JP6468562B2 - Proteasome inhibitory compounds - Google Patents

Description

  The present invention relates to a novel compound having proteasome inhibitory activity, a proteasome inhibitor containing the compound, a pharmaceutical composition containing the compound as an active ingredient, particularly a pharmaceutical composition for treating a proteasome-related disease such as cancer.

  Proteolysis called ubiquitin-proteasome system is used to break down proteins that are no longer necessary for eukaryotic cells, such as they cannot be folded normally, become inactivated by external stress such as active oxygen, or reach the end of their lives. It is known that a system exists. The ubiquitin-proteasome system is a marker that is formed by modifying a protein that is no longer needed with a protein consisting of 76 amino acids called ubiquitin, and the protein is recognized and decomposed by a huge enzyme complex called a proteasome. .

  Recent research has shown that the ubiquitin-proteasome system not only processes the degradation of unwanted proteins, but also quantitatively regulates cell cycle-related factors, signal transduction factors, transcription factors, etc. It has been clarified that it is also involved in the control of apoptosis, and it has been found that it is a system related to the basis of life activity. And as abnormalities occur in this ubiquitin-proteasome system, it is known that various modulations are caused to cells, and by controlling the function of this ubiquitin-proteasome system, it is thought that abnormalities in ubiquitin-proteasome are involved. The possibility of treating various diseases and malignant cells in which apoptosis is difficult to induce is pointed out, and proteasome inhibitors are attracting attention as therapeutic agents for various diseases.

  The proteasome is a huge enzyme complex that recognizes and degrades all proteins labeled with ubiquitin. This complex is called a 19S complex or 11S complex at both ends of a cylindrical structure called a core particle (CP, 20S proteasome) in which four ring structures composed of two α rings and two β rings are stacked. It has a shape in which the structure is bound, and proteolysis is performed in the cavity portion in the 20S proteasome. Among the molecules constituting the core particle, molecules called β1, β2, and β5 are known to have different catalytic activities such as caspase-like, trypsin-like, and chymotrypsin-like, respectively.

By inhibiting the activity of this proteasome, it is thought that abnormal activation of the proteasome can be brought close to normal, or apoptosis can be induced by accumulating unnecessary proteins in the cell to treat the disease. . As proteasome inhibitors, for example, compounds such as bortezomib or ester compounds thereof (for example, Patent Documents 1 to 5), compounds having a peptide-like structure such as belactocin, and the like are known.
Bortezomib (trade name: Velcade), whose compound name is N-2-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid, was first approved in Japan as a treatment for refractory multiple myeloma Proteasome inhibitor. Although the mechanism of action on cancer cells has not been completely elucidated, it has been found that cancer cells stop abnormal cell division and induce apoptosis by accumulation of unwanted proteins in the cells.

US Pat. No. 6,083,903 US Pat. No. 6,297,217 US Pat. No. 6,617,317 US Pat. No. 6,713,446 US Pat. No. 6,958,319

Kawamura et al., J. Med. Chem. 2013, 56, 3689-3700 Kawamura et al., J. Med. Chem. 2014, 57, 2726-2735

  The biggest problem when using a conventional proteasome inhibitor as a pharmaceutical preparation is that it has a strong side effect. For example, with bortezomib, peripheral neuropathy, gastrointestinal disorders such as vomiting and diarrhea, and myelosuppression are recognized as side effects. This is considered to occur because the proteasome is present in both normal cells and abnormal cells in common, thereby inhibiting the proteasome in normal cells. Therefore, development of a proteasome inhibitor that selectively exerts an effect only in abnormal cells is desired.

  On the other hand, recent biological studies have shown that important abnormal signaling pathways that are affected by proteasome inhibition in blood cancer also exist in solid tumors. It is also known that cancer cells have higher proteasome expression than normal cells and are more sensitive to proteasome inhibitors. Therefore, proteasome inhibitors are expected to be effective therapeutic agents not only in currently approved multiple myeloma but also in other solid tumors. However, as described above, side effects are a problem when proteasome inhibitors are used in the treatment of diseases. Therefore, development of a novel proteasome inhibitor having an inhibitory action and protease selectivity different from those of conventional proteasome inhibitors is required.

  Therefore, the object of the present invention is to solve the problems of the prior art, is highly proteasome-selective, and is more irreversible, so there are few side effects even at high doses, and it is equivalent to or equivalent to conventional proteasome-inhibiting compounds. A novel compound having the above-mentioned proteasome inhibitory activity, a pharmaceutical composition containing the compound, particularly a pharmaceutical composition for treating a proteasome-related disease such as cancer, a method for treating a proteasome-related disease using the compound, etc. It is to provide.

  In order to solve the above problems, the present inventors have sought to find a novel proteasome-inhibiting compound, which improves the currently known proteasome-inhibiting compound, belactocin, and has a proteasome-inhibiting activity higher than that of belactocin (Non-Patent Document 1). However, such a compound has a large number of asymmetry points in its skeleton, and further improvement is necessary in terms of practicality as a pharmaceutical from the viewpoint of complexity of synthesis, membrane permeability, biological stability, and the like. I faced a new challenge. Therefore, by further improving the peptide backbone of such compounds, making it non-peptide while retaining proteasome activity, and substituting the peptide boronate for the β-lactone moiety, sufficient proteasome inhibitory activity and good inhibition selection The present invention has been completed by discovering that it is possible to synthesize and to easily synthesize cancer cells and to synthesize them more easily.

That is, the present invention relates to:
[1] The following formula I
Where
A ¹ and A ² are each independently a 5-membered or 6-membered aromatic ring, and one or more hydrogen atoms present in the aromatic ring are optionally halogen, hydroxyl group, nitro group, amino group , May be substituted with a group selected from a cyano group or a C1-10 alkyl group,
S ¹ and S ² are, independently of each other, C 1-5 alkylene, and any one of carbon atoms not adjacent to X and A ¹ or A ² is arbitrary among the carbon atoms present in the alkylene. May be replaced with an oxygen atom,
X is C or N;
R ¹ is a C1-5 alkyl group,
R ² is a C1-10 hydrocarbon group,
Here, one or two or more carbon atoms present in R ¹ and R ² are replaced with oxygen atoms, nitrogen atoms or sulfur atoms, provided that when two or more atoms are replaced, these atoms are Not adjacent to each other,
One or more hydrogen atoms present in R ¹ and R ² may be optionally substituted with a substituent selected from halogen, hydroxyl group, carboxyl group, nitro group, amino group or cyano group,
Y ¹ and Y ² are each independently a hydrogen atom or a boronyl protecting group, and Y ¹ and Y ² may be combined to form a ring structure;
m and n are independently of each other, 1, 2 or 3.
Or a pharmaceutically acceptable salt thereof.

[2] The structure of the -B (OY ¹ ) OY ² moiety of formula I is represented by the following formula II
In the formula, Y is a C1-10 alkylene group, and among the carbon atoms present in the alkylene, any one of the carbon atoms not adjacent to O is arbitrarily replaced with an oxygen atom, a nitrogen atom or a sulfur atom. One or two or more hydrogen atoms of the alkylene group may be optionally substituted with a group selected from halogen, hydroxyl group, nitro group, amino group or cyano group, and Two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group,
Or formula III
In the formula, ring Y ′ is a C4-10 cycloalkenyl group or arylene group, and one or more ring member carbon atoms constituting Y ′ are optionally replaced with an oxygen atom, a nitrogen atom or a sulfur atom. 1 or 2 or more hydrogen atoms of each ring member atom may optionally be a group selected from halogen, C1-4 alkyl group, hydroxyl group, carboxyl group, nitro group, amino group, or cyano group May be substituted, or two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group.
Or a pharmaceutically acceptable salt thereof.

[3] The structure of the -B (OY ¹ ) OY ² moiety of formula I is
The compound of [1] or [2], or a pharmaceutically acceptable salt thereof.
[4] The compound of [1] to [3] or a pharmaceutically acceptable salt thereof, wherein R ² is an isobutyl group, a cyclohexylmethyl group or a benzyl group.
[5] The compound of [1] to [4] or a pharmaceutically acceptable salt thereof, wherein S ¹ and S ² are both —C—C—C— or —C—O—C—.
[6] The compound of [1] to [5], wherein A ¹ and A ² are both phenyl, or a pharmaceutically acceptable salt thereof.
[7] The compound of [1] to [6], wherein X is C, or a pharmaceutically acceptable salt thereof.

[8] The following formula IV
Where
m and n are independently of each other, 1, 2 or 3.
Or a pharmaceutically acceptable salt thereof.
[9] A proteasome inhibitor comprising an effective amount of the compound of [1] to [8] or a pharmaceutically acceptable salt thereof.
[10] A pharmaceutical composition for treating a proteasome-related disease, comprising at least one compound of [1] to [8] or a pharmaceutically acceptable salt thereof.
[11] The pharmaceutical composition according to [10], wherein the proteasome-related disease is a tumor.

  ADVANTAGE OF THE INVENTION According to this invention, the novel compound which has the proteasome inhibitory activity which is comparable to the conventional compound which has proteasome inhibitory activity, and exhibits suitable protease selectivity and the stability (biostability) in a biological environment is provided. . Therefore, the compound of the present invention can be used as an excellent antitumor agent compared with an antitumor agent containing a conventional proteasome inhibitor compound, such as sufficient antitumor activity and few side effects.

FIG. 1 is a graph showing the results of a reversibility test of the compound of the present invention (bottom) and bortezomib (top), which is a known proteasome inhibitory compound. It can be seen that when bortezomib is washed, the inhibitory activity is greatly reduced, whereas the inhibitory activity of the compound of the present invention is hardly reduced. FIG. 2 is a graph comparing the results of a reversibility test of Compound 4a of the present invention, Compound 11a having a structure similar to the compound of the present invention, and bortezomib, which is a known proteasome inhibitory compound. The vertical axis of the graph shows the ratio when the control chymotrypsin-like proteasome activity is taken as 100%. It can be seen that the similar compound 11a and bortezomib greatly recovers chymotrypsin-like proteasome activity when washed, whereas compound 4a of the present invention keeps chymotrypsin-like proteasome activity low.

The present invention will be described in detail below.
(1) Compound of the present invention In one aspect, the present invention relates to a novel compound having proteasome inhibitory activity.
In one embodiment, the compound of this aspect has the following general formula:
It is a compound which has a structure represented by these.

  In the present invention, the term “proteasome inhibitory compound” means a compound having an activity of inhibiting the enzymatic reaction of the proteasome. The proteasome is an enzyme complex, and is considered to have a plurality of enzyme activities such as chymotrypsin-like activity, trypsin-like activity, and caspase-like activity. If any one of them can be inhibited, the “proteasome of the present invention is used. It corresponds to “inhibitory compound”. Of course, it may be possible to simultaneously inhibit a plurality of enzyme activities. In addition, the proteasome-inhibiting compound of the present invention is a compound suitable as a pharmaceutical composition as described herein. Thus, when referring simply to a “compound” or “proteasome inhibiting compound”, it is intended to include “pharmaceutically acceptable salts thereof” unless otherwise indicated.

In the present invention, the term “alkyl” or “alkyl group” includes saturated linear or branched alkyl groups, and unsaturated linear or branched alkyl groups such as alkenyl and alkynyl groups. In the present invention, “alkylene” or “alkylene group” means a group in which one hydrogen atom is eliminated from the alkyl.
In the present invention, the term “cycloalkyl” or “cycloalkyl group” includes cyclic saturated alkyl groups and unsaturated cyclic alkyl groups such as alkenyl and alkynyl groups. In addition, for example, a group in which a ring member atom is further substituted with an alkyl group or a cycloalkyl group, such as 2-methylcyclohexyl, or a hydrogen atom in an alkyl group, such as 2-cyclohexylethyl, is substituted with a cycloalkyl group Groups are also encompassed by “cycloalkyl”. Therefore, the number of carbon atoms in the cycloalkyl in which the number of carbon atoms is specified means the number of carbon atoms in the whole group including the number of carbon atoms contained in these substituted alkyl groups.

In the present invention, “aryl” or “aryl group” means an aromatic hydrocarbon group, for example, a group in which a ring member atom is further substituted with an alkyl group or a cycloalkyl group, such as 2-methylphenyl, A group in which a hydrogen atom of an alkyl group or a cycloalkyl group is substituted with an aryl group is also included in the “aryl group”. Therefore, the number of carbon atoms in the aryl group in which the number of carbon atoms is specified means the number of carbon atoms in the whole group including the number of carbon atoms contained in these substituted alkyl groups.
In the present invention, the term “hydrocarbon group” is a term encompassing all the “alkyl group”, “cycloalkyl group” and “aryl group”.

  In the present invention, the term “protecting group” is a group for inactivating the reaction activity of a functional group to be protected, and can be easily eliminated by an appropriate reaction to yield the original functional group. Means a group that can. For example, “boronyl group protecting group” includes an ether protecting group, an acetal protecting group, an acyl protecting group, etc., which are protecting groups for the hydroxy group of the boronyl group. Examples of the protecting group for the boronyl group include Although not limited thereto, a pinanediol group, a pinacol group, a methyl group, a phenyl group and the like can be mentioned.

In the above general formula, S ¹ and S ² may be the same or different independently from each other, and mean alkylene having 1 to 5 carbon atoms. Both S ¹ and S ² may be saturated alkylene or unsaturated alkylene, but are preferably saturated alkylene. The number of carbon atoms may be any as long as it is 1 to 5 (C1 to 5), preferably 2 or 3, and most preferably 3.
Here, when the number of carbon atoms of S ¹ and S ² is 3 or more, any ^one of carbon atoms at both ends, that is, a carbon atom adjacent to A ¹ or A ² and a carbon atom other than a carbon atom adjacent to X is , Optionally may be replaced by S or O, preferably O. Thus, S ¹ and S ² are most preferably —CH ₂ —O—CH ₂ —.

In the above general formula, A ¹ and A ² are each independently a 5-membered or 6-membered aromatic ring, and one or more hydrogen atoms present in the aromatic ring are optionally halogen, hydroxyl group, It may be substituted with a group selected from a nitro group, an amino group, a cyano group, and a C1-10 alkyl group. Such an aromatic ring is preferably phenyl, pyridyl, furanyl, thiophenyl, naphthyl, biphenyl and the like, and most preferably phenyl.
In the above general formula, X represents C or N, preferably C. Further, from the viewpoint of ease of synthesis and the like, it is preferable that the number of asymmetric points in the skeleton of the compound is small. Accordingly, X is not an asymmetric center, that is, it is preferable that -S ¹ -A ¹ and -S ² -A ² are the same.
Thus, in a most preferred embodiment, the structure in the general formula
Is
Having the structure

In the above general formula, m and n may be any integer as long as they do not adversely affect the proteasome inhibitory activity of the whole compound, but if too large, the compound itself becomes too large to bind to the proteasome, and is small. Number is preferred. Thus, in a preferred embodiment, m and n are 1, 2 or 3. m and n are independent of each other, and may be the same number or different numbers.
In the above general formula, R ¹ and R ² may be each independently any hydrocarbon group having 1 to 12 carbon atoms, and one or more carbon atoms present in the hydrocarbon group are oxygen atoms May be replaced by a nitrogen atom or a sulfur atom, but when two or more atoms are replaced, these atoms are not adjacent to each other. In addition, one or more hydrogen atoms of the hydrocarbon group may be optionally substituted with a substituent such as halogen, hydroxyl group, carboxyl group, nitro group, amino group, cyano group.

R ¹ may be any hydrocarbon group as long as it does not adversely affect the proteasome inhibitory activity of the whole compound, but is preferably a C1-6 hydrocarbon group, more preferably a C1-5 alkyl group. is there. Examples of R ¹ include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, isopentyl, t-butyl, vinyl, 2-propen-2-yl, and the like. .
R ² may be any hydrocarbon group as long as it does not adversely affect the proteasome inhibitory activity of the whole compound, and is a C1-7 hydrocarbon group in a preferred embodiment, and C1-5 in a more preferred embodiment. It is an alkyl group. In another preferred embodiment, R ² is a natural amino acid side chain.

In the above general formula, In represents a proteasome inhibitory active site. A known proteasome-inhibiting compound, belactocin A, has a portion that binds to a portion called S1 pocket present in the proteasome CP, and in contrast to threonine 1 (Thr1) that is a proteasome active site, It is considered that the β-lactone ring is opened to bind by acylating the threonine residue, thereby inhibiting the enzyme activity. In one embodiment of the compounds of the present invention, the structure in the above general formula
Is presumed to exhibit proteasome inhibitory activity by binding to the S1 pocket present in the CP of the proteasome and binding of In to Thr1, which is the proteasome active site. Therefore, In preferably has a site that easily forms a bond such as a hydrogen bond or an electrophilic attack on the hydroxyl group present in the side chain of threonine. In addition, proteasome inhibitory active sites present in compounds known as proteasome inhibitory compounds are also preferred as In of the present invention. Examples of In include, but are not limited to, a β-lactone ring, a boronic acid, a boronic acid ester, an epoxide, and the like, and more preferably a structure having high in vivo stability such as a boronic acid and a boronic acid ester. It is. Thus, in a preferred embodiment of the invention, In is a boronic acid or boronic ester.

Accordingly, in a preferred embodiment, the proteasome inhibitory compound of the present invention has the following formula I
It is a compound represented by these.

In the above formula I, A ¹ , A ² , S ¹ , S ² , X, R ¹ , R ² , m and n are as defined above.
In the above formula I, Y ¹ and Y ² are each independently a hydrogen atom or a protecting group for a boronyl group. Y ¹ and Y ² may be combined to form a ring structure. Examples of Y ¹ and Y ² include, but are not limited to, the above-described protecting groups for the boronyl group.

In addition, in -B (OY ¹ ) OY ² which is the structure in the above formula I, when Y ¹ and Y ² are combined to form a ring structure, for example, the following general formulas II and III
It can take the structure of.

  In the above formula II, Y is a C1-10 alkylene group, and among the carbon atoms present in the alkylene, any one of the carbon atoms not adjacent to O is optionally an oxygen atom, a nitrogen atom or a sulfur atom. And one or more hydrogen atoms of the alkylene group may be optionally substituted with a group selected from a halogen, a hydroxyl group, a nitro group, an amino group, or a cyano group. In addition, two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group.

  In the above formula III, ring Y ′ is a C4-10 cycloalkenyl group or arylene group, and one or more ring carbon atoms constituting Y ′ are optionally oxygen atom, nitrogen atom or sulfur 1 or 2 or more hydrogen atoms which each ring member atom may be substituted with an atom is a group selected from halogen, C1-4 alkyl group, hydroxyl group, carboxyl group, nitro group, amino group or cyano group And two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group.

In a more preferred embodiment, -B (OY ¹ ) OY ² is
It is. These structures are also known as inhibitory active sites of known proteasome inhibitory compounds.

In view of the above, in a highly preferred embodiment of the compounds of the present invention, the compounds of the present invention have the formula IV
Where m and n are as defined above.
It is a compound represented by these.

  Any known synthesis method can be used alone or in combination for the synthesis of the compound of the present invention. It will be understood by those skilled in the art that an optimum synthesis method for the compound of the present invention can be appropriately selected and necessary conditions can be appropriately determined. In the following examples, some examples of the synthesis of specific compounds included in the present invention are shown, but it is understood by those skilled in the art that alternative synthetic methods may be used. It is.

(2) Proteasome inhibitor containing the compound of the present invention The compound of the present invention is a compound having proteasome inhibitory activity as described above. Accordingly, proteasome inhibitors comprising an effective amount of a compound of the invention are also included in the invention.
In the present invention, the “effective amount” means the amount of an active ingredient necessary for achieving the use of the composition, that is, for the composition to function effectively, and there is an adverse effect exceeding the benefit of the use of the active ingredient. An amount that does not occur is preferred. The amount varies depending on the use of the composition, the method of use, the type of compound used as an active ingredient, and the conditions of the intended use. For example, in vitro tests using cultured cells, model animals such as mice and rats, etc. It can be appropriately determined by a test method well known to those skilled in the art, such as a test in the above.

  The proteasome inhibitor of the present invention contains at least one compound of the present invention as an active ingredient, but further contains other compounds of the present invention and / or known proteasome inhibitory compounds as long as the proteasome inhibitory activity is not adversely affected. It's okay. In addition, for example, a component for effectively achieving the proteasome inhibitory activity effect of the compound of the present invention such as a pharmaceutically acceptable carrier and other optional components such as excipients may be included. These other components are well known in the art, and those skilled in the art can appropriately select these components according to the purpose and method of use.

(3) Pharmaceutical composition containing the compound of the present invention The compound of the present invention is a novel compound, has the property of inhibiting the activity of the proteasome, and also has in vivo stability. It is suitable as. Accordingly, pharmaceutical compositions comprising an effective amount of a compound of the present invention are also encompassed by the present invention. The pharmaceutical composition of the present invention can be suitably used particularly for treating a proteasome-related disease, that is, a disease whose pathological condition is improved by inhibiting the function of the proteasome.

  In the present invention, the “proteasome-related disease” means a disease in which the activity of the proteasome is related to the onset or exacerbation of the disease. The ubiquitin-proteasome system is considered to perform protein quality control in vivo, and homeostasis cannot be maintained due to an abnormality in this system, which leads to cell abnormalities. Examples of proteasome-related diseases include, but are not limited to, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, polyglutamine disease, prion disease, amyotrophic lateral sclerosis, tumors such as cancer, rheumatism And other immune system diseases.

Proteasome-inhibiting compounds fail by inhibiting abnormal degradation of normal proteins in cells that cannot maintain homeostasis as a result of excessive degradation of normal proteins due to abnormal ubiquitination of normal proteins and increased proteasome expression Can be eliminated and lead to apoptosis. Therefore, the pharmaceutical composition of the present invention can be suitably used particularly for the treatment of proteasome-related diseases.
Proteasome-inhibiting compounds are known to have therapeutic effects, particularly in cancer, and are approved as pharmaceuticals. For example, in tumor cells, it is known that various normal proteins such as p53 are abnormally ubiquitinated, and excessive degradation of these normal proteins plays a part in the cause of tumorigenesis. Conceivable. Proteasome-inhibiting compounds can lead tumor cells to apoptosis by inhibiting abnormal degradation of normal cells. Accordingly, in a preferred embodiment, the pharmaceutical composition of the present invention is used to treat cancer.

In the present invention, “tumor” includes benign tumors and malignant tumors (cancer, malignant neoplasm). “Cancer” includes hematopoietic tumors, including blood cancers, epithelial malignant tumors (cancer, carcinoma) and non-epithelial malignant tumors (sarcoma, sarcoma).
Examples of cancer treated with the pharmaceutical composition of the present invention include, but are not limited to, blood cancer, colon cancer, skin cancer and the like.

  The pharmaceutical composition of the present invention contains at least one compound of the present invention as an active ingredient. However, as long as there is no adverse effect on the pharmacological effect and an adverse effect exceeding the benefits of use does not occur, other pharmaceutical compounds of the present invention and // Known proteasome inhibitory compounds may be included. Further, it may contain other optional components such as a component for effectively achieving the proteasome inhibitory activity effect of the compound of the present invention, such as a pharmaceutically acceptable carrier and a surfactant, and an excipient. . These other components are well known in the art, and those skilled in the art can appropriately select these components according to the purpose and method of use.

In addition, the dosage form of the pharmaceutical composition containing the proteasome-inhibiting compound of the present invention as an active ingredient is not particularly limited, but tablets, solutions, oil emulsions (emulsion formulations), polymer nanoparticles, liposome formulations, Particulate preparations bound to beads having a diameter of several μm, preparations attached with lipids, microsphere preparations, microcapsule preparations and the like can be mentioned, and those skilled in the art can appropriately select a suitable dosage form.
Examples of the administration method of the pharmaceutical composition of the present invention include any known administration method such as intradermal administration, subcutaneous administration, intramuscular administration, and intravenous administration. The dosage of the proteasome-inhibiting compound of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient, but is usually 1 mg to 1000 mg, preferably 10 mg to 100 mg per day. Preferably, it is administered once.

(4) Method for preventing and / or treating cancer The present invention is also a method for preventing and / or treating cancer in a subject, comprising an effective amount of one or more proteasome-inhibiting compounds of the present invention. In a method comprising administering to a subject in need thereof.
The “subject” in the present invention may be any individual organism as long as it can suffer from cancer, but preferably human and non-human mammals (eg, mouse, rat, guinea pig, hamster, etc.) Dentates, primates such as chimpanzees, cloven-hoofed animals such as cows, goats, and sheep, terridae such as horses, rabbits, dogs, and cats), and more preferably human individuals. In the present invention, the subject may be healthy or afflicted with some disease, but when cancer prevention and / or treatment is intended, the subject is typically afflicted with cancer. Means a subject who has or is at risk of suffering from. In one embodiment of the present invention, the cancer is a cancer in which abnormally increased proteasome expression is observed.

Examples of the proteasome inhibitory compound of the present invention used in the prevention / treatment method of the present invention include any of those described herein. The specific dose of the active ingredient depends on various conditions relating to the subject in need thereof, such as severity of symptoms, general health status of the subject, age, weight, subject sex, diet, timing and frequency of administration, It can be determined in consideration of the drug used in combination, the response to treatment, the dosage form, compliance with the treatment, and the like.
As a specific dose, for example, in the case of the proteasome inhibitory compound of the present invention, it is preferable to administer 1 mg to 1000 mg, preferably 10 mg to 100 mg once a day. As the administration method, any known appropriate administration method such as intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration and the like can be used.

  The prophylactic / therapeutic method of the present invention may further include a step of selecting, as the prophylactic / therapeutic subject, a subject for whom administration of the proteasome inhibitory compound is effective before the administering step. This aspect of the present invention may further include the step of determining the presence or absence of abnormally enhanced proteasome expression in the subject tumor cells prior to the selecting step. Presence / absence of abnormally increased proteasome expression in the subject can be determined by any known technique.

All patents, applications and other publications mentioned herein are hereby incorporated by reference in their entirety.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

Example 1. Synthesis of Compounds 3a-11a and 3b-5b An overview of the synthesis of compounds 3a-11a and 3b-5b is shown in Scheme 1 below. Since these pinanediol esters of peptide boronic acid are unstable under silica gel column chromatography conditions, all target compounds were purified by reverse phase column chromatography.

The reagents and conditions in Scheme 1 are as follows.
(A) Boc-Asp (OBn) —OH, PivCl, Et ₃ N, CH ₂ Cl ₂ , 0 ° C. to room temperature (b) Boc-Glu (OBn) —OH, PivCl, Et ₃ N, CH ₂ Cl ₂ , 0 ° C. to room temperature (c) TFA, CH ₂ Cl ₂ , 0 ° C. to room temperature (d) Ac ₂ O, Et ₃ N, CH ₂ Cl ₂ , 0 ° C.
(E) Pd / C, H ₂ , THF

(F) Compound 15, EDC, HOAt, DIEA, CH ₂ Cl ₂ , −18 ° C. to room temperature (g) Compound 16, EDC, HOAt, DIEA, CH ₂ Cl ₂ , −18 ° C. to room temperature (h) Compound 17, EDC, HOAt, DIEA, CH ₂ Cl ₂ , −18 ° C. to room temperature (i) R—Cl, Et ₃ N, CH ₂ Cl ₂ , 0 ° C.
(J) R—OH, PivCl, Et ₃ N, CH ₂ Cl ₂ , 0 ° C. to room temperature (k) CH ₃ NH ₂ .HCl, EDC, HOAt, DIEA, CH ₂ Cl ₂ , 0 ° C. to room temperature (l) 4- (Benzyloxy) butan-1-amine.HCl, EDC, HOAt, DIEA, CH ₂ Cl ₂ , from −18 ° C. to room temperature

The pinanediol ester of leucine boronic acid (Compound 12) was condensed with Boc-Asp (OBn) -OH or Boc-Glu (OBn) -OH by the acid anhydride method to obtain Compound 13a or 13b, respectively. After removing the Boc group of compounds 13a and 13b by treatment with TFA / CH ₂ Cl ₂ , the resulting amino group was acetylated with Ac ₂ O to give compounds 14a and 14b, respectively. The Bn groups of compounds 14a and 14b were hydrocracked and then condensed with compounds 15 to 17 to obtain compounds 3a to 5a and 3b to 5b, respectively.

The target compounds 6a-11a were also synthesized by a similar method. The Bn group of compound 13a was removed and then condensed with 16 to give compound 18a. The Boc group of 18a was removed, and then condensed with the corresponding carboxylic acid or acyl chloride to obtain the target compounds 6a to 9a. The Bn group of compound 14a was removed, and then condensed with CH ₃ NH ₂ or 4- (benzyloxy) butan-1-amine to obtain the target compound 10a or 11a, respectively. Details of each synthesis reaction are shown below.

(1) (S) -Benzyl 3-acetamido-4-(((R) -3-methyl-1-((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6 -Methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) amino) -4-oxobutanoate (compound 14a)
To a solution of Boc-Asp (OBn) -OH (3.84 g, 11.9 mmol, 2.0 eq) dissolved in 120 ml of dichloromethane (DCM), triethylamine (1.66 ml, 11.9 mmol, 2.0 eq) was added. ) And PivCl (1.47 ml, 11.9 mmol, 2.0 eq) were added at 0 ° C. After 1 hour keeping 0 ° C., the reaction mixture is concentrated in vacuo, the residue is dissolved in ethyl acetate (AcOEt), washed with water and brine, dried over Na ₂ SO ₄ and the solvent is removed under reduced pressure. Removal gave the corresponding acid anhydride as a colorless oil.

To a solution of amine hydrochloride compound 12 (1.79 g, 5.95 mmol) in DCM (150 ml) was added triethylamine (2.48 ml, 17.9 mmol, 3.0 eq) and the acid anhydride in DCM (30 ml). The solution dissolved in was added at 0 ° C. After 5 minutes keeping 0 ° C., the reaction mixture was allowed to warm to room temperature. After 14 hours keeping room temperature, the reaction mixture is concentrated in vacuo, the residue is dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent is removed under reduced pressure. To give compound 13a as a white amorphous solid.
To a solution of the amorphous solid in DCM (60 ml) was added trifluoroacetic acid (TFA, 12 ml) at 0 ° C. After 5 minutes keeping 0 ° C., the reaction mixture was allowed to warm to room temperature. After 15 hours at room temperature, the reaction mixture was concentrated in vacuo to give the corresponding amine as a brown viscous oil.

To a solution of the viscous oil in DCM (60 ml), triethylamine (2.48 ml, 17.9 mmol, 3.0 eq) and Ac ₂ O (1.12 ml, 11.9 mmol, 2.0 eq) Was added at 0 ° C. After 5 minutes keeping 0 ° C., the reaction mixture was allowed to warm to room temperature. After 20 minutes at room temperature, the reaction mixture was concentrated in vacuo, the residue was dissolved in ethyl acetate (AcOEt), washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ , and The solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (n-hexane / AcOEt 1: 1-1: 2) to give compound 14a (2.07 g, 4.04 mmol, 68% yield over 3 steps) as a white amorphous solid. Obtained.

(2) (S) -Benzyl 4-acetamido-5-(((R) -3-methyl-1-((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6 -Methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) amino) -5-oxopentanoate (compound 14b)
As in (1) above, using Boc-Glu (OBn) -OH instead of Boc-Asp (OBn) -OH, amine hydrochloride 12 (1.25 g, 4.15 mmol) to compound 14b (1.84 g) 3.49 mmol, yield of 3 steps 84%, white amorphous solid).

(3) (S)-2-acetamido ^-N 4 - (3- (benzyloxy) -2 - ((benzyloxy) methyl) ^propyl) -N 1 - ((R)-3-methyl-1 - (( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 3a)
Pd / C (50 mg) was added to a solution of compound 14a (77.5 mg, 0.151 mmol, 1.1 equivalent) dissolved in tetrahydrofuran (THF, 6.0 ml). The flask was purged with hydrogen and the reaction mixture was stirred under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give the corresponding carboxylic acid as a white amorphous solid.

A solution of the amine compound 15 (39.1 mg, 0.137 mmol, 1.0 equivalent) dissolved in DCM (2.0 ml) and the amorphous solid obtained above in DCM (1.5 ml), 1-hydroxy-7-azabenzotriazole (HOAt, 20.5 mg, 0.151 mmol, 1.1 eq), N, N-diisopropylethylamine (DIEA, 25.6 μl, 0.151 mmol, 1.1 eq) And 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl, 28.9 mg, 0.151 mmol, 1.1 eq) was added at -18 ° C. After 10 minutes maintaining -18 ° C, the reaction mixture was warmed to 0 ° C. After 2 hours at 0 ° C., the reaction mixture was diluted with AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The crude product was purified by reverse phase HPLC (Mightysil RP-18 250-20 (5 μm), acetonitrile / H ₂ O 70:30) to yield compound 3a (64.5 mg, 0.0935 mmol, yield from compound 15). 68%) as a colorless viscous oil.

(4) (S)-2-acetamido ^-N 4 - (4-(benzyloxy) -3 - ((benzyloxy) methyl) ^butyl) -N 1 - ((R)-3-methyl-1 - (( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 4a)
Analogously to (3) above, compound 4a (93.9 mg, 0.133 mmol, 77% yield, colorless viscous oil) was synthesized from amine compound 16 (51.7 mg, 0.173 mmol).

(5) (S)-2-acetamido ^-N 4 - (5-(benzyloxy) -4 - ((benzyloxy) methyl) ^pentyl) -N 1 - ((R)-3-methyl-1 - (( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 5a)
In the same manner as in the above (3), compound 5a (98.6 mg, 0.137 mmol, yield 71%, colorless viscous oily substance) was synthesized from amine compound 17 (61.0 mg, 0.195 mmol).

(6) (S) -2-acetamido-N ^5- (3- (benzyloxy) -2-((benzyloxy) methyl) propyl) -N ¹ -((R) -3-methyl-1-(( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) pentanediamide (compound 3b)
Similar to (3) above, compound 14b was used instead of compound 14a, and amine compound 15 (36.8 mg, 0.129 mmol) to compound 3b (71.1 mg, 0.101 mmol, 78% yield, colorless A viscous oily substance) was synthesized.

(7) (S) -2-acetamido-N ^5- (4- (benzyloxy) -3-((benzyloxy) methyl) butyl) -N ¹ -((R) -3-methyl-1-(( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) pentanediamide (compound 4b)
Similar to (3) above, compound 14b was used instead of compound 14a, and amine compound 16 (50.2 mg, 0.168 mmol) to compound 4b (101 mg, 0.141 mmol, 84% yield, colorless viscous oil) Material) was synthesized.

(8) (S)-2-acetamido ^-N 5 - (5-(benzyloxy) -4 - ((benzyloxy) methyl) ^pentyl) -N 1 - ((R)-3-methyl-1 - (( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) pentanediamide (compound 5b)
Similar to (3) above, compound 14b was used instead of compound 14a, and amine compound 17 (61 mg, 0.195 mmol) to compound 5b (107 mg, 0.146 mmol, 75% yield, colorless viscous oil) Was synthesized.

(9) ^(S) -N 4 - (4-(benzyloxy) -3 - ((benzyloxy) methyl) butyl) -2- ^{(2,5-dichloro-benzamide)} -N 1 - ((R) -3 -Methyl-1-((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (Compound 6a)
To a solution of Boc-Asp (OBn) -OH (25.6 mg, 0.0792 mmol, 1.0 equivalent) in DCM (1.0 ml) was added triethylamine (11.0 μl, 0.0792 mmol, 1.0 equivalent). Amount) and PivCl (9.75 μl, 0.0792 mmol, 1.0 eq) were added at 0 ° C. After 1 hour keeping 0 ° C., the reaction mixture was concentrated in vacuo, the residue was dissolved in AcOEt, washed with water and brine, dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The corresponding acid anhydride was obtained as a colorless oil.

To a solution of compound 12 (23.9 mg, 0.0792 mmol) in DCM (1.0 ml), triethylamine (27.6 μl, 0.198 mmol, 2.5 eq) and the acid anhydride were added to DCM (1. 0 ml) was added at 0 ° C. After 5 minutes maintaining 0 ° C., the reaction mixture was warmed to room temperature and stirred for 30 minutes. The reaction mixture is concentrated in vacuo, the residue is dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent is removed under reduced pressure to remove compound 13a. Obtained as a white amorphous solid.
Pd / C (45 mg) was added to a solution of the viscous oily substance in THF (1.0 ml). The flask was purged with hydrogen and the reaction mixture was stirred for 90 minutes under a hydrogen atmosphere. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give the corresponding carboxylic acid as a white amorphous solid.

To a solution of the amorphous solid dissolved in DCM (1.0 ml), amine compound 16 (23.7 mg, 0.0792 mmol, 1.0 equivalent) HOAt (10.8 mg, 0.0792 mmol, 1.0 equivalent) , DIEA (13.5 μl, 0.0792 mmol, 1.0 eq), and EDC · HCl (15.2 mg, 0.0792 mmol, 1.0 eq) were added at −18 ° C. After 5 minutes maintaining -18 ° C, the reaction mixture was warmed to room temperature and stirred for 16 hours. The reaction mixture is concentrated in vacuo, the residue is dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ , and the solvent is removed under reduced pressure to provide compound 18a ( 2.07 g, 4.04 mmol, 68% yield over 3 steps) was obtained as a colorless viscous oil.

  TFA (500 μl) was added at 0 ° C. to a solution of the viscous oil in DCM (500 μl). After 5 minutes maintaining 0 ° C., the reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction mixture was concentrated in vacuo to give the corresponding amine as a brown viscous oil.

To a solution of the viscous oil in DCM (1.0 ml), triethylamine (22.0 μl, 0.158 mmol, 2.0 eq) and 2,5-dichlorobenzoyl chloride (16.6 mg, 0.0792 mmol, 1.0 equivalent) was added at 0 ° C. After 3 hours keeping 0 ° C., the reaction mixture is concentrated in vacuo, the residue is dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent is reduced in vacuo. Removed below. The crude product was purified by reverse phase HPLC (Mightysil RP-18 250-20 (5 μm), acetonitrile / H ₂ O 70:30) to yield compound 6a (33.7 mg, 0.0404 mmol, 49% yield over 5 steps). ) Was obtained as a colorless viscous oil.

(10) (S)-2-benzamido ^-N 4 - (4-(benzyloxy) -3 - ((benzyloxy) methyl) ^butyl) -N 1 - ((R)-3-methyl-1 - (( 3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 7a)
Similar to (9) above, using benzoyl chloride instead of 2,5-dichlorobenzoyl chloride, compound 12 (25.2 mg, 0.0835 mmol) to compound 7a (33.9 mg, 0.0443 mmol) A 53% yield, colorless viscous oily substance) was synthesized.

(11) ^(S) -N 4 - (4-(benzyloxy) -3 - ((benzyloxy) methyl) ^butyl) -N 1 - ((R)-3-methyl -1 - ((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) -2- (pyrazine-2-carboxamido) succinamide ( Compound 8a)
Similar to (9) above, using pyrazinecarboxylic acid and PivCl instead of 2,5-dichlorobenzoyl chloride, compound 12 (23.7 mg, 0.0787 mmol) to compound 8a (28.4 mg, 0.0370 mmol, 5 steps yield 47%, colorless viscous oily substance) was synthesized.

(12) ^(S) -N 4 - (4-(benzyloxy) -3 - ((benzyloxy) methyl) ^butyl) -N 1 - ((R)-3-methyl -1 - ((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) -2- (6-phenylpicolinamide) succinamide ( Compound 9a)
As in (9) above, using pyrazinecarboxylic acid and PivCl instead of 2,5-dichlorobenzoyl chloride, compound 12a (23.7 mg, 0.0787 mmol) to compound 9a (35.0 mg, 0.0415 mmol, 5 steps yield 54%, colorless viscous oil).

(13) (S) -2-acetamido-N ⁴ -methyl-N ¹ -((R) -3-methyl-1-((3aS, 4S, 6S, 7aR) -3a, 5,5-trimethylhexahydro -4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 10a)
To a solution of benzyl ester 14a (93.5 mg, 0.1825 mmol) dissolved in THF (2.0 ml), Pd / C (90.0 mg) was added. The flask was purged with hydrogen and the reaction mixture was stirred under a hydrogen atmosphere for 2 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give the corresponding carboxylic acid 19a as a white amorphous solid.

In a solution of the amorphous solid in DCM (2.0 ml), CH ₃ NH ₂ .HCl (43.9 mg, 0.650 mmol, 3.5 eq), HOAt (25.2 mg, 0.185 mmol, 1. 0 eq), DIEA (252 μl, 1.48 mmol, 8.0 eq), and EDC.HCl (39.1 mg, 0.204 mmol, 1.1 eq) were added at 0 ° C. After 5 minutes maintaining 0 ° C., the reaction mixture was warmed to room temperature and stirred for 19 hours. The reaction mixture was concentrated in vacuo, the residue was dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The crude product was purified by reverse phase HPLC (Mightysil RP-18 250-20 (5 μm), acetonitrile / H ₂ O 60:40) to give compound 10a (14.3 mg, 0.0329 mmol, 18% yield over 2 steps). ) Was obtained as a colorless viscous oil.

(14) (S) -2- acetamido ^-N 4 - (4- (benzyloxy) ^{butyl) -N 1 - ((R)} -3- methyl -1 - ((3aS, 4S, 6S, 7aR) -3a , 5,5-trimethylhexahydro-4,6-methanobenzo [d] [1,3,2] dioxaborol-2-yl) butyl) succinamide (compound 11a)
To a solution of benzyl ester 14a (89.2 mg, 0.174 mmol) in THF (2.0 ml) was added Pd / C (90.0 mg). The flask was purged with hydrogen and the reaction mixture was stirred under a hydrogen atmosphere for 2 hours. The reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo to give the corresponding carboxylic acid 19a as a white amorphous solid.

Tert-butyl (4- (benzyloxy) butyl) carbamate (48.6 mg, 0.174 mmol, 1.0 equivalent) was dissolved in AcOEt (2 ml) containing 4M HCl and stirred at room temperature for 1 hour. The reaction mixture was concentrated in vacuo to give the corresponding amine as a colorless viscous oil.
To a solution of compound 19a in DCM (2.0 ml), the above amine, DIEA (88.8 μl, 0.522 mmol, 3.0 eq), HOAt (23.7 mg, 0.174 mmol, 1.0 eq) ) And EDC.HCl (33.4 mg, 0.174 mmol, 1.0 equiv) were added at -18 ° C. After 10 minutes maintaining -18 ° C, the reaction mixture was warmed to room temperature and stirred for 3 hours. The reaction mixture was concentrated in vacuo, the residue was dissolved in AcOEt, washed with 1M HCl, saturated NaHCO ₃ and brine, dried over Na ₂ SO ₄ and the solvent removed under reduced pressure. The crude product was purified by reverse phase HPLC (Mightysil RP-18 250-20 (5 μm), acetonitrile / H ₂ O 65:35) to give compound 11a (77.2 mg, 0.132 mmol, 76% yield over 2 steps). ) Was obtained as a colorless viscous oil.

The structural characteristics of each compound are shown in Table 1 below.

Example 2. Proteasome Inhibition Assay The inhibitory activity of various compounds synthesized in Example 1 and other proteasome inhibitory compounds on the human 20S proteasome is shown in Asai et al., Biochem Pharmacol. 2004 Jan 15; 67 (2): 227-34. The procedure was as described. Briefly, 3 nM human 20S proteasome was prepared by reacting various compounds and DMSO in reaction buffer (50 mM Tris-HCl (pH 7.5), 25 mM KCl, 10 mM NaCl, 1 mM MgCl ₂ , 0.018% SDS). For 5 minutes and then reacted with 75 μM Suc-LLVY-AMC, which is a chymotrypsin-like activity-selective substrate, at 25 ° C. for 60 minutes, and the fluorescence intensity of the degradation product AMC was measured with a microplate reader. . The activity value when treated with DMSO alone was taken as 100%, and the inhibitory activity of various compounds was determined.

  The results are shown in Table 2 and Table 3. As can be seen from the table below, all the compounds showed a sufficient inhibitory effect on the chymotrypsin-like activity of proteasome CP. Inhibiting activity equivalent to

Example 3 Cell growth inhibition assay (1) Colon cancer cell line HCT 116
The growth inhibitory effect on the cells of the compound of the present invention synthesized above was verified using HCT 116 strain, which is a human colon cancer cell line. The experiment was performed according to the product protocol using Cell counting kit-8 (Dojindo Laboratories). Cells were seeded in a 96-well microtiter plate at 3000 cells / well and treated with the test compound for 72 hours.
The results are shown in Table 4. As can be seen from Table 4, any compound other than Compound 10a was able to effectively inhibit the growth of HCT 116.

(2) Multiple myeloma cell line MM. Which is a multiple myeloma cell line instead of the colon cancer cell line HCT116. 1S and MM. Using 1R, the growth inhibitory effect on the cells of the compound of the present invention was verified by the same method as in (1) above. MM. The 1S cell line is dexamethasone-sensitive human multiple myeloma cells, MM. 1R is dexamethasone-resistant human multiple myeloma cells, all of which were obtained from ATCC.

The results are shown in Table 5. As can be seen from Table 5, the compound of the present invention was able to suppress cell proliferation to an extent comparable to that of existing proteasome inhibitors. Moreover, like existing proteasome inhibitors, cell proliferation could be suppressed regardless of the presence or absence of resistance to dexamethasone. MM. 1S cells and MM. The concentration of each compound showing growth inhibition for 1R cells tended to be lower than the concentration showing growth inhibition for HCT 116 cells.

Example 4 Inhibitory Activity Reversibility Assay In order to confirm the reversibility (stability) of the inhibitory activity, a reversibility assay was performed using compounds 4a, 11a and bortezomib. 15 nM 20S proteasome was preincubated with each inhibitory compound at 25 ° C. for 12 hours. 37.5 μM Suc-LLVY-amc was then added as a substrate to initiate the “standard assay” reaction of the 20S proteasome. After incubation at 25 ° C. for 1 hour, fluorescence (excitation light: 390 nm, emission light: 450 nm) of each reaction was measured with a VersaMax Pro microplate reader (Molecular Devices). For each “wash-out assay” reaction, each pre-incubated reaction mixture was subjected to a Microcon Centrifugal Filter (100 kDa molecular weight cut) prior to reaction with substrate to remove inhibitors not bound to the 20S proteasome. Off) and washed 5 times with assay buffer by centrifuging at 11,000 × g for 3 min. Thereafter, the reaction was carried out by adding a substrate in the same manner as in the “standard assay”.

The results are shown in FIG. 1 and FIG. As can be seen from FIG. 1 and FIG. 2, compound 11a and bortezomib showed a significant increase in chymotrypsin-like activity when washed, whereas the compound of the present invention hardly increased after washing. Therefore, it is considered that the compound of the present invention strongly binds to the 20S proteasome and does not easily decrease the inhibitory activity. In particular, the compounds 4a and 11a differ only in the structure of the R ³ portion, and from this, it is considered that the R ³ portion has a branched structure, thereby strengthening the binding to the 20S proteasome and exhibiting stable inhibitory activity. It is done.

Example 5. Protease selectivity assay To verify the proteasome specificity of the inhibitory activity of the compounds of the present invention, a protease selectivity assay was performed using compound 4a and bortezomib. It was performed as described in Arastu-Kapur et al., Clin. Cancer Res .. 2011, 17 (9), 2734-2743. Briefly, for cathepsin A, the activity of 22 ng / mL cathepsin A (R & D systems) was used with 40 μM Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys (Dnp)- After incubation with OH (R & D systems) mixed with assay buffer (50 mM MOPS, pH 5.5) at 25 ° C. for 2 hours, fluorescence (excitation light: 320 nm, emission light: 405 nm) of each reaction was obtained. Measured. For cathepsin B, the activity of 40 ng / mL cathepsin B (R & D systems) was mixed with 10 μM Z-Leu-Arg-AMC (R & D systems) as a substrate in assay buffer (25 mM MES, pH 5.0). Then, each reaction product was measured for fluorescence (excitation light: 380 nm, emission light: 460 nm). For cathepsin G, the activity of 8.0 μU / mL cathepsin G (R & D systems) and 200 μM Suc-Ala-Ala-Pro-Phe-AMC (Bachem) as an assay buffer (50 mM Tris-HCl, pH 7) were used. .5 150 mM NaCl) was incubated at 25 ° C. for 1 hour, and then fluorescence (excitation light: 380 nm, emission light: 460 nm) of each reaction was measured. For HtrA2 / Omi, the activity of 7.5 μg / mL of HtrA2 / Omi (R & D systems) was used, and 40 μM of Mca-IRRVSYSF (K-Dnp) K (INNOVAGEN) was used as an assay buffer (50 mM Tris-HCl, pH 8). 0.0) was incubated at 37 ° C. for 1 hour, and then fluorescence (excitation light: 320 nm, emission light: 405 nm) of each reaction was measured.

The results are shown in Table 6. As can be seen from Table 6, bortezomib showed inhibitory activity against cathepsin A and cathepsin G, whereas the compound of the present invention showed no inhibitory activity against any protease. Therefore, the compound of the present invention is considered to have high proteasome selectivity.

  The present invention provides a novel proteasome inhibitory compound. Such a compound has irreversible inhibitory activity, high proteasome inhibitory selectivity, and high in vivo stability while having inhibitory activity equivalent to previously known proteasome inhibitory compounds. It can be said that it has many advantages compared to conventional proteasome inhibitory compounds in its use as an active ingredient. Therefore, it is particularly useful as a therapeutic agent for proteasome-related diseases such as cancer.

Claims (11)

The following formula I
Where
A ¹ and A ² are each independently a 5-membered or 6-membered aromatic ring, and one or more hydrogen atoms present in the aromatic ring are optionally halogen, hydroxyl group, nitro group, amino group , May be substituted with a group selected from a cyano group or a C1-10 alkyl group,
S ¹ and S ² are, independently of each other, C 1-5 alkylene, and any one of carbon atoms not adjacent to X and A ¹ or A ² is arbitrary among the carbon atoms present in the alkylene. May be replaced with an oxygen atom,
X is C or N;
R ¹ is a C1-5 alkyl group,
R ² is a C1-10 hydrocarbon group,
Here, one or two or more carbon atoms present in R ¹ and R ² are replaced with oxygen atoms, nitrogen atoms or sulfur atoms, provided that when two or more atoms are replaced, these atoms are Not adjacent to each other,
One or more hydrogen atoms present in R ¹ and R ² may be optionally substituted with a substituent selected from halogen, hydroxyl group, carboxyl group, nitro group, amino group or cyano group,
Y ¹ and Y ² are each independently a hydrogen atom or a boronyl protecting group, and Y ¹ and Y ² may be combined to form a ring structure;
m and n are independently of each other, 1, 2 or 3.
Or a pharmaceutically acceptable salt thereof. The structure of the -B (OY ¹ ) OY ² moiety of formula I is
In the formula, Y is a C1-10 alkylene group, and among the carbon atoms present in the alkylene, any one of the carbon atoms not adjacent to O is arbitrarily replaced with an oxygen atom, a nitrogen atom or a sulfur atom. One or two or more hydrogen atoms of the alkylene group may be optionally substituted with a group selected from halogen, hydroxyl group, nitro group, amino group or cyano group, and Two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group,
Or formula III
In the formula, ring Y ′ is a C4-10 cycloalkenyl group or arylene group, and one or more ring member carbon atoms constituting Y ′ are optionally replaced with an oxygen atom, a nitrogen atom or a sulfur atom. 1 or 2 or more hydrogen atoms of each ring member atom may optionally be a group selected from halogen, C1-4 alkyl group, hydroxyl group, carboxyl group, nitro group, amino group, or cyano group May be substituted, or two hydrogen atoms of one carbon atom may be substituted with one oxygen atom to form a carbonyl group.
The compound of Claim 1 which is a structure represented by these, or its pharmaceutically acceptable salt. The structure of the -B (OY ¹ ) OY ² moiety of formula I is
The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof. R ² is an isobutyl group, a cyclohexylmethyl group or benzyl group, the compound according to claim 1 or a pharmaceutically acceptable salt thereof. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein S ¹ and S ² are both -C-C-C- or -C-O-C-. salt. The compound according to any one of claims 1 to 5, wherein A ¹ and A ² are both phenyl, or a pharmaceutically acceptable salt thereof.   The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein X is C. Formula IV
Where
m and n are independently of each other, 1, 2 or 3.
Or a pharmaceutically acceptable salt thereof.   A proteasome inhibitor comprising an effective amount of the compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition for treating a proteasome-related disease, comprising at least one compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.   The pharmaceutical composition according to claim 10, wherein the proteasome-related disease is a tumor.