like.
The term "heteroaromatic ring" in the present invention denotes a monovalent aromatic radical of a5, 6 or 7 membered ring and includes a fused ring system of 5 to 20 atoms (at least one of which is aromatic) containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur. Typical heteroaromatic ring groups include, but are not limited to, those selected from pyridyl (pyridyl) including, for example, 2-hydroxypyridyl, imidazolyl (imidazopyridinyl), imidazopyridyl (imidazopyridyl), 1-methyl-1H-benzo [ d ] imidazole, [1,2,4] triazolo [1,5-a ] pyridine, pyrimidinyl (pyrimidinyl) including, for example, 4-hydroxypyrimidinyl, pyrazolyl (pyrazoyl), triazolyl (triazolyl), pyrazinyl (pyrazoyl), tetrazolyl (tetrazolyl), furyl (furyl), thienyl (thienyl), isoxazolyl, thiazolyl (thiazolyl), oxadiazolyl (oxazoyl), oxazolyl (oxazoyl), isothiazolyl (isothiazolyl), pyrrolyl (pynoliyl), quinolyl (quinolyl), isoquinolyl (isoquinolyl), cinnolinyl (tetrahydroisoquinolyl), tetrahydroisoquinolyl (quinolyl), benzoimidazolyl (indolyl), benzimidazolyl (imidazolyl) (benzimidazolyl), indazolyl (indolizinyl), indolizinyl (indolizinyl), phthalazinyl (phthalazinyl), pyridazinyl (pyridazyl), triazinyl (triazinyl), isoindolyl (isoindolyl), pteridinyl (pteridinyl), purinyl (purinyl), oxadiazolyl (oxydiazolinyl), thiadiazolyl (thiadiazolyl), furazanyl (furazanyl), benzofurazanyl (benzofurazanyl), benzothiophenyl (benzothiophenyl), benzothiazolyl (benzothiazolyl), benzoxazolyl (benzoxazolinyl), quinolinyl (quinolyl), naphthyridinyl (naphthyridinyl), pyranopyridinyl (pyridopyridinyl), and the like.
The term "pharmaceutical composition" in the present invention denotes a combination of at least one drug, optionally together with a pharmaceutically acceptable carrier or adjuvant, combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical compositions include temporally and/or spatially separated combinations, so long as they are capable of acting together to achieve the objectives of the present invention. For example, the ingredients contained in the pharmaceutical composition may be administered to the subject in bulk, or separately. When the ingredients contained in the pharmaceutical composition are administered separately to a subject, the ingredients may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, aqueous buffered solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may comprise wetting agents, emulsifiers or buffer substances as additives.
The term "antibody drug conjugate" in the present invention refers to a compound in which an antibody/antibody functional fragment, a linker and a drug moiety are chemically linked together, and the drug moiety of the antibody drug conjugate according to the present invention is a conjugate of a duocarmycin analog and a nitrogen mustard compound according to the present invention, and may be other compounds comprising a conjugate of a duocarmycin analog and a nitrogen mustard compound. The antibody/antibody functional fragment and the linker of the antibody drug conjugate can be any antibody/antibody functional fragment and linker.
The pharmaceutical composition, antibody drug conjugate according to the present invention may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.
[ specific examples ]
EXAMPLE 1 Synthesis of Compound 3
Figure BDA0002283861970000111
Compound 1(4.23g, 10mmol, available from Nonbutero technologies, Inc., Beijing) was placed in a round-bottomed flask and dissolved in 100mL of tetrahydrofuran. Pd/C (1.0g) was carefully added, 12.5mL of a 25% (m/m) aqueous solution of ammonium formate was added, and the reaction was stirred while the temperature was raised to 50 ℃. After 2h, the reaction was terminated, and the reaction solution was filtered through celite and washed with 50mL of × 2 tetrahydrofuran. The filtrates were combined, concentrated to a small volume under reduced pressure, and extracted with 100mL of ethyl acetate and 100mL of water. The organic phase was washed twice with 50mL of water, 50mL of saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate. Filtration and spin-drying gave 3.28g of 2 as pale yellow solids with a yield of 98.5%. LC-MS, [ M + H ]: 334.4.
Compound 2(33.3mg, 0.1mmol) was placed in a round bottom flask, dissolved by adding 1mL of 4M HCl/dioxane solution, and the reaction was stirred at room temperature. After 3h, the solvent was removed by concentration under reduced pressure to give 27mg of 3 as a dark yellow solid with a yield of 100%. LC-MS, [ M + H ]: 234.3.
EXAMPLE 2 Synthesis of Compound 5
Compound 4(45.8mg, 0.15mmol, purchased from Dalian Meiren Biotechnology Ltd.) was dissolved in anhydrous methanol, and triethylamine (45.5mg, 0.45mmol) was added. Boc2O (65.5mg, 0.3mmol) was added dropwise to the reaction solution, and the reaction was stirred at room temperature for 1 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and the residue was diluted with 20mL of ethyl acetate, washed with 20mL of 0.01N diluted hydrochloric acid, 20mL of water, 20mL of saturated sodium chloride and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave 72mg of pale yellow oil 5 which was directly subjected to the next reaction without purification. LC-MS (LC-MS) shows that [ M + H ] is 405.2.
EXAMPLE 3 Synthesis of Compound I-01
Figure BDA0002283861970000122
Compounds 5(72mg) and 6(30.6mg, 0.15mmol, purchased from Shaoshima chemical technology, Inc.) were dissolved in 1mL of DMF, HATU (68.4mg, 0.18mmol), DIPEA (74.4. mu.L, 0.45mmol) were added, and the reaction was stirred at room temperature for 2 h. After completion of the reaction, the reaction mixture was diluted with 10mL of ethyl acetate, washed with 5mL of 1N diluted hydrochloric acid, 10mL of water, 10mL of saturated brine and dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure to remove the solvent, and column chromatography of the crude product (PE: EA 2:1) gave 54.9mg of compound 7 as a white solid in 62.0% yield. LC-MS, [ M + H ]: 592.9.
Compound 7(54.9mg, 0.093mmol) was dissolved in 2mL of tetrahydrofuran, and 1mL of an aqueous solution of lithium hydroxide (22.3mg, 0.93mmol) was added, and the reaction was stirred at 40 ℃. After 18 hours, the reaction was terminated, and the reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran. 1.5mL of 1N diluted hydrochloric acid was added to precipitate a large amount of milky precipitate, which was extracted with 20mL of ethyl acetate and 15mL of water, and the organic layer was washed with 20mL of water, 20mL of saturated sodium chloride and dried over anhydrous sodium sulfate. Filtration and concentration under reduced pressure removed the solvent, and the residue was subjected to column chromatography (PE: EA ═ 2:3) to give 42.2mg of compound 8as a pale yellow solid in 81.0% yield. LC-MS, [ M + H ]: 563.8.
Compound 3(20.2mg, 0.075mmol) and 8(42.2mg, 0.075mmol) were placed in a round bottom flask, dissolved by addition of 1mL DMF and EDCI (43.1mg, 0.225mmol) was added and the reaction stirred at room temperature for 18 h. After completion of the reaction, the reaction mixture was diluted with 1mL of acetonitrile and separated by Pre-HPLC to obtain 29.5mg of a pale yellow solid compound 9 with a yield of 51.0%. LC-MS, [ M + H ]: 778.6.
Compound 9(29.5mg, 0.038mmol) was placed in a round bottom flask, 1.5mL of dichloromethane was added, 0.5mL of trifluoroacetic acid was added, and the solid was dissolved. After stirring the reaction at room temperature for 1 hour, the solvent was removed by concentration under reduced pressure. The residual oil was dissolved in 10mL of a mixed system of methylene chloride and petroleum ether (1:1), and the solvent was removed by concentration under reduced pressure. The above steps were repeated 3 times to obtain 28.5mg of a yellow solid compound I-01 with a yield of 95.0%. LC-MS, [ M + H ]: 678.2.
Example 4: synthesis of Compound I-03
Figure BDA0002283861970000131
Synthesis of compound 11 referring to the synthesis of compound 7, the starting material was changed from 5 to 10 (purchased from carbofuran technologies ltd.). 64.7mg of compound 11 was obtained as a white solid with a yield of 66.0%. LC-MS, [ M + H ]: 489.3.
Synthesis of compound 12 reference was made to the synthesis of compound 8. 48.2mg of compound 12 was obtained as a white solid with a yield of 78.8%. LC-MS, [ M + H ]: 462.1.
Synthesis of Compound I-03 reference is made to the Synthesis of Compound 9. 34.6mg of pale yellow solid compound I-03 was obtained with a yield of 49.0%. LC-MS, [ M + H ]: 677.2.
EXAMPLE 5 Synthesis of Compound I-05
Figure BDA0002283861970000141
Synthesis of compound 14 referring to the synthesis of compound 7, the starting material was changed from 5 to 13 (purchased from gangrenm biotechnology limited). 67.5mg of compound 14 was obtained as a white solid with a yield of 62.0%. LC-MS, [ M + H ]: 544.2.
Synthesis of compound 15 reference was made to the synthesis of compound 8. 24.9mg of compound 15 as a white solid was obtained with a yield of 38.7%. LC-MS, [ M + H ]: 516.2.
Synthesis of Compound I-05 reference was made to the Synthesis of Compound 9. 18.1mg of a pale yellow solid compound I-05 was obtained with a yield of 51.6%. LC-MS, [ M + H ]: 730.2.
EXAMPLE 6 Synthesis of Compound I-07
Figure BDA0002283861970000151
Synthesis of compound 17 referring to the synthesis of compound 7, the starting material was changed from 6 to 16 (purchased from tokyo chemical industry co., ltd.). 455mg of compound 17 as a white solid was obtained with a yield of 56.0%. LC-MS, [ M + H ]: 594.5.
Synthesis of compound 18 reference was made to the synthesis of compound 8. 519mg of the crude compound 18 are obtained and directly used in the next step without further treatment. LC-MS, [ M + H ]: 565.7.
Synthesis of compound 19 reference was made to the synthesis of compound 9. 33.7mg of crude pale yellow solid compound 19 were obtained in 43.0% yield. LC-MS, [ M + H ]: 781.0.
Synthesis of Compound I-07 reference is made to the method of Synthesis of Compound I-01. 23.8mg of pale yellow solid compound I-07 was obtained with a yield of 85.0%. LC-MS, [ M + H ]: 681.3.
Example 7: synthesis of Compound I-09
Figure BDA0002283861970000152
Synthesis of compound 20 reference is made to the synthesis of compound 7, starting from 5 to 10 and 6 to 16. 322.2mg of crude white solid compound 20 was obtained and directly charged into the next step without further treatment. LC-MS, [ M + H ]:493.2
Synthesis of compound 21 reference was made to the synthesis of compound 8. 235.6mg of compound 21 was obtained as a white solid, which was directly used in the next step without further treatment. LC-MS, [ M + H ]: 465.1.
Synthesis of Compound I-09 reference is made to the Synthesis of Compound 9. 23.3mg of pale yellow solid compound I-09 was obtained with a yield of 34.0%. LC-MS, [ M + H ]: 680.4.
Example 8: synthesis of Compound I-13
Figure BDA0002283861970000161
Synthesis of Compound 23 referring to the synthesis of Compound 7, the starting material was changed from 6 to 22 (Shanghai Bidebao pharmaceutical science Co., Ltd.). 60.4mg of compound 23 was obtained as a white solid with a yield of 68.0%. LC-MS, [ M + H ]: 592.2.
Synthesis of compound 24 reference was made to the synthesis of compound 8. Yield 45.7mg of compound 24 as a white solid, 79.4%. LC-MS, [ M + H ]: 565.8.
Synthesis of compound 25 reference was made to the synthesis of compound 9. 31.2mg of crude pale yellow solid compound 25 was obtained in 49.4% yield. LC-MS, [ M + H ]: 779.6.
Synthesis of Compound I-13 reference is made to the method of Synthesis of Compound I-01. 26.5mg of pale yellow solid compound I-13 was obtained with a yield of 97.5%. LC-MS, [ M + H ]: 679.2.
Example 9: synthesis of Compound I-15
Figure BDA0002283861970000171
Synthesis of compound 26 reference is made to the synthesis of compound 7, starting from 5 to 10 and 6 to 22. 67.8mg of compound 26 was obtained as a white solid with a yield of 69.0%. LC-MS, [ M + H ]: 491.2.
Synthesis of compound 27 reference was made to the synthesis of compound 8. 51.8mg of compound 27 was obtained as a white solid with a yield of 81.2%. LC-MS, [ M + H ]: 463.1.
Synthesis of Compound I-15 reference was made to the Synthesis of Compound 9. 39.1mg of pale yellow solid compound I-15 were obtained with a yield of 51.5%. LC-MS, [ M + H ]: 678.2.
Example 10: synthesis of Compound I-19
Figure BDA0002283861970000172
Compound 9(41.6mg, 0.053mmol) was dissolved in 0.5mL tetrahydrofuran, and a solution of tetrazole (46.1mg, 0.66mmol) in 1mL acetonitrile was added, followed by addition of 28(180.7mg, 0.65 mmol). After 1h, hydrogen peroxide (0.2mL, 1.2mmol) was added to the reaction solution, and after 2h, the reaction was terminated. The solvent was evaporated to dryness by concentration under reduced pressure, the crude product was taken up in 1mL of dichloromethane, 10mL of petroleum ether was added dropwise for crystallization, and filtration was carried out to obtain 20(25.8mg, 0.026mmol) as a pale yellow solid with a yield of 49.1%. LC-MS, [ M + H ]: 971.3.
Compound 29(25.8mg, 0.026mmol) was placed in a round bottom flask and 1.5mL of dichloromethane and then 0.5mL of trifluoroacetic acid were added and the solid dissolved. After stirring the reaction at room temperature for 1h, dichloromethane and trifluoroacetic acid were removed by concentration under reduced pressure. The residue was dissolved in a mixed system of methylene chloride and petroleum ether (1:1), and the solvent was removed by concentration under reduced pressure. The above procedure was repeated 3 times to give 18.8mg of yellow solid compound I-19 in 95% yield. LC-MS, [ M + H ]: 759.0.
Example 11: synthesis of Compound I-22
Figure BDA0002283861970000181
Compound 9(39.0mg, 0.050mmol) was dissolved in 1mL of tetrahydrofuran, a solution of compound 31(16.00mg, 0.080mmol) in 1mL of dichloromethane was added, DIPEA (16. mu.L, 0.06mmol) was added thereto, and the reaction was stirred at room temperature. After 1h, LC-MS detection shows that compound 9 has reacted. To the reaction mixture was added compound 33 (12. mu.L, 0.08mmol), and the reaction was stirred at room temperature. After 2 hours, the reaction was terminated, and the reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was dissolved in 30mL of ethyl acetate, the organic phase was washed with saturated sodium carbonate solution until the aqueous phase was colorless, the organic layer was washed with 30mL of water, 30mL of saturated sodium chloride, and dried over anhydrous sodium sulfate. Spin-dry to give 20.8mg of compound 34 as a pale yellow solid in 46% yield. LC-MS, [ M + H ]: 904.3.
Compound 34(20.8mg, 0.023mmol) was placed in a round bottom flask and 1.5mL of dichloromethane was added and the temperature was reduced to-5 ℃. 0.5mL of trifluoroacetic acid was then added and the solid dissolved. After stirring the reaction at-5 ℃ for 1h, dichloromethane and trifluoroacetic acid were removed by rotary evaporation. The residue was dissolved in a mixed system of methylene chloride and petroleum ether (1:1), and the solvent was removed by concentration under reduced pressure. The residue was repeated the above procedure 3 times to give 17.2mg of pale yellow solid I-22 in 93% yield. LC-MS, [ M + H ]: 804.3.
EXAMPLE 12 Synthesis of other Compounds of similar Structure
The synthesis of the following compounds was accomplished by a synthetic method similar to examples 3 and 4.
Figure BDA0002283861970000201
Example 13 cytotoxicity assay
Detecting I-01, I-03, I-05, I-07, I-09, I-13, I-15, I-19, I-23, I-24, I-25 and I-26, i-27 and I-28 on SK-BR-3 (human breast cancer cells and adherent cells), and I-01, I-03, I-05, I-13 and I-15 on SMMC7721 (human liver cancer cells and adherent cells), SK-OV-3 (human ovarian adenocarcinoma cells and adherent cells), A549 (human non-small cell lung cancer cells and adherent cells), NCI-N87 (human gastric cancer cells and adherent cells), Raji (human lymphoma cells and suspension cells) and Jurkat (acute T cell leukemia cells and suspension cells) respectively.
(1) Group of adherent cells:
① digestion and cell counting: continuously culturing SK-BR-3, SMMC7721, SK-OV-3, A549 and NCI-N87 cells for 1-3 days after passage or liquid change, discarding supernatant, and washing with 5ml PBS once; taking 2ml pancreatin to digest the cells until the cells become round or observing the clear separation between the cells under a microscope, blowing off the cells by using 2ml complete culture medium, and uniformly mixing; and counting by using a cell counter.
② plank: add 200. mu.l of sterile PBS per well a week outside the 96-well plate; SK-BR-3, SMMC7721SK-OV-3, A549 and NCI-N87 cells at a density of 5 × 1045X 10 pieces/ml45X 10 pieces/ml45X 10 pieces/ml41X 10 pieces/ml5Adding the mixture into a negative group hole and a drug administration group hole in sequence, wherein each volume is 100 mu l; the blank wells were filled with 100. mu.l/well of cell complete medium. Standing for 15 min after the plate paving is finished, and placing at 37 ℃ and 5% CO2The cells were cultured overnight in a cell incubator.
③ sample application: adding the diluted samples (the dilution concentrations are respectively shown in tables 1,2,3,4 and 5) into the administration group of wells according to 100 mul/well in sequence, and paralleling the two wells; adding 100 μ l/well of cell complete culture medium into the negative group and the blank group at 37 deg.C and 5% CO2The culture was continued in the cell incubator for 3 days.
④ color development: the supernatant from the 96-well cell culture plate was aspirated, and CCK-8 developer diluted 10-fold with cell completion medium was added to 100. mu.l/well, followed by culturing in a 5% CO2 cell culture chamber at 37 ℃ for 4 hours.
⑤ reading: the Soft Max5.2 software was opened using a microplate reader, the absorbance at 450nm was measured and the OD values of the blank set of wells were automatically subtracted by the software.
TABLE 1 Effect concentration of Small molecules on SK-BR-3 cells (unit: ng/ml)
Figure BDA0002283861970000211
TABLE 2 concentration of effect of small molecules on SMMC7721 cells (unit: ng/ml)
Figure BDA0002283861970000221
TABLE 3 concentration of effect of Small molecules on SK-OV-3 cells (unit: ng/ml)
Figure BDA0002283861970000222
TABLE 4 concentration of effect of Small molecules on A549 cells (unit: ng/ml)
Figure BDA0002283861970000223
TABLE 5 concentration of effect of Small molecules on NCI-N87 cells (unit: ng/ml)
(2) Suspension cell group:
① cell count: sucking the Raji and Jurkat cell suspension into a 15ml centrifuge tube, centrifuging at 800rpm/min for 5 minutes, and removing the supernatant; the cells were thoroughly flushed with 2ml of cell culture medium and mixed well, and counted by an automatic cell counter.
② adding medicine: adding 200 mu l of sterile PBS per well in a mode of adding medicine and then adding cells on the outer side of a 96-well plate for one week; adding diluted samples (the dilution concentrations are respectively shown in the table 6 and the table 7) into the administration group holes according to 100 mul/hole; negative and blank wells were filled with 100. mu.l/well of cell complete medium.
③ plank: raji and Jurkat cells were plated at a density of 1X 1051X 10 pieces/ml5Adding each/ml of the mixture into a negative group hole and a drug administration group hole in sequence, wherein each hole is 100 mu l, and the two holes are parallel; the blank wells were filled with 100. mu.l/well of cell complete medium. The Raji cell plates were further cultured in a 5% CO2 cell culture chamber at 37 ℃ for 5 days, and the Jurkat cells were further cultured for 3 days.
④ color development: adding CCK-8 color developing agent into 96-well cell culture plate at 23 μ l/well in sequence, and adding 5% CO at 37 deg.C2The cell culture chamber was continued for 4 hours.
⑤ reading: the Soft Max5.2 software was opened using a microplate reader, the absorbance at 450nm was measured and the OD values of the blank set of wells were automatically subtracted by the software.
TABLE 6 concentration of effect of Small molecules on Raji cells (unit: ng/ml)
Figure BDA0002283861970000231
TABLE 7 concentration of effect of Small molecules on Jurkat cells (unit: ng/ml)
Figure BDA0002283861970000232
Figure BDA0002283861970000241
The evaluation of the cell activities of the compounds I-01, I-03, I-05, I-07, I-09, I-13, I-15, I-19, I-23, I-24, I-25, I-26, I-27 and I-28 on SK-BR-3 (human breast cancer cell, adherent cell) cells is shown in Table 8. As can be seen from the table, the compounds I-01, I-03, I-05, I-07, I-09, I-13, I-15, I-19, I-23, I-24, I-25, I-26, I-27 and I-28 all showed good inhibitory effect on SK-BR-3 (human breast cancer cells, adherent cells) cells. The maximum inhibition rate of the compounds is more than 99 percent, which indicates that the compounds have the capability of completely killing dividing or dormant tumor cells. In general, these compounds have potent cytotoxic activity, and the IC of most compounds50Usually between 0.1-10nM, and can exert antitumor effect at very low concentrations.
TABLE 8 result of evaluation of SK-BR-3 cytotoxic Activity
Figure BDA0002283861970000242
To further validate the broad spectrum of killing or inhibition of each type of tumor cells by the above compounds, we performed validation in multiple tumor models. Wherein, the compounds I-01, I-03, I-05, I-13 and I-15 respectively act on SK-BR-3 (human breast cancer cells and adherent cells), SMMC7721 (human liver cancer cells and adherent cells), SK-OV-3 (human ovarian adenocarcinoma cells and adherent cells), A549 (human non-small cell lung cancer cells and adherent cells), NCI-N87 (human gastric cancer cells and adherent cells), Raji (human lymphoma cells and suspension cells), JuThe evaluation of the cell activity of rkat (acute T-cell leukemia cells, suspension cells) cells is shown in table 9. As can be seen from the table, the maximum inhibition rates of these compounds were all above 97%, indicating that these compounds have the ability to completely kill dividing or dormant tumor cells. In general, these compounds have potent cytotoxic activity, and the IC of most compounds50Usually between 0.1-10nM, and can exert antitumor effect at very low concentrations. In addition, the compounds have stronger inhibitory activity on SK-Br-3 cells, SK-OV-3 cells, Raji cells and JuaKat cells, which indicates that the compounds have certain cell and tissue difference when killing tumor cells; meanwhile, the I-01 and the I-13 show unexpected broad killing or inhibition spectrum of tumor cells, have excellent killing or inhibition activity on various tumor cells, and the toxins in the two compounds are
Figure BDA0002283861970000254
Combinations of (a) and (b).
TABLE 9 evaluation results of cytotoxic Activity of Compounds I-01, I-03, I-05, I-13, and I-15
Figure BDA0002283861970000253
Figure BDA0002283861970000261
The growth inhibition rate graphs of I-01, I-03, I-05, I-13 and I-15 for SK-BR-3 (human breast cancer cell, adherent cell), SMMC7721 (human liver cancer cell, adherent cell), SK-OV-3 (human ovarian adenocarcinoma cell, adherent cell), A549 (human non-small cell lung cancer cell, adherent cell), NCI-N87 (human gastric cancer cell, adherent cell), Raji (human lymphoma cell, suspension cell) and Jurkat (acute T cell leukemia cell, suspension cell) cell are respectively shown in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6 and FIG. 7. FIG. 8 shows the scatter plot of the IC50 values of I-01, I-03, I-05, I-13, I-15 against SK-BR-3, SMMC7721, SK-OV-3, A549, NCI-N87, Raji, Jurkat cells, respectively, and it can be seen from FIG. 8 that I-01, I-03, I-05, I-13, I-15 have growth inhibitory effects against SK-BR-3, SMMC7721, SK-OV-3, A549, NCI-N87, Raji, Jurkat cells, especially NCI-N87, SK-BR-3, Raji, I-01, I-03, I-05, I-13, I-15.
EXAMPLE 14 preparation of antibody drug conjugates
The preparation of the antibody drug conjugate adopts a general preparation method, firstly, a linker part and a drug part are coupled to form a linker-drug conjugate, and then the linker-drug conjugate and the antibody part are coupled to form the antibody drug conjugate, wherein the specific preparation method comprises the following steps:
the linker (i.e., the compound provided by the invention) is dissolved in a proper amount of DMF, and a proper amount of drug moieties (such as MMAE, MMAF or cytotoxins such as the compound provided by the invention), HOBt, DIPEA and pyridine are added under the protection of nitrogen. Stir at rt for 24 h and monitor the progress of the reaction by TLC. After the reaction is finished, purifying by using a preparative high performance chromatography, and carrying out reduced pressure rotary evaporation on the prepared solution to obtain a linker-drug conjugate; in a buffer containing 10% DTPA in 20mM borax borate pH 9.0, the four pairs of interchain disulfide bonds of the antibody were reduced by the addition of 4-6 equivalents of tris (2-carboxyethyl) phosphine hydrochloride (TCEP). After reaction for about 1h at room temperature, DMSO (20% V/V) dissolved linker-drug conjugate (5-10 equiv) was added and the reaction was allowed to proceed overnight at room temperature. After the reaction is finished, performing centrifugal ultrafiltration for 3 times by using PBS buffer solution, purifying to remove residual unreacted medicines and free small molecules such as DMSO, and detecting the coupling condition by using SDS-PAGE electrophoresis and a hydrophobic high performance liquid chromatography (HIC-HPLC) method.
The structure of the prepared antibody drug conjugate is shown as follows (the following examples are the structure example of connecting one toxin, and more than 2 toxins can be simultaneously connected to one antibody according to the requirements of cytotoxic loading and different linkers adopted by the antibody drug conjugate):
the invention has been illustrated by various specific embodiments. However, it is understood by those skilled in the art that the present invention is not limited to the respective embodiments, and that various changes or modifications may be made by those skilled in the art within the scope of the present invention, and various technical features mentioned throughout the present specification may be combined with each other without departing from the spirit and scope of the present invention. Such modifications and variations are intended to be within the scope of the present invention.

Claims (7)

1. A compound as described in formula (I),
Q－L－T (I)
wherein:
the structure of Q is
Figure FDA0002283861960000011
The structure of the T is selected from
Figure FDA0002283861960000012
The structure of the L is selected from
Figure FDA0002283861960000014
Figure FDA0002283861960000015
2. The compound of claim 1, wherein said compound is selected from the structures:
Figure FDA0002283861960000016
Figure FDA0002283861960000021
Figure FDA0002283861960000031
Figure FDA0002283861960000041
3. a pharmaceutical composition comprising a compound according to any one of claims 1 to 2.
4. An antibody drug conjugate, wherein the compound of any one of claims 1 to 2 is a prodrug of the antibody conjugate.
5. Use of a compound according to any one of claims 1-2 for the manufacture of a medicament.
6. The use of claim 5, wherein the drug is an antibody drug conjugate.
7. Use of a compound according to any one of claims 1 to 2 or a pharmaceutical composition according to claim 3 or an antibody drug conjugate according to claim 4 in the manufacture of a medicament for the treatment of cancer, an autoimmune disease, an inflammatory disease or an infectious disease.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101616911A (en) * 2007-02-21 2009-12-30 梅达莱克斯公司 Chemical linkers and conjugate thereof with single amino acids
CN101939028A (en) * 2007-11-30 2011-01-05 百时美施贵宝公司 Monoclonal antibody partner molecule conjugates directed to protein tyrosine kinase 7 (PTK7)
CN105636591A (en) * 2014-01-29 2016-06-01 上海恒瑞医药有限公司 Ligand-cytotoxicity drug conjugate, preparation method therefor, and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101616911A (en) * 2007-02-21 2009-12-30 梅达莱克斯公司 Chemical linkers and conjugate thereof with single amino acids
CN101939028A (en) * 2007-11-30 2011-01-05 百时美施贵宝公司 Monoclonal antibody partner molecule conjugates directed to protein tyrosine kinase 7 (PTK7)
CN105636591A (en) * 2014-01-29 2016-06-01 上海恒瑞医药有限公司 Ligand-cytotoxicity drug conjugate, preparation method therefor, and uses thereof

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