Protected HDAC (histone deacetylase) inhibitors

Abstract

The present invention relates to protected HDAC inhibitor compounds of formula I, 1 , Ar 2 , X, R 1 and R 2 is as defined herein. In aspects, the invention relates to uses of the compounds and methods of deprotecting the compounds.

Description

[Background technology]

[0001] Histone acetylation / deacetylation plays a crucial role in the transcriptional regulation of eukaryotic cells. The acetylation status of histone and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyltransferases (HATs). Histone deacetylases (HDACs) belong to a family of enzymes that remove acetyl groups from the ε-amino moiety of the lysine group of histone and non-histone proteins. HDAC inhibitors (HDACi) inhibit the activity of HDAC enzymes. Due to the biological importance of HDACs, their inhibition has clinical significance, and HDAC inhibition has emerged as a key therapeutic strategy, particularly in the treatment of cancer, neurodegenerative diseases, inflammatory diseases, and neuropathy.

[0002] HDAC enzymes are classified based on the homology of their accessory domains to yeast histone deacetylases and are currently divided into four main groups. Class I HDAC1, -2, -3, and -8 are associated with yeast RPD3 deacetylase; Class IIA includes HDAC4, -5, -7, and -9; Class IIB-6 and -10 are associated with the yeast Hda1 (histone deacetylase 1) gene; Class III (also known as sirtuins) are associated with the Sir2 gene and include SIRT1-7; Class IV, which includes only Class IV HDAC11, has the functionality of both Class I and Class II.

[0003] Typical HDAC inhibitors act only on class I, II, and IV HDACs by binding to the zinc-containing catalytic domain of HDACs. These HDAC inhibitors can be further classified based on the chemical site that binds to the zinc ion (excluding cyclic tetrapeptides that bind to the zinc ion via a thiol group). Examples include hydroxamic acids (or hydroxamate salts) such as trichostatin A; cyclic tetrapeptides (such as trapoxin B) and depsipeptides; benzamides; electrophilic ketones; and fatty acid compounds such as phenylbutyric acid and valproic acid.

[0004] Hydroxamic acid constitutes the largest class of HDAC inhibitors.

[0005] Examples of hydroxamic acid-based HDAC inhibitors include vorinostat (suberoylanilide hydroxamic acid, SAHA), bellinostat, panobinostat, zivinostat, prasinostat, xinostat, and abexinostat.

[0006] While HDAC inhibitors play an important therapeutic role, the instability of HDAC inhibitors such as hydroxamic acid poses challenges in terms of storage stability, solubility, formulation, and manufacturing.

[0007] One object of the present invention is to address or mitigate one or more of these problems. One object of the present invention is to provide a protected HDAC inhibitor, in particular a protected hydroxamic acid. One object of the present invention is to provide a protected HDAC inhibitor that may exhibit improved stability. One object of the present invention is to provide a protected HDAC inhibitor that may exhibit improved solubility. Embodiments of the present invention relate to methods for protecting and / or deprotecting HDAC inhibitors, such as hydroxamic acid-based HDAC inhibitors. In embodiments, the deprotection step may be carried out in situ within cells and tissues, i.e., by endogenous enzymes. [Overview of the project]

[0008] This invention relates to the use of esters as protecting groups. Specifically, it relates to the use of esters as protecting groups for HDAC inhibitors. In this process, functional groups such as the hydroxam group of the HDAC inhibitor are protected as esters. These esters can then be removed in the presence of an enzyme, which may be an endogenous enzyme. Upon removal of the ester, the functional group is deprotected. This deprotection can occur in situ, meaning the enzyme can be an endogenous enzyme, and deprotection can occur when the HDAC inhibitor enters a cell. The cell may be a mammalian cell. In embodiments, deprotection occurs via the action of an endogenous enzyme on the protected ester group when the HDAC inhibitor enters a target cell. This can result in a "clean" activation of HDAC inhibition in situ.

[0009] Accordingly, the present invention encompasses protected HDAC inhibitors. Protected HDAC inhibitors can be protected by an ester group. This can function as a prodrug. It can function as an HDAC inhibitor that can be deprotected in situ. In this embodiment, the protected HDAC inhibitor is metabolized to its active form after administration. This has significant advantages in terms of storage, formulation and manufacture, etc. As used herein, the term “prodrug” means a compound that is administered in an inactive form and converted to its active form in vivo through chemical, biochemical or physiological processes.

[0010] Advantageously, the ester can be selected to enable activation of HDAC inhibition only in the target tissue through enzymatic removal. That is, the protecting group can be selected so that enzymatic removal occurs only in the cells, tissues, or regions of the body in question, i.e., in the cells, tissues, or regions of the body containing the complementary enzyme. As will be understood by those skilled in the art, the protecting group can be further modified to adjust properties such as half-life, solubility, and targeting to suit the properties of the target tissue.

[0011] Suitable protecting groups include, but are not limited to, acetate (C1-C9) esters, hydroxyacetate (C1-C9) esters, methoxyacetate (C1-C9) esters, phenylacetate, propionate, butyrate, salicylate, pyruvate, lactate ester, citrate ester, PEG ester, glycerol ester, peptide ester, for example mono-, di-, and triglycine esters, phosphate esters, sulfonate esters, and carbonates, for example tetraethylene glycol, O-glycosyl ether, and O-glycosyl ester.

[0012] In one embodiment, the protecting group is an acetate (C1-C9) ester. In another embodiment, the protecting group is an acetate C1 ester.

[0013] Enzymes suitable for deprotection include, but are not limited to, lipases, lactases, esterases, amylases, cytochrome P450s, glycosidases, such as β-glucuronidase, sucrase, and hyaluronidase, peptidases, phosphatases, and sulfatases.

[0014] In one embodiment, the enzyme is lipase or lactase.

[0015] In one embodiment, the HDAC inhibitor is a photoactive HDAC inhibitor, and it will be understood that the present invention is not limited thereto and is more broadly applicable to HDAC inhibitors and HDAC inhibitors.

[0016] In one embodiment, the HDAC inhibitor is a photoactive HDAC inhibitor. "Photoactive HDAC inhibitor" means an HDAC inhibitor that has dual cytomodulatory activity, i.e., photoactivated cell killing in parallel with HDAC inhibitory activity such as biochemical effects and / or targeting.

[0017] Exemplary forms of such compounds are shown below. [ka]

[0018] Examples of such HDAC inhibitors are the compounds disclosed herein. The inventors have demonstrated that when such photoactive HDAC inhibitors are protected according to the present invention, their cytotoxic function is maintained. A delay in biological activity has been observed during deprotection.

[0019] In one aspect of the present invention, a compound of formula I is provided. [ka] During the ceremony, R 1 is H or an alkyl group having 1 to 10 carbon atoms; R 2 PZ is an alkyl group with 1 to 15 carbon atoms. Ren It is a base and arbitrary N atom, -C ( =O )- and -NH ( C=O ) - one or more of the following It has been replaced with; Z is as follows: [ka] In the formula, R 3 These are H, C1-C9 alkyl, -CH2OH, -CH2OCH3, -Ph, -C6H4OH, -CH(CH3)OH, -C(CH2COOH)2OH, -C(=O)CH3, -CH2NH2, CH2NH(C=O)CH2NH2, or -CH2NH(C=O)CH2NH(C=O)CH2NH2; or R 1 and R 2 It has 5 or 6 members and forms part of a heterocyclic group Y that is substituted with PZ, where P is as defined above and Z is as defined above; Ar1 and Ar2 are independently selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole groups; X is -C=CC(=O)OR 4 And R 4 This is an alkyl group having 1 to 10 carbon atoms, which may be optionally substituted with one or more oxygen atoms.

[0020] The compound of the present invention has the general structure shown in Formula I above.

[0021] The term "heterocyclic group having 5 or 6 members" refers to a monocyclic ring group that includes 5 or 6 ring members and optionally contains one or more heteroatoms selected from the group consisting of N, S, SO, SO2, O2, and O, in addition to the nitrogen atom of formula I. The term "heterocyclic group" includes aromatic, partially unsaturated, and saturated ring systems. Examples of non-aromatic groups, but not limited to these, include piperazinyl, morpholinyl, thiomorpholinyl, dioxidethiomorpholinyl, pyrrolidine-1-yl, and pyrrolidine-3-yl groups. Examples of aromatic (heteroaryl) groups, but not limited to these, include pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, indolyl, and benzothiadiazolyl groups. In one embodiment, the heterocyclic group is a saturated ring system. According to formula I, the ring system is substituted with PZ. In one embodiment, PZ is located at position 4 relative to the nitrogen atom of formula I.

[0022] As used herein, the term “alkyl” refers to a fully saturated, branched, unbranched, or cyclic hydrocarbon moiety, i.e., a primary, secondary, or tertiary alkyl, or, where appropriate, a cycloalkyl or a cycloalkyl-substituted alkyl. Unless otherwise stated, an alkyl group contains 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably 1 to 4 carbon atoms. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl; 2,2-dimethylpentyl; 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0023] In one embodiment, R 3 is C1-C6 alkyl.

[0024] In one embodiment, R 3 is C1-C3 alkyl. In one embodiment, R 3 is -CH3.

[0025] In one embodiment, Ar1 is a thiazole or phenyl group.

[0026] In one embodiment, Ar2 is a pyridine, thiophene or furan group.

[0027] In one embodiment, Ar1 is selected from a thiazole or phenyl group, and Ar2 is selected from a pyridine, thiophene or furan group.

[0028] In one embodiment, R 1 and R 2 form part of a heterocyclic group Y. The heterocyclic group Y is substituted with P-Z, where P is an alkyl group having 1 to 15 carbon atoms, optionally Ren with an atom, - 、N C=O ( C=O )- and -NH ( C=O ) - one or more of the following substituted; Z is as follows,

Chemical formula

[0029] In one embodiment, the substituent P-Z is at the 4-position relative to the nitrogen atom of formula I.

[0030] R1 and R 2 In embodiments where Y forms part of a heterocyclic group Y, Y is piperazine.

[0031] Y may be any of the following: [ka]

[0032] If Y is piperazine, then P is -C (=O). - C1~C replaced by 15 Alki Ren It may be a base. In one embodiment, P is -C(=O)(CH2)6 - That is the case.

[0033] In equation I, X is -C=CC(=O)OR 4 And R 4 This is an alkyl group containing 1 to 10 carbon atoms, which are optionally substituted with one or more oxygen atoms.

[0034] In one embodiment, R 4 These are C1-C6 alkyl groups.

[0035] In one embodiment, R 4 These are -CH3, -C(CH3)3, or -CH2CH(CH3)2.

[0036] Optionally, R 1 and R 2 It does not form a heterocyclic group Y. In this embodiment, R 1 R is H or an alkyl group having 1 to 10 carbon atoms; 2 PZ is arbitrary, where P is arbitrary. N atom,- ( C=O )- and -NH ( C=O ) - one or more of the following Alkyl compounds containing 1 to 15 carbon atoms that are substituted with Ren It is the basis; Z is as follows: [ka] In the formula, R 3 is H, C1-C9 alkyl, -CH2OH, -CH2OCH3, -Ph, -C6H4OH, -CH(CH3)OH, -C(CH2COOH)2OH, -C(=O)CH3, -CH2NH2, -CH2NH(C=O)CH2NH2, or -CH2NH(C=O)CH2NH(C=O)CH2NH2. In this embodiment, R 1 R may be a C1-C3 alkyl group. In one embodiment, R 1 It is -CH3.

[0037] R 2 If P is PZ, then P is -NHC (=O) - C1~C replaced by 15 Alki Ren That's fine.

[0038] In one embodiment, P is -(CH2)5NHC(=O)(CH2)6 - That is the case.

[0039] In one embodiment, R 4 is -(CH2CH2O) n CH3 is an integer from 1 to 8. In one embodiment, R 4 It is -(CH2CH2O)3CH3.

[0040] In one embodiment, R 4 is -(CH2CH2O) n CH3 is an integer from 1 to 8, preferably R 4 It is -(CH2CH2O)3CH3, and R 1 and R 2 It does not form a heterocyclic group.

[0041] In one embodiment, the compound of formula I is selected from compounds 92, 93, 101, and 102. [ka] [ka]

[0042] The compounds according to the present invention are inherently fluorescent. According to the present invention, the compounds can be used in fluorescence imaging.

[0043] In one embodiment, the present invention relates to the use of a compound of formula I in the generation of reactive oxygen species (ROS) when the compound of formula I is activated by light.

[0044] Triplet-state photosensitizers (PSs) typically possess a light-harvesting region that performs a dual function of light harvesting and intersystem crossing, where single-state electrons non-radiatively transition to the triplet state. When the triplet excited state disappears, it can lead to the formation of reactive oxygen species (ROS), radicals from ground-state molecular oxygen, or direct chemical reactions with surrounding molecules. Localized ROS production is an immunodefense strategy employed in both animal and plant systems in response to pathogen attacks. Within animal, plant, fungal, and bacterial cells, ROS elicit various regulatory effects depending on the rate and extent of their production; apoptosis is observed at high concentrations, while stimulus responses are often observed at low concentrations (Guo et al., Stem Cells Dev. 2010, 19, 1321-1331).

[0045] Photodynamic therapy (PDT) utilizes the ability of photosensitizers to generate reactive oxygen species (ROS) to destroy cancer cells, pathogenic microorganisms, and / or unwanted tissues, usually through apoptosis. Typically, photosensitizing compounds are excited near or within specific target tissues or conditions (e.g., microbial infections, neoplasia, tumors), triggering the generation of large amounts of ROS and subsequent destruction of that tissue. Low ROS levels can induce cell proliferation, leading to applications in wound healing or more general tissue regeneration therapies.

[0046] Therefore, PDT relies on targeting photosensitive compounds to accumulate in desired locations, such as cells in affected tissue, and then locally delivering light to activate ROS production. While compounds used in PDT are well known, they often have various drawbacks, including small absorption peaks, which make photoactivation difficult, especially in the case of large tumors where light penetration is difficult; long biological half-lives; low pharmacological properties such as low water solubility; and low targeting ability (i.e., poor ability to target and accumulate in specific tissues or cells, leading to significant off-target damage).

[0047] Advantageously, the compounds of the present invention are biologically inactive in their inactive state, but generate ROS when irradiated with low-to-medium energy short-wavelength visible light.

[0048] Therefore, compounds of formula I can be used to generate reactive oxygen species (ROS), thereby controlling cell development, i.e., cell proliferation, differentiation, and apoptosis, leading to a variety of therapeutic and non-therapeutic applications. Compounds of formula I are particularly advantageous for use in applications influenced by ROS control because they exhibit efficient targeting and can minimize off-target effects. Furthermore, they can be formulated to suit different cell types, enabling selective targeting effects.

[0049] Therefore, in some embodiments, the present invention relates to the use of the compounds or conjugates of the present invention in photodynamic therapy (PDT).

[0050] ROS generation can be controlled based on therapeutic needs, such as inducing apoptosis for cell excision, causing proliferation in wound healing, or a combination of these. For example, in wound care, high levels of ROS may be initially induced, leading to apoptosis of bacterial and / or fungal cells, followed by lower levels of ROS to aid in skin regeneration.

[0051] Accordingly, the present invention relates to a method for treating a patient having a disease or condition that would benefit from HDAC inhibition, the method comprising administering a therapeutically effective amount of a compound of formula I or a conjugate thereof to the patient, wherein the compound of formula I is metabolized to an active form in vivo.

[0052] In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of formula I in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0053] Advantageously, compounds of formula I are protected as esters, thereby giving them stability. Protected compounds are easier to store. Protected compounds may exhibit improved solubility. Activation of the compound involves the removal of the protecting group.

[0054] One aspect of the present invention relates to a method for deprotecting a compound of formula I, the method comprising contacting the compound of formula I with an enzyme.

[0055] The enzyme may be an endogenous enzyme. Suitable enzymes for deprotection include, but are not limited to, lipases, lactases, esterases, amylases, cytochrome P450s, glycosidases, such as β-glucuronidase, sucrase, and hyaluronidase, peptidases, phosphatases, and sulfatases.

[0056] In one embodiment, the enzyme is lipase or lactase.

[0057] Advantageously, the ester protecting group can be selected so as to enable activation of HDAC inhibition only in the target tissue by enzymatic removal; that is, the protecting group can be selected so that enzymatic removal occurs only in those cells, tissues, or regions of the body in question, i.e., in the cells, tissues, or regions of the body containing the complementary enzyme. As will be understood by those skilled in the art, the protecting group can be further modified to adjust properties such as half-life, solubility, and targeting to suit the properties of the target tissue.

[0058] Suitable protecting groups include, but are not limited to, acetate (C1-C9) esters, hydroxyacetate (C1-C9) esters, methoxyacetate (C1-C9) esters, phenylacetate, propionate, butyrate, salicylate, pyruvate, lactate ester, citrate ester, PEG ester, glycerol ester, peptide ester, for example mono-, di-, and triglycine esters, phosphate esters, sulfonate esters, and carbonates, for example tetraethylene glycol, O-glycosyl ether, and O-glycosyl ester.

[0059] In one embodiment, the protecting group is an acetate (C1-C9) ester. When the ester protecting group is a C1 acetate ester, the R of the compound of formula I 3 It is -CH3.

[0060] According to one aspect of the present invention, a method for deprotecting a compound of formula I is provided, the method comprising reacting the compound of formula I with a base in the presence of a solvent.

[0061] Suitable bases for use in the method of the present invention include NaOH, LiOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, LiOMe, NaOMe, KOMe, LiOEt, NaOEt, KOEt, and LiO t Bu and KO t Bu is one example.

[0062] The solvent may be a polar solvent. Suitable solvents include methanol, ethanol, propanol, butanol, n-alcohols, isopropanol, isobutanol, sec-butanol, tert-butanol, water, tetrahydrofuran, 1,4-dioxane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0063] The method can be carried out at temperatures between 5°C and 100°C or between 15°C and 30°C. Advantageously, this method can be carried out at room temperature.

[0064] The reaction can be carried out for 30 minutes to 48 hours. In one embodiment, the reaction is carried out for 1 to 12 hours or 2 to 7 hours.

[0065] Once the reaction has occurred, the reaction mixture can be post-treated using techniques known to those skilled in the art. For example, the reaction mixture can be diluted, the combined organic matter can be washed, dried, and evaporated to obtain the deprotected compound as a crude solid.

[0066] Therefore, the present invention relates to protected HDAC inhibitors, and examples thereof include the following compounds. [ka] [ka]

[0067] In one embodiment, the protected HDAC inhibitor is compound 92, compound 93, compound 101, or compound 102.

[0068] In one embodiment, the protected HDAC inhibitor is compound 92, compound 93, or compound 101.

[0069] The following compounds 12, 13, and 14 are useful as control compounds. [ka]

[0070] One aspect of the present invention relates to a method for deprotecting a compound of formula I, the method comprising reacting the compound of formula I with a base in the presence of a solvent. The method optionally includes a purification step.

[0071] The present invention relates to a method for activating HDAC inhibition by protecting an HDAC inhibitor as an ester and removing the ester with an esterase. The esterase may be an endogenous esterase. Ester removal by the esterase can be performed in vivo.

[0072] The present invention relates to a method for treating a disorder or condition mediated by HDAC inhibition, the method comprising administering a protected HDAC inhibitor as described herein, the HDAC inhibitor being subsequently deprotected in situ.

[0073] The compound of formula I can advantageously demonstrate increased solubility, improved chemical stability and storage stability, and ease of manufacture compared to its deprotected counterpart (Figure 1).

[0074] Next, the present invention will be described as merely an example with reference to the attached drawings. [Brief explanation of the drawing]

[0075] [Figure 1] Figure 1 shows a schematic diagram of the present invention, where PG represents a protecting group. [Figure 2] Figure 2 shows an exemplary synthesis of acetate-protected hydroxamic acid (88). [Figure 3] Figure 3 shows an exemplary synthesis of a protected HDAC inhibitor (92) having photoactivatable cell-killing activity according to the present invention. [Figure 4] Figure 4 shows an exemplary synthesis of an alternative protected HDAC inhibitor (93) having photoactivatable cell-killing activity according to the present invention. [Figure 5] Figure 5 shows the synthesis of building block compound (96). [Figure 6] Figure 6 shows the synthesis of building block compound (99). [Figure 7] Figure 7 shows the synthesis of the photoactivating compound (100). [Figure 8] Figure 8 shows the synthesis of exemplary compound (101). [Figure 9] Figure 9 shows the results of a fluorescein diacetate cell viability assay measuring the viability of HaCaT keratinocytes in response to treatment with compound 92, with and without irradiation. [Figure 10] Figure 10 shows immunofluorescence imaging of HaCaT keratinocytes treated with compound 92 and EtOH, and co-treated with an anti-acetyl H3 primary antibody to detect the presence of acetylated H3 histones. [Figure 11] Figure 11 shows the amount of acetyl-H3 present in SCC-4 cells in response to treatment with compound 93 and a control compound according to the present invention at 15 minutes and 1 hour later. [Figure 12] Figure 12 shows the amount of acetyl-H3 present in SCC-4 cells in response to treatment with compounds 93 and 101 according to the present invention, as well as a control compound, after 1 hour. [Figure 13] Figure 13 shows the amount of acetyl-H3 in SCC-4 cells in response to treatment with compound 92 and the control compound according to the present invention after 15 minutes. [Figure 14] Figure 14 shows the expression levels of caspase-3 in HaCaT cells before and after photoactivation of 50 nM compound 93 and 100 nM compound 93, as well as after treatment with the control. [Figure 15] Figure 15 shows a fluorescence microscope image illustrating the colocalization of compound 101. [Figure 16] Figure 16 shows a fluorescence microscope image illustrating the colocalization of compound 93. [Figure 17] Figure 17 shows an exemplary deprotection of compound 93. [Examples]

[0076] General methodology: All light irradiation was at 405 nm and 29 mW / cm². 2 The experiment was run using an improved PhotoReact 365™ that emits light for 5 minutes at the output of [specific output]. All image analysis was performed using ImageJ software, and all graphs were created using Prism.

[0077] Cell viability assay: SCC-4 cells were seeded at 20,000 cells per well in a 96-well plate with opaque walls. The following day, before irradiating the "light-treated" plate, the plate was treated with the target compound at a certain concentration range (100 pM to 1 μM) for 1 hour. The following day, the plate was treated in one of the following ways: • After 10 minutes of treatment with propidium iodide (PI) and fluorescein diacetate (FDA), followed by washing with PBS, fluorescence was measured at 535 / 617 nm for PI and at 485 / 520 nm for FDA. After treating with a 12 mM MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) solution for 2 hours, the sample is dissolved in DMSO using an orbital shaker, and the absorbance at 540 nm is measured. After treating with XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) labeling reagent and electron coupling reagent for 4 hours, the samples were washed with PBS and the absorbance was measured at 650 nm. Cells were lysed with CellTitre-Glo® reagent on an orbital shaker for 10 minutes, and then luminescence was measured.

[0078] Microscopic examination procedure:

[0079] colocalization SCC-4 cells were seeded at 50,000 cells per well in 8-well chamber slides. The following day, the cells were treated with 1 μM of the target compound for 1 hour, then fixed with 4% paraformaldehyde (PFA) or the medium was changed to Live Cell Imaging solution. Co-staining (e.g., MitoTracker, Bodipy ER Tracker, LipidSpot 610) was applied for 30 minutes, followed by imaging with Zeiss LSCM 880.

[0080] Immunofluorescence HaCaT cells or SCC-4 cells were seeded at 50,000 cells per well in sterile coverslips containing 6-well plates. The following day, the cells were treated with the target compound for 30 minutes, and then irradiated with "light". The next day, the cells were fixed with 4% PFA, permeabilized using Triton X-100 / Tween 20, blocked with BSA / goat serum (depending on the antibody), stained with primary antibody, then secondary antibody, and then mounted on coverslips for imaging with Zeiss LSCM 880.

[0081] Immunoprecipitation SCC-4 cells were seeded at 350,000 cells per well in a 6-well plate, and the following day, the cells were treated with the target compound. After the necessary incubation, the "light" treated cells were irradiated, and all cells were lysed using RIPA buffer. SDS-PAGE was performed to transfer proteins to a nitrocellulose / PVDF membrane, followed by blocking and primary antibody staining, then secondary antibody staining. Chemiluminescent signals were imaged using the iBright Imaging System.

[0082] Example 1: Synthesis of acetate-protected hydroxamic acid 88 An exemplary synthesis of acetate-protected hydroxamic acid 88 is shown in Figure 2, and is further explained in Examples 1.1 to 1.4 below.

[0083] Example 1.1 Synthesis of 1-tert-butyl methyl 8-octanedioate 85 Compound 47 (33.0 g, 175 mmol) was dissolved in tert-butanol (250 mL), cooled to 0°C, and then di-tert-butyl dicarbonate (57.3 g, 262.5 mmol) and 4-dimethylaminopyridine (6.4 g, 52.5 mmol) were added. The resulting suspension was rapidly stirred at room temperature for 2 hours. The solution was diluted with 5% HCl and extracted with dichloromethane (DCM) (3 times). The organic matter was washed with saturated NH4Cl and H2O, dried (MgSO4), and evaporated to obtain crude red oil (58.9 g). This was purified by SiO2 chromatography (hexane / ethyl acetate (siRNA), 9:1) to obtain compound 85 as a colorless oil (29.06 g, 68%). 1 H NMR (400 MHz, CDCl3) δ 1.28 - 1.35 (m, 4H), 1.43 (s, 9H), 1.54 - 1.65 (m, 4H), 2.19 (t, J = 7.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 24.7, 24.9, 28.1, 28.7, 28.8, 34.0, 35.5, 51.4, 79.9, 173.1, 174.2.

[0084] Example 1.2 Synthesis of tert-butyl 86 7-(hydroxycarbamoyl)heptanoate Compound 85 (6.1 g, 25.0 mmol) was dissolved in methanol (MeOH) (21 mL), and then 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (11.2 mL, 75.0 mmol) and hydroxylamine (NH2OH4) (50% aqueous solution, 15.3 mL, 250 mmol) were added. The resulting solution was stirred at room temperature (RT) for 3 hours. The mixture was diluted with dichloromethane (DCM), the organic matter was washed with 5% HCl and H2O, dried (MgSO4), and evaporated to obtain crude yellow oil (3.23 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 9:1) to obtain compound 86 as a colorless oil (2.34 g, 38%).1 H NMR (400 MHz, CDCl3) δ 1.26 - 1.37 (m, 4H), 1.43 (s, 9H), 1.52 - 1.68 (m, 4H), 2.14 (s, 2H), 2.19 (t, J = 6.9 Hz, 2H)

[0085] Example 1.3 Synthesis of 7-[(acetyloxy)carbamoyl]heptanoate tert-butyl 87 Compound 86 (2.3 g, 9.37 mmol) was dissolved in dichloromethane (40 mL), then acetyl chloride (0.8 mL, 11.24 mmol) and triethylamine (1.56 mL, 11.24 mmol) were added, and the resulting solution was stirred at room temperature for 3 hours. The solution was diluted with dichloromethane, the organic matter was washed with saturated NH4Cl and H2O, dried (MgSO4), and evaporated to obtain a crude pale yellow oil (2.75 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 99:1) to obtain compound 87 as a colorless oil (1.65 g, 61%). 1 H NMR (400 MHz, CDCl3) δ 1.24 - 1.37 (m, 4H), 1.39 (s, 9H), 1.48 - 1.58 (m, 2H), 1.59 - 1.69 (m, 2H), 2.13 - 2.19 (m, 2H), 2.17 (s, 3H), 2.18 - 2.23 (m, 2H), 9.64 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 18.2, 24.7, 28.0, 28.4, 28.5, 32.6, 35.3, 80.1, 168.7, 173.3; MS(ES): m / z = 288.2 [M+H] + HRMS (ES) Calculated Value C 14 H 26 NO5 [M+H] + : 288.1805, Measured value 288.1801

[0086] Example 1.4 Synthesis of 7-[(acetyloxy)carbamoyl]heptanoic acid 88 Compound 87 (1.65 g, 5.74 mmol) was dissolved in dichloromethane (60 mL), then trifluoroacetic acid (TFA) (5 mL, 65 mmol) was added, and the resulting solution was stirred at room temperature for 18 hours. The solution was evaporated, and the crude residue was purified by SiO2 chromatography (dichloromethane / methanol, 95:5) to obtain compound 88 as a white solid (1.10 g, 83%). 1 H NMR (700 MHz, DMSO-d6) δ 1.23 - 1.28 (m, 4H), 1.45 - 1.52 (m, 4H), 2.09 (t, J = 7.4 Hz, 2H), 2.13 (s, 3H), 2.18 (t, J = 7.4 Hz, 2H), 11.53 (br, 1H), 11.95 (br, 1H); 13 C NMR (176 MHz, DMSO-d6) δ 18.1, 24.3, 24.6, 28.1, 28.2, 31.8, 33.6, 168.5, 169.7, 174.4; MS(ES): m / z = 232.1 [M+H] + HRMS (ES) Calculated Value C 10 H 18 NO5 [M+H] + : 232.1179, Measured value 232.1167

[0087] Example 2: Synthesis of protected HDAC inhibitor 92 The synthesis of an exemplary protected HDAC inhibitor 92 with photoactivatable cell-killing activity is shown in Figure 3 and further explained in Examples 2.1 to 2.4 below.

[0088] Example 2.1: Synthesis of 4-(5-bromo-1,3-thiazole-2-yl)piperazine-1-carboxylate tert-butyl 89 2,5-Dibromo-1,3-thiazole (10 g, 41.2 mmol) was dissolved in N,N-dimethylformamide (DMF) (100 mL), and 1-Boc-piperazine (10 g, 53.5 mmol) and K2CO3 (7.40 g, 53.5 mmol) were added. The resulting mixture was stirred at 70°C for 72 hours. The mixture was cooled, diluted with H2O, and extracted with ethyl acetate. The organic matter was washed with H2O and brine, dried (MgSO4), and evaporated to obtain crude oil. This was purified by SiO2 chromatography (petroleum ether / ethyl acetate, 8:2) to obtain compound 89 as a pale yellow solid (10.5 g, 74%). 1 H NMR (400 MHz, CDCl3) δ 1.46 (s, 9H), 3.38 - 3.41 (m, 4H), 3.52 - 3.55 (m, 4H), 7.06 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 28.3, 48.0, 80.4, 95.2, 140.4, 154.5, 171.5

[0089] Example 2.2: Synthesis of 1-(5-bromo-1,3-thiazole-2-yl)piperazine 90 Compound 89 (10.45 g, 30.0 mmol) was dissolved in dichloromethane (100 mL), then trifluoroacetic acid (9.2 mL, 120.0 mmol) was added, and the resulting solution was stirred overnight at room temperature. The solution was evaporated to obtain crude yellow oil (23 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 95:5) to obtain the trifluoroacetate salt (10.7 g) of the desired compound. Subsequently, this was dissolved in dichloromethane and rapidly stirred with saturated NaHCO3 for 0.5 hours. The organic matter was washed with H2O, dried (MgSO4), and evaporated to obtain compound 90 as a white solid (6.15 g, 83%). 1 H NMR (400 MHz, DMSO-d6) δ 2.73 - 2.77 (m, 4H), 3.24 - 3.27 (m, 4H), 7.18 (s, 1H); MS(ES): m / z = 248.0, 250.0 [M + H] +HRMS (ES) calculated value C7H 11 N3SBr [M + H] + : 247.9852, Measured value 247.9850

[0090] Example 2.3: Synthesis of (2E)-3-(5-{2-[2-(piperazin-1-yl)-1,3-thiazole-5-yl]ethinyl}pyridine-2-yl)prop-2-enoate methyl 91 Triethylamine (Et3N) (200 mL) was degassed by bubbling with Ar for 1 hour. Compound 90 (2.80 g, 11.3 mmol), compound 42 (2.32 g, 12.41 mmol), Pd(PPh3)2Cl2 (390 mg, 0.18 mmol), and CuI (107 mg, 0.18 mmol) were then added under Ar, and the resulting suspension was stirred at 60°C for 72 hours. The solvent was then evaporated to obtain a crude solid, which was purified twice by SiO2 chromatography (95:5 to 9:1, dichloromethane / methanol, 1% triethylamine) to obtain compound 91 as a bright orange solid (2.28 g, 57%). 1 H NMR (400 MHz, DMSO-d6) δ 2.75 - 2.82 (m, 4H), 3.35 - 3.41 (m, 4H), 3.75 (s, 3H), 6.91 (d, J = 15.7 Hz, 1H), 7.58 (s, 1H), 7.69 (d, J = MS (ES) m / z = 355.1 [M+H] + HRMS (ES) Calculated Value C 18 H 19 N4O2S [M+H] + : 355.1223, Measured value 355.1223

[0091] Example 2.4: Synthesis of (2E)-3-(5-{2-[2-(4-{7-[(acetyloxy)carbamoyl]heptanoyl}piperazine-1-yl)-1,3-thiazole-5-yl]ethinyl}pyridine-2-yl)prop-2-enoate methyl 92 Compound 88 (0.39 g, 1.69 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.32 g, 1.85 mmol) were dissolved in dichloromethane (30 mL) at 0°C. Then, 4-methylmorpholine (0.20 mL, 1.85 mmol) was added dropwise over 5 minutes. The resulting mixture was stirred at 0°C for 2 hours, then compound 91 (0.5 g, 1.41 mmol) and 4-methylmorpholine (0.19 mL, 1.68 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The mixture was diluted with dichloromethane, washed with H2O, dried, and evaporated to obtain a crude yellow solid (1.7 g). This was purified by SiO2 chromatography (99:1, dichloromethane / methanol), and further recrystallized from acetonitrile (MeCN) to obtain compound 92 as a yellow solid (0.53 g, 66%): 1 H NMR (700 MHz, CDCl3) δ 1.35 - 1.44 (m, 4H), 1.64 - 1.68 (m, 2H), 1.69 - 1.73 (m, 2H), 2.22 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 3.50 (t, J = 5.4 Hz, 2H), 3.58 - 3.65 (m, 4H), 3.78 (t, J = 5.4 Hz, 2H), 3.82 (s, 3H), 6.93 (d, J = 15.6 Hz, 1H), 7.38 (dd, J = 8.2, 0.9 Hz, 1H), 7.45 (s, 1H), 7.66 (d, J = 15.6 Hz, 1H), 7.74 (dd, J = 8.1, 2.1 Hz, 1H), 8.69 (dd, J = 2.1, 0.8 Hz, 1H), 9.32 (s, 1H); 13C NMR (176 MHz, CDCl3) δ 18.3, 24.7, 28.1, 28.3, 32.5, 32.8, 40.6, 44.7, 48.0, 48.4, 51.9, 85.4, 91.2, 106.6, 120.7, 122.5, 123.5, 138.3, 142.7, 146.0, 151.3, 151.9, 167.1, 171.5, 171.8; MS(ES): m / z = 568.2 [M+H] + HRMS (ES) Calculated Value C 28 H 34 N5O6S [M+H] + : 568.2224, Measured value 568.2220

[0092] Example 3: Synthesis of protected HDAC inhibitor 93 The synthesis of Tert-butyl(2E)-3-(5-{2-[4-(4-{7-[(acetyloxy)carbamoyl]heptanoyl}piperazine-1-yl)phenyl]ethynyl}thiophen-2-yl)propa-2-enoate 93 is shown in Figure 4 and will be explained in detail below.

[0093] Compound 88 (2.89 g, 12.5 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (2.39 g, 13.6 mmol) were dissolved in dichloromethane (100 mL) at 0°C, and then 4-methylmorpholine (1.5 mL, 13.6 mmol) was added dropwise over 5 minutes. The resulting mixture was stirred at 0°C for 2 hours, then compound 27 (4.11 g, 10.41 mmol) and 4-methylmorpholine (1.36 mL, 12.4 mmol) were added, and the mixture was stirred at room temperature for 18 hours. The mixture was diluted with dichloromethane, washed with H2O, dried with (MgSO4), and evaporated to obtain a crude yellow solid (1.7 g). This was purified by SiO2 chromatography (97:3, dichloromethane / methanol), and further recrystallized from acetonitrile to obtain compound 93 as a yellow solid (2.51 g, 40%). 1H NMR (600 MHz, CDCl3) δ 1.32 - 1.45 (m, 4H), 1.50 (s, 9H), 1.63 (p, J = 7.1 Hz, 2H), 1.69 (p, J = 7.1 Hz, 2H), 2.19 (s, 3H), 2.25 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 3.21 (t, J = 5.3 Hz, 2H), 3.24 (t, J = 5.3 Hz, 2H), 3.55 - 3.66 (m, 2H), 3.75 (t, J = 5.2 Hz, 2H), 6.11 (dd, J = 15.6, 1.1 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 7.03 - 7.14 (m, 2H), 7.36 - 7.44 (m, 2H), 7.58 (d, J = 15.6 Hz, 1H), 9.91 (s, 1H); 13 C NMR (176 MHz, CDCl3) δ 18.3, 24.8, 28.1, 28.2, 28.4, 32.4, 32.7, 41.1, 45.2, 48.1, 48.4, 53.4, 80.6, 81.3, 96.0, 112.9, 115.3, 119.2, 126.1, 130.6, 132.0, 132.7, 135.4, 140.2, 150.6, 165.9, 168.7, 171.8

[0094] Example 4: Synthesis of building block compound 96 The synthesis of N-(5-aminopentyl)-4-iodo-N-methylaniline 96 is shown in Figure 5 and will be explained in detail below.

[0095] Example 4.1: Synthesis of 5-chloro-N-(4-iodophenyl)-N-methylpentanamide 94 N-methyl-4-iodoaniline (24.04 g, 103 mmol) was dissolved in dichloromethane (300 mL), and the solution was cooled to 0°C. 5-chlorovaleryl chloride (14.6 mL, 113.3 mmol), followed by pyridine (9.16 mL, 113.3 mmol), was added, and the resulting solution was stirred at room temperature for 16 hours. The solution was diluted with dichloromethane, the organic matter was washed with saturated NH4Cl and H2O, dried (MgSO4), and evaporated to obtain crude brown oil (40 g). This was purified by SiO2 chromatography (7:3 cyclohexane / ethyl acetate) to obtain compound 94 as a yellow oily substance (35.16 g, 97%). 1 H NMR (400 MHz, CDCl3) δ 1.57 - 1.79 (m, 4H), 1.98 - 2.19 (m, 2H), 3.24 (s, 3H), 3.35 - 3.52 (m, 2H), 6.93 (d, J = 7.9 Hz, 2H), 7.75 (d, J = 8.0Hz, 2H)

[0096] Example 4.2: Synthesis of 5-azido-N-(4-iodophenyl)-N-methylpentanamide 95 Compound 94 (35.0 g, 99.5 mmol) was dissolved in N,N-dimethylformamide (200 mL), sodium azide (13.53 g, 208.95 mmol) was added, and the solution was stirred at 80°C for 18 hours. The suspension was cooled, diluted with H2O, and then extracted with RINKAN. The organic matter was washed with H2O and brine, dried (MgSO4), and evaporated to obtain crude orange oil (37.6 g). This was purified by SiO2 chromatography (7:3 cyclohexane / ethyl acetate) to obtain compound 95 as orange oil (33.4 g, 94%). 1 H NMR (400 MHz, CDCl3) δ 1.45 - 1.59 (m, 2H), 1.61 - 1.69 (m, 2H), 1.91 - 2.20 (m, 2H), 3.10 - 3.36 (m, 5H), 6.85 - 7.02 (m, 2H), 7.67 - 7.84 (m, 2H)

[0097] Example 4.3: Synthesis of N-(5-aminopentyl)-4-iodo-N-methylaniline 96 Compound 95 (5.24 g, 14.6 mmol) was dissolved in toluene (80 mL), and BH3·Me2S (2.0 M in toluene, 16.8 mL, 33.6 mmol) was added. The solution was stirred under reflux for 16 hours. The mixture was cooled and then stirred with a 10% w / v aqueous Na2CO3 solution for 0.5 hours. The mixture was diluted with ethyl acetate, the organic matter was washed with H2O and brine, dried (MgSO4), and evaporated to obtain crude yellow oil (4.21 g). This was purified by SiO2 chromatography (9:1 dichloromethane / methanol, 2% triethylamine) to obtain compound 96 as a clear oil (1.76 g, 38%), which was immediately used in the next step: 1 H NMR (400 MHz, CDCl3) δ 1.28 - 1.39 (m, 2H), 1.43 - 1.53 (m, 2H), 1.53 - 1.61 (m, 2H), 1.97 (s, 2H), 2.70 (t, J = 7.0 Hz, 2H), 2.88 (s, 3H), 3.21 -3.32 (m, 2H), 6.41 - 6.47 (m, 2H), 7.39 - 7.46 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 24.3, 26.4, 33.1, 38.2, 41.9, 52.5, 76.4, 114.3, 137.6, 148.7

[0098] Example 5: Synthesis of building block compound 99 The synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl(2E)-3-(5-ethynylthiophen-2-yl)prop-2-enoate is shown in Figure 6 and will be explained in detail below.

[0099] Example 5.1: Synthesis of 5-iodothiophene-2-carbaldehyde 24 To a solution of 2-thiophenecarboxaldehyde (9.34 mL, 100.0 mmol) in ethanol (50 mL), N-iodosuccinimide (24.75 g, 110.0 mmol) and p-toluenesulfonic acid monohydrate (1.90 g, 10.0 mmol) were added at 50°C, and the resulting solution was stirred at 50°C for 1 hour. 1.0 M HCl (80 mL) was added, and the mixture was extracted with ethyl acetate, washed with saturated Na2S2O3, H2O, and brine, dried (MgSO4), and evaporated to obtain compound 24 as a yellow oil (25.26 g, >100%), which was slowly crystallized: 1 H NMR (400 MHz, CDCl3) δ 7.39 (s, 2H), 9.77 (s, 1H)

[0100] Example 5.2: Synthesis of 5-[2-(trimethylsilyl)ethynyl]thiophene-2-carbaldehyde 97 Triethylamine (300 mL) was degassed by bubbling with argon for 1 hour. Then, compound 24 (25 g, 105 mmol), trimethylsilylacetylene (16.0 mL, 115.5 mmol), Pd(PPh3)2Cl2 (740 mg, 1.05 mmol), and CuI (200 mg, 1.05 mmol) were added under argon, and the resulting suspension was stirred at room temperature for 18 hours. The mixture was diluted with diethyl ether and passed through Celite / SiO2 to obtain crude brown oil (17.7 g). This was purified by SiO2 chromatography to obtain compound 97 as an orange oil (12.79 g, 58%), which was slowly crystallized: 1 H NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 7.24 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 9.83 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ -0.5, 26.9, 96.3, 104.6, 132.5, 133.1, 135.7, 143.8, 182.4; IR (ATR) v max / cm -12960w, 2899w, 2833w, 2148m, 1666s, 1438s, 1249s, 1223s, 1207s, 838s; MS (ES) m / z = 209.0 [M+H] + ; HRMS (ES) calculated value C 10 H 13 SOSi [M+H] + : 209.0451, found 209.0454

[0101] Example 5.3: (2E)-3-{5-[2-(Trimethylsilyl)ethynyl]thiophen-2-yl}prop-2-enoic acid methyl 98 synthesis Trimethylphosphonoacetate (14.0 mL, 86.4 mmol) and LiCl (3.66 g, 86.4 mmol) were added to anhydrous tetrahydrofuran (250 mL) at 0 °C. The resulting solution was stirred for 15 minutes, then compound 97 (15.0 g, 72 mmol) was added. To this solution, 1,8-diazabicyclo[5.4.0]undec-7-ene (12.9 mL, 86.4 mmol) was slowly added, and the resulting slurry was stirred at room temperature for 16 hours. This was poured onto crushed ice and extracted with ethyl acetate. The organic matter was washed with H2O and brine, dried (MgSO4), and evaporated to give a crude brown oil (21 g). This was purified by SiO2 chromatography (9:1 cyclohexane / ethyl acetate) to give compound 98 as a pale yellow solid (17.23 g, 91%): 1 H NMR (400 MHz, CDCl3) δ 0.24 (s, 9H), 3.78 (s, 3H), 6.19 (d, J = 15.7 Hz, 1H), 7.05 - 7.14 (m, 2H), 7.67 (d, J = 15.7 Hz, 1H); 13 C NMR (75 MHz, CDCl3) δ -0.3, 51.7, 97.0, 101.9, 117.3, 125.8, 130.6, 133.3, 136.5, 140.4, 166.9; IR (ATR) v max / cm -12953w, 2899w, 2144m, 1715s, 1621s, 1516w, 1432m, 1391w, 1301s, 1269s, 1202s, 1161s, 838s; MS(ES): m / z = 265.1 [M+H] + ; HRMS (ES) calculated value C 13 H 17 O2SSi [M+H] + : 265.0713, found 265.0713

[0102] Example 5.4: Synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-(5-ethynylthiophen-2-yl)prop-2-enoate 99 Compound 98 (13.03 g, 49.3 mmol) was dissolved in triethylene glycol monomethyl ether (50 mL), and 20% w / v aqueous NaOH solution (1.3 mL) was added. The resulting mixture was stirred at room temperature for 16 h, and then the solution was diluted with ethyl acetate. The organic matter was washed with H2O and brine, dried (MgSO4), and evaporated to give a crude dark oil (IS g). This was purified by SiO2 chromatography (1:1 cyclohexane / ethyl acetate) to give compound 99 as a yellow oil (8.18 g, 51%), which darkened rapidly: 1 H NMR (400 MHz, CDCl3) δ 3.36 (s, 3H), 3.46 (s, 1H), 3.50 - 3.56 (m, 2H), 3.61 - 3.69 (m, 6H), 3.72 - 3.79 (m, 2H), 4.26 - 4.38 (m, 2H), 6.24 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 3.8 Hz, 1H), 7.17 (d, J = 3.8 Hz, 1H), 7.68 (dd, J = 15.7, 0.6 Hz, 1H); 13IR (ATR) v max / cm -1 MS(ES): m / z = 325.1 [M+H] + HRMS (ES) Calculated Value C 16 H 21 O5S [M+H] + : 325.1104, Measured value 325.1100

[0103] Example 6: Synthesis of photoactivating compound 100 The synthesis of photoactivating compound 100 is shown in Figure 7 and will be explained in detail below.

[0104] Synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl(2E)-3-[5-(2-{4-[(5-aminopentyl)(methyl)amino]phenyl}ethynyl)thiophen-2-yl]propa-2-enoate 100] Compound 96 (1.70 g, 5.34 mmol) and Compound 99 (2.42 g, 7.48 mmol) were dissolved in triethylamine (80 mL), and the solution was degassed by bubbling argon for 1 hour. Then, Pd(PPh3)2Cl2 (372 mg, 0.53 mmol) and CuI (100 mg, 0.53 mmol) were added under argon, and the resulting suspension was stirred at 60°C for 72 hours. The resulting suspension was diluted with dichloromethane, washed with saturated NaHCO3 and water, dried (MgSO4), and evaporated to obtain a crude dark-colored oil (3.75 g). This was purified by SiO2 chromatography (95:5 dichloromethane / methanol, 1% triethylamine) to obtain compound 100 as a bright orange solid (0.47 g, 17%). 1H NMR (400 MHz, CDCl3) δ 1.32 - 1.41 (m, 2H), 1.54 - 1.64 (m, 4H), 2.81 (t, J = 7.2 Hz, 2H), 2.95 (s, 3H), 3.30 - 3.35 (m, 2H), 3.37 (s, 3H), 3.53 - 3.56 (m, 2H), 3.64 - 3.69 (m, 6H), 3.75 - 3.79 (m, 2H), 4.29 - 4.39 (m, 2H), 6.20 (d, J = 15.7 Hz, 1H), 6.56 - 6.65 (m, 2H), 7.07 - 7.14 (m, 2H), 7.31 - 7.40 (m, 2H), 7.70 (dd, J = 15.7, 0.6 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 24.2, 26.5, 31.0, 38.3, 41.1, 52.2, 59.0, 63.7, 69.2, 70.6, 70.6, 71.9, 80.6, 97.7, 108.3, 111.4, 116.4, 127.6, 131.3, 131.5, 132.8, 137.0, 139.4, 149.2, 166.7; IR (ATR) max / cm -1 2925m, 2871m, 2189w, 1738m, 1712m, 1604s, 1530s, 1511m, 1376m, 1196m; MS(ASAP): m / z = 515.2 [M+H] + HRMS (ASAP) calculated value C 28 H 39 N2O5S [M+H] + : 515.2574, Measured value 515.2569

[0105] Example 7: Synthesis of protected HDAC inhibitor compound 101 The synthesis of protected HDAC inhibitor compound 101, which has photoactivatable cell-killing activity, is shown in Figure 8 and is described in detail below.

[0106] Synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl(2E)-3-[5-(2-{4-[(5-{7-[(acetyloxy)carbamoyl]heptanamide}pentyl)(methyl)amino]phenyl}ethynyl)thiophen-2-yl]prop-2-enoate 101 Compound 100 (128 mg, 0.25 mmol) was dissolved in dichloromethane (10 mL), the solution was cooled to 0°C, and then 4-methylmorpholine (0.055 mL, 0.5 mmol), compound 88 (76 mg, 0.33 mmol), and propylphosphonic anhydride (50% by weight in ethyl acetate, 0.32 mL, 0.5 mmol) were added. The resulting mixture was stirred at room temperature for 16 hours. The mixture was diluted with dichloromethane, washed with H2O, dried with (MgSO4), and evaporated to obtain crude yellow oil. This was purified by SiO2 chromatography (95:5, dichloromethane / methanol) to obtain compound 101 as yellow oil (141 mg, 77%). 1 H NMR (400 MHz, CDCl3) δ 1.30 - 1.42 (m, 6H), 1.46 - 1.55 (m, 2H), 1.55 - 1.63 (m, 4H), 1.64 - 1.72 (m, 2H), 2.11 - 2.17 (m, 2H), 2.20 (s, 3H), 2.23 (t, J = 7.3 Hz, 2H), 2.95 (s, 3H), 3.17 -3.26 (m, 2H), 3.33 (t, J = 7.3 Hz, 2H), 3.37 (s, 3H), 3.51 - 3.57 (m, 2H), 3.63 - 3.70 (m, 6H), 3.71 - 3.80 (m, 2H), 4.26 - 4.40 (m, 2H), 5.64 (s, 1H), 6.20 (d, J = 15.6 Hz, 1H), 6.61 (d, J = 5.4 Hz, 2H), 7.09 (d, J = 3.8 Hz, 1H), 7.11 (d, J = 3.9 Hz, 1H), 7.31 - 7.41 (m, 2H), 7.70 (dd, J = 15.7, 0.6 Hz, 1H), 9.55 (s, 1H); 13C NMR (101 MHz, CDCl3) δ 18.3, 24.3, 24.7, 25.3, 26.5, 26.9, 28.0, 28.2, 29.5, 32.5, 36.2, 38.3, 39.3, 52.2, 53.4, 59.0, 63.7, 69.2, 70.5, 70.6, 70.6, 71.9, 77.0, 80.7, 97.6, 111.4, 116.4, 127.5, 131.3, 131.5, 132.8, 137.0, 139.4, 148.2, 166.7, 168.8, 173.3; IR (ATR) v max / cm -1 MS(ES): m / z = 728.3 [M+H] + HRMS (ES) Calculated Value C 38 H 54 N3O9S [M+H] + : 728.3575, Measured value 728.3578

[0107] Example 8: Fluorescein diacetate cell viability assay A fluorescein diacetate cell viability assay was performed as follows to measure the viability of HaCaT keratinocytes treated with compound 92 in the absence of irradiation (no light) and with irradiation (light).

[0108] HaCaT keratinocyte cells were seeded in two 96-well plates and incubated at 37°C and 5% CO2 for 24 hours. The incubation medium was removed, compound 92 was added at various concentrations along with a dimethyl sulfoxide (DMSO) control, and the cells were incubated at 37°C and 5% CO2 for 1 hour. Subsequently, one plate was irradiated at 405 nm for 5 minutes (72 mW / cm²). 2Next, both plates were incubated at 37°C and 5% CO2 for 24 hours. The medium was removed, the cells were washed with 1X phosphate-buffered saline (PBS), then fluorescein diacetate (FDA) was added, and the cells were incubated at room temperature in the dark for 10 minutes. The fluorescein diacetate stain was then removed, and the cells were washed with 1X phosphate-buffered saline. The plates were read at 485 / 520 nm, and cell viability was measured at different treatment concentrations of compound 92 with and without light exposure. The results are shown in Figure 9. This indicates that photoactivation causes cell death at low concentrations (IC). 50 (=0.69 μM). Without photoactivation, high concentrations (IC) are reached due to HDAC inhibitory activity in response to enzyme metabolism. 50 At a concentration of 5.50 μM, cell viability decreases.

[0109] Example 9: Immunofluorescence imaging Immunofluorescence imaging of HaCaT keratinocytes treated with compound 92 (5 μM) and ethanol (EtOH), and co-treated with an anti-acetyl H3 primary antibody to detect the presence of acetylated H3 histones, was performed as follows:

[0110] 50,000 HaCaT cells were plated onto coverslips and grown for 2 days. Compound 92 (5 μM) was added, and the cells were incubated at room temperature for 30 minutes. The cells were washed with phosphate-buffered saline and then fixed with 4% PFA. After washing the cells again with phosphate-buffered saline, they were blocked and permeabilized with 0.3% Triton 100-X / 5% goat serum in phosphate-buffered saline for 60 minutes. The cells were then washed with phosphate-buffered saline, anti-acetylhistone 3 antibody was added, and the cells were incubated overnight at 4°C. After adding a secondary antibody (Alexa-594 anti-rabbit) for 45 minutes, the cells were washed again and mounted for imaging.

[0111] The results are shown in Figure 10. This indicates that compound 92 shows limited activity after 10 minutes, but after 1 hour, the cells exhibit a characteristic nuclear ring phenotype, showing accumulation of acetylated H3 in response to HDAC enzyme inhibition. This delayed behavior is an indicator of the initial delay in the enzymatic metabolism of compound 92 to its active form. Ethanol-treated cells do not exhibit this nuclear ring phenotype at any point in time.

[0112] Example 10: Abundance of acetyl H3 in treated SCC-4 cells To measure the abundance of acetyl-H3 protein in SCC-4 cells, cells were seeded at 350,000 cells per well in a 6-well plate. The following day, cells were treated with a compound or control. After 15 minutes and / or 1 hour of treatment, the cells were lysed in RIPA buffer. SDS-PAGE was performed on the cell lysates using Any kD® Mini-PROTEAN® TGX® Precast Protein Gels to isolate the proteins. The proteins were then transferred to a PVDF membrane (Macherey-Nagel) and blocked with TBST containing 5% milk and 2.5% fish skin gelatin. Primary antibodies (acetyl-H3, 9677S, CST and α-tubulin, T5168, Sigma) and secondary antibodies (goat anti-rabbit IgG, A6154, Sigma and goat anti-mouse, SA00001-1, Proteintech) were diluted in blocking buffer and stained at room temperature for 1 hour, with TBST washing between steps. Chemiluminescence signals were measured using an iBright imager (Invitrogen). Acetyl-H3 levels were normalized, and the resulting concentration measurements (measured using ImageJ software) are shown in Figures 11-13. Image analysis was performed using ImageJ software.

[0113] Figure 11 shows the concentration measurements of acetyl-H3 after 15 minutes and 1 hour of treatment with culture medium, DMSO (dimethyl sulfoxide), SAHA (suberoyl anilide hydroxamic acid), compound 27, and compound 93. Acetyl-H3 levels were normalized to α-tubulin (15 min) and AC-40 (1 hour).

[0114] Figure 12 shows the concentration measurements of acetyl-H3 after treatment for 1 hour with culture medium, DMSO (dimethyl sulfoxide), SAHA (suberoyl anilide hydroxamic acid), compound 27, compound 93, and compound 101. Acetyl-H3 levels were normalized relative to α-tubulin.

[0115] Figure 13 shows the concentration measurements of acetyl-H3 after treatment with culture medium, DMSO (dimethyl sulfoxide), SAHA (suberoyl anilide hydroxamic acid), compound 27, and compound 92 for 15 minutes. Acetyl-H3 levels were normalized relative to α-tubulin.

[0116] Example 11: Caspase-3 expression in treated HaCaT cells HaCaT cells were seeded at 50,000 cells per well on coverslips of 6-well plates. The following day, cells were treated with 50 nM and 100 nM of compound 93 for 30 minutes. DMSO was used as a control. Photoactivation was performed at 405 nm and 29 mW / cm². 2The procedure was performed by irradiation using a PhotoReact 365 modified to emit light for 5 minutes. The following day, cells were fixed with 4% PFA, permeabilized with Triton X-100 / Tween20, and blocked with blocking buffer (5% BSA and 0.1% Tween20 in PBS). Primary antibody (caspase-3, ab13847, Abcam) and secondary antibody (goat anti-rabbit IgG Alexa Fluor 594) were diluted in PBS containing 5% goat serum and 0.1% Tween20. Coverslips were added to slides and imaged with Zeiss LSCM 880. Image analysis was performed using ImageJ software. Figure 14 shows caspase-3 expression and quantification of expression levels in HaCaT cells treated with DMSO, 50 nM compound 93, and 100 nM compound 93.

[0117] Example 12: Co-localization of compounds in SCC-4 cells SCC-4 cells were seeded at 50,000 cells / well on 8-well chamber slides. The following day, the cells were treated with 1 μM compound 101 and 1 μM compound 93 for 1 hour. The cells were replaced with live cell imaging medium. Co-staining (MitoTracker, Bodipy ER Tracker, LipidSpot 610, or LysoTracker) was added to the cells 30 minutes before imaging. Imaging was performed with a Zeiss LSCM 880, and image analysis was performed using ImageJ software.

[0118] Figure 15 shows the co-localization of compound 101 with mitochondria (MitoTracker), lipid droplets (LipidSpot610), and acidic organelles (LysoTracker) in SCC-4 cells. Pearson coefficients were calculated to demonstrate the correlation between compound localization and co-staining localization. The results are shown in Table 1.

[0119] [Table 1]

[0120] Figure 16 shows the co-localization of compound 93 with mitochondria (MitoTracker), endoplasmic reticulum (Bodipy ER Tracker), lipid droplets (LipidSpot610), and acidic organelles (LysoTracker) in SCC-4 cells. Pearson coefficients were calculated to demonstrate the correlation between compound localization and co-staining localization. The results are shown in Table 2.

[0121] [Table 2]

[0122] Example 13: Deprotection of compound 93 As shown in Figure 17, deprotection of compound 93 yielded Tert-butyl(2E)-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazine-1-yl}phenyl)ethynyl]thiophen-2-yl}prop-2-enoate 103. This is explained below.

[0123] Compound 93 (500 mg, 0.82 mmol) was dissolved in methanol (30 mL), then NaOH (32 mg, 0.82 mmol, 1 mL as a solution in H2O) was added, and the resulting solution was stirred at room temperature for 5 hours. The mixture was diluted with dichloromethane, the organic matter was washed with H2O, dried (MgSO4), and evaporated to obtain a crude yellow solid. This was purified by recrystallization from acetonitrile to obtain compound 103 as a yellow solid (170 mg, 36%). 1H NMR (700 MHz, ジメチルスルホキシド-d6) δ 1.22 - 1.29 (m, 4H), 1.47 (s, 9H), 1.47 - 1.52 (m, 4H), 1.94 (t, J = 7.4 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 3.20 - 3.25 (m, 2H), 3.25 - 3.29 (m, 2H), 3.55 - 3.60 (m, 4H), 6.18 (d, J = 15.7 Hz, 1H), 6.91 - 7.00 (m, 2H), 7.31 (d, J = 3.8 Hz, 1H), 7.37 - 7.44 (m, 2H), 7.48 (d, J = 3.8 Hz, 1H), 7.66 (d, J = 15.8 Hz, 1H), 8.64 (s, 1H), 10.32 (s, 1H); 13 C NMR (176 MHz, ジメチルスルホキシド-d6) δ 24.6, 25.0, 27.8, 28.4, 28.5, 32.1, 32.2, 40.5, 44.4, 46.8, 47.2, 80.1, 80.9, 96.6, 110.2, 114.7, 118.9, 125.3, 132.1, 132.4, 132.7, 135.5, 139.6, 150.7, 165.0, 169.1, 170.7; IR (ATR) v max / cm -1 3207br, 2977w, 2930w, 2858w, 2194w, 1698m, 1619s, 1603s, 1526m, 1508m, 1231s, 1145s, 754m; MS (ASAP) m / z = 566.2 [M+H] + ; HRMS (ASAP) calculation value C 31 H 40 N3O5S [M+H] + : 566.2683, measured value 566.2683

[0124] All features and / or all steps of the methods or processes disclosed herein (including the attached claims, abstract and drawings) can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed herein (including the attached claims, abstract and drawings) can be replaced by an alternative feature that serves the same, equivalent, or similar purpose, unless otherwise stated. Thus, unless otherwise stated, each feature disclosed is merely a general example of a set of equivalent or similar features. The present invention is not limited to the details of the embodiments described above. The present invention extends to novel features or novel combinations of features disclosed herein (including the attached claims, abstract and drawings), or novel steps or novel combinations of the methods or processes disclosed herein.

Claims

1. A compound of formula I or formula II. 【Chemistry 1】 (In formula I, R ２ is H, C １ ~C ９ alkyl, -CH ２ OH, -CH ２ OCH ３ , -Ph, -C ６ H ４ OH, -CH(CH ３ ), -C(CH ２ COOH) ２ OH, -C(=O)CH ３ , -CH ２ NH ２ , -CH ２ NH(C=O)CH ２ NH ２ or -CH ２ NH(C=O)CH ２ NH(C=O)CH ２ NH ２ ; P is an alkylene group having 1 to 15 carbon atoms, optionally substituted with one or more of -C(=O)- and -NHC(=O)-; Ar １ and Ar ２ Each of these groups is independently selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole groups; X is -C=C-C(=O)OR ４ And R ４ R is an alkyl group having 1 to 10 carbon atoms, or R 4 is -(CH2CH2O)nCH3 (where n is an integer from 1 to 8). In formula II, R ３ H, C １ ~C ９ Alkyl, -CH ２ OH, -CH ２ OCH ３ -Ph, -C ６ H ４ OH, -CH(CH ３ )OH, -C(CH ２ COOH) ２ OH, -C(=O)CH ３ ien-CH ２ NH ２ ien-CH ２ NH(C=O)CH ２ NH ２ or -CH ２ NH(C=O)CH ２ NH(C=O)CH ２ NH ２ And; P is an alkylene group having 1 to 15 carbon atoms, optionally substituted with one or more of -C(=O)- and -NHC(=O)-; R １ is H or an alkyl group having 1 to 10 carbon atoms; Ar １ and Ar ２ Each of these groups is independently selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole groups; X is -C=C-C(=O)OR ４ And R ４ R is an alkyl group having 1 to 10 carbon atoms, or R 4 is -(CH₂CH₂O)nCH₃ (where n is an integer from 1 to 8).

2. R ３ However, C １ ~C ３ A compound of formula I or formula II according to claim 1, which is alkyl.

3. R ３ However, -CH ３ The compound of formula I or formula II as described in claim 2.

4. Ar １ A compound of formula I or formula II according to any one of claims 1 to 3, wherein the compound is thiazole or phenyl.

5. Ar ２ A compound of formula I or formula II according to any one of claims 1 to 4, wherein the compound is pyridine, thiophene, or furan.

6. P is replaced by -C (=O)- １ ~C １５ A compound of formula I or formula II according to any one of claims 1 to 5, wherein the compound is an alkylene group.

7. P is -C(=O)(CH ２ ) ６ The compound of formula I or formula II according to claim 6, wherein -C(=O)- is bonded to the N atom of formula I or formula II.

8. R ４ A compound of formula I or formula II according to any one of claims 1 to 7, wherein the compound is an alkyl group having 1 to 10 carbon atoms.

9. R ４ However, -CH ３ , -C(CH ３ ) ３ or -CH ２ CH (CH ３ ) ２ The compound of formula I or formula II as described in claim 8.

10. R １ However, C １ ~C ３ A compound of formula II according to any one of claims 1 to 9, wherein the compound is alkyl.

11. P is replaced by -NHC (=O)- in C １ ~C １５ A compound of formula I or formula II according to any one of claims 1 to 9, which is an alkylene.

12. P is - (CH ２ ) ５ NHC(=O)(CH ２ ) ６ - A compound of formula I or formula II according to claim 11.

13. R ４ However, - (CH ２ CH ２ O) ｎ CH ３ A compound of formula I or formula II according to any one of claims 1 to 9, 11, or 12, wherein n is an integer from 1 to 8.

14. The compound of formula I is selected from the following compounds 92, 93, and 102. The compound of formula I or formula II according to any one of claims 1 to 9, 11, 12, or 13, wherein the compound of formula II is the following compound 101. 【Chemistry 2-1】 【Chemistry 2-2】

15. Use of a compound of formula I or formula II according to any one of claims 1 to 9, 11, 12, 13, or 14 in photodynamic therapy.

16. A pharmaceutical composition comprising, optionally, one or more pharmaceutically acceptable excipients, diluents, or carriers, a compound of formula I according to any one of claims 1 to 9, 11, 12, 13, or 14, or a compound of formula II according to any one of claims 1 to 14.

17. A method for deprotecting a compound of formula I according to any one of claims 1 to 9, 11, 12, 13, or 14, or a compound II of formula II according to any one of claims 1 to 14, comprising contacting the compound of formula I or formula II with an enzyme (excluding methods for surgically treating or diagnosing a human being).

18. A method for deprotecting a compound of formula I or a compound of formula II according to claim 17, wherein the enzyme is an endogenous enzyme.

19. A method for deprotecting a compound of formula I according to any one of claims 1 to 9, 11, 12, 13, or 14, or a compound of formula II according to any one of claims 1 to 14, comprising reacting the compound of formula I or formula II with a base in the presence of a solvent.

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