TW202235408A - Compounds - Google Patents

Abstract

The invention provides compounds of formula (I), that are peptidase enhanced cytotoxics. The invention further provides pharmaceutical compositions comprising the compounds, and the use of the compounds or pharmaceutical compositions as a medicament, particularly for use in the treatment or prophylaxis of cancers such as multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukaemia and lymphoma.

Description

compound

The present invention relates to peptidase enhanced cytotoxic (PEnC) compounds and the use of such compounds in the treatment of diseases such as cancer.

Increased expression of various hydrolases such as peptidases, esterases and proteases has been described in several types of human malignancies, especially those characterized by slow growth and aggressive phenotypes (Pharmacol Ther. 1999; 83 : 67-123). The Zn ²⁺ -dependent membrane-bound exopeptidase aminopeptidase N (APN, also known as CD13), widely expressed in mammalian cells, plays important roles in cancer development, including, for example, tumor cell invasion, differentiation, proliferation, cell Processes of apoptosis, motility and angiogenesis (Cancer Lett. 2006; 243; 135-43; Curr Med Chem. 2007; 14: 639-47; Curr Med Chem. 2008; 15: 2850-65). Multiple functions of APN have designated it as a "moonlighting ectoenzyme" (Trends Mol Med. 2008; 14: 361-71). Taken together, these capabilities suggest APN as a potential therapeutic target in cancer therapy. Novel drugs targeting this target have been developed using different approaches, including enzyme inhibitors and APN-targeting carrier constructs (Cancer Sci. 2011; 102: 501-8).

Peptidase-enhanced cytotoxic agents (PEnC) are a class of lipophilic peptides that exhibit increased activity via peptidase cleavage and have the potential to treat many cancers. The present inventors have discovered a new family of peptidase-enhanced cytotoxic agents that find utility in the treatment of cancer.

（I） 其中， R ₁為H或-C _1-6烷基； R ₂為-C _1-6烷基；-CH ₂-吲哚基；-CH ₂-苯基；或包含1、2、3或4個氮或硫原子之-CH ₂-5員雜芳基；其中-C _1-6烷基視需要經-OH取代；且苯基視需要經1、2或3個獨立地選自由以下組成之群的取代基取代：鹵素；-NH ₂；-OH；-O-C _1-6烷基；視需要經1、2或3個鹵素取代之-N(C _1-6烷基) ₂；及-NO ₂；且 R ₃為H或-C(O)R ^a；其中R ^a為-C _1-6烷基或-CH ₂-苯基，其視需要經1、2或3個鹵素取代。 The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, ester, amide or carbamate, including salts of such ester, amide or carbamate,

(I) Among them, R ₁ is H or -C _1-6 alkyl; R ₂ is -C _1-6 alkyl; -CH ₂ -indolyl; -CH ₂ -phenyl; or contains 1, 2, -CH ₂ -5 membered heteroaryl with 3 or 4 nitrogen or sulfur atoms; wherein -C _1-6 alkyl is optionally substituted by -OH; and phenyl is optionally 1, 2 or 3 independently selected from -NH ₂ ; -OH; -OC _1-6 alkyl; -N(C _1-6 alkyl) ₂ optionally substituted with 1, 2 or 3 halogens; and -NO ₂ ; and R ₃ is H or -C(O)R ^a ; wherein R ^a is -C _1-6 alkyl or -CH ₂ -phenyl, optionally substituted by 1, 2 or 3 halogens .

The present inventors have found that compounds of formula (I) are effective anticancer agents. In particular, the present inventors have found using an in vitro cytotoxicity assay that the compound of formula (I) exhibits cytotoxic activity against blood and solid cancer cells. The present inventors also found that the compound of formula (I) is particularly effective in inhibiting tumor growth and metastasis in the chorioallantoic allantoic membrane (CAM) model of human osteosarcoma.

The present invention further provides a pharmaceutical composition, which comprises the compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may optionally contain additional therapeutic agents, such as protease inhibitors (protease inhibitors; PIs), immunomodulatory drugs (IMiDs) or alkylating agents.

The invention further provides a compound or a pharmaceutical composition according to the invention for use as a medicine. In addition, there is also provided a compound or pharmaceutical composition according to the present invention for treating or preventing cancer, such as multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

The present invention further provides a method of treating a patient comprising administering a pharmaceutically effective amount of a compound or pharmaceutical composition according to the present invention.

The invention further provides the use of the compounds according to the invention for the manufacture of a medicament for the treatment or prevention of cancer.

The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester, amide or carbamate, including salts of such ester, amide or carbamate.

As described in the Examples section, the inventors have found that compounds of formula (I) are surprisingly cytotoxic to blood and solid cancer cells. It is worth noting that the inventors have proved that the compound of formula (I) is particularly effective in inhibiting the growth and metastasis of human osteosarcoma tumors by using the extra-ovo CAM model of human osteosarcoma.

In the compound of formula (I), R ¹ is selected from H or -C _1-6 alkyl. Preferably, R ¹ is H or -C _1-4 alkyl. For example, ^R can be H, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or secondary butyl. More preferably, R ¹ is H, ethyl or isopropyl.

In the compound of formula (I), R ² is selected from the group consisting of: -C _1-6 alkyl; -CH ₂ -indolyl; -CH ₂ -phenyl; and comprising 1, 2, 3 or 4 -CH ₂ -5 membered heteroaryl of nitrogen or sulfur atom; wherein -C _1-6 alkyl is optionally substituted by -OH; and phenyl is optionally 1, 2 or 3 independently selected from the group consisting of Substituent substitution by: halogen (for example, F, Cl, Br or I); -NH ₂ ; -OH; -OC _1-6 alkyl; Br or I) substituted -N(C _1-6 alkyl) ₂ ; and -NO ₂ . For example, R ² can be -C _1-4 alkyl; -CH ₂ -indolyl; -CH ₂ -phenyl; or -CH ₂ -5 membered heteroaryl containing 1, 2 or 3 nitrogen or sulfur atoms wherein -C _1-4 alkyl is optionally substituted with -OH; and phenyl is optionally substituted with 1 or 2 substituents independently selected from the group consisting of: chlorine; fluorine; -NH ₂ ; -OH ; -OC _1-4 alkyl; -N(C _1-4 alkyl) ₂ optionally substituted with 1 or 2 chlorine or fluorine atoms; and -NO ₂ .

As used herein, the term "heteroaryl containing 1, 2, 3 or 4 nitrogen or sulfur atoms" refers to an aromatic cyclic group of carbon atoms in which one, two, three or four carbon atoms are One, two, three or four heteroatoms independently selected from nitrogen and sulfur are substituted. Examples of 5-membered heteroaryl groups containing 1, 2, 3 or 4 nitrogen or sulfur atoms include thiophene, thiazole, isothiazole, pyrrole, dihydropyrrole, pyrazole, pyrazoline, imidazole, imidazoline, triazole, Thiadiazoles and tetrazoles.

Preferably, R ² is a -CH ₂ -5 membered heteroaryl group containing a nitrogen or sulfur atom; or optionally 1 or 2 substituents independently selected from chlorine, fluorine and -OC _1-4 alkyl Substituted _-CH2 -phenyl. For example, R2 can be ^-CH2 -thiophene; _-CH2 -pyrrole; _{-CH2 -} dihydropyrrole; or -CH optionally substituted with 1 or 2 chlorine or fluorine atoms or 1 or 2 methoxy ₂ -Phenyl. More preferably, R ² is -CH ₂ -thiophene; or -CH ₂ -phenyl optionally substituted with 1 or 2 chlorine or fluorine atoms or a methoxy group. For example, R2 can be ^-CH2 _- thiophene; methoxybenzyl; fluorobenzyl; chlorobenzyl; Even more preferably, R2 is ^-CH2 _- thiophene; 4-methoxybenzyl; 4-fluorobenzyl; 4-chlorobenzyl; 2,4-difluorobenzyl; benzyl.

In compounds of formula (I), R ³ is selected from the group consisting of H and -C(O)R _a . Preferably, R ³ is H or -C(O)R _a . More preferably, R ³ is H.

In the compound of formula (I), R _a is -C _1-6 alkyl or -CH ₂ -phenyl optionally substituted with 1, 2 or 3 halogens (eg, F, Cl, Br or I). Preferably, R _a is -C _1-4 alkyl or -CH ₂ -phenyl, which is optionally substituted with 1 or 2 chlorine or fluorine atoms. For example, _Ra can be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, phenyl, fluorophenyl, chlorophenyl, difluorophenyl, or dichlorobenzene base. Thus, for example, in compounds where R ³ is -C(O) _Ra , R ³ may be -C(O)CH ₃ , -C(O)CH ₂ CH ₃ , -C(O)CH ₂ ( CH ₃ ) ₂ , -C(O)CH(CH ₃ )CH ₂ CH ₃ ; -C(O)CH ₂ CH ₂ (CH ₃ ) ₂ ; -C(O)-fluorophenyl (i.e. 2-fluorobenzene formyl, 3-fluorobenzoyl or 4-fluorobenzoyl), -C(O)-chlorophenyl (i.e. 2-chlorobenzoyl, 3-chlorobenzoyl or 4- Chlorobenzoyl).

The present inventors have found that the compound of formula (I) is particularly cytotoxic to blood and solid cancer cells. In the compound of formula (I), R ¹ is H or -C _1-4 alkyl, and R ² is a compound containing a nitrogen or sulfur atom. -CH ₂ -5 membered heteroaryl; or -CH ₂ -phenyl optionally substituted with 1 or 2 chlorine or fluorine atoms or one -OC _1-4 alkyl; and R ³ is H. Therefore, in some preferred embodiments, R ¹ is H or -C _1-4 alkyl; R ² is -CH ₂ -5 membered heteroaryl containing a nitrogen or sulfur atom, or optionally modified by 1 or -CH ₂ -phenyl substituted by 2 chlorine, fluorine or -OC _1-4 alkyl; and R ³ is H.

In a further preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfanyl)phenyl ]propionyl]amino]-3-(4-fluorophenyl)propionic acid ethyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(4-Fluorophenyl) propionate ethyl ester hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(4-fluorophenyl)propionate isopropyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(4-Fluorophenyl)propionic acid isopropyl ester hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(4-chlorophenyl)propionic acid ethyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(4-chlorophenyl) ethyl propionate hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(2,4-dichlorophenyl)propionic acid ethyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(2,4-dichlorophenyl) ethyl propionate hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(4-methoxyphenyl)propionate ethyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(4-Methoxyphenyl) ethyl propionate hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(2-thienyl)ethyl propionate:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(2-thienyl) ethyl propionate hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-phenyl-propionic acid ethyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-Phenyl-propionic acid ethyl ester hydrochloride.

In another preferred embodiment, the compound of the present invention is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylhydrogensulfuryl)phenyl ]propionyl]amino]-3-(2,4-dichlorophenyl)propionate isopropyl ester:

For example, the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino ]-3-(2,4-Dichlorophenyl) propionate isopropyl hydrochloride.

In particular instances, compounds of the invention may contain isotopic atoms. As defined herein, an isotopic atom is an atom of an element that is not the most common naturally occurring isotope. Deuterium is a safe and stable isotope of hydrogen. In one embodiment, the compounds of the invention have a deuterium abundance level greater than the naturally occurring abundance of deuterium. The naturally occurring abundance of deuterium is 0.0156 mol%, where mol% is the total molar percentage of hydrogen (which is deuterium) in the sample. Thus, in 1 mole of naturally occurring hydrogen, 0.156 mmol is deuterium, or in a sample of 6.022×10 ²³ naturally occurring hydrogen atoms, there are 9.39×10 ¹⁹ deuterium atoms, or in 6413 naturally occurring hydrogen In a sample of atoms, there is one deuterium atom. Deuterium abundance levels greater than the naturally occurring abundance of deuterium can be at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium. In certain embodiments, compounds of the invention have a deuterium abundance level of at least 1 mol%, 5 mol%, 10 mol%, 50 mol%, 90 mol%, or 98 mol% deuterium.

Depending on the substituents present in the compounds of the invention, the compounds may form esters, amides, carbamates and/or salts. Salts of the compounds of the invention suitable for use in medicine are those wherein the counterion is pharmaceutically acceptable. However, salts with non-pharmaceutically acceptable counterions are within the scope of the present invention, eg for use as intermediates in the preparation of compounds of the present invention and pharmaceutically acceptable salts and physiologically functional derivatives thereof. The term "physiologically functional derivatives" refers to chemical derivatives of the compounds of the invention which have the same physiological function as the compounds of the invention, for example by being transformable in vivo. Esters, amides and carbamates are examples of physiologically functional derivatives.

Suitable salts according to the invention include those formed with organic or inorganic acids or bases. In particular, suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as those having 1 to 4 carbon atoms, unsubstituted or modified, for example Halogen-substituted alkanecarboxylic acids, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids; or organic sulfonic acids, such as (C ₁ -C ₄ )-alkyl-sulfonic acids or aryl- Sulfonic acids, unsubstituted or substituted, for example with halogen. Pharmaceutically acceptable acid addition salts include those acid addition salts formed from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoro Acetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxalic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid , malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, isethionic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid and glutamic acid, lysine and arginine. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, may be suitable as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.

啉；硫代

啉；哌啶；吡咯啶；單-低碳數烷基胺、二-低碳數烷基胺或三-低碳數烷基胺，例如乙基-丙胺、三級丁基-丙胺、二乙基-丙胺、二異丙基-丙胺、三乙基-丙胺、三丁基-丙胺或二甲基-丙胺；或單羥基-低碳數烷基胺、二羥基-低碳數烷基胺或三羥基-低碳數烷基胺，例如單-乙醇胺、二-乙醇胺或三乙醇胺。此外，可形成相應內鹽。Pharmaceutically acceptable base salts include ammonium salts; alkali metal salts such as those of potassium and sodium; alkaline earth metal salts such as those of calcium and magnesium; and salts with organic bases such as dicyclohexylamine ; N-methyl-D-glucosamine;

phylloline; thio

morphine; piperidine; pyrrolidine; mono-lower alkylamine, di-lower alkylamine or tri-lower alkylamine, such as ethyl-propylamine, tertiary butyl-propylamine, diethyl base-propylamine, diisopropyl-propylamine, triethyl-propylamine, tributyl-propylamine or dimethyl-propylamine; or monohydroxy-lower alkylamine, dihydroxy-lower alkylamine or Trihydroxy-lower alkylamines such as mono-ethanolamine, di-ethanolamine or triethanolamine. In addition, corresponding inner salts can be formed.

Preferred salts of the compounds of the present invention include acid addition salts such as those formed from hydrochloric acid, hydrobromic acid, acetic acid, p-toluenesulfonic acid, tartaric acid, sulfuric acid, succinic acid, phosphoric acid, oxalic acid, Nitric acid, methanesulfonic acid, malic acid, maleic acid and citric acid. More preferably, the salts of the compounds of the present invention are hydrochlorides (ie addition salts formed with hydrochloric acid).

The compounds of the invention may have suitable groups for conversion into esters, amides or carbamates. Thus, typical ester and amide groups formed from acid groups in the compounds of the present invention include -COOR ^G , -CONR ^G ₂ , -SO ₂ OR ^G or -SO ₂ N(R ^G ) ₂ , and in the compounds of the present invention Typical ester and amide and carbamate groups formed from -OH or -NHR ^G groups include -OC(O)R ^G , -NR ^G C(O)R ^G , -NR ^G CO ₂ R ^G , -OSO ₂ R ^G and -NR ^G SO ₂ R ^G , wherein R ^G is selected from the group consisting of: C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _3-8 ring Alkyl and C _3-8 cycloalkyl C _1-8 alkyl, halo C _1-8 alkyl, dihalogen C _1-8 alkyl, trihalogen C _1-8 alkyl, phenyl and benzene C _1-4 alkyl; more preferably R ^G is selected from the group consisting of: C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cycloalkyl and C _3-8 cycloalkyl C _1-6 alkyl.

Those of ordinary skill in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. Such complexes are called "solvates". For example, complexes with water are called "hydrates". The complex can be incorporated into the solvent in stoichiometric or non-stoichiometric amounts. Solvates are described in Water-Insoluble Drug Formulation, 2nd Edition, R. Lui, CRC Press, p. 553 and Byrn et al., Pharm. Res., 12(7), 1995 , 945-954. The compounds of the invention, as well as their esters, amides, carbamates and/or salts, may be in solvated form prior to their preparation in solution. Solvates of the compounds of the invention which are suitable as pharmaceuticals are those wherein the relevant solvent is pharmaceutically acceptable. For example, hydrates are pharmaceutically acceptable solvates.

Compounds that, once administered to a recipient, are capable of being converted into a compound of the invention as described above, or an active metabolite or residue thereof, are termed "prodrugs." Prodrugs can be converted, for example, in vivo into their active form which has a medicinal effect, for example by hydrolysis in the blood. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, A. C. S. Symposium Series (1976), Volume 14; "Design of Prodrugs". H. Bundgaard, ed., Elsevier, 1985; and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.

Unless otherwise limited in a particular instance, the following definitions apply to the terms as used throughout this specification.

As used herein, the term "alkyl" means straight chain and branched chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, di-butyl, pentyl and hexyl. Among the unbranched alkyl groups methyl, ethyl, propyl, isopropyl, n-butyl are preferably present. In branched chain alkyl, tertiary butyl, isobutyl, 1-ethylpropyl and 1-ethylbutyl may be referred to.

As used herein, the term "cycloalkyl" means a saturated group in a ring system. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are preferred, and fluorine and chlorine are particularly preferred.

As mentioned above, the compounds of the present invention are peptidase-enhanced cytotoxic agents (PEnC). The present invention also provides a compound according to the present invention or a composition comprising a compound according to the present invention and a pharmaceutically acceptable carrier for use as a medicine.

The compounds, compositions and pharmaceutical compositions according to the present invention can be used to treat and/or prevent cancer, reduce tumor growth and/or kill tumor cells. Therefore, the compounds of the present invention are useful for curing and/or prolonging the survival of patients suffering from cancer diseases. The present invention is especially suitable for treating and/or preventing multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma.

Although it is possible for the compounds according to the invention to be administered alone, they are preferably present in compositions and especially in pharmaceutical compositions. The pharmaceutical composition includes oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus injection or infusion) and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including transdermal, buccal, sublingual and intraocular) administration of these pharmaceutical compositions, although the most suitable route may depend, for example, on the condition and condition of the individual under treatment.

Pharmaceutical compositions of the invention suitable for oral administration may be presented as discrete units, such as capsules, cachets, or lozenges, each containing a predetermined amount of the active ingredient; in powder or granule form; in aqueous or non-aqueous liquids in the form of solutions or suspensions; or in the form of oil-in-water liquid emulsions or water-in-oil liquid emulsions. The compounds of the invention may also be presented in the form of boluses, elixirs or pastes. Various pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, eg, Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Suppl. 42:2S, 1988.

Pharmaceutical compositions for parenteral administration include aqueous and nonaqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended recipient; and may include suspensions. Aqueous and non-aqueous sterile suspensions of agents and thickeners. Preferably, the formulations are presented in unit-dose or divided-dose containers, such as sealed ampoules and vials. The formulations can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as physiological saline or water for injection, immediately before use. Ready-to-use injection and infusion solutions and suspensions can be prepared from sterile powders, granules and other dry compositions. Exemplary compositions for parenteral administration include injectable solutions or suspensions, which may contain, for example, suitable nontoxic parenterally acceptable solutes, diluents or solvents, such as mannitol, 1,3-butane Glycols, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor .

Pharmaceutical compositions for nasal, aerosol or inhalation administration include solutions in physiological saline which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability and/or other dissolving or dispersing agents. Reagents, such as those known in the art.

Compositions for rectal administration may be in the form of suppositories with common carriers such as cocoa butter, synthetic glycerides or polyethylene glycols. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal lumen to release the drug.

Pharmaceutical compositions for topical (buccal or sublingual) administration in the mouth include lozenges containing the active ingredient in a flavored base such as sucrose and acacia or tragacanth, and in a flavored base such as gelatin and glycerin or sucrose and acacia. Tablets containing the active ingredient in a gum base. Exemplary compositions for topical administration include topical carriers such as Plastibase (mineral oil gelled with polyethylene).

The amount of a compound of the invention required to achieve therapeutic efficacy will vary with the particular route of administration and the characteristics of the individual being treated, such as species, age, weight, sex, medical condition, the particular disease and its severity, and others. Relevant medical and physical factors. A physician with ordinary knowledge can readily determine and administer the effective amount of a compound of the invention required to treat or prevent cancer.

Depending on the individual to be treated and the form of cancer, the compounds of the invention are administered in high single doses every day, every two or three days, every week, every two weeks, every three weeks or every four weeks or even longer.

Preferably, the compound of the present invention or its salt and/or solvate (excluding the mass of any relative ion or solvent) can be administered in an amount of about 1 mg to 150 mg per administration. For example, 1, 2, 3, 5, 10, 15, 20, 25, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 mg. Alternatively, a compound of the present invention, or a salt and/or solvate thereof (excluding any counter ion or solvent mass) may be administered in a single high dose. A single high dose may be about 150 mg to 800 mg. For example, 150, 200, 300, 400, 500, 600, 700 and 800 mg.

Although the compound of the present invention can be used as the sole active ingredient in the present invention, it is also possible to use it in combination with one or more other therapeutic agents, and the use of such combination provides a preferred embodiment of the present invention. Such other therapeutic agents may be agents or other pharmaceutically active substances suitable for the treatment or prevention of cancer. Such agents are known in the art. Examples of other therapeutic agents useful in the present invention include steroids (prednisone and dexamethasone), IMiDs (thalidomide, lenalidomide and polidomide ( pomalidomide), PIs (bortezomib and carfilzomib), histone deacetylase (HDAC) inhibitors (panobinostat), PEnC (eg Melflufen) and conventional chemotherapy (alkylating agents (eg melphalan, cyclophosphamide) and doxorubicin). In a preferred embodiment, the other therapeutic agent is doxorubicin.

The one or more additional therapeutic agents may be administered simultaneously, sequentially, or separately from the administration of the compounds of the invention. The individual components of such combinations may be administered separately at different times during the course of therapy or administered concurrently in fractional or single combinations. Example General Experimental Details

方案 1試劑及條件：a）2-巰基乙醇、Pd ₂(dba) ₃、Xantphos、1,4-二

烷（dioxane），130℃，1小時定量；b）惠尼格氏鹼（Hünig's base）、HATU、DMF，15分鐘，室溫，72%；c）POCl ₃、DCM，室溫，18小時，83%。 材料及方法 通用方法 A synthetic route for the preparation of example compound 1 is shown in Scheme 1 :

Scheme 1 Reagents and conditions: a) 2-mercaptoethanol, Pd ₂ (dba) ₃ , Xantphos, 1,4-di

Dioxane, 130°C, quantified in 1 hour; b) Hünig's base (Hünig's base), HATU, DMF, 15 minutes, room temperature, 72%; c) POCl ₃ , DCM, room temperature, 18 hours, 83%. Materials and Methods General Methods

步驟 1 ： (2S)-3-[3,5- 雙 (2- 羥乙基氫硫基 ) 苯基 ]-2-( 三級丁氧基羰胺基 ） 丙酸之製備

Preparative HPLC was performed on a Gilson system equipped with a UV detector using an XBridge preparative C-18 5 µm OBD, 19×50 mm column. Analytical HPLC-MS was performed using an Agilent 1100 Series Liquid Chromatography/Mass Selective Detector (MSD) (single quadrupole) equipped with an electrospray interface and a UV diode array detector. By two methods, an ACE 3 C8 (3.0 mm×50 mm) column with a gradient of acetonitrile in 0.1% TFA in water over 3 minutes and a flow rate of 1 mL/min was used, or with a 3-minute gradient of acetonitrile in A gradient in 10 mM ammonium bicarbonate and an Xbridge C18 (3.0 mm×50 mm) column with a flow rate of 1 mL/min were used for analysis. ¹ H-NMR spectra were recorded on a 400 MHz instrument at 25°C. Compounds have been named using the software MarvinSketch 5.6.0.1. In addition, commercial or common names are used for commercial starting materials and reagents. Example Compound 1 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of ethyl 3-(4- fluorophenyl ) propionate hydrochloride

Step 1 : Preparation of (2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propionic acid

烷（3 mL）中之(2S)-2-(三級丁氧基羰胺基)-3-(3,5-二溴苯基)丙酸（300 mg，0.71 mmol）、Pd ₂(dba) ₃（39 mg，0.06 mmol）、xantphos（82 mg，0.14 mmol）及惠尼格氏鹼（0.49 mL，2.84 mmol）之微波管中添加2-巰基乙醇（149 µL，2.13 mmol）。蓋上管，且混合物用N ₂氣體沖洗並且在130℃下加熱1小時。在矽藻土墊上濾出固體，且蒸發溶劑。使用DCM:MeOH:AcOH（95:4:1至93:6:1）之梯度在矽膠上急驟層析粗殘餘物。洗提份在真空下濃縮且與甲苯共蒸發，以得到呈油狀之標題化合物（318 mg，定量產率）。 to contain in 1,4-di

(2S)-2-(tertiary butoxycarbonylamino)-3-(3,5-dibromophenyl)propionic acid (300 mg, 0.71 mmol), Pd ₂ (dba ) ₃ (39 mg, 0.06 mmol), xantphos (82 mg, 0.14 mmol) and Winiger's base (0.49 mL, 2.84 mmol) were added with 2-mercaptoethanol (149 µL, 2.13 mmol). The tube was capped, and the mixture was flushed with _N2 gas and heated at 130 °C for 1 h. The solid was filtered off on a pad of celite, and the solvent was evaporated. The crude residue was flash chromatographed on silica gel using a gradient of DCM:MeOH:AcOH (95:4:1 to 93:6:1). Fractions were concentrated in vacuo and co-evaporated with toluene to give the title compound (318 mg, quantitative yield) as an oil.

HPLC purity: 89%; MS (ESI ⁺ ) m/z 318 [M+H-BOC] ⁺ . Step 2 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of ethyl] acyl ] amino ]-3-(4- fluorophenyl ) propionate

Add HATU (323 mg, 0.85 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (296 mg, 0.71 mmol), ethyl (2S)-2-amino-3-(4-fluorophenyl)propionate hydrochloride (211 mg, 0.85 mmol) and Winiger’s base (613 µL, 3.54 mmol) in DMF (7 mL). The mixture was stirred at room temperature for 15 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (DCM:acetone 5:1). Fractions were concentrated under vacuum. The residue was redissolved in EtOAc and washed with 0.2 M NaHSO ₄ solution, dried over MgSO ₄ and concentrated in vacuo to give the title compound (314 mg, 72% yield) as a white solid.

HPLC purity: 100%; MS (ESI ⁺ ) m/z 511 [M+H-BOC] ⁺ . ¹ H NMR (400 MHz, DMSO-d6) δ ppm 1.11 (3H, t), 1.14-1.32 (9H, m), 2.56-2.66 (1H, m), 2.80-2.88 (1H, m), 2.92-3.10 (6H, m), 3.57 (4H, q), 4.05 (2H, q), 4.17 (1H, m) 4.43-4.54 (1H, m) 4.93 (t, 2H) 6.89 (1H, d) 7.03-7.13 ( m, 5H) 7.24-7.31 (m, 2H) 8.36 (d, 1H). Step 3 : Example compound 1 ( (2S)-2-[[(2S)-2- amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amine base ]-3-(4- fluorophenyl ) ethyl propionate hydrochloride ) preparation

烷（184 µL，0.74 mmol）。在真空下濃縮溶液，以得到呈白色固體狀之標題化合物（120 mg，84%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with POCl ₃ (69 µL, 0.74 mmol) at room temperature -Ethyl (tertiary butoxycarbonylamino)-propionyl]amino]-3-(4-fluorophenyl)propanoate (150 mg, 0.25 mmol) in DCM (5 mL) overnight . The reaction mixture was diluted with DCM and washed by 0.2 M Na ₂ CO ₃ solution (11 mL, 2.21 mmol). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkanes (184 µL, 0.74 mmol). The solution was concentrated under vacuum to afford the title compound (120 mg, 84% yield) as a white solid.

步驟 1 ： (2S)-2- 胺基 -3-(4- 氟苯基 ) 丙酸異丙酯鹽酸鹽之製備

HPLC purity: 93%; MS (ESI ⁺ ) m/z 547 [M+H] ⁺ . ¹ H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (3H, t), 2.85-2.94 (1H, m), 2.97-3.12 (2H, m), 3.14-3.22 (1H, m), 3.34-3.41 (4H, m), 3.73-3.80 (4H, m), 4.06-4.16 (3H, m), 4.51-4.59 (1H, m) 7.09-7.37 (7H, m) 8.18 (br.s, 3H) 9.24 ( 1H, d). Example Compound 2 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of isopropyl 3-(4- fluorophenyl ) propionate hydrochloride

Step 1 : Preparation of (2S)-2- amino- 3-(4- fluorophenyl ) propionic acid isopropyl ester hydrochloride

Thionyl chloride (0.299 mL, 4.09 mmol) was slowly added to (2S)-2-amino-3-(4-fluorophenyl)propanoic acid (0.500 g, 2.73 mmol) at 0 °C under nitrogen in 2-propanol (20.0 mL) in the mixture. The reaction was then heated at 80 °C overnight. The reaction mixture was cooled to room temperature and then concentrated. Ether was added and the solid was filtered off, washed with ether and dried to give 685 mg of crude product as a white solid. The crude product was used in the next step without further purification. MS (ESI ⁺ ) m/z 226 [M+H] ⁺ . Step 2 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of Acyl ] Amino ]-3-(4- Fluorophenyl ) propionate Isopropyl Ester

Add HATU (33 mg, 0.09 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (30 mg, 0.07 mmol) (synthesis described in Example 1), (2S)-2-Amino-3-(4-fluorophenyl)propanoic acid isopropyl ester-hydrochloride (20 mg, 0.08 mmol) and Winig's base (37 μL, 0.22 mmol) in DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (20% to 100% EtOAc/petroleum ether). Fractions were concentrated in vacuo to afford the title compound (44 mg, 98% yield) as a white solid. HPLC purity: 93%; MS (ESI ⁺ ) m/z 525 [M+H-BOC] ⁺ . Step 3 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of -(4- fluorophenyl ) isopropyl propionate hydrochloride

烷（53 μL，0.21 mmol）。攪拌溶液1小時，在真空下濃縮且藉由製備型HPLC（10%至80%乙腈/0.1% TFA水溶液）純化殘餘物，以得到呈白色固體狀之標題化合物（13 mg，31%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (19 μL, 0.21 mmol) at room temperature -Isopropyl (tertiary butoxycarbonylamino)propionyl]amino]-3-(4-fluorophenyl)propanoate (44 mg, 0.07 mmol) in DCM (2 mL) overnight . The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃ solution (10 mL). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkane (53 μL, 0.21 mmol). The solution was stirred for 1 h, concentrated in vacuo and the residue was purified by preparative HPLC (10% to 80% acetonitrile/0.1% TFA in water) to give the title compound (13 mg, 31% yield) as a white solid .

步驟 1 ： (2S)-2-( 三級丁氧基羰胺基 )-3-(4- 氯苯基 ) 丙酸乙酯之製備

HPLC purity: 98+ %; MS (ESI ⁺ ) m/z 561 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 2.59 (d, 3H), 2.63 (d, 3H) , 2.87 (dd, 1H), 3.03 (dq, 2H), 3.15 (dd, 1H), 3.37 (t, 4H), 3.76 (t, 4H), 4.09 (br s, 1H), 4.52 (q, 1H) , 4.89 (septet, 1H), 7.14 (t, 2H), 7.19 (d, 2H), 7.31 (m, 3H). Example compound 3 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]- Preparation of ethyl 3-(4- chlorophenyl ) propionate hydrochloride

Step 1 : Preparation of ethyl (2S)-2-( tertiary butoxycarbonylamino )-3-(4- chlorophenyl ) propionate

To a solution of (2S)-2-(tertiary butoxycarbonylamino)-3-(4-chlorophenyl)propionic acid (0.500 g, 1.67 mmol) in absolute ethanol (20.0 mL) under nitrogen DCC (0.413 g, 2.00 mmol) and DMAP (0.0204 g, 0.167 mmol) were added. The reaction was stirred for 2 hours and then the reaction mixture was concentrated. DCM (5 mL) was added and solid DHU was filtered off and the filtrate was concentrated. The crude material was then purified by flash chromatography (ISCO; SiO ₂ , 12 g column; petroleum ether/EtOAc 0%-20%) to afford 387 mg (71%) of the product as a colorless oil which, after standing Solidified to a white solid. MS (ESI ⁺ ) m/z 228 [M+H-BOC] ⁺ . Step 2 : Preparation of ethyl (2S)-2- amino- 3-(4- chlorophenyl ) propionate hydrochloride

烷（4.00 M，2.35 mL，9.40 mmol）且攪拌反應物6小時。剩餘起始物質，因此額外添加0.5 mL含4 M HCl之二

烷，且攪拌反應物隔夜。濃縮反應混合物且脫水，以得到0.305 g（98%）呈白色固體狀之標題化合物。MS (ESI ⁺) m/z228 [M+H] ⁺。 步驟 3 ： (2S)-2-[[(2S)-3-[3,5- 雙 (2- 羥乙基氫硫基 ) 苯基 ]-2-( 三級丁氧基羰胺基 ) 丙醯基 ] 胺基 ]-3-(4- 氯苯基 ) 丙酸乙酯之製備

To a solution of (2S)-2-(tertiary butoxycarbonylamino)-3-(4-chlorophenyl)propionate (0.385 g, 1.17 mmol) in DCM (7.00 mL) was added HCl containing of two

(4.00 M, 2.35 mL, 9.40 mmol) and the reaction was stirred for 6 hours. Starting material remains, so add an additional 0.5 mL of 4 M HCl bis

alkanes, and the reaction was stirred overnight. The reaction mixture was concentrated and dried to afford 0.305 g (98%) of the title compound as a white solid. MS (ESI ⁺ ) m/z 228 [M+H] ⁺ . Step 3 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of ethyl] acyl ] amino ]-3-(4- chlorophenyl ) propionate

Add HATU (33 mg, 0.09 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (30 mg, 0.07 mmol) (synthesis described in Example 1), ethyl (2S)-2-amino-3-(4-chlorophenyl)propanoate-hydrochloride (23 mg, 0.08 mmol) and Winig's base (37 μL, 0.22 mmol) in DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (20% to 100% EtOAc/petroleum ether). Fractions were concentrated in vacuo to afford the title compound (54 mg, 99% yield) as a white solid. HPLC purity: 97.8%; MS (ESI ⁺ ) m/z 527 [M+H-BOC] ⁺ . Step 4 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of -(4- chlorophenyl ) ethyl propionate hydrochloride

烷（45 μL，0.18 mmol）。攪拌溶液1小時且在真空下濃縮，以得到呈白色固體狀之標題化合物（34 mg，66%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (24 μL, 0.26 mmol) at room temperature - A solution of ethyl (tertiary butoxycarbonylamino)propionyl]amino]-3-(4-chlorophenyl)propanoate (54 mg, 0.09 mmol) in DCM (2 mL) overnight. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃ solution (10 mL). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkane (45 μL, 0.18 mmol). The solution was stirred for 1 hour and concentrated under vacuum to give the title compound (34 mg, 66% yield) as a white solid.

步驟 1 ： (2S)-2-( 三級丁氧基羰胺基 )-3-(2,4- 二氯苯基 ) 丙酸乙酯之製備

HPLC purity: 96.6%; MS (ESI ⁺ ) m/z 565 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.15 (t, 3H), 2.88 (dd, 1H), 3.01 (dd, 1H), 3.08 (dd, 1H), 3.16 (dd, 1H), 3.37 (t, 4H), 3.76 (t, 4H), 4.10 (q, 3H), 4.57 (q, 1H), 7.19 (d, 2H), 7.29 (t, 1H), 7.34 (q, 4H), 9.19 (d, 1H). Example compound 4 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of ethyl 3-(2,4- dichlorophenyl ) propionate hydrochloride

Step 1 : Preparation of ethyl (2S)-2-( tertiary butoxycarbonylamino )-3-(2,4- dichlorophenyl ) propionate

Prepared according to the procedure described above for ethyl (2S)-2-(tertiary-butoxycarbonylamino)-3-(4-chlorophenyl)propanoate to afford 342 mg (63%) of the title compound . MS (ESI ⁺ ) m/z 262 [M+H-Boc] ⁺ . Step 2 : Preparation of ethyl (2S)-2- amino- 3-(2,4- dichlorophenyl ) propionate hydrochloride

Prepared according to the method described above for ethyl (2S)-2-amino-3-(4-chlorophenyl)propionate hydrochloride to give 280 mg (quantitative) of the title compound. MS (ESI ⁺ ) m/z 262 [M+H] ⁺ . Step 3 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of ethyl] acyl ] amino ]-3-(2,4- dichlorophenyl ) propionate

Add HATU (33 mg, 0.09 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (30 mg, 0.07 mmol) (synthesis described in Example 1), ethyl (2S)-2-amino-3-(2,4-dichlorophenyl)propanoate-hydrochloride (25 mg, 0.08 mmol) and Winig's base (37 μL, 0.22 mmol) in DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (20% to 100% EtOAc/petroleum ether). Fractions were concentrated in vacuo to afford the title compound (40 mg, 84% yield) as a white solid. HPLC purity: 98.4%; MS (ESI ⁺ ) m/z 561 [M+H-BOC] ⁺ . Step 4 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of ethyl (2,4- dichlorophenyl ) propionate hydrochloride

烷（45 μL，0.18 mmol）。攪拌溶液1小時，在真空下濃縮且藉由製備型HPLC（10%至80%乙腈/0.1% TFA水溶液）純化殘餘物，以得到呈白色固體狀之標題化合物（12 mg，31%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (17 μL, 0.18 mmol) at room temperature -(tertiary butoxycarbonylamino)propionyl]amino]-3-(2,4-dichlorophenyl)propanoic acid ethyl ester (40 mg, 0.06 mmol) in DCM (2 mL) solution overnight. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃ solution (10 mL). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkane (45 μL, 0.18 mmol). The solution was stirred for 1 h, concentrated in vacuo and the residue was purified by preparative HPLC (10% to 80% acetonitrile/0.1% TFA in water) to give the title compound (12 mg, 31% yield) as a white solid .

步驟 1 ： (2S)-2-( 三級丁氧基羰胺基 )-3-(4- 甲氧基苯基 ) 丙酸乙酯之製備

HPLC purity: 97.9%; MS (ESI ⁺ ) m/z 599 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.13 (t, 3H), 2.89 (dd, 1H), 3.09 (dd, 1H), 3.17 (dd, 1H), 3.25 (dd, 1H), 3.37 (t, 4H), 3.76 (t, 4H), 4.10 (q, 3H), 4.65 (q, 1H), 7.19 (d, 2H), 7.29 (t, 1H), 7.40 (dd, 1H), 7.45 (d, 1H), 7.63 (d, 1H), 9.30 (d, 1H). Example Compound 5 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of ethyl 3-(4 -methoxyphenyl ) propionate hydrochloride

Step 1 : Preparation of ethyl (2S)-2-( tertiary butoxycarbonylamino )-3-(4 -methoxyphenyl ) propionate

Prepared according to the procedure described above for ethyl (2S)-2-(tertiary-butoxycarbonylamino)-3-(4-chlorophenyl)propanoate to afford 351 mg (64%) of the title compound . MS (ESI ⁺ ) m/z 224 [M+H-BOC] ⁺ . Step 2 : Preparation of ethyl (2S)-2- amino- 3-(4 -methoxyphenyl ) propionate hydrochloride

Prepared according to the method described above for ethyl (2S)-2-amino-3-(4-chlorophenyl)propanoate-hydrochloride to afford 265 mg (94%) of the title compound. MS (ESI ⁺ ) m/z 224 [M+H] ⁺ . Step 3 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of ethyl] acyl ] amino ]-3-(4 -methoxyphenyl ) propionate

Add HATU (33 mg, 0.09 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (30 mg, 0.07 mmol) (synthesis described in Example 1), ethyl (2S)-2-amino-3-(4-methoxyphenyl)propanoate hydrochloride (22 mg, 0.08 mmol) and Winig's base (37 μL, 0.22 mmol) in DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (20% to 100% EtOAc/petroleum ether). Fractions were concentrated in vacuo to afford the title compound (28 mg, 63% yield) as a white solid. HPLC purity: 97.9%; MS (ESI+) m/z 523 [M+H-BOC]+. Step 4 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of ethyl (4 -methoxyphenyl ) propionate hydrochloride

烷（34 μL，0.14 mmol）。攪拌溶液1小時且在真空下濃縮，以得到呈白色固體狀之標題化合物（26 mg，97%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (12 μL, 0.14 mmol) at room temperature -(tertiary butoxycarbonylamino)propionyl]amino]-3-(4-methoxyphenyl)propanoic acid ethyl ester (28 mg, 0.05 mmol) in DCM (2 mL) overnight. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃ solution (10 mL). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkane (34 μL, 0.14 mmol). The solution was stirred for 1 hour and concentrated under vacuum to give the title compound (26 mg, 97% yield) as a white solid.

步驟 1 ： (2S)-2-[[(2S)-3-[3,5- 雙 (2- 羥乙基氫硫基 ) 苯基 ]-2-( 三級丁氧基羰胺基 ) 丙醯基 ] 胺基 ]-3-(2- 噻吩基 ) 丙酸乙酯

HPLC purity 97.0%; MS (ESI+) m/z 559 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (t, 3H), 2.93 (m, 4H), 3.12 (m, 1H), 3.38 (m, 3H), 3.72 (s, 3H), 3.76 (t, 4H), 4.03 (br s, 1H), 4.10 (q, 2H), 4.52 (q, 1H), 6.86 (d, 2H), 7.17 (m, 4H), 7.29 (m ,1H), 8.97 (m, 1H). Example compound 6 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of ethyl 3-(2- thienyl ) propionate hydrochloride

Step 1 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Acyl ] amino ]-3-(2- thienyl ) propionic acid ethyl ester

Add HATU (40 mg, 0.11 mmol) to (2S)-3-[3,5-bis(2-hydroxyethylmercapto)-phenyl]-2-(tertiary butoxycarbonylamino) Propionic acid (37 mg, 0.09 mmol) (synthesis described in Example 1), [(1S)-2-ethoxy-2-oxo-1-(2-thienylmethyl)ethyl]chloride Ammonium chloride (obtained from a commercial source, 25 mg, 0.11 mmol) and Winig's base (45 μL, 0.26 mmol) in a mixture in DMF (0.5 mL). The mixture was stirred at room temperature for 30 minutes, after which time the solvent was evaporated. The crude residue was flash chromatographed on silica gel (20% to 100% EtOAc/petroleum ether). Fractions were concentrated in vacuo to afford the title compound (48 mg, 92% yield) as a white solid. HPLC purity: 95+ %; MS (ESI ⁺ ) m/z 499 [M+H-BOC] ⁺ . Step 2 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of -(2- thienyl ) ethyl propionate hydrochloride

烷（60 μL，0.24 mmol）。攪拌溶液1小時，在真空下濃縮且藉由製備型HPLC（10%至70%乙腈/0.1% TFA水溶液）純化殘餘物，以得到呈白色固體狀之標題化合物（8.5 mg，19%產率）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (22 μL, 0.24 mmol) at room temperature - A solution of ethyl (tertiary butoxycarbonylamino)propionyl]amino]-3-(2-thienyl)propanoate (48 mg, 0.08 mmol) in DCM (2 mL) overnight. The reaction mixture was diluted with DCM and washed with saturated NaHCO ₃ solution (10 mL). Brine was added to separate the phases. The organic phase was dried over _MgSO4 and filtered. Add bis containing 4 M HCl to the filtrate

alkane (60 μL, 0.24 mmol). The solution was stirred for 1 h, concentrated in vacuo and the residue was purified by preparative HPLC (10% to 70% acetonitrile/0.1% TFA in water) to give the title compound (8.5 mg, 19% yield) as a white solid .

步驟 1 ： (2S)-2-[[(2S)-3-[3,5- 雙 (2- 羥乙基氫硫基 ) 苯基 ]-2-( 三級丁氧基羰胺基 ) 丙醯基 ] 胺基 ]-3- 苯基 - 丙酸乙酯之製備

HPLC purity: 98+%; MS (ESI ⁺ ) m/z 535 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ ppm 1.18 (t, 3H), 2.90 (dd, 1H) , 3.15 (dd, 1H), 3.27 (m, 2H), 3.36 (t, 4H), 3.76 (t, 4H), 4.13 (q, 2H), 4.13 (m, 1H), 4.59 (m, 1H), 6.98 (m, 2H), 7.18 (d, 2H), 7.29 (t, 1H), 7.39 (m, 1H), 9.16 (d, 1H). Example Compound 7 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]- Preparation of 3- phenyl - propionic acid ethyl ester hydrochloride

Step 1 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of Acyl ] Amino ]-3 -Phenyl - Propionic Ethyl Ester

To (2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2-(tertiary butoxycarbonylamino)propionic acid (0.0350 g, 0.0838 mmol) ( The synthesis is described in Example 1), (2S)-2-Amino-3-phenyl-propionic acid ethyl ester hydrochloride (obtained from a commercial source, 0.0212 g, 0.0922 mmol), 1-hydroxybenzotriazole (0.0113 g, 0.0838 mmol) and TEA (0.0351 mL, 0.251 mmol) was added EDC HCl (0.0193 g, 0.101 mmol). The reaction was stirred overnight to give an overall conversion of starting material to product. The reaction was dissolved in toluene and washed twice with 1 M HCl and finally brine, dried over MgSO ₄ , filtered and the solvent was evaporated to give (2S)-2-[[(2S)-3-[3,5 -Bis(2-hydroxyethylmercapto)phenyl]-2-(tertiary butoxycarbonylamino)propionyl]amino]-3-phenyl-propionic acid ethyl ester (0.0430 g, yield rate: 86%). MS (ESI ⁺ ) m/z 529 [M+H-BOC] ⁺ . Step 2 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 - Preparation of phenyl - propionic acid ethyl ester - hydrochloride

烷（4.00 M，0.00725 mL，2當量）來製備HCl鹽。將HCl鹽凍乾，以得到(2S)-2-[[(2S)-2-胺基-3-[3,5-雙(2-氯乙基氫硫基)苯基]丙醯基]胺基]-3-苯基-丙酸乙酯-鹽酸鹽（0.00600 g，產率15%）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (0.0199 mL, 0.218 mmol) at room temperature - A solution of ethyl (tertiary butoxycarbonylamino)propionyl]amino]-3-phenyl-propionate (0.0430 g, 0.0725 mmol) in DCM (2 mL) overnight. The solvent was concentrated under vacuum and the residue was redissolved in a mixture of DMF (1.5 mL) and water (0.5 mL) and eluted by 30% to 60% acetonitrile containing 0.1% TFA (3 injections). Purified by preparative HPLC. During purification, pure fractions were taken and immediately frozen on an ice bath; when all material was collected, the solvent was removed under lyophilization to give pure TFA salt (7.5 mg, 19.5% yield). By dissolving the material in a mixture of acetonitrile (0.6 mL) and water (1.4 mL) and then adding 4 M HCl in bis

Alkanes (4.00 M, 0.00725 mL, 2 equiv) to prepare the HCl salt. The HCl salt was lyophilized to give (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl] Amino]-3-phenyl-propionic acid ethyl ester-hydrochloride (0.00600 g, 15% yield).

步驟 1 ： (2S)-2-( 三級丁氧基羰胺基 )-3-(2,4- 二氯苯基丙酸異丙酯之製備

MS (ESI ⁺ ) m/z 529 [M+H] ⁺ ; ¹ H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 7.5 Hz, 1H), 8.25 - 8.01 (m, 3H), 7.35 - 7.22 (m, 6H), 7.19 (d, J = 1.7 Hz, 2H), 4.57 (td, J = 8.1, 6.0 Hz, 1H), 4.10 (q, J = 7.0 Hz, 3H), 3.76 (t, J = 7.2 Hz, 4H), 3.37 (t, J = 7.2 Hz, 4H), 3.20 - 2.96 (m, 3H), 2.88 (dd, J = 14.1, 8.7 Hz, 1H), 1.13 (t, J = 7.1 Hz, 3H). Example Compound 8 - (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylsulfuryl ) phenyl ] propionyl ] amino ]- Preparation of isopropyl 3-(2,4- dichlorophenyl ) propionate hydrochloride

Step 1 : Preparation of (2S)-2-( tertiary butoxycarbonylamino )-3-(2,4- dichlorophenylpropionic acid isopropyl ester

Add (2S)-2-(tertiary butoxycarbonylamino)-3-(2,4-dichlorophenyl)propionic acid (0.300 g, 0.898 mmol) to TEA (0.250 mL, 1.80 mmol) To a solution in dichloromethane (6 mL) was added propan-2-ol (0.138 mL, 1.80 mmol), followed by EDC HCl (0.189 g, 0.987 mmol) and DMAP (0.0110 g, 0.0898 mmol). The mixture was stirred for 24 hours and then concentrated to about 20 mL on a rotary evaporator. Toluene (80 mL) was added and the organic phase was washed with 1 M HCl, NaHCO ₃ (aq), brine, dried over MgSO ₄ , filtered and the solvent was evaporated to give (2S)-2-(tertiary butoxycarbonylamine phenyl)-3-(2,4-dichlorophenyl)propanoic acid isopropyl ester (0.273 g, 0.726 mmol, 81% yield). MS (ESI ⁺ ) m/z 276 [M+H-BOC] ⁺ . Step 2 : Preparation of isopropyl (2S)-2- amino- 3-(2,4- dichlorophenyl ) propionate

To (2S)-2-(tertiary butoxycarbonylamino)-3-(2,4-dichlorophenyl)propanoic acid isopropyl ester (0.273 g, 0.726 mmol) in dichloromethane (2 mL) To the solution in was added TFA (2.00 mL). The reaction was stirred at room temperature for 1 hour after which time the solvent was evaporated. The residue was partitioned into a mixture of ethyl acetate and saturated _NaHCO3 (aq). The organic phase was washed with brine, dried _over MgSO, filtered and the solvent was evaporated to give isopropyl (2S)-2-amino-3-(2,4-dichlorophenyl)propanoate as an oil ( 0.150 g, yield 75%). The material was used in the next step without further purification. MS (ESI ⁺ ) m/z 276 [M+H] ⁺ . Step 3 : (2S)-2-[[(2S)-3-[3,5 -bis (2- hydroxyethylmercapto ) phenyl ]-2-( tertiary butoxycarbonylamino ) propane Preparation of Acyl ] Amino ]-3-(2,4- Dichlorophenyl ) propionate Isopropyl Ester

To (2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2-(tertiary butoxycarbonylamino)propionic acid (0.106 g, 0.253 mmol) (synthetic described in Example 1), (2S)-2-amino-3-(2,4-dichlorophenyl)propanoic acid isopropyl ester (70.0 mg, 0.253 mmol), 1-hydroxybenzotriazole ( 0.0343 g, 0.253 mmol) and TEA (0.106 mL, 0.760 mmol) was added EDC HCl (0.0583 g, 0.304 mmol). The reaction was stirred overnight to obtain total conversion of starting material to product. The reaction was dissolved in toluene and washed twice with 1 M HCl and finally brine, dried over MgSO ₄ , filtered and the solvent was evaporated to give (2S)-2-[[(2S)-3-[3,5 -Bis(2-hydroxyethylmercapto)phenyl]-2-(tertiary butoxycarbonylamino)propionyl]amino]-3-(2,4-dichlorophenyl)propanoic acid Isopropyl ester (0.0680 g, 40% yield). MS (ESI ⁺ ) m/z 611 [M+H-BOC] ⁺ . Step 4 : (2S)-2-[[(2S)-2- Amino- 3-[3,5 -bis (2 -chloroethylmercapto ) phenyl ] propionyl ] amino ]-3 Preparation of -(2,4- dichlorophenyl ) propionate isopropyl hydrochloride

烷（4.00 M，0.010 mL，2當量）來製備HCl鹽。將HCl鹽凍乾，以得到(2S)-2-[[(2S)-2-胺基-3-[3,5-雙(2-氯乙基氫硫基)苯基]丙醯基]胺基]-3-(2,4-二氯苯基)丙酸異丙酯-鹽酸鹽（0.0103 g，產率16%）。 Treat (2S)-2-[[(2S)-3-[3,5-bis(2-hydroxyethylmercapto)phenyl]-2 with _POCl3 (0.0276 mL, 0.302 mmol) at room temperature -(tertiary butoxycarbonylamino)propionyl]amino]-3-(2,4-dichlorophenyl)propanoic acid isopropyl ester (0.0680 g, 0.101 mmol) in DCM (3 mL) solution overnight. The solvent was concentrated under vacuum and the residue was redissolved in a mixture of DMF (1.5 mL) and water (0.5 mL) and eluted by 40% to 65% acetonitrile containing 0.1% TFA (3 injections). Purified by preparative HPLC. During purification, pure fractions were taken and immediately frozen on an ice bath; when all was collected, the solvent was lyophilized to give the TFA salt. By dissolving the material in a mixture of acetonitrile (0.6 mL) and water (1.4 mL) and then adding 4 M HCl in bis

Alkanes (4.00 M, 0.010 mL, 2 equiv) to prepare the HCl salt. The HCl salt was lyophilized to give (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl] Amino]-3-(2,4-dichlorophenyl)propanoic acid isopropyl ester-hydrochloride (0.0103 g, 16% yield).

MS (ESI ⁺ ) m/z 611 [M+H] ⁺ ; 1H NMR (400 MHz, DMSO-d6) δ 9.34 (d, J = 7.3 Hz, 1H), 8.13 (s, 2H), 7.63 (d, J = 2.1 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 8.3, 2.2 Hz, 1H), 7.29 (t, J = 1.7 Hz, 1H), 7.24 - 7.19 ( m, 2H), 4.88 (hept, J = 6.2 Hz, 1H), 4.59 (q, 1H), 4.09 (s, 1H), 3.77 (t, J = 7.2 Hz, 4H), 3.37 (t, J = 7.2 Hz, 5H), 3.21 (td, J = 14.0, 5.5 Hz, 2H), 3.08 (dd, J = 14.0, 8.5 Hz, 1H), 2.89 (dd, J = 14.2, 8.7 Hz, 1H), 1.18 (d , J = 6.2 Hz, 3H), 1.07 (d, J = 6.2 Hz, 3H). Cell Analysis Example 1 : Cytotoxicity Protocol in Test Tube - Fluorometric Microculture Cytotoxicity Assay ( FMCA ) Analysis Protocol

Cells: The following cell lines were tested: RPMI-8226/GFP (multiple myeloma) and CCRF-CEM/RFP (leukemia) obtained from AntiCancer were supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 U/100 µg/ml) and L-glutamine 2 mM (both from Sigma St Louis, MO, USA) in RPMI-1640 (Sigma # R0883). ● The HepG2 (human liver cancer) cell line obtained from ATCC was supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 U/100 µg/ml) and L-glutamine 2 mM (both from Sigma St Louis, MO, USA) in Eagle's Minimum Essential Medium (ATCC). HepG2 cells were subcultured twice a week with a maximum of 20 subcultures. Cells were seeded in Nunc 384-well assay plates at a density of 1000 cells/well. ● MCF-7 (breast cancer, adenocarcinoma) obtained from ATCC was supplemented with 10% fetal calf serum, penicillin/streptomycin (100 U/100 µg/ml), L-glutamine 2 mM and 1 mM Sodium pyruvate (all from Sigma St Louis, MO, USA) was cultured in Eagle's minimal essential medium (Sigma #M5650).

Procedure: The Fluorescence Microculture Cytotoxicity Assay (FMCA) is based on the measurement of the fluorescence of luciferin by hydrolysis of luciferin diacetate (FDA) by cells with intact plasma membranes and is described in detail in Lindhagen E, Nygren P and Larsson R., Nat Protoc. 2008;3(8):1364-1369. doi:10.1038/nprot.2008.114.

For each cell line, cells were seeded in Nunc 384-well plates at the densities shown in Table 1 using a pipetting robot Biomek 4000 (Beckman Coulter, Fullerton CA, USA) and cultured overnight. Table 1 cell line Density (cells/well) RPMI-8226/GFP 5000 CCRF-CEM/RFP 650 HepG2 1000 MCF-7 2500

A 10 mM stock solution of Example Compound 1 in DMSO was prepared. Compounds were added to cells by acoustic dispensing (Echo 550 from Labcyte Corporation, CA, USA) directly from 384-well source culture plates (Labcyte) containing 10 mM stock solutions or dilutions thereof.

Compounds were distributed in a dose-response format in duplicate from eight two-fold dilutions at top concentrations of 32 or 64 µM to generate _IC50 values. Bortezomib (2 µM) was used as a positive control and dose-response testing of doxorubicin (maximum concentration 2 µM) was repeated to track assay performance over time.

After incubating with the compounds in the incubation chamber for 72 hours (at 37°C, 95% humidity, 5% CO ₂ ), the 384-well discs were centrifuged (200 g, 60 seconds) and washed with a Biotech ELX washer (Biotek, Winooski, VT, USA) the medium was removed and 70 µl of phosphate-buffered saline (PBS, Sigma) was added to the wells. Repeat this procedure once. Subsequently, the plates were centrifuged (200 g, 30 s) and the PBS was removed by an ELX washer, after which 50 µL/well assay buffer ( HEPES buffered saline with 0.5 mM MgCl ₂ and 0.5 mM CaCl ₂ , pH 7.4), and finally 1 µL of luciferin diacetate ( 0.5 mg/mL in DMSO). Plates were then incubated in a Cytomat (37°C, 95% humidity, 5% _CO2 , Thermo Fischer Scientific) before analysis in a Fluostar Omega (BMG Technologies, Germany) with wavelengths set at ex 485/em 530 nm 50 minutes.

Cytotoxicity was assessed after 72 hours, where cell viability was presented as a viability index (SI, %) and fluorescence in test wells was defined as the percentage of the control culture minus the blank value. Criteria for a successful molecule included a signal-to-noise ratio of >10 for the control culture, a CV of <30%, and a SI of the positive control (bortezomib) <5%. Half maximal inhibitory concentrations ( _IC50 ) were determined from logarithmic concentration-response curves in Graph Pad Prism (GraphPad software Inc., CA, USA) using nonlinear regression analysis. result

The _IC50 values for Example Compound 1 in each cell line are shown in Table 2 below: Table 2 cell line Concentration of Example Compound 1 ( mM ) _IC50 ( μM ) CCRF-CEM/RFP 10 0.12 RPMI-8226/GFP 10 0.83 HepG2 10 1.71 MCF-7 10 1.12

As can be seen from the _IC50 values in Table 2, Example Compound 1 is a highly active cytotoxic agent with high activity in both blood and solid cancer cell lines.

The _IC50 values of example compounds 1-8 and meflufen in the HepG2 and 8226S cell lines are shown in Table 3 below: Table 3 compound HepG2 _IC50 ( μM ) 8226S _IC50 ( μM ) meflufen 0.9291 0.6792 Example compound 1 1.425 0.275 Example compound 2 1.791 0.3772 Example compound 3 1.899 0.4901 Example compound 4 1.533 0.5185 Example compound 5 1.547 0.5199 Example compound 6 1.43 0.2511 Example compound 7 1.216 0.276 Example compound 8 1.813 0.6365 Example 2 : Efficacy Study of Meflufen-HCL and Example Compound 1 against Osteosarcoma Induced by 143B or MNNG/HOS Cell Lines

abbreviation: CAM Chorioallantoic membrane p p-value ℃ Celsius PBS Phosphate Buffered Saline DMSO Dimethyridine Sol. the solution SD standard deviation FBS fetal bovine serum SEM mean standard error Neg Ctrl negative control E. gestational age Pos Ctrl positive control mg mg Cpd compound kg Kilogram PFA Paraformaldehyde (paraformaldehyde) ml ml no Number of eggs/group µl microliter RQ Relative Amount of Cancer Metastasis mol mole [C] concentration µM micromole Vol. volume nM Nemol Tt. deal with Materials and methods

The study timeline is illustrated in Figure 1.

Materials: Fertilized White Leghorn eggs were incubated at 37.5°C for 9 days at 50% relative humidity. On day E9, the chorioallantoic membrane (CAM) was lowered by drilling a small hole through the eggshell to the air sac, and a 1 cm ² window was cut in the eggshell above the CAM. At least twenty-five eggs (depending on survival rate during the first 9 days of development) were used for each condition. Data could be collected in fewer than 21 eggs per group (minimum 15 eggs per group) due to some early egg death occurring just after transplantation or some poor tumor grafts.

Tumor induction: MNNG/HOS cell line (Homo sapiens osteosarcoma cell line, obtained from ATCC (MNNG/HOS Cl #5 [R-1059-D] (ATCC® CRL-1547™))) was cultured in supplemented with 10% FBS and 1% penicillin/streptomycin in DMEM medium. On day E9, cells were trypsinized, washed with complete medium and suspended in graft medium. An inoculum of ¹ x 106 cells was added to the CAM of each egg. Eggs were subsequently randomly divided into 8 groups (see Table 4a below).

The 143B cell line (Homo sapiens osteosarcoma cell line; obtained from ATCC (143B (ATCC® CRL-8303™))) was cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. On day E9, cells were trypsinized, washed with complete medium and suspended in graft medium. An inoculum of 0.5 x ¹⁰⁶ cells was added to the CAM of each egg. Eggs were subsequently randomly divided into 8 groups (see Table 4b below).

Treatment: On day 10 (E10), tumors became detectable. They were then treated 4 times (at E11, E13, E15 and E17) under different conditions: vehicle (PBS with 1% DMSO), Ref compound (doxorubicin) at 50 µM, three different doses (12.5 µM , 50 µM and 200 µM) of meflufen-HCL and three different doses (12.5 µM, 50 µM, 200 µM) of example compound 1. Details of the treatments are presented in Tables 4a and 4b below. For all conditions, the injection volume was always 100 µl/egg, and the molecules were dropped onto the tumor.

。 Melfluphen-HCL is the hydrochloride salt of melphalan flufenamide and is a peptidase-enhanced cytotoxic agent (PEnC). Mefluphen-HCL has the following structure:

.

。 Doxorubicin is a known chemotherapeutic agent for the treatment of osteosarcoma and has the following structure:

.

The storage conditions used and the final formulation are as follows: • Store Mefluphen-HCL powder at -20°C; • Store Example Compound 1 at -20°C • Prepare an intermediate stock solution in 20 mM pure DMSO and store at - at 80°C; • Prepare final solutions in PBS (for concentrations, see Tables 4a and 4b below) just before use and store at room temperature (to ensure no heat shock to eggs or cells) for less than one hour ( time required to process all eggs). The concentrations of meflufen and Example Compound 1 stated in Tables 4a and 4b are calculated assuming complete solubility in PBS. Table 4a : Concentrations of final solutions for the MNNG/HOS cell line panel group description molecular name injection concentration Concentration in eggs (µmol/L ) Group 1 Negative Control (Vehicle) DMSO PBS with 1% DMSO 0.002% Group 2 Ref compound Adriamycin 50 µM 0,11 group 3 Mefluphen[1] Mefluphen-HCL 12.5 µM 0,03 Group 4 Mefluphen[2] Mefluphen-HCL 50 µM 0,11 Group 5 Mefluphen[3] Mefluphen-HCL 200 µM 0,44 Group 6 Example Compound 1 [1] Example compound 1 12.5 µM 0,03 Group 7 Example Compound 1 [2] Example compound 1 50 µM 0,11 Group 8 Example compound 1 [3] Example compound 1 200 µM 0,44 Table 4b : Concentrations of final solutions for the 143B cell line panel group description molecular name injection concentration Concentration in egg (µmol/L ) Group 1 Negative Control (Vehicle) DMSO PBS with 1% DMSO 0.002% Group 2 Ref compound Adriamycin 50 µM 0,11 group 3 Mefluphen[1] Mefluphen-HCL 12.5 µM 0,03 Group 4 Mefluphen[2] Mefluphen-HCL 50 µM 0,11 Group 5 Mefluphen[3] Mefluphen-HCL 200 µM 0,44 Group 6 Example Compound 1 [1] Example compound 1 12.5 µM 0,03 Group 7 Example Compound 1 [2] Example compound 1 50 µM 0,11 Group 8 Example compound 1 [3] Example compound 1 200 µM 0,44

Tumor growth analysis: On day 18 (E18), for each egg, the upper part of the CAM (with the tumor) was removed, washed in PBS and then directly transferred in PFA (fixed for 48 hours). Tumors were then washed (PBS), carefully dissected from normal CAM tissue, and weighed. These data were subjected to one-way analysis of variance using post hoc tests.

Cancer metastasis and invasion: At the same time, a 1 cm ² portion of the lower CAM of each egg was collected to evaluate the number of cancer metastasis cells. Genome DNA was extracted from the CAM and analyzed by qPCR with specific primers for the Alu sequence.

The calculation of the Cq of each sample, the average Cq and the relative amount of cancer metastasis in each group was directly processed by Bio-Rad® CFX Maestro® software.

According to the Real-Time PCR Data Markup Language (RDML) data standard (http://www.rdml.org), Cq is defined as the cycle where the curvature of the amplification curve is maximum (partial PCR cycle). Cq is obtained during the exponential phase, where the qPCR curve is linear. It is a fundamental result of qPCR: a lower Cq value means a higher initial copy number of the target gene. When the PCR efficiency is 100%, a difference of 1 cycle between two reactions means that there are 2-fold more copies (genes) in the reaction with the lower Cq value than in the reaction with the higher Cq value.

Statistical analysis was performed directly on data from Bio-Rad CFX Maestro® software using GraphPad Prism® software.

Toxicity : Egg viability was checked at each treatment or at least every two days. Abnormalities were also checked at the end of the study during sample collection. On day E18, the number of stillbirths was fully counted and combined with the final observation of macroscopic abnormalities to assess toxicity.

Significance of statistical analyzes: For all statistical analyses, statistical differences between groups are visible on the graphs in the presence of significant stars with the following: No star: no statistical difference (p-value >0.05); One star (*): 0.05≥p value＞0.01; ● Two stars (**): 0.01≥p value＞0.001; ● Three stars (***): 0.001≥p value.

Results: ( a ) MNNG/HOS cell line tumor growth: Table 5 and Figure 2 present the average tumor weights of different MNNG/HOS cell line experimental groups at the end of the study. Figure 5 shows representative pictures of tumors (ex ovo). Table 5 : Mean, SD , SEM and p -value of tumor weight ( mg ) of each MNNG/HOS cell line experimental group tumor analysis p -value comparison no Weight ( mg ) SD SEM % reg. negative control Adriamycin Mefluphen [1] Mefluphen [2] Mefluphen [3] Example Compound 1 [1] Example Compound 1 [2] negative control 15 222.92 76.54 19.76 N/A / / / / / / / Adriamycin 15 158.12 92.98 24.01 29.07 0.1805 / / / / / / Mefluphen [1] 15 186.80 65.08 16.80 16.21 0.6629 0.9881 / / / / / Mefluphen [2] 13 166.37 64.70 17.95 25.37 0.3858 >0.9999 0.9995 / / / / Mefluphen [3] 13 130.17 66.50 18.44 41.61 0.0135 0.9623 0.5414 0.8837 / / / Example Compound 1 [1] 14 191.04 84.51 22.59 14.30 0.9178 0.9039 0.9998 0.9828 0.3119 / / Example Compound 1 [2] 14 79.86 42.34 11.32 64.17 <0.0001 0.057 0.0035 0.0325 0.5619 0.0012 / Example compound 1 [3] 13 49.38 26.78 7.43 77.85 <0.0001 0.0017 <0.0001 0.0009 0.068 <0.0001 0.9453

Cancer metastatic invasion: Table 6 and Figure 3 present the data analysis of cancer metastatic invasion measured by qPCR for human Alu sequences in the lower CAM. Table 6 : Relative amount of cancer metastasis in lower CAM of each group compared to negative control ( arbitrary value of cancer metastasis is 1 ) Cancer Metastasis Analysis P value comparison no RQ SEM Average Cq fade % negative control Adriamycin Mefluphen [1] Mefluphen [2] Mefluphen [3] Example Compound 1 [1] Example Compound 1 [2] negative control twenty four* 1 0.09687 21.76 N/A / / / / / / / Adriamycin 8 1.24336 0.07151 21.45 -24.34 0.9652 / / / / / / Mefluphen [1] 8 0.80215 0.08953 22.08 19.79 0.9707 0.732 / / / / / Mefluphen [2] 8 0.79659 0.23315 22.09 20.34 0.9707 0.732 0.9912 / / / / Mefluphen [3] 8 1.14329 0.25305 21.57 -14.33 0.9912 0.9912 0.9236 0.9236 / / / Example Compound 1 [1] 8 0.96157 0.17534 21.82 3.84 0.9912 0.9652 0.9912 0.9912 0.9912 / / Example Compound 1 [2] 8 0.50895 0.15341 22.74 49.11 0.258 0.068 0.9652 0.9652 0.2012 0.7307 / Example compound 1 [3] 8 0.35512 0.10731 23.26 64.49 0.0367 0.0102 0.732 0.732 0.0367 0.2554 0.9912 *Negative control group has been applied to all plates (3 plates) of qPCR used for the study. For analysis, 8 samples in this group were considered n=24 by Bio-Rad CFX Maestro® software.

Toxicity: Table 7 and Figure 4 present the percentage of dead and surviving embryos at the end of the study (at E18) for all experimental groups. Table 7. Number of dead and surviving embryos in different experimental groups total survive die Survival% die% negative control 17 15 2 88 12 Adriamycin 18 15 3 83 17 Mefluphen[1] 17 15 2 88 12 Mefluphen[2] 17 13 4 76 twenty four Mefluphen[3] 17 13 4 76 twenty four Example Compound 1 [1] 17 14 3 82 18 Example Compound 1 [2] 18 14 4 78 twenty two Example compound 1 [3] 16 13 3 81 19

( b ) 143B cell line tumor growth: Table 8 and Figure 6 present the mean values of tumor weights of different 143B cell line experimental groups at the end of the study. Figure 9 shows representative pictures of tumors (ex ovo). Table 8 : Mean, SD , SEM and p -value of tumor weight ( mg ) of each 143B cell line experimental group tumor analysis P value comparison no Weight ( mg ) SD SEM % reg. negative control Adriamycin Mefluphen [1] Mefluphen [2] Mefluphen [3] Example Compound 1 [1] Example Compound 1 [2] negative control 15 191.36 57.19 14.77 N/A / / / / / / / Adriamycin 18 58.58 47.42 11.18 69.39 <0.0001 / / / / / / Mefluphen [1] 14 230.45 77.78 20.79 -20.43 0.6498 <0.0001 / / / / / Mefluphen [2] 11 227.48 77.11 23.25 -18.88 0.7951 <0.0001 >0.9999 / / / / Mefluphen [3] 17 120.56 61.18 14.84 36.99 0.0243 0.0536 <0.0001 0.0003 / / / Example Compound 1 [1] 15 203.55 62.91 16.24 -6.37 0.9993 <0.0001 0.9277 0.9726 0.0037 / / Example Compound 1 [2] 15 87.69 69.17 17.86 54.17 0.0002 0.8596 <0.0001 <0.0001 0.7786 <0.0001 / Example compound 1 [3] 19 39.93 25.05 5.75 79.13 <0.0001 0.9806 <0.0001 <0.0001 0.0024 <0.0001 0.2972

Cancer metastatic invasion: Table 9 and Figure 7 present the data analysis of cancer metastatic invasion measured by qPCR for human Alu sequences in the lower CAM. Table 9 : Relative amount of cancer metastasis in lower CAM of each group compared to negative control ( arbitrary value of cancer metastasis is 1 ) Cancer Metastasis Analysis P value comparison no RQ SEM Average Cq fade % negative control Adriamycin Mefluphen [1] Mefluphen [2] Mefluphen [3] Example Compound 1 [1] Example Compound 1 [2] negative control twenty four* 1 0.14962 22.09 N/A / / / / / / / Adriamycin 8 0.21249 0.08204 24.33 78.75 0.0267 / / / / / / Mefluphen [1] 8 0.92163 0.20298 22.21 7.84 0.9989 0.2539 / / / / / Mefluphen [2] 8 1.51694 0.35853 21.49 -51.69 0.3827 0.0006 0.4625 / / / / Mefluphen [3] 8 0.39651 0.10914 23.43 60.35 0.2085 0.9987 0.6103 0.0049 / / / Example Compound 1 [1] 8 0.45186 0.02963 23.24 54.81 0.3078 0.9967 0.7328 0.0090 0.9989 / / Example Compound 1 [2] 8 0.36005 0.07844 23.57 63.99 0.1483 0.9989 0.5333 0.0033 0.9989 0.9989 / Example compound 1 [3] 8 0.23409 0.05371 24.19 76.59 0.0340 0.9989 0.2863 0.0007 0.9989 0.9974 0.9989 *Negative control group has been applied to all plates (3 plates) of qPCR used for the study. For analysis, 8 samples in this group were considered n=24 by Bio-Rad CFX Maestro® software.

Toxicity : Table 10 and Figure 8 present the percentage of dead and surviving embryos at the end of the study (at E18) for all experimental groups. Table 10. Number of dead and surviving embryos in different experimental groups total survive die Survival% die% negative control 20 15 5 75 25 Adriamycin 19 18 1 95 5 Mefluphen[1] 17 14 3 82 18 Mefluphen[2] 17 11 6 65 35 Mefluphen[3] 20 17 3 85 15 Example Compound 1 [1] 19 15 4 79 twenty one Example Compound 1 [2] 18 15 3 83 17 Example compound 1 [3] 19 19 0 100 0

Discussion of results: The purpose of this example was to characterize and compare the efficacy of mefluphene-HCL and example compound 1 on human osteosarcoma induced by MNNG/HOS and 143B osteosarcoma cell lines in the model described above. Both compounds were evaluated by their effects on tumor growth and metastasis as well as by their embryotoxicity.

Tumor Growth: For the MNNG/HOS cell line, in terms of tumor growth inhibition, Example Compound 1 exhibited significant tumor inhibition (vs. For the negative control, p<0.001 for both doses), meflufen inhibited tumor growth only at the higher dose (200 µM) (p<0.05).

For the 143B cell line, in terms of tumor growth inhibition, Example Compound 1 exhibited a significant tumor inhibitory effect at a medium dose (50 µM) and a higher dose (200 µM) compared to the negative control group (compared to the negative control group, p < 0.001 for both doses), but meflufen inhibited tumor growth only at the higher dose (200 µM) (p < 0.05).

Between meflufen and example compound 1, example compound 1 demonstrated superior potency over mefluphen at both effective doses in both cell lines: ● MNNG/HOS cell line: p<0.001 for Example Compound 1 50 µM versus Mefluphen 50 µM and Example Compound 1 200 µM versus Mefluphen 200 µM. • 143B cell line: p<0.001 for Compound 1 50 µM vs. Mefluphen 50 µM and p<0.01 for Compound 1 200 µM vs. Mefluphen 200 µM.

Inhibition of cancer metastasis: For the MNNG/HOS cell line, there was no significant difference between the meflufen-treated group and the negative control in terms of cancer metastasis inhibition, while treatment with a higher dose of Example Compound 1 (200 µM) There was a decrease in the group.

For the 143B cell line, in terms of cancer metastasis inhibition, only 200 µM of Example Compound 1 compound exhibited a significant cancer metastasis regression effect compared to the negative control group.

Toxicity: With regard to toxicity, no abnormal death was observed in any of the experimental groups compared with the negative control group.

none

[ Fig. 1 ] is a schematic research timeline of Experimental Example 2. [ FIG. 2 ] shows the mean ± SEM of tumor weights measured for each MNNG/HOS cell line experimental group. [ FIG. 3 ] Shows the relative amount of cancer metastasis in low CAM of each MNNG/HOS cell line experimental group compared with the negative control group (arbitrary value of cancer metastasis is 1). [ FIG. 4 ] shows the number of dead and surviving embryos in each MNNG/HOS cell line experimental group. [Fig. 5] Representative pictures of tumors (out of ovo) in the negative control group and the MNNG/HOS cell line experimental group in the 200 µM example compound 1 group. [ FIG. 6 ] shows the mean ± SEM of tumor weights measured for each 143B cell line experimental group. [ Fig. 7 ] Shows the relative amount of cancer metastasis in low CAM of each 143B cell line experimental group compared with the negative control group (arbitrary value of cancer metastasis is 1). [ FIG. 8 ] shows the number of dead and surviving embryos of each 143B cell line experimental group. [Fig. 9] is a representative picture of the tumor (out of egg) of the negative control group and the 143B cell line experimental group in the 200 µM example compound 1 group.

Claims (14)

A compound of formula (I), or a pharmaceutically acceptable salt, ester, amide or carbamate, including salts of such esters, amide or carbamate,

(I) Among them, R ₁ is H or -C _1-6 alkyl; R ₂ is -C _1-6 alkyl; -CH ₂ -indolyl; -CH ₂ -phenyl; or contains 1, 2, -CH ₂ -5 membered heteroaryl with 3 or 4 nitrogen or sulfur atoms; wherein -C _1-6 alkyl is optionally substituted by -OH; and phenyl is optionally 1, 2 or 3 independently selected from Substituent substitution of the group consisting of: halogen; -NH ₂ ; -OC _1-6 alkyl; -N(C _1-6 alkyl) ₂ optionally substituted with 1, 2 or 3 halogens; and -NO ₂ ; and R ₃ is H or -C(O) ^Ra ; wherein ^Ra is C _1-6 alkyl or -CH ₂ -phenyl, which is optionally substituted by 1, 2 or 3 halogens. A compound as claimed in claim 1, wherein R ² is -CH ₂ -5 membered heteroaryl containing a nitrogen or sulfur atom; or substituted by 1 or 2 chlorine or fluorine atoms or an -OC _1-4 alkyl as required -CH ₂ -phenyl; preferably, R ² is -CH ₂ -thiophene or -CH ₂ -phenyl, which is optionally substituted by 1 or 2 chlorine or fluorine atoms or a methoxy group. The compound as claimed in item 1 or 2, wherein R ¹ is H, ethyl or isopropyl. A compound as claimed in any one of claims 1 to 3, wherein R ³ is -C(O)R ^a , wherein R ^a is -C _1-4 alkyl or -CH ₂ -phenyl, which is optionally modified by 1 or substituted by 2 chlorine or fluorine atoms. The compound according to any one of claims 1 to 3, wherein R ³ is H. The compound according to any one of claims 1 to 5, wherein the compound is (2S)-2-[[(2S)-2-amino-3-[3,5-bis(2-chloroethyl hydrogen sulfide Base) phenyl] propionyl] amino] -3-(4-fluorophenyl) ethyl propionate:

or a pharmaceutically acceptable salt, amide or carbamate thereof, including salts of such amide or carbamate; for example, wherein the compound is (2S)-2-[[(2S)- 2-Amino-3-[3,5-bis(2-chloroethylmercapto)phenyl]propionyl]amino]-3-(4-fluorophenyl)propionic acid ethyl ester hydrochloride . A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 6 and a pharmaceutically acceptable carrier, and optionally additional therapeutic agents such as protease inhibitors (protease inhibitor; PI), immune modulators drug (IMiD), alkylating agent, or doxorubicin. A compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use as a medicine. A compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for treating or preventing cancer. A compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for the treatment or prevention of multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma. The compound for use according to any one of claims 8 to 10, wherein the compound is administered together with one or more additional therapeutic agents (e.g. simultaneously, sequentially or separately), the one or more additional therapeutic agents For example protease inhibitors (PIs), immunomodulatory drugs (IMiDs), alkylating agents or doxorubicin, and in particular doxorubicin. A method for treating a patient, comprising administering a pharmaceutically effective amount of the compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7. A method for treating or preventing cancer, comprising administering an effective amount of the compound as claimed in any one of items 1 to 6 or the pharmaceutical composition as claimed in item 7, for example, wherein the cancer is multiple myeloma, osteosarcoma Cancer, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma. A use of a compound according to claims 1 to 6 for the manufacture of a medicament for the treatment or prevention of cancer, for example wherein the cancer is multiple myeloma, osteosarcoma, breast cancer, lung cancer, ovarian cancer, leukemia and lymphoma either of them.

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