KR101621418B1 - A mitochondria-targeted anti-tumor composition - Google Patents

Abstract

The present invention relates to a mitochondrial targeted anti-tumor composition, and more particularly to a purine-based 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine - (6-Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine was used as a molecular chaperone inhibitor and mitochondria-penetrating moiety Tumor composition which is excellent in the inhibitory effect of molecular chaperone (e.g., Hsp90 and / or TRAP1) increasing in tumor cells and is more stable and acting at a low concentration by linking triphenylphosphate (TPP) .

Description

A mitochondria-targeted anti-tumor composition < RTI ID = 0.0 >

The present invention relates to a composition for use as an anti-tumor agent, and more particularly to a composition which is excellent in the inhibition of molecular chaperones (eg, Hsp90 and / or TRAP1) that are increased in tumor cells, RTI ID = 0.0 > anti-tumor < / RTI >

Tumor cells exhibit enhanced ability to survive and proliferate in very unfriendly environments. They have been shown to down-regulate a number of cellular pathways that prevent normal (ie, non-cancerous) cells from dividing in harmful environments, and they also inactivate the apoptotic pathway leading to apoptosis in many normal tissues under adverse conditions. Tumor cells are also believed to upregulate the pathways necessary to maintain vigorous proliferation. For example, many tumor cells activate the cell stress-response pathway, which allows them to synthesize and maintain the protein machinery necessary to sustain proliferation. Activated stress responses in tumors include up-regulation of heat-shock proteins (Hsps), which are ATPase-inducible molecular chaperones.

Among these chaperone proteins, the Hsp90 family (heat shock protein 90) has four recognized members in mammalian cells, specifically Hsp90α and β, Grp94 and TRAP-1 (Tumor necrosis factor receptor -Associated Protein-1).

Hsp90 is a ubiquitous chaperone protein involved in the folding, activation and assembly of a wide variety of proteins, including key proteins involved in signal transduction, cell cycle regulation, and transcriptional regulation. Researchers have reported that the HSP90 chaperone protein is associated with important signal proteins such as steroid hormone receptors and protein kinases, including Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 1996, 10, 1491-502; Dai, K. et al. J. Biol. Chem. 1996, 271, 22030-4 ]). That is, Hsp90 is upregulated in a number of cancerous tissues. Hsp90 regulates the balance between folding / maturing and proteasome destruction of a limited number of client proteins, some of which are involved in signal transduction and cell proliferation.

In addition, Hsp90 is an evolutionarily well-preserved molecular chaperone that is frequently found in normal cells and overexpressed in cancer cells. Hsp90 is also expressed in the mitochondria of cancer cells, and expression of TRAP1, a mitochondrial Hsp90 homolog, is also increased. These chaperones protect mitochondria from oxidative stress and play an important role in preventing cells from dying. When inhibitors are used to inhibit these chaperones, mitochondrial function modulation is induced and cancer cell selective cell death is followed. This is a logical basis for the ability to be used as a target protein in the development of anticancer drugs. Several drug candidates have been developed that inhibit mitochondrial TRAP1 and Hsp90, and studies of their cytotoxic cytotoxicity have been reported.

In this regard, the inventor has invented a composition or pharmaceutically acceptable salt thereof, according to US Patent Publication No. 2009/0099080 (titled: Mitochondria-targeted anti-tumor agent) Where A is a molecular chaperone suppressor and B is a mitochondria-penetrating moiety, and A and B are connected by a linking moiety or not. As the molecular chaperone inhibitor (A), Hsp90 inhibitor of the Ansamycin class or Geldanamycin class of gamitrinib was used as the molecular chaperone inhibitor (A) (US Patent Publication No. 2009/0099080 15A). However, such ansamycin or geldanamycin compounds have excellent anticancer efficacy, but have been reported to exhibit many side effects with it, and have a disadvantage in that they act at a relatively high concentration.

Recently, Korean Patent Publication No. 2012-0083898 (name: purine derivative useful as an HSP90 inhibitor) is a compound that inhibits heat shock protein 90 (Hsp90) and has a purine derivative Lt; RTI ID = 0.0 > Hsp90 < / RTI > For example, in Korean Patent Publication No. 2012-0083898, a purine-based compound (PU-H71) as shown in the following Chemical Formula 7 and 9- (3- (isopropylamino) propyl) -8- - phenylbenzo [d] [1,3] dioxol-5-ylthio) -9H-purin-6-amine and the like (Korean Patent Publication No. 2012-0083898, page 67, etc.).

(7)

However, such a purine-based compound (PU-H71) has a disadvantage that its anticancer efficacy is lower than that of the existing antitrynib.

Accordingly, there is always a need to develop a new anti-tumor composition which is basically excellent in anticancer efficacy and stable and low in side effects.

Disclosure of the Invention The object of the present invention is to solve the above problems and to provide an anti-tumor composition which is excellent in inhibiting the molecular chaperone (eg, Hsp90 and / or TRAP1 etc.) to be.

In order to accomplish the above object, a mitochondrial-targeted anti-tumor composition according to an embodiment of the present invention comprises a compound in which a mitochondria-penetrating moiety is linked to a molecular chaperone inhibitor, Or a pharmaceutically acceptable salt thereof, wherein the molecular chaperone inhibitor is 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- [1,3] dioxol-5-ylsulfanyl) adenine), and the mitochondria-penetrating moiety is triphenylphosphate (TPP).

Meanwhile, another embodiment of the present invention includes a composition as described above, wherein the 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine binds to Hsp90, Of the Hsp90 inhibitor.

Yet another embodiment of the present invention is a mitochondrial TRAP1 inhibitor comprising the above composition, wherein the compound inhibits TRAP1 protein existing in the mitochondria through the mitochondria.

Still another embodiment of the present invention is a method for inducing the death of cancer or tumor cells, which comprises the step of administering the above composition to a subject.

Further, another embodiment of the present invention is a method for producing 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- 1,3,5-dioxol-5-ylsulfanyl) adenine by linking moieties or linking moieties with a mitochondria-penetrating moiety, triphenylphosphate (TPP) Lt; RTI ID = 0.0 > anti-tumor < / RTI >

The details of other embodiments are included in the detailed description and drawings.

The present invention relates to purine-based 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- 5-ylsulfanyl) adenine as a molecular chaperone inhibitor and linking triphenylphosphate (TPP) as a mitochondria-penetrating moiety to the tumor cells There is an effect that an anti-tumor composition which is excellent in the inhibitory effect of an increasing molecular chaperone (e.g., Hsp90 and / or TRAP1 and the like) and more stable and operates at a low concentration can be provided.

1 is a nuclear magnetic resonance (H-NMR) graph of a TPP-PU compound and a precursor thereof according to a preferred embodiment of the present invention,
2 shows the activity of the TPP-PU compound (SMTIN-P01) according to a preferred embodiment of the present invention,
(A) The drug affects the mitochondrial membrane potential. TMRM-loaded HeLa cells were cultured with 20 μM of PU-H71, gamitrinib, SNTIN-P01, and TPP-BIIB for 30 min and analyzed by confocal microscope. White bar, 20μm.
(B) Hsp70 upregulation and client depletion. Cells were treated with 5 μM of drug for 24 h and analyzed by Western blot.
(C) Induces the stress response of mitochondria. Cells were treated with 2 [mu] M of drug for 24 hours and analyzed by Western blot.
(D) Cytotoxic activity of SMTIN-P01. Gamitrinib, SMTIN-P01, PU-H71 and TPP-BIIB were immunized with kidney (ACHN), cervix (HeLa), ovary (SK-OV3), prostate (22Rv1) various human cancer cell lines derived from liver (SK-HEP-1), brain (A172), lung (H460), and breast (MDA-MB-231) Lt; / RTI > Cell viability was analyzed by MTT assay. Data are mean ± SEM obtained from two independent triplicated experiments.
FIG. 3 shows the in vivo activity of a TPP-PU compound (SMTIN-P01) according to a preferred embodiment of the present invention,
(A) Indicates inhibition of tumor growth in vivo. 10 mg / kg of drug was injected daily into nude mice transplanted with 22Rv1 cells. Tumor size was measured with a caliper. Eight tumors and four mice per group were tested. Data are mean ± SEM.
(B) tumor weight. (A) At the end of the experiment, the tumor tissue was recovered and weighed. Data are mean ± SEM.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

The present invention relates to a mitochondrial targeted anti-tumor composition comprising a compound in which a mitochondria-penetrating moiety is linked to a molecular chaperone inhibitor, or a pharmaceutically acceptable salt thereof .

As used herein, the term " molecular chaperone " is a group of any proteins involved in correct intracellular folding and assembly of polypeptides, found in bacteria, mitochondria, and eukaryotic cytoplasm. Molecular chaperones, particularly heat shock protein (Hsp) gene families (such as HSPA9, Hsp60, Hsp90 and / or TRAP-1) are involved in protein folding quality control, proteolysis, It assists in trafficking. Molecular chaperones are involved in compartmentalization within subcellular microdomains, including cyclic cycling of ATPase activity, recruitment of additional chaperones, and mitochondria. Molecular chaperones are often associated with increased cell viability (Beere, J Cell Sci 2004; 117: 2641-51), increased stability of survival effectors (Sato et al., Proc Natl Acad Sci USA 2000; 97: 10832-7), and inactivation of p53 (Wadhwa et al., J Biol Chem 1998; 273: 29586-91).

In the present specification, 'molecular chaperone suppressor' means having a function of suppressing the above-mentioned molecular chaperone. Among them, it is preferable to be a molecular chaperone suppressor that targets mitochondria, that is, accumulates in the membrane through the mitochondrial membrane. Chaperone inhibitors can target mitochondria through a combination with a mitochondrial penetration moiety. For example, the molecular chaperone inhibitor may be a protein, or a chaperone binding fragment thereof, that binds to a chaperone protein. It is preferred that the inhibitors useful in the methods and compositions described herein act directly on the chaperone protein itself, that is, they do not act upstream or downstream. Many such inhibitors, such as peptide inhibitors and small molecule inhibitors, are already known in the art. In some embodiments, molecular chaperone inhibitors useful in the present invention inhibit ATPase activity of a chaperone, e.g., Hsp60, HspA9, Hsp90, and / or TRAP-1. In some embodiments, molecular chaperone inhibitors useful in the present invention inhibit the binding of Hsp60, HspA9, Hsp90, or TRAP-1 to cyclophilin D. In some embodiments, molecular chaperone inhibitors useful in the present invention inhibit the binding of Hsp60, HspA9, Hsp90, or TRAP-1 to male vivin (male vivin). In some embodiments, the molecular chaperone inhibitor binds to the chaperone and induces proteasome degradation of the client protein of the chaperone.

Said molecular chaperone suppressor is linked to a mitochondrial through-going moiety. The molecular chaperone suppressor may be modified by conjugation with a mitochondrial penetration moiety using methods known in the art. The mitochondrial penetration-enhancing moiety is a chemical moiety or function that increases the mitochondrial localization of a molecular chaperone inhibitor (e. G., Chemically conjugated) coupled to the mitochondrial penetration moiety when compared to a single molecular chaperone inhibitor (E.g., peptides, peptidomimetics, or other compounds).

The linkage between the above-mentioned molecular chaperone suppressor and the mitochondrial through-going moiety can be made by a covalent bond, for example, a peptide bond or a thioether bond. For example, when one or both of the chaperone suppressor and the mitochondrial penetration moiety are non-peptidic, it is preferred that the small molecule, the molecular chaperone inhibitor and the mitochondrial penetration moiety are connected via a chemical linker, for example. In some embodiments, the linkage between the molecular chaperone inhibitor and the mitochondrial penetration moiety can be made by a non-transmissive interaction, for example, an ionic interaction.

In some embodiments, the mitochondrial through-going moiety can be linked to a molecular chaperone inhibitor via a linker. Means a linking of mitochondria-penetrating moiety and chaperone inhibitor via a covalent or non-covalent bond or linkage. A number of different linkers may be used to link the chaperone inhibitor to the mitochondria-penetrating moiety.

Useful general methods for preparing the compositions described herein are known in the art. For example, the contents of US Pat. No. 2009/0099080 (titled: Mitochondria-targeted anti-tumor agent) invented by the present inventor can be applied to understand the present invention. That is, what is not described in the detailed description of the present invention to be described later may include contents described in the aforementioned U.S. patent.

In the mitochondria-targeted anti-tumor composition as described above, the present invention relates to the above-mentioned anti-tumor composition wherein the molecular chaperone inhibitor is 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- 6-Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine), and the mitochondria-penetrating moiety is triphenylphosphate (TPP).

In one embodiment, the 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine is preferably represented by the following formula (4).

[Chemical Formula 4]

And, the molecular chaperone suppressor and the mitochondria-penetrating moiety may be connected by a connecting moiety or may be connected without a connecting moiety, but more preferably, they are connected by a connecting moiety.

In addition, the linking moiety is preferably selected from the group consisting of a peptide linker and a chemical linker, more preferably a carbon chain including 6 carbon atoms. For example, the linking moiety may be a hexyl group in which six carbon atoms are connected in series.

It is also possible that the triphenylphosphoric acid is linked to the 9th carbon of the purine ring of the 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine. That is, the present inventors confirmed that when the mitochondria-penetrating moiety (TPP) and / or the connecting moiety is linked to imidazole 1 carbon of the purine ring as shown in the following formula (6) Respectively.

Accordingly, the compound according to the present invention is preferably represented by the following formula (6).

[Chemical Formula 6]

The present inventors have studied and developed a novel purine-based 8- (6-iodo-benzo [1,3] dioxol (6-Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine was used as a molecular chaperone suppressor, and triphenylphosphoric acid as a mitochondria-penetrating moiety (TPP), the present inventors have accomplished the present invention after confirming that the inhibitory effect of molecular chaperone (eg, Hsp90 and / or TRAP1) increasing in tumor cells is excellent and more stable and works at a low concentration.

In other words, the biochemical / cell physiological characteristics of the inhibitory substances targeted to mitochondria and the extensive information on the functional groups in animals are collected and based on this, the structure / function analysis of the Hsp90 inhibitor using computer modeling, Lt; RTI ID = 0.0 > Hsp90 < / RTI > inhibitors and / or mitochondrial TRAP1 inhibitors that can be delivered.

Specifically, a purine-based compound, which is more stable and inferior to the ansamycin or geldanamycin-based inhibitors used in conventional antitumin gamitrinib, is used as a molecular chaperone inhibitor Respectively. For example, based on the PU-H71 and BIIB-021, which are used as Hsp90 inhibitors, as shown in the following flowcharts 1 and 2, and based on the computer modeling, an optimum position , TPP-PU and TPP-BIIB, which are targeted to mitochondria, were prepared by linking triphenylphosphoric acid (TPP) among various mitochondria-penetrating moieties to PU-Br and BIIB-Br as intermediates .

[Flowchart 1]

[Flowchart 2]

However, based on the same or similar purine-based compounds as TPP-PU and TPP-BIIB described above, the same triphenyl phosphoric acid (TPP) is used here with the same or similar linker (linking moiety) Was used as a mitochondria-penetrating moiety, the TPP-PU was excellent in anticancer efficacy, but the TPP-BIIB had poor anticancer efficacy.

Namely, as can be seen from the examples and experimental examples to be described later, 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- TPO-PU (SMTIN-P01), which utilizes -Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine, was tested for activity of PU-H71, which is used as a conventional Hsp90 inhibitor, (Fig. 2D), which was superior to TPP-BIIB designed on the basis of the antitumor effect, and had a similar or higher effect than the antitumor activity of gamitrinib.

According to this, even if triphenyphosphoric acid (TPP) is connected to a purine-based compound, all compounds do not exhibit excellent activity, and linkers and / Or its connection sites are different from each other. That is, the TPP-BIIB was targeted to mitochondria using triphenylphosphate (TPP) as a mitochondria-penetrating moiety, but the inhibitory activity against the target protein was weak, It is judged that the linker and / or the connection site thereof did not match BIIB-021.

The present invention has the effect of providing an anti-tumor composition that is superior in inhibiting the molecular chaperone (e.g., Hsp90 and / or TRAP1) that is increased in tumor cells and is more stable and acting at low concentration.

Meanwhile, another embodiment of the present invention includes a composition as described above, wherein the 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine binds to Hsp90, Of the Hsp90 inhibitor.

The compounds and / or compositions according to the present invention are useful as a mitochondrial-targeted chaperone inhibitor in the treatment of diseases associated with unregulated cell proliferation during unregulated cell proliferation (e.g., at tumor formation and in cancer) . By way of example, tumors treated by the present invention may be associated with common cancers, and it is possible that the present invention is an inhibitor that binds to Hsp90 and inhibits said Hsp90.

And, the inhibitor according to the present invention may be included in a pharmaceutical composition, and such composition may typically comprise an active compound and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" may include solvents, suspending media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, which are compatible with pharmaceutical administration. Supplementary active compounds may also be included in the compositions. Also included are pharmaceutical compositions per se, and pharmaceutically acceptable salts of the compounds described herein. It is well known that non-toxic addition salts of pharmacologically active amine compounds are not different in their activity from their free bases.

Yet another embodiment of the present invention is a mitochondrial TRAP1 inhibitor comprising the above-mentioned composition, wherein the compound inhibits TRAP1 protein existing in the mitochondria through the mitochondria.

The TPP-PU according to the present invention as described above is designed to target PU-H71, which is used as an Hsp90 inhibitor, to mitochondria, but has a target protein inhibitory activity remarkably superior to that of PU-H71. In addition, most of the conventional Hsp90 inhibitors such as PU-H71 inhibit the targets in the cytoplasm, and there was almost no inhibitory activity against TRAP1 and the like inside the mitochondria. However, TPP-PU according to the present invention efficiently And inhibits TRAP1 and the like present therein, so that the drug mechanism and drug efficacy are significantly different from the conventional inhibitors such as PU-H71.

Still another embodiment of the present invention is a method for inducing the death of cancer or tumor cells, which comprises the step of administering the above composition to a subject.

The method comprises administering to a subject in need thereof a therapeutically effective amount of a compound, composition and / or inhibitor according to the present invention, or a subject determined to be in need of such treatment.

Further, another embodiment of the present invention is a method for producing 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- 1,3,5-dioxol-5-ylsulfanyl) adenine by linking moieties or linking moieties with a mitochondria-penetrating moiety, triphenylphosphate (TPP) Lt; RTI ID = 0.0 > anti-tumor < / RTI >

Mitochondria-penetrating moieties and chaperone suppressors may be joined by synthetic linkers so that they can be made as a single protein chain, using recombinant methods known in the art. For example, a chaperone inhibitor of the invention can be functionally linked to one or more mitochondrial penetration moieties (by chemical coupling, gene fusion, non-covalent linkage, or other methods).

Here, the linking may be performed by reacting 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine represented by the following formula 4 with 1-bromo- (1-Bromo-6-triphenylphosphonium-hexyl bromide) is preferably reacted.

[Chemical Formula 4]

The present invention may be better understood by the following examples, which are for the purpose of illustrating the invention and are not intended to limit the scope of protection defined by the appended claims.

Example  One: TPP - PU  Preparation of compounds

The TPP-PU compound according to the present invention was prepared according to the following reaction formula. The TPP-PU compound (SMTIN-P01) according to the present invention is designed to conjugate between PU-H71 and the mitochondrial target moiety triphenylphosphonium by comparing the cocrystal structure of PU-H71 with Hsp90 and TRAP1 .

[Reaction Scheme]

Example  1-1: Purchasing materials

4,5,6-triaminopyrimidine sulfate was purchased from Eastern Sources. Sodium bicarbonate, acetic acid, hydrochloric acid, sodium nitrate, urea, potassium iodide, magnesium sulfate, sodium metabisulfite and cesium carbonate were purchased from Samchun Pure Chemical Co., LTD. N-dimethylforamide, copper (I) iodide, sodium tert-butoxide, N-iodosuccinimide and 1,6-dibromohexane were purchased from Sigma-Aldrich . 2,9-Dimethyl-1,10-phenanthroline hemihydrates were purchased from Alfa Aesar. 1,6-Dibromohexane was purchased from Tokyo Chemical Industry Co., LTD.

Example  1-2: 8- Mercaptoadenine Manufacturing

In the above reaction formula, a compound of formula (8-Mercaptoadenine) was prepared. Namely, 4,5,6-triaminopyrimidine sulfate (5.35 g, 24 mmol), NaHCO ₃ (10.08 g, 120 mmol), H ₂ O (90 mL), CS ₂ (14.43 mL, 240 mmol), and EtOH (45 mL) was refluxed for 3 days. After cooling, excess CS ₂ and solvent were removed with a rotary evaporator. The resulting mixture was dissolved in ethanol and H ₂ O and filtered. 4 ml of acetic acid was added to the filtrate. The white precipitate was collected, dried (2.34 g, 58.33%) and used further. ^{1 H NMR (400MHz, DMSO-} d 6) δ 13.00 (s, 1H), 12.02 (s, 1H), 8.04 (s, 1H), 6.73 (bs, 2H)

Example  1-3: 5- Iodo - benzo [1,3] dioxole

In the above reaction scheme, the compound of formula (5-Iodo-benzo [1,3] dioxole) was prepared. In other words, 3,4- (methylenedioxy) aniline (5 g, 36.46 mmol) was placed in an Erlenmeyer flask and dissolved in 35% HCl (32 mL) and ice (96 g). To this was added a chilled solution of NaNO ₂ (5.3 g, 76.8 mmol) mixed in water (30 mL) over 10 min and stirred for an additional 10 min. Urea (1.66 g, 27.6 mmol) was added to the mixture followed by the addition of cooled KI (17.9 g) mixed with water (20 mL) after 10 min. The mixture was stirred overnight. The reacted mixture was then extracted with CH ₂ Cl ₂ , dried over MgSO ₄ and concentrated. The crude product was purified by silica gel column chromatography using hexane as an eluent to obtain the desired aryliodide (4.29 g, 47.43%). The remaining color was removed by washing with Na ₂ S ₂ O ₄ . _{^{1 H NMR (400MHz, CDCl 3}} ) δ 7.11-7.13 (m, 2H), 6.58 (d, 2H), 5.94 (s, 2H)

Example  1-4: 1- Bromo -6- triphenylphosphonium - hexyl bromide Manufacturing

(1-Bromo-6-triphenylphosphonium-hexyl bromide) was prepared in the above reaction scheme. In other words, Triphenylphosphine (6.45 g, 24.59 mmol) and 1,6-dibromohexane (18.9 mL, 122.9 mmol) were mixed in a round-bottom flask fitted with a reflux condenser and heated at 90 & And heated in an oil bath. The flask was cooled to room temperature and after 3 hours two layers were obtained. The monophosphonium salt was formed as a sticky solid on the floor, and excess liquid dibromide remained on top. The dibromide was decanted off and the monophosphonium salt was dissolved in CH2Cl2 and then precipitated with ether to remove unreacted triphenylphospinine. The paste-like solid was dried in vacuo and then purified by column chromatography on silica gel (EtOAc / CH ₂ Cl ₂ / MeOH at 4: 4: 1). _{^{1 H NMR (400MHz, CDCl 3}} ) δ 7.65-7.81. (m, 15H), 3.99-3.91 (m, 2H), 1.38-1.81 (m, 9H)

Example  1-5: 8- ( Benzo [1,3] dioxol-5- ylsulfanyl ) adenine Manufacturing

In the above reaction formula, a compound of formula (8- (Benzo [1,3] dioxol-5-ylsulfanyl) adenine) was prepared. To a solution of 8-Mercaptoadenine (500 mg, 2.99 mmol), 2,9-Dimethyl-1,10-phenanthroline hemihydrate (65 mg, 0.299 mmol), CuI (60 mg, 0.299 mmol), NaO- mmol), 5-iodo-benzo [1,3] dioxole (2.23 g, 8.97 mmol) and DMF (30 mL) were mixed in a round bottom flask. The reaction vessel was sealed with parafilm, placed in an oil bath (110 ° C) and stirred for 24 hours. The solvent was removed on a rotary evaporator and the crude product was purified by column chromatography on silica gel (EtOAc / CH ₂ Cl ₂ / MeOH at 4: 4: 1). ^{1 H NMR (400MHz, DMSO-} d 6) δ 8.05 (s, 1H), 7.11 (bs, 2H), 7.03 (s, 1H), 6.95 (s, 1H), 6.06 (s, 2H)

Example  1-6: 8- (6- Iodo - Benzo [1,3] dioxol-5- ylsulfanyl ) adenine Manufacturing

(8- (6-Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine) was prepared in the above reaction scheme. To a mixture of 8- (Benzo [1,3] dioxol-5-ylsulfanyl) adenine (500 mg, 1.736 mmol), acetonitrile (3.5 mL), TFA (675 L), and NIS (2.34 g, 10.41 mmol) And stirred for 24 hours. The solvent was removed with a rotary evaporator. The mixture was directly purified by column chromatography on silica gel (EtOAc / CH ₂ Cl ₂ / MeOH at 4: 4: 1). _{^{1 H NMR (400MHz, CDCl 3}} ) δ 8.19 (s, 1H), 7.44 (s, 1H), 7.20 (s, 1H), 7.03 (bs, 1H), 6.09 (s, 2H)

Example  1-7: TPP - PU Manufacturing

(TPP-PU) was prepared in the above reaction scheme. Namely, 8- (6-Iodo-Benzo [1,3] dioxol-5-ylsulfanyl) adenine (400 mg, 0.97 mmol), dried Cs ₂ CO ₃ (1.26 g, 3.86 mmol) and 1-Bromo-6-triphenylphosphonium-hexyl bromide (1.95 g, 3.86 mmol) were mixed in a round bottom flask. The reaction vessel was dried in vacuo. To this was added anhydrous DMF. The mixture was stirred at 80 < 0 > C for 24 hours. The solvent was removed with a rotary evaporator. The mixture was purified by column chromatography (EtOAc / CH ₂ Cl ₂ / MeOH at 4: 4: 1 followed by EtOAc / CH ₂ Cl ₂ / MeOH at 2: 2: 1). Then, it was further purified using high performance liquid chromatography. ^{1 H NMR (400MHz, MeOD)} δ 8.28 (s, 1H), 7.89-7.71 (m, 15H), 7.45 (s, 1H), 7.25 (s, 1H), 6.08 (s, 2H), 4.29 (m, 2H), 3.34-3.42 (m, 3H), 1.87 (m, 2H), 1.67 (m, 4H), 1.42 (m, 2H); MS m / z 758.07 (M ^< ^{+ &} gt ^; ) [

Comparative Example  One: TPP - BIIB Manufacturing

After preparing BIIB-Br from BIIB-021 according to the above-mentioned flowchart 2, TPP-BIIB was prepared. That is, Chloroadenine 1 and methylbromopyridine were reacted to obtain intermediate 2 after regioisomer separation. Intermediate 3 was obtained by reacting with 1-bromohexanol to introduce the linker hexyl group. To introduce TPP (triphenylphosphate) group, BIIB-Br was synthesized by substituting alcohol with bromide and TPP-BIIB was synthesized by reacting with TPP.

Experimental Example  1: Analysis method

In the following experimental examples, the TPP-PIB compound (SMTIN-P01) prepared in Example 1 and the TPP-BIIB prepared in Comparative Example 1, DMSO, antitrynip Gamitrinib) and PU-H71, respectively.

Experimental Example  1-1: Cell viability ( cell viability ) analysis

Cells (5 × 10 ³ cells / well) were cultured overnight in 96-well plates and treated with drug for 24 hours. To measure the cell viability, the cells were exposed to 3, 4,5-dimethyl-thyzoyl-2-yl) 2,5 diphenyltetrazolium bromide (MTT) and absorbed at 595 nm using an Infinity M200 microplate reader (TECAN) Crystallized formazan was quantified by measurement. Absorbance data were compared to those of the vehicle control and expressed as percent viability.

Experimental Example  1-2: Xenograft Tumor Model ( Xenograft tumor models )

All animal-related experiments were approved by UNIST (IACUC-12-003-A). Sterilized (suspended) was suspended in (sterile) PBS (200㎕) 22Rv1 (7 × 10 6) subcutaneously injecting the cells into 6-week-old BALB / c nu / nu male mice (Japan SLC Inc.) on both sides (both flanks) And grown to an average volume of about 100 mm < ^{3 &} gt ;. Mice were randomly divided into 4 groups (2 tumors / mouse, 4 mice / group). The drug dissolved in PBS 20% Cremophor EL (Sigma) was intraperitoneally injected. The mice were given 10 mg / kg of drug every day. Tumors were measured daily with a caliper and the volume of the tumor was calculated as: V = 1/2 × (width) ² × length.

Experimental Example  1-3: Mitochondrial membrane potential imaging ( Imaging mitochondrial membrane potential )

Using HeLa cells 37 ℃ and 5% CO ₂ In the condition 10% FBS and 1% penicillin / a streptomycin supplemented DMEM (Lonza), the Lab Tek II slide chamber to 40-80% confluency when (confluency) Seeded. Cells were incubated with 100 nM tetramethylrhodomine methyl ester (TMRM) and drug for 6 h and analyzed using a FV1000 laser confocal scanning microsope (Olympus). All analyzes were performed under the same conditions.

Experimental Example  2: Analysis results

Experimental Example  2-1: SMTIN - P01  Wow Gametrinib  Tumor activity comparison

2 shows the activity of the TPP-PU compound (SMTIN-P01) according to a preferred embodiment of the present invention,

(A) The drug affects the mitochondrial membrane potential. TMRM-loaded HeLa cells were cultured with 20 μM of PU-H71, gamitrinib, SNTIN-P01, and TPP-BIIB for 30 min and analyzed by confocal microscope. White bar = 20 μm.

(B) Hsp70 upregulation and client depletion. Cells were treated with 5 μM of drug for 24 h and analyzed by Western blot.

(C) Induces the stress response of mitochondria. Cells were treated with 2 [mu] M of drug for 24 hours and analyzed by Western blot.

(D) SMTIN-P01. Gamitrinib, SMTIN-P01, PU-H71 and TPP-BIIB were immunized with kidney (ACHN), cervix (HeLa), ovary (SK-OV3), prostate (22Rv1) various human cancer cell lines derived from liver (SK-HEP-1), brain (A172), lung (H460), and breast (MDA-MB-231) Lt; / RTI > Cell viability was analyzed by MTT assay. Data are mean ± SEM obtained from two independent triplicated experiments.

As shown in Figure 2, inactivation of mitochondrial Hsp90 and TRAP1 activates cyclophilin-D and opens a permeability transition pore (KPP) (Kang et al., 2007; Kang et al., 2009 ). SMTIN-P01, similar to camitri nip, was able to induce mitochondrial membrane depolarization, while PU-H71 and TPP-BIIB gave no effect on the mitochondrial membrane potential (ΔΨm) (FIG. 2A).

This indicates that none of the mitochondrial targeting moiety (TPP) and the cytoplasmic Hsp90 inhibitor significantly affect the mitochondrial membrane potential ([Delta] [tau] m). PU-H71 treatment induced depletion of client proteins and up-regulation of Hsp70, and the response to Hsp90 inhibition was typical signature (Guo et al., 2005 ; Trepel et al., 2010) and no activity by SMTIN-P01 treatment (Fig. 2B).

Compared with Gamitrinib, SMTIN-P01 not only initiates mitochondrial unfolded protein responses that lower the cell death threshold in many cancer cells (Fig. 2C), but also exhibits remarkable activity in cytotoxicity against various cancer cell types (Fig. 2D). Interestingly, SMTIN-P01 is a parental Hsp90 inhibitor PU-H71; TPP-BIIB exhibits stronger cytotoxicity in all cancer cells than in marginal cytotoxic (Fig. 2D).

These results indicate that the mitochondrial targeting of PU-H71 produces a cytotoxic agent with a very different mechanism compared to the parental compound.

Experimental Example  2-2: SMTIN - P01 of In vivo Tumor activity and pharmacokinetics pharmacokinectics )

FIG. 3 shows the in vivo activity of a TPP-PU compound (SMTIN-P01) according to a preferred embodiment of the present invention,

(A) Indicates inhibition of tumor growth in vivo. 10 mg / kg of drug was injected daily into nude mice transplanted with 22Rv1 cells. Tumor size was measured with a caliper. Eight tumors and four mice per group were tested. Data are mean ± SEM.

(B) tumor weight. (A) At the end of the experiment, the tumor tissue was recovered and weighed. Data are mean ± SEM.

As shown in FIG. 3, SMTIN-P01 inhibited tumor growth more than gamitrinib (Fig. 3A) in xenotransplantation experiments using prostate cancer cells (22Rv1), and the tumor weighed PU-H71 / gamitrinib (Fig. 3B). ≪ tb > < TABLE >

in Considering the undefined activity in vitro (Fig. 2), the results indicate that SMTIN-P01 is more in vivo activity, which may be related to the behavior of the parental compounds, PU-H71 and the geldanamycin derivative (the former accumulating more in the tumor and the latter accumulating more in normal cells) (Caldas-Lopes et al., 2009; Eiseman et al., 2005).

While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be apparent to those skilled in the art that changes may be made.

Claims (12)

The molecular chaperone inhibitor 8- (6-Iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine (8- (6-Iodo-Benzo [ 5-ylsulfanyl) adenine and mitochondria-penetrating moiety triphenylphosphate (TPP) are linked by a linker which is a carbon chain containing 6 carbon atoms, Or a pharmaceutically acceptable salt thereof,
The compound is represented by the following general formula (6)
[Chemical Formula 6]

The 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine binds to Hsp90 (heat shock protein 90)
Wherein said compound is a mitochondrial TRAP1 inhibitor that inhibits TRAP1 (Tumor Necrosis Factor Receptor-Associated Protein-1) protein that penetrates mitochondria and is present inside said mitochondria.
delete delete delete delete delete delete delete delete delete (6-Iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine as a molecular chaperone inhibitor ) With a mitochondria-penetrating moiety, triphenylphosphate (TPP), by a linker that is a carbon chain comprising six carbon atoms, to the mitochondria-targeted anti-tumor composition ≪ / RTI >
12. The method of claim 11,
The step of coupling by the linker comprises reacting 8- (6-iodo-benzo [1,3] dioxol-5-ylsulfanyl) adenine represented by the following formula 4 with 1-bromo (1-Bromo-6-triphenylphosphonium-hexyl bromide) is reacted with the compound of formula (I).
[Chemical Formula 4]

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