Protected HDAC (histone deacetylase) inhibitors

Abstract

The present invention relates to a protected HDAC inhibitor compound of formula I, wherein Y, Ar1, Ar2, X, R 1 and R 2 is as defined herein. In aspects, the invention relates to the use of said compounds and methods of deprotecting said compounds.

Description

[0001] Acetylation / deacetylation of histones plays an important role in transcriptional regulation in eukaryotic cells. The acetylation state 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 lysine groups on histone and non-histone proteins. HDAC inhibitors (HDACi) inhibit the activity of HDAC enzymes - due to the biological importance of HDACs, their inhibition has important clinical implications, and HDAC inhibition has become an important therapeutic strategy for the treatment of cancer, neurodegenerative diseases, inflammatory diseases and neurological disorders, among others.

[0002] HDAC enzymes are classified based on their homology to the accessory domain of yeast histone deacetylase and are currently divided into four major groups:

[0003] Class I, which includes HDAC1, HDAC2, HDAC3, and HDAC8, is related to the yeast RPD3 deacetylase;

[0004] Class IIA, which includes HDAC4, HDAC5, HDAC7, and HDAC9; Class IIB, which includes HDAC6 and HDAC10, is related to the yeast Hda1 (histone deacetylase 1) gene;

[0005] Class III (also known as sirtuins) are associated with the Sir2 gene and include SIRT1-7;

[0006] • Class IV, which contains only HDAC11, has features of both class I and class II.

[0007] Typical HDAC inhibitors act specifically on class I HDAC, class II HDAC, and class IV HDAC by binding to the zinc-containing catalytic domain of HDAC. These HDAC inhibitors can be further classified based on the chemical part that binds to the zinc ion (except for the cyclic tetrapeptide that binds to the zinc ion with a thiol group). Examples include hydroxamic acid (or hydroxamate), such as trichostatin A; cyclic tetrapeptides (such as trapoxin B), and depsipeptides; benzamide; electrophilic ketones, and fatty acid compounds such as phenylbutyrate and valproic acid.

[0008] Hydroxamic acids constitute the largest class of HDAC inhibitors.

[0009] Examples of hydroxamic acid-based HDAC inhibitors include vorinostat (suberoylanilide hydroxamic acid, SAHA), belinostat, panobinostat, givinostat, pracinostat, quisinostat, and abexinostat.

[0010] Although HDAC inhibitors have important therapeutic effects, the instability of HDAC inhibitors such as hydroxamic acids poses difficulties in terms of storage stability, solubility, formulation and manufacturing.

[0011] The object of the present invention is to solve or alleviate one or more of these problems. The object of the present invention is to provide a protected HDAC inhibitor, and particularly a protected hydroxamic acid. The object of the present invention is to provide a protected HDAC inhibitor that can present improved stability. The object of the present invention is to provide a protected HDAC inhibitor that can present improved solubility. Embodiments of the present invention relate to methods for protecting and / or deprotecting HDAC inhibitors such as HDAC inhibitors based on hydroxamic acid. In embodiments, the deprotection step can be carried out in situ in cells and tissues, i.e., by endogenous enzymes. SUMMARY OF THE INVENTION

[0013] The present invention relates to the use of esters as protecting groups. The present invention relates to the use of esters as protecting groups of HDAC inhibitors. In the process, functional groups of HDAC inhibitors such as hydroxamic groups are protected as esters. Then, the ester can be removed in the presence of an enzyme. The enzyme can be an endogenous enzyme. The removal of the ester leads to the deprotection of the functional group. This deprotection can occur in situ, i.e., the enzyme can be an endogenous enzyme, and the deprotection can occur when the HDAC inhibitor enters a cell. The cell can be a mammalian cell. In an embodiment, the deprotection occurs when the HDAC inhibitor enters a target cell via the action of an endogenous enzyme on the protecting ester group. This can lead to the in situ "clean" activation of HDAC inhibition.

[0014] Therefore, the present invention encompasses protected HDAC inhibitors. Protected HDAC inhibitors can be protected by ester groups. This can serve as a prodrug. It can be used 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, preparation and manufacture. As used herein, the term "prodrug" means one or more compounds administered in an inactive form, and it is converted into its active form in vivo via a chemical process, a biochemical process or a physiological process.

[0015] Advantageously, the ester can be selected to allow 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 body regions of interest, i.e., in those cells, tissues or body regions that contain the complementary enzyme. As will be appreciated by those skilled in the art, the protecting group can be further adjusted to adjust properties such as half-life, solubility, targeting, etc., to suit the properties of the target tissue.

[0016] Suitable protecting groups include, but are not limited to, acetate (C1-C9), glycolate (C1-C9), methoxyacetate (C1-C9), phenylacetate, propionate, butyrate, salicylate, pyruvate, lactate, citrate, PEG esters, glycerides, peptide esters such as monoglycinate, diglycinate and triglycinate, phosphates, sulfonates, carbonates such as tetraethylene glycol, O-glycosyl ethers, O-glycosyl esters.

[0017] In an embodiment, the protecting group is (C1-C9) acetate. In an embodiment, the protecting group is C1 acetate.

[0018] Suitable enzymes for deprotection include, but are not limited to, lipases, lactases, esterases, amylases, cytochrome P450, glycosidases such as β-glucuronidase, sucrase and hyaluronidase, peptidases, phosphatases and sulfatases.

[0019] In embodiments, the enzyme is lipase or lactase.

[0020] In an embodiment, the HDAC inhibitor is a photoactive HDAC inhibitor, although the invention is not so limited and it will be appreciated that the invention is more broadly applicable to HDAC inhibitors and HDAC inhibitor drugs.

[0021] In an embodiment, the HDAC inhibitor is a photoactive HDAC inhibitor. "Photoactive HDAC inhibitor" means an HDAC inhibitor that has dual cell modulating activities, ie, photoactivated cell killing and HDAC inhibitory activities such as biochemical effects and / or targeting.

[0022] Exemplary forms of such compounds are shown below:

[0023]

[0024] Examples of such HDAC inhibitors are compounds as disclosed herein. The inventors have demonstrated that when such photoactive HDAC inhibitors are protected according to the invention, the cell killing function is maintained. It has been observed that biological activity is delayed when deprotection occurs.

[0025] In an aspect of the present invention, there is provided a compound of formula I:

[0026]

[0027] in:

[0028] R 1 is H or an alkyl group containing 1 to 10 carbon atoms;

[0029] R 2 is PZ, wherein P is an alkyl group containing from 1 to 15 carbon atoms, said alkyl group being optionally substituted with one or more of a N atom, -C=O and -NHC=O;

[0030] and Z is:

[0031]

[0032] Where 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;

[0033] or

[0034] R 1 and R 2 forming part of a heterocyclic group Y having 5 or 6 members and substituted by PZ, wherein P is as defined above, and wherein Z is as defined above;

[0035] Ar1 and Ar2 are each independently selected from a phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran or thiazole group; and

[0036] X is -C=CC(=O)OR 4 , where R 4 is an alkyl group containing from 1 to 10 carbon atoms, said alkyl group being optionally substituted by one or more O atoms.

[0037] The compounds of the present invention have the general structure shown in Formula I above.

[0038] The term heterocyclic group with 5 or 6 members means such a monocyclic group, which includes 5 or 6 ring members and optionally includes 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 include piperazinyl, morpholinyl, thiomorpholinyl, dioxythiomorpholinyl, pyrrolidin-1-yl and pyrrolidin-3-yl groups, but are not limited thereto. Examples of aromatic (heteroaryl) groups include pyrrole, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, indolyl and benzothiadiazolyl groups, but are not limited thereto. In an embodiment, the heterocyclic group is a saturated ring system. According to formula I, the ring system is substituted by PZ. In an embodiment, PZ is in the 4th position relative to the nitrogen atom of formula I.

[0039] As used herein, the term "alkyl" refers to a fully saturated, branched, unbranched or cyclic hydrocarbon moiety, i.e., a primary alkyl, a secondary alkyl or a tertiary alkyl, or, where appropriate, a cycloalkyl or an alkyl substituted with a cycloalkyl. In the absence of additional indications, the alkyl group comprises from 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, or 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.

[0040] In an embodiment, R 3 It is a C1-C6 alkyl group.

[0041] In an embodiment, R 3 is C1-C3 alkyl. In an embodiment, R 3 It is -CH3.

[0042] In an embodiment, Ar1 is a thiazole or phenyl group.

[0043] In an embodiment, Ar2 is a pyridine, thiophene or furan group.

[0044] In an embodiment, Ar1 is selected from a thiazole or phenyl group, and Ar2 is selected from a pyridine, thiophene or furan group.

[0045] In an embodiment, R 1 and R 2 Forming part of the heterocyclic group Y. The heterocyclic group Y is substituted with PZ, wherein P is an alkyl group containing from 1 to 15 carbon atoms, the alkyl group being optionally substituted with one or more of N atoms, -C=O and -NHC=O; and Z is:

[0046]

[0047] Where R 3 It 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.

[0048] In an embodiment, the substituent PZ is in the 4 position relative to the nitrogen atom of formula I.

[0049] In which R 1 and R 2 In embodiments forming part of a heterocyclic group Y, Y is piperazine.

[0050] Y can be:

[0051]

[0052] When Y is piperazine, P may be a C1-C 15 Alkyl groups. In an embodiment, P is -C(=O)(CH2)6.

[0053] In Formula I, X is -C=CC(=O)OR 4 , where R 4 is an alkyl group containing from 1 to 10 carbon atoms, said alkyl group being optionally substituted by one or more O atoms.

[0054] In an embodiment, R 4 It is a C1-C6 alkyl group.

[0055] In an embodiment, R 4 It is -CH3, -C(CH3)3 or -CH2CH(CH3)2.

[0056] Optionally, R 1 and R 2 does not form a heterocyclic group Y. In this embodiment, R 1 is H or an alkyl group containing 1 to 10 carbon atoms; and R 2 is PZ, wherein P is an alkyl group containing from 1 to 15 carbon atoms, said alkyl group being optionally substituted with one or more of a N atom, -C=O and -NHC=O;

[0057] and Z is:

[0058]

[0059] Where 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 It can be a C1-C3 alkyl group. In the embodiment, R 1 It is -CH3.

[0060] When R 2 When PZ, P may be a C1-C 15 alkyl.

[0061] In an embodiment, P is -(CH2)5NHC(=O)(CH2)6.

[0062] In an embodiment, R 4 Yes -(CH2CH2O) n CH3, wherein n is an integer between 1 and 8. In an embodiment, R 4 It is -(CH2CH2O)3CH3.

[0063] In an embodiment, R 4 Yes -(CH2CH2O) n CH3, wherein n is an integer between 1 and 8, preferably R 4 is -(CH2CH2O)3CH3, and R 1 and R 2 No heterocyclic group is formed.

[0064] In an embodiment, the compound of Formula I is selected from Compound 92, Compound 93, Compound 101 and Compound 102:

[0065]

[0066]

[0067] The compounds according to the invention are intrinsically fluorescent. According to the invention, the compounds can be used for fluorescence imaging.

[0068] In one aspect, the present invention relates to the use of a compound of formula I to generate reactive oxygen species (ROS) when said compound is activated by light.

[0069] Triplet photosensitizers (PS) generally include a light collection region that is responsible for the dual functions of light collection and intersystem crossing, where electrons in the singlet state are transferred to the triplet state non-radiatively. The quenching of the triplet excited state can lead to the formation of reactive oxygen species (ROS) (free radicals from ground state molecular oxygen) or direct chemical reactions with surrounding molecules. Local ROS production is an immune defense strategy that is used in both animal and plant systems to respond to pathogen attacks. In the cells of animals, plants, fungi and bacteria, ROS triggers a variety of regulatory effects depending on the rate and degree of its production; at high concentrations, apoptosis is observed, while at low concentrations, irritant reactions are often observed (Guo et al. Stem Cells Dev. 2010, 19, 1321-1331).

[0070] Photodynamic therapy (PDT) exploits the ability of photosensitizers to generate ROS, usually to destroy cancer cells, pathogenic microorganisms, and / or unwanted tissues through apoptosis. Typically, photosensitizing compounds are excited near / inside a specific target tissue or condition (e.g., microbial infection, neoplasia, tumor, etc.), resulting in the generation of large amounts of ROS and subsequent destruction of the tissue. At low levels of ROS, cell proliferation can be triggered, leading to applications in wound healing or more general tissue regeneration therapies.

[0071] Therefore, PDT relies on the targeting of photosensitive compounds to accumulate at desired locations (such as cells of diseased tissue), and local light delivery to activate ROS production. Although compounds for PDT are known, they generally have a variety of disadvantages, including small absorption peaks, resulting in difficulty in photoactivation, especially for bulky tumors where light penetration may be difficult to achieve; long biological half-life, resulting in skin photosensitivity that persists for extended periods of time after treatment; poor pharmacological properties, such as poor water solubility; and poor targeting ability (i.e., poor ability to target and accumulate in specific tissues or specific cells, resulting in significant off-target damage).

[0072] Advantageously, the compounds of the invention are biologically inert in the inactive state, but generate ROS when irradiated with short wavelength visible light of low to moderate energy.

[0073] Therefore, the compounds of formula I can be used to generate reactive oxygen species (ROS), and thereby control cell development, i.e., control cell proliferation, differentiation and apoptosis, leading to a variety of therapeutic and non-therapeutic uses. The compounds of formula I are particularly advantageous for use in applications mediated by the control of ROS, because they exhibit effective targeting, which can result in less off-target effects. They can also be adjusted to different cell types, allowing for selective targeting.

[0074] Thus, in an aspect, the invention relates to the use of a compound or conjugate of the invention in photodynamic therapy (PDT).

[0075] The generation of ROS can be controlled based on therapeutic needs, for example, to induce apoptosis for cell ablation, to cause proliferation in wound healing, or a combination of these. For example, in wound care, high levels of ROS can be initially triggered, leading to apoptosis of bacterial and / or fungal cells, followed by low levels of ROS to help skin regeneration.

[0076] Thus, the present invention relates to a method of treating a patient suffering from 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 its active form.

[0077] In one aspect, the invention relates to a pharmaceutical composition comprising a compound of formula I, optionally in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0078] Advantageously, the compound of formula I is protected as an ester, which imparts stability to the compound. The protected compound is easier to store. The protected compound may exhibit improved solubility. Activation of the compound involves removal of the protecting group.

[0079] Aspects of the invention relate to a method of deprotecting a compound of formula I, the method comprising contacting the compound of formula I with an enzyme.

[0080] The enzyme may be an endogenous enzyme. Suitable enzymes 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.

[0081] In embodiments, the enzyme is lipase or lactase.

[0082] Advantageously, the ester protecting group can be selected to allow 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 body regions of interest, i.e., in those cells, tissues or body regions that contain the complementary enzyme. As will be appreciated by those skilled in the art, the protecting group can be further adjusted to regulate properties such as half-life, solubility, targeting, etc., to accommodate the properties of the target tissue.

[0083] Suitable protecting groups include, but are not limited to, acetate (C1-C9), glycolate (C1-C9), methoxyacetate (C1-C9), phenylacetate, propionate, butyrate, salicylate, pyruvate, lactate, citrate, PEG esters, glycerides, peptide esters such as monoglycinate, diglycinate and triglycinate, phosphates, sulfonates, carbonates such as tetraethylene glycol, O-glycosyl ethers, O-glycosyl esters.

[0084] In an embodiment, the protecting group is an acetate (C1-C9) ester. When the ester protecting group is a C1 acetate, R in the compound of Formula I 3 It is -CH3.

[0085] According to an aspect of the present invention, there is provided 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.

[0086] Suitable bases for use in the process of the present invention include NaOH, LiOH, KOH, Li2CO3, Na2CO3, K2CO3, Cs2CO3, LiOMe, NaOMe, KOMe, LiOEt, NaOEt, KOEt, LiO t Bu and KO t Bu.

[0087] The solvent may be a polar solvent. Suitable solvents include methanol, ethanol, propanol, butanol, isopropanol, isobutanol, sec-butanol, tert-butanol, water, tetrahydrofuran, 1,4-dioxane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

[0088] The process may be carried out at from 5° C. to 100° C. or from 15° C. to 30° C. Advantageously, the process may be carried out at room temperature.

[0089] The reaction may be carried out for a duration of from 30 minutes to 48 hours. In embodiments, the reaction is carried out for a duration of from 1 hour to 12 hours or from 2 hours to 7 hours.

[0090] After the reaction occurs, 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 and dried and evaporated to give the deprotected compound as a crude solid.

[0091] Therefore, the present invention relates to protected HDAC inhibitors, examples of which include the following compounds:

[0092]

[0093]

[0094] In embodiments, the protected HDAC inhibitor is Compound 92, Compound 93, Compound 101 or Compound 102.

[0095] In embodiments, the protected HDAC inhibitor is Compound 92, Compound 93, or Compound 101.

[0096] Related compounds used as reference compounds include Compound 12, Compound 13 and Compound 14 below:

[0097]

[0098] Aspects of the present invention relate to 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. The method optionally comprises a purification step.

[0099] The present invention relates to a method for protecting an HDAC inhibitor as an ester and removing the ester with an esterase to activate HDAC inhibition. The esterase can be an endogenous esterase. Removal of the ester with an esterase can be performed in vivo.

[0100] The present invention relates to a method of treating a disorder or condition mediated by HDAC inhibition, the method comprising administering a protected HDAC inhibitor as described herein, wherein the HDAC inhibitor is subsequently deprotected in situ.

[0101] The compounds of Formula I can advantageously exhibit increased solubility, improved chemical and storage stability, and ease of manufacture compared to their deprotected counterparts ( Figure 1 ). Example:

[0102] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0103] Figure 1 A schematic diagram of the present invention is shown, wherein PG indicates a protecting group;

[0104] Figure 2 An exemplary synthesis of acetate protected hydroxamic acid (88) is shown;

[0105] Figure 3 An exemplary synthesis of a protected HDAC inhibitor (92) having photoactivated cell-killing activity according to the present invention is shown;

[0106] Figure 4 An exemplary synthesis of an alternative protected HDAC inhibitor (93) having photoactivated cell killing activity according to the present invention is shown;

[0107] Figure 5The synthesis of a building block compound (96) is shown;

[0108] Figure 6 The synthesis of building block compound (99) is shown;

[0109] Figure 7 The synthesis of photoactivated compound (100) is shown;

[0110] Figure 8 The synthesis of exemplary compound (101) is shown;

[0111] Fig. 9 Shown are 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;

[0112] Fig.10 Shown are immunofluorescence imaging of HaCaT keratinocytes treated with compound 92 and EtOH and co-treated with an anti-acetyl-H3 primary antibody that detects the presence of acetylated H3 histones;

[0113] Fig.11 shows the abundance of acetyl-H3 in SCC-4 cells in response to treatment with compound 93 according to the present invention and with a control compound after 15 minutes and one hour;

[0114] Fig.12 shows the abundance of acetyl-H3 in SCC-4 cells in response to treatment with compound 93 and compound 101 according to the present invention and with a control compound after 1 hour;

[0115] Fig.13 shows the abundance of acetyl-H3 in SCC-4 cells in response to treatment with compound 92 according to the present invention and with a control compound after 15 minutes;

[0116] Fig.14 Shown are the expression levels of caspase-3 in HaCaT cells after treatment with 50 nM of compound 93 and 100 nM of compound 93 before and after photoactivation relative to control;

[0117] Fig.15 Fluorescence microscopy images showing co-localization of Compound 101 are shown;

[0118] Fig.16 Fluorescence microscopy images showing co-localization of compound 93 are shown;

[0119] Fig.17An exemplary deprotection of compound 93 is shown.

[0120] Experimental Example:

[0121] General approach:

[0122] All photoirradiation was performed using a modified PhotoReact 365 TM The improved PhotoReact 365 TM 29mW / cm in 5 minutes 2 All images were analyzed using ImageJ software and all graphs were created using Prism.

[0123] Cell viability assay:

[0124] Opaque wall 96-well plates were seeded with 20,000 SCC-4 cells per well. The next day, the plates were treated with a range of concentrations (100 pM–1 μM) of the compound of interest for 1 hour prior to irradiating the “light” treated plates. The following day, the plates were:

[0125] • Treatment with propidium iodide (PI) and fluorescein diacetate (FDA) lasted for 10 minutes, followed by washing in PBS, after which the fluorescence of PI was measured at 535 / 617 nm and that of FDA at 485 / 520 nm.

[0126] • Treatment with 12 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution for 2 hours, then lysis in DMSO on an orbital shaker and measurement of absorbance at 540 nm.

[0127] Treatment with XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) labeling reagent and electron coupling reagent was continued for 4 hours, followed by washing with PBS and measuring absorbance at 650 nm.

[0128] Using CellTitre-Glo on an orbital shaker TM Reagent treatment lasted for 10 minutes to allow cell lysis, and then luminescence was measured.

[0129] Microscopy procedures:

[0130] Colocalization

[0131] SCC-4 cells were seeded at 50,000 cells per well on 8-well chamber slides and the following day, treated with 1 μM of the compound of interest for 1 hour, then fixed with 4% paraformaldehyde (PFA) or the medium was changed to live cell imaging solution. Co-staining agents (such as MitoTracker, Bodipy ER Tracker or LipidSpot 610) were applied for 30 minutes and then imaged on a Zeiss LSCM 880.

[0132] Immunofluorescence

[0133] HaCaT cells or SCC-4 cells were seeded at 50,000 cells per well in sterile coverslips containing 6-well plates. The next day, the cells were treated with the compound of interest for 30 minutes, and then the "light" treated cells were irradiated. The following day, the cells were then fixed with 4% PFA, permeabilized with Triton X-100 / Tween 20, blocked in BSA / goat serum (depending on the antibody), and stained with primary antibodies followed by secondary antibodies, and then the coverslips were mounted on slides and imaged on a Zeiss LSCM 880.

[0134] Immunoprecipitation

[0135] SCC-4 cells were seeded at 350,000 cells per well in 6-well plates and the following day, the cells were treated with the compounds of interest. After the desired 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 prior to blocking and primary antibody and then secondary antibody staining. Chemiluminescent signals were imaged using the iBright imaging system.

[0136] Example 1: Synthesis of acetate protected hydroxamic acid 88

[0137] The synthesis of an exemplary acetate protected hydroxamic acid 88 is described in Figure 2 and further described in Examples 1.1 to 1.4 below.

[0138] Example 1.1

[0139] Synthesis of 1-tert-butyl 8-methylsuberate 85

[0140] Compound 47 (33.0g, 175mmol) is dissolved in the tert-butyl alcohol (250mL) and is cooled to 0 ℃, then di-tert-butyl dicarbonate (57.3g, 262.5mmol) and 4-dimethylaminopyridine (6.4g, 52.5mmol) are added, and the resulting suspension is stirred rapidly at room temperature for 2h. The solution is diluted with 5%HCl and extracted (3x) with dichloromethane (DCM). Organic matter is washed with saturated NH4Cl and H2O, dried (MgSO4) and evaporated to provide a rough red oil (58.9g). This passes through SiO2 chromatography (hexane / ethyl acetate (EtOAc), 9:1) purifying to provide compound 85 (29.06g, 68%) as a colorless oil: 1 H NMR (400MHz, CDCl3) δ1.28–1.35(m,4H),1.43(s,9H),1.54–1.65(m,4H),2.19(t,J=7.5Hz,2H),2.29(t,J=7.5Hz,2H),3.65(s,3H); 13 C NMR (101MHz, CDCl3) δ24.7,24.9,28.1,28.7,28.8,34.0,35.5,51.4,79.9,173.1,174.2.

[0141] Example 1.2

[0142] Synthesis of tert-butyl 7-(hydroxycarbamoyl)heptanoate 86

[0143] Compound 85 (6.1 g, 25.0 mmol) was dissolved in methanol (MeOH) (21 mL), followed by the addition of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) (11.2 mL, 75.0 mmol) and hydroxylamine (NH2OH) (50% aqueous solution, 15.3 mL, 250 mmol), and the resulting solution was stirred at room temperature (RT) for 3 h. The mixture was diluted with dichloromethane (DCM), and the organic matter was 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 (2.34 g, 38%) as a colorless oil: 1 H NMR (400MHz, CDCl3) δ1.26–1.37(m,4H),1.43(s,9H),1.52–1.68(m,4H),2.14(s,2H),2.19(t,J=6.9Hz,2H).

[0144] Example 1.3

[0145] Synthesis of tert-butyl 7-[(acetyloxy)carbamoyl]heptanoate 87

[0146] Compound 86 (2.3 g, 9.37 mmol) was dissolved in dichloromethane (40 mL), followed by addition of acetyl chloride (0.8 mL, 11.24 mmol) and triethylamine (1.56 mL, 11.24 mmol), and the resulting solution was stirred at room temperature for 3 h. The solution was diluted with dichloromethane, and organic matter was washed with saturated NH4Cl and H2O, dried (MgSO4) and evaporated to give a crude light yellow oil (2.75 g). This was purified by SiO2 chromatography (dichloromethane / methanol, 99:1) to give compound 87 (1.65 g, 61%) as a colorless oil: 1 H NMR (400MHz, CDCl3) δ1.24–1.37(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 (101MHz, CDCl3) δ18.2,24.7,28.0,28.4,28.5,32.6,35.3,80.1,168.7,173.3; MS (ES): m / z=288.2[M+H] + ; HRMS (ES) for C 14 H 26 NO5[M+H] + Calculated value: 288.1805, measured value: 288.1801.

[0147] Example 1.4

[0148] Synthesis of 7-[(Acetyloxy)carbamoyl]heptanoic acid 88

[0149] Compound 87 (1.65 g, 5.74 mmol) was dissolved in dichloromethane (60 mL), followed by addition of trifluoroacetic acid (TFA) (5 mL, 65 mmol), and the resulting solution was stirred at room temperature for 18 h. The solution was evaporated, and the crude residue was purified by SiO chromatography (dichloromethane / methanol, 95:5) to give compound 88 (1.10 g, 83%) as a white solid: 1H NMR (700MHz, DMSO-d6) δ1.23–1.28(m,4H),1.45–1.52(m,4H),2.09(t,J=7.4Hz,2H),2.13(s,3H),2.18(t,J=7.4Hz,2H),11.53(br,1H),11.95(br,1H); 13 C NMR (176MHz, DMSO-d6) δ18.1, 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) for C 10 H 18 NO5[M+H] + Calculated value: 232.1179, measured value: 232.1167.

[0150] Example 2: Synthesis of protected HDAC inhibitor 92

[0151] The synthesis of an exemplary protected HDAC inhibitor 92 with photoactivated cell killing activity is Figure 3 and further described in Examples 2.1 to 2.4 below.

[0152] Example 2.1:

[0153] Synthesis of tert-butyl 4-(5-bromo-1,3-thiazol-2-yl)piperazine-1-carboxylate 89

[0154] 2,5-dibromo-1,3-thiazole (10 g, 41.2 mmol) was dissolved in N,N-dimethylformamide (DMF) (100 mL), followed by 1-Boc-piperazine (10 g, 53.5 mmol) and K2CO3 (7.40 g, 53.5 mmol), and the resulting mixture was stirred at 70 ° C for 72 h. The mixture was cooled, diluted with H2O and extracted with ethyl acetate. Organic matter was washed with H2O and brine, dried (MgSO4) and evaporated to give a crude oil. This was purified by SiO2 chromatography (petroleum ether / ethyl acetate, 8:2) to give compound 89 (10.5 g, 74%) as a light yellow solid: 1 H NMR (400MHz, CDCl3) δ1.46 (s, 9H), 3.38–3.41 (m, 4H), 3.52–3.55 (m, 4H), 7.06 (s, 1H); 13 C NMR (101MHz, CDCl3) δ28.3, 48.0, 80.4, 95.2, 140.4, 154.5, 171.5.

[0155] Example 2.2:

[0156] Synthesis of 1-(5-bromo-1,3-thiazol-2-yl)piperazine 90

[0157] Compound 89 (10.45g, 30.0mmol) is dissolved in dichloromethane (100mL), trifluoroacetic acid (9.2mL, 120.0mmol) is added subsequently, and the resulting solution is stirred at room temperature overnight. The solution is evaporated to give a crude yellow oil (23g). This passes through SiO chromatography (dichloromethane / methanol, 95:5) purifying, to give the trifluoroacetate (10.7g) of the desired compound. It is then dissolved in dichloromethane and with saturated NaHCO rapid stirring continues 0.5h. With organic H o washing, drying (MgSO ) and evaporation, to give compound 90 (6.15g, 83%) as a white solid: 1 HNMR (400MHz, DMSO-d6) δ2.73–2.77(m,4H),3.24–3.27(m,4H),7.18(s,1H); MS(ES): m / z=248.0,250.0[M+H] + ; HRMS (ES) for C7H 11 N3SBr[M+H] + Calculated value: 247.9852, measured value: 247.9850.

[0158] Example 2.3:

[0159] (2E)-3-(5-{2-[2-(piperazin-1-yl)-1,3-thiazol-5-yl]ethynyl}pyridin-2-yl)prop-2-enoic acid Synthesis of Methyl Ester 91

[0160] Triethylamine (Et3N) (200mL) was degassed by bubbling with Ar for 1h. Then compound 90 (2.80g, 11.3mmol), compound 42 (2.32g, 12.41mmol), Pd(PPh3)2Cl2 (390mg, 0.18mmol) and CuI (107mg, 0.18mmol) were added under Ar, and the resulting suspension was stirred at 60°C for 72h. 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 (2.28g, 57%) as a bright orange solid: 1H NMR (400MHz, DMSO-d6) δ2.75–2.82(m,4H),3.35–3.41(m,4H),3.75(s,3H),6.91(d,J=15.7Hz,1H),7.58(s,1H),7.69(d,J= 15.7Hz, 1H), 7.79 (dd, J=8.1, 0.9Hz, 1H), 7.96 (dd, J=8.1, 2.2Hz, 1H), 8.72 (dd, J=2.2, 0.9Hz, 1H); MS(ES) m / z=355.1[M+H] + ; HRMS (ES) for C 18 H 19 N4O2S[M+H] + Calculated value: 355.1223, measured value: 355.1223.

[0161] Example 2.4:

[0162] (2E)-3-(5-{2-[2-(4-{7-[(acetyloxy)carbamoyl]heptanoyl}piperazin-1-yl)-1,3-thiophene Synthesis of methyl 2-oxazol-5-yl]ethynyl}pyridin-2-yl)prop-2-enoate 92

[0163] 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, followed by dropwise addition of 4-methylmorpholine (0.20 mL, 1.85 mmol) over 5 min. The resulting mixture was stirred at 0 ° C for 2 h, followed by addition of compound 91 (0.5 g, 1.41 mmol) and 4-methylmorpholine (0.19 mL, 1.68 mmol), 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 (0.53 g, 66%) as a yellow solid: 1H NMR (700MHz, CDCl3) δ1.35–1.44(m,4H),1.64–1.68(m,2H),1.69–1.73(m,2H),2.22(s,3H),2 .26(t,J=7.4Hz,2H),2.39(t,J=7.4Hz,2H),3.50(t,J=5.4Hz,2H),3.58–3.65(m,4H),3.78(t, J=5.4Hz,2H),3.82(s,3H),6.93(d,J=15.6Hz,1H),7.38(dd,J=8.2,0.9Hz,1H),7.45(s,1H), 7.66(d,J=15.6Hz,1H),7.74(dd,J=8.1,2.1Hz,1H),8.69(dd,J=2.1,0.8Hz,1H),9.32(s,1H); 13 C NMR (176MHz, CDCl3) δ18.3,24.7,28.1,28.3,32.5,32.8,40.6,44.7,48.0,48.4,51.9,85.4,91.2,106.6 ,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) for C 28 H 34 N5O6S[M+H] + Calculated value: 568.2224, measured value: 568.2220.

[0164] Example 3: Synthesis of protected HDAC inhibitor 93

[0165] 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 Figure 4 and described in detail below.

[0166] 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, followed by dropwise addition of 4-methylmorpholine (1.5 mL, 13.6 mmol) over 5 min. The resulting mixture was stirred at 0 ° C for 2 h, followed by addition of compound 27 (4.11 g, 10.41 mmol) and 4-methylmorpholine (1.36 mL, 12.4 mmol), 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 (2.51 g, 40%) as a yellow solid: 1 H NMR (600MHz, CDCl3) δ1.32–1.45(m,4H),1.50(s,9H),1.63(p,J=7.1Hz,2H),1.69(p,J=7.1Hz ,2H),2.19(s,3H),2.25(t,J=7.2Hz,2H),2.37(t,J=7.5Hz,2H),3.21(t,J=5.3Hz,2H),3.24( t,J=5.3Hz,2H),3.55–3.66(m,2H),3.75(t,J=5.2Hz,2H),6.11(dd,J=15.6,1.1Hz,1H),6.84 (d,J=8.7Hz,2H),7.03–7.14(m,2H),7.36–7.44(m,2H),7.58(d,J=15.6Hz,1H),9.91(s,1H); 13 C NMR (176MHz, CDCl3) δ18.3,24.8,28.1,28.2,28.4,32.4,32.7,41.1,45.2,48.1,48.4,53.4,80.6,81 .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.

[0167] Example 4: Synthesis of building block compound 96

[0168] Synthesis of N-(5-aminopentyl)-4-iodo-N-methylaniline 96 Figure 5 and described in detail below.

[0169] Example 4.1:

[0170] Synthesis of 5-chloro-N-(4-iodophenyl)-N-methylpentanamide 94

[0171] N-methyl-4-iodoaniline (24.04g, 103mmol) is dissolved in dichloromethane (300mL), and the solution is cooled to 0 ° C. Add 5-chlorovaleryl chloride (14.6mL, 113.3mmol), then add pyridine (9.16mL, 113.3mmol), and the resulting solution is stirred at room temperature for 16h. The solution is diluted with dichloromethane, and the organic matter is washed with saturated NH4Cl and H2O, dried (MgSO4) and evaporated to give a crude brown oil (40g). This is by SiO2 chromatography (7:3 cyclohexane / ethyl acetate) purification, to give compound 94 (35.16g, 97%) as a yellow oil: 1 H NMR (400MHz, CDCl3) δ1.57–1.79(m,4H),1.98–2.19(m,2H),3.24(s,3H),3.35–3.52(m,2H),6.93(d,J=7.9Hz,2H),7.75(d,J=8.0Hz,2H).

[0172] Example 4.2:

[0173] Synthesis of 5-azido-N-(4-iodophenyl)-N-methylpentanamide 95

[0174] Compound 94 (35.0 g, 99.5 mmol) was dissolved in N,N-dimethylformamide (200 mL), and sodium azide (13.53 g, 208.95 mmol) was added, and the solution was subsequently stirred at 80 ° C for 18 h. The suspension was cooled, diluted with H o, and then extracted with EtOAc. Organic matter was washed with H o and brine, dried (MgSO ) and evaporated to give a crude orange oil (37.6 g). This was purified by SiO chromatography (7: 3 cyclohexane / ethyl acetate) to give compound 95 (33.4 g, 94%) as an orange oil: 1 H NMR (400MHz, CDCl3) δ1.45–1.59(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).

[0175] Example 4.3:

[0176] Synthesis of N-(5-aminopentyl)-4-iodo-N-methylaniline 96

[0177] Compound 95 (5.24g, 14.6mmol) is dissolved in toluene (80mL), and BH is added.MeS (2.0M in toluene, 16.8mL, 33.6mmol), then the solution is stirred under reflux for 16h. The mixture is cooled, then 10%w / v NaCOThe aqueous solution is stirred for 0.5h. The mixture is diluted with ethyl acetate, and organic HO and salt water washing, dried (MgSO) and evaporated to give a crude yellow oil (4.21g). This is by SiOChromatography (9:1 dichloromethane / methanol, 2% triethylamine) purification, to give compound 96 as a clear oil, which directly proceeds to the next step (1.76g, 38%): 1 H NMR (400MHz, CDCl3) δ1.28–1.39(m,2H),1.43–1.53(m,2H),1.53–1.61(m,2H),1.97(s,2H), 2.70(t,J=7.0Hz,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 (101MHz, CDCl3) δ24.3, 26.4, 33.1, 38.2, 41.9, 52.5, 76.4, 114.3, 137.6, 148.7.

[0178] Example 5: Synthesis of Building Block Compound 99

[0179] Synthesis of 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-(5-ethynylthiophen-2-yl)prop-2-enoate Figure 6 and described in detail below.

[0180] Example 5.1:

[0181] Synthesis of 5-iodothiophene-2-carboxaldehyde 24

[0182] To a solution of 2-thiophenecarboxaldehyde (9.34 mL, 100.0 mmol) in ethanol (50 mL) at 50° C., N-iodosuccinimide (24.75 g, 110.0 mmol) and p-toluenesulfonic acid monohydrate (1.90 g, 10.0 mmol) were added 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, H2O and brine, dried (MgSO4) and evaporated to give compound 24 (25.26 g, >100%) as a slowly crystallizing yellow oil: 1H NMR (400MHz, CDCl3) δ7.39 (s, 2H), 9.77 (s, 1H).

[0183] Example 5.2:

[0184] Synthesis of 5-[2-(Trimethylsilyl)ethynyl]thiophene-2-carboxaldehyde 97

[0185] Triethylamine (300mL) is degassed by bubbling with argon for 1h. Then add compound 24 (25g, 105mmol), trimethylsilyl acetylene (16.0mL, 115.5mmol), Pd (PPh3) 2Cl2 (740mg, 1.05mmol) and CuI (200mg, 1.05mmol) under argon, and the resulting suspension is stirred at room temperature for 18h. The mixture is diluted with diethyl ether and passed through diatomite / SiO2, to give a crude brown oil (17.7g). This is by SiO2 chromatography purification, to give compound 97 (12.79g, 58%) as a slowly crystallized orange oil: 1 H NMR (400MHz, CDCl3) δ0.25 (s, 9H), 7.24 (d, J = 4.0Hz, 1H), 7.60 (d, J = 4.0Hz, 1H), 9.83 (s, 1H); 13 C NMR(101MHz, 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 -1 2960w, 2899w, 2833w, 2148m, 1666s, 1438s, 1249s, 1223s, 1207s, 838s; MS (ES) m / z=209.0[M+H] + ; HRMS (ES) for C 10 H 13 SOSi[M+H] + Calculated value: 209.0451, measured value: 209.0454.

[0186] Example 5.3:

[0187] Synthesis of (2E)-3-{5-[2-(trimethylsilyl)ethynyl]thiophen-2-yl}prop-2-enoic acid methyl ester 98

[0188] At 0 ° C, trimethylphosphonoacetate (14.0 mL, 86.4 mmol) and LiCl (3.66 g, 86.4 mmol) were added to anhydrous tetrahydrofuran (250 mL), and the resulting solution was stirred for 15 min, followed by the addition of compound 97 (15.0 g, 72 mmol). 1,8-diazabicyclo [5.4.0] undec-7-ene (12.9 mL, 86.4 mmol) was slowly added to the solution, and the resulting slurry was stirred at room temperature for 16 h. It was poured into 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 (17.23 g, 91%) as a light yellow solid: 1 H NMR (400MHz, CDCl3) δ0.24(s,9H),3.78(s,3H),6.19(d,J=15.7Hz,1H),7.05–7.14(m,2H),7.67(d,J=15.7Hz,1H); 13 C NMR(75MHz, 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 –1 2953w, 2899w, 2144m, 1715s, 1621s, 1516w, 1432m, 1391w, 1301s, 1269s, 1202s, 1161s, 838s; MS (ES): m / z=265.1[M+H] + ; HRMS (ES) for C 13 H 17 O2SSi[M+H] + Calculated value: 265.0713, measured value: 265.0713.

[0189] Example 5.4:

[0190] 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-(5-ethynylthiophen-2-yl)prop-2-enoate Synthesis of 99

[0191] Compound 98 (13.03g, 49.3mmol) is dissolved in triethylene glycol monomethyl ether (50mL), and 20%w / v NaOH aqueous solution (1.3mL) is subsequently added. The resulting mixture is stirred at room temperature for 16h, and the solution is subsequently diluted with ethyl acetate. Organic matter H o ​​and salt water washing, dry (MgSO ) and evaporate to give a crude dark oil (16g). This passes through SiO chromatography (1: 1 cyclohexane / ethyl acetate) purification, to give compound 99 (8.18g, 51%) as a rapidly darkening yellow oil: 1 H NMR (400MHz, CDCl3) δ3.36(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=15.7Hz,1H),7.09(d,J=3.8Hz,1H),7.17(d,J=3.8Hz,1H),7.68(dd,J=15.7,0.6Hz,1H); 13 C NMR (101MHz, CDCl3) δ59.0,63.8,69.1,70.5,70.6,71.9,83.7,117.7,124.6,130.5,133.8,136.5,140.8,166.3; IR(ATR)v max / cm –1 3241br, 3089w, 2874br, 2098w, 1705s, 1622s, 1516m, 1445m, 1342m, 1301m, 1264s, 1165s, 1100s, 807s; MS (ES): m / z=325.1[M+H] + ; HRMS (ES) for C 16 H 21 O5S[M+H] + Calculated value: 325.1104, measured value: 325.1100.

[0192] Example 6: Synthesis of Photoactivated Compound 100

[0193] The synthesis of photoactivated compound 100 was Figure 7 and described in detail below.

[0194] 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-[5-(2-{4-[(5-aminopentyl)(methyl)amino Synthesis of 2-(4-(4-(2-(phenyl)ethynyl)thiophen-2-yl)prop-2-enoate 100

[0195] Compound 96 (1.70g, 5.34mmol) and compound 99 (2.42g, 7.48mmol) are dissolved in triethylamine (80mL), and solution is degassed by bubbling with argon for 1h. Then Pd (PPh is added under argon) Cl (372mg, 0.53mmol) and CuI (100mg, 0.53mmol), and the resulting suspension is stirred at 60 DEG C for 72h. The resulting suspension is diluted with dichloromethane, and washed with saturated NaHCO and water, dried (MgSO) and evaporated to give a rough dark oil (3.75g). This passes through SiO Chromatography (95:5 dichloromethane / methanol, 1% triethylamine) purifying, to give compound 100 (0.47g, 17%) as a light orange solid: 1 H NMR (400MHz, CDCl3) δ1.32–1.41(m,2H),1.54–1.64(m,4H),2.81(t,J=7.2Hz,2 H),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=15.7Hz,1H),6.56–6. 65(m,2H),7.07–7.14(m,2H),7.31–7.40(m,2H),7.70(dd,J=15.7,0.6Hz,1H); 13 C NMR (101MHz, CDCl3) δ24.2,26.5,31.0,38.3,41.1,52.2,59.0,63.7,69.2,70.6,70.6,71.9,80. 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) for C 28 H 39 N2O5S[M+H] + Calculated value: 515.2574, measured value: 515.2569.

[0196] Example 7: Synthesis of protected HDAC inhibitor 101

[0197] Synthesis of protected HDAC inhibitor compound 101 with photoactivated cell killing activity Figure 8 and described in detail below:

[0198] 2-[2-(2-methoxyethoxy)ethoxy]ethyl (2E)-3-[5-(2-{4-[(5-{7-[(acetoxy)amino Synthesis of 2-(4 ...

[0199] Compound 100 (128mg, 0.25mmol) is dissolved in dichloromethane (10mL), and the solution is cooled to 0 ℃, then 4-methylmorpholine (0.055mL, 0.5mmol), compound 88 (76mg, 0.33mmol) and propylphosphonic anhydride (50%wt. in ethyl acetate, 0.32mL, 0.5mmol) are added, and the resulting mixture is stirred at room temperature for 16h. The mixture is diluted with dichloromethane, washed with H o, dried (MgSO ) and evaporated to give a crude yellow oil. This is by SiO chromatography (95:5, dichloromethane / methanol) purification, to give compound 101 (141mg, 7%) as a yellow oil: 1 H NMR (400MHz, CDCl3) δ1.30–1.42(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=7.3Hz,2H),2.95(s,3H),3.17–3.26(m,2H),3.33(t,J=7.3Hz,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=15.6Hz,1H),6.61(d,J=5. 4Hz,2H),7.09(d,J=3.8Hz,1H),7.11(d,J=3.9Hz,1H),7.31–7.41(m,2H),7.70(dd,J=15.7,0.6Hz,1H),9.55(s,1H); 13C NMR (101MHz, CDCl3) δ18.3,24.3,24.7,25.3,26.5,26.9,28.0,28.2,29.5,32.5,36.2,38.3,39.3,52.2,53.4,59.0,63.7,69.2,70. 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 3304br, 2927m, 2860m, ​​2189m, 1708m, 1645m, 1619s, 1603s, 1529s, 1512m, 1367m, 1242m, 1193s, 1137s, 1110m, 852m; MS (ES): m / z=728.3[M+H] + ; HRMS (ES) for C 38 H 54 N3O9S[M+H] + Calculated value: 728.3575, measured value: 728.3578.

[0200] Example 8: Fluorescein diacetate cell viability assay

[0201] The fluorescein diacetate cell viability assay, which measures the viability of HaCaT keratinocytes in response to treatment with Compound 92 in the absence of irradiation (no light) and upon irradiation (light), was performed as follows:

[0202] HaCaT keratinocytes were seeded in two 96-well plates and incubated at 37°C, 5% CO2 for 24 h. Before incubating the cells at 37°C, 5% CO2 for one hour, the incubation medium was removed and a series of concentrations of compound 92 and a dimethyl sulfoxide (DMSO) control were added, and then one plate was irradiated at 405 nm for 5 min (72 mW / cm 2 ). Both plates were then incubated at 37°C, 5% CO2 for 24h. The culture 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 10min. The fluorescein diacetate stain was then removed and the cells were washed with 1X phosphate buffered saline. The plates were read at 485nm / 520nm to determine the cell viability of compound 92 at different treatment concentrations with and without light exposure. The results are in Fig. 9and demonstrates that photoactivation occurs at low concentrations (IC 50 =0.69 μM) caused cell death. Due to the HDAC inhibitory activity in response to enzymatic metabolism, cell viability was reduced at higher concentrations (IC 50 =5.50 μM) decreased without photoactivation.

[0203] Example 9: Immunofluorescence imaging

[0204] Immunofluorescence imaging of HaCaT keratinocytes treated with compound 92 (5 μM) and ethanol (EtOH) and co-treated with an anti-acetyl-H3 primary antibody that detects the presence of acetylated H3 histones was performed as follows:

[0205] 50,000 HaCaT cells were plated on coverslips and grown for 2 days, then compound 92 (5 μM) was added and incubated at room temperature for 30 min. The cells were washed in phosphate-buffered saline and then fixed in 4% PFA. The 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. The cells were then washed with phosphate-buffered saline, and anti-acetyl histone 3 antibodies were added and incubated overnight at 4 ° C. Secondary antibodies (Alexa-594 anti-rabbit) were added for 45 min, and the cells were then washed again and mounted for imaging.

[0206] The results are Fig.10 As shown in Figure 2, compound 92 exhibited limited activity after ten minutes, but after one hour, the cells exhibited a characteristic nuclear ring phenotype, which indicates that acetylated H3 accumulates in response to inhibition of the HDAC enzyme. This delayed behavior indicates an initial lag phase when compound 92 is enzymatically metabolized to the active form. Ethanol-treated cells did not exhibit this nuclear ring phenotype at any time point.

[0207] Example 10: Acetyl-H3 Abundance in Treated SCC-4 Cells

[0208] To measure acetyl-H3 protein abundance in SCC-4 cells, cells were seeded at 350,000 cells / well in 6-well plates. The following day, cells were treated with compounds or controls. After 15 minutes and / or 1 hour of treatment, cells were lysed in RIPA buffer. Any kD was used. TM TGX TMPrecast protein gels were performed on cell lysates for SDS-PAGE to separate proteins. Proteins were subsequently transferred to PVDF membranes (Macherey-Nagel) and then blocked in 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 each staining lasted for 1 hour 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 densitometry measurements (measured using ImageJ software) were analyzed using the ELISA kit. Figures 11 to 13 Image analysis was performed using ImageJ software.

[0209] Fig.11 Shown are densitometry measurements of acetyl-H3 after 15 min and 1 h treatment with culture 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).

[0210] Fig.12 Shown are densitometry measurements of acetyl-H3 after 1 hour treatment with culture medium, DMSO (dimethyl sulfoxide), SAHA (suberoylanilide hydroxamic acid), Compound 27, Compound 93, and Compound 101. Acetyl-H3 levels were normalized to α-tubulin.

[0211] Fig.13 Shown are densitometry measurements of acetyl-H3 after 15 min treatment with culture medium, DMSO (dimethyl sulfoxide), SAHA (suberoylanilide hydroxamic acid), Compound 27, and Compound 92. Acetyl-H3 levels were normalized to α-tubulin.

[0212] Example 11: Expression of caspase-3 in treated HaCaT cells

[0213] HaCaT cells were seeded on coverslips in 6-well plates at 50,000 cells per well. The following 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 by irradiation using PhotoReact 365, which was modified to irradiate at 29 mW / cm 2The light at 405 nm was emitted. The following day, the cells were then fixed with 4% PFA, permeabilized with Triton X-100 / Tween 20, and blocked in blocking buffer (5% BSA and 0.1% Tween 20 in PBS). The primary antibody (caspase-3, ab13847, Abcam) and the secondary antibody (goat anti-rabbit IgG Alexa Fluor 594) were diluted in PBS containing 5% goat serum and 0.1% Tween 20. A cover glass was added to the slide and imaged on a Zeiss LSCM 880. Image analysis was performed using ImageJ software. Fig.14 Shown are caspase-3 expression in HaCaT cells treated with DMSO, 50 nM of compound 93, and 100 nM of compound 93 and quantification of expression levels.

[0214] Example 12: Colocalization of compounds in SCC-4 cells

[0215] SCC-4 cells were seeded on 8-well chamber slides at 50,000 cells / well. The following day, cells were treated with 1 μM of compound 101 and 1 μM of compound 93 for 1 hour. Cells were switched to live cell imaging solution medium. Co-staining agents (MitoTracker, Bodipy ER Tracker, LipidSpot 610 or LysoTracker) were added to cells 30 minutes before imaging. Imaging was performed using a Zeiss LSCM 880 and image analysis was performed on ImageJ.

[0216] Fig.15 Co-localization of Compound 101 with mitochondria (MitoTracker), lipid droplets (LipidSpot610), and acidic organelles (LysoTracker) of SCC-4 cells is shown. Pearson coefficients were calculated to demonstrate the correlation between compound localization and co-stainer localization, and the results are shown in Table 1.

[0217]

[0218]

[0219] Table 1: Correlation between compound localization and co-staining agent localization.

[0220] Fig.16The co-localization of compound 93 with mitochondria (MitoTracker), endoplasmic reticulum (Bodipy ERTracker), lipid droplets (LipidSpot610) and acidic organelles (LysoTracker) of SCC-4 cells is shown. Pearson coefficients were calculated to demonstrate the correlation between compound localization and co-staining agent localization, and the results are shown in Table 2.

[0221]

[0222] Table 2: Correlation between compound localization and co-staining agent localization.

[0223] Example 13: Deprotection of compound 93

[0224] Deprotection of compound 93 to produce tert-butyl (2E)-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazin-1-yl}phenyl)ethynyl]thiophen-2-yl}prop-2-enoate 103 Fig.17 and described in detail below.

[0225] Compound 93 (500 mg, 0.82 mmol) was dissolved in methanol (30 mL), followed by addition of NaOH (32 mg, 0.82 mmol, as a solution in H o, 1 mL), and the resulting solution was stirred at room temperature for 5 h. The mixture was diluted with dichloromethane, and organic matter was washed with H o, dried (MgSO ) and evaporated to give a crude yellow solid. This was purified by recrystallization from acetonitrile to give compound 103 (170 mg, 36%) as a yellow solid: 1 H NMR (700 MHz, dimethyl sulfoxide-d6) δ1.22–1.29 (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=15.7Hz,1H),6.91–7.00(m,2H),7.31(d,J=3.8Hz,1H),7.37–7. 44(m,2H),7.48(d,J=3.8Hz,1H),7.66(d,J=15.8Hz,1H),8.64(s,1H),10.32(s,1H); 13C NMR (176 MHz, dimethyl sulfoxide-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) for C 31 H 40 N3O5S[M+H] + Calculated value: 566.2683, measured value: 566.2683.

[0226] All features disclosed in this specification (including any attached claims, abstracts and drawings) and / or all steps of any method or process disclosed in this specification can be combined in any combination, except for at least some mutually exclusive combinations of such features and / or steps. Unless otherwise expressly stated, each feature disclosed in this specification (including any attached claims, abstracts and drawings) can be used as an optional feature replacement for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a series of universal equivalent features or similar features. The present invention is not limited to the details of the aforementioned embodiments. The present invention extends to any novel feature or any novel combination of features disclosed in this specification (including any attached claims, abstracts and drawings), or to any novel step or any novel combination of steps in the steps of any method or process disclosed in this specification.

Claims

1. A compound of formula I: in: R 1 is H or an alkyl group containing 1 to 10 carbon atoms; R 2 is PZ, wherein P is an alkyl group containing from 1 to 15 carbon atoms, said alkyl group being optionally substituted with one or more of a N atom, -C=O and -NHC=O; and Z is: Where R 3 is H or C1-C9 alkyl; or R 1 and R 2 forming part of a heterocyclic group Y having 5 or 6 members and substituted by PZ, wherein P is as defined above, and wherein Z is as defined above; Ar1 is thiazole or phenyl, and Ar2 is pyridine, thiophene or furan; and X is -C=CC(=O)OR 4 , where R 4 is an alkyl group containing from 1 to 10 carbon atoms, said alkyl group being optionally substituted by one or more O atoms.

2. The compound of formula I according to claim 1, wherein R 3 It is a C1-C3 alkyl group.

3. A compound of formula I according to claim 2, wherein R 3 It is -CH3.

4. A compound of formula I according to any preceding claim, wherein R 1 and R 2 Forming part of the heterocyclic group Y.

5. A compound of formula I according to claim 4, wherein Y is piperazine.

6. The compound of formula I according to claim 4, wherein P is C1-C substituted by -C(=O) 15 Alkyl group.

7. A compound of formula I according to claim 6, wherein P is -C(=O)(CH2)6.

8. A compound of formula I according to any one of claims 1 to 3, 6 and 7, wherein R 4 is an alkyl group.

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

10. The compound of formula I according to claim 8, wherein R 4 It is -CH3, -C(CH3)3 or -CH2CH(CH3)2.

11. The compound of formula I according to claim 9, wherein R 4 It is -CH3, -C(CH3)3 or -CH2CH(CH3)2.

12. A compound of formula I according to any one of claims 1 to 3, wherein R 1 It is a C1-C3 alkyl group.

13. A compound of formula I according to claim 12, wherein R 1 It is -CH3.

14. A compound of formula I according to any one of claims 1 to 3, wherein R 2 is PZ, wherein P is a C1-C 15 alkyl.

15. A compound of formula I according to claim 12, wherein R 2 is PZ, wherein P is a C1-C 15 alkyl.

16. A compound of formula I according to claim 14, wherein P is -(CH2)5NHC(=O)(CH2)6.

17. A compound of formula I according to claim 15, wherein P is -(CH2)5NHC(=O)(CH2)6.

18. A compound of formula I according to any one of claims 1 to 3, 13 or 15 to 17, wherein R 4 Yes -(CH2CH2O) n CH3, wherein n is an integer between 1 and 8.

19. A compound of formula I according to claim 12, wherein R 4 Yes -(CH2CH2O) n CH3, wherein n is an integer between 1 and 8.

20. The compound of formula I according to claim 14, wherein R 4 Yes -(CH2CH2O) n CH3, wherein n is an integer between 1 and 8.

21. A compound of formula I according to claim 18, wherein R 4 It is -(CH2CH2O)3CH3.

22. A compound of formula I according to claim 19 or 20, wherein R 4 It is -(CH2CH2O)3CH3.

23. The compound of formula I according to claim 1, wherein the compound is selected from Compound 92, Compound 93, Compound 101 and Compound 102:

24. Use of a compound of formula I according to any preceding claim in the preparation of a medicament for use in photodynamic therapy.

25. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 23, optionally in combination with one or more pharmaceutically acceptable excipients, diluents or carriers.

26. A method of deprotecting a compound of formula I according to any one of claims 1 to 23, the method comprising contacting the compound of formula I with an enzyme.

27. The method for deprotecting a compound of formula I according to claim 26, wherein the enzyme is an endogenous enzyme.

28. A method for deprotecting a compound of formula I according to any one of claims 1 to 23, the method comprising reacting the compound of formula I with a base in the presence of a solvent.

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