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 an important role in transcriptional regulation in eukaryotic cells. The acetylation status of histone and nonhistone 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 moieties of lysine groups in histone and nonhistone proteins. HDAC inhibitors (HDACi) inhibit the activity of HDAC enzymes. The biological importance of HDACs makes their inhibition of interest clinically, and HDAC inhibition has emerged as an important therapeutic strategy in the treatment of cancer, neurodegenerative and inflammatory diseases, and neurological disorders, among others.

[0002] HDAC enzymes have been classified based on accessory domain homology to yeast histone deacetylases and are currently divided into four major groups. · Class I Class I, which includes HDAC1, -2, -3, and -8, is related to the yeast RPD3 deacetylase; Class IIA includes HDAC4, -5, -7, and -9; class IIB-6 and -10 are related to the yeast Hda1 (histone deacetylase 1) gene; · Class III (also known as sirtuins) is related to the Sir2 gene and includes SIRT1-7; Class IV Class IV, which contains only HDAC11, has both class I and class II functions.

[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 (except for cyclic tetrapeptides, which bind to the zinc ion at 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, fatty acid compounds such as phenylbutyric acid and valproic acid.

[0004] The hydroxamic acids constitute the largest class of HDAC inhibitors.

[0005] Examples of hydroxamic acid HDAC inhibitors include vorinostat (suberoylanilide hydroxamic acid, SAHA), belinostat, panobinostat, divinostat, pracinostat, xinostat, and abexinostat.

[0006] Although HDAC inhibitors play an important therapeutic role, the instability of HDAC inhibitors such as hydroxamic acids creates difficulties in terms of storage stability, solubility, formulation and manufacturing.

[0007] It is an object of the present invention to address or alleviate one or more of these problems. It is an object of the present invention to provide protected HDAC inhibitors, particularly protected hydroxamic acids. It is an object of the present invention to provide protected HDAC inhibitors that may exhibit improved stability. It is an object of the present invention to provide protected HDAC inhibitors that may exhibit improved solubility. Embodiments of the present invention relate to methods of protecting and / or deprotecting HDAC inhibitors, such as hydroxamic acid HDAC inhibitors. In embodiments, the deprotection step may be carried out in situ, i.e. by endogenous enzymes, in cells and tissues. Summary of the Invention

[0008] The present invention relates to the use of esters as protecting groups. The present invention relates to the use of esters as protecting groups for HDAC inhibitors. In this process, a functional group, such as a hydroxamic group, of an HDAC inhibitor is protected as an ester. The ester can then be removed in the presence of an enzyme. The enzyme may be an endogenous enzyme. Removal of the ester results in deprotection of the functional group. This deprotection can occur in situ, that is, the enzyme can be an endogenous enzyme, and deprotection can occur when the HDAC inhibitor enters the cell. The cell can be a mammalian cell. In an embodiment, deprotection occurs via the action of an endogenous enzyme on the protecting ester group when the HDAC inhibitor enters the target cell. This can result in a "clean" activation of HDAC inhibition in situ.

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

[0010] Advantageously, ester can be selected so that it can only activate HDAC inhibition in target tissue through enzymatic removal.That is, protective group can be selected so that enzymatic removal occurs only in the target body cell, tissue or area, that is, in the body cell, tissue or area that contains complementary enzyme.As will be understood by those skilled in the art, protective group can be further adjusted to adjust properties such as half-life, solubility, targeting, etc. to match the properties of 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 esters, citrate esters, PEG esters, glycerol esters, peptide esters such as mono-, di- and triglycine esters, phosphate esters, sulfonate esters, carbonates such as tetraethylene glycol, O-glycosyl ethers, O-glycosyl esters.

[0012] In one embodiment, the protecting group is an acetate (C1-C9) ester. In one 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 P450, glycosidases such as β-glucuronidase, sucrase and hyaluronidase, peptidases, phosphatases, sulfatases.

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

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

[0016] In one embodiment, the HDAC inhibitor is a photoactivated HDAC inhibitor. By "photoactivated HDAC inhibitor" is meant an HDAC inhibitor that has dual cell-modulating 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 such photoactive HDAC inhibitors maintain cell-killing function when protected according to the present invention.A delay in biological activity is observed during deprotection.

[0019] In one aspect of the invention, there is provided a compound of formula I: [ka] During the ceremony, R 1 is H or an alkyl group having 1 to 10 carbon atoms; R 2 is PZ, where P is an alkyl group having 1 to 15 carbon atoms, optionally substituted with one or more N atoms, -C=O and -NHC=O; Z is: [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; or R 1 and R 2 has 5 or 6 members and forms part of a heterocyclic group Y substituted with PZ, P being as defined above and Z being as defined above; Ar1 and Ar2 are each independently selected from a phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole group; X is -C=CC(=O)OR 4 and R 4is an alkyl group having 1 to 10 carbon atoms which may be optionally substituted with one or more O atoms.

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

[0021] The term heterocyclic group having 5 or 6 members means a monocyclic ring group containing 5 or 6 ring members and optionally containing, in addition to the nitrogen atom of formula I, one or more heteroatoms selected from the group consisting of N, S, SO, SO2, O2 and O. The term "heterocyclic group" includes aromatic, partially unsaturated and saturated ring systems. Examples of non-aromatic groups include, but are not limited to, piperazinyl, morpholinyl, thiomorpholinyl, dioxidothiomorpholinyl, pyrrolidin-1-yl and pyrrolidin-3-yl groups. Examples of aromatic (heteroaryl) groups include, but are not limited to, 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 in the 4-position 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., primary, secondary, or tertiary alkyl, or, where appropriate, cycloalkyl or cycloalkyl-substituted alkyl. Unless otherwise specified, alkyl groups contain 1-10 carbon atoms, preferably 1-6 carbon atoms, and more preferably 1-4 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, n-decyl.

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

[0024] In one embodiment, R3 is C1-C3 alkyl. In one embodiment, R3 is -CH3.

[0025] In one embodiment, Ar 1 is a thiazole or phenyl group.

[0026] In one embodiment, Ar 2 is a pyridine, thiophene or furan group.

[0027] In one embodiment, Ar1 is selected from a thiazole or phenyl group; Ar 2 is selected from a pyridine, thiophene or furan group.

[0028] In one embodiment, R 1 and R 2 forms part of the heterocyclic group Y. The heterocyclic group Y is substituted with PZ, where P is an alkyl group having 1 to 15 carbon atoms, optionally substituted with one or more N atoms, -C=O and -NHC=O; Z is: [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.

[0029] In one embodiment, the substituent PZ is in the 4-position relative to the Formula I nitrogen atom.

[0030] R 1 and R 2 In embodiments in which forms part of the heterocyclic group Y, Y is piperazine.

[0031] Y can also be: [ka]

[0032] When Y is piperazine, P is a C1-C 15 It may be an alkyl group. In one embodiment, P is -C(=O)(CH2)6.

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

[0034] In one embodiment, R 4 is a C1 to C6 alkyl group.

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

[0036] Optionally, R 1 and R 2 does not form a heterocyclic group Y. In this embodiment, R 1 is H or an alkyl group having 1 to 10 carbon atoms; R 2 is PZ, where P is an alkyl group containing 1 to 15 carbon atoms, optionally substituted with one or more N atoms, -C=O and -NHC=O; Z is: [ka] In the formula, R 3is H, C1-C9 alkyl, -CH2OH, -CH2OCH3, -Ph, -CH4OH, -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 may be C1-C3 alkyl. In one embodiment, R 1 is -CH3.

[0037] R 2 When is PZ, P is C1-C substituted with -NHC(=O). 15 It may be alkyl.

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

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

[0040] In one embodiment, R 4 is -(CH2CH2O) n CH3, n is an integer from 1 to 8, preferably R 4 is -(CH2CH2O)3CH3, R 1 and R 2 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 invention are intrinsically fluorescent. According to the invention, the compounds can be used for fluorescence imaging.

[0043] In one aspect, 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 (PS) usually contain a light-harvesting region that performs the dual functions of light harvesting and intersystem crossing, where single electrons are non-radiatively transferred to the triplet state. Disappearance of the triplet excited state can result in 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 immune defense strategy employed in both animal and plant systems in response to pathogen attack. Within animal, plant, fungal and bacterial cells, ROS trigger different regulatory effects depending on the rate and extent of their production; at high concentrations apoptosis is observed, whereas at low concentrations irritation responses are often observed (Guo et al., Stem Cells Dev. 2010, 19, 1321-1331).

[0045] Photodynamic therapy (PDT) exploits the ability of photosensitizers to generate ROS to destroy cancer cells, pathogenic microorganisms and / or unwanted tissues, usually by apoptosis. Typically, photosensitizing compounds are excited near or within a specific target tissue or condition (e.g., microbial infection, neoplasia, tumor, etc.), causing the generation of large amounts of ROS and subsequent destruction of that tissue. Low ROS levels induce cell proliferation, leading to applications in wound healing or more general tissue regeneration therapy.

[0046] Thus, PDT relies on targeting and accumulating photosensitive compounds in desired locations, such as cells in diseased tissues, and delivering light locally to activate ROS generation. Although compounds for use in PDT are known, they often suffer from various drawbacks, including small absorption peaks, which make photoactivation difficult, especially for large tumors where light penetration is difficult; long biological half-lives, which lead to prolonged skin photosensitivity after treatment; poor pharmacological properties, such as low aqueous solubility; and poor targeting ability (i.e., poor ability to target and accumulate in specific tissues or cells, causing significant off-target damage).

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

[0048] Thus, the compounds of formula I can be used to generate reactive oxygen species (ROS) and thereby control cell development, i.e., cell proliferation, differentiation and apoptosis, leading to a variety of therapeutic and non-therapeutic applications. The compounds of formula I are particularly advantageous for use in applications affected by the control of ROS, since they can exhibit efficient targeting and have fewer off-target effects. They can also be tailored to different cell types, thus achieving selective targeting effects.

[0049] Thus, in some aspects, the present invention relates to the use of a compound or conjugate of the invention in photodynamic therapy (PDT).

[0050] ROS production can be controlled based on therapeutic needs, e.g., to induce apoptosis for cell ablation, to induce proliferation in wound healing, or a combination of these. For example, in wound care, high levels of ROS can be induced first, leading to apoptosis of bacterial and / or fungal cells, and then low levels of ROS can be triggered to aid in skin regeneration.

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

[0052] In one aspect, the invention relates to a pharmaceutical composition comprising a compound of formula I, optionally in combination with one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0053] Advantageously, compounds of formula I are protected as esters, which provide stability to the compounds. Protected compounds are more easily stored. Protected compounds may exhibit improved solubility. Activation of the compounds involves removal of the protecting group.

[0054] One aspect of the invention relates to a method of 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. Enzymes suitable for deprotection include, but are not limited to, lipase, lactase, esterase, amylase, cytochrome P450, glycosidases, such as β-glucuronidase, sucrase and hyaluronidase, peptidases, phosphatases, sulfatases.

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

[0057] Advantageously, the ester protecting group can be selected to allow activation of HDAC inhibition only in the target tissue by enzymatic removal, i.e., the protecting group can be selected so that enzymatic removal occurs only in those cells, tissues or regions of the subject's body, i.e., cells, tissues or regions of the body that contain the complementary enzyme.As will be appreciated by those skilled in the art, the protecting group can be further tailored to tailor properties such as half-life, solubility, targeting, etc. 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 esters, citrate esters, PEG esters, glycerol esters, peptide esters such as mono-, di- and triglycine esters, phosphate esters, sulfonate esters, carbonates such as tetraethylene glycol, O-glycosyl ethers, O-glycosyl esters.

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

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

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

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

[0063] The process may be carried out at a temperature between 5° C. and 100° C. or between 15° C. and 30° C. Advantageously, the process may 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 hour to 12 hours or 2 hours to 7 hours.

[0065] Once the reaction has taken place, the reaction mixture can be worked up using techniques known to those skilled in the art, for example, the reaction mixture can be diluted and the combined organics washed, dried and evaporated to provide the deprotected compound as a crude solid.

[0066] Thus, the present invention relates to protected HDAC inhibitors, examples of which 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] Related compounds useful as control compounds include compounds 12, 13 and 14 below. [ka]

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

[0071] The present invention relates to a method of protecting HDAC inhibitors as esters and removing the esters with esterases to activate HDAC inhibition. The esterases may be endogenous esterases. Removal of the esters by esterases 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, which is subsequently deprotected in situ.

[0073] Compounds of formula I can advantageously exhibit increased solubility, improved chemical and storage stability, and ease of manufacture compared to their deprotected counterparts (FIG. 1).

[0074] The invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0075] [Figure 1] FIG. 1 shows a schematic diagram of the present invention, where PG represents a protecting group. [Diagram 2] FIG. 2 shows an exemplary synthesis of acetate-protected hydroxamic acid (88). [Diagram 3] FIG. 3 shows an exemplary synthesis of a protected HDAC inhibitor (92) with photoactivated cell killing activity in accordance with the present invention. [Figure 4] FIG. 4 shows an exemplary synthesis of an alternative protected HDAC inhibitor (93) with photoactivated cell killing activity in accordance with the present invention. [Diagram 5] FIG. 5 shows the synthesis of building block compound (96). [Figure 6] FIG. 6 shows the synthesis of building block compound (99). [Figure 7] FIG. 7 shows the synthesis of photoactivatable compound (100). [Figure 8] FIG. 8 shows the synthesis of exemplary compound (101). [Figure 9] FIG. 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] FIG. 10 shows immunofluorescence imaging of HaCaT keratinocytes treated with Compound 92 and EtOH and co-treated with anti-acetyl-H3 primary antibody to detect the presence of acetylated H3 histone. [Figure 11] FIG. 11 shows the abundance of acetyl-H3 in SCC-4 cells in response to 15 minutes and 1 hour treatment with compound 93 according to the invention and a control compound. [Figure 12] FIG. 12 shows the abundance of acetyl-H3 in SCC-4 cells in response to one hour treatment with compound 93 and compound 101 according to the invention and a control compound. [Figure 13] FIG. 13 shows the abundance of acetyl-H3 in SCC-4 cells in response to treatment with compound 92 according to the invention and a control compound after 15 minutes. [Figure 14] FIG. 14 shows the expression levels of caspase-3 in HaCaT cells after treatment with 50 nM Compound 93 and 100 nM Compound 93 and controls before and after photoactivation. [Figure 15] FIG. 15 shows fluorescence microscopy images demonstrating the colocalization of Compound 101. [Figure 16] FIG. 16 shows fluorescence microscopy images demonstrating the colocalization of Compound 93. [Figure 17] FIG. 17 shows an exemplary deprotection of compound 93. EXAMPLES

[0076] General methodology: All light exposure was at 405 nm and 29 mW / cm 2 The images were analyzed using a modified PhotoReact 365™ with an output of 1000 Hz for 5 min. All image analysis was performed using ImageJ software and all graphs were generated using Prism.

[0077] Cell viability assay: Opaque-walled 96-well plates were seeded with 20,000 SCC-4 cells per well. The following day, plates were treated with a range of concentrations (100 pM to 1 μM) of the compound of interest for 1 hour before illuminating the "light" treated plates. The following day, plates were treated with either: After 10 min with propidium iodide (PI) and fluorescein diacetate (FDA) and subsequent washing with PBS, fluorescence was measured at 535 / 617 nm for PI and 485 / 520 nm for FDA. After treating with 12 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution for 2 hours, dissolve in DMSO on an orbital shaker and measure absorbance at 540 nm. · Treated with XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) labeling reagent and electron coupling reagent for 4 hours, washed with PBS, and measured absorbance at 650 nm. Luminescence was measured after cell lysis with CellTitre-Glo™ Reagent for 10 min on an orbital shaker.

[0078] Microscopy procedure:

[0079] colocalization SCC-4 cells were seeded at 50,000 cells per well in 8-well chamber slides and treated the following day with 1 μM of the compound of interest for 1 h before fixation with 4% paraformaldehyde (PFA) or media exchange with Live Cell Imaging solution. Co-stains (e.g., MitoTracker, Bodipy ER Tracker, LipidSpot 610) were applied for 30 min and then imaged on a Zeiss LSCM 880.

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

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

[0082] Example 1: Synthesis of acetate-protected hydroxamic acid 88 The synthesis of an exemplary acetate-protected hydroxamic acid 88 is shown in FIG. 2 and further described below in Examples 1.1-1.4.

[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) and cooled to 0° C., after which 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 h. The solution was diluted with 5% HCl and extracted with dichloromethane (DCM) (3×). The organics were washed with saturated NH4Cl and H2O, dried (MgSO4), and evaporated to give a crude red oil (58.9 g). This was purified by SiO2 chromatography (hexanes / ethyl acetate (EtOAc), 9:1) to give 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 7-(hydroxycarbamoyl)heptanoate 86 Compound 85 (6.1 g, 25.0 mmol) was dissolved in methanol (MeOH) (21 mL), then 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (11.2 mL, 75.0 mmol) and hydroxylamine (NH2OH4) (50% in water, 15.3 mL, 250 mmol) were added, and the resulting solution was stirred at room temperature (RT) for 3 h. The mixture was diluted with dichloromethane (DCM), and the organics were washed with 5% HCl and H2O, dried (MgSO4), and evaporated to give a crude yellow oil (3.23 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 9:1) to give 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 tert-butyl 7-[(acetyloxy)carbamoyl]heptanoate 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 h. The solution was diluted with dichloromethane and the organics were washed with saturated NH4Cl and H2O, dried (MgSO4) and evaporated to give a crude pale yellow oil (2.75 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 99:1) to give 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) calcd. 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 h. The solution was evaporated and the crude residue was purified by SiO2 chromatography (dichloromethane / methanol, 95:5) to give 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) calcd. 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 photoactivated cell killing activity is shown in FIG. 3 and further described below in Examples 2.1-2.4.

[0088] Example 2.1: Synthesis of tert-butyl 4-(5-bromo-1,3-thiazol-2-yl)piperazine-1-carboxylate 89 2,5-Dibromo-1,3-thiazole (10 g, 41.2 mmol) was dissolved in N,N-dimethylformamide (DMF) (100 mL), 1-Boc-piperazine (10 g, 53.5 mmol) and K2CO3 (7.40 g, 53.5 mmol) were added, and the mixture was stirred at 70 °C for 72 h. The mixture was cooled, diluted with H2O, and extracted with ethyl acetate. The organics were washed with H2O and brine, dried (MgSO4), and evaporated to give a crude oil, which was purified by SiO2 chromatography (petroleum ether / ethyl acetate, 8:2) to give 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-thiazol-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 at room temperature overnight. The solution was evaporated to give a crude yellow oil (23 g), which was purified by SiO2 chromatography (dichloromethane / methanol, 95:5) to give the trifluoroacetate salt of the desired compound (10.7 g). This was then dissolved in dichloromethane and rapidly stirred with saturated NaHCO3 for 0.5 h. The organics were washed with H2O, dried (MgSO4) and evaporated to give 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) calcd. C7H 11 N3SBr [M + H] + : 247.9852, measured value 247.9850

[0090] Example 2.3: Synthesis of methyl (2E)-3-(5-{2-[2-(piperazin-1-yl)-1,3-thiazol-5-yl]ethynyl}pyridin-2-yl)prop-2-enoate 91 Triethylamine (Et3N) (200 mL) was degassed by bubbling Ar for 1 h. 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 h. The solvent was then evaporated to give a crude solid, which was purified twice by SiO2 chromatography (95:5 to 9:1, dichloromethane / methanol, 1% triethylamine) to give compound 91 as a light 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) calcd. C 18 H 19 N4O2S [M+H] + : 355.1223, measured value 355.1223

[0091] Example 2.4: Synthesis of methyl (2E)-3-(5-{2-[2-(4-{7-[(acetyloxy)carbamoyl]heptanoyl}piperazin-1-yl)-1,3-thiazol-5-yl]ethynyl}pyridin-2-yl)prop-2-enoate 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 min. The resulting mixture was stirred at 0° C. for 2 h, 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 h. The mixture was diluted with dichloromethane, washed with H2O, dried (MgSO4), and evaporated to give a crude yellow solid (1.7 g). This was purified by SiO2 chromatography (99:1, dichloromethane / methanol) and further recrystallized from acetonitrile (MeCN) to give 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) calcd. 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}piperazin-1-yl)phenyl]ethynyl}thiophen-2-yl)prop-2-enoate 93 is shown in FIG. 4 and described 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 min. The resulting mixture was stirred at 0° C. for 2 h, after which 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 h. The mixture was diluted with dichloromethane, washed with H2O, dried (MgSO4), and evaporated to give a crude yellow solid (1.7 g). This was purified by SiO2 chromatography (97:3, dichloromethane / methanol) and further recrystallized from acetonitrile to give 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 FIG. 5 and described 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) was added followed by pyridine (9.16 mL, 113.3 mmol) and the resulting solution was stirred at room temperature for 16 h. The solution was diluted with dichloromethane and the organics were washed with saturated NH4Cl and H2O, dried (MgSO4) and evaporated to give a crude brown oil (40 g). This was purified by SiO2 chromatography (7:3 cyclohexane / ethyl acetate) to give compound 94 as a yellow oil (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 h. The suspension was cooled, diluted with H2O, and then extracted with EtOAc. The organics were washed with H2O and brine, dried (MgSO4), and evaporated to give a crude orange oil (37.6 g). This was purified by SiO2 chromatography (7:3 cyclohexane / ethyl acetate) to give compound 95 as an 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), BH3·Me2S (2.0 M in toluene, 16.8 mL, 33.6 mmol) was added, and the solution was stirred at reflux for 16 h. The mixture was cooled and then stirred with 10% w / v aqueous Na2CO3 for 0.5 h. The mixture was diluted with ethyl acetate, and the organics were washed with H2O and brine, dried (MgSO4), and evaporated to give a crude yellow oil (4.21 g). This was purified by SiO2 chromatography (9:1 dichloromethane / methanol, 2% triethylamine) to give compound 96 as a clear oil (1.76 g, 38%), which was used immediately 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 FIG. 6 and described 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) at 50° C. was added N-iodosuccinimide (24.75 g, 110.0 mmol) and paratoluenesulfonic acid monohydrate (1.90 g, 10.0 mmol), and the resulting solution was stirred at 50° C. for 1 h. 1.0 M HCl (80 mL) was added and the mixture was extracted with ethyl acetate, washed with saturated Na2S2O3, HO, and brine, dried (MgSO4), and evaporated to give compound 24 as a yellow oil (25.26 g, >100%), which 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 argon through for 1 h. 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 then added under argon and the resulting suspension was stirred at room temperature for 18 h. The mixture was diluted with diethyl ether and passed through Celite / SiO2 to give a crude brown oil (17.7 g). This was purified by SiO2 chromatography to give compound 97 as an orange oil (12.79 g, 58%) that 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) calcd. C 10 H 13 SOSi [M+H] + : 209.0451, measured value 209.0454

[0101] Example 5.3: Synthesis of methyl (2E)-3-{5-[2-(trimethylsilyl)ethynyl]thiophen-2-yl}prop-2-enoate 98 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 min, and 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 added slowly, and the resulting slurry was stirred at room temperature for 16 h. It was poured onto crushed ice and extracted with ethyl acetate. The organics were 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; 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) calcd. C 13 H 17 O2SSi [M+H] + : 265.0713, measured value 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 (1.3 mL) was added. The resulting mixture was stirred at room temperature for 16 h, after which the solution was diluted with ethyl acetate. The organics were washed with H2O and brine, dried (MgSO4), and evaporated to give a crude dark oil (16 g). This was purified by SiO2 chromatography (1:1 cyclohexane / ethyl acetate) to give compound 99 as a yellow oil (8.18 g, 51%) that 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) calcd. C 16 H 21 O5S [M+H] + : 325.1104, measured value 325.1100

[0103] Example 6: Synthesis of photoactivatable compound 100 The synthesis of photoactivatable compound 100 is shown in FIG. 7 and described 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]prop-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 h. Pd(PPh3)2Cl2 (372 mg, 0.53 mmol) and CuI (100 mg, 0.53 mmol) were then added under argon and the resulting suspension was stirred at 60° C. for 72 h. The resulting suspension was diluted with dichloromethane, washed with saturated NaHCO3 and water, dried (MgSO4) and evaporated to give a crude dark oil (3.75 g). This was purified by SiO2 chromatography (95:5 dichloromethane / methanol, 1% triethylamine) to give compound 100 as a light 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) v 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 the protected HDAC inhibitor compound 101 with photoactivated cell killing activity is shown in FIG. 8 and described in detail below.

[0106] Synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-[5-(2-{4-[(5-{7-[(acetyloxy)carbamoyl]heptanamido}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) and the solution was cooled to 0° C., after which 4-methylmorpholine (0.055 mL, 0.5 mmol), compound 88 (76 mg, 0.33 mmol) and propylphosphonic anhydride (50 wt % in ethyl acetate, 0.32 mL, 0.5 mmol) were added and the resulting mixture was stirred at room temperature for 16 h. The mixture was diluted with dichloromethane, washed with HO, dried (MgSO4) and evaporated to give a crude yellow oil, which was purified by SiO2 chromatography (95:5, dichloromethane / methanol) to give compound 101 as a 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) calcd. 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 to measure the viability of HaCaT keratinocytes in response to treatment with Compound 92 in the absence (no light) and upon illumination (light) was performed as follows.

[0108] HaCaT keratinocyte cells were seeded in two 96-well plates and incubated at 37°C and 5% CO2 for 24 h. The incubation medium was removed and compound 92 was added at various concentrations along with a dimethyl sulfoxide (DMSO) control, after which the cells were incubated at 37°C and 5% CO2 for 1 h, after which one plate was irradiated at 405 nm for 5 min (72 mW / cm2). 2). Both plates were then incubated at 37°C, 5% CO2 for 24 hours. The medium was removed and 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 to measure cell viability at different treatment concentrations of compound 92 with and without light exposure. The results are shown in Figure 9, which indicates that light activation causes cell death at low concentrations (IC 50 = 0.69 μM). In the absence of photoactivation, HDAC inhibitory activity in response to enzyme metabolism resulted in a high concentration (IC 50 = 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 anti-acetyl-H3 primary antibody to detect the presence of acetylated H3 histone was performed as follows.

[0110] 50,000 HaCaT cells were plated on coverslips and grown for 2 days, after which compound 92 (5 μM) was added and incubated at room temperature for 30 min. Cells were washed with phosphate-buffered saline and then fixed with 4% PFA. Cells were washed again with phosphate-buffered saline, then blocked and permeabilized with 0.3% Triton 100-X / 5% goat serum in phosphate-buffered saline for 60 min. Cells were then washed with phosphate-buffered saline, anti-acetyl histone 3 antibody was added, and incubated overnight at 4 °C. Secondary antibody (Alexa-594 anti-rabbit) was added for 45 min, after which cells were washed again and mounted for imaging.

[0111] The results are shown in Figure 10. It can be seen that compound 92 shows limited activity after 10 minutes, but after 1 hour the cells display a characteristic nuclear ring phenotype indicative of accumulation of acetylated H3 in response to inhibition of HDAC enzymes. This delayed behavior is indicative of an early lag phase as compound 92 is enzymatically metabolized to an active form. Ethanol-treated cells do not display this nuclear ring phenotype at any time point.

[0112] Example 10: Acetyl H3 Abundance 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 next day, cells were treated with compounds or controls. After 15 min and / or 1 h of treatment, the cells were lysed in RIPA buffer. SDS-PAGE was performed on cell lysates using Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels to separate proteins. Proteins were then transferred to PVDF membranes (Macherey-Nagel) and then 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 for 1 h each at room temperature with TBST washes between steps. Chemiluminescent signals were measured using an iBright imager (Invitrogen). Acetyl-H3 levels were normalized and the resulting densitometric values ​​(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 min and 1 h treatment with medium, DMSO (dimethyl sulfoxide), SAHA (suberoylanilide hydroxamic acid), compound 27 and compound 93. Acetyl-H3 levels were normalized to α-tubulin (15 min) and AC-40 (1 h).

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

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

[0116] Example 11: Expression of Caspase-3 in Treated HaCaT Cells HaCaT cells were seeded on cover slips in 6-well plates at 50,000 cells per well. The next day, cells were treated with 50 nM and 100 nM of compound 93 for 30 min. DMSO was used as a control. Photoactivation was performed at 405 nm, 29 mW / cm 2Imaging was performed by illumination with a PhotoReact 365 modified to emit light at 37 °C for 5 min. The next day, cells were fixed using 4% PFA, permeabilized using 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. A coverslip was added to the slide and imaged on a Zeiss LSCM 880. Image analysis was performed using ImageJ software. Figure 14 shows the expression of caspase-3 in HaCaT cells treated with DMSO, 50 nM compound 93, and 100 nM compound 93, as well as quantification of the expression levels.

[0117] Example 12: Colocalization of compounds in SCC-4 cells SCC-4 cells were seeded at 50,000 cells / well on 8-well chamber slides. The next day, cells were treated with 1 μM compound 101 and 1 μM compound 93 for 1 h. Cells were replaced with live cell imaging solution medium. Co-stains (MitoTracker, Bodipy ER Tracker, LipidSpot 610, or LysoTracker) were added to cells 30 min prior to imaging. Images were taken on 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. The Pearson coefficient was calculated to demonstrate the correlation between the localization of the compound and the localization of the co-staining. 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's coefficient was 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 gave Tert-butyl (2E)-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazin-1-yl}phenyl)ethynyl]thiophen-2-yl}prop-2-enoate 103, as explained below.

[0123] Compound 93 (500 mg, 0.82 mmol) was dissolved in methanol (30 mL), then NaOH (32 mg, 0.82 mmol, as a solution in HO, 1 mL) was added and the resulting solution was stirred at room temperature for 5 h. The mixture was diluted with dichloromethane and the organics were washed with HO, dried (MgSO4) and evaporated to give a crude yellow solid, which was purified by recrystallization from acetonitrile to give 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 disclosed in this specification (including the accompanying claims, abstract and drawings) and / or all steps of the disclosed method or process may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including the accompanying claims, abstract and drawings) may be replaced by an alternative feature serving the same, equivalent or similar purpose, unless otherwise stated. Thus, unless otherwise stated, each feature disclosed is merely a generic example of a series of equivalent or similar features. The invention is not limited to the details of the foregoing embodiments. The invention extends to any novel or novel combination of features disclosed in this specification (including the accompanying claims, abstract and drawings) or any novel or novel combination of steps of the method or process disclosed herein.

Claims

1. A compound of formula I. 【Chemistry 1】 (In the formula, R １ is H or an alkyl group having 1 to 10 carbon atoms; R ２ is P-Z, where P is an alkyl group having 1 to 15 carbon atoms, optionally substituted with one or more N atoms, -C=O, and -NHC=O; Z is as follows: 【Chemistry 2】 In the formula, R ３ is 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 , -CH ２ N.H. ２ , -CH ２ NH(C=O)CH ２ N.H. ２ or -CH ２ NH(C=O)CH ２ NH(C=O)CH ２ N.H. ２ and or R １ and R ２ forms part of a heterocyclic group Y having 5 or 6 members and substituted with P-Z, P being as defined above and Z being as defined above; Ar １ and Ar ２ are each independently selected from a phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole group; X is -C=C-C(=O)OR ４ and R ４ is an alkyl group having 1 to 10 carbon atoms, optionally substituted with one or more O atoms.

2. R ３ But, C １ ~C ３ 2. The compound of formula I according to claim 1, wherein:

3. R ３ But -CH ３ 3. The compound of formula I according to claim 2, wherein

4. Ar １ 4. A compound of formula I according to any preceding claim, wherein is thiazole or phenyl.

5. Ar ２ 4. A compound of formula I according to any preceding claim, wherein is pyridine, thiophene or furan.

6. R １ and R ２ 4. A compound of formula I according to any preceding claim, wherein forms part of the heterocyclic group Y, optionally Y being piperazine.

7. P is C substituted with -C(=O) １ ~C １５ 7. The compound of formula I according to claim 6, which is an alkyl group.

8. -C(=O)(CH ２ ) ６ 8. A compound of formula I according to claim 7.

9. R ４ 4. A compound of formula I according to any preceding claim, wherein is an alkyl group.

10. R ４ But -CH ３ , -C(CH ３ ) ３ or -CH ２ CH (CH ３ ) ２ 10. The compound of formula I according to claim 9, wherein

11. R １ But, C １ ~C ３ alkyl, optionally R １ Ga-CH ３ 6. A compound of formula I according to any one of claims 1 to 5,

12. R ２ is P-Z, P is replaced by -NHC(=O) １ ~C １５ A compound of formula I according to any one of claims 1 to 5 or claim 11, which is alkyl.

13. P is -(CH ２ ) ５ NHC(=O)(CH ２ ) ６ 13. The compound of formula I according to claim 12, wherein

14. R ４ But -(CH ２ CH ２ O) ｎ CH ３ where n is an integer from 1 to 8, and optionally R ４ But -(CH ２ CH ２ O) ３ CH ３ 14. A compound of formula I according to any of claims 1 to 5 or 11 to 13,

15. 2. A compound of formula I according to any preceding claim, wherein the compound is selected from the following compounds: Compound 92, Compound 93, Compound 101 and Compound 102. 【Chemistry 3-1】 【Chemistry 3-2】

16. 10. The use of a compound of formula I as defined in any preceding claim in photodynamic therapy.

17. A pharmaceutical composition comprising a compound of formula I as defined in any one of claims 1 to 15, optionally in combination with one or more pharma- ceutically acceptable excipients, diluents or carriers.

18. 1. A method for deprotecting a compound of formula I (except for a method of surgery, therapy or diagnosis of a human), comprising contacting said compound of formula I with an enzyme.

19. 20. The method for deprotecting a compound of formula I according to claim 18, wherein the enzyme is an endogenous enzyme.

20. A method for deprotecting a compound of formula I, comprising reacting the compound of formula I with a base in the presence of a solvent.

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