CN101357893B - Ethylene diamine metalloid protease inhibitor and use thereof - Google Patents

Abstract

The invention relates to ethylenediamine metalloproteinase inhibitors and applications thereof. The ethylenediamine metalloprotease inhibitor is a peptidoid compound with a structure of general formula (I), and various optical isomers, pharmaceutically acceptable salts, solvates and prodrugs thereof. The present invention also relates to a pharmaceutical composition containing the structural peptoid compound of formula (I) and its pharmaceutical application. The invention provides a class of potent peptide-like metalloprotease inhibitors, which can effectively treat diseases with abnormal expression of metalloproteinase activity.

Description

Ethylenediamine Metalloproteinase Inhibitors and Their Applications

technical field

The present invention relates to a preparation method, an activity test, a composition containing the peptidoid compound and the application of the peptidoid compound with an ethylenediamine skeleton to selectively inhibit the action of metalloprotease.

Background technique

1. Matrix metalloproteinases (MMPs)

MMPs are a class of endopeptidases dependent on calcium ions and zinc ions, which play an important role in the degradation of extracellular matrix, tissue reconstruction and the regulation of various soluble factors between cells. The activity of MMPs is strictly controlled by the expression level of genes and the secretion level of zymogen activators/inhibitors. In many pathological processes, such as arthritis, tissue ulceration, growth and metastasis of malignant tumors, MMPs also play an important role.

At present, 28 members of the MMPs family have been found in mammals (Szabo, K.A. et al. Clinical and Applied Immunology Reviews. 2004, 4, 295), which are divided into different groups according to their structure, specific substrate and different cell locations. Subtypes, including 1 collagenase (MMP-1, -8, -13, -18), 2 gelatinases (MMP-2, -9), 3 matrix degrading enzymes (MMP-3, -10, -11 ), 6 membrane-type-matrix metalloproteinases (MMP-14, -15, -16, -17, -24, -25), and other unclassified ones such as stromelysin (MMP-7 and -26) and Macrophage metalloelastin (MMP-12) and so on. Among them, gelatinase (MMP-2 and -9) has been proved to be closely related to the malignant phenotype of aggressive tumors and the poor prognosis of cancer patients. They are involved in the invasion of tumor cells to the basement membrane and matrix, and the penetration of blood vessel walls. , and metastasis of tumor cells. Studies in recent years have shown that MMPs are also related to the growth of primary tumors and secondary tumors, as well as angiogenesis, and even promote the process of tumor proliferation. Therefore, therapeutic strategies targeting these enzymes have been rapidly developed, and MMPs inhibitors have become a hot spot in the research of cancer therapeutic drugs.

The examples that can be treated with MMPs inhibitors include: rheumatoid arthritis (Mullins, DE; et al.Biochim.Biophys.Acta.1983,695,117); osteoarthritis (Henderson, D.; et al.Drugs of the Future, 1990, 15, 495); cancer; tumor cell metastasis (Deryugina, EI; et al. Cancer Metastasis Rev.2006, 25, 9); multiple sclerosis (Rosenberg , GA et al. Ann Neurol. 2001, 50, 431 ); and various tissue ulcers or tissue ulcerative conditions. Corneal ulcers may be caused by alkali burns, or by infection with Pseudomonas aeruginosa, Acanthamoeba, herpes simplex, and vaccinia viruses.

Other examples of conditions characterized by hyperactivity of metalloproteases include periodontal disease, epidermolysis bullosa, fever, inflammation and scleritis (Cf. Decicco, et al WO95/29892).

2. Aminopeptidase N

Aminopeptidase N (APN, CD13) is a type II membrane-bound glycoprotein with a molecular weight of about 150Kd, belonging to the Gluzincins subfamily of the zinc ion-dependent metalloproteinase and aminopeptidase Ml family, and exists in the form of homodimers In the cell membrane, it participates in the degradation of the N-terminal amino acid of the substrate. APN is widely expressed in kidney and intestinal brush border cells, bone marrow progenitor cell membranes, monocyte membranes, central nervous system synaptic cell membranes, fibroblasts, endothelial cell membranes, and placental cell membranes, participating in the physiological regulation of the body. Studies have proved that APN plays an important role in tumorigenesis, immune function regulation and virus infection.

1) APN is highly expressed on the surface of tumor cells. This enzyme can degrade the main components of the extracellular matrix (ECM), destroy the natural barrier of the body, and participate in the formation of tumor new blood vessels as a new signal transduction molecule, thereby promoting the invasion and metastasis of tumor cells (Sato Y, Biol.Pharm .Bull., 2004, 27(6): 772-776; Saiki, I.; et al. Int. J. Cancer., 1993, 54, 137; Menrad A., Speicher D., Wacker J., et al. . Cancer Res., 1993, 53(6): 1450-1455). 2) APN is abundantly expressed on the surface of granulocytes and lymphocytes, and is also involved in T lymphocyte-dependent inflammatory responses; it can also be expressed on the surface of antigen-presenting cells to degrade immune active substances (such as interleukin-8); it is involved in antigen processing and The major histocompatibility complex type II (MHC-II) adhesion epitope on the cell surface depends on the recognition of antigens by T cells, which reduces the ability of T cells to recognize their antigens, and at the same time weakens macrophages and NK The ability of cells to recognize and kill tumor cells reduces the body's immunity. 3) APN, as the receptor on the surface of human coronavirus HCoV-229E and transmissible gastroenteritis virus (TGEV), plays an important role in upper respiratory tract infection (such as: SARS) and acute enteritis, and its function is related to the activity of enzyme Correlation (Delmas, B., et al. Nature, 1992, 357, 417; Yeager, C.L.; et al. Nature, 1992, 357, 420). 4) APN is involved in T lymphocyte-dependent inflammatory response and the process of HIV virus particles entering host cells. Studies have shown that the activity of APN in HIV-infected patients is much higher than that in healthy volunteers. When HIV-1 invades host cells, the highly expressed APN degrades the chemokine fMLP that can desensitize the HIV-1 co-receptor CCR5, thereby reducing the natural immune function of the cells and sensitizing CCR5 to promote the entry of the virus into the host cell. (ShenW, Li B, et al.Blood, 2000, 96(8), 2887; Shipp MA, et al.Blood, 1993, 82(4), 1052) 5) APN participates in the endogenous analgesic substance endorphin And the degradation of enkephalin, which causes the excessive release of substance P, leading to pain. 6) APN degrades angiotensin and participates in the regulation of blood pressure (Mitsui, T.; et al. Biol. Pharm. Bull., 2004, 27, 768.).

For more than ten years, the research and development of MMPs inhibitors has been extremely rapid, but so far no one has been listed. Most MMPs inhibitors are peptides or peptide analogs, which are sensitive to enzyme degradation. In addition, because MMPs exhibit a broad spectrum of action characteristics, in addition to ECM, they also cleave other substrates such as enzymes that can promote the expression of growth factors. Or activate integrin by inhibiting proteolysis to indirectly promote tumor growth, which is why most MMPs inhibitors are shot down in the clinical stage. In addition, most of the inhibitors against APN are natural products. The only drug on the market, Ubenimex, has a dipeptide-like structure containing β-amino acids. It is currently used as an immune enhancer for the treatment of leukemia, but because it is derived from olive It is isolated from the culture medium of Streptomyces olivorecticuli, and the source is limited.

Studies have shown that MMPs and APN are closely related to the invasion of malignant tumors and the occurrence and development of metastasis (Sounni N.E., Janssen M., Foidart J.M., et al.. Matrix Biol., 2003, 22(1), 55-61). Both use collagen, the main component of the extracellular matrix, as a substrate, and destroy the body's natural barrier to tumor cells, leading to the infiltration and metastasis of tumor cells. However, the difference between the two lies in their different substrate degradation sites: the former is endopeptidase, which can degrade the substrate from the middle of the peptide; the latter is ectopeptidase, which is characterized by the substrate The terminal amino group begins to degrade the substrate. The present invention combines MMPs and APN for research. This patent involves the selectivity of the designed peptoid compound to the two, and it is expected to develop specific and selective inhibitors by optimizing the structure of the peptoid compound. The peptoid compounds designed in the present invention are screened for APN activity and found that several drug molecules have weaker activity than the currently only marketed ubenimex.

Contents of the invention

Aiming at the deficiencies of the prior art, the invention provides an ethylenediamine metalloproteinase inhibitor and a preparation method thereof.

The design of the compound containing the general formula (I) shown below in the present invention adopts the strategy of peptoid and isostere design. Peptoids and isosteres have been widely used in the design and development of antiviral and antitumor drugs. Their structures are composed of natural or unnatural amino acids similar to peptide structures, but the overall conformation is different from natural polypeptide substances. On the one hand, peptoids have the intrinsic activity of substrates, which can inhibit the activity of enzymes by recognizing the active center of enzymes, and at the same time improve the selectivity and efficiency of the target site; in addition, there are structural differences between peptoids and natural peptides. The difference is not easy to be degraded by peptidase, the biological stability and availability are improved, and the action time of the compound is long.

Specifically, the present invention uses optically pure amino acids as raw materials, through methyl esterification under acidic conditions, the resulting compound is then subjected to Boc or Cbz protected amino protection, reduction, mesylation, nucleophilic substitution and other steps to synthesize key intermediates , and then condensed with different biologically active acid chlorides (such as gallic acid, caffeic acid, phenylacetic acid, ferulic acid, non-steroidal anti-inflammatory drug organic acids) and amino acid derivatives, and then deprotected to obtain different series of dipeptides Or tripeptide-like, the purpose is to enhance the affinity and metabolic stability of the compound with the enzyme or receptor.

The present invention designs and synthesizes a class of metalloprotease inhibitors with a new structure core. In vitro tests show that it has no cytotoxic activity but exhibits significant in vitro enzyme inhibitory activity, and is expected to become a class of non-cytotoxic anticancer drug candidates.

Technical scheme of the present invention is as follows:

Peptoid compounds having the general formula I, as well as optical isomers, diastereomers and racemate mixtures thereof, pharmaceutically acceptable salts, solvates or prodrugs thereof.

in,

_R is aryl, heteroaryl, arylC1-6alkyl, heteroarylC1-9alkyl, arylC2-6alkenyl, heteroarylC2-6alkenyl, arylC2-6alkyne radical, heteroaryl C2-6 alkynyl, C1-6 alkyl, optionally substituted by one or more of the following groups: hydroxyl, halogen, nitro, cyano, halogen C1-8 alkyl, C1-8 alkane Oxygen, C1-6 alkylcarbonyl, C1-8 alkoxycarbonyl, aryl C1-8 alkoxycarbonyl;

_R2 is hydrogen, aryl, heteroaryl, arylC1-6alkyl, heteroarylC1-9alkyl, arylC2-6alkenyl, heteroarylC2-6alkenyl, arylC2- 6 alkynyl, heteroaryl C2-6 alkynyl, optionally substituted by one or more of the following groups: halogen, nitro, cyano, halogen C1-8 alkyl, C1-8 alkoxy, C1-6 Alkylcarbonyl, C1-8 alkoxycarbonyl;

R ₃ is aryl C1-6 alkyl, heteroaryl C1-9 alkyl, aryl C2-6 alkenyl, heteroaryl C2-6 alkenyl, aryl C2-6 alkynyl, heteroaryl C2- 6 alkynyl, optionally substituted by one or more of the following groups: halogen, nitro, cyano, halogen C1-8 alkyl, C1-8 alkoxy, C1-6 alkylcarbonyl, C1-8 alkoxy carbonyl.

*The carbons shown have the R configuration.

Preferably, R ₁ is aryl C1-6 alkyl, aryl C2-6 alkenyl, heteroaryl C1-9 alkyl; R ₂ is hydrogen; R ₃ is aryl C1-6 alkyl, aryl C2 -6 alkenyl, heteroaryl C1-9 alkyl, most preferably _R3 is benzyl.

The above-mentioned peptoid compound (I) specifically includes the following compounds:

(2S)-2,6-diamino-N-[(2R)-2-amino-3-phenylpropyl]hexanamide,

(2S)-2-Amino-4-methylthio-N-[(2R)-2-amino-3-phenylpropyl]butanamide,

4-Amino-N-[(2R)-2-amino-3-phenylpropyl]butanamide,

6-Amino-N-[(2R)-2-amino-3-phenylpropyl]hexanamide,

(2S)-2-amino-3-hydroxy-N-[(2R)-2-amino-3-phenylpropyl]propionamide,

N-[(2R)-2-amino-3-phenylpropyl]-3-phenylpropanamide,

(2S)-2,5-Diamino-N-[(2R)-2-amino-3-phenylpropyl]pentanamide,

2-Amino-N-[(2R)-2-amino-3-phenylpropyl]acetamide,

(2S)-2-amino-4-methyl-N-[(2R)-2-amino-3-phenylpropyl]-3-hydroxyl-L-prolinamide,

(2S)-2-amino-N-[(2R)-2-amino-3-phenylpropyl]-3-phenylpropanamide,

(2S)-2-Amino-4-methyl-N-[(2R)-2-amino-3-phenylpropyl]pentanamide,

(2S)-2-Amino-3-methyl-N-[(2R)-2-amino-3-phenylpropyl]pentanamide,

N-[(2R)-2-amino-3-phenylpropyl]-L-pyrrolidineamide,

(3S)-3-amino-N-[(2R)-2-amino-3-phenylpropyl]propionamide,

(2S)-2-amino-3-methyl-N-[(2R)-2-amino-3-phenylpropyl]propionamide,

N-[(2R)-2-amino-3-phenylpropyl]-L-histidineamide,

N-[(2R)-2-amino-3-phenylpropyl]-4-methyl-benzenesulfonamide,

(2S)-2-amino-3-mercapto-N-[(2R)-2-amino-3-phenylpropyl]propionamide,

(2E)-N-[(2R)-2-Amino-3-phenylpropyl]-3-phenylacrylamide,

N-[(2R)-2-amino-3-phenylpropyl]-3,4,5-trimethoxy-benzamide,

N-[(2R)-2-amino-3-phenylpropyl]-2-phenylacetamide,

(2E)-N-[(2R)-2-amino-3-phenylpropyl]-3-(3,4-dimethoxy)phenylacrylamide,

(2E)-N-[(2R)-2-Amino-3-phenylpropyl]-3-(3-methoxy-4-hydroxy)phenylacrylamide,

(2E)-N-[(2R)-2-amino-3-phenylpropyl]-3-(3,4-dihydroxy)phenylacrylamide,

N-[(2R)-2-amino-3-phenylpropyl]-4-nitro-benzamide,

N-[(2R)-2-amino-3-phenylpropyl]-3,5-dinitro-benzamide,

Methyl 4-{N-[(2R)-2-amino-3-phenylpropyl]carboxamido}benzoate,

The peptoid compound intermediate for preparing the above-mentioned general formula (I) is: (2R)-2-tert-butoxycarbonyl amido-3-phenyl-propylamine, or 2-tert-butoxycarbonyl amido-N-[(2R )-2-tert-butoxycarbonylamino-3-phenylpropyl]acetamide.

The use of these peptoid compounds in the prevention or treatment of mammalian diseases related to the abnormal expression of metalloproteinases, including matrix metalloproteinases or aminopeptidase N, activity. The mammalian diseases related to the abnormal expression of metalloproteinase activity include: inflammation, cancer, multiple sclerosis, various tissue ulcers or tissue ulcerative diseases, periodontal disease, epidermolysis bullosa, leukemia and the like. Therefore, the present invention also relates to pharmaceutical compositions containing the compound of structure (I).

A pharmaceutical composition comprising (1) the peptoid compound according to any one of claims 1-3, and (2) one or more pharmaceutically acceptable carriers or excipients.

In addition, the present invention also includes a pharmaceutical composition suitable for oral administration to mammals, comprising (1) a peptide compound according to any one of claims 1-3, and (2) a pharmaceutically acceptable carrier, optionally comprising (3) One or more pharmaceutically acceptable excipients.

In addition, the present invention also includes a pharmaceutical composition suitable for parenteral administration to mammals, comprising (1) a peptide compound according to any one of claims 1-3, and (2) a pharmaceutically acceptable carrier, optionally comprising ( 3) One or more pharmaceutically acceptable excipients.

Detailed description of the invention

Definitions and terms used

The terms and definitions used in this document have the following meanings:

"Heteroalkyl" means a saturated or unsaturated chain comprising carbon atoms and at least one heteroatom, none of which are adjacent. The heteroalkyl group contains 2-15 atoms (carbon atoms), preferably 2-10 atoms. Heteroalkyl groups can be straight or branched, substituted or unsubstituted.

"Aryl" means an aromatic carbocyclic group. Preferred aromatic rings contain 6-10 carbon atoms.

"Halo", or "halogen" includes fluorine, chlorine, bromine or iodine, preferably fluorine and chlorine.

"Cycloalkyl" is a substituted or unsubstituted, saturated or unsaturated cyclic group containing carbon atoms and/or one or more heteroatoms. The ring may be a monocyclic or fused, bridged or spiro ring system. Monocyclic rings generally contain 3-9 atoms, preferably 4-7 atoms, and polycyclic rings contain 7-17 atoms, preferably 7-13 atoms.

"Heteroaryl" is an aromatic heterocyclic ring, which may be a monocyclic or bicyclic group. Preferred heteroaryl groups include, for example, thienyl, furyl, pyrrolyl, pyridyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, and tetrazolyl, benzothiazolyl, benzofuryl , indolyl, etc.

"Pharmaceutically acceptable salt" refers to the curative and non-toxic salt form of the compound of formula (I). It can form an anionic salt from any acidic group (such as carboxyl group), or form a cationic salt from any basic group (such as amino group). Many such salts are known in the art. A cationic salt formed on any acidic group such as carboxyl, or an anionic salt formed on any basic group such as amino. Such salts are known in the art from a number of formulas, such as cationic salts including alkali metal (such as sodium and potassium) and alkaline earth metal (such as magnesium and calcium) salts and organic salts (such as ammonium salts). Anionic salts are also conveniently obtained by treating the basic form of (I) with the corresponding acid, such acids include mineral acids such as sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxy Propionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-propanetriic acid, methanesulfonic acid, Ethylsulfonic acid, benzenemethanesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, etc. Such salts are well known to the skilled artisan and the skilled artisan can prepare any salt given by the knowledge in the art. Furthermore, the skilled artisan may prefer one salt to another based on solubility, stability, ease of formulation, and the like. Determination and optimization of these salts are within the experience of the skilled artisan.

A "solvate" is a complex formed by combining a solute (eg, a metalloproteinase inhibitor) and a solvent (eg, water). See J. Honig et al., The Van Nostrand Chemist's Dictionary, p. 650 (1953). The pharmaceutically acceptable solvent that the present invention adopts includes those solvents (such as water, ethanol, acetic acid, N,N-dimethylformamide, dimethyl sulfoxide and the technology in this field that do not interfere with the biological activity of metalloproteinase inhibitors. known or readily identifiable solvents).

As used herein, "optical isomers", "enantiomers", "diastereomers", "racemates" and the like define all possible stereoisomeric forms of the compounds of the present invention or their physiological derivatives . Unless otherwise indicated, the chemical designations of the compounds of the invention include mixtures of all possible stereochemical forms comprising all diastereomers and enantiomers of the basic structural molecule, as well as substantially pure individual isomers of the compounds of the invention form, ie containing less than 10%, preferably less than 5%, especially less than 2%, most preferably less than 1% of other isomers. The various stereoisomeric forms of the peptoids of the invention are expressly included within the scope of the invention.

The peptide compound of formula (I) can also exist in other protected forms or derivative forms, which are obvious to those skilled in the art, and should be included in the scope of the present invention.

The substituents mentioned above may themselves be substituted by one or more substituents. Such substituents include those listed in C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology (1979). Preferred substituents include, for example, alkyl, alkenyl, alkoxy, hydroxy, oxy, nitro, amino, aminoalkyl (such as aminomethyl, etc.), cyano, halo, carboxyl, carbonylalkoxy ( Such as carbonylethoxy, etc.), thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl (such as piperidinyl, morpholinyl, pyrrolyl, etc.), imino, hydroxyalkyl, aryl Oxy, arylalkyl, and combinations thereof.

synthesis

The target compound was synthesized via the following route.

In short, using optical amino acids as raw materials, here we choose D-phenylalanine as an example, successively undergo methylation, Boc protection, reduction, mesylation, and _SN2 substitution reaction with sodium azide. Magnesium is reduced in a protic solvent, and then condensed by a polypeptide to remove the protecting group to obtain the target compound.

synthetic route:

Reagents: (a) methanol, hydrochloric acid; (b) di-tert-butyl carbonate, dichloromethane, triethylamine, 0°C; (c) lithium aluminum tetrahydride, ether; (d) methanesulfonyl chloride, tetrahydrofuran, 0°C ; (e) sodium azide, dimethyl sulfoxide; (f) magnesium, methanol; (g) di-tert-butyl carbonate, dichloromethane, triethylamine, 0 ° C; (h) isobutyl chloroformate, N - Methylmorpholine, tetrahydrofuran, -15°C; (i) ethyl acetate saturated with hydrogen chloride; (j) oxalyl chloride, dichloromethane, 0°C; (k) triethylamine, tetrahydrofuran, 0°C.

Those skilled in the art can change the above-mentioned steps to improve the yield. They can determine the synthetic route according to the basic knowledge in the art, such as selecting reactants, solvent and temperature, and can avoid side reactions by using various conventional protecting groups. occur thereby increasing the yield. These conventional conservation methods can be found, for example, in T. Greene, Protecting Groups in Organic Synthesis.

Obviously, the above route is a stereoselective synthesis, and its optically active peptoid compound can also be prepared through the above route. For example, the raw material D-phenylalanine is replaced by its optical isomer (L configuration). Various other isomers of ethylenediamine derivatives can be easily obtained by those skilled in the art, and can be purified by conventional separation means, such as chiral salt or chiral chromatography column.

The assay of MMPs inhibitory activity is described in Vijaykumar, M.B. et al., Matrix Biol. 2000, 19, 26. Succinylated gelatin has been proven to be hydrolyzed by gelatinases (including MMP-2, -9), and the concentration of free amino groups produced by hydrolysis of peptide bonds is positively correlated with the enzyme activity. Succinic anhydride protects the free amino groups in gelatin, and the exposed primary ammonia after hydrolysis reacts with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to develop color, and the amino group content is determined by detecting the absorbance at 450nm, thereby determining gelatinase activity.

The test for APN inhibitory activity is described in Lejczak, B et al. Biochemistry, 1989, 28, 3549. The substrate L-leucyl-p-nitroaniline is degraded by APN to produce p-nitroaniline which absorbs at 405nm, and the concentration of p-nitroaniline is positively correlated with the enzyme activity. The content of p-nitroaniline is determined by detecting the absorbance at 405nm, so as to determine the activity of aminopeptidase, which indirectly reflects the degree of inhibition of the enzyme activity by the inhibitor.

The cell activity of the compound was tested using the MTT assay method, HL-60 cell suspension was seeded in a 96-well plate), medium containing different concentrations of the compound was added to each well, after incubation, stained with MTT, after continued incubation, the enzyme The absorbance OD value of each well was measured at 570nm on the standard instrument, and the cell growth inhibition rate was calculated to determine the activity of the compound.

The in vitro enzyme inhibition test of the peptoid compound of general formula (I) proves that this peptoid compound is a kind of ethylenediamine peptide metalloprotease inhibitor

The ethylenediamine derivative of the present invention is spatially matched with the active site of the metalloprotease, so it shows higher inhibitory activity in vitro. Moreover, it can be metabolized into active fragments in vivo, such as caffeic acid and cinnamic acid derivatives, which still have anti-tumor activity, so they also show high anti-tumor activity in vivo

Formulation, pharmaceutical composition, dosage and administration

The ethylenediamine derivatives of the present invention may exist in free form or in salt form. Pharmaceutically acceptable salts of many compound types and methods for their preparation are known to those skilled in the art. Pharmaceutically acceptable salts include conventional non-toxic salts, including quaternary ammonium salts of bases of such compounds with inorganic or organic acids.

The compounds of the present invention may form hydrates or solvates. Methods are known to those skilled in the art to form hydrates of the compounds when lyophilized with water or to form solvates when they are concentrated in solution with a suitable organic solvent.

The present invention comprises a pharmaceutical composition comprising a therapeutic amount of the compound of the present invention, and one or more pharmaceutically acceptable carriers and/or excipients. Carriers include, for example, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, discussed in more detail below. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition may be a liquid, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Formulations can be designed to mix, granulate and compress or dissolve ingredients, depending on the formulation required. In another approach, the composition can be formulated as nanoparticles.

The pharmaceutical carrier used can be, for example, solid or liquid.

Typical solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; Can be encapsulation material. In powders, the carrier is a finely divided solid, which is in admixture with the finely divided active ingredient. In tablets the active ingredient is mixed with the carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Powders and tablets preferably contain up to 99% active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone Ketones, low melting waxes and ion exchange resins.

Typical liquid carriers include syrup, peanut oil, olive oil, water, and the like. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, tinctures and sealed compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators agent. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, such as cellulose derivatives, preferably carboxymethylcellulose sodium salt solution), alcohols (including monohydric and polyhydric alcohols) , such as glycols) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration the carrier can also be an oil or fat such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant. Sterile solutions or suspensions. Liquid pharmaceutical compositions can be administered, for example, for intravenous, intramuscular, intraperitoneal or subcutaneous injection. When injecting, it can be pushed in once or gradually injected into the meridian infusion for 30 minutes. The compound can also be administered orally in the form of liquid or solid compositions.

Carriers or excipients may include time delay materials known in the art such as glyceryl monostearate or glyceryl distearate, and may also include waxes, ethylcellulose, hydroxypropylmethylcellulose, isobutylene methyl ester, etc. When formulations are used for oral administration, it is recognized that 0.01% Tween 80 in PHOSALPG-50 (phospholipid and 1,2-propanediol concentrate, A. Nattermann & Cie. GmbH) is used for the preparation of acceptable oral formulations of other compounds, which can be adapted to Formulation of various compounds of the invention.

A wide variety of pharmaceutical forms can be used in administering the compounds of the present invention. If a solid carrier is used, the preparation can be in tablet, powder or pellet form placed into a hard capsule or in the form of a troche or lozenge. The amount of solid carrier will vary widely, but preferably will be from about 25 mg to about 1.0 g. If a liquid carrier is used, the preparation can be a syrup, emulsion, soft capsule, sterile injectable solution or suspension in ampoules or vials or non-aqueous liquid suspension.

To obtain stable water-soluble dosage forms, the compound or a pharmaceutically acceptable salt thereof can be dissolved in an aqueous solution of organic or inorganic acid, 0.3M succinic acid or citric acid solution. Alternatively, the acidic derivatives can be dissolved in a suitable basic solution. If a soluble form is not available, the compound can be dissolved in a suitable co-solvent or combination thereof. Examples of such suitable co-solvents include, but are not limited to, ethanol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerol, polyoxyethylene fatty acid esters, Fatty alcohols or glycerol hydroxy fatty acid esters and the like.

Various delivery systems are known and can be used to administer the compound or other formulations including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, and the like. Methods of introduction include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (usually preferred) oral routes . The compounds may be administered by any convenient or other suitable route, such as by infusion or bolus injection, absorption via epithelial or mucosal lines (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or via drug-loaded stents and It can be administered together with other bioactive agents. Administration can be systemic or local. For the treatment or prevention of nasal, bronchial or pulmonary diseases, the preferred route of administration is oral, nasal or bronchial aerosol or nebulizer.

Description of drawings

Figure 1 and Figure 2 are schematic diagrams of the docking results of compound 15n and the active region of APN.

Detailed ways

The present invention will be further described below in conjunction with the examples, but not limited thereto.

Example 1. Synthesis of compounds of the invention.

1) (2R)-Phenylalanine methyl ester hydrochloride (2). In a 250ml three-neck flask, suspend phenylalanine (20g, 0.121mol) in 150ml of anhydrous methanol, pass through dry HCl gas until the solution becomes clear, continue to pass HCl gas until the solution becomes cloudy. The reaction solution was evaporated to dryness to obtain a white solid. The methanol was dissolved and then evaporated to dryness, repeated three times to remove the HCl gas. After dissolving with a small amount of methanol, anhydrous ether was added, and a large amount of white precipitate was precipitated, which was kept overnight in the refrigerator. It was filtered, dried and weighed to obtain 22.7 g of crystalline solid. Yield 86.90%, mp 158-160.5°C.

2) (2R)-2-tert-butoxycarbonyl amido-phenylpropanoic acid methyl ester (3). Phenylalanine methyl ester hydrochloride (10.8g, 0.02mol) was suspended in 200ml of anhydrous dichloromethane, and 16.3ml of triethylamine (0.12mol) and di-tert-butyl carbonate (( Boc) ₂ 0, 9.6g, 0.04mol), stirred at room temperature for 12 hours. TLC monitors until there is no raw material point, stop the reaction, wash with 1M phosphoric acid aqueous solution and saturated sodium bicarbonate aqueous solution successively, remove water with anhydrous sodium sulfate, filter, and rotate the chlorine solution to dryness to obtain a colorless oily product.

3) (2R)-2-tert-butoxycarbonylamido-3-phenyl-propanol (4). Compound 3 (2.8 g, 0.01 mol) was dissolved in anhydrous ether, and LiAlH ₄ (0.76 g, 0.02 mol) was added in batches under ice-bath conditions, and the reaction was maintained at room temperature for 8 hours. Add hydrochloric acid solution (concentrated hydrochloric acid: water 1:1) to the reaction solution to quench the reaction, react for 10 min, filter to remove the sticky residue, and spin the filtrate to dryness to obtain a white solid. Yield 78.68%, mp: 92-93°C.

4) (2R)-Propyl 2-tert-butoxycarbonylamido-3-phenyl-methanesulfonate (5). Compound 4 (2.5g, 0.01mol) was dissolved in anhydrous tetrahydrofuran, cooled to 0°C in an ice bath, triethylamine (2.08ml, 0.015mol) was added, and methanesulfonyl chloride (1.7g , 0.01mol) of tetrahydrofuran solution, dropped in about 0.5h. Removed the ice bath, reacted at room temperature for 5h, poured the reaction solution into cold water, a white solid precipitated, filtered and dried to obtain 2.85g, yield 86.63%. mp: 105-108°C.

5) tert-butyl(2-azidomethyl-1-phenethyl)carbamate (6). The compound (10 g, 0.030 mol) was dissolved in DMF, and the internal temperature was controlled at 55-60° C., and sodium azide (3.96 g, 0.060 mol) was added in batches to react at constant temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was poured into 300ml of cold water, placed in the refrigerator to cool and stand still, a large amount of yellow solid was precipitated, filtered and dried to obtain 6.51g of crude product, which was separated and purified by column chromatography (eluent: petroleum ether-ethyl acetate 10:1), 5.24g of pure product was obtained, and the yield was 63.52%. mp: 112-114°C.

6) (2R)-2-tert-butoxycarbonylamido-3-phenyl-propylamine (7). Compound 6 (4g, 0.015mol) was dissolved in anhydrous methanol, cooled to 0°C in an ice bath, and magnesium strips (1.08g, 0.045mol) were added in batches. After about 0.5h of reaction, the internal temperature of the reaction solution rose, with A large amount of gas was evolved, and the progress of the reaction was monitored by TLC. After the reaction, the reaction solution was evaporated, the residue was diluted with cold water, and its pH value was adjusted to 9-10. Extracted with ether, washed the organic phase with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to remove the solvent to obtain 2.13 g of white solid with a yield of 56.35%. mp: 71-74°C. ESI-MS m/z: 251.1 (M+H); ¹ H-NMR (MeOD) δppm 1.41 (s, 9H), 2.59-2.65 (J ₁ =6, J ₂ =12, dd, 1H), 2.71 -2.81 (m, 3H), 3.79 (m, 1H), 7.18-7.32 (m, 5H).

7) Preparation of tert-butoxycarbonylglycine (9). Add 0.1mol glycine to a 500ml three-neck flask equipped with two constant pressure dropping funnels and stirring paddles on both sides, dissolve it with 110ml sodium hydroxide solution (1N), and cool it to zero in an ice bath. Slowly add 50 ml of a THF solution containing di-tert-butyl carbonate ((Boc) ₂ O, 21.8 g, 0.11 mol). At the same time, use 1N sodium hydroxide solution to control the pH between 8 and 9. The dropwise addition was completed in about 1 hour, and stirred for 1 hour in an ice bath. The ice bath was removed, and stirred at room temperature for 18 hours. During the whole process, the pH was controlled at 8~9 with 1N NaOH. After the reaction was completed, the reaction solution was concentrated to remove THF, the residual solution was extracted with petroleum ether (100ml×3), the aqueous phase was adjusted to pH 2~3 with 1N KHSO ₄ (or KHSO ₄ solid), and then extracted with ethyl acetate ( 150ml×10), the organic phase was dried with anhydrous sodium sulfate for 12 hours. Filter and concentrate. mp: 87-88°C.

8) 2-tert-butoxycarbonylamino-N-[(2R)-2-tert-butoxycarbonylamino-3-phenylpropyl]acetamide (10a). At -15°C, tert-butoxycarbonylglycine (1.54g, 0.088mol) and N-methylmorpholine (1.05ml, 0.096mol) were dissolved in THF (15ml), and isobutyl chloroformate ( 1.23ml, 0.096mol) of THF (10ml) solution, reacted for 0.5h, added dropwise the THF solution of compound 7 (2g, 0.08mol), continued to react for 1h, removed the cold bath, reacted at room temperature for 5h, filtered, evaporated under reduced pressure Solvent, the residue was dissolved in ethyl acetate, and the organic phase was washed successively with 5% NaHCO ₃ aqueous solution, 10% citric acid aqueous solution, and saturated brine, dried over anhydrous sodium sulfate, filtered, and recrystallized with ethyl acetate to obtain 2.45g white solid. Yield 73.84%. mp=103-105°C. ESI-MS m/z: 408.3 (M+H); ¹ H-NMR (DMSO) δppm 1.22 (s, 9H), 1.38 (s, 9H), 2.58-2.61 (m, 1H), 2.67-2.74 (J ₁ =5.4, J ₂ =13.8, dd, 1H), 3.01-3.07 (m, 1H), 3.16-3.19 (m, 1H), 3.50-3.56 (m, 2H), 3.66 -3.68 (m, 1H) 7.17-7.27 (m, 5H).

9) 2-Amino-N-[(2R)-2-amino-3-phenylpropyl]acetamide hydrochloride (11a). Compound 10 (1.0 g, 0.0024 mol) was dissolved in ethyl acetate (15 ml) saturated with hydrochloric acid, and the reaction progress was monitored by TLC. After 5 hours of reaction at room temperature, a white solid was precipitated, filtered and washed to obtain 0.38 g of a white solid, and the yield was 78.35 %. mp = 195.5-197.8°C. ESI-MS m/z: 208.3 (M+H); ¹ H-NMR (D ₂ O) δppm 2.81-2.89 (J ₁ =6, J ₂ =15, dd, 1H), 2.94-3.01 (J ₁ =6, J ₂ =15, dd, 1H), 3.38-3.48 (J ₁ =4.5, J ₂ =15, dd, 1H), 3.49-3.56 (J ₁ =4.5, J ₂ =15, dd, 1H) , 3.62-3.73 (m, 1H), 3.80 (s, 2H), 7.23-7.37 (m, 5H).

Embodiment 2 target compound inhibits gelatinase activity test (In vitro)

For the test principle and detailed test steps, refer to CN1528745A pyrrolidine matrix metalloproteinase inhibitor and its preparation method, and the test results are shown in Table 1.

Embodiment 3 target compound inhibits the activity test of aminopeptidase N (In vitro)

For the test principle and detailed test steps, refer to CN1974554A Cyclic imide peptide metalloprotease inhibitor and its application, and the test results are shown in Table 1.

Table 1. In vitro enzyme inhibition test results

The values in the table are the average values of three experiments, and the values after "±" represent standard deviations.

Embodiment 4 target compound suppresses the active test of cell proliferation (In vitro)

1. [Materials] HL-60 cell line, tetramethylazolazolium blue MTT, 10% fetal bovine serum, 96-well plate

2. [Method]

Cell culture human acute myeloid leukemia cells HL60, introduced by the Shanghai Cell Institute of the Chinese Academy of Sciences. Adopt conventional culture. Cells in logarithmic growth phase were used in the experiments.

Cell growth assay (MTT method) HL-60 cell suspension was adjusted to 1×10 ⁵ /ml, seeded in 96-well plate (50 μl/well), 10 ⁴ cells/well. After 4 hours of plating, add 50ul of medium containing different concentrations of compounds to each well, so that the final concentrations of the compounds in the wells are: 800, 600, 400, 200, 100ug/ml, and set up three replicate wells for each concentration without adding cells. When the wells were read, they were used as blank wells, the wells with cells added and no compound added were used as compound blank wells, and bestatin was used as compound positive control. Incubate at 37°C, 5% CO ₂ for 48h, add 10μl of 0.5% MTT staining solution to each well, continue to incubate for 4h, centrifuge at 2500rpm for 30min, discard the medium in the well, add DMSO, 100ul/well. Measure the absorbance OD value of each well at 570nm on a microplate reader, and the cell growth inhibition rate is calculated according to the following formula:

Table 2 Cell Proliferation Experiment Results

compound _IC50 (mM) compound _IC50 (mM) Ubenimex 1.65 11h 2.29 11n 1.09 11i 3.2 15n 0.63

The structure-activity relationship research of embodiment 5 target compound

The application software Sybyl7.0 was used to dock the target compound with the active region of APN, and Figure 1 shows the docking results of the compound 15n with the best activity in this series.

As shown in Figure 1, the configuration of compound 15n is similar to that of ubenimex (dark structure in the left figure) when it binds to the active region of APN, and 15n can well occupy the hydrophobic area of APN. Sex pockets A, C, C', while the carbonyl and amino groups in the structure can chelate zinc ions in the active region of APN. Figure 2 is a two-dimensional schematic diagram of the docking of 15n and APN simulated by Ligplot. From the figure, we can get that the three amino acids His ²⁹⁷ , Glu ²⁹⁸ , and His ³⁰¹ of the amino acid conservative sequence (HEXXHX ₁₈ E) of 15n and the catalytic center of APN Hydrophobic bonds were formed respectively; in addition, 15n and Tyr ³⁸¹ also formed a strong hydrophobic interaction. These structural features provide a basis for us to conduct structure-activity relationship studies to reasonably expect and design APN inhibitors with better inhibitory activity.

Claims (6)

1. A compound of formula I, and optical isomers, diastereomers and racemic mixtures thereof, and pharmaceutically acceptable salts thereof:

the compound of formula I is one of the following compounds:

2-amino-N- [ (2R) -2-amino-3-phenylpropyl ] acetamide,

(2S) -2-amino-N- [ (2R) -2-amino-3-phenylpropyl ] -3-phenylpropionamide,

(2S) -2-amino-4-methyl-N- [ (2R) -2-amino-3-phenylpropyl ] pentanamide,

(2S) -2-amino-3-methyl-N- [ (2R) -2-amino-3-phenylpropyl ] pentanamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -L-pyrrolidinamide,

(3S) -3-amino-N- [ (2R) -2-amino-3-phenylpropyl ] propanamide,

(2S) -2-amino-3-methyl-N- [ (2R) -2-amino-3-phenylpropyl ] propanamide,

(2S) -2-amino-4-methyl-N- [ (2R) -2-amino-3-phenylpropyl ] -3-hydroxy-L-prolinamide,

(2S) -2, 6-diamino-N- [ (2R) -2-amino-3-phenylpropyl ] hexanamide,

(2S) -2-amino-4-methylsulfanyl-N- [ (2R) -2-amino-3-phenylpropyl ] butanamide,

4-amino-N- [ (2R) -2-amino-3-phenylpropyl ] butanamide,

6-amino-N- [ (2R) -2-amino-3-phenylpropyl ] hexanamide,

(2S) -2-amino-3-hydroxy-N- [ (2R) -2-amino-3-phenylpropyl ] propanamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -3-phenylpropionamide,

(2S) -2, 5-diamino-N- [ (2R) -2-amino-3-phenylpropyl ] pentanamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -L-histidinamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -4-methyl-benzenesulfonamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -4-chloro-butyramide,

(2S) -2-amino-3-mercapto-N- [ (2R) -2-amino-3-phenylpropyl ] propanamide,

(2E) -N- [ (2R) -2-amino-3-phenylpropyl ] -3-phenylpropenamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -3, 4, 5-trimethoxy-benzamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -2-phenylacetamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -4-phenylbutanamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -3, 5-dichlorobenzamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -2- (2-naphthyl) -carboxamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -2- [2- (2, 6-dichlorophenyl) amino ] phenylacetamide,

n- [ (2R) -2-amino-3-phenylpropyl ] benzamide,

(2E) -N- [ (2R) -2-amino-3-phenylpropyl ] -3- (3, 4-dimethoxy) phenylacrylamide,

(2E) -N- [ (2R) -2-amino-3-phenylpropyl ] -3- (3-methoxy-4-hydroxy) phenylpropenamide,

(2E) -N- [ (2R) -2-amino-3-phenylpropyl ] -3- (3, 4-dihydroxy) phenylpropenamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -4-nitro-benzamide,

n- [ (2R) -2-amino-3-phenylpropyl ] -3, 5-dinitro-benzamide,

4- { N- [ (2R) -2-amino-3-phenylpropyl ] carboxamido } benzoic acid methyl ester.

2. The compound of claim 1, wherein: the carbon represented by formula I has the R configuration.

3. Use of a compound of claim 1 or 2 for the preparation of a medicament for the prevention or treatment of a mammalian disease associated with aberrant expression of metalloprotease activity.

4. The pharmaceutical use of a compound according to claim 3, wherein the metalloprotease is a matrix metalloprotease or aminopeptidase N.

5. The pharmaceutical use of a compound according to claim 3, wherein the mammalian disease associated with aberrant expression of metalloprotease activity is: inflammation, cancer, multiple sclerosis, various tissue ulcers or ulcerative conditions, periodontal disease, epidermolysis bullosa and leukemia.

6. A pharmaceutical composition comprising a compound of claim 1 or 2 and one or more pharmaceutically acceptable carriers or excipients.

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