Peptide drug conjugates and their uses

Abstract

This invention discloses compounds of the general formula (I): L1, L2, LD1, LD2, R5, and R6 are defined in the specification. This invention provides pharmaceutical compositions covering such compounds and methods for treating cancer using these compound complexes.

Description

Technical Field

[0001] The present invention generally refers to a compound complex of formula (I), a pharmaceutical composition containing the compound, and the use of the compound complex in treating cancer. Prior Art

[0002] Luteinizing hormone releasing hormone (LHRH) is a useful ligand targeting LHRH receptor (LHRH-R), which is overexpressed in the plasma membrane of various types of cancer cells but is undetectable in many normal organ tissues.

[0003] According to journal reports, LHRH-R is highly expressed in various cancers. About 86% of prostate cancer, about 80% of endometrial cancer and ovarian cancer, about 80% of kidney cancer, about 50% of breast cancer, and 32-50% of pancreatic cancer all express this receptor. See Li et al., Mini Rev Med Chem 17(3), 258-267(2017).

[0004] Since LHRH-R receptors provide highly specific and selective tumor targeting, methods that leverage this property to target cancer have attracted much attention. See Reubi, 2003 and Vhora et al., 2014.

[0005] A major challenge in developing new and highly effective drug complexes is to selectively deliver drugs to tumor sites while sparing healthy tissue. One strategy is to covalently bind chemotherapeutic agents to antibodies that help recognize specific tumor antigens. However, antibody-drug complexes are limited by poor in vitro and in vivo stability, high antigenicity, complex binding chemistry, relatively high manufacturing costs, and limited penetration into solid tumors. Therefore, the present invention has developed another type of strategy, which is to design and form effective LHRH-drug complexes with sufficient stability and safety to transport therapeutic agents to the tumor site and release the drugs at the appropriate time and space to kill cancer cells, thereby achieving safe and effective treatment with reduced side effects.

[0006] There is a need to develop new LHRH-drug complexes for effectively delivering therapeutic agents to cancer cells without the above-mentioned drawbacks. Summary of the invention

[0007] To meet the above needs, LHRH compounds are designed to be covalently combined with cancer treatment drugs to form drug conjugates.

[0008] Therefore, the compound of one of the present invention has the following derivation trend schematic formula (I):

[0009] In the above formula (I), L1 and L2 are each independently C1-C6 alkyl, aryl or aralkyl; LD1 is -N(R1)(R2) or a first linker-drug moiety represented by -LK1-DG1, wherein R1 is H, R2 is H or a substituted carbonyl, or R1 and R2 together with the nitrogen atom to which they are attached form -N3, a substituted or unsubstituted guanidino, a 5- to 14-membered heteroaryl, or a 5- to 14-membered heterocycloalkyl; LK1 is a first linker; and DG1 is a first drug carrier moiety; LD2 is -N(R3)(R4) or a second linker-drug moiety represented by -LK2-DG2, wherein R3 is H, R4 is a substituted carbonyl group, or R3 and R4 together with the nitrogen atom to which they are connected form a substituted or unsubstituted guanidine group, a 5- to 14-membered heteroaryl group, or a 5- to 14-membered heterocycloalkyl group; LK2 is a second linker; DG2 is a second drug carrier moiety; R5 is an aryl group, a C1-C6 alkyl group, or a third linker-drug moiety represented by -LK3-DG3, wherein LK3 is a third linker and DG3 is a third drug carrier moiety; and R6 is a C1-C6 alkyl group.

[0010] The compounds of formula (I) may have any combination of one or more of the following characteristics:

[0011] (i) LD1 and LD2 are each independently -NH2, -N3, -NHC(O)NH2, -NHC(=NH)NH2, or -N=C(NHCH2CH3)2;

[0012] (ii) LD2 is -NHC(O)NH2, -NHC(=NH)NH2, or -N=C(NHCH2CH3)2;

[0013] (iii) one of LD1 and LD2 is NHC(O)NH2, -NHC(=NH)NH2, or -N=C(NHCH2CH3)2, and the other is a first linker-drug moiety or a second linker-drug moiety;

[0014] (iv) one of LD1, LD2 and R5 is selected from and The group formed;

[0015] (v) R5 is a substituted phenyl group or a substituted C1-C6 alkyl group;

[0016] (vi) R5 is aminopropyl, 4-hydroxyphenyl or ;

[0017] (vii) R6 is isobutyl or aminobutyl;

[0018] (viii) L1 is ethylene, propylene or phenylene;

[0019] (ix) n is 3 or 4.

[0020] A subset of compounds of formula (I) includes the following compounds of formula (II) or formula (III): and wherein n is 1 to 6, and R1 to R6 and L1 are each as defined above.

[0021] Another compound derivative of the invention includes a pharmaceutical composition comprising any one of the above compounds and a pharmaceutically acceptable carrier.

[0022] The scope of the present invention encompasses a method for treating cancer using any of the above compounds, and comprises administering an effective dose to any patient in need thereof.

[0023] Schematic diagram of compound derivation trend The compounds of formula (I) include compounds 1 to 32, and their structures are shown below.

[0024] Compound 1

[0025] Compound 2

[0026] Compound 3

[0027] Compound 4

[0028] Compound 5

[0029] Compound 6

[0030] Compound 7

[0031] Compound 8

[0032] Compound 9

[0033] Compound 10

[0034] Compound 11

[0035] Compound 12

[0036] Compound 13

[0037] Compound 14

[0038] Compound 15

[0039] Compound 16

[0040] Compound 17

[0041] Compound 18

[0042] Compound 19

[0043] Compound 20

[0044] Compound 21

[0045] Compound 22

[0046] Compound 23

[0047] Compound 24

[0048] Compound 25

[0049] Compound 26

[0050] Compound 27

[0051] Compound 28

[0052] Compound 29

[0053] Compound 30

[0054] Compound 31

[0055] Compound 32

[0056] Preferred compounds are Compound 8, Compound 16, Compound 27 and Compound 28.

[0057] The term "alkyl" herein refers to a linear or branched monovalent or divalent hydrocarbon containing 1 to 20 (e.g., 1 to 10 and 1 to 6) carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. Alkyl includes its halogen-substituted derivatives, i.e., haloalkyl, which refers to an alkyl substituted by one or more halogen (chlorine, fluorine, bromine, or iodine) atoms. Examples include trifluoromethyl, bromomethyl, and 4,4,4-trifluorobutyl. The term "alkoxy" refers to -O-alkyl. Examples include methoxy, ethoxy, propoxy, and isopropoxy. Alkoxy includes haloalkoxy, which refers to an alkoxy substituted by one or more halogen atoms. Examples include -O-CH2Cl and -O-CHClCH2Cl.

[0058] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, bicyclic, tricyclic or tetracyclic monovalent or divalent hydrocarbon radical having 3 to 12 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl.

[0059] The term "heterocycloalkyl" refers to a non-aromatic monovalent or divalent 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having one or more heteroatoms such as O, N, P, and S. Examples of heterocycloalkyl include, but are not limited to, piperazinyl, imidazolidinyl, azepanyl, pyrrolidinyl, dihydrothiadiazolyl, dioxanyl, morpholinyl, tetrahydropuranyl, and tetrahydrofuranyl.

[0060] The term "aryl" refers to a monovalent or divalent 6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic aromatic ring system, wherein each ring may have 1 to 5 substituents. Examples of aryl groups include phenyl, naphthyl, and anthracenyl. The term "arylene" refers to a divalent aromatic group. The term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0061] The term "heteroaryl" refers to a monovalent or divalent aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having one or more heteroatoms (e.g., O, N, P, and S). Examples include triazolyl, oxazolyl, thiadiazolyl, tetrazolyl, pyrazolyl, pyridinyl, furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl, and benzothiazolyl. The term "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group.

[0062] The term "carbonyl" refers to -C(O)-.

[0063] The term "halogen" refers to a fluorine, chlorine, bromine or iodine free radical. The term "amino" refers to a free radical derived from an amine, which is unsubstituted or mono- / di-substituted by an alkyl, aryl, cycloalkyl, heterocycloalkyl or heteroaryl group. The term "alkylamino" refers to an alkyl-NH-. The term "dialkylamino" refers to an alkyl-N(alkyl)-.

[0064] The alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkoxy and aryloxy mentioned herein include substituted and unsubstituted moieties. Examples of substituents include, but are not limited to, halogen, hydroxyl, amino, cyano, nitro, mercapto, alkoxycarbonyl, amido, carboxy, alkylsulfonyl, alkylcarbonyl, carbamido, carbamyl, carboxyl, thiourea, thiocyanato, sulfonamide, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl and heterocycloalkyl, wherein alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, cycloalkyl and heterocycloalkyl may be further substituted.

[0065] The compound of formula (I), formula (II) or formula (III) may contain anions. Examples of anions include Cl-, Br-, I-, SO4 2-, PO4 3-, ClO4 -, CH3CO2 - and CF3CO2 -.

[0066] The term "compound" when referring to a compound of formula (I), (II) or (III) also encompasses salts, solvates and prodrugs thereof. Salts can be formed between anions and positively charged groups on the compound (e.g., amine groups). Examples of suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, acetate, malate, tosylate, tartrate, fumarate, glutamate, glucuronide, lactate, glutarate and maleate. Salts can also be formed between cations and negatively charged groups. Examples of suitable cations include sodium, potassium, magnesium, calcium and ammonium cations such as tetramethylammonium. In addition, salts may also contain quaternary nitrogen atoms. Solvates are complexes formed between an active compound and a pharmaceutically acceptable solvent. Examples of pharmaceutically acceptable solvents include water, ethanol, isopropanol, ethyl acetate, acetic acid and ethanolamine. Prodrugs are compounds that are metabolized into pharmaceutically active drugs after administration. Examples of prodrugs include esters and other pharmaceutically acceptable derivatives which, upon administration to a subject, are capable of providing the active compounds of the invention.

[0067] The present invention is described in detail in the following figures, definitions and detailed description. Through the following practical examples and claims, the features, objectives and advantages of the present invention will be more clearly presented. Implementation

[0068] As mentioned above, LHRH-drug complexes are used to treat certain cancers.

[0069] Luteinizing hormone releasing hormone (also called "gonadotropin-releasing hormone" or "GNRH") or "LHRH" is a decapeptide hormone with the following structure: (Pyr)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2.

[0070] LHRH acts as a ligand in the LHRH-complex of the present invention and is linked to the drug moiety via one or more linker groups.

[0071] LHRH analogs are also suitable as ligands of the LHRH-drug complex of the present invention. Such analogs include LHRH (or GNRH) agonists, LHRH (or GNRH) antagonists, or any combination of LHRH analogs, LHRH agonists, or LHRH antagonists that can bind to LHRH receptors. Preferably, LHRH analogs, LHRH agonists, LHRH antagonists, or any combination thereof can bind to one or more LHRH receptors and are gonadotropin secretion inhibitors or gonadotropin receptor effect blockers.

[0072] LHRH agonists that can be used in the present invention include, for example, peptides described in Treatment with LHRH analogs: Controversies and perspectives, The Parthenon Publishing Group Ltd. (1996), JP-A-3-503165, JP-A-3-101695, JP-A-7-97334, and JP-A-8-259460. Exemplary peptides that can be used in the present invention have the following chemical formula: (Pyr)Glu-R1a-Trp-Ser-R2a-R3a-R4a-Arg-Pro-R5a (IA) Wherein, R1a is His, Tyr, Trp or p-NH2-Phe; R2a is Tyr or Phe; R3a is Gly or a D-type amino acid residue which may optionally have one or more substituents; R4a is Leu, Ile or Nle; R5a is Gly-NH-R6a (R6a is a hydrogen atom or an alkyl group which may optionally have a hydroxyl group), NH-R7a (R7a is a hydrogen atom, an amine group, an alkyl group which may optionally have a hydroxyl group or a urea group (-NH-CO-NH2)) or a salt thereof.

[0073] In the above formula (IA), when R3a is a D-amino acid residue, the D-amino acid may be an α-D-amino acid having up to 9 carbon atoms (i.e., D-Leu, Ile, Nle, Val, Nval, Abu, Phe, Phg, Ser, Thr, Met, Ala, Trp, α-Aibu), etc. Examples of substituents that can be used with R3a include, but are not limited to, tert-butyl, tert-butoxy, tert-butoxycarbonyl, methyl, dimethyl, trimethyl, 2-naphthyl, indol-3-yl, 2-methylindolyl, benzyl-imidazole-2-yl, etc. In addition, in formula (I), examples of alkyl groups of R6a or R7a include, but are not limited to, C1-4 alkyl groups, examples of which are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl.

[0074] In addition, salts of the peptide represented by formula (IA) (also referred to herein as peptide (IA)) include, but are not limited to, acid salts (i.e., carbonates, bicarbonates, acetates, trifluoroacetates, propionates, succinates, etc.) and metal complexes (i.e., copper complexes, zinc complexes, etc.). Peptide (IA) or its salts can be prepared by any method known to a person of ordinary skill in the art to which the invention belongs, such as U.S. Pat. No. 3,853,837, U.S. Pat. No. 4,008,209, U.S. Pat. No. 3,972,859, British Patent No. GB1,423,083, Proceedings of the National Academy of Sciences of the United States of America, vol. 78, pp. 6509-6512 (1981), or methods similar thereto.

[0075] Preferably, the peptide (IA) may have the structure shown in US 2012 / 0129773 A1, in particular, formula (a) to formula (j).

[0076] Examples of LHRH antagonists that can be used in the present invention include, for example, those disclosed in U.S. Pat. Nos. 4,086,219, 4,124,577, 4,253,997 and 4,317,815 or peptides represented by Formula II of US 2012 / 0129773 A1. These peptides include their salts and optical isomers or mixtures of optical isomers can be used. Pharmaceutically acceptable salts are preferably used. Examples of such salts include, but are not limited to, salts of inorganic acids (i.e., hydrochloric acid, sulfuric acid, nitric acid, etc.), salts of organic acids (i.e., carbonic acid, bicarbonic acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.), etc. Preferably, the salt of the peptide is a salt of an organic acid (i.e., carbonic acid, bicarbonic acid, succinic acid, acetic acid, propionic acid, trifluoroacetic acid, etc.). Most preferably, the salt of the peptide is acetate. More specifically, these salts may be mono-, di- or tri-salts.

[0077] The peptide or its salt can be prepared by any method known to those skilled in the art, such as the method described in JP-A-3-101695 (EP-A 413209), Journal of Medicinal Chemistry, Vol. 35, p. 3942 (1992), or a method similar thereto.

[0078] In addition, linear peptides (U.S. Pat. Nos. 5,140,009 and 5,171,835), cyclic hexapeptide derivatives (JP-A-61-191698), bicyclic peptide derivatives (Journal of Medicinal Chemistry, Vol. 36, pp. 3265-3273 (1993)), etc., which are LHRH derivatives, can be used. As examples of non-peptide compounds having LHRH antagonistic activity, compounds described in JP-A-62-116514, WO 95 / 28405 (JP-A-8-295693), WO 97 / 14697 (JP-A-9-169767), WO 97 / 14682 (JP-A-9-169735), WO 96 / 24597 (JP-A-9-169768), J. Med. Chem., Vol. 32, pp. 2036-2038 (1989), etc. can be used.

[0079] Examples of suitable LHRH antagonists include, but are not limited to, abarelix, ganirelix, cetrorelix, 5-(N-benzyl-N-methyl-aminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthiophene-[2,3-d]pyrimidine-2,4(1H,3H)-dione (5-(N-benzyl-N-methyl-aminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthiophene-[2,3-d]pyrimidine-2,4(1H,3H)-dione, yl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno-[2,3-d]pyrimidine-2,4(1H,3H)-dione), 5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-ethylureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1 pyrimidine-2,4(1H,3H)-dione and 5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-ethylureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione hydrochloride.

[0080] Any anticancer drug can be linked to the LHRH ligand via a cross-linking agent. Examples of anticancer drugs include oxaliplatin platinum salts, vinca alkaloids, eribulin, epothilones (e.g., ixabepilone available as Ixempra® from Bristol-Myers Squibb, New York, New York), arsenic trioxide (Trisenox®, Teva Pharmaceuticals USA, Parsippany, New York, USA), and arsenic trioxide (Trisenox®, Teva Pharmaceuticals USA, Parsippany, New York, USA). Jersey), cytarabine (Cytosar-U® and Depocyt®), etoposide, hexamethylmelamine, ifosfamide (Ifex®), methotrexate (Trexall®), procarbazine (Matulane®), vinblastine, platinum compounds (cisplatin, carboplatin, oxaliplatin), vincristine, taxanes (docetaxel, paclitaxel), bortezomib (Velcade®), thalidomide (Thalomid®), and lenalidomide.

[0081] LHRH-drug complexes can be used to treat different cancers (eg, colorectal cancer, renal cell carcinoma, lung cancer, pancreatic cancer, endometrial cancer, ovarian cancer, breast cancer, and prostate cancer).

[0082] LHRH (or its analog) is covalently linked to the drug carrier via one or more linkers. A crosslinker is a chemical reagent with two or more reactive groups at its ends. Examples of reactive groups found in crosslinkers include, but are not limited to, amines for binding to carboxylate groups of target molecules; NHS-esters for binding to amine groups of target molecules; maleimides for binding to thiol groups of target molecules; isocyanates for binding to hydroxyl groups of target molecules; alkynes (-C≡CH) for binding to azide groups (-N3) of target molecules; hydrazine or amine groups for binding to aldehyde or ketone groups of target molecules. When the crosslinker reacts with LHRH (or its analog) and drug molecules, one end of it binds to LHRH (or its analog) and the other end forms a covalent bond with the drug.

[0083] The cross-linker portion of the LHRH-drug complex preferably contains a cleavable group, such as -NHC(O)O-, -NHC(O)N-, -NHC(O)-, -C(O)O-, -C(O)S-, -SS- and an amino acid moiety. Examples are shown above for the drug-linker moiety.

[0084] The preparation of the compounds of formula (I), (II) or (III) can be achieved by reacting the cross-linking agent, the drug molecule and LHRH (or the like) stepwise or in one pot using conventional methods, for example, the procedures provided in the references cited above. Their anti-tumor activity can then be evaluated using methods known in the art.

[0085] The compounds of the present invention contain non-aromatic double bonds or one or more asymmetric centers. They each exist in the form of a racemate or a racemic mixture, a single R mirror image isomer, a single S mirror image isomer, a single diastereomer, a diastereomer mixture, a cis isomer or a trans isomer. Such isomeric compounds are within the scope of the present invention. They may exist as a mixture, or may be separated using chiral synthesis or chiral separation techniques.

[0086] Preferably, the compound of formula (I), (II) or (III) is formulated into a pharmaceutical composition containing a pharmaceutical carrier, and then the composition is administered to a subject in need thereof to treat LHRH receptor-related cancer.

[0087] To practice the methods of the present invention, compositions having one or more of the above-described complexes may be administered parenterally, orally, nasally, rectally, topically, or buccally.

[0088] The term "parenteral" as used herein encompasses subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection of a sterile injectable composition. Indeed, the term refers to any suitable infusion technique.

[0089] Sterile injectable compositions may be solutions or suspensions in nontoxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable carriers and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are often used as solvents or suspending media (e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives, may be used to prepare injectables, as may natural pharmaceutically acceptable oils, such as olive oil and castor oil, especially their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, carboxymethyl cellulose or similar dispersants. Other commonly used surfactants such as Tweens and Spans or other similar emulsifiers or bioavailability enhancers, which are commonly used to make pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0090] The composition for oral administration can be in any orally acceptable dosage form, including capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oil phase in combination with an emulsifier or suspending agent. If desired, certain sweeteners, flavorings or coloring agents can be added. Oral solid dosage forms can be prepared by spray drying technology; hot melt extrusion strategies, micronization and nano-milling technology.

[0091] Nasal aerosol or inhalation compositions can be prepared according to techniques well known in the art of pharmaceutical preparation. For example, such compositions can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and / or other solubilizing agents or dispersants known in the art. Compositions with active compounds can also be administered in the form of suppositories for rectal administration.

[0092] The carrier in a pharmaceutical composition must be "acceptably compatible," meaning that it is compatible with the active ingredient of the composition (and preferably capable of stabilizing the active ingredient) and not deleterious to the subject being treated. One or more solubilizing agents may be used as pharmaceutical excipients for delivery of the active compound. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

[0093] The term "treatment" refers to the administration or administration of a compound to a subject for the purpose of curing, alleviating, relieving, altering, remedying, improving or affecting a disease, symptom or tendency. "Effective dose" refers to the amount of a compound required to give a subject the desired effect. As recognized by those of ordinary skill in the art to which the invention pertains, the effective dose will vary depending on the route of administration, the use of excipients, and the possibility of co-administration with other treatment methods (e.g., the use of other active agents). The dosage content of the compound of formula (I), (II) or (III) is 0.01 mg / kg body weight to 500 mg / kg body weight per day (e.g., 0.05 mg / kg body weight to 300 mg / kg body weight, 0.1 mg / kg body weight to 200 mg / kg body weight, and 1 mg / kg body weight to 100 mg / kg body weight). The specific dosage content for a particular patient will depend on many factors, including age, weight, general health, sex, diet, time of administration, excretion rate, and severity of the disease. To enhance the efficacy of treatment, the compound may be administered simultaneously with one or more other orally active anti-tumor compounds.

[0094] Without further elaboration, it is believed that a person skilled in the art will be able to utilize the present invention to the maximum extent based on the above description. Therefore, the following specific embodiments should be interpreted as merely illustrative and not limiting the rest of the present disclosure in any way.

[0095] All publications cited herein, including patent documents, are incorporated by reference in their entirety.

[0096] Example

[0097] Compounds 1-32 of the present application were prepared according to the following procedures.

[0098] The materials and reagents used in the preparations are commercially available from various suppliers. They are listed below.

[0099] Protective agent: Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-hArg(Et)2-OH, Fmoc-D-hArg(Et)2-OH, Fmoc-Arg(pbf)-OH, Fmoc-D-Arg(pbf)-OH, Fmoc-Le uOH, Fmoc-D-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH-Fmoc-Ser(tBu)-OH-Fmoc-(D-3-Pal]-OH, Fmoc-D-Phe(4-Cl)-OH and Fmoc-D-2-NAL-OH.

[0100] Coupling reagents: hydroxybenzotriazole (HOBT), N,N'-diisopropylcarbodiimide (DIC), diisopropylethylamine (DIEA), acetone (ACE), CHOH, dichloromethane (DCM) and dimethylformamide (DMF).

[0101] Deprotection reagent: 20% Pip / DMF.

[0102] Resins used for peptide synthesis and purification: Rink Resin.

[0103] Method: General production steps

[0104] 1. Resin swelling

[0105] Add the resin into the reaction bottle, add appropriate amount of DCM, stir and swell for 15-30 minutes.

[0106] 2. Deprotection

[0107] Drain the DCM, add 20% Pip / DMF and stir for 20-30 min.

[0108] 3. Deprotection washing

[0109] The deprotecting reagent was drained off and washed with 5-8 equivalents of DMF.

[0110] 4. Deprotection Detection

[0111] Place 15-30 pieces of resin in a test tube, add 1-2 ml of detection reagent, place the test tube in a water bath above 95°C for 30-60 seconds, take out the test tube and observe the color of the resin. If the color of the resin becomes darker, it means that the deprotection is successful.

[0112] 5. Coupling of the first amino acid

[0113] Add appropriate amount of DM to dissolve 3-5 equivalents of FMOC-D-ALA-OH and HOBI, then add 3-5 equivalents of DIC and stir to react for 1-2 hours.

[0114] 6. Resin sealing surface

[0115] Drain the coupling solution and add DMF into the reaction flask, stir acetic anhydride and N,N-diisopropylethylamine (DIEA) for 20-30 min.

[0116] 7. Reaction washing

[0117] The reagent was drained and washed with 3-5 equivalents of DMF.

[0118] 8. Go to Protection

[0119] Add 20% Pip / DMF and stir for 20-30 minutes.

[0120] 9. Deprotection washing

[0121] The deprotecting reagent was drained off and washed with 5-8 equivalents of DMF.

[0122] 10. Deprotection Detection

[0123] Place 15-30 pieces of resin in a test tube, add 1-2 ml of detection reagent, place the test tube in a water bath above 95°C for 30-60 seconds, take out the test tube, and observe the color of the resin. A darker color of the resin indicates successful deprotection.

[0124] 11. Coupling

[0125] Add an appropriate amount of DM to dissolve 3-5 equivalents of Fmoc-Pro-OH and HOBT, then add 3-5 equivalents of DIC and stir the reaction for 1-2 hours.

[0126] 12. Coupling detection

[0127] Put 15-30 pieces of resin into a test tube, add 1-2 ml of detection reagent, put the test tube into a water bath above 95°C for 30-60 seconds, take out the test tube and observe the color of the resin. No obvious change in the color of the resin indicates successful coupling.

[0128] Repeat steps 7 to 12 to condense the amino acids in the sequence from the C side to the N side.

[0129] After the last amino acid is coupled, the deprotection is successful, and the resin is washed with an appropriate amount of methanol and drained for 3-5 hours.

[0130] Lysis step

[0131] Reagent formula: trifluoroacetic acid (TFA) / ethylenedithiol (EDT) / water = 90 / 5 / 5, stir and lyse at room temperature for 2-3 hours, filter out the resin, add ether to the lysate to precipitate, centrifuge to remove the upper liquid reagent, then add ether and stir to disperse evenly. Centrifuge 2-3 times, transfer the solid coarse polypeptide to a clean tray, and place it in a 35°C vacuum drying oven for 3-5 hours. Analyze a small portion of the sample by HPLC and MS for purification.

[0132] Purification steps

[0133] Take out a certain amount of crude peptide and dissolve it by adding a certain amount of pure water and acetonitrile mixture through ultrasound. After the sample is clear and transparent, filter it through a vacuum filter and purify it through a DAC system.

[0134] Lyophilization Steps

[0135] After purification, the qualified components were placed in a freeze drying dish, covered with a lid and placed in a freeze dryer for freeze drying. After freeze drying, the freeze drying plate was taken out, weighed, and the peptide samples were divided and stored at -20°C.

[0136] Synthesis of Peptide-Alkyne Compounds

[0137] Compounds 2A, 6A, 10A, 12A, 13A and 15A were prepared according to the following procedures.

[0138] General Procedure: A solution of ((1R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(4-nitrophenyl) carbonate B) (1.05 eq) in DMF (1 mL) was added to LHRH analog or Degarelix (1 eq) and N,N-diisopropylethylamine (DIPEA) (3 eq) and the reaction mixture was stirred at room temperature for 30 min. The completion of the reaction was checked by HPLC. The reaction mixture was concentrated under reduced pressure. The residual oil was treated with ethyl acetate to precipitate a solid, which was purified by washing with diethyl ether, ethyl acetate (EA), dichloromethane (CH2Cl2) to give the peptide acetylene compound as a white solid.

[0139] Synthesis of Peptide-Azide Compounds:

[0140] Compound 32 was prepared according to the procedure described in Gironda-Martínez et al., J. Org. Lett. 2019, 21, 9555. as shown below.

[0141] More specifically, LHRH or an analog (1 equivalent) was dissolved in DMF (1 mL) at 0°C. Imidazole-1-sulfonyl azide hydrochloride (10 equivalents) and DIPEA (3 equivalents) were added. The reaction mixture was stirred for 0.5 hours. The solvent was removed under vacuum to give an oil, which was treated with ethyl acetate. The resulting solid was collected and purified by washing with ether, EA, and CH2Cl2 to give a white solid peptide azide compound (68.4 mg, 67.5%). LC-MS: [M+H+]+=1429.

[0142] Synthesis of Linker-Drug A to L

[0143] Linker-Drug A

[0144] Linker-Drug A was prepared according to the procedure described in U.S. Application Publication No. 2022 / 0401592 A1.

[0145] Linker-Drug B

[0146] Linker-drug B was prepared according to the procedure described in International Application Publication No. WO2021262628 A1.

[0147] Linker-drug A (40 mg, 1 eq) was dissolved in DMF (1.0 mL), 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine (8.15 mg, 1 eq) and triethylamine (Et3N) (12.9 mg, 3 eq). The resulting reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under vacuum. The crude product was extracted with CH2Cl2 and 1M hydrochloric acid (HCl) (aq.). The aqueous layer was neutralized. The product was then extracted with CH2Cl2. The organic layers were combined, dried over sodium sulfate (Na2SO4) and filtered. The solvent was evaporated to give linker-drug B (yield = 92.4%).

[0148] Linker-Drug C

[0149] Linker-drug C was prepared according to the procedure described in Tsai et al., Front Chem. 2022, 9, 822587.

[0150] Linker-Drug D

[0151] Linker-drug D was prepared according to the procedure described in Shih et al., WO2021262628 A1.

[0152] More specifically, DM1 (200 mg, 0.271 mmol, 0.95 eq.), EDCI (81.5 mg, 0.425 mmol, 1.5 eq.) were added to a stirred solution of carboxylic acid C (150 mg, 0.284 mmol, 1 eq.) in 5 ml of DMF at room temperature, and DMAP (6.92 mg, 0.06 mmol, 0.2 eq.) was slowly added. The resulting reaction mixture was stirred at room temperature for 1 hour and then extracted with CH2Cl2 (20 mL). Subsequently, the CH2Cl2 solution was washed with a saturated sodium bicarbonate (NaHCO3) aqueous solution (10 mL) and water (3×10 mL), dried over magnesium sulfate (MgSO4), and concentrated under reduced pressure. The residue thus obtained was purified by column chromatography (silica gel; EA:hexane=9:1 to 100% EA) to obtain linker-drug D (280.5 mg, 83%).

[0153] Linker-Drug E

[0154] Compound 1 (154 mg, 1 eq.) (Tsou et al., WO2021262628 A1) and DIPEA (3 eq.) were added to a solution of ((1R, 8S, 9r)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(4-nitrophenyl)carbonate B (93.9 mg, 1 eq.) in DMF (1 mL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction was checked for completion by HPLC and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EA:hexane=1:6 to 1:1) to give compound 2 (yield=80.6%). The product was then dissolved in methanol (MeOH) and 6N lithium hydroxide (LiOH(aq)) was added at room temperature. The solvent was removed and concentrated under reduced pressure. The residue was redissolved in CH2Cl2. The insoluble residue was filtered off. The filtrate was washed with water, dried over MgSO4(s) and the solvent was removed under vacuum. The acid 3 was obtained as a white gummy product and checked by LC-MS ([M+Na+]+=702).

[0155] To a stirred solution of carboxylic acid 3 (120 mg, 0.177 mmol, 1 eq.) in 5 ml DMF at room temperature, DM1 (195.65 mg, 0.265 mmol, 1.5 eq.) and EDCI (67.7 mg, 0.353 mmol, 2.0 eq.) were added, and DMAP (23.7 mg, 0.194 mmol, 1.1 eq.) was slowly added and stirred at room temperature for 1 hour. The resulting residue was extracted with CH2Cl2 (10.0 mL). The CH2Cl2 solution was then washed with saturated aqueous NaHCO3 solution (5.0 mL) and water (3×5.0 mL), dried over MgSO4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel; EA:hexane=9:1 to 100% EA) to obtain linker-drug E (113.6 mg, 45.8%) and checked by LC-MS ([M+H+]+=1400).

[0156] Linker-Drug F

[0157] Linker-drug F was prepared according to the procedure described in Wagner et al., Bioconjugate Chem. 2015, 26, 197-200.

[0158] More specifically, CBTF (22.85 mg, 1 eq.) and DM1 (40 mg, 1 eq.) were dissolved in DMSO (1 mL). The reaction mixture was stirred at room temperature for 1 hour. The crude product was extracted with CH2Cl2 and 1N NH4Cl(aq). The organic layers were combined, dried over Na2SO4 and filtered. The solvent was evaporated to obtain linker-drug F.

[0159] LC-MS: [M+Na+]+=1182.

[0160] Linker-Drug G

[0161] Linker-Drug G was prepared according to the procedure described in WO2021262628 A1.

[0162] More specifically, DM4 (40 mg, 0.051 mmol, 1 eq.) was slowly added to a stirred solution of acetylene (17.1 mg, 0.054 mmol, 1 eq.) in 1 mL of DCM / Et3N (1 / 1) at room temperature. The reaction mixture was stirred at room temperature for 1 hour and then diluted with CH2Cl2 (10 mL). The CH2Cl2 solution was washed with saturated aqueous NaHCO3 solution (5 mL) and water (3×5 mL), dried over MgSO4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel; EA:hexane=9:1 to 100% EA) to obtain linker-drug G (22.1 mg, 39.5%).

[0163] LC-MS: [M+2H+]2+=550.

[0164] Linker-Drug H

[0165] Linker-Drug H was prepared according to the procedure described in Lee et al., Journal of Medicinal Chemistry 2008, 51, 6442-49.

[0166] Linker-Drug I

[0167] Linker-drug I was prepared according to the procedure described in Cheng et al., WO2015179299 A1.

[0168] Linker-Drug J

[0169] Linker-drug J was prepared according to the procedure described in Wang et al., Cancers (2019), 11, 957-974.

[0170] 3-azidopropan-1-ol (1 g, 9.89 mmol, 1 eq) was dissolved in DCM (30 mL) and triethylamine (2 g, 20 mmol, 2 eq) and cooled to 0 °C. A solution of dichlorodiisopropylsilane (1.83 g, 10 mmol, 1 eq) in DCM (20 mL) was slowly added dropwise. After 30 minutes, a solution of 4-hydroxybenzaldehyde (1.2 g, 10 mmol, 1 eq) in DCM (10 mL) was slowly added. The reaction mixture was stirred for 1 hour. The solvent was concentrated to give a crude product, which was purified by column chromatography to give the intermediate aldehyde (2.32 g, 70.1%).

[0171] The aldehyde (1.01 g, 3.03 mmol, 1 eq) was dissolved in anhydrous THF (10 mL) and cooled to -5 °C. Sodium borohydride (114.6 mg, 3.03 mmol, 1 eq) was added. The mixture was stirred at this temperature for 2 hours and then extracted with DCM / NH4Cl. The solvent was removed under reduced pressure to give the crude alcohol, which was purified by column chromatography to give the alcohol (0.75 g, 73%) as a colorless oil.

[0172] The alcohol (548.3 mg, 1.62 mmol, 1 eq) was dissolved in anhydrous DCM (15 mL), 4-nitrophenyl carbonochloridate (988.5 mg, 3.2 mmol, 2 eq) and DIPEA (315 mg, 2.44 mmol, 1.5 eq) and stirred at room temperature for 2 hours. The reaction mixture was extracted with DCM / NH4Cl. The organic layer was separated and concentrated under reduced pressure to give a crude product, which was further purified by column chromatography to give PNP-carbonate product (702.7 g, 86.3%) as a white solid.

[0173] To a stirred solution of PNP-carbonate compound (250 mg, 0.497 mmol, 1 eq) in 16 ml of DCM was added DM1 (367.2 mg, 0.497 mmol, 1 eq) and DMAP (60.8 mg, 0.4897 mmol, 1 eq) at room temperature. The resulting mixture was stirred at room temperature for 1 hour and then diluted with CH2Cl2 (10 mL). The CH2Cl2 solution was washed with saturated NH4Cl aqueous solution (5 mL) and water (3×5 mL), dried over MgSO4 and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel; EA:hexane=1:1 to 100% EA) to obtain linker-drug J (365.8 mg, 66.8%).

[0174] LC-MS: [M+Na+]+=1124.

[0175] Linker-Drug K

[0176] Linker-drug K was prepared according to the procedure described in Tang et al., CN110152013 A.

[0177] Linker-Drug L

[0178] A solution of compound B (50 mg, 0.159 mmol, 1 eq.), DM-1 (117 mg, 0.16 mmol), DIPEA (48 mg, 0.48 mmol, 3 eq.) and DMAP (0.97 mg, 0.008 mmol) in DMF (1 ml) was stirred at room temperature for 16 hours, then diluted with DCM and washed several times with dilute aqueous NaHCO3. The combined organic layers were dried over Na2SO4. The solvent was removed. The residue was purified by column chromatography (silica gel; EA: hexane = 9:1 to 100% EA) and preparative LC / MS to obtain linker-drug L (106.3 mg, 0.083 mmol, 73.1%).

[0179] LC-MS: [M+Na+]+=936.

[0180] Synthesis of Peptide Drug Complex 7-31

[0181] The drug conjugate of the present invention is prepared by reacting a linker-drug (A to L) with a peptide alkyne compound (2A, 6A, 10A, 12A, 13A and 15A) or a peptide azide compound 32.

[0182] To a stirred solution of the peptide alkyne compound (1 eq.) in DMF was added the linker-drug (1.05 eq.) at room temperature. The resulting reaction mixture was stirred at room temperature for 30 min. The completion of the reaction was monitored by HPLC. The solvent was removed under reduced pressure to give an oil. The crude solid was precipitated by trituration with EA, which was filtered and washed three times with EA, ether and DCM to give a high purity peptide drug-complex.

[0183] According to the above general procedure, the complex of the present invention was prepared. Please refer to Table 1 below, which contains its mass, purity determined by HPLC, and binding affinity to the LHRH receptor.

[0184] Compound 14 was prepared from 6 (18 mg, 0.012 mmol, 1 eq) and linker-drug A (11.48 mg, 0.012 mmol, 1 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to obtain compound 14 (6.81 mg, 24.7%) as a white solid.

[0185] Compound 15 was prepared from 15A (139 mg, 0.0768 mmol, 1 eq) and linker-drug C (104 mg, 0.0108 mmol, 1.5 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 15 (118 mg, 57%) as a white solid.

[0186] Compound 7 was prepared from compound 4 (49 mg, 0.032 mmol, 1 eq.) and linker-drug H (33.54 mg, 0.032 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to obtain compound 7 (45.6 mg, 57.6%) as a white solid.

[0187] Compound 8 was prepared from compound 2 (25.2 mg, 0.018 mmol, 1 eq) and linker-drug A (16.88 mg, 0.018 mmol, 1 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to obtain 8 (36.4 mg, 90.8%) as a white solid.

[0188] Compound 9 was prepared from compound 15A (320 mg, 0.177 mmol, 1 eq.) and linker-drug D (277 mg, 222 mmol, 1.3 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 9 (228 mg, 42%) as a white solid.

[0189] Compound 10 was prepared from 10A (226.0 mg, 0.146 mmol, 1 eq) and linker-drug C (92 mg, 0.102 mmol, 0.7 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 10 (164 mg, 46%) as a white solid.

[0190] Compound 11 was prepared from 6A (30 mg, 0.018 mmol, 1 eq.) and linker-drug D (23.86 mg, 0.019 mmol, 1.05 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 11 (22.3 mg, 42.8%) as a white solid.

[0191] Compound 16 was prepared from 2A (38 mg, 0.024 mmol, 1 eq.) and linker-drug D (30.06 mg, 0.024 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 16 (27.4 mg, 40.4%) as a white solid.

[0192] Compound 18 was prepared from degarelix (133 mg, 0.078 mmol, 1 eq.) and linker-drug I (90 mg, 0.086 mmol, 1.1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 18 (128 mg, 64%) as a white solid.

[0193] Compound 25 was prepared from 2A (30 mg, 0.019 mmol, 1 eq.) and linker-drug B (18.98 mg, 0.019 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 25 (21.2 mg, 43.3%) as a white solid.

[0194] Compound 26 was prepared from 6A (30 mg, 0.018 mmol, 1 eq) and linker-drug B (18.18 mg, 0.018 mmol, 1 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 26 (24.5 mg, 51.4%) as a white solid.

[0195] Compound 29 was prepared from 6A (60 mg, 0.036 mmol, 1 eq.) and linker-drug J (40.1 mg, 0.036 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 29 (71.3 mg, 72%) as a white solid.

[0196] Compound 30 was prepared from 2A (60 mg, 0.038 mmol, 1 eq.) and linker-drug J (41.8 mg, 0.038 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 30 (61.7 mg, 60.6%) as a white solid.

[0197] Compound 17 was prepared from 2A (33 mg, 0.021 mmol, 1 eq.) and linker-drug K (24.1 mg, 0.021 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 17 (24.4 mg, 42.5%) as a white solid.

[0198] Compound 19 was prepared from 15A (60 mg 0.033 mmol 1 eq.) and linker-drug J (36.5 mg, 0.033 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 19 (68.2 mg, 71%) as a white solid.

[0199] Compound 20 was prepared from 32 (10 mg, 0.007 mmol, 1 eq) and linker-drug L (6.4 mg, 0.007 mmol, 1 eq) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 20 (11.6 mg, 70.7%) as a white solid.

[0200] Compound 21 was prepared from 32 (28 mg, 0.02 mmol 1 eq.) and linker-drug E (27.44 mg, 0.02 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 21 (32.2 mg, 57%) as a white solid.

[0201] Compound 22 was prepared from 32 (35 mg, 0.025 mmol 1 eq.) and linker-drug G (26.9 mg, 0.025 mmol eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 22 (26.1 mg, 41.3%) as a white solid.

[0202] Compound 23 was prepared from 12 (60 mg, 0.041 mmol, 1 eq.) and linker-drug A (38.28 mg, 0.041 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 23 (31.7 mg, 33.6%) as a white solid.

[0203] Compound 24 was prepared from 13 (30 mg, 0.021 mmol, 1 eq.) and linker-drug A (20.1 mg, 0.021 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 24 (27.5 mg, 58.8%) as a white solid.

[0204] Compound 27 was prepared from 13A (40 mg, 0.025 mmol, 1 eq.) and linker-drug D (34.81 mg, 0.028 mmol, 1.1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 27 (35.1 mg, 49.1%) as a white solid.

[0205] Compound 28 was prepared from 12A (30 mg, 0.018 mmol, 1 eq.) and linker-drug D (23.89 mg, 0.019 mmol, 1.05 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to afford 28 (36.2 mg, 69.4%) as a white solid.

[0206] Compound 31 was prepared from abarelix (20 mg, 0.015 mmol, 1 eq.) and linker-drug F (16.8 mg, 0.015 mmol, 1 eq.) by the general procedure described for peptide drug-conjugates. Final purification was performed by washing the residue with solvent to obtain 31 (18.3 mg, 53.9%) as a white solid.

[0207] Compound 2A was prepared from 2 (100 mg, 0.071 mmol 1 eq.) and compound B (22.5 mg, 0.019 mmol, 1.05 eq.) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 2A (86.5 mg, 77.3%) as a white solid. LC-MS: [M+2H+]2+=790.

[0208] Compound 10A was prepared from abarelix (200 mg, 0.146 mmol 1 eq) and compound B (69 mg, 0.218 mmol, 1.5 eq) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 10A (226 mg, 99%) as a white solid. MS: [M+2H+]2+=769.

[0209] Compound 6A was prepared from 6 (90 mg, 0.061 mmol, 1 eq.) and compound B (20 mg, 0.064 mmol 1.05 eq.) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 6A (61.7 mg, 61.4%) as a white solid. LC-MS: [M+2H+]2+=825.

[0210] Compound 12A was prepared from 12 (100 mg, 0.068 mmol 1 eq.) and compound B (21.4 mg, 0.068 mmol, eq.) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 12A (108.8 mg, 97.1%) as a white solid. LC-MS: [M+2H+]2+=825.

[0211] Compound 13A was prepared from 13 (90 mg, 0.064 mmol, 1 eq.) and compound B (20.2 mg, 0.064 mmol, 1 eq.) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 13A (66.2 mg, 65.6%) as a white solid. LC-MS: [M+2H+]2+=790.

[0212] Compound 15A was prepared from degarelix (20 mg, 0.012 mmol 1 eq) and compound B (10 mg, 0.032 mmol, 2.5 eq) by the general procedure described for peptide alkyne adduct. Final purification was performed by washing the residue with solvent to give 15A (21.7 mg, 99%) as a white solid. LC-MS: [M+2H+]2+=905.

[0213] Compound 32 was prepared from 2 (10 mg, 0.007 mmol, 1 eq) by the general procedure described for peptide azide adduct. Final purification was performed by washing the residue with solvent to afford 32 (8.6 mg, 84.4%) as a white solid.

[0214] Radioligand binding assay

[0215] 2 μg of purified membrane containing LHRHR (also known as GnRHR) was incubated with 0.18 nM [125I]-[D-Trp6]-LHRH and the target compound in incubation buffer (50 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2 and 0.2% BSA). The reaction mixture was incubated at 25°C for 1 hour and then transferred to a 96-well GF / B filter plate (Millipore Corp., Billerica, MA, USA), terminated by filtration through a manifold and washed six times with wash buffer (50 mM HEPES, pH 7.4 and 100 mM NaCl). The radioactivity bound to the filter was measured by a Topcount® system (PerkinElmer Inc., Waltham, MA, USA). IC50 values ​​were determined from the concentration of compound required to inhibit 50% of the specific binding of [125I]-[D-Trp6]-LHRH and were calculated by nonlinear regression (GraphPad Software, San Diego, CA, USA).

[0216] These drug conjugates are designed to actively target LHRH-receptors at tumor sites and promote the accumulation of drug conjugates in situ. Therefore, the selective and strong binding of synthetic peptide ligands to receptors is very important for enriching these conjugates and then releasing the drug payload in the tumor microenvironment.

[0217] The results are shown in Table 1. The compounds of the present invention had binding as low as 0.17 nM.

[0218] Table 1 Note that + indicates IC50 greater than 100 nM, ++: 10 nM to 100 nM, +++: 1 nM to 10 nM, ++++: 0.1 nM to 1 nM.

[0219] Pharmacokinetic properties

[0220] The pharmacokinetic properties of the compounds of the present invention were measured according to the procedure described in Liu et al., Bioconjugate Chem. 2017, 28, 7, 1878-1892.

[0221] To extend the life of the intact complex during systemic circulation in vivo, single intravenous dose pharmacokinetic studies showed that the clearance (CL) and volume distribution Vss of these complexes were reduced, indicating increased system stability and improved distribution. In particular, complexes 14, 8 and 9 showed a wide range of AUC coverage. In addition, the results of complexes 11 and 16 demonstrated that the complexes of the present invention advantageously achieve similar exposure using different dosing frequencies.

[0222] Table 2 In vivo pharmacokinetic studies of peptide drug-complexes

[0223] In vivo antitumor activity

[0224] The in vivo antitumor activity of compound 16 was evaluated in the ovarian OVCAR-3 tumor model. OVCAR-3 cells were suspended in RPMI 1640 medium (without phenol red) and MatrigelTM at a ratio of 1:1 and implanted subcutaneously (1×106 cells / flank) into the left flank of immunodeficient (NOD / SCID) mice. Tumor volume (mm3) was calculated by the following formula: volume = (length × width^2) / 2. When the average tumor volume was approximately 190 mm3 on day 49 after inoculation, tumor-bearing mice were randomly divided into groups (n=8 per group). Compound 16 was dissolved in DMA, cremorphor, and 5% glucose solution (1:2:27, v / v / v). Vehicle control (5 mL / kg) and compound 16 (1.5 or 2.5 mg / kg) were intravenously administered to tumor-bearing mice once a week on days 1, 8, 15, 22, 29, and 36 for 6 weeks. Tumor size and body weight of each animal were measured twice weekly during the study.

[0225] The in vivo antitumor activity of compound 16 was evaluated in the ovarian A2780 tumor model. A2780 cells were suspended in RPMI 1640 medium (without phenol red) and MatrigelTM at a ratio of 1:1 and implanted subcutaneously (1×106 cells / flank) into the left flank of immunodeficient nude mice. Tumor volume (mm3) was calculated by the following formula: volume = (length × width^2) / 2. When the average tumor volume was approximately 390 mm3 on day 13 after inoculation, the tumor-bearing mice were randomly divided into groups (n=8 per group). Compound 16 was dissolved in DMA, cremorphor, and 5% glucose solution (1:2:7, v / v / v). Vehicle control (2.5 mL / kg) and compound 16 (1.5 or 2 mg / kg) were intravenously administered to tumor-bearing mice twice a week on days 1, 4, 8, and 11 for two weeks. The tumor size and body weight of each animal were measured twice a week during the study.

[0226] The in vivo antitumor activity of compound 16 was evaluated in the triple-negative breast HCC1806 tumor model. HCC1806 cells were suspended in RPMI 1640 medium (without phenol red) and MatrigelTM at a 1:1 ratio and implanted subcutaneously (1×106 cells / flank) into the left flank of immunodeficient nude mice. Tumor volume (mm3) was calculated by the following formula: volume = (length × width^2) / 2. When the average tumor volume was approximately 210 mm3 on day 10 after inoculation, the tumor-bearing mice were randomly divided into groups (n=8 per group). Compound 16 was dissolved in DMA, cremorphor, and 5% glucose solution (1:2:27, v / v / v). Tumor-bearing mice were intravenously administered multiple doses of vehicle control (5 mL / kg) and DBPR376 (2.5 mg / kg) once a week (qw) or every two weeks (b2w). Tumor size and body weight of each animal were measured twice a week during the study.

[0227] Compounds 8, 9, 11, 14, 16, 25, 26, and 27 were evaluated according to the above procedure. Each compound effectively inhibited tumor growth of HCC1806-resistant triple-negative breast cancer in vivo when 1 mg / kg to 2.5 mg / kg of each compound was administered to mice every other week, once a week, or twice a week. Surprisingly, when 1 mg / kg of compounds 9 and 14 were administered to mice twice a week, the growth of anti-HCC1806 triple-negative breast cancer was 100% inhibited within 15 days (i.e., no cancer progression); when 1.5 mg / kg of compound 16 was administered to mice twice a week, the growth of anti-HCC1806 triple-negative breast cancer was 100% inhibited within 30 days; when 2 mg / kg of compounds 25 and 26 were administered to mice twice a week, the growth of anti-HCC1806 triple-negative breast cancer was 100% inhibited within 30 days; when 2.5 mg / kg of compounds 16 and 27 were administered to mice once a week, the growth of anti-HCC1806 triple-negative breast cancer was 100% inhibited within 30 days or up to 100 days; when 2.5 mg / kg of compound 16 was administered to mice once a week, the growth of anti-HCC1806 triple-negative breast cancer was 100% inhibited within 60 days.

[0228] Other embodiments

[0229] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only a general example of a series of equivalent or similar features.

[0230] In addition, based on the above description, a person with ordinary knowledge in the technical field of the invention can easily determine the essential characteristics of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the protection requested.

Claims

1. A compound of formula (I), 1. Among them, the compound of formula (I) is one of compounds 7-11 and 14-31:

2. The compound as claimed in claim 1, wherein, The compound of formula (I) is one of compounds 8, 9, 11, 14, 16, 21 and 25 to 28.

3. A pharmaceutical composition comprising the compound as described in claim 1 and a pharmaceutically acceptable carrier.

4. Use of a compound as claimed in claim 1 for the preparation of a medicament for treating cancer, wherein, The cancer mentioned is pancreatic cancer, ovarian cancer, breast cancer, or prostate cancer.

5. The use as described in claim 4, wherein, The cancer is either ovarian cancer or breast cancer.