JPWO2020094471A5 - - Google Patents

Description

The present invention provides novel pharmaceutical compounds comprising an α _v β ₃ integrin antagonist, a linking unit comprising an elastase-cleavable L-Val-L-Pro-L-Asp, a polyethylene glycol (PEG) spacer, and a cytotoxic component, the It relates to processes for its preparation, its use to treat, prevent, or manage diseases and conditions, including hyperproliferative disorders such as cancer, in humans and other mammals.

Chemotherapy for cancer is usually associated with serious side effects, which are due to the toxicity of the chemotherapeutic agents to proliferating cells in other tissues than the tumor tissue. For many years scientists have been working on the problem of improving the selectivity of the active compounds used. Frequent approaches are, for example, changes in pH (US 2005/0130001), enzymes (eg glucuronidase, US 2004/0023000 and US 2004/0020300), or antibody-enzyme conjugates (US 2005/0001000, US 2004/0020003, 3) is the synthesis of prodrugs that are more or less selectively released in the target tissue. A problem with these approaches is, inter alia, the lack of stability of the conjugates in other tissues and organs, especially the ubiquitous distribution of the active compound following its extracellular release in tumor tissue.

20(S)-camptothecin is a pentacyclic alkaloid isolated by Wall et al. in 1966 (Non-Patent Document 1). It has shown highly active antitumor properties in many in vitro and in vivo tests. Unfortunately, however, the realization of this promising potential in clinical studies has failed due to toxicity and solubility problems.

Ring-opening of the E-ring lactone to form the sodium salt afforded a water-soluble compound in pH-dependent equilibrium with the closed ring form. Again, clinical trials have not yet been successful.

About 20 years later, it became clear that the biological activity was due to enzymatic inhibition of topoisomerase I. Since then, there has been a resurgence of research efforts to find camptothecin derivatives that are more water soluble, well tolerated and active in vivo.

Salts of camptothecin derivatives modified with A- and B-rings and salts of 20-O-acyl derivatives with ionizable groups have been described to improve water solubility (Patent Document 6). The latter prodrug concept was later taken over by modified camptothecin derivatives (Patent Document 7). However, the 20-O-acyl prodrugs described have very short in vivo half-lives and are very rapidly cleaved to yield the parent structure.

Integrins are heterodimeric transmembrane proteins found on the surface of cells and play an important role in cell adhesion to the extracellular matrix. Integrins encode extracellular glycoproteins such as fibronectin and vitronectin on the extracellular matrix by coding the RGD sequence (RGD is a one-letter code representing the amino acid sequence of arginine-glycine-aspartic acid) generated in these proteins. recognize through.

In general, integrins, e.g., the vitronectin receptor, also called the α _v β ₃ receptor, alternatively the α _v β ₅ receptor, or the GpIIb/IIIa receptor, are involved in cell migration, angiogenesis, and cell-matrix adhesion. play an important role in the biological processes of and are therefore also involved in diseases in which these processes are important steps. Examples include cancer, osteoporosis, arteriosclerosis, restenosis, and eye disease.

For example, α _v β ₃ receptors are abundant on growing endothelial cells, allowing them to adhere to the extracellular matrix. Thus, α _v β ₃ receptors play an important role in angiogenesis, the formation of new blood vessels that are a critical prerequisite for tumor growth and metastasis formation in cancerous diseases.

Blocking the above receptors could prove to be an important starting point for the treatment of this type of disorder. When the adhesion of growing endothelial cells to the extracellular matrix is inhibited by blocking the corresponding integrin receptors with, for example, cyclic peptides or monoclonal antibodies, angiogenesis does not occur and tumor growth is halted. Or it regresses (see, for example, Non-Patent Document 2).

WO 2005/010300 describes conjugates in which a tumor-targeting molecule is linked to a functional unit, such as a cytostatic agent, or a detectable label, such as a radionuclide. Inter alia, integrin antagonists, such as, for example, peptides with the RGD sequence described above, have been described as molecules that target tumors and tumor stroma. Doxorubicin has been described as an example of a cytostatic drug linked to this type of molecule for tumors.

In the case of the compounds of WO 2005/010000, the linkage is such that the tumor-corresponding molecule and the functional unit are directly linked to each other while retaining their respective properties (eg, pp. 56, 1.17-p. 58, l.10 and Ex.6). As a result, these compounds, in the case of radicals with α _v β ₃ integrin antagonism, express α _v Through binding to the _β3 integrin receptor), it selectively concentrates in the immediate vicinity of tumor cells, but direct binding allows functional units such as cytostatics to enter the intracellular space of tumor tissue. cannot be released to

Basically, the conjugates are selectively localized to tumor tissue by the effect of the moieties corresponding to the α _v β ₃ or α _v β ₅ integrin receptors found in the conjugates on the one hand and released from the conjugates on the other hand. The cytostatic agent may act more directly on tumor cells, and thus the conjugate may have an increased effect on tumor tissue compared to the conjugates described in US Pat. Should have toxic effects. In particular, such toxic effects and tumor selectivity should be even higher if the release of the cytostatic agent takes place in close proximity to the tumor tissue or within the tumor cells.

US Pat. No. 5,300,001 discloses enzyme-activated anti-tumor prodrug compounds that are specifically cleavable by collagenase (IV) and elastase. For elastase-cleavable linking units, the application states that the specific tetrapeptide sequences Ala-Ala-Pro-Val and Ala-Ala-Pro-Nva are suitable. Furthermore, this document does not mention any conjugates comprising a portion corresponding to the α _v β ₃ integrin receptor and a cytostatic agent.

Y. Liu et al. ( ₃ ) describe conjugates of auristatin linked to an α _v β3 integrin targeting moiety via a legumain cleavable linker.

US Pat. No. _5,300,003 discloses conjugates with cytotoxic agents that target the α _v β3 integrin and have peptide linkers that can be specifically cleaved by elastase. With respect to elastase-cleavable linking units, peptide sequences containing Pro-Val and Pro-Leu are described in this application and are suitable. Camptothecins and quinolone carboxylic acids are exemplified as toxophore moieties.

Such conjugates have the following problems.
Sufficient solubility to allow intravenous administration in appropriate vehicles,
high tumor penetrance of intact conjugates,
high stability in plasma to avoid systemic uncoupling;
efficient binding to target receptors in the tumor microenvironment,
efficient cleavage by enzymes present in the tumor microenvironment,
• Higher stability and cell permeability of the cleaved toxophile moiety for improved uptake versus redistribution into tumor cells.

DE-A 42 29 903 EP-A 511 917 EP-A 595 133 WO 88/07378 US 4 975 278 US 4 943 579 WO 96/02546 WO 98/10795 WO 00/69472 EP 1 238 678

J. Am. Chem. Chem. Soc. 88, 3888 (1966) Brooks et al. in Cell 79, 1157-1164 (1994) Mol. Pharmaceutics 2012, 9, 168

The present invention relates to pharmaceutical compounds that are conjugates comprising an α _v β ₃ integrin antagonist, a linking unit selectively cleavable by elastase, a polyethylene glycol (PEG) spacer, and a cytotoxic element (toxophore). The conjugate is tumor-specific as a result of linkage to the α _{v β3} integrin antagonist via a preferred linking unit that can be selectively cleaved by elastase, that is, by an enzyme that can be found especially in the tumor stroma. works. Preferred linking units provide sufficient stability of the conjugate of the cytostatic agent and the α _{v β3} integrin antagonist in a biological medium, such as culture medium or serum, while simultaneously releasing the cytostatic agent. As a result of its specific enzymatic or hydrolytic cleavability with , it provides the desired intracellular action within the tumor tissue.

In particular, the compounds of the invention exhibit favorable characteristics:
• Improved stability of the conjugate after replacement of thiourea with a urea bond • Employment of 7-ethylcamptothecin as a particularly suitable toxophore moiety.
- Beneficial effects on e.g. the stability of the lactone ring (Drugs Fut 2002, 27(9), 869)
High cell permeability and low excretion (e.g. compared to SN38 )
Modified spacers with beneficial effects on solubility, integrin binding affinity and elastase cleavability Tumor accumulation of toxic carriers after conjugated versus direct administration Superior therapeutic efficacy in various tumor models

For this purpose, 7-ethylcamptothecin is particularly preferred as the toxophore moiety in the conjugates described above.

The present invention provides compounds of formula (I) and salts, solvates and solvates of salts thereof,

ここで、
ＣＴは、それぞれヒドロキシル基、カルボキシル基、またはアミノ基を追加的に運ぶことができる、細胞毒性ラジカル、細胞増殖抑制剤のラジカル、および細胞増殖抑制剤誘導体のラジカルの群からの一価ラジカルであり、
ＬＩは、式－Ｌ－Ｖａｌ－Ｌ－Ｐｒｏ－Ｌ－Ａｓｐ－の二価ペプチドラジカルであり、
ＳＰは、式－Ｃ＝Ｏ－（ＣＨ_２）_ｘ－Ｏ－（ＣＨ_２－ＣＨ_２－Ｏ）_ｙ－ＣＨ_２－ＣＨ_２－ＮＨ－Ｃ＝Ｏ－の基であり、ｘ＝１－５であり、および、ｙ＝０－１５であり、
ＩＡは、α_ｖβ_３インテグリン受容体に対応する一価ラジカルである。

here,
CT is a monovalent radical from the group of cytotoxic radicals, cytostatic radicals, and cytostatic derivative radicals, which can additionally carry a hydroxyl, carboxyl, or amino group, respectively. ,
LI is a divalent peptide radical of the formula -L-Val-L-Pro-L-Asp-
SP is a group of formula -C=O-(CH ₂ ) _x -O-(CH ₂ -CH ₂ -O) _y -CH ₂ -CH ₂ -NH-C=O-, where x=1-5 and y=0-15,
IA is the monovalent radical corresponding to the α _{v β3} integrin receptor.

The bivalent peptide radial LI can be attached to CT or SP via its N-terminal or C-terminal positions. Preferably, LI is attached to CT via its C-terminal position and to SP via its N-terminal position.

The present invention further provides compounds of formula (Ia) and salts, solvates and solvates of salts thereof,

ここで、ｘは１－５であり、および、ｙ＝０－１５である。

where x is 1-5 and y=0-15.

Compounds of formula (I) or (Ia) where x is 1-4 are preferred, compounds of formula (Ia) where x is 1-2 are more preferred, and compounds of formula (Ia) where x is 2 are most preferred. preferable.

Compounds of formula (I) or (Ia) in which y is 0-10 are preferred, compounds of formula (Ia) in which y is 0-5 are more preferred, and compounds of formula (Ia) in which y is 2 are most preferred. preferable.

Compounds of formula (II) and their salts, solvates and solvates of their salts are preferred.

Preferred salts in the context of the invention are physiologically acceptable salts of the compounds of the invention. Also included are salts which are not suitable per se for pharmaceutical uses, but which can be used, for example, in the isolation, purification, or preservation of the compounds of the invention.

Physiologically acceptable salts of the compounds of the invention include inter alia acid addition salts of mineral, carboxylic and sulfonic acids, for example hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, succinic acid, fumaric acid, maleic acid, lactic acid, tartaric acid, Examples include salts of malic acid, citric acid, gluconic acid, benzoic acid, and embonic acid.

In addition, physiologically acceptable salts of the compounds of this invention also include salts derived from conventional bases, by way of example, and if desired, alkali metal salts such as sodium and potassium salts. , alkaline earth metal salts (e.g. calcium and magnesium salts), zinc salts and ammonia or organic amines having 1 to 20 carbon atoms, for example and optionally ethylamine, diethylamine, triethylamine, N , N-ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, choline, benzalkonium, procaine, dibenzylamine, dicyclohexylamine, N-methyl Included are ammonium salts derived from morpholine, N-methylpiperidine, arginine, lysine, and 1,2-ethylenediamine.

A preferred salt is the disodium salt of the compound of formula (II).

Solvates in the context of the invention describe those forms of the compounds of the invention which form a complex in the solid or liquid state by coordination with solvent molecules. Hydrates are a specific form of solvates in which the coordination is with water. Preferred solvates in the context of the present invention are hydrates.

The invention also includes all suitable isotopic variants of the compounds of the invention. An isotopic variant of a compound of the invention, as used herein, is one in which at least one atom in the compound of the invention is replaced with another atom of the same atomic number, but which is commonly or predominantly occurring in nature. is understood to mean a compound having an atomic mass different from that of the atomic mass of the compound. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine, and iodine, e.g., ² H (deuterium), ³ H (tritium), ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ¹⁸ F, ³⁶ Cl, ⁸² Br, ¹²³ I, ¹²⁴ I, ¹²⁹ I, and ¹³¹ I and the like. Certain isotopic variants of the compounds of the present invention, particularly those incorporating one or more radioactive isotopes, are relatively easy to prepare and detectable, and thus, e.g. It may also be useful to study the biology or distribution of an active ingredient in the body, and ³ H, ¹⁴ C, and/or ¹⁸ F isotopically labeled compounds are particularly suitable for this purpose. In addition, the incorporation of isotopes such as deuterium can significantly improve the metabolic stability of the compounds, leading to certain therapeutic effects, e.g., increased half-life in the body or reduced required effective dosage. Thus, such modifications of the compounds of the invention may constitute preferred embodiments of the invention. Isotopic variants of the compounds of the present invention may be prepared using the respective reagents and/or starting compounds by commonly used processes known to those skilled in the art, such as the methods described further below and the procedures described in the Examples. It can be prepared using the corresponding isotopic modification of the compound.

The synthesis of conjugates of the invention (eg, Example 1) is outlined in the scheme below.

Scheme 1: Synthesis of α _v β ₃ integrin ligands:

Separation of enantiomers can also be achieved in various steps by chromatography using chiral columns.

Scheme ₂ : Synthesis of α _v β3 integrin conjugates using 7-ethylcamptothecin:

Method of treatment:
The present invention also relates to methods for using the compounds and compositions thereof to treat hyperproliferative disorders in mammals. The method comprises administering to a mammal in need thereof, including humans, an effective amount of the compound to treat the disorder. Hyperproliferative disorders include, but are not limited to breast cancer, respiratory cancer, brain cancer, genital cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer. , and distant metastatic cancers thereof. These disorders also include lymphoma, sarcoma, and leukemia.

Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.

Examples of cancers of the respiratory system include, but are not limited to, small cell and non-small cell lung cancer, as well as bronchial adenoma and pleuroalveolar tumor.

Examples of brain cancers include, but are not limited to, brainstem and pituitary glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumors.

Tumors of the male reproductive organs include, but are not limited to, prostate cancer and testicular cancer.

Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancers, as well as uterine sarcoma.

Tumors of the gastrointestinal tract include, but are not limited to, anal cancer, colon cancer, colon cancer, esophageal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small bowel cancer, and salivary gland cancer.

Tumors of the urinary system include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvic cancer, ureteral cancer, and urethral cancer.

Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.

Head and neck cancers include, but are not limited to laryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lip and oral cavity cancer.

Lymphomas include, but are not limited to, AIDS-related lymphomas, non-Hodgkin's lymphomas, cutaneous T-cell lymphomas, Hodgkin's disease, and lymphomas of the central nervous system.

Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

These disorders are well characterized in humans, but also exist with a similar etiology in other mammals and can be treated by administering the pharmaceutical compositions of the invention.

Treatment of the conditions identified above in mammals is determined by standard toxicity tests and based on standard laboratory techniques known to evaluate compounds useful in the treatment of hyperproliferative disorders. Effective dosages of the compounds of the invention can be determined by standard pharmacological assays for the treatment of It can be readily determined for treatment of each desired indication. The amount of active ingredient to be administered in the treatment of one of these conditions will depend on the particular compound and dosage unit employed, the mode of administration, the duration of treatment, the age and sex of the patient to be treated, and the condition being treated. It can vary greatly, depending on considerations such as nature and degree.

The total amount of active ingredient administered will generally range from about 0.001 mg/kg to about 200 mg/kg body weight per day, preferably from about 0.01 mg/kg to about 20 mg/kg body weight per day. Clinically useful dosing schedules range from one to three times daily dosing to once every four weeks dosing. In addition, a "drug holiday," during which the patient is not administered drug for a period of time, is believed to be beneficial in balancing overall pharmacological efficacy and tolerability. A single unit dose can contain from about 0.5 mg to about 1500 mg of active ingredient, and can be administered more or less than once a day. For administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injection, and for the use of infusion techniques, the average daily dosage is preferably from 0.01 to 200 mg/kg of total body weight. be. The average daily rectal dosage regimen is preferably 0.01-200 mg/kg of total body weight. The average daily intravaginal dosage regimen is preferably 0.01-200 mg/kg of total body weight. The average daily topical dosage regimen is preferably 0.1 to 200 mg administered 1 to 4 times daily. The transdermal concentration is preferably that concentration necessary to maintain a daily dose of 0.01-200 mg/kg. The average daily inhaled dose is preferably 0.01-100 mg/kg of total body weight.

Of course, the initial and ongoing dosing regimen for each patient will depend on the nature and severity of the condition as judged by the attending diagnostician, the activity of the particular compound used, the age and general condition of the patient, the time of administration, and the route of administration. , drug excretion rate, drug combination, etc. The desired mode of treatment and frequency of administration of a compound or pharmaceutically acceptable salt or ester thereof or composition of the invention can be ascertained by one of ordinary skill in the art using conventional treatment trials.

The invention further provides the use of the compounds of the invention for the preparation of pharmaceutical compositions for the treatment of the aforementioned disorders.

Administration The compounds of the invention can have systemic and/or local activity. Suitable for this purpose, for example, via oral, parenteral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, cutaneous, transdermal, conjunctival, auris routes or as implants, stents, etc. can be administered in any manner.

These routes of administration allow the compounds of the present invention to be administered in suitable dosage forms.

For oral administration, the compounds of the invention can be formulated into dosage forms known in the art that deliver the compounds of the invention rapidly and/or in a modified manner, such as , tablets (uncoated tablets or coated tablets with e.g. slow dissolving or insoluble enteric coatings or controlled release coatings), orally disintegrating tablets, film/wafer, film/freeze They include lyophylisates, capsules (eg hard or soft gelatin capsules), dragees, granules, pellets, powders, emulsions, suspensions, aerosols or solutions and the like. It is possible to incorporate the compounds of the present invention in crystalline and/or amorphous and/or dissolved form into said dosage forms.

Parenteral administration avoids (e.g., intravenous, intraarterial, intracardiac, intraspinal, or intralumbar) absorption processes, or includes (e.g., intramuscular, subcutaneous, intradermal, transdermal, or intraperitoneal) can be performed. Dosage forms suitable for parenteral administration are, inter alia, injection and infusion preparations in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.

Examples of suitable other routes of administration include pharmaceutical forms for inhalation [especially powder inhalers, nebulizers], nasal drops, nasal solutions, nasal sprays; Tablets/Films/Wafers/Capsules; Suppositories; Eye Drops, Eye Ointments, Eye Baths, Eye Inserts, Ear Drops, Ear Sprays, Ear Powders, Ear Washes, Ear Tampons; Vaginal Capsules, Aqueous Suspensions (lotions, mixurae agitandae), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (such as patches), milks, pastes, foams, dusting powders, implants, or a stent.

The compounds according to the invention can be incorporated into prescribed dosage forms. This can be achieved in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, among others:
• Fillers and carriers such as cellulose, microcrystalline cellulose (eg Avicel®, etc.), lactose, mannitol, starch, calcium phosphates (eg, Di-Cafos®, etc.).
• Ointment bases (eg petrolatum, paraffin, triglycerides, waxes, wool wax, wool wax alcohol, lanolin, hydrophilic ointments, polyethylene glycol).
• Suppository bases (eg, polyethylene glycol, cocoa butter, hard fats).
• Solvents (eg water, ethanol, isopropanol, glycerol, propylene glycol, medium chain triglyceride fatty oils, liquid polyethylene glycols, paraffin).
- Surfactants, emulsifiers, dispersants or wetting agents (e.g. sodium dodecyl sulfate), lecithins, phospholipids, fatty alcohols (e.g. Lanette®), sorbitan fatty acid esters (e.g. Span®) , polyoxyethylene sorbitan fatty acid ester (e.g., Tween (registered trademark)), polyoxyethylene fatty acid glyceride (e.g., Cremophor (registered trademark), etc.), polyoxyethylene fatty acid ester, polyoxyethylene fatty acid Alcohol ethers, glycerol fatty acid esters, poloxamers (such as Pluronic®).
- Buffers, acids and bases (eg phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, trometamol, triethanolamine).
• Isotonic agents (eg, glucose, sodium chloride).
• Adsorbents (eg, highly disperse silica).
Viscosity increasing agents, gel formers, thickeners and/or binders (e.g. polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropyl-cellulose, carboxymethylcellulose-sodium, starch, carbomer, polyacrylic acid (Carbopol ( Carbopol®), alginate, gelatin).
Disintegrants (e.g. modified starch, carboxymethylcellulose-sodium, sodium starch glycolate (e.g. Explotab®, etc.), cross-linked polyvinylpyrrolidone, croscarmellose-sodium (e.g. AcDiSol®) )Such)).
• Flow control agents, lubricants, glidants and release agents (eg magnesium stearate, stearic acid, talc, highly disperse silica (eg Aerosil®, etc.)).
- Coating materials (e.g. sugar, shellac) and film formers for film or diffusion membranes that dissolve rapidly or in a modified manner (e.g. polyvinylpyrrolidone (e.g. Kollidon®, etc.) ), polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate, cellulose acetate phthalate, polyacrylate, polymethacrylate (eg Eudragit®, etc.)).
• Capsule materials (eg gelatin, hydroxypropyl methylcellulose).
- Synthetic polymers (e.g. polylactides, polyglycolides, polyacrylates, polymethacrylates (e.g. Eudragit®, etc.), polyvinylpyrrolidones (e.g. Kollidon®, etc.), polyvinyl alcohols, polyvinyls acetates, polyethylene oxides, polyethylene glycols, and their copolymers and block copolymers).
- Plasticizers (eg polyethylene glycol, propylene glycol, glycerol, triacetin, triacetyl citrate, dibutyl phthalate).
● Penetration enhancer.
- Stabilizers (eg, antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, etc.).
• Preservatives (eg, parabens, sorbic acid, thiomersal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate).
- Coloring agents (eg inorganic pigments, eg iron oxide, titanium dioxide, etc.).
• Flavors, sweeteners, flavor masking agents and/or odor masking agents.

The invention further relates to pharmaceutical compositions comprising at least one compound according to the invention, conventionally together with one or more pharmaceutically suitable excipients, and uses thereof according to the invention.

Combinations According to another aspect, the invention provides at least one compound of general formula (I) or (Ia) according to the invention and at least one or more compounds, especially for the treatment and/or prevention of hyperproliferative disorders. It includes pharmaceutical combinations with the specified agents, including additional active ingredients.

The term "combination" in the present invention is used as known to those skilled in the art, said combination being a fixed combination, a non-fixed combination or a kit-of-parts It is possible.

A "fixed combination" in the context of the present invention is used as known to those skilled in the art, e.g. Ingredients are defined as a combination present together in one unit dosage or one single entity. An example of a "fixed combination" is a pharmaceutical composition in which a first active ingredient and a further active ingredient are present in admixture for co-administration, such as in a formulation. Another example of a "fixed combination" is a pharmaceutical combination in which the first active ingredient and the additional active ingredient are present in one unit without being mixed.

A loose combination or "kit of parts" in the context of the present invention is used as known to those skilled in the art and is defined as a combination in which the first active ingredient and further active ingredients are present in more than one unit. . An example of a non-fixed combination or kit of parts is a combination in which a first active ingredient and a further active ingredient are present separately. The components of a non-fixed combination or kit-of-parts can be administered separately, sequentially, simultaneously, simultaneously, or chronologically staggered.

The compounds of the invention can be administered as the sole pharmaceutical agent or in combination with one or more other pharmaceutically active ingredients, the combination of which does not cause unacceptable side effects. The invention also includes such pharmaceutical combinations. For example, the compounds of the invention can be combined with known active ingredients for the treatment and/or prevention of hyperproliferative disorders.

Examples of active ingredients for the treatment and/or prevention of hyperproliferative disorders include: 131I-chTNT, abarelix, abemaciclib, abiraterone, akarabtinib, aclarubicin, adalimumab, ad-trastuzumab emtansine, afatinib, aflibercept, aldesleukin, alectinib, alemtuzumab, alendronate, alitretinoin, altretamine, amifostine, aminoglutethimide, hexyl aminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anetholedithiorethione, anez Mavrabutansine, angiotensin II, antithrombin III, apalutamide, aprepitant, alcitumomab, argravine, diarsenic trioxide, asparaginase, atezolizumab, avelumab, axicabtagene ciloleucel, axitinib, azacytidine, basiliximab, berotecan, bendamustine, besilesomab , bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin, blinatumomab, bortezomib, bosutinib, buserelin, brentuximab vedotin, brigatinib, busulfan, cabazitaxel, cabozantinib, calcitonin, calcium folinate, calcium levofolinate, capecitabine, capromab, carbamazepine, carboplatin , carfilzomib, carmofur, carmustine, catumaxomab, celecoxib, thermoleukin, ceritinib, cetuximab, chlorambucil, chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin, cladribine, clodronic acid, clofarabine, cobimetinib, copanlisib, crisantaspase, crizotinib, cyclophos Famide, cyproterone, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine, degarelix, denileukin diftitox, denosumab, depreotide, deslorelin, dianhydrogalactitol, dexrazoxane, dibulose chloride Pygeum, dianhydrogalactitol, diclofenac, dinutuximab, docetaxel, dolasetron, doxifluridine, doxorubicin, doxo rubicin + estrone, dronabinol, durvalumab, eculizumab, edrecolomab, elliptinium acetate, elotuzumab, eltrombopag, enacidenib, endostatin, enocitabine, enzalutamide, epirubicin, epithiostanol, epoetin alfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib, esomeprazole, estradiol, estramustine, ethinyl estradiol, etoposide, everolimus, exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone, floxridine, fludarabine, fluorouracil, flutamide, folinic acid, formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol, gadoteridol, meglumine gadoteric acid, gadobecetamide, gadoxetate, gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab, glucarpidase, glutoxime, GM-CSF, goserelin, granisetron, granulocyte colony stimulating factor, histamine hydrochloride, histrelin, hydroxycarbamide, I-125 seed, lansoprazole, ibandronic acid, ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib, imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate, inotuzumab ozogamicin , interferon alpha, interferon beta, interferon gamma, iobitridol, iobenguane (123I), iomeprol, ipilimumab, irinotecan, itraconazole, ixabepilone, ixazomib, lanreotide, lansoprazole, lapatinib, iasocoline, lenalidomide, lenvatinib, lenograstim, lentinan, letrozole, leuprorelin, levamisole, levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine, lonidamine, lutetium Lu 177 dotatate, masoprocol, medroxyprogesterone, megestrol, melarsoprol, melphalan, mepitiostane, mercaptopurine, mesna, Methadone, methotrexate, methoxalen, methyl aminolevulinate, methylprednisolone, methyltestosterone, methylosine, midostaurin, mi famultide, miltefosine, miriplatin, mitobronitol, mitoguazone, mitractol, mitomycin, mitotane, mitoxantrone, mogamulizumab, molgramostim, mopidamole, morphine hydrochloride, morphine sulfate, mbasi, nabilone, nabiximol, nafarelin, naloxone + pentazocine, naltrexone, naltoglas tim, necitumumab, nedaplatin, nelarabine, neratinib, neridronic acid, netupitant/palonosetron, nivolumab, pentetreotide, nilotinib, nilutamide, nimorazole, nimotuzumab, nimustine, nintedanib, niraparib, nitracrine, nivolumab, obinutuzumab, octreotide, ofatumumab, olaparimab, olaparib taxine mepesuccinate, omeprazole, ondansetron, oprelvekin, orgotein, orilotimod, osimertinib, oxaliplatin, oxycodone, oxymetholone, ozogamicin, p53 gene therapy, paclitaxel, palbociclib, palyfermin, palladium-103 seed, palonosetron, pamidronate , panitumumab, panobinostat, pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxy PEG-epoetin beta), pembrolizumab, pegfillastim, peginterferon alfa-2b, pembrolizumab, pemetrexed, pentazocine, pentostatin, pepromycin, Perflubutane, perfosfamide, pertuzumab, picibanil, pilocarpine, pirarubicin, pixantrone, plelixafor, plicamycin, polyglutam, polyestradiol phosphate, polyvinylpyrrolidone + sodium hyaluronate, polysaccharide-K, pomalidomide, ponatinib, porfimer sodium, pralatrex sart, prednimustine, prednisone, procarbazine, procodazole, propranolol, quinagolide, rabeprazole, lacotsumomab, radium-223 chloride, radtinib, raloxifene, raltitrexed, ramosetron, ramucirumab, ranimustine, rasburicase, lazoxan, lefamethinib, regorafenib, ribociclib, risedronic acid, rhenium- 186 etidronate, rituximab, lorapitant, romidepsin, romiplostim, romurtide, rucaparib, samarium (153Sm) lexidronam, sargramostim, sarilumab, satumomab, secretin, siltuximab, sipuleucel-T, schizophyllan, sovzoxan, glycidazole sodium, sonidegib, sorafenib, stanozolol, streptozocin, sunitinib, talaporfin, talimodinlaherparepvec, tamibarotene , tamoxifen, tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomab merpentane, 99mTc-HYNIC-[Tyr3]-octreotide, tegafur, tegafur + gimeracil + oteracil, temoporfin, temozolomide, temsirolimus, teniposide, testosterone, tetrofosmin, thalidomide, thiotepa , thymalfasine, thyrotropine alfa, thioguanine, tisagenlecureucel, tocilizumab, topotecan, toremifene, tositumomab, trabectedin, trametinib, tramadol, trastuzumab, trastuzumab emtansine, treosulfan, tretinoin, trifluridine + tipiracil, trilostane, triptorelin , trametinib, trofosfamide, thrombopoietin, tryptophan, ubenimex, baratinib, barbicin, vandetanib, vapreotide, vemurafenib, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, bismodegib, vorinostat, vorozole, yttrium-90 glass microspheres, dinostatin, dinostatin stima Lamar, zoledronic acid, zorubicin.

Abbreviations:
The table below describes the abbreviations used herein.
Abu - γ-aminobutyric acid ACN - acetonitrile Boc - tert. - Butyloxycarbonyl Bzl - Benzyl DCM - Dichloromethane DIEA - Diisopropylethylamine (Hunig base)
DMAP - dimethylaminopyridine DMF - dimethylformamide DMSO - dimethylsulfoxide EDCI - 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ee - enantiomeric excess FCS - fetal bovine serum Fmoc - fluorenyl-9-methoxycarbonyl HATU - 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HPLC - high performance liquid chromatography MTBE - methyl tert. - butyl ether NMP - N-methylpyrrolidone RP - reverse phase rt - room temperature RTV - relative tumor volume TFA - trifluoroacetic acid THF - tetrahydrofuran TLC - thin layer chromatography

Various aspects of the invention described in this application are illustrated by the following examples, which are not intended to limit the invention in any way.

The exemplary test experiments described herein serve to illustrate the invention and the invention is not limited to the examples given.

Experimental Part All reagents whose synthesis is not described in the experimental part are either commercially available or are known compounds or may be formed from known compounds by methods known to those skilled in the art.

Compounds and intermediates produced according to the methods of the invention may require purification. Purification of organic compounds is well known to those skilled in the art and there may be several methods of purifying the same compound. In some cases, no purification may be required. In some cases, compounds may be purified by crystallization. In some cases, impurities may be stirred using a suitable solvent. In some cases, compounds are packaged in, for example, prepacked silica gel cartridges, such as the Biotage autopurifier system (SP4® or Isolera Four® ) , Biotage SNAP cartridges KP-Sil® or KP It may be purified by chromatography, particularly flash column chromatography, using —NH ® , and eluents such as hexane/ethyl acetate or DCM/methanol gradients in combination. In some cases, compounds are purified, for example, by running a Waters autopurifier equipped with a diode array detector and/or an on-line electrospray ionization mass spectrometer to an appropriate pre-packed reversed-phase column and trifluoroacetic acid, formic acid, or ammonia. It may be purified by preparative HPLC using a combined eluent such as a gradient of water and acetonitrile which may contain additives such as water.

In some cases, the purification methods described above may be used in the form of salts of compounds of the invention having sufficiently basic or acidic functionality, e.g., for compounds of the invention that are sufficiently basic, e.g. Acetate or formate salts may be provided, or, in the case of compounds of the invention which are sufficiently acidic, for example, ammonium salts. Salts of this type can be converted into their free base or free acid forms, respectively, by various methods known to those skilled in the art, or used as salts in subsequent biological assays. can be done. The specific forms (e.g., salts, free bases, etc.) of the compounds of the present invention described and isolated herein are not necessarily used to quantify a specific biological activity of said compound. It should be understood that this is not the only form that can be applied to biological assays.

Standard procedure for UPLC-MS:
Analytical UPLC-MS was performed as described below. Masses (m/z) are reported from positive mode electrospray ionization unless negative mode is indicated (ESI-). Method 1 is used for the most part. otherwise indicate.

HPLC-MS method and LC-MS method:
Method 0:
Mass determinations were performed by high performance liquid chromatography-mass spectrometry (HPLC-MS) using electrospray ionization (ESI) or by FAB or MALDI mass spectrometry.

Method 1 (LC-MS):
Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8μ 50 x 1 mm; Eluent A: 1 L water + 0.25 mL 99% formic acid; % formic acid; Gradient: 0.0 min 90% A → 1.2 min 5% A → 2.0 min 5% A; Stove: 50°C; Flow rate: 0.40 mL/min; UV detection: 208-400 nm.

Intermediate 1
(3R)-3-(3-aminophenyl)-3-[(tert-butoxycarbonyl)amino]propanoic acid

A mixture of 151 g 3-nitrobenzaldehyde, 94 g ammonium acetate, 127 g malonic acid and 1 L 2-propanol was heated under reflux for 5 hours. The solution was filtered and the precipitate was washed with 0.7 L of hot 2-propanol. The crude product was dried in vacuo, suspended in 1.5 L of water, treated with 1N hydrochloric acid and filtered. The filtrate was concentrated to give 146g. NMR (400 MHz, D ₄ -methanol): δ = 3.09 (m, 2H), 4.88 (m, 1H), 7.74 (t, 1H), 7.90 (d, 1H), 8. 33 (d, 1H), 8.43 (s, 1H).

20 g (95 mmol) of this intermediate and 31.2 g of di-tert-butyl dicarbonate were dissolved in 150 mL of a dioxane/water mixture (1:1) and 33 mL of DIEA was added. The mixture was stirred for about 90 minutes until complete dissolution was observed. After evaporation of the solvent, the remaining residue was dissolved in 1 L DCM and extracted 3 times with 500 mL 5% citric acid. The organic phase was concentrated and the product was precipitated with a mixture of DCM/diethyl ether/petrolether (1:1:1) and filtered. After drying, 23.5 g (80%) of the desired product was obtained.

5 g (16.1 mmol) of this intermediate and 3.095 g (23 mmol) of (2R)-2-amino-2-phenylethanol were dissolved in acetonitrile and left at 0° C. for 3 days. The precipitate was filtered, dissolved in DCM and extracted twice with 5% citric acid. The organic phase was dried over sodium sulphate and evaporated. This procedure was repeated twice. 1.52 g (30%) of the desired product was obtained with an ee of 95% and [α] _D ²⁵ =+34.4°/methanol.

1500 mg (0.243 mmol) of this intermediate was dissolved in 100 mL of methanol and hydrogenated over palladium/carbon under atmospheric pressure for 30 minutes. The catalyst was separated, the solution was concentrated, digested with diethyl ether, filtered and the residue dried in vacuo. 1334 mg (98%) of the title compound were obtained. [DC: (dichloromethane/methanol/ammonia (17%) (15:4:0.5); _Rf = 0.18].

Intermediate 2
(3R)-3-[(tert-butoxycarbonyl)amino]-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid

8300 mg (29.6 mmol) of intermediate 1 and 9843 mg (44.4 mmol) of 3-nitrobenzenesulfonyl chloride were dissolved in 400 ml of DCM/DMF (1:1) and 7.2 mL of pyridine were added. The mixture was stirred overnight at room temperature. The mixture was then diluted with 200 mL DCM and extracted three times with 50 mL 5% citric acid. The organic phase was concentrated. After drying the remaining residue, 13.8 g (quantitative) of (3R)-3-[(tert-butoxycarbonyl)amino]-3-(3-{[(3-nitrophenyl)sulfonyl]amino} phenyl)propanoic acid was obtained. [DC: (dichloromethane/methanol/ammonia (17%) (15:4:0.5); _Rf = 0.2]).

13800 mg (29.65 mmol) of this intermediate was dissolved in 1000 mL of methanol and hydrogenated at normal pressure over palladium/carbon for 5 hours. The catalyst was separated, the solution was concentrated and the residue was washed twice with diethyl ether and then dried in vacuo. 12240 mg (95%) of (3R)-3-(3-{[(3-aminophenyl)sulfonyl]amino}phenyl)-3-[(tert-butoxycarbonyl)amino]propanoic acid were obtained.

12200 mg (28 mmol) of this intermediate were dissolved in 600 mL of dioxane, 5722 mg (67 mmol) of 1-isocyanatopropane were added and the mixture was stirred overnight. The solution was concentrated in vacuo and the remaining residue was purified by flash chromatography using an eluent mixture of DCM/methanol/NH4OH (17%) (15/4/0.5). Relevant fractions were pooled and concentrated in vacuo. After drying the residue in vacuo, 11220 mg (67%) of the title compound were obtained. LC-MS (Method 1): Rt = 0.9 min; MS (ESIpos): m/z = 521 (M+H) ⁺ .

Intermediate 3
(3R)-3-{[(4-aminophenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid

400 mg (0.768 mmol) of intermediate 2 was dissolved in 10 mL DCM and 2 mL trifluoroacetic acid was added. After stirring for 90 minutes at room temperature, the reaction mixture was concentrated in vacuo. The residue was treated with a 5% solution of disodium carbonate and then dissolved in a mixture of DCM/methanol. After precipitation with diethyl ether, filtration and drying in vacuo, 260 mg (81%) of (3R)-3-amino-3-{3-[({3-[(propylcarbamoyl)amino]phenyl} Sulfonyl)amino]phenyl}propanoic acid was obtained. LC-MS (Method 0): Rt = 4.11 min; MS: m/z = 421 = (M+H) ⁺ .

250 mg (0.595 mmol) of this intermediate were dissolved in 15 mL of DMF, 117 mg (0.713 mmol) of 1-isocyanate-4-nitrobenzene were added and the solution was stirred at room temperature for 30 minutes. A further 30 mg of 1-isocyanate-4-nitrobenzene was added and stirring was continued for 30 minutes. The solution was concentrated in vacuo and the remaining residue purified by flash chromatography. After concentrating the relevant fractions in vacuo, 160 mg (46%) of (3R)-3-{[(4-nitrophenyl)carbamoyl]amino}-3-{3-[({3-[(propyl) Carbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid was obtained. LC-MS (method 0): Rt = 5.61 min; MS: m/z = 585 = (M+H) ⁺ .

142 mg (0.243 mmol) of this intermediate was dissolved in 20 mL of methanol/DCM (10:1) and hydrogenated under normal pressure over palladium/carbon for 30 minutes. The catalyst was separated, the solution was concentrated, digested with diethyl ether, filtered and the residue dried in vacuo. 103 mg (76%) of the title compound were obtained. LC-MS (method 0): Rt = 4.31 min; MS: m/z = 555 = (M+H) ⁺ . ¹ H-NMR (500 MHz, D _-methanol ): δ = 0.93 (t, 3H), 1.5 (m, 2H), 2.74 (d, 2H), 3.1 (dt, 2H) , 5.15 (t, 1H), 6.68 (d, 2H), 6.85 (d, 1H), 7.05 (d, 1H), 7.1 (d, 1H), 7.13 ( t, 1H), 7.28-7.4 (m, 3H), 7.6 (s, 1H), 7.66 (d, 1H).

Intermediate 4
(4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline- 4-yl L-valinate trifluoroacetate (1:1)

2.59 g (10.6 mmol) of N-(tert-butoxycarbonyl)-valine-N-carboxyanhydride and 0.5 g of 4-(N,N-dimethylamino)-pyridine were dissolved in 150 ml of anhydrous dichloromethane. 2 g (5.3 mmol) of (4S)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H ,12H)-dione (7-ethylcamptothecin, synthesized as described by S. Sawada et al. in Chem. Phar. Bull 1991-39(6)-1445) was added to a stirred suspension. The mixture was stirred at room temperature for 20 hours and then concentrated in vacuo. 8 ml of ACN was added to the residue followed by 5 mL of diethyl ether. The mixture was filtered and the remaining residue was dried in vacuo. 2964 mg (92%) of the protected intermediate were obtained. LC-MS (method 1): Rt = 1.19 min; MS (ESIpos): m/z = 576 (M+H) ⁺ .

2964 mg (5.15 mmol) of this Boc-protected intermediate compound in 6 ml of dichloromethane and 60 ml of trifluoroacetic anhydride was stirred at room temperature for 30 minutes and then sonicated for 1 hour. After concentration in vacuo, the product was lyophilized from an acetonitrile/water mixture. 3.622 g (quantitative) of the title compound were obtained. LC-MS (Method 1): Rt = 0.68 min; MS (ESIpos): m/z = 476 (M+H) ⁺ .

Intermediate 5
(2S)-1-[(19S)-19-(2-tert-butoxy-2-oxoethyl)-2,2-dimethyl-4,17,20-trioxo-3,8,11,14-tetraoxa-5 , 18-diazaicosan-20-yl]pyrrolidine-2-carboxylic acid

This intermediate 5 was synthesized according to classical methods known in peptide chemistry, in which first benzyl L-prolinate hydrochloride and 4-tert-butyl hydrochloride in DMF in the presence of DIEA. Coupling with 1-(2,5-dioxopyrrolidin-1-yl)N-(tert-butoxycarbonyl)-L-aspartate (1:1) followed by hydrogenation over palladium/carbon Cleavage of the benzyl ester was performed. (2S)-1{(2S)-4-tert-butoxy-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}pyrrolidine- By stirring the solution of the 2-carboxylic acid for 15 minutes, tert. -Butoxycarbonyl protecting group was removed followed by purification via flash chromatography using DCM/methanol 3:1 as eluent. This intermediate was dissolved in DMF and treated with tert-butyl{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxo) in the presence of DIEA. Propoxy}ethoxy)ethoxy]ethyl}carbamate (2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5- in DMF with 1-hydroxypyrrolidine-2,5-dione and EDCI) (obtained previously by transformation of azaheptadecane-17-oic acid to the activated ester). LC-MS (Method 1): Rt = 0.86 min; MS (ESIpos): m/z = 590 (M+H) ⁺ .

Intermediate 6
(3R)-3-{[(4-{[(4-nitrophenoxy)carbonyl]amino}phenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl ) amino]phenyl}propanoic acid

8.99 g (43.3 mmol) of 4-nitrophenyl carbonochloridate was dissolved in 1300 mL of THF and 12 g (21.64 mmol) of (3R)-3-{[(4-aminophenyl)carbamoyl]amino }-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid was added. The mixture was heated and stirred under reflux for 45 minutes, then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure to a volume of 100 mL. The solution was poured into diethyl ether and the precipitate was filtered. After drying in vacuum overnight, 11.6 g of the title compound were obtained. LC-MS (Method 1): Rt = 0.97 min; MS (ESIpos): m/z = 720 (M+H) ⁺ .

Intermediate 7
Reference compound for integrin ligand (S epimer of intermediate 3):
(3S)-3-{[(4-aminophenyl)carbamoyl]amino}-3-{3-[({3-[(propylcarbamoyl)amino]phenyl}sulfonyl)amino]phenyl}propanoic acid

This compound was synthesized analogously to Intermediate 3 described above using the epimer of Intermediate 1 that was found in the mother liquor during the optical resolution step.

Example 1: α _v β ₃ Integrin Conjugate Disodium (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6 ,7]indolizino[1,2-b]quinolin-4-yl 1-{(2S)-2-(carboxylatomethyl)-17-[4-({[(1R)-2-carboxylato-1- {3-[({3-[(propyl-carbamoyl)amino]phenyl}sulfonyl)amino]phenyl}ethyl]carbamoyl}amino)anilino]-4,17-dioxo-7,10,13-trioxa-3,16 -diazaheptadecane-1-oil}-L-prolyl-L-valinate

40 mg (68 μmol) of intermediate 4 and 48 mg (81 μmol) of intermediate 5 were dissolved in 6.4 mL of DMF and 33.5 mg (88 μmol) of HATU and 35 μL of DIEA were added. The mixture was stirred at room temperature for 30 minutes. The mixture was evaporated and the remaining residue was purified by HPLC. 28 mg (39%) of the protected intermediate were obtained. LC-MS (Method 1): Rt = 1.15 min; MS (ESIpos): m/z = 1047 (M+H) ⁺ .

28 mg of this intermediate were dissolved in 2 ml of dichloromethane. 2 ml of trifluoroacetic anhydride was added and the mixture was stirred at room temperature for 30 minutes and then sonicated for 1 hour. After concentration in vacuo, the product was lyophilized from an acetonitrile/water mixture. Obtained 30 mg (quantitative) of the deprotected intermediate as an orange solid. LC-MS (Method 1): Rt = 0.72 min; MS (ESIpos): m/z = 891 (M+H) ⁺ .

1900 mg (1.89 mmol) of this intermediate were dissolved in 60 mL of DMF, 1361 mg (1.89 mmol) of intermediate 6 were added and the mixture was stirred at room temperature for 2 hours. The solution was concentrated in vacuo and the remaining residue was treated with water and 5% citric acid and filtered. The remaining residue was dissolved in DCM/methanol and diethyl ether was added. The precipitate was filtered and purified by flash chromatography using an eluent mixture of DCM/methanol/NH4OH (17%) (15/2/0.2->15/4/0.4). Relevant fractions were pooled and concentrated in vacuo. After drying the residue in vacuo, 942 mg (34%) of the title compound were obtained. LC-MS (Method 1): Rt = 0.97 min; MS (ESIpos): m/z = 1471 (M+H) ⁺ .

20 mg (14 μmol) of this intermediate was dissolved in 4 mL of dioxane/water (1:1), 30 μL (30 μmol) of 1 n aqueous sodium hydroxide solution was added, the mixture was sonicated at room temperature for 5 minutes and frozen. dried. 21 mg (quantitative) of the title compound were obtained. LC-MS (Method 1): Rt = 0.97 min; MS (ESIpos): m/z = 1471 (M-2Na ⁺ +2H ⁺ +H) ⁺ .

Example 2: Reference compound (S epimer) of Example 1:
Disodium (4S)-4,11-diethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3′,4′:6,7]indolizino[1,2-b] quinolin-4-yl 1-{(2S)-2-(carboxylatomethyl)-17-[4-({[(1S)-2-carboxylato-1-{3-[({3-[(propyl -carbamoyl)amino]phenyl}sulfonyl)amino]phenyl}ethyl]carbamoyl}amino)anilino]-4,17-dioxo-7,10,13-trioxa-3,16-diazaheptadecan-1-oyl}- L-prolyl-L-valinate

This compound was synthesized analogously to Example 1 using intermediate 7, an epimer of the α _v β ₃ ligand.

Biological evaluation of 7-ethylcamptothecin, a preferred toxophore, and the conjugate of Example 1
In vitro test to determine cell permeability

-

Caco-2:
The cell permeability of substances can be investigated by in vitro testing in a flux assay using Caco-2 cells. [M. D. Troutman andD. R. Thakker, Pharm. Res. 20(8), 1210-1224 (2003)]. For this purpose, cells were cultured on 24-well filterplates for 15-16 days. To determine permeation, each test substance was applied to either apical (A) or basal (B) cells in HEPES buffer and incubated for 2 hours. After 0 and 2 hours samples were taken from the cis and trans compartments. Samples were separated by HPLC (Agilent 1200, Boblingen, Germany) using a reverse-phase column. The HPLC system was linked to a triple quadrupole mass spectrometer API 4000 (AB SCIEX Deutschland GmbH, Darmstadt, Germany) via a Turbo Ion Spray Interface. Permeability was assessed based on Papp values calculated using the formula published by Schwab et al. [D. Schwab et al. , J. Med. Chem. 46, 1716-1725 (2003)]. A substance was classified as actively transported if the ratio of P _app (B−A) to P _app (A−B) (efflux ratio) was >2 or <0.5.

In this assay, the carrier (4S)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2] used in the conjugate of Example 1 -b]quinoline-3,14(4H,12H)-dione(7-ethyl-camptothecin) shows very good permeability of P _app A->B=171 nm/s and low emission ratio of 1 rice field. This compares favorably with the profile of SN38, the toxic carrier released from irinotecan, which exhibits a significantly lower permeability of P _app A−>B=8 nm/s and an elimination ratio of 36. New data for SN38: a permeability of P _app A->B=20 nm/s and an emission ratio of 9.

P-glycoprotein (p-GP) assay:
Many tumor cells express transport proteins for drugs and this is often accompanied by the development of resistance to cytostatics. Thus, substances that are not substrates for such transport proteins, such as P-glycoprotein (P-gp) or BCRP, for example, may exhibit improved activity profiles.

Substrate properties of P-gp (ABCB1) substances were determined by flux assays using LLC-PK1 cells overexpressing P-gp (L-MDR1 cells) [A. H. Schinkel et al. , J. Clin. Invest. 96, 1698-1705 (1995)]. For this purpose, LLC-PK1 cells or L-MDR1 cells were cultured on 96-well filterplates for 3-4 days. To determine permeation, each test substance, alone or in the presence of an inhibitor (e.g., ivermectin or verapamil), is applied either apically (A) or basally (B) to cells in HEPES buffer. applied and incubated for 2 hours. After 0 and 2 hours samples were taken from the cis and trans compartments. Samples were separated by HPLC using a reverse phase column. The HPLC system was linked to a triple quadrupole mass spectrometer API 3000 (Applied Biosystems Applera, Darmstadt, Germany) via a Turbo Ion Spray Interface. Permeability was assessed based on P _app values calculated using the formula published by Schwab et al. [D. Schwab et al. , J. Med. Chem. 46, 1716-1725 (2003)]. Substances were classified as P-gp substrates if the efflux ratio of P _app (B−A) to P _app (A−B) was >2.

In this assay, the carrier (4S)-4,11-diethyl-4-hydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2] used in the conjugate of Example 1 -b]quinoline-3,14(4H,12H)-dione(7-ethyl-camptothecin) has excellent permeability of P _app A->B=196 nm/s and low emission ratio of 0.6 showed that. This compares favorably with the profile of SN38, the released toxoid carrier from irinotecan, which exhibits a significantly lower permeability of P _app A−>B=10 nm/s and an elimination ratio of 16.

In vitro cytotoxicity against NCI-H1975 and its transporter mutants When tumor cells NCI-H1975 are transfected with drug transporter p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) , 7-ethylcamptothecin's cytotoxic activity was not adversely affected, in sharp contrast to SN38.

α _v β ₃ Binding Assay α _v β ₃ from human A375 cells was assayed according to Wong et al. in Molecular Pharmacology 50, 529-537 (1996). In each case 10 μL of α _v β ₃ (5 ng) in TBS (pH 7.6), 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1% n-octylglucopyranoside (Sigma); ), 10 μL of test substance in 0.1% DMSO and 45 μL of TBS (pH 7.6), 2 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM MnCl ₂ were incubated for 1 hour at room temperature. In each case, 25 μL of WGA SPA beads (Amersham, 4 mg/ml) and 10 μL of Ecstatin (0.1 μCi, Amersham, chloramine T labeled) were then added. After 16 hours at room temperature, samples were measured in a scintillation counter (Wallac 1450). The test results are shown in Table 2 below.

In Vitro Cytotoxicity in the Presence or Absence of Elastase-Cleaving Elastase Cells were cultured according to standard procedures using media recommended by the provider. Cells in a total volume of 100 μL were seeded into white-bottomed 96-well plates (#3610). After a 24 hour incubation period at 37° C. and 5% CO ₂ the medium was changed by adding 90 μL of fresh medium. Treatment is initiated by adding test compound to cells in 10 μl of medium. Concentrations from 10 ⁻⁵ M to 10 ⁻¹³ M were chosen in triplicate, followed by incubation at 37° C. and 5% carbon dioxide. One set of samples was treated with test compound only, and 10 nM elastase was additionally pipetted into a second set of otherwise identically treated samples. After 72 hours proliferation is detected using the MTT assay (ATCC). At the end of the incubation period, MTT reagent was added to all samples for 4 hours, after which cells were lysed overnight by addition of detergent. The dye formed was detected at 570 nm. The proliferation of cells not treated with the test substance but otherwise treated identically was defined as the value of 100%. Dose-response curves allowed the determination of respective _IC50 values, which are summarized in Table 3. (Figure 1 and Table 4).

The presence of neutrophil elastase causes a significant improvement in compound cytotoxicity using the renal carcinoma cell line 786-O. The compounds also demonstrate a clear dependence on elastase using the colon cancer cell line HT29. Furthermore, elastase-induced cleavage causes a dramatic increase in the cytotoxic effects of the compounds.

Solubility of the conjugate of Example 1 compared to the conjugate of Example 1 of EP 1 238 678:
Method: For each vehicle tested, 0.5-1.0 mg of test compound was weighed into a 2 ml Eppendorf vial. A few Glas perls (Φ3 mm) and 1.0 ml vehicle were added. The vials were shaken at 1400 rpm at room temperature (25° C.) for 24 hours. After this period, the supernatant (approximately 230 μl) was transferred to centrifuge tubes. After 30 minutes at 42000 rpm, the solute was transferred to another vial and diluted with DMSO (1:5 and 1:50). These two diluted liquids were analyzed by HPLC (readout: area).

HPLC method:
Eluent A: 1 ml trifluoroacetic acid/L water
Eluent B: 1 ml trifluoroacetic acid/L acetonitrile
Slope:
Time [min] A [%] B [%] Flow rate: [ml/min]
0.0 98 2 1.5
0.2 98 2 1.5
3.3 10 90 1.5
4.0 10 90 1.5
4.1 98 2 2.5
4.7 98 2 2.5
5.0 98 2 1.5
Column: ZORBAX Extend-C18, 3.0×50 mm, 3,5 μm
Oven temperature: 30°C
Detection: 214 and 254 nm
Injection volume: 20 μL

For quantification, standard curves were obtained from DMSO solutions of test compounds (100 μl/ml, 20 μg/ml and 2.5 μg/ml) by using the same HPLC method.

Stability in pH 4 citrate buffer of the conjugate of Example 1 compared to the conjugate of Example 1 of EP 1 238 678:
Method: 0.15 mg of test compound was dissolved in 0.1 ml dimethylsulfoxide and 0.4 ml acetonitrile. The HPLC vial containing the sample solution was shaken and sonicated for complete dissolution. Then 1.0 ml of the respective buffer solution (citrate buffer pH 4; citric acid/sodium hydroxide/sodium chloride Fluka 33643) was added and the samples were stirred. Sample solutions were analyzed by HPLC to determine the amount of test compound and up to two side products at specified times (0, 1, 2, 4, and 24 hours) over 24 hours at 37°C. t(0) values were obtained from samples taken immediately after vortexing with buffer at room temperature. Quantified using peak area (percentage).
LC & LC/MS Purity Analysis: Starting material was analyzed for purity by LC; a 24 hour sample was further analyzed by LC/MS (Waters Quattro Micro).

Plasma Stability of the Conjugates of Example 1 Determining Release of Parent Compound in Rat Plasma:
1 mg of the test compound of Example 1 was dissolved in a mixture of 1.5 mL of dimethylsulfoxide and 1 ml of water. The HPLC vial was shaken and sonicated for complete dissolution. 500 μl of this solution was added to 0.5 mL of rat plasma while vortexing at a temperature of 37°C. Aliquots (10 μL each) were taken at each time point and analyzed by HPLC to determine the amount of test compound. All data are presented as percent area of initial compound at t0.

The compound of Example 1 is stable in rat plasma for >24 hours.

1 mg of test compound was dissolved in 0.5 ml of acetonitrile/dimethylsulfoxide (1:1). The HPLC vial was shaken and sonicated for complete dissolution. 20 μl of this solution was added to 1 ml of warm plasma at 37° C. while vortexing. After 0.17, 0.5, 1, 1.5, 2, and 4 hours, add 100 μl plasma solution of compound at room temperature to a vial containing 300 μl acetonitrile/buffer pH 3 (80:20). This stopped the enzymatic reaction. The mixture was centrifuged for 10 minutes at 5000 rpm. Supernatants were analyzed by HPLC to determine the amount of test compound and up to two side products. t(0) values were obtained from processed samples taken immediately after vortexing with plasma at room temperature. Quantification was performed using peak area (percentage).

Under assay conditions, 7-ethylcamptothecin is stable for at least 4 hours, while camptothecin is degraded by about 50% during the same period.

Pharmacokinetics 4 mg of the conjugate of Example 1 was dissolved in saline and administered iv to female 786-O tumor-bearing NMRI nu/nu mice. Tumor and plasma samples were taken at various time points to determine the levels of intact conjugate and the carrier 7-ethyl-camptothecin cleaved from the conjugate.

For comparison, 1 mg/kg of 7-ethylcamptothecin was dissolved in a mixture of 5% aqueous dextrose/solutol/DMSO (85/10/5) and administered to female 786-O tumor-bearing NMRI nu/nu mice. administered iv. In addition, tumor and plasma samples were taken at various time points to determine levels of 7-ethyl-camptothecin.

Finally, for comparison, 4 mg of the epimer reference conjugate of Example 23 (which has weak α _v β ₃ binding affinity) was dissolved in saline and tested on female 786-O tumor-bearing NMRI nu/nu. Mice were administered iv. Tumor and plasma samples were taken at various time points to determine the levels of intact conjugate and the carrier 7-ethyl-camptothecin cleaved from the conjugate.

Table 7 summarizes the tumor/plasma ratio of 7-ethylcamptothecin detected in each of these experiments. Enhanced delivery of 7-ethylcamptothecin to tumors by α _v β ₃ integrin conjugates is demonstrated compared to direct administration of the toxin carrier and administration of the weakly binding epimer control conjugate.

In Vivo Xenograft Testing The anti-tumor activity of Example 1 was tested in a mouse xenograft model of human cancer. For this purpose, immunocompromised mice were subcutaneously implanted with tumor cells or tumor fragments. At an average tumor size of 20-40 mm ² , mice were randomized into treatment and control groups (n=8 mice/group) and treatment initiated with vehicle alone or Example 1 (formulation: phosphate Buffered saline (“PBS”); route of administration: intravenous (“iv”) into the tail vein. Intravenous treatment was performed once daily for 3 consecutive days, followed by a 4-day drug holiday. Tumor size and body weight were measured at least weekly. Tumor area was detected by electronic calipers [length (mm) x width (mm)]. Experimental groups were terminated when pre-defined ethical endpoints based on German and European animal welfare regulations were reached. In vivo anti-tumor efficacy was expressed as the T/C ratio of the mean tumor areas measured for the treated and control groups on the last day when vehicle controls remained in the study (treated/control; mean tumor area of treated groups /mean tumor area of control group). A compound with a T/C below 0.5 is defined as active (ie, effective). Statistical analysis was evaluated using SigmaStat software. A one-way ANOVA was performed and differences from controls were compared by a pairwise comparison procedure (Dunn's method).

result:
Example 1 showed potent anti-tumor effects in various xenograft models of human tumors when treated with monotherapy. Specifically, Example 1 was effective in reducing tumor area in breast cancer, colon cancer, lung cancer, and renal cancer models.

Claims (18)

A compound of formula (I), or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof,

During the ceremony ,
CT is a monovalent radical of a cytostatic or cytotoxic element ;
LI is a divalent peptide radical of the formula -L-Val-L-Pro-L-Asp-;
SP is a group of formula —C═O—(CH ₂ ) _x —O—(CH ₂ —CH ₂ —O) _y —CH ₂ —CH ₂ —(NH) _z —C═O—, where x= 1-5, y=0-15, and z=0-1,
A compound , or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, wherein IA is a monovalent radical corresponding to the α _v β ₃ integrin receptor. 2. The compound of Claim 1, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, wherein CT is the monovalent radical of 7-ethylcamptothecin. CT is

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, which is 4. The compound of any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, wherein IA is conjugated to SP through a urea linkage. the structure of formula (Ia),

or having a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof,
A compound according to any one of claims 1-4, or a pharmaceutically acceptable salt or solvate thereof, wherein x is 1-5 and y=0-15 , or solvates of salts. The compound of any one of claims 1-5, or a pharmaceutically acceptable salt, solvate, or salt thereof, wherein x = 1-4 and y = 0-10. Solvate. The compound of any one of claims 1-6, or a pharmaceutically acceptable salt, solvate or salt thereof, wherein x = 1-2 and y = 0-5. Solvate. structure of formula (II),

A compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, alternatively with a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, Solvates, or solvates of salts. 9. The compound of any one of claims 1-8, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, which is the disodium salt.

The disodium salt of the compound of any one of claims 1-9, which is Use of a compound according to any one of claims 1-10, or a pharmaceutically acceptable salt, solvate or solvate of a salt thereof, in the manufacture of a medicament for treating a disease. A compound according to any one of claims 1-10, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof, in the manufacture of a medicament for treating a hyperproliferative disorder. Use of. (i) a compound of any one of claims 1-10, or a pharmaceutically acceptable salt, solvate, or solvate of a salt thereof; medicament, including in combination with excipients acceptable to 14. Use of an agent according to claim 13 for treating hyperproliferative disorders. Use of a compound as defined in any one of claims 1-12 or a medicament as defined in claim 13 for use in treating ophthalmic diseases. The hyperproliferative disorder is a solid tumor of the breast, respiratory, brain, genital, gastrointestinal, urinary tract, eye, liver, skin, head and neck, thyroid, or parathyroid cancer, and/or distant metastases thereof. The use according to claim 12 or 14. 15. Use according to claim 12 or 14, wherein said hyperproliferative disorder is cancer. 18. Use according to claim 17, wherein the hyperproliferative disorder is breast cancer, colon cancer, lung cancer, or renal cancer.