TRAP1 inhibitors and their uses

Abstract

The present invention provides a compound or a pharma- ceutically acceptable salt thereof that inhibits TRAP1. In this case, the compound or a pharma- ceutically acceptable salt thereof can inhibit the binding of TRAP1 to a client protein. The present invention also provides a pharmaceutical composition for treating cancer or ophthalmic disease, comprising a compound or a pharma- ceutically acceptable salt thereof that inhibits TRAP1, and its use.

Description

[Technical Field]

[0001] The present specification relates to compounds for inhibiting TRAP1 and uses thereof, and more particularly to TRAP1 inhibitors and compositions containing the same for treating cancer or ophthalmic diseases. [Background technology]

[0002] Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial protein that exists exclusively in mitochondria and is a paralog of the chaperone protein heat shock protein-90 (Hsp90). The present inventors synthesized compounds for inhibiting TRAP1, and further recognized the use of the compounds in pharmaceutical compositions for treating ophthalmic diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] In one embodiment, the present specification provides a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof.

[0004] In addition, the present specification provides, as another embodiment, a compound or a pharmaceutically acceptable salt thereof that inhibits the binding of TRAP1 to a client protein.

[0005] In addition, as another embodiment, the present specification provides a pharmaceutical composition comprising a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof.

[0006] Furthermore, in another embodiment, the present specification provides a method for treating a disease, comprising administering to a subject in need thereof a pharmaceutical composition comprising a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof.

[0007] In one embodiment, the present specification provides use of a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof for treating an ophthalmic disease.

[0008] In one embodiment, the present specification provides a composition for treating an ophthalmic disease, comprising a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof.

[0009] In one embodiment, the present specification provides a use of a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof for producing a composition for treating an ophthalmic disease.

[0010] In one embodiment, the present specification provides a method for treating an ophthalmic disease, comprising administering a compound that inhibits TRAP1 or a pharmaceutically acceptable salt thereof to a subject in need thereof. [Means for solving the problem]

[0011] The present specification provides a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the same for treating cancer or ophthalmic diseases. [Chemical formula 1] JPEG0007766885000001.jpg25170In this case, L is (CH2) n Including, The n is an integer of 7 or more and 40 or less, A pharmaceutical composition is provided comprising a compound, or a pharmaceutically acceptable salt thereof, wherein A is selected from methyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.

[0012] In this case, the present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, wherein A is selected from aryl, cycloalkyl, and heterocyclyl, each substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy.

[0013] In this case, A is a substituted or unsubstituted aryl; The present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, wherein the substituted or unsubstituted aryl is phenyl substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy.

[0014] In this case, the A is: JPEG0007766885000002.jpg93170JPEG0007766885000003.jpg47170

[0015] In this case, A is a substituted or unsubstituted aryl; The substituted or unsubstituted aryl is naphthalene substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same are provided.

[0016] JPEG0007766885000004.jpg42170

[0017] In this case, A is a substituted or unsubstituted aryl; The substituted or unsubstituted aryl is a benzodioxole substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same are provided.

[0018] JPEG0007766885000005.jpg31170

[0019] wherein A is a substituted or unsubstituted cycloalkyl; The substituted or unsubstituted cycloalkyl is cyclohexyl substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same are provided.

[0020] In this case, the present invention provides a pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof, wherein A is unsubstituted cyclohexyl.

[0021] wherein A is a substituted or unsubstituted heterocyclyl; The substituted or unsubstituted heterocyclyl is a pyrrolidine or chroman substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same are provided.

[0022] In this case, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein A is pyrrolidine substituted with one or more groups selected from oxycarbonyl, C1-5 alkyl, and ═O, and a pharmaceutical composition comprising the same.

[0023] JPEG0007766885000006.jpg37170

[0024] In this case, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein A is chroman-2-yl substituted with one or more groups selected from C1-5 alkyl and hydroxyl, and a pharmaceutical composition comprising the same.

[0025] JPEG0007766885000007.jpg32170

[0026] In this case, the present invention provides a compound in which n is an integer of 9 or more, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the same.

[0027] In this case, there is provided a method for treating cancer or an ophthalmic disease, comprising administering the pharmaceutical composition to a subject in need thereof.

[0028] In this case, the pharmaceutical composition provides a method for treating cancer or ophthalmic diseases by oral administration.

[0029] In this case, the pharmaceutical composition provides a method for treating ophthalmic diseases by topically administering it using eye drops.

[0030] In this case, a TRAP1 inhibitor comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is provided.

[0031] In this case, the TRAP1 inhibitor is provided as an inhibitor characterized by binding to CBS.

[0032] In this case, there is provided an inhibitor of the binding of TRAP1 to a client protein, comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof. [Effects of the Invention]

[0033] The compounds provided herein, or pharmaceutically acceptable salts thereof, can be used to inhibit TRAP1.

[0034] The compounds provided herein, or pharmaceutically acceptable salts thereof, can be used to inhibit the binding of TRAP1 to a client protein.

[0035] The pharmaceutical composition comprising the compound provided herein or its pharmaceutically acceptable salt can be used to treat certain diseases related to the TRAP1 mechanism.For example, the pharmaceutical composition comprising the compound provided herein or its pharmaceutically acceptable salt can be used to treat cancer or ophthalmological diseases.

[0036] The compounds provided herein have the advantages of not causing side effects on normal cells and of allowing non-invasive treatment such as administration via eye drops by selectively inhibiting TRAP1. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1a shows the overall structure of the zTRAP1 dimer after binding of MitoQ and AMPPNP. Protomers A and B are displayed in light gray and dark gray, respectively. Figure 1b shows in detail the recognition of MitoQ by TRAP1. The side chains of the residues interacting with MitoQ can be seen. The Fo-Fc map (gray mesh) was calculated in the absence of MitoQ. Figure 1c shows the MitoQ binding pocket structure. The top image shows the Ub-binding pocket, and the bottom image shows the TPP-binding pocket. [Figure 2] The crystal structure of TRAP1 and MitoQ bound together is shown. [Figure 3] The structure of alkyl-TPP is shown. [Figure 4] The graph shows the results of an analysis of the binding ability of alkyl-TPP to the CBS (Client binding site) in TRAP1. [Figure 5]1 shows the antioxidant-TPP conjugate structure. [Figure 6] 1 shows the results of an analysis of the CBS binding ability of antioxidant-TPP conjugates in TRAP1. [Figure 7] Other synthetic compound structures are shown. [Figure 8] Figures 8a and 8b show the results of analyzing the CBS binding ability of other synthetic compounds in TRAP1, respectively. [Figure 9] Figure 9(a) shows the results of analyzing the TRAP1 ATPase activity of alkyl-TPP. Figure 9(b) shows the results of analyzing the binding ability of alkyl-TPP to the ATP binding pocket of TRAP1. mP stands for millipolarization. [Figure 10] Results of TRAP1 and Hsp90 inhibition in cancer cells are shown. [Figure 11] Figure 11(a) shows the results of analyzing TRAP1 ATPase activity depending on the linker length, and Figure 11(b) shows the results of analyzing millipolarization (mP) depending on the linker length. [Figure 12] 1 shows the effect of antioxidant-TPP conjugates on cells. [Figure 13] 1 shows the effects of MitoQS, Mito-VitEL, and Mito-CPS on Hsp90 and TRAP1. [Figure 14] The results of the TRAP1 ATPase activity analysis of other synthetic substances are shown. [Figure 15] The results of TRAP1 inhibition by other synthetic substances are shown. [Figure 16] The results of the in vivo tumor growth inhibitory effects of other synthetic substances are shown. [Figure 17] Western blot results of tumors are shown. [Figure 18-19]This study demonstrates increased TRAP1 expression in diabetic retinopathy models. Figure 18(a) shows Western blot analysis of OIR retinas compared to age-matched room air retinas. TRAP1 protein levels are normalized to β-actin. Figure 18(b) shows qPCR analysis of TRAP1 expression in room air and OIR retinas. Figure 19(a) shows Western blot analysis of STZ-DM retinas compared to age-matched controls. Figure 19(b) shows qPCR analysis of TRAP1 expression in STZ-DM retinas compared to controls. Figure 18(c) and Figure 19(c) show staining for TRAP1 (TRAP1), glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and DAPI (middle gray in merge) in STZ-DM and age-matched controls or OIR and room air retinas. [Figure 20] Figure 20 shows that TRAP1 knockout in the OIR model exacerbates pathological retinal neovascularization. Panel a in Figure 20 shows whole-mount staining of P17 OIR retinas with CD31. Panels b and c in Figure 20 show quantification of neovascular bundles and avascular areas normalized to TRAP1+ / +. [Figure 21] Figure 21(a) shows the results of HIF1α immunofluorescence staining of STZ mouse retinas, and Figure 21(b) shows the results of HIF1α immunofluorescence staining of P17 OIR mouse retinas. [Figure 22] Figure 22(a) shows the results of qPCR analysis of VEGF-A and ANGPTL4 expression in STZ-DM retinas, and Figure 22(b) shows the results of qPCR analysis of VEGF-A and ANGPTL4 expression in P17 OIR retinas. [Figure 23-24]Figures 23(a) and 24(a) show that OIR mice at P12 were treated with intravitreal injections of MitoQ or eye drops to investigate the HIF1α inhibitory activity of MitoQ in vivo. Figures 23(b) and 24(b) show the results of whole-mount staining of the retina with CD31. Figures 23(c) and 24(c) show the ratios of avascular and neovascular areas in OIR retinas after MitoQ administration. [Figure 25] This shows the results of analyzing the HIF1α inhibitory activity of alkyl-TPP using the MIO-MI HRE GFP cell line. [Figure 26] This shows the results of analyzing the HIF1α inhibitory activity of TPP-antioxidant conjugates using the MIO-MI HRE GFP cell line. [Figure 27] This shows the results of analyzing the HIF1α inhibitory activity of other synthetic compounds using the MIO-MI HRE GFP cell line. DETAILED DESCRIPTION OF THE INVENTION

[0038] (Definition of terms) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0039] The specific contents of the invention will be disclosed below.

[0040] (TRAP1) TRAP1 is one of the paralogs of Hsp90 and is a mitochondrial protein found only in mitochondria. Hsp90, including TRAP1, has a structure consisting of two protomers. In this case, each protomer is designated the first and second protomers. When a specific client protein binds to Hsp90, the protomers move closer together, ATP binds, and the protomers are degraded, forming the three-dimensional structure of the client protein. Each protomer consists of an N-terminal domain, a middle domain, and a C-terminal domain. The N-terminal domain binds ATP to generate energy for Hsp90s activity. The middle domain is where specific client proteins bind to each Hsp90s. The C-terminal domain connects the two protomers. Hsp90s are characterized by high homology between paralogs in the N-terminal region but low homology in the middle domain. Thus, compounds targeting the N-terminal region can act non-selectively on Hsp90 paralogs, whereas compounds targeting other regions with low homology can act selectively on Hsp90 paralogs.Unless otherwise specified, Hsp90 includes cytoplasmic Hsp90s, Hsp90-α1, Hsp90-α2, and Hsp90-β.

[0041] It is also known that inhibition of TRAP1 reduces the expression of SIRT3 and SDHB (see Interplay between TRAP1 and Sirtuin-3 Modulates Mitochondrial Respiration and Oxidative Stress to Maintain Stemness of Glioma Stem Cells. Cancer Res 79, 1369-1382). Therefore, in order to confirm the presence or absence of TRAP1 inhibition, the expression of SIRT3 and SDHB can be confirmed herein.

[0042] (aryl) The term "aryl," as used herein, includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" includes polycyclic ring systems having two or more cyclic rings, in which two adjacent rings share two or more carbon atoms, and at least one of the rings is aromatic. The other cyclic ring may be, for example, a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0043] (cycloalkyl) A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Unless otherwise defined, monocyclic cycloalkyl groups typically have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The second ring of a bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings.

[0044] (heterocyclyl) The term "heterocyclyl" refers to a substituted or unsubstituted non-aromatic ring system, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, containing at least one heteroatom, preferably 1 to 4 heteroatoms, and more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocycle" include polycyclic ring systems having two or more rings, where two adjacent rings share two or more carbon atoms and at least one of the rings is heterocyclic. Illustratively, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0045] (hydroxyalkyl) "Hydroxyalkyl" refers to an alkyl group having at least one hydroxy substituent, e.g., a straight-chain monovalent hydrocarbon radical of 1 to 6 carbon atoms or a branched-chain monovalent hydrocarbon radical of 3 to 6 carbon atoms, substituted with one or two hydroxyl groups, provided that if two hydroxyl groups are present, they are not both on the same carbon atom. Specific examples include hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl, and 2-(hydroxymethyl)-3-hydroxypropyl, and the like.

[0046] (ophthalmological disease) As used herein, the term "ophthalmic disease" includes neovascular ophthalmic diseases characterized by choroidal neovascularization, retinal neovascularization, subretinal neovascularization, corneal neovascularization, iris neovascularization, or neovascular glaucoma. The term "neovascular ophthalmic disease" may also refer to retinal neovascularization, and may also refer to neovascular ophthalmic diseases characterized by diabetic retinopathy, retinopathy of prematurity, or retinal vein occlusion. The term "choroidal neovascularization" may also refer to age-related wet macular degeneration (wet AMD).

[0047] (Pharmaceutically acceptable) As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage formulations that, within the scope of sound medical judgment, do not exhibit excessive toxicity, irritation, allergic response, or other problem or side effect, are suitable for contact with the tissues of a subject, and have a reasonable benefit / risk ratio.

[0048] I. Compound of Formula 1 1.Chemical formula 1 [Chemical formula 1] JPEG0007766885000008.jpg25170 The present specification provides a compound having the structure of Chemical Formula 1.

[0049] A can be selected from methyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.

[0050] L is (CH2) n Including, (CH2) n In one embodiment, n may be an integer of 7 or more and 10 or less, 7 or more and 20 or less, 7 or more and 30 or less, 7 or more and 40 or less, or 7 or more and 50 or less. nIn a specific example, n may be an integer of 7 to 40, but is not limited thereto. In this specification, L may be understood as a concept corresponding to a connecting portion or a linker.

[0051] 2. Structure of A 1) Methyl As an example, A may be methyl.

[0052] 2) Substituted or unsubstituted aryl As an example, A may be substituted or unsubstituted aryl. As a specific example, A may be aryl substituted or unsubstituted with one or more selected from halogen, ═O, hydroxy, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be phenyl substituted or unsubstituted with one or more selected from halogen, ═O, hydroxy, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be JPEG0007766885000009.jpg168170

[0053] As another specific example, A may be naphthalene unsubstituted or substituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be naphthalen-2-yl substituted with one or more selected from C1-5 alkyl and ═O. As a more specific example, A may be JPEG0007766885000010.jpg37170

[0054] As another specific example, A may be a substituted or unsubstituted benzodioxole substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be 1,3-benzodioxole or 2,2-difluoro-1,3-benzodioxole. As a specific example, A may be 1,3-benzodioxole or 2,2-difluoro-1,3-benzodioxole. JPEG0007766885000011.jpg27170

[0055] However, A is not limited to this.

[0056] 3) Substituted or unsubstituted cycloalkyl As one example, A may be substituted or unsubstituted cycloalkyl. As a specific example, A may be a monocyclic cycloalkyl having 3 to 10 carbon atoms substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be cyclohexyl substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. However, A is not limited thereto.

[0057] 4) Substituted or unsubstituted heterocyclyl As an example, A may be substituted or unsubstituted heterocyclyl. As a specific example, A may be pyrrolidine or chroman substituted or unsubstituted with one or more selected from halogen, ═O, hydroxyl, oxycarbonyl, hydroxyC1-5 alkyl, C1-5 alkyl, C1-5 alkenyl, C1-5 alkynyl, and C1-5 alkoxy. As a more specific example, A may be pyrrolidine substituted with one or more selected from oxycarbonyl, C1-5 alkyl, and ═O. As another specific example, A may be chroman-2-yl substituted with one or more selected from C1-5 alkyl and hydroxyl. As a specific example, A may be JPEG0007766885000012.jpg32170

[0058] 3. Structure of L In Formula 1, L may be a structure having a specific length. For example, it may be a structure containing alkyl, alkenyl, alkynyl, and / or ethyleneoxy. For example, L may be (CH2) n In this case, n may be an integer of 7 to 40, inclusive, as an example. n is a factor that determines the length of the linker or connecting portion in the compound of the present application, and may play an important role in the binding of the compound having the structure of Chemical Formula 1 to TRAP1.

[0059] However, the structure of L is not limited thereto. In this case, the length of L may be, as an example, 10, 15, 20, or 25 angstroms or more. In this case, as an example, the length of L may be a maximum of 50, 60, 70, 80, or 90 angstroms. However, the maximum length of L is not limited thereto. In an example, L may be a structure containing alkyl, alkenyl, alkynyl, and / or ethyleneoxy having the above-mentioned lengths. As a specific example, L may be an alkyl structure having the above-mentioned lengths.

[0060] 1) Binding ability depending on distance from TPP The crystal structure of TRAP1 and MitoQ (interchangeably referred to as SMX herein) in Figure 2 confirms that effective binding is possible when the distance between the two TRAP1 protomers is approximately 25 Å and the distance between the Ub and TPP moieties of MitoQ is appropriate. Furthermore, experimental results for alkyl-TPP (the structure of alkyl-TPP is shown in Figure 3) confirm that competitive binding between octyl-TPP and SB-TM2 is possible.

[0061] That is, in the compound structure of the present application, the alkyl chain (CH2) bonded to TPP n When the alkyl chain length of the TPP is C8 or longer, the compound can effectively bind to TRAP1. More specifically, when the alkyl chain length of the TPP is C8 or longer, the compound can bind to the CBS (client binding site) of TRAP1. Furthermore, the binding strength increases as the distance from the TPP increases, for example, as the alkyl chain length increases. In one example, a comparison of the binding strength with SMX (MitoQ) showed that dodecyl-TPP, tetradecyl-TPP, and hexadecyl-TPP had superior binding strength compared to SMX. In one example, hexadecyl-TPP, which has the longest alkyl chain length, in particular, showed a binding strength that was twice as strong as SMX (see Table 1 and Figure 4).

[0062] [Table 1]

[0063] 2) Antioxidant-TPP conjugate Antioxidant-TPP conjugates (MitoQ, Mito-CP, SkQ1, Mito-VitE) L ,Mito-TEMPO,MitoQ s ,Mito-VitE,Mito-CPs The structure of Mito-TEMPO and Mito-CP (the structure of which is shown in Figure 5) was used. s It was confirmed that Mito-VitE had no TRAP1 binding ability (see Table 2 and Figure 6), which indicates that an appropriate distance between the linkers is essential for TRAP1 binding.

[0064] [Table 2]

[0065] 3) Other synthetic compounds Based on the results of binding experiments using other compounds with n = 10 (the structures of the synthesized compounds are shown in Figure 7), it can be confirmed that when TPP and various compound structures are linked via hydrocarbons of a specific length, they have binding ability to TRAP1 (see Tables 3 and 4 and Figure 8).

[0066] [Table 3]

[0067] [Table 4]

[0068] That is, for a compound having the structure of Formula 1 to bind to TRAP1, it is important that n is an integer of at least 7. It has also been confirmed that the binding strength to TRAP1 increases as the value of n increases, and accordingly, n may be an integer of up to 20, 30, 40, or 50, as an example. However, the maximum value of n is not limited thereto.

[0069] 4. Salt of Formula 1 The compounds disclosed herein may be considered in the form of a salt. In this case, the salt includes a pharmaceutically acceptable salt. The salts disclosed herein include acid addition salts or basic addition salts. Exemplary acids that form such salts include hydrochloric acid, sulfuric acid, phosphoric acid, glycolic acid, lactic acid, pyruvic acid, citric acid, succinic acid, glutaric acid, etc., and exemplary bases that form salts include lithium, sodium, potassium, calcium, magnesium, methylamine, trimethylamine, etc. However, the salts are not limited thereto and can be easily selected by those skilled in the art.

[0070] 5. Examples of specific compounds [Table 5] JPEG0007766885000018.jpg237170JPEG0007766885000019.jpg244170JPEG0007766885000020.jpg112170

[0071] II. Uses of the Compound of Formula 1 1. TRAP1 inhibition One invention disclosed herein provides the use of the above compounds to inhibit TRAP1.

[0072] The compounds disclosed herein bind to and inhibit TRAP1 function, thereby reducing the expression of SDHB and SIRT3. Additionally, they increase p-AMPK and CHOP (see Control of tumor bioenergetics and survival stress signaling by mitochondrial Hsp90s, Cancer Cell 22, 331-344; Mitochondrial Hsp90s suppress calcium-mediated stress signals propagating from mitochondria to the ER in cancer cells, Molecular Cancer 13, 148), which are known markers of TRAP1 inhibition (see Figures 10, 12, 13, 15, and 17).

[0073] Unlike PU-H71, which is known to bind to the ATP-pocket binding site, the compounds provided herein do not inhibit ATPase activity in a concentration-dependent manner in the case of TPP-alkyl. That is, the compounds provided herein are not associated with ATPase activity inhibition. Furthermore, the compounds provided herein do not bind to the ATP binding site. This was confirmed in one embodiment of the present invention by ATP pocket binding strength analysis (see Figures 9, 11, and 14).

[0074] Therefore, the compounds provided herein can bind to and inhibit TRAP1. More specifically, the compounds provided herein can inhibit TRAP1 without binding to the ATP binding site of TRAP1.

[0075] That is, the present specification provides a TRAP1 inhibitor comprising a compound having a structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof. In this case, the compound or a pharmaceutically acceptable salt thereof may be characterized in that it does not bind to the ATP binding site.

[0076] Furthermore, the present specification provides that a compound having the structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof can be used to produce a TRAP1 inhibitor.

[0077] 2. Inhibition of TRAP1 binding to client proteins The compounds may inhibit the activity of TRAP1 by binding to the intermediate units of TRAP1. More specifically, the compounds inhibit the activity of TRAP1 by binding to the client binding site of TRAP1 but not to the ATP binding site.

[0078] To confirm this, we analyzed the binding structure of TRAP1 and MitoQ, prepared fluorescent probes that bind to the client binding site, and then used these to analyze the binding strength of compounds (see Figures 1, 2, 4, 6, and 8). The experimental results confirmed that TPP-alkyls, antioxidant-TPP conjugates, and other compounds all bind to the client binding site of TRAP1. Furthermore, analysis of ATPase activity, confirmation of ATP binding site binding, and confirmation of cytosolic Hsp90 inhibition confirmed that the compounds described herein inhibit TRAP1 activity without binding to the ATP binding site (see Figures 9, 11, and 14).

[0079] Specifically, the compounds described herein selectively inhibit TRAP1 without affecting cytoplasmic Hsp90 client proteins (Akt, Cdk4) or Hsp70, a marker of Hsp90 inhibition (see Evidence for Efficacy of New Hsp90 Inhibitors Revealed by Ex Vivo Culture of Human Prostate Tumors. Clinical Cancer Research 18, 3562-3570) (see Figures 10, 12, 13, 15, and 17).

[0080] That is, the compounds herein bind to the intermediate unit of TRAP1 and do not affect the N-terminal site where ATP binds to generate energy for the activity of Hsp90s.

[0081] Therefore, the compounds provided herein can bind to the intermediate unit of TRAP1 and inhibit the binding of client proteins known to bind to the intermediate unit, i.e., the compounds provided herein can be used to inhibit the binding of TRAP1 to client proteins.

[0082] Provided herein is a TRAP1-client protein binding inhibitor comprising a compound having a structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0083] As used herein, a compound having the structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof can be used to manufacture a TRAP1-client protein binding inhibitor.

[0084] 3. Pharmaceutical Compositions The compound or its pharmaceutically acceptable salt provided herein can be used as a pharmaceutical composition.That is, the present specification provides a pharmaceutical composition comprising the compound or its pharmaceutically acceptable salt.The present specification provides that the compound or its pharmaceutically acceptable salt can be used to manufacture a pharmaceutical composition.

[0085] The pharmaceutically acceptable salts include salts of compounds derived from various physiologically acceptable organic and inorganic counterions.The counterions are well known in the art, and include, for example, sodium, potassium, calcium, magnesium, aluminum, lithium, and ammonium (when the molecule contains an acidic functional group), such as tetraalkylammonium.In addition, when the molecule contains a basic functional group, the counterions include salts of organic or inorganic acids, such as hydrochloride, sulfate, phosphate, diphosphate, nitric acid hydrobromide, tartrate, mesylate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, pamoate, salicylate, stearate, methanesulfonate, p-toluenesulfonate, and oxalate. Suitable pharmaceutically acceptable salts include those listed in the literature [Remington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985) and P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002]. Examples of acid addition salts include salts formed from acids or organic acids, such as hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid. Such organic acids include alginic acid, ascorbic acid, anthranilic acid, benzoic acid, camphorsulfuric acid, citric acid, embonic acid (pamoic acid), ethanesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gentisic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, isonicotinic acid, isothionic acid, lathic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, pantothenic acid, phenylacetic acid, propionic acid, saccharic acid, salicylic acid, stearic acid, succinic acid, sulfinic acid, trifluoroacetic acid, and arylsulfonic acids (benzenesulfonic acid and p-toluenesulfonic acid).Examples of base addition salts formed from alkali metals, alkaline earth metals, and organic bases include chloroprocaine, choline, N,N-dibenzylethylenediamine, diethanolamine, ethylenediamine, lysine, meglumine (N-methylglucamine), and crocaine, as well as salts formed therein. Salts having non-physiologically acceptable anions or cations are included within the scope of the present invention as useful intermediates for the preparation of physiologically acceptable salts and / or for use in non-therapeutic situations, such as in vitro. Pharmaceutically acceptable salts according to the present application include halide salts, i.e., fluoride salts, bromide salts, iodide salts, and the like.

[0086] The pharmaceutical composition can be used to treat diseases that can be treated by inhibiting TRAP1.

[0087] An example of such a disease is cancer. TRAP1 is well known to be associated with cancer, and TRAP1 may be a target for cancer therapy (see, for example, Regulation of Tumor Cell Mitochondrial Homeostasis by an Organelle-Specific Hsp90 Chaperone Network, 2007, Kang et al.; Control of Tumor Bioenergetics and Survival Stress Signaling by Mitochondrial Hsp90s, 2012, Chae et al.; The mitochondrial chaperone TRAP1 as a candidate target of oncotherapy, 2001, Xie et al.; TRAP1: a viable therapeutic target for future cancer treatments, 2017, Lettini et al.). Furthermore, experimental results have confirmed that the compounds provided herein inhibit TRAP1 in cancer cells, thereby reducing the size of cancer.

[0088] Therefore, the compounds herein can be used in the manufacture of pharmaceutical compositions for the treatment of cancer.

[0089] The present specification may provide a pharmaceutical composition for treating cancer, comprising a compound having a structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0090] In this case, the cancer may include thyroid cancer, gastric cancer, colon cancer, lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, etc. However, the disease is not limited to cancer and includes all diseases known to be associated with TRAP1.

[0091] In one embodiment, the disease includes an ophthalmic disease. In this case, the ophthalmic disease may include a neovascularized ophthalmic disease characterized by choroidal neovascularization disease, retinal neovascularization disease, subretinal neovascularization disease, corneal neovascularization disease, iris neovascularization disease, or neovascular glaucoma. The neovascularized ophthalmic disease may also refer to a retinal neovascularization disease, which may also refer to a neovascularized ophthalmic disease characterized by diabetic retinopathy, retinopathy of prematurity, or retinal vein occlusion. Furthermore, the choroidal neovascularization disease may also refer to age-related wet macular degeneration (wet AMD).

[0092] 1) Ophthalmological diseases The ophthalmic diseases described herein can be classified according to the ocular structure in which the abnormality occurs. In this case, the ocular structure may be an ocular component including the conjunctiva, sclera, cornea, iris, ciliary body, lens, choroid, retina, vitreous body, optic nerve, or ocular muscles. In this case, an ophthalmic disease occurring in the retina among the above structures is called a retinopathy or retinal disease.

[0093] Ophthalmological diseases can also be classified according to the presence or absence of neovascularization. Neovascularization refers to the physical phenomenon of new blood vessels forming around malformed blood vessels. Neovascularization can occur abnormally due to abnormal vascular weakening, ischemia, or excessive production of neovascularization factors. In this case, abnormal neovascularization can cause vascular structures to become crowded, preventing blood vessels from growing thick enough, which can lead to increased vascular pressure and abnormal symptoms such as blood vessels separating from the ocular structures.

[0094] The ophthalmological diseases associated with neovascularization include choroidal neovascularization, retinal neovascularization, subretinal neovascularization, corneal neovascularization, and iris neovascularization. In addition, retinal neovascularization can cause diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, etc. Furthermore, subretinal neovascularization can cause wet age-related macular degeneration (wet AMD).

[0095] In this specification, the ophthalmic disease in the use for treating ophthalmic diseases may refer to the ophthalmic diseases associated with neovascularization described above.

[0096] 2) Relationship between ophthalmological diseases and TRAP1 TRAP1 expression in retinal disease models To confirm the relationship between ophthalmic diseases and TRAP1, we examined the expression level of TRAP1 using a retinal disease model.

[0097] The experimental results confirm that TRAP1 expression is increased in both the oxygen-induced retinopathy model and the STZ (streptozotocin)-induced diabetic retinopathy model (see Figures 18 and 19).

[0098] In other words, it can be confirmed that there is a correlation between ophthalmic diseases and increased TRAP1 expression.

[0099] Furthermore, in oxygen-induced retinopathy and streptozotocin (STZ)-induced diabetic retinopathy models, we confirmed that TRAP1 levels increased along with the hypoxia marker HIF1α and downstream angiogenic factor VEGF-A (see Figures 18a and 19a). Furthermore, staining results confirmed that TRAP1 was colocalized with the staining of glutamine synthase (GS), a marker of Muller cells responsible for the production of various angiogenic factors during the progression of retinal disease (see Figures 18c and 19c).

[0100] That is, these results confirm that the expression of TRAP1 in Müller cells is increased in a hypoxic environment.

[0101] TRAP1 knockout in retinal disease models The experimental results show that in the TRAP1 knockout OIR model, the neovascular and avascular regions were reduced (see FIG. 20).

[0102] In other words, this indicates that inhibiting TRAP1 can improve pathological retinal neovascularization.

[0103] Furthermore, by further referring to the results of retinal HIF1α staining, it can be confirmed that HIF1α is reduced when TRAP1 is knocked out in the STZ or OIR model (see FIG. 21).

[0104] That is, it can be confirmed that inhibiting TRAP1 may decrease HIF1α.

[0105] Furthermore, the mRNA levels of VEGF-A and ANGPTL4 were elevated in STZ TRAP1+ / + compared with Con TRAP1+ / +, but not in STZ-TRAP1- / -. This indicates that TRAP1 inhibition does not increase VEGF-A and ANGPTL4 expression (Fig. 22a).

[0106] Furthermore, VEGF-A and ANGPTL4 mRNA levels were decreased in TRAP1+ / - and TRAP1- / - compared with TRAP1+ / + in the OIR model retina, indicating that TRAP1 inhibition reduces the expression of VEGF-A and ANGPTL4 (see Figure 22b).

[0107] In conclusion, it is no exaggeration to say that TRAP1 inhibition destabilizes HIF1α and reduces various angiogenic factors that induce retinal pathology under hypoxic conditions.

[0108] TRAP1 inhibitors and retinal disease Treatment with a TRAP1 inhibitor can reduce the avascular and neovascular areas of the retina. The experimental results confirm that treatment with MitoQ significantly reduced both the avascular and neovascular areas in the retina of the OIR model. This confirms that MitoQ inhibits abnormal angiogenesis by inhibiting TRAP1 and stimulates normal angiogenesis, thereby suppressing the onset of vascular disease. These experimental results were consistent when MitoQ was injected intravitreally and administered as eye drops. This indicates that MitoQ effectively penetrates the tissue and exerts its effects even when administered as an eye drop (see Figures 23 and 24).

[0109] Furthermore, to confirm whether other TRAP1 inhibitor compounds can be used to treat retinal diseases, we investigated whether the compounds can inhibit HIF1α. As a result, we confirmed that alkyl-TPP, TPP-antioxidant conjugates (with appropriate linker lengths), and other synthetic compounds that were confirmed to inhibit TRAP1 all had HIF1α inhibitory activity (see Figures 25-27 and Tables 6-9).

[0110] [Table 6]

[0111] [Table 7]

[0112] [Table 8]

[0113] [Table 9]

[0114] 3) Pharmaceutical compositions for treating ophthalmic diseases The present disclosure provides a pharmaceutical composition for treating ophthalmic diseases, comprising a TRAP1 inhibitor. The TRAP1 inhibitor includes a compound having the structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof. The present disclosure also provides a use of a TRAP1 inhibitor in the preparation of a pharmaceutical composition for treating ophthalmic diseases. Specifically, the present disclosure provides a use of a compound having the structure of Chemical Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for treating ophthalmic diseases. As described above, ophthalmic diseases are associated with TRAP1. Furthermore, experimental results have confirmed that inhibiting TRAP1 can destabilize HIF1α and reduce various angiogenesis factors that induce retinal disease under hypoxic conditions. Therefore, ophthalmic diseases can be treated by inhibiting TRAP1.

[0115] 4.Treatment method The compound or a pharmaceutically acceptable salt thereof provided herein can be administered to a subject in need thereof to treat a specific disease. That is, the present specification provides a method for treating a specific disease, which comprises administering a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0116] The diseases include all diseases that can be treated through inhibition of TRAP1. Examples of the diseases include, but are not limited to, cancer, and include all diseases known to be associated with TRAP1. In this case, the cancer may include thyroid cancer, gastric cancer, colon cancer, lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, gallbladder cancer, biliary tract cancer, etc.

[0117] In addition, as a specific example, there is provided a method for treating an ophthalmic disease, comprising administering a pharmaceutical composition containing a compound provided herein or a pharmaceutically acceptable salt thereof to a subject in need thereof, wherein the ophthalmic disease includes all of the ophthalmic diseases described in "3. Pharmaceutical Compositions 1) Ophthalmic Diseases."

[0118] The pharmaceutical composition can be administered to a subject in need thereof via various routes, including oral (e.g., drenches such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including prinkle capsules and gelatin capsules), loaves, powders, granules, and pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingual); anal, rectal, or vaginal (e.g., pessaries, creams, or foams); parenteral (intramuscular, intravenous, subcutaneous, or intraspinal, e.g., via a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; transdermal (e.g., a patch applied to the skin); and topical (e.g., creams, ointments, or sprays applied to the skin, or eye drops). In one example, the pharmaceutical composition can be administered orally. In another example, the pharmaceutical composition can be administered topically using eye drops. However, this is not limiting.

[0119] The subject may be a mammal, such as a human, or a non-human mammal. When administered to a subject, such as a human, the composition or compound may preferably be administered as a pharmaceutical composition comprising, for example, a compound of the present application and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters.

[0120] The actual dosage of the pharmaceutical composition may vary for a particular patient, composition, and method of administration to obtain an effective amount of the active ingredient to achieve the desired therapeutic response without toxicity to the patient.

[0121] The selected dosage will depend on a variety of factors, including the activity of the particular compound or combination of compounds used, or their esters, salts, or amides, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in conjunction with the particular compound used, the age, sex, weight, condition, general health, and medical history of the subject being treated, and other factors well known in the medical arts.

[0122] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the required therapeutically effective amount of pharmaceutical composition.For example, a physician or veterinarian can start the dosage of pharmaceutical composition or compound at a lower level than necessary to achieve the desired therapeutic effect, and slowly increase the dosage until the desired effect is achieved.A "therapeutically effective amount" refers to the concentration of a compound sufficient to induce the desired therapeutic effect.

[0123] Generally, it is understood that the effective amount of a compound may vary depending on the subject's weight, sex, and medical history.Other factors that affect the effective amount include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, other types of therapeutic agents administered together with the compound of the present application.A large total dose can be delivered by administering the formulation multiple times.Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th ed., 1814-1882, incorporated herein by reference).

[0124] In certain embodiments, the compounds provided herein can be administered alone or in combination with other types of therapeutic agents. As used herein, the term "conjoint administration" refers to any form of administration of two or more different therapeutic compounds such that a second compound is administered while a previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective on the subject simultaneously, and may include a synergistic effect of the two compounds). For example, different therapeutic compounds can be administered concomitantly or sequentially in the same or separate formulations. In certain embodiments, different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other. Thus, subjects receiving such treatment can benefit from the combined effects of different therapeutic compounds.

[0125] In certain embodiments, the combined administration of a compound provided herein with one or more additional therapeutic agents (e.g., one or more additional chemotherapeutic agents) provides improved efficacy compared to the separate administration of the compound of the present application or the one or more additional therapeutic agents. In certain such embodiments, the combined administration provides an additive effect, where additive effect refers to the combined effect of the separate administration of the compound of the present application and the one or more additional therapeutic agents.

[0126] III. Experimental Example 1. Compound synthesis The present inventors produced SB-TM2, a fluorescent probe that binds to TRAP1, to confirm the TRAP1 binding ability of the compounds, and synthesized and purchased the compounds for use in other experiments.

[0127] Methyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A15878)), ethyltriphenylphosphonium bromide (Alfa Aesar (Cat.# B23096)), butyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A10504)), hexyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A13826)), octyltriphenylphosphonium bromide (Alfa Aesar (Cat.# L02412)), decyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A11021)), dodecyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A14295)), tetradecyltriphenylphosphonium bromide (Alfa Aesar (Cat.# L04311)), hexadecyltriphenylphosphonium bromide (Alfa Aesar (Cat.# A15180), MitoQ (BioVision, Cat.#: B1309), SkQ1 (MedchemExpress Cat.#: HY-100474), and Mito-TEMPO (SIGMA Cat.#: SML0737) were purchased and used in the experiments.

[0128] 1-1.SB-TM2 probe synthesis SB-TM2: 6-(11-oxo-2,3,5,6,7,11-hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamido)hexyl)triphenylphosphonium methanesulfonate. JPEG0007766885000025.jpg61170

[0129] Step A N-(6-hydroxyhexyl)-20-oxo-27-oxa-23-azatetracycloheptadeca-(14),1(16),15(18),17(19)-tetraene-16-carboxamide

[0130] To a solution of compound 14-oxo-20-oxa-16-azatetracycloheptadeca-(8),1(10),9(12),11(13)-tetraene-10-carboxylic acid (376.5 mg, 1.31 mmol), 6-aminohexan-1-ol (170.12 mg, 1.38 mmol), and HATU (602.15 mg, 1.58 mmol) in DMF (30 mL) was added DIPEA (511.67 mg, 3.95 mmol), followed by stirring at 25 °C for 12 h. LCMS indicated that the starting material had been consumed and the desired product had formed. The reaction mixture was poured into 60 mL of H2O and then extracted with ethyl acetate (50 mL × 2). The combined ethyl acetate was washed with saturated brine (50 mL × 2) and dried over Na2SO4. The organic layer was evaporated to dryness to afford the crude product as a yellow foam, which was purified by column chromatography (SiO, Petroleum ether / EtOAc = 1:0 to 1:4) to give the desired product as a yellow foam (465 mg, 1.17 mmol, 92.4% yield).

[0131] MS(ESI):mass calcd.for C 22 H 28 N2O4,384.2;m / z found, 385.2[M+H]+.

[0132] 1H NMR(400MHz,CDCl3)δ1.36-1.50(m,4H),1.51-1.72(m,4H),1.92-2.06(m,4H),2.78(t,J=6.0Hz,2H),2.90(t,J=6.3Hz,2H),3.28 - 3.39(m,4H),3.45(q,J=6.8Hz,2H),3.65(t,J=6.5Hz,2H),7.02(s,1H),8.61(s,1H),8.88(br s,1H).

[0133] Step B. 6-[(21-oxo-29-oxa-24-azatetracycloheptadeca-1(15),2(17),16(19),18(20)-tetraene-17-carbonyl)amino]hexyl methanesulfonate N-(6-hydroxyhexyl)-20-oxo-27-oxa-23-azatetracycloheptadeca-(14),1(16),15(18),17(19)-tetraene-16-carboxamide (400 mg, 1.04 mmol) was dissolved in DCM (25 mL), then DMAP (1.27 mg, 10.40 mmol) and DIEA (1.34 g, 10.40 mmol) were added, and the resulting mixture was cooled to 0 °C. MsCl (106 mg, 9.25 mmol) was added dropwise, followed by stirring at 0 °C for 2 h. TLC (petroleum ether: EtOAc = 1:1, Rf = 0.43) indicated that the starting alcohol had been consumed and one major new spot had formed. The mixture was partitioned between dichloromethane (35 mL) and saturated aqueous NaHCO (35 mL). The organic layer was collected, dried over Na2SO4, filtered, and concentrated in vacuo to give the crude product as a yellow solid, which HNMR showed to be sufficiently pure for the next step reaction.

[0134] 1H NMR(400MHz,CDCl3)δ1.36-1.56(m,4H),1.60-1.85(m,4H),1.99(m,4H),2.78(m,2H),2.90(m,2H),3.01( s,3H),3.26-3.39(m,4H),3.44(q,J=6.6Hz,2H),4.23(t,J=6.5Hz,2H),7.02(s,1H),8.61(s,1H),8.88(br d,J=4.9Hz,1H).

[0135] Step C. (6-(11-oxo-2,3,5,6,7,11-hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxamido)hexyl)triphenylphosphonium methanesulfonate 6-[(21-oxo-29-oxa-24-azatetracycloheptadeca-1(15),2(17),16(19),18(20)-tetraene-17-carbonyl)amino]hexyl methanesulfonate (97 mg, 203.41 μmol) was thoroughly mixed with triphenylphosphane (266.76 mg, 1.02 mmol) in 5 mL of toluene (5 mL), and the resulting solution was stirred at 130 °C for 18 h under N2. LCMS indicated that the sulfonate had been consumed and the desired product had formed as the major constituent. The reaction mixture was cooled to room temperature and evaporated to dryness. TLC (DCM:MeOH = 10:1, Rf = 0.24) showed the formation of one major new spot below OPPh3. The crude product was purified by flash column chromatography on silica gel (initial elution with 50–100% EtOAc in petroleum over 20 min, held at 100% for 15 min to remove any impurities on the desired product, then switched to 0–10% MeOH in DCM over 20 min and held at 10% for 20 min) to give the desired product as the triphenylphosphonium methanesulfonate salt. The product was further lyophilized to remove residual solvent to give a yellow solid (106 mg, 145.55 μmol, 23.85% yield, 98.24% purity).

[0136] MS(ESI):mass calcd.for C 40 H 42 N2O3P+, 629.29;m / z found, 629.5[M+H]+.

[0137] 1H NMR(400MHz,MeOD)δ1.45(m,2H),1.52-1.78(m,6H),1.90-2.04(m,4H),2.69(s,3H),2.7 4-2.87(m,4H),3.34-3.50(m,8H),7.08(s,1H),7.62-7.97(m,15H),8.44(s,1H),9.04(br s,1H);31P NMR (162MHz,METHANOL-d4)δppm 23.81(s,1P).

[0138] 1-2. Synthesis of (10-(3-bromo-4,5,6-trimethoxy-2-methylphenyl)decyl)triphenylphosphonium bromide JPEG0007766885000026.jpg74170

[0139] Step A. 10-Bromo-1-(2-hydroxy-3,4-dimethoxy-6-methyl-phenyl)decan-1-one Freshly powdered AlCl3 (457.89 mg, 3.43 mmol) was added to 10-bromodecanoyl chloride (0.536 g, 1.89 mmol) and 1,2,3-trimethoxy-5-methyl-benzene (312.86 mg, 1.72 mmol) in dry DCE (10 mL) and stirred at 25 °C for 40 h. LCMS showed that the desired product was formed as the major component. The mixture was poured into ice water and extracted with CHCl2 (50 mL * 2). The combined extracts were washed with water, dried over NaSO4, and concentrated to give an oil, which was purified by column chromatography (SiO2, 10:0 to 10:1 petroleum ether / EtOAc) to give a colorless oil (520 mg, 66.56% yield).

[0140] MS(ESI):mass calcd.for C 19 H 29 BrO4,400.12;m / z found, 402.8[M+H]+.

[0141] 1H NMR(400MHz,CDCl3)δ1.21-1.55(m,10H),1.56-1.78(m,2H),1.85(m,2H),2.46(s,3H),2.89 (t,J=7.4Hz,2H),3.41(t,J=6.8Hz,2H),3.88(d,J=12.3Hz,6H),6.31(s,1H),10.38(s,1H).

[0142] Step B. 2-(10-Bromodecyl)-5,6-dimethoxy-3-methyl-phenol 10-Bromo-1-(2-hydroxy-3,4-dimethoxy-6-methyl-phenyl)decan-1-one (520 mg, 1.14 mmol) was dissolved in TFA (10 mL), followed by the addition of EtSiH (2 mL), followed by stirring at 80 °C for 12 h. LCMS showed that the starting ketone was consumed and one new peak formed. The reaction mixture was evaporated to dryness, which was purified by column chromatography (SiO, 5:0 to 5:1 petroleum ether / EtOAc) to give a colorless oil (410 mg, 82.34% yield).

[0143] MS(ESI):mass calcd.for C 19 H 31 BrO3,386.15;m / z found, 388.9[M+H]+.

[0144] 1H NMR(400MHz,CDCl3)δ1.22-1.55(m,14H),1.86(quin,J=7.1Hz,2H),2.26(s,3H), 2.51-2.65(m,2H),3.42(t,J=6.9Hz,2H),3.86(m,6H),5.82(s,1H),6.29(s,1H).

[0145] Step C. 4-Bromo-2-(10-bromodecyl)-5,6-dimethoxy-3-methyl-phenol 2-(10-Bromodecyl)-5,6-dimethoxy-3-methyl-phenol (410 mg, 940.98 μmol) and NaBr (145.23 mg, 1.41 mmol) were dissolved in AcOH (10 mL), and then HO (160.04 mg, 1.41 mmol, 30%) was added at 25 °C, followed by stirring for 2 h. LCMS showed that the starting material was consumed and a new peak had formed. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (40 mL × 2). The combined organic layers were washed with saturated NaHCO (40 mL) until pH > 7, then dried over NaSO, and concentrated to a colorless oil (300 mg, crude).

[0146] MS(ESI):mass calcd.for C19 H 30 Br2O3,464.06;m / z found, 466.9[M+H]+.

[0147] 1H NMR(400MHz,CDCl3)δ1.22-1.55(m,14H),1.86(quin,J=7.1Hz,2H),2.26(s,3H), 2.51-2.65(m,2H),3.42(t,J=6.9Hz,2H),3.85(s,3H),3.93(s,3H),5.77(s,1H).

[0148] Step D. (10-(3-bromo-4,5,6-trimethoxy-2-methylphenyl)decyl)triphenylphosphonium bromide 4-Bromo-2-(10-bromodecyl)-5,6-dimethoxy-3-methyl-phenol (300 mg, 597.11 μmol) and PPh3 (939.68 mg, 3.58 mmol) were dissolved in toluene (1 mL) and then stirred at 130 °C for 18 h under N2. TLC (DCM:MeOH = 10:1, Rf = 0.2) showed that one main peak formed under OPPh3. The reaction mixture was evaporated to give a brown residue, which was purified by prep-HPLC (Column: 3_Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [water (0.2% FA)-ACN]; B%: 52%-82%, 6 min). The desired product was obtained as a white solid after lyophilization (16 mg, 12.24% yield, 97.2% purity).

[0149] MS(ESI):mass calcd.for C 37 H 45 BrO3P+, 647.23;m / z found, 649.3[M+H]+.

[0150] 1H NMR(400MHz,CHLOROFORM-d)δ1.13-1.70(m,16H),2.34(s,3H),2.56-2.76(m,2H),3.65-3.7 9(m,2H),3.68-3.77(m,1H),3.83(s,3H),3.88(s,3H),7.61-7.93(m,15H),8.76(s,1H);31P NMR (162MHz, CHLOROFORM-d) δ24.47(s,1P).

[0151] 1-3. Synthesis of (10-(2-bromo-5-hydroxy-3,4-dimethoxy-6-methylphenyl)decyl)triphenylphosphonium formate JPEG0007766885000027.jpg71170

[0152] Step A. 5-(10-bromodecyl)-1,2,3-trimethoxy-benzene To a solution of 5-bromo-1,2,3-trimethoxy-benzene (1.3 g, 5.26 mmol, 1 eq) in THF (20 mL) was added n-BuLi (2.5 M, 2.10 mL, 1 eq) dropwise at −78 °C. After the addition, the mixture was stirred at that temperature for 1 h, and then a solution of 1,10-dibromodecane (3.16 g, 10.52 mmol, 2 eq) in THF (10 mL) was added dropwise at −78 °C. The resulting mixture was stirred at 20 °C for 11 h. LCMS showed that 50.6% of the desired mass was detected. The residue was diluted with saturated NH 4 Cl (10 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 100 / 0 to 95 / 5). The compound 5-(10-bromodecyl)-1,2,3-trimethoxy-benzene (580 mg, 1.02 mmol, 19.35% yield, 68% purity) was obtained as a colorless oil.

[0153] MS(ESI):mass calcd.for C 19 H 31BrO3,387.4;m / z found, 387.1[M+H]+.

[0154] 1H NMR(400MHz,CDCl3)δ=6.40(s,2H),3.86(s,6H),3.83(s,3H),3.42(t,J=6.8Hz,2H),2.59-2.52(m,2H),1.86(quin,J=7.2Hz,2H),1.60(br d,J=5.5 Hz,2H),1.48-1.38(m,2H),1.38-1.26(m,10H).

[0155] Step B. 6-(10-Bromodecyl)-2,3,4-trimethoxy-benzaldehyde A solution of 5-(10-bromodecyl)-1,2,3-trimethoxybenzene (580 mg, 1.02 mmol, 1 eq) in dry CHCl (2 mL) was slowly added dropwise to a solution of AlCl (239 mg, 1.79 mmol, 97.95 μL, 1.76 eq) in dry CHCl (8 mL) at 0 °C. The mixture was stirred at the same temperature for 45 min, and then a solution of dichloro(methoxy)methane (188.97 mg, 1.64 mmol, 145.36 μL, 1.61 eq, 68% purity) in dry CHCl (2 mL) was slowly added dropwise over 10 min. The mixture was stirred at 0 °C for 2 h 5 min. LCMS indicated the reaction was complete. The reaction mixture was poured into 30 mL of ice water, the methylene chloride phase was separated, and the aqueous phase was extracted twice with 50 mL of methylene chloride. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was used in the next step without further purification. Compound 6-(10-bromodecyl)-2,3,4-trimethoxy-benzaldehyde (510 mg, 858.27 μmol, 84.29% yield, 69.9% purity) was obtained as a colorless oil.

[0156] MS(ESI):mass calcd.for C 20 H 31 BrO4,415.4;m / z found, 415.1[M+H]+.

[0157] 1H NMR(400MHz,CDCl3)δ=10.41(s,1H),6.53(s,1H),4.00(s,3H),3.95(s,3H),3.89(s,3H) ,3.43(t,J=6.9Hz,2H),2.99-2.92(m,2H),1.93-1.82(m,2H),1.49-1.39(m,4H),1.32(br s,10H).

[0158] Step C. 6-(10-bromodecyl)-2-hydroxy-3,4-dimethoxy-benzaldehyde BCl3 (1 M, 1.9 mL, 2.21 eq) was added dropwise to a solution of 6-(10-bromodecyl)-2,3,4-trimethoxybenzaldehyde (510.00 mg, 858.27 μmol, 1 eq, 69.9% purity) in CHCl2 (10 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min and then at 20 °C for 30 min. LCMS showed the reaction was complete. The residue was poured into ice water (30 mL) and extracted with CHCl2 (50 mL * 3). The combined organic layers were dried over NaSO4, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 0 to 95 / 5). The compound 6-(10-bromodecyl)-2-hydroxy-3,4-dimethoxy-benzaldehyde (300 mg, 583.06 umol, 67.93% yield, 78% purity) was obtained as a colorless oil.

[0159] MS(ESI):mass calcd.for C 19 H 29 BrO4,401.3;m / z found, 401.1[M+H]+.

[0160] 1H NMR(400MHz,CDCl3)δ=12.30-12.20(m,1H),10.24-10.03(m,1H),6.34(s,1H),3.96(s,3H),3.89(s,3H),3.43(t,J =6.9Hz,2H),2.90-2.83(m,2H),1.88(quin,J=7.1Hz,2H),1.70-1.60(m,2H),1.50-1.38(m,3H),1.49-1.29(m,1H).

[0161] Step D. 3-Bromo-2-(10-bromodecyl)-6-hydroxy-4,5-dimethoxy-benzaldehyde To a solution of 6-(10-bromodecyl)-2-hydroxy-3,4-dimethoxy-benzaldehyde (250 mg, 622.92 μmol, 1 eq) in CHCl3 (2.5 mL) and CCl4 (2.5 mL) was added NBS (133.04 mg, 747.51 μmol, 1.2 eq) at 0 °C. The mixture was stirred at 0 °C for 1 h. The mixture was then stirred at 20 °C for 11 h. LCMS showed the reaction was complete. The mixture was diluted with saturated NaHCO3 (10 mL) and extracted with EtOAc (20 mL * 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO2, Petroleum ether / Ethyl acetate = 4:1). The compound 3-bromo-2-(10-bromodecyl)-6-hydroxy-4,5-dimethoxy-benzaldehyde (200 mg, 307.77 umol, 49.41% yield, 73.9% purity) was obtained as a yellow oil.

[0162] MS(ESI):mass calcd.for C 19 H 28 Br2O4,480.2;m / z found, 481.0[M+H]+.

[0163] Step E. 5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol To a solution of 3-bromo-2-(10-bromodecyl)-6-hydroxy-4,5-dimethoxy-benzaldehyde (190 mg, 292.38 μmol, 1 eq, 73.9% purity) and EtSiH (169.99 mg, 1.46 mmol, 233.50 μL, 5 eq) in CHCl (4 mL) at 0 °C, TFA (708.40 mg, 6.21 mmol, 460 μL, 21.25 eq) was added dropwise over 5 min via addition funnel. The reaction mixture was stirred at 0 °C for 2 h. LCMS indicated the reaction was complete. The mixture was slowly poured into saturated NaHCO (50 mL) and extracted with 100 mL of CHCl (100 mL * 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO, Petroleum ether: Ethyl acetate = 4:1) to obtain the compound 5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol (130 mg, 241.64 μmol, 82.65% yield, 72% purity) as a colorless oil.

[0164] Step F. 4-Bromo-5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol To a stirred solution of 5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol (130 mg, 241.64 μmol, 1 eq, 72% purity) and NaBr (37.29 mg, 362.46 μmol, 11.65 μL, 1.5 eq) in AcOH (5 mL) was added HO (41.09 mg, 362.46 μmol, 34.82 μL, 30% purity, 1.5 eq), followed by stirring at 20 °C for 3 h. LCMS showed the reaction was complete. The residue was diluted with 30 mL of saturated NaHCO:NaSO = 10:1 and extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give the residue. The crude product was used in the next step without further purification. The compound 4-bromo-5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol (140 mg, 195.18 umol, 80.77% yield, 65% purity) was obtained as a yellow oil.

[0165] MS(ESI):mass calcd.for C 19 H 30 Br2O3,466.3;m / z found, 466.9[M+H]+.

[0166] 1H NMR(400MHz,CDCl3)δ=5.73(s,1H),3.86(s,3H),3.78(s,3H),3.34(t,J=6.9Hz,2H),2.71-2.64(m,2H),2.14(s,3H),1.84-1.76(m,2H),1.37(br d,J=4.1Hz,7H),1.24(br s,7H).

[0167] Step G. (10-(2-bromo-5-hydroxy-3,4-dimethoxy-6-methylphenyl)decyl)triphenylphosphonium formate A stirred solution of 4-bromo-5-(10-bromodecyl)-2,3-dimethoxy-6-methyl-phenol (140 mg, 195.18 μmol, 1 eq, 65% purity) and PPh3 (255.96 mg, 975.88 μmol, 5 eq) in toluene (2 mL) was heated at 125 °C for 8 h under N2. LCMS showed the reaction was complete. The solvent was removed in vacuo to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 100 / 0 to 0 / 100; Ethyl acetate:MeOH = 100 / 0 to 92 / 8). The residue was purified by prep-HPLC (FA condition; column: Xtimate C18 100*30mm*3um; mobile phase: [water (0.225% FA)-ACN]; B%: 40%-70%, 8 min) to obtain the compound (10-(2-bromo-5-hydroxy-3,4-dimethoxy-6-methylphenyl)decyl)triphenylphosphonium formate (6 mg, 8.61 umol, 4.41% yield, 99.54% purity) as a colorless gum.

[0168] MS(ESI):mass calcd.for C 37 H 45 BrO3P+, 648.6;m / z found, 649.2[M+H]+.

[0169] 1H NMR(400MHz,CDCl3)δ=8.56(br s,1.309H),7.78-7.59(m,15H),3.84(s,3H),3.76(s,3H),3.44(br s, 2H), 2.70-2.59 (m, 2H), 2.13 (s, 3H), 1.50 (br s, 4H), 1.40-1.12 (m, 12H).

[0170] 31P NMR (162MHz, CDCl3) δ=24.17(s, 1P).

[0171] 1-4. Synthesis of (10-(3-bromo-4,5,6-trimethoxy-2-methylphenyl)decyl)triphenylphosphonium bromide JPEG0007766885000028.jpg41170

[0172] Step A. 10-Bromo-1-(2,3,4-trimethoxy-6-methyl-phenyl)decan-1-one To a stirred solution of 4-bromo-1,2,3-trimethoxy-5-methyl-benzene (449.11 mg, 1.72 mmol) and 10-bromodecanoyl chloride (536.94 mg, 1.89 mmol) in DCE (10 mL) was added AlCl (206.41 mg, 1.55 mmol), followed by stirring at 25 °C for 18 h. LCMS indicated the formation of the desired product as the major component. TLC (Petroleum ether: EtOAc = 3:1, Rf = 0.4) indicated the formation of one major new spot. The reaction mixture was poured into ice water and extracted with DCM (30 mL × 3), then dried over NaSO and concentrated to give a yellow oil, which was purified by flash column chromatography on silica gel (0–100% EtOAc in petroleum ether over 30 min) to give a colorless oil (215 mg, 29.1% yield).

[0173] MS(ESI):mass calcd.for C 20 H 31 BrO4,414.14;m / z found, 416.8[M+H]+.

[0174] 1H NMR(400MHz,CDCl3)δ1.32(m,8H),1.38-1.49(m,2 H),1.67(m,2H),1.86(quin,J=7.2Hz,2H),2.19(s,3H),2.75(t,J=7.4Hz,2H),3.41(t,J=6.9Hz,2H),3.77-3.92(m,9H),6.48(s,1H).

[0175] Step B. 4-(10-bromodecyl)-1,2,3-trimethoxy-5-methylbenzene To a stirred solution of 10-bromo-1-(2,3,4-trimethoxy-6-methyl-phenyl)decan-1-one (210 mg, 455.03 mmol) in TFA (10 mL) was added EtSiH (1.46 g, 12.52 mmol, 2 mL) at 25 °C, followed by stirring at 80 °C for 2 h. LCMS showed the desired product as the major component. TLC (petroleum ether: EtOAc = 4:1, Rf = 0.45) showed the formation of one major new spot. The reaction mixture was evaporated to dryness in vacuo to give a colorless oil, which was further purified by flash column chromatography on silica gel (25 g, 0–50% EtOAc in petroleum ether over 30 min). The desired product 4-(10-bromodecyl)-1,2,3-trimethoxy-5-methyl-benzene (118 mg, 250.93 umol, 55.15% yield) was obtained as a colorless oil.

[0176] MS(ESI):mass calcd.for C 20 H 33 BrO3,400.16;m / z found, 403.0[M+H]+.

[0177] 1H NMR(400MHz,CDCl3)δ1.20-1.54(m,14H),1.77-1.96(m,2 H),2.27(s,3H),2.46-2.64(m,2H),3.42(t,J=6.8Hz,2H),3.76-3.97(m,9H),6.49(s,1H).

[0178] Step C. 1-Bromo-5-(10-bromodecyl)-2,3,4-trimethoxy-6-methyl-benzene To a stirred solution of 4-(10-bromodecyl)-1,2,3-trimethoxy-5-methyl-benzene (118 mg, 250.93 μmol) and NaBr (38.73 mg, 376.39 μmol) in AcOH (5 mL) was added HO (42.68 mg, 376.39 μmol), followed by stirring at 25 °C for 2 h. LCMS indicated the desired product had formed as the major component. The reaction mixture was partitioned between EtOAc / HO (80 mL / 60 mL). The organic layer was washed with saturated aqueous NaHCO (60 mL) until pH > 7. The combined organic layers were dried over NaSO and concentrated to give a yellow oil (140 mg, crude). HNMR indicated a purity consistent with the desired product for the next step.

[0179] MS(ESI):mass calcd.for C 20 H 32 Br2O3,478.07;m / z found, 481.0[M+H]+.

[0180] 1H NMR (400MHz, CDCl3) δ1.20-1.52(m,14H),1.78-1.94(m,2H),2.36(s,3H),2.62(m,2H),3.42(t,J=6.9 Hz,2H),3.81-3.98(m,9H).

[0181] Step D. (10-(3-bromo-4,5,6-trimethoxy-2-methylphenyl)decyl)triphenylphosphonium bromide A stirred solution of 1-bromo-5-(10-bromodecyl)-2,3,4-trimethoxy-6-methyl-benzene (140 mg, 279.13 μmol) and PPh3 (366.06 mg, 1.40 mmol) in toluene (1 mL) was heated at 130 °C for 18 h under N2. LCMS indicated the formation of the desired product. TLC (DCM:MeOH = 10:1, Rf = 0.2) showed the formation of a major new peak under OPPh3. The reaction mixture was evaporated to give a brown residue, which was purified by flash column chromatography on silica gel (25 g, 0–15% MeOH in DCM over 30 min). The desired product was obtained as a white solid after lyophilization (108.5 mg, 51.41% yield, 98.2% purity).

[0182] MS(ESI):mass calcd.for C 38 H 47 BrO3P+, 661.24;m / z found, 663.3[M+H]+.

[0183] 1H NMR(400MHz,CHLOROFORM-d)δ1.12-1.50(m,12H),1.64(m,4H),2.34(s,3H),2.52-2.71(m,2H),3.77-3.97(m,11H),7.60-7.97(m,15H);31P NMR (162MHz, CHLOROFORM-d) δ24.53(s,1P).

[0184] 1-5. Synthesis of (10-(2-bromo-3,4,5-trimethoxy-6-methylphenyl)decyl)triphenylphosphonium bromide JPEG0007766885000029.jpg87170

[0185] Step A. 5-(10-bromodecyl)-1,2,3-trimethoxy-benzene To a solution of 5-bromo-1,2,3-trimethoxy-benzene (2 g, 8.09 mmol, 1 eq) in THF (30 mL) was added n-BuLi (2.5 M, 3.24 mL, 1 eq) dropwise at −78 °C. After the addition, the mixture was stirred at that temperature for 1 h, and then a solution of 1,10-dibromodecane (4.86 g, 16.19 mmol, 2 eq) in THF (10 mL) was added dropwise at −78 °C. The resulting mixture was stirred at 20 °C for 11 h. LCMS showed that 20% of the desired mass was detected. The residue was diluted with saturated NH 4 Cl (10 mL) and extracted with EtOAc (50 mL*3). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to give the residue. The residue was purified by column chromatography (SiO 2 , petroleum ether / ethyl acetate = 100 / 0 to 95 / 5). The compound 5-(10-bromodecyl)-1,2,3-trimethoxy-benzene (430 mg, 395.86 umol, 4.89% yield, 35.66% purity) was obtained as a colorless oil.

[0186] MS(ESI):mass calcd.for C 19 H 31 BrO3,387.4;m / z found, 389.1[M+H]+.

[0187] Step B. 6-(10-Bromodecyl)-2,3,4-trimethoxy-benzaldehyde To a solution of 5-(10-bromodecyl)-1,2,3-trimethoxybenzene (430 mg, 395.86 μmol, 1 eq, 35.66% purity) in dry CHCl (2 mL) was slowly added dropwise at 0 °C a solution of AlCl (178 mg, 1.33 mmol, 72.95 μL, 3.37 eq) in dry CHCl (6 mL). The mixture was stirred at the same temperature for 45 min, and a solution of dichloro(methoxy)methane (140 mg, 1.22 mmol, 107.69 μL, 3.08 eq) in dry CHCl (2 mL) was slowly added dropwise over 10 min. The mixture was stirred at 0 °C for 2 h 5 min. LCMS indicated the reaction was complete. The reaction mixture was poured into 30 mL of ice water, the methylene chloride phase was separated, and the aqueous phase was extracted twice with 50 mL of methylene chloride. The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to obtain a residue. The crude product was used in the next step without further purification. Compound 6-(10-bromodecyl)-2,3,4-trimethoxy-benzaldehyde (410 mg, 384.97 μmol, 97.25% yield, 39% purity) was obtained as a colorless oil.

[0188] MS(ESI):mass calcd.for C 20 H 31 BrO4,415.4;m / z found, 415.2[M+H]+.

[0189] Step C. 1-(10-bromodecyl)-3,4,5-trimethoxy-2-methyl-benzene To a mixture of 6-(10-bromodecyl)-2,3,4-trimethoxybenzaldehyde (410 mg, 384.97 μmol, 1 eq, 39% purity) and EtSiH (447.64 mg, 3.85 mmol, 614.89 μL, 10 eq) was added TFA (3 mL). The mixture was stirred at 20 °C for 12 h. LCMS showed the reaction was complete. The mixture was slowly poured into saturated NaHCO (50 mL) and extracted with CHCl (50 mL * 3). The combined organic layers were dried over NaSO, filtered, and concentrated under reduced pressure to give the residue. The residue was purified by prep-TLC (SiO, Petroleum ether / Ethyl acetate = 4:1). The compound 1-(10-bromodecyl)-3,4,5-trimethoxy-2-methyl-benzene (120 mg, 152.18 umol, 39.53% yield, 50.9% purity) was obtained as a colorless oil.

[0190] MS(ESI):mass calcd.for C 20 H 33 BrO3,401.4;m / z found, 402.8[M+H]+.

[0191] Step D. 1-Bromo-2-(10-bromodecyl)-4,5,6-trimethoxy-3-methyl-benzene To a stirred solution of 1-(10-bromodecyl)-3,4,5-trimethoxy-2-methyl-benzene (120 mg, 152.18 μmol, 1 eq, 50.9% purity) and NaBr (15.66 mg, 152.18 μmol, 4.89 μL, 1 eq) in AcOH (4 mL) was added HO (17.25 mg, 152.18 μmol, 14.62 μL, 30% purity, 1 eq), followed by stirring at 20 °C for 12 h. LCMS showed the reaction was complete. The residue was diluted with 30 mL of sat. NaHCO:NaSO = 10:1 and extracted with EtOAc (30 mL * 3). The combined organic layers were washed with brine (10 mL), dried over NaSO, filtered, and concentrated under reduced pressure to give the residue. The crude product was used in the next step without further purification. The compound 1-bromo-2-(10-bromodecyl)-4,5,6-trimethoxy-3-methyl-benzene (130 mg, crude) was obtained as a yellow oil.

[0192] MS(ESI):mass calcd.for C 20 H 32 Br2O3,480.3;m / z found, 480.9[M+H]+.

[0193] Step E. 1-Bromo-2-(10-BLAH-decyl)-4,5,6-trimethoxy-3-methyl-benzene A stirred solution of 1-bromo-2-(10-bromodecyl)-4,5,6-trimethoxy-3-methyl-benzene (130 mg, 162.41 μmol, 1 eq, 60% purity) and PPh3 (212.99 mg, 812.04 μmol, 5 eq) in toluene (2 mL) was heated at 125 °C for 12 h under N2. LCMS showed the reaction was complete. The solvent was removed in vacuo to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 100 / 0 to 0 / 100; Ethyl acetate:MeOH = 100 / 0 to 92 / 8). The compound 1-bromo-2-(10-BLAHdecyl)-4,5,6-trimethoxy-3-methyl-benzene (30 mg, 39.69 umol, 24.44% yield, 98.234% purity) was obtained as a colorless oil.

[0194] MS(ESI):mass calcd.for C 38 H 47 BrO3P+, 662.7;m / z found, 663.2[M+H]+.

[0195] 1H NMR(400MHz,CDCl3)δ=7.93-7.66(m,15H),3.94-3.78(m,11H),2.78-2.67(m,2H),2.22(s,3H),1.64(br s,4H),1.50-1.34(m,4H),1.25(br d,J=10.1Hz,8H)31P NMR(162MHz,CDCl3)δ=24.54(s,1P).

[0196] 1-6. Synthesis of formic acid, triphenyl(10-(2,3,4,5-tetramethoxy-6-methylphenyl)decyl)phosphonium salt JPEG0007766885000030.jpg77170

[0197] Step A. 10-Bromo-1-(2,3,4,5-tetramethoxy-6-methyl-phenyl)decan-1-one To a stirred solution of 1,2,3,4-tetramethoxy-5-methyl-benzene (710 mg, 3.35 mmol, 1 eq) and 10-bromodecanoyl chloride (992.09 mg, 3.68 mmol, 1.1 eq) in DCE (15 mL) was added AlCl (401.45 mg, 3.01 mmol, 164.53 µL, 0.9 eq), followed by stirring at 25 °C for 18 h. LCMS showed complete consumption of the starting material. The reaction mixture was extracted with DCM (15 mL * 3) and 10 mL of H2O. The combined organic layers were then evaporated to dryness to give the product. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 15-20% ethyl acetate / petroleum ether gradient @ 45 mL / min). The compound 10-bromo-1-(2,3,4,5-tetramethoxy-6-methyl-phenyl)decan-1-one (800 mg, crude) was obtained as a yellow oil.

[0198] MS(ESI):mass calcd.for C 21 H 33 BrO5,444.15;m / z found, 445.2[M+H]+.

[0199] Step B. 1-(10-bromodecyl)-2,3,4,5-tetramethoxy-6-methyl-benzene To a stirred solution of 10-bromo-1-(2,3,4,5-tetramethoxy-6-methyl-phenyl)decan-1-one (350 mg, 785.83 μmol, 1 eq) in TFA (15 mL) was added triethylsilane (2.55 g, 21.91 mmol, 3.50 mL, 27.89 eq) at 25 °C, followed by stirring at 80 °C for 2 h. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 40 g SepaFlash Silica Flash Column, eluting with a 15–20% ethyl acetate / petroleum ether gradient at 45 mL / min). The compound 1-(10-bromodecyl)-2,3,4,5-tetramethoxy-6-methyl-benzene (150 mg, 347.70 umol, 44.25% yield) was obtained as a colorless oil.

[0200] MS(ESI):mass calcd.for C 21 H 35 BrO4,430.17;m / z found, 433.2[M+3]+.

[0201] Step C. Formic Acid, Triphenyl(10-(2,3,4,5-tetramethoxy-6-methylphenyl)decyl)phosphonium Salt A stirred solution of 1-(10-bromodecyl)-2,3,4,5-tetramethoxy-6-methyl-benzene (130 mg, 301.34 μmol, 1 eq) and PPh3 (344.51 mg, 1.31 mmol, 4.36 eq) in toluene (1 mL) was heated at 130 °C for 18 h under N2. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo to give the crude product. The residue was purified by prep-HPLC (FA condition). Column: Xtimate C18 100*30 μm*10 μm; Mobile phase: [water (0.225% FA)-ACN]; B%: 40%-70%, 10 min. The compound triphenyl-[10-(2,3,4,5-tetramethoxy-6-methyl-phenyl)decyl]phosphonium (64.7 mg, 103.30 μmol, 34.28% yield, 98% purity) was obtained as a yellow oil.

[0202] MS(ESI):mass calcd.for C 39 H 50 O4P+, 613.34;m / z found, 613.6[M+H]+.

[0203] 1H NMR(400MHz,DMSO-d6)δppm1.17-1.37(m,12H)1.40-1.59(m,4H)2.04-2.11(m,3H)2.49(br s,2H)3.55-3.60(m,2H) 3.65-3.68(m,3H)3.69-3.72(m,3H)3.76-3.81(m,6H)7.72-7.84(m,12H)7.87-7.97(m,3H)8.21-8.43(m,1H).

[0204] 1-7. Synthesis of formic acid, (10-(4,5-dimethoxy-2-methylphenyl)decyl)triphenylphosphonium salt JPEG0007766885000031.jpg85170

[0205] Step A. 10-Bromodecanoyl chloride To a mixture of 10-bromodecanoic acid (500 mg, 1.99 mmol, 1 eq) in DCM (4 mL) was added SOCl2 (947.36 mg, 7.96 mmol, 577.66 uL, 4 eq). The reaction mixture was stirred at 25 °C for 2 h. TLC showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo. The crude product was not purified. Compound 10-bromodecanoyl chloride (500 mg, crude) was obtained as an orange oil.

[0206] Step B. 10-Bromo-1-(4,5-dimethoxy-2-methylphenyl)decan-1-one To a solution of 1,2-dimethoxy-4-methyl-benzene (250 mg, 1.64 mmol, 1 eq) and 10-bromodecanoyl chloride (487.17 mg, 1.81 mmol, 1.1 eq) in DCE (10 mL) was added AlCl3 (197.13 mg, 1.48 mmol, 80.79 uL, 0.9 eq). The mixture was stirred at 25 °C for 16 h. LCMS showed complete consumption of starting material. The reaction mixture was quenched with H2O (10 mL) and the mixture was filtered. The reaction filtrate was extracted with DCM (20 mL * 3). The organic layer was separated, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO; 12 g SepaFlash Silica Flash Column, eluent of 0 to 20% ethyl acetate / petroleum ether gradient @ 80 mL / min) to obtain the compound 10-bromo-1-(4,5-dimethoxy-2-methyl-phenyl)decan-1-one (300 mg, 737.59 μmol, 44.90% yield, 94.740% purity) as a white solid.

[0207] MS(ESI):mass calcd.for C 19 H 29 BrO3,384.13;m / z found, 387.0[M+3]+.

[0208] 1H NMR(400MHz,CHLOROFORM-d)δppm1.32-1.38(m,2H)1.52-1.69(m,4H)1.78(quin,J=7.13Hz,4H)2.28(t,J=7.50Hz,2H )2.42(s,3H)2.79(t,J=7.38Hz,2H)3.33(t,J=6.82Hz,2H)3.84(d,J=4.25Hz,6H)6.59-6.70(m,1H)7.10-7.17(m,1H).

[0209] Step C. 1-(10-bromodecyl)-4,5-dimethoxy-2-methylbenzene To a solution of 10-bromo-1-(4,5-dimethoxy-2-methyl-phenyl)decan-1-one (250 mg, 648.79 μmol, 1 eq) in TFA (10 mL) was added EtSiH (1.82 g, 15.65 mmol, 2.50 mL, 24.13 eq). The mixture was stirred at 80 °C for 2 h. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo to give the crude product. The residue was purified by flash silica gel chromatography (ISCO; 20 g SepaFlash Silica Flash Column, eluent of 0–15% ethyl acetate / petroleum ether gradient at 80 mL / min). The compound 1-(10-bromodecyl)-4,5-dimethoxy-2-methyl-benzene (130 mg, 350.07 μmol, 53.96% yield) was obtained as a colorless oil.

[0210] MS(ESI):mass calcd.for C 19 H 31 BrO2,370.15;m / z found, 371.2[M+H]+.

[0211] 1H NMR(400MHz,CHLOROFORM-d)δppm1.14-1.35(m,12H)1.41(dt,J=15.10,7.65Hz,2H)1.73(quin,J=7.16 Hz,2H)2.09-2.19(m,3H)2.36-2.43(m,2H)3.28(t,J=6.82Hz,2H)3.72(d,J=3.13Hz,6H)6.43-6.62(m,2 H).

[0212] Step D. Formic Acid, (10-(4,5-Dimethoxy-2-methylphenyl)decyl)triphenylphosphonium Salt To a solution of 1-(10-bromodecyl)-4,5-dimethoxy-2-methyl-benzene (100 mg, 269.29 μmol, 1 eq) in toluene (5 mL) was added PPh3 (353.16 mg, 1.35 mmol, 5 eq). The mixture was stirred at 130 °C for 24 h. LCMS showed complete consumption of the starting material. The reaction mixture was concentrated in vacuo to give the crude product. The residue was purified by prep-HPLC (FA condition: column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 35% to 70%, 35 min) to give the desired compound (40 mg, purity 93.747%) as a white solid, which was further separated by prep-HPLC (condition: column: Phenomenex Luna C18 75*30 mm*3 μm; mobile phase: [water (0.225% FA)-ACN]; B%: 35% to 70%, 35 min). Compound 10-(4,5-dimethoxy-2-methyl-phenyl)decyl-triphenyl-phosphonium (17 mg, 27.41 μmol, 10.18% yield, 96.703% purity, FA) was obtained as a colorless oil.

[0213] MS(ESI):mass calcd.for C 37 H 46 O2P+, 553.32;m / z found, 553.5[M+H]+.

[0214] 1H NMR(400MHz,DMSO-d6)δppm1.16-1.31(m,10H)1.38-1.60(m,6H)2.16(s,3H)2.40-2.48(m,2H)3.56-3.6 0(m,2H)3.69(d,J=2.75Hz,6H)6.68(s,1H)6.72(s,1H)7.74-7.85(m,12H)7.87-7.94(m,3H)8.44(s,1H).

[0215] 1-8. Synthesis of (10-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)decyl)triphenylphosphonium JPEG0007766885000032.jpg68170

[0216] Preparation procedure for compound 2 Cpd.1 (10.0 g, 58.0 mmol, 1.00 eq) and Cpd.2B (12.9 g, 63.8 mmol, 1.10 eq) were added to a round-bottom flask filled with AgNO (9.87 g, 58.0 mmol, 1.00 eq) and ACN (100 mL) and HO (100 mL). A solution of (NH)SO (15.9 g, 69.6 mmol, 15.1 mL, 1.20 eq) in HO (100 mL) was added dropwise to the mixture. The mixture was stirred at 75 °C in the dark for 4 h. LCMS (ET36187-5-P1L, Cpd.2: RT = 1.431 min) showed partial consumption of Cpd.1 and formation of Cpd.2. The mixture was cooled to 20 °C. The mixture was extracted with EtOAc (100.0 mL × 3). The organic layer was washed with NaHCO3 (50.0 mL) and brine (50.0 mL). The organic layer was concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 1 / 0 to 0 / 1). Cpd.2 (5.91 g, 17.9 mmol, 30.9% yield) was obtained as a white solid.

[0217] Preparation procedure for compound 3 Cpd.2 (2.00 g, 6.09 mmol, 1.00 eq) was added to a three-neck round-bottom flask charged with DCM (200 mL). CBr4 (2.42 g, 7.31 mmol, 1.20 eq) and PPh3 (1.92 g, 7.31 mmol, 1.20 eq) were added to the mixture. The reaction was stirred at 20 °C for 2 h. TLC (Petroleum ether / Ethyl acetate = 15 / 1 Rf = 0.53) showed Cpd.2 was consumed and Cpd.3 was formed. LCMS (ET36249-7-p1a, Cpd.3: RT = 1.678 min) showed Cpd.3 was formed. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 2 / 1 to 15 / 1). Cpd.3 (1.10 g, 2.81 mmol, 46.1% yield) was obtained as a yellow oil.

[0218] 1H NMR: ET36249-7-P1a (400MHz, CDCl3).

[0219] δ8.07~8.09(m,2H),7.68~7.70(m,2H),3.41(t,J=4Hz,2H),2.63(t,J=8Hz,2H),2.20(s,3H),1.48~1.87(m,2H),1.30~1.46(m,15H).

[0220] Target compound manufacturing procedure Cpd.3 (0.20 g, 511 μmol, 1.00 eq) was added to a one-neck round-bottom flask filled with Toluene (1.40 mL). PPh3 (160 mg, 613 μmol, 1.20 eq) was added to the mixture. The mixture was degassed with N2 three times. The mixture was stirred at 120 °C for 16 h. TLC (dichloromethane / methanol = 10 / 1, Rf = 0.50) indicated that Cpd.3 had been consumed and the reaction was complete. HPLC (ET36249-13-p1e) indicated that the target was 91.2% pure. The mixture was concentrated in vacuo to remove Toluene (1.40 mL). The residue was filled with MeOH (4.00 mL). The residue was purified by prep-TLC (dichloromethane / methanol = 10 / 1). The target (12.0 mg, 18.3 umol, 3.59% yield) was obtained as an orange gum.

[0221] 1H NMR: ET36249-13-P1d (400MHz, MeOD-d4).

[0222] δ7.88~8.04(m,2H),7.75~7.87(m,15H),3.42~3.35(m,2H),2.63(t,J=8Hz,2H),2.16(s,3H),1.28~1.69(m,16H).

[0223] 1-9. Synthesis of triphenyl(10-phenyldecyl)phosphonium chloride JPEG0007766885000033.jpg58170

[0224] Preparation procedure for compound 2 Cpd.1 (3.00 g, 10.0 mmol, 1.00 eq) was added to THF (7.50 mL) at 0 °C. The mixture was degassed with N2 three times. PhLi (1.80 M, 1.83 mL, 0.33 eq) was added dropwise to the solution at 0 °C. The mixture was stirred at 0 °C for 1 h and warmed to 15 °C. The mixture was stirred at 15 °C for 48 h. TLC (Petroleum ether:Ethyl acetate = 1:0, Rf (Cpd.2) = 0.60) indicated that Cpd.1 had been consumed and the reaction was complete. The mixture was concentrated in vacuo. No further purification was performed. Cpd.2 (2.30 g, crude) was obtained as a colorless oil.

[0225] 1H NMR:ET41362-1-P1C1(400MHz,CDCl3) δ7.27-7.26(m,2H),7.19-7.17(d,J=8Hz,2H),1.89-1.82(m,15H),1.59-1.52(m,9H),1.30(s,32H),0.94-0.90(t,J=8Hz,12H).

[0226] Preparation procedure for triphenyl(10-phenyldecyl)phosphonium chloride Cpd.2 (0.70 M, 11.0 mL, 1.00 eq) was added to toluene (27.0 mL) at 15 °C. PPh3 (4.06 g, 15.4 mmol, 2.00 eq) was added to the solution. The mixture was degassed with N2 three times and heated to 100 °C. The mixture was stirred at 100 °C for 12 h. TLC (dichloromethane:methanol = 20:1, Rf (target 1) = 0.30) indicated that Cpd.2 had been consumed and the reaction was complete. The mixture was concentrated in vacuo. The crude product was purified by silica gel chromatography (dichloromethane:methanol = 100:0 to 20:1). Target 1 (27.0 mg, 54.8 µmol, 7.09 e-1% yield, 97.5% purity by LCMS (ET41362-3-P1J1)) was obtained as a yellow oil.

[0227] 1H NMR:ET41362-3-P1J1(400MHz,CDCl3) δ7.79-7.71(d,J=32Hz,15H),7.28-7.16(m,6H),3.95-3.74(m,4H),2.59-2.55(t,J=8Hz,2H),1.62-1.56(m,4H),1.25-1.20(d,J=20Hz,10H).

[0228] 1-10.Synthesis of (10-cyclohexyldecyl)triphenylphosphonium chloride JPEG0007766885000034.jpg59170

[0229] Preparation procedure for compound 3 Cpd.1 (3.00 g, 10.0 mmol, 1.00 eq) was added to THF (3.00 mL) at 0 °C. The mixture was degassed with N2 three times. CuLi2Cl4 (0.10 M, 999 μL, 0.01 eq) was added to the solution. Cpd.1a (1.00 M, 12.0 mL, 1.20 eq) was added dropwise to the solution at 0 °C. The mixture was stirred at 0 °C for 1 h and warmed to 15 °C. The mixture was stirred at 15 °C for 20 h. TLC (Petroleum ether:Ethyl acetate = 1:0, Rf (Cpd.3) = 0.80) indicated that Cpd.1 was consumed and the reaction was complete. The mixture was concentrated in vacuo. No further purification was performed. Cpd.3 (3.90 g, crude) was obtained as a colorless oil.

[0230] 1H NMR:ET41362-2-P1C1(400MHz,CDCl3) δ3.43-3.40(t,J=8Hz,1H),1.70-1.67(m,15H),1.20-1.12(m,14H),0.90-0.84(m,8H).

[0231] Preparation procedure for (10-cyclohexyldecyl)triphenylphosphonium chloride Cpd.3 (0.70 M, 18.3 mL, 1.00 eq) was added to toluene (16.0 mL) at 15 °C. PPh3 (6.74 g, 25.7 mmol, 2.00 eq) was added to the solution. The mixture was degassed with N2 three times and heated to 100 °C. The mixture was stirred at 100 °C for 12 h. TLC (dichloromethane:methanol = 20:1, Rf (target 2) = 0.20) indicated that Cpd.3 had been consumed and the reaction was complete. The mixture was concentrated in vacuo. The crude product was purified by silica gel chromatography (dichloromethane:methanol = 100:0 to 20:1). Target 2 (0.02 g, 40.4 µmol, 3.14 e-1% yield, 98.1% purity by LCMS (ET41362-4-P1J1)) was obtained as a yellow oil.

[0232] 1H NMR:ET41362-4-P1C1(400MHz,CDCl3) δ7.89-7.70(m,15H),3.83(s,2H),2.60(s,2H),1.68-1.62(m,9H),1.18(s,18H),0.87-0.79(m,2H).

[0233] 1-11. Preparation of (10-(3,4-dimethylphenyl)decyl)triphenylphosphonium bromide JPEG0007766885000035.jpg82170

[0234] Step A. 10-Bromodecanoyl chloride To a solution of 10-bromodecanoic acid (30.0 g, 119 mmol, 1.00 eq) in DCM (210 mL) was added SOCl2 (56.8 g, 478 mmol, 34.7 mL, 4.00 eq). The mixture was stirred at 25 °C for 1 h. TLC (Petroleum ether:ethyl acetate = 5:1, Rf (start material) = 0.30, Rf (product) = 0.52) showed complete consumption of the starting material. The mixture was concentrated in vacuo. 10-Bromodecanoyl chloride (30.0 g, crude) was obtained as a yellow oil.

[0235] Step B. 10-Bromo-1-(3,4-dimethylphenyl)decan-1-one To a solution of o-xylene (5.00 g, 47.1 mmol, 5.69 mL, 1.00 eq) and 10-bromodecanoyl chloride (14.0 g, 51.8 mmol, 1.10 eq) in DCE (35.0 mL) was added AlCl3 (5.65 g, 42.4 mmol, 2.32 mL, 0.90 eq). The mixture was stirred at 25 °C for 16 h. TLC (Petroleum ether:Ethyl acetate = 5:1, starting material Rf = 0.70, product Rf = 0.88) indicated complete consumption of the starting material. The mixture was poured into ice water (50.0 mL) and extracted with DCM (50.0 mL). The organic phase was separated and concentrated in vacuo. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 1:0 to 1:1). 10-Bromo-1-(3,4-dimethylphenyl)decan-1-one (6.00 g, 13.4 mmol, 28.3% yield, 75.5% purity) was obtained as a yellow solid, identified by HNMR (ET47086-1-P1A2) and LCMS (ET47086-1-P1A1, T=0.996, M+1=339.2).

[0236] 1H NMR(400MHz,CHLOROFORM-d)δ=7.74(s,1H)7.70(dd,J=7.60,1.64Hz,1H)7.21(d,J=7.60 Hz,1H)3.41(t,J=6.80Hz,2H)2.93(t,J=7.60Hz,2H)2.32(s,6H)1.86(quin,J=7.20Hz,2H)1.73(quin,J=7.20Hz,2H)1.26-1.49(m,10H).

[0237] Step C. 4-(10-bromodecyl)-1,2-dimethyl-benzene To a solution of 10-bromo-1-(3,4-dimethylphenyl)decan-1-one (1.00 g, 2.95 mmol, 1.00 eq) in TFA (7.00 mL) was added EtSiH (8.57 g, 73.7 mmol, 11.8 mL, 25.0 eq). The mixture was stirred at 80 °C for 2 h. TLC (Petroleum ether:ethyl acetate = 5:1, Rf (start material) = 0.7, Rf (product) = 0.6) indicated complete consumption of the starting material. The mixture was poured into water (10.0 mL) and then extracted with EtOAc (5.00 mL × 3). The combined organic layers were washed with brine (10.0 mL). The residue was purified by column chromatography (SiO, Petroleum ether:Ethyl acetate = 1:1 to 0:1). 4-(10-Bromodecyl)-1,2-dimethyl-benzene (0.20 g, 615 umol, 20.8% yield) was obtained as a colorless oil.

[0238] Step D: 10-(3,4-dimethylphenyl)decyltriphenylphosphonium bromide salt To a solution of 4-(10-bromodecyl)-1,2-dimethylbenzene (0.20 g, 618 μmol, 1.00 eq) in Tol (7.00 mL) was added PPh3 (806 mg, 3.07 mmol, 5.00 eq). The mixture was stirred at 130 °C for 18 h. TLC1 (Dichloromethane:Methanol = 5:1, Rf (product) = 0.4) and TLC2 (Petroleum ether:Ethyl acetate = 5:1, Rf (start material) = 0.8) indicated complete consumption of the starting material. The mixture was concentrated in vacuo. The residue was triturated with PE:MTBE = 2:1 (1 mL) for 30 min at 25 °C. 10-(3,4-Dimethylphenyl)decyltriphenylphosphonium bromide salt (0.05 g, 84.2 μmol, 13.6% yield, 98.9% purity, Br) was obtained as a white solid, which was confirmed by HNMR (ET46959-5-P1B1), LCMS (ET46959-5-P1B1, T = 2.958, M + = 507.2), and HPLC (ET46959-5-P1A3, T = 4.121, Purity = 98.9%).

[0239] 1H NMR (400MHz, DMSO-d6) δ=7.93-7.86(m,3H),7.84-7.73(m,12H),7.00(d,J=7.60Hz,1H),6.92(s,1H),6.86(br d,J=7.60Hz,1H),3.56(br t,J=14.4Hz,2H),2.48-2.43(m,2H),2.16(d,J=5.20Hz,6H),1.57-1.38(m,6H),1.20(br d,J=15.2Hz,10H).

[0240] 1-12. Preparation of 10-(2,5-dimethoxy-3,4-dimethyl-phenyl)decyl-triphenyl-phosphonium JPEG0007766885000036.jpg91170

[0241] Step A. 10-Bromo-1-(2,5-dimethoxy-3,4-dimethyl-phenyl)decan-1-one To a solution of 2,3-dimethylbenzene-1,4-diol (11.0 g, 79.6 mmol, 1.00 eq) in EtOH (70.0 mL) was added KOH (11.2 g, 199 mmol, 2.50 eq) and dimethyl sulfate (25.1 g, 199 mmol, 18.9 mL, 2.50 eq). The mixture was stirred at 0 °C for 3.5 h. TLC (Petrileum ether:ethyl acetate = 5:1, Rf (start material) = 0.10, Rf (product) = 0.70) indicated complete consumption of the starting material. The mixture was poured into 3 M HCl (100 mL) and then extracted with PE (50.0 mL × 3). The combined organic layers were washed with 1 M HCl (50.0 mL), water (50.0 mL), and brine (50.0 mL). The residue was purified by column chromatography (SiO, Petroleum ether:Ethyl acetate = 1:0 to 0:1). 10-Bromo-1-(2,5-dimethoxy-3,4-dimethyl-phenyl)decan-1-one (6.00 g, 36.1 mmol, 45.3% yield) was obtained as a brown solid, which was identified by LCMS (ET46959-3-P1A1, T = 0.795, M + H = 167.3) and HNMR (ET46959-3-P1A1).

[0242] 1H NMR(ET46959-3-P1A1,400MHz,DMSO-d6) δ=6.71(s,2H),3.70(s,6H),2.06(s,6H).

[0243] Step B. 10-Bromo-1-(2,5-dimethoxy-3,4-dimethyl-phenyl)decan-1-one To a solution of 1,4-dimethoxy-2,3-dimethylbenzene (1.00 g, 6.02 mmol, 1.14 mL, 1.00 eq) and 10-bromodecanoyl chloride (1.78 g, 6.62 mmol, 1.10 eq) in DCE (7.00 mL) was added AlCl (722 mg, 5.41 mmol, 296 μL, 0.90 eq). The mixture was stirred at 25 °C for 2 h. TLC (Petroleum ether:ethyl acetate = 5:1, Rf (start material) = 0.83, Rf (product) = 0.72) indicated complete consumption of the starting material. The reaction mixture was quenched with H O (10.0 mL), and the mixture was filtered. The reaction filtrate was extracted with DCM (20.0 mL × 3). The organic phase was separated, dried over Na SO , filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate = 1:0 to 0:1) to give 10-bromo-1-(2,5-dimethoxy-3,4-dimethyl-phenyl)decan-1-one (500 mg, 1.25 mmol, 20.8% yield) as a yellow solid, which was identified by HNMR (ET46959-6-P1A1) and LCMS (ET46985-6-P1A2, T = 1.011, M+H = 399.3).

[0244] 1H NMR:(ET46959-6-P1A1,400MHz,DMSO-d6) δ=6.89(s,1H),3.76(s,3H),3.59(s,3H),2.93(t,J=7.20Hz,2H),2.16(s,3H),2.11(s,3H),1.39-1.24(m,16H).

[0245] Step C. 1-(10-bromodecyl)-2,5-dimethoxy-3,4-dimethyl-benzene To a solution of 10-bromo-1-(2,5-dimethoxy-3,4-dimethyl-phenyl)decan-1-one (500 mg, 1.25 mmol, 1.00 eq) in TFA (5.00 mL) was added EtSiH (3.64 g, 31.3 mmol, 5.00 mL, 25.0 eq). The mixture was stirred at 80 °C for 2 h. TLC (Petroleum ether:ethyl acetate = 5:1, Rf (start material) = 0.7, Rf (product) = 0.6) indicated complete consumption of the starting material. The mixture was poured into water (10.0 mL) and then extracted with EtOAc (5.00 mL × 3). The combined organic layers were washed with brine (10.0 mL). The residue was purified by column chromatography (SiO, Petroleum ether:Ethyl acetate = 1:0 to 0:1). 1-(10-Bromodecyl)-2,5-dimethoxy-3,4-dimethyl-benzene (0.20 g, 519 μmol, 41.4% yield) was obtained as a pale yellow oil, which was identified by HNMR (ET46959-8-P1A) and LCMS (ET46959-8-P1A, T=1.055. M+H=385.3).

[0246] 1H NMR (ET46959-8-P1A, 400MHz, DMSO-d6) δ=6.63-6.56(m,1H),3.70(s,3H),3.54(s,3H),2.10(s,3H),2.02(s,3H),1.77(q,J=7.20Hz,2H),1.59-1.48(m,2H),1.41-1.21(m,14H).

[0247] Step D. Formic acid, 10-(2,5-dimethoxy-3,4-dimethyl-phenyl)decyl-triphenyl-phosphonium salt To a solution of 1-(10-bromodecyl)-2,5-dimethoxy-3,4-dimethylbenzene (0.20 g, 519 μmol, 1.00 eq) in Tol (3.50 mL) was added PPh3 (272 mg, 1.04 mmol, 2.00 eq). The mixture was stirred at 130 °C for 18 h. TLC (Dichloromethane:Methanol = 5:1, Rf (product) = 0.4) and TLC (Petroleum ether:Ethyl acetate = 5:1, Rf (start material) = 0.8) indicated complete consumption of the starting material. The mixture was concentrated in vacuo. The residue was purified by prep-HPLC (column: Phenomene x Luna C18 75 × 30 mm × 3 μm; mobile phase: [water(FA)-ACN]; B%: 40% to 75%, 8 min). Formic acid, 10-(2,5-dimethoxy-3,4-dimethyl-phenyl)decyl-triphenyl-phosphonium salt (30.0 mg, 52.5 µmol, 10.1% yield, 99.3% purity) was obtained as a yellow gum, which was identified by HNMR (ET46959-9-P1A), LCMS (ET46959-9-P1A, T=2.938, M+=567.2), and HPLC (ET46959-9-P1B, T=4.052, Purity=99.3%).

[0248] 1H NMR (ET46959-9-P1A, 400MHz, DMSO-d6) δ=8.52(s,1H),7.92-7.86(m,3H),7.83-7.74(m,12H),6.61-6.56(m,1H),3.70(s, 3H), 3.54 (s, 5H), 2.10 (s, 3H), 2.02 (s, 3H), 1.56-1.38 (m, 7H), 1.30-1.15 (m, 11H).

[0249] 1-13. Preparation of (10-(3,4-dimethoxyphenyl)decyl)triphenylphosphonium bromide JPEG0007766885000037.jpg58170

[0250] Step 1: 10-Bromo-1-(3,4-dimethoxyphenyl)decan-1-one To a solution of 1,2-dimethoxybenzene (5.00 g, 36.2 mmol, 4.63 mL, 1.00 eq) and 10-bromodecanoyl chloride (10.7 g, 39.8 mmol, 1.10 eq) in DCE (35.0 mL) was added AlCl3 (4.34 g, 32.6 mmol, 1.78 mL, 0.90 eq). The mixture was stirred at 25 °C for 16 h. The mixture was poured into ice water (50.0 mL) and extracted with DCM (50.0 mL). The organic phase was separated and concentrated in vacuo. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 1:0 to 1:1). 10-Bromo-1-(3,4-dimethoxyphenyl)decan-1-one (3.00 g, 7.92 mmol, 21.8% yield, 98.0% purity) was obtained as a white solid.

[0251] MS(ESI):mass calcd.for C 18 H 27 BrO3,370.11;m / z found, 371.2[M+H]+.

[0252] 1H NMR (400MHz, CDCl3-d) δ=7.59(dd,J=8.32,1.96Hz,1H),7.54(d,J=1.96Hz,1H),6.89(d,J=8.44Hz,1H),3.95(d,J=3.32Hz,6H),3.41 (t,J=6.84Hz,2H),2.92(t,J=7.40Hz,2H),1.85(quin,J=7.16Hz,2H),1.66-1.78(m,2H),1.27-1.50(m,10H).

[0253] Step 2: 4-(10-bromodecyl)-1,2-dimethoxybenzene To a solution of 10-bromo-1-(3,4-dimethoxyphenyl)decan-1-one (2.00 g, 5.39 mmol, 1.00 eq) in TFA (15.0 mL) was added EtSiH (15.7 g, 134 mmol, 21.5 mL, 25.0 eq). The mixture was stirred at 80 °C for 1 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO, petroleum ether: ethyl acetate = 1:0 to 10:1). 4-(10-bromodecyl)-1,2-dimethoxybenzene (1.00 g, 2.79 mmol, 51.7% yield, 99.5% purity) was obtained as a pale yellow oil.

[0254] MS(ESI):mass calcd.for C 18 H 29 BrO2,356.14;m / z found, 357.2[M+H]+.

[0255] Step 3: (10-(3,4-dimethoxyphenyl)decyl)triphenylphosphonium bromide To a solution of 4-(10-bromodecyl)-1,2-dimethoxybenzene (200 mg, 559 μmol, 1.00 eq) in Tol. (1.40 mL) was added PPh3 (161 mg, 615 μmol, 1.10 eq). The mixture was stirred at 130 °C for 12 h. The mixture was concentrated in vacuo. The crude product was triturated with MeCN (2.00 mL) at 20 °C for 1 h and then purified under reversed phase (neutral conditions). 10-(3,4-Dimethoxyphenyl)decyl-triphenyl-phosphonium (50.0 mg, 92.3 μmol, 16.4% yield, 99.6% purity) was obtained as a yellow gum.

[0256] MS(ESI):mass calcd.for C 36 H 44 BrO2P, 618.23;m / z found, 539.2[M]+.

[0257] 1H NMR (400MHz, DMSO-d6) δ=7.85-7.95(m,3H),7.73-7.84(m,12H),6.82(d,J=8.16Hz,1H),6.75(d,J=1.84Hz,1 H),6.66(dd,J=8.08,1.83Hz,1H),3.70(d,J=7.96Hz,6H),3.48-3.63(m,2H),2.47(br s, 2H), 2.07 (s, 1H), 1.37-1.59 (m, 6H), 1.10-1.32 (m, 10H).

[0258] 1-14. Mito-CP (2,2,5,5-tetramethyl-3-(((10-(triphenylphosphonio)decyl)oxy)carbonyl)pyrrolidine-1-oleic acid) and Mito-CP S Preparation of (2,2,5,5-tetramethyl-3-((2-(triphenylphosphonio)ethoxy)carbonyl)pyrrolidine-1-oleic acid) JPEG0007766885000038.jpg48170

[0259] (2-Hydroxyethyl)triphenylphosphonium (6a).

[0260] A mixture of 2-bromoethanol (1.5 mmol), PPh3 (1.0 mmol), and acetonitrile (20 mL) was refluxed for 24 h. After cooling to room temperature, the solvent was removed by rotary evaporation. The remaining pale yellow oil was washed twice with ethyl ether to give the phosphonium salt 6a. Yield: 77%; 1H NMR (400 MHz, CDCl3) δ 7.95-7.56 (m, 15H), 5.21 (s, 1H), 4.14 (d, J = 16.6 Hz, 2H), 3.83 (s, 2H).

[0261] (10-Hydroxydecyl)triphenylphosphonium (6b).

[0262] The synthetic procedure for 6a was applied with 10-bromodecanol (1.5 mmol) to give the product as a brown oil. Yield: 85%; H NMR (400 MHz, CDCl) δ 7.92-7.83 (m, 6H), 7.79 (dd, J = 7.7, 5.8 Hz, 3H), 7.70 (m, 6H), 3.91-3.81 (m, 2H), 3.63 (t, J = 6.6 Hz, 2H), 1.69-1.50 (m, 12H), 1.31 (m, 4H).

[0263] 2,2,5,5-Tetramethyl-3-((2-(triphenylphosphonio)ethoxy)carbonyl)pyrrolidine-1-oleic acid (Mito-CPs; 8).

[0264] Pyridine (1.0 mmol) was added to a solution of 3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oleic acid (7, 1.0 mmol) in benzene. The flask was kept cooled in an ice bath, and thionyl chloride (2.0 mmol) was added dropwise over 1 hour. The solvent was removed by evaporation under vacuum. The resulting residue and (2-hydroxyethyl)triphenylphosphonium (6a, 1.0 mmol) were dissolved in dichloromethane (10 mL). Pyridine (1.0 mmol) was added dropwise to this solution in an ice bath, and the mixture was stirred at room temperature for 6 hours. The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated in vacuo. The crude product was purified by MPLC (MeOH 5% in DCM) to give compound (21%); HRMS (ESI, m / z) calculated for C 29 H 34 NO3P[M]+475.2276,found 475.2260.

[0265] 2,2,5,5-Tetramethyl-3-(((10-(triphenylphosphonio)decyl)oxy)carbonyl)pyrrolidine-1-oleic acid (Mito-CP; 9).

[0266] The synthetic procedure for 8 was applied with (10-hydroxydecyl)triphenylphosphonium (6b, 1.0 mmol) to give 9 as a brown oil. Yield: 24%; HRMS (ESI, m / z) calculated for C 37 H 50 NO3P[M]+587.3528,found 587.3524.

[0267] 1-15.MitoQ S Preparation of ((4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl)triphenylphosphonium bromide JPEG0007766885000039.jpg24170

[0268] 1-(Bromomethyl)-2,3,4,5-tetramethoxy-6-methylbenzene (2).

[0269] 1,2,3,4-Tetramethoxy-5-methylbenzene (2.721 g, 12.7 mmol) and paraformaldehyde (0.763 g, 25.4 mmol) were dissolved in 47% HBr (10 mL). The mixture was then stirred at 40 °C for 2 hours and allowed to stand at room temperature. After completion of the reaction, the product was extracted with hexane (40 mL), and the combined organic layer was dried over sodium sulfate. The remaining solvent was removed in vacuo to give a light yellow oil 2 (yield: 88%); H NMR (400 MHz, CDCl) δ 4.61 (s, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 3.79 (s, 3H), 2.27 (s, 3H); C NMR (100 MHz, CDCl) δ 148.5, 148.0, 147.8, 144.7, 126.6, 124.9, 61.3, 61.1, 61.1, 60.8, 26.6, 11.1.

[0270] 2-(Bromomethyl)-5,6-dimethoxy-3-methylcyclohexa-2,5-diene-1,4-dione (3).

[0271] Intermediate 2 (211 mg, 0.687 mmol) was dissolved in THF (10 mL). Then, ammonium cerium(IV) nitrate (1.5 g, 2.74 mmol) dissolved in water (10 mL) was added to the reaction mixture. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the resulting mixture was extracted with DCM (20 mL), and the extract was washed with brine until neutral. The combined organic layer was then dried over sodium sulfate, and the remaining solvent was removed in vacuo. The residue was purified by silica gel chromatography to give 3 as a yellow oil (yield: 45%); 1H NMR (400 MHz, CDCl3) δ 5.39 (s, 2H), 4.03 (s, 3H), 4.02 (s, 3H), 2.17 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 183.9, 181.6, 145.0, 144.5, 141.7, 137.6, 61.4, 61.3, 21.5, 12.0; MS (ESI, m / z) calculated for C 10 H 12 BrO4[M+H]+274.99,found 275.00.

[0272] ((4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methyl)triphenylphosphonium (MitoQs; 4).

[0273] 3 (22.31 mg, 0.0726 mmol) and triphenylphosphine (57.1 mg, 0.218 mmol) were dissolved in ACN (10 mL), and the reaction mixture was refluxed overnight. After cooling to room temperature, the remaining solvent was removed in vacuo. The residue was purified by silica gel chromatography to give a white solid 4 (yield: 18%); 1H NMR (400 MHz, CD3OD) δ 7.88-7.81 (m, 3H), 7.70-7.64 (m, 6H), 7.62-7.55 (m, 6H), 3.80 (s, 3H), 3.52 (s, 3H), 3.34 (s, 2H), 1.81 (d, J = 1.5 Hz, 3H); 13C NMR(101MHz,MeOD)δ182.74(d,J=3.3Hz),182.65(d,J=2.3Hz),145.80,145.70,145.21,143.96,135.27(d,J=3.0Hz),134.30(d, J=10.1Hz),130.37(d,J=12.8Hz),118.14(d,J=85.9Hz),61.31,61.26,24.81(d,J=49.8Hz),14.68(d,J=2.7Hz);HRMS(ESI,m / z) calculated for C 28 H 26 O4P[M]+457.1563,found 457.1566.

[0274] 1-16. Mito-VitE: Preparation of 2-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)ethyl)triphenylphosphonium JPEG0007766885000040.jpg501704-(Tetrahydro-2H-pyran-2-yl)butan-2-one (11).

[0275] To a solution of 4-hydroxybutan-2-one (11.35 mmol) and 3,4-dihydro-2H-pyran (15.89 mmol) in CHCl (15 mL), pyridinium p-toluenesulfonate (1.135 mmol, 0.1 eq) was added and stirred at room temperature for 4 hours. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in EtO (50 mL). This was washed with saturated aqueous NaCl (40 mL) and HO (10 mL), dried over anhydrous MgSO, filtered, and concentrated in vacuo to give compound 11. Yield:64%;1H NMR(400MHz,CDCl3)δ4.60(t,J=3.4Hz,1H),4.00(dt,J=10.2,6.2Hz,1H),3.89-3.80(m,1H),3.69(dt,J=10 .1,6.3Hz,1H),3.55-3.45(m,1H),2.71(t,J=6.2Hz,2H),2.20(s,3H),1.83-1.64(m,3H),1.60-1.54(m,3H).

[0276] 3-Methyl-5-((tetrahydro-2H-pyran-2-yl)oxy)pent-1-en-3-ol (12).

[0277] Vinylmagnesium bromide (13.35 mmol) was added to a solution of 11 (5.81 mmol) in THF (25 mL) at −78° C. This was stirred for 2 h and then warmed to room temperature over 30 min. Saturated aqueous NH4Cl (50 mL) was added dropwise, and this was extracted with Et2O. The combined organic phase was washed with saturated aqueous NaCl, dried over anhydrous MgSO4, filtered, and concentrated to give a pale yellow oil. The crude product was purified by MPLC. Yield:93%;1H NMR(400MHz,CDCl3)δ5.90(ddd,J=16.7,10.7,5.8Hz,1H),5.38-5.27(m,2H),5.10(ddd,J=10.3,8.7,1.4Hz,1H),4.67-4.53(m,1H),3.97(dt, 1H)

[0278] 2-(2-hydroxyethyl)-2,5,7,8-tetramethylchroman-6-ol (13).

[0279] A solution of 12 (5.39 mmol) and freshly prepared 2,3,5-trimethylbenzene-1,4-diol (4.49 mmol) in formic acid (10 ml) was refluxed under nitrogen for 3.5 h. The reaction mixture was poured onto crushed ice, and the organic material was extracted with Et2O. The combined organic phases were washed with H2O, dried over anhydrous MgSO4, and concentrated in vacuo. The brown oily residue was dissolved in MeOH and concentrated HCl and refluxed under argon for 30 min. After removing the solvent in vacuo, the residue was dissolved in Et2O. It was washed again under argon with H2O, saturated aqueous NaHCO3, and H2O, dried over anhydrous MgSO4, filtered, and concentrated to give a brown oil. The crude product was purified by MPLC to give the compound. Yield:35%;1H NMR(400MHz, CDCl3)δ5.30(s,1H),4.23(s,1H),3.97-3.86(m,2H),2.66(dd,J=9.8,6.2Hz ,2H),2.17(s,3H),2.12(s,3H),2.09(s,3H),1.97(m,2H),1.93-1.82(m,2H),1.29(s,3H).

[0280] 2,5,7,8-Tetramethyl-2-(2-((methylsulfonyl)oxy)ethyl)chroman-6-yl methanesulfonate (14).

[0281] To a solution of 13 (0.479 mmol) and triethylamine (2.88 mmol) in CHCl (2 mL) was added methanesulfonyl chloride (1.055 mmol) and stirred at room temperature for 1 h. The reaction mixture was diluted with CHCl (20 mL). It was washed with H2O, dried over anhydrous MgSO4, filtered, and concentrated in vacuo to give a white solid. This was recrystallized from EtOH to give the product. Yield:43%;1H NMR(400MHz,CDCl3)δ4.56-4.45(m,2H),4.41(dd,J=14.8,8.7Hz,2H),3.25(s,3H),3.00(s,3H) ,2.63(t,J=7.0Hz,2H),2.23(d,J=13.2Hz,6H),2.09(s,3H),1.86(t,J=6.8Hz,2H),1.31(s,3H).

[0282] Triphenyl(2-(2,5,7,8-tetramethyl-6-((methylsulfonyl)oxy)chroman-2-yl)ethyl)phosphonium (15).

[0283] A mixture of 14 (0.256 mmol), sodium iodide (192 mg, 1.279 mmol), and triphenylphosphine (1.279 mmol) was flushed with argon in a Kimax tube, then sealed, and the reaction was stirred in the melt at 90 °C for 48 h. The crude product was dissolved in CHCl and precipitated three times from petroleum ether. The product was dissolved in methanol and passed through an anion exchange column packed with -Oms. Residual solvent was removed in vacuo to give the pure product 15. Yield:39%;1H NMR(400MHz,CDCl3)δ7.87-7.72(m,9H),7.66(td,J=7.9,3.4Hz,6H),4.12(m,2H),3.28(s,3H),2.61- 2.49(m,2H),2.25(s,3H),2.15(s,3H),2.05(s,2H),2.03(s,3H),2.00(d,J=6.5Hz,2H),1.49(s,3H).

[0284] (2-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)ethyl)triphenylphosphonium (Mito-VitE; 16).

[0285] Lithium diisopropylamide (0.119 mmol, 1 M solution in THF) was added to a solution of 15 (0.100 mmol) in THF (2 mL) at 0 °C. After 30 min, the solution was warmed to room temperature, and then aqueous saturated NH4OMs (10 mL) was added. The aqueous layer was extracted three times with CHCl2, and the combined organic phases were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by MPLC to give product 16. Yield:9%;1H NMR(400MHz,CDCl3)δ7.85-7.73(m,4H),7.71-7.58(m,11H),3.77-3.64(m,2H),3.41-3.28(m ,2H),2.17(s,3H),2.07(d,J=11.7Hz,6H),1.94(m,4H),1.34(d,J=7.6Hz,3H);HRMS(ESI,m / z) calculated for C 33 H 36 O2P[M]+495.2447,found 495.2445.

[0286] 1-17.Mito-VitE L Preparation of (10-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)decyl)triphenylphosphonium iodide JPEG0007766885000041.jpg83170

[0287] 11-((tetrahydro-2H-pyran-2-yl)oxy)undecylic acid (18).

[0288] A solution of 11-hydroxyundecylic acid (17, 11.35 mmol), 3,4-dihydro-2H-pyran (15.89 mmol), and pyridinium p-toluenesulfonate (1.135 mmol) in CHCl (15 mL) was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo, and the residue was dissolved in EtO (50 mL). This was washed with saturated aqueous NaCl (40 mL) and HO (10 mL), dried over anhydrous MgSO, filtered, and concentrated in vacuo to give 18. Yield:46%;1H NMR(400MHz,CDCl3)δ4.63-4.48(m,1H),3.88(ddd,J=11.1,7.6,3.3Hz,1H),3.75-3.64(m,1H),3.55-3.4 8(m,1H),3.38(dt,J=9.6,6.7Hz,1H),2.34(t,J=7.5Hz,2H),1.91-1.44(m,14H),1.32(d,J=24.9Hz,8H).

[0289] N-Methoxy-N-methyl-11-((tetrahydro-2H-pyran-2-yl)oxy)undecanamide (19).

[0290] To a solution of 18 (5.23 mmol) in CHCl (10 mL) were added N,O-dimethylhydroxylamine hydrochloride (5.75 mmol) and N-methylmorpholine (5.75 mmol) sequentially at −15 °C. After 10 min, N-(3-dimethylaminopropyl)-N′-decylcarbodiimide hydrochloride (1.62 g, 5.75 mmol) was added portionwise over 15 min and stirred at −15 °C for 3 h. The reaction was quenched by the addition of 1 M HCl (5 mL), and the organics were extracted with CHCl (3 × 50 mL). The organic extracts were combined, washed with saturated NaHCO solution (50 mL), dried over MgSO, filtered, and evaporated to give the desired amide 19 (yield: 80%), which was used in the next step without further purification. 1H NMR(400MHz,CDCl3)δ4.60(dd,J=20.5,17.9Hz,1H),3.87(ddd,J=11.1,7.4,3.5Hz,1H),3.79-3.65(m,4H),3.50(dt,J=5.1,4. 5Hz,1H), 3.38(dt,J=9.6,6.7Hz,1H),3.18(s,3H),2.41(t,J=7.6Hz,2H),1.90-1.78(m,1H),1.76-1.47(m,14H),1.29(s,8H).

[0291] 12-((tetrahydro-2H-pyran-2-yl)oxy)dodecan-2-one (20).

[0292] To a solution of 19 (4.18 mmol) in EtO (13 mL) was added MeMgI (10.45 mmol) at 0 °C. After stirring at the same temperature for 3 h, saturated aqueous NHCl (30 mL) was added to the reaction mixture. The organic layer was separated, and the aqueous layer was extracted with tBuOMe (3 × 30 mL). The combined organic layers were dried over NaSO and concentrated under reduced pressure. The residue was purified by flash chromatography to give compound 20. Yield:80%;1H NMR(400MHz,CDCl3)δ4.61-4.54(m,1H),3.87(ddd,J=11.1,7.4,3.4Hz,1H),3.77-3.66(m,1H),3.55-3.46(m,1H),3.38(dt,J=9.6,6.7H z,1H),2.41(t,J=7.5Hz,2H),2.13(s,3H),1.90-1.76(m,1H),1.72(ddd,J=11.9,6.0,3.2Hz,1H),1.63-1.47(m,14H),1.37-1.20(m,6H).

[0293] 3-Methyl-13-((tetrahydro-2H-pyran-2-yl)oxy)tridec-1-en-3-ol (21).

[0294] Vinylmagnesium bromide (8.3 mmol, 2.5 eq) was added to a solution of 20 (3.32 mmol) in THF (15 ml) at −78° C. The solution was stirred for 2 h and then warmed to room temperature over 30 min. Saturated aqueous NH4Cl (50 mL) was added dropwise and the organic material was extracted with Et2O. The combined organic layers were washed with saturated aqueous NaCl, dried over anhydrous MgSO4, filtered, concentrated, and purified by MPLC to give compound 21. Yield: 93%; 1H NMR(400MHz,CDCl3)δ5.91(dd,J=17.4,10.8Hz,1H),5.20(dd,J=17.4,1.2Hz,1 H),5.04(dd,J=10.8,1.2Hz,1H),4.64-4.51(m,2H),3.87(ddd,J=11.0,7.4,3. 3Hz,2H),3.77-3.65(m,2H),3.56-3.46(m,2H),3.38(dt,J=9.5,6.7Hz,2H),1. 90-1.77 (m, 2H), 1.71 (tt, J=16.3, 7.0Hz, 2H), 1.64-1.46 (m, 10H), 1.27 (s, 8H).

[0295] 2-(10-hydroxydecyl)-2,5,7,8-tetramethylchroman-6-ol (22).

[0296] A solution of 21 (3.08 mmol) and freshly prepared 2,3,5-trimethylbenzene-1,4-diol (2.56 mmol) in formic acid (10 ml) was refluxed under nitrogen for 3.5 h. The reaction mixture was poured onto crushed ice, and the organic material was extracted with Et2O under argon. The combined organic phases were washed with H2O, dried over anhydrous MgSO4, and concentrated in vacuo. The brown residue was dissolved in MeOH and conc. HCl, and the reaction mixture was refluxed under argon for an additional 30 min. The solvent was removed in vacuo, and the residue was dissolved in Et2O. It was washed again under argon with H2O, saturated aqueous NaHCO3, and H2O, dried over anhydrous MgSO4, filtered, and concentrated to give a brown oil, which was purified by MPLC to give compound 22. Yield:35%;1H NMR(400MHz,CDCl3)δ4.38(s,1H),3.62(t,J=6.6Hz,2H),2.59(t,J=6.8Hz,2H),2.15 (s,3H),2.10(s,6H),1.76(qq,J=13.9,7.1Hz,2H),1.64-1.24(m,17H),1.22(s,3H).

[0297] 2-(10-Iododecyl)-2,5,7,8-tetramethylchroman-6-ol (23).

[0298] To a solution of triphenylphosphine (0.348 mmol), 1H-imidazole (0.348 mmol), and iodine (0.182 mmol) in CHCl (2 mL) was added a solution of 22 (0.165 mmol) in CHCl (2 mL) via syringe at 0 °C. The reaction mixture was stirred at room temperature for an additional 12 h. The mixture was washed with NaSO, HO, brine solution, dried over NaSO, filtered, and concentrated under reduced pressure. The resulting residue was purified by MPLC to give compound 23. 1H NMR(400MHz,CDCl3)δ4.19(d,J=2.7Hz,1H),3.19(t,J=4.0Hz,2H),2.60(t,J=6.8Hz,2H),2.16(m,3H),2.1 1(s,6H),1.90-1.69(m,5H),1.63-1.47(m,4H),1.44-1.21(m,10H),0.99-0.95(m,1H),0.86-0.83(m,3H).

[0299] (10-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)decyl)triphenylphosphonium iodide (Mito-VitE; 24).

[0300] A mixture of 23 (0.25 mmol) and triphenylphosphine (327 mg, 1.25 mmol) was flushed with argon in a Kimax tube, sealed, and stirred in the melt at 90 °C for 48 h. The crude product was dissolved in CHCl and precipitated three times from petroleum ether. Residual solvent was removed in vacuo to give 24. Yield:12%;1H NMR(400MHz,CDCl3)δ7.86-7.79(m,9H),7.73-7.68(m,6H),3.76-3.71(m,2H),2.59(t,J=6.8Hz,2H),2.15(s,3H),2. 10(s,3H),2.09(s,3H),1.81-1.72(m,2H),1.63-1.62(m,4H),1.37-1.36(m,2H),1.21-1.18(m,15H);HRMS(ESI,m / z) calculated for C 41 H 52 O2P[M]+607.3699,found 607.2697.

[0301] 1-18.SB-U141: Preparation of triphenyl(8-(2,3,4,5-tetramethoxy-6-methylphenyl)octyl)phosphonium JPEG0007766885000042.jpg94170

[0302] Methyl 8-chloro-8-oxotanate (2) was obtained by reacting 8-methoxy-8-oxooctanoic acid (1) with SOCl2 at 50 °C for 4 hours. Methyl 9-oxo-9-(2,3,4,5-tetramethoxy-6-methylphenyl)anoate (3) was obtained by reacting 2 with 1,2,3,4-tetramethoxy-5-methylbenzene in AlCl3 and dichloromethane (DCM) at 40 °C. 1-(2,3,4,5-tetramethoxy-6-methylphenyl)octan-1,8-diol (4) was obtained by reacting 3 with LAH in THF at room temperature. 8-(2,3,4,5-tetramethoxy-6-methylphenyl)octan-1-ol (5) was obtained by reacting 4 with Pd / C and H2 in MeOH. 1-(8-bromooctyl)-2,3,4,5-tetramethoxy-6-methylbenzene (6) was obtained by reacting 5 with CBr4PPh3 in DCM. Triphenyl(8-(2,3,4,5-tetramethoxy-6-methylphenyl)octyl)phosphonium (SB-U141) was obtained by reacting 6 with PPh3 in ACN at 90 °C.

[0303] 1-19.SB-U142: Preparation of triphenyl(12-(2,3,4,5-tetramethoxy-6-methylphenyl)dodecyl)phosphonium JPEG0007766885000043.jpg78170

[0304] Methyl 12-chloro-12-oxododecanoate (2) was synthesized by reacting 12-methoxy-12-oxododecanoic acid (1) with SOCl2 at 50 °C for 4 hours. Methyl 12-oxo-12-(2,3,4,5-tetramethoxy-6-methylphenyl)dodecanoate (3) was obtained by reacting 2 with 1,2,3,4-tetramethoxy-5-methylbenzene and AlCl3 in DCM at 40 °C. 1-(2,3,4,5-tetramethoxy-6-methylphenyl)dodecane-1,12-diol (4) was obtained by reacting 3 with LAH in THF. 12-(2,3,4,5-tetramethoxy-6-methylphenyl)dodecan-1-ol (5) was obtained by reacting 4 with H2 and Pd / C in MeOH. 1-(12-Bromododecyl)-2,3,4,5-tetramethoxy-6-methylbenzene (6) was synthesized by reacting 5 with CBr and PPh in DCM at room temperature. Triphenyl(12-(2,3,4,5-tetramethoxy-6-methylphenyl)dodecyl)phosphonium (SB-U142) was synthesized by reacting 6 with PPh in ACN at 90 °C.

[0305] 1-20.SB-U151: Preparation of (8-(4,5-dimethoxy-2-methylphenyl)octyl)triphenylphosphonium JPEG0007766885000044.jpg69170

[0306] Methyl 8-chloro-8-oxooctanoate (2) was obtained by reacting 8-methoxy-8-oxooctanoic acid with SOCl2 at 50 °C. Methyl 8-(4,5-dimethoxy-2-methylphenyl)-8-oxooctanoate (3) was obtained by reacting 2 with AlCl3 in DCM at 40 °C. 1-(4,5-dimethoxy-2-methylphenyl)octane-1,8-diol (4) was obtained by reacting 3 with LAH in THF at room temperature. 8-(4,5-dimethoxy-2-methylphenyl)octan-1-ol (5) was obtained by reacting 4 with Pd / C and H2 in MeOH. 1-(8-bromooctyl)-4,5-dimethoxy-2-methylbenzene (6) was obtained by reacting 5 with CBr4 and PPh3 in DCM at room temperature. (8-(4,5-dimethoxy-2-methylphenyl)octyl)triphenylphosphonium (SB-U151) was obtained by reacting 6 with PPh3 in ACN at 90 °C.

[0307] 1-21.SB-U152: Preparation of (12-(4,5-dimethoxy-2-methylphenyl)dodecyl)triphenylphosphonium JPEG0007766885000045.jpg67170

[0308] Methyl 12-chloro-12-oxododecanoate (2) was synthesized by reacting 12-methoxy-12-oxododecanoic acid with SOCl2 at 50 °C for 4 hours. Methyl 12-(4,5-dimethoxy-2-methylphenyl)-12-oxododecanoate (3) was synthesized by reacting 2 with 1,2-dimethoxy-4-methylbenzene. 1-(4,5-dimethoxy-2-methylphenyl)dodecane-1,12-diol (4) was synthesized by reacting 3 with LAH. 12-(4,5-dimethoxy-2-methylphenyl)dodecan-1-ol (5) was synthesized by reacting 4 with Pd / C / H2. 1-(12-bromododecyl)-4,5-dimethoxy-2-methylbenzene (6) was synthesized by reacting 5 with CBr4 / PPh3. (12-(4,5-dimethoxy-2-methylphenyl)dodecyl)triphenylphosphonium (SB-U152) was synthesized by reacting 6 with PPh3.

[0309] Example 2. Recombinant Protein Production The genes encoding zTRAP1 and hTRAP1 were cloned into a modified pET-Duet vector containing an N-terminal hexa-histidine tag followed by a TEV protease cleavage site and expressed in E. coli BL21(DE3) cells. After 15 h of induction with 0.4 mM IPTG at 20°C, cells were harvested and lysed by sonication. The soluble fraction of the lysate was applied to a Ni2+ affinity chromatography column (GE Healthcare). The hexa-histidine tag was cleaved by TEV protease, and the proteins were further purified by gel filtration chromatography in a buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 μM beta-mercaptoethanol (beta-ME).

[0310] Example 3. Structural analysis The present inventors analyzed the binding structure of TRAP1 and MitoQ in order to derive the structure of compounds that bind to TRAP1.

[0311] Purified zTRAP1 was mixed with AMPPNP at a molar ratio of 1:1.5. Crystallization was performed as described in Lavery et al., 2014. After crystal growth, 0.1 mM MitoQ was added to the crystallization drop and incubated for 24 hours. For X-ray diffraction experiments, the crystals were transferred to a well solution containing 20% ​​glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at beamline 5C at the Pohang Accelerator Laboratory (PAL) and processed using HKL-2000 software (Otwinowski and Minor, 1997). The electron density of MitoQ was calculated using the difference Fourier method. Model building and refinement were performed using the Coot and Phenix programs, respectively (Adams et al., 2010; Emsley et al., 2010).

[0312] Structural analysis revealed that the distance between the two protomers of TRAP1 is approximately 25 Å, and that if the distance between the Ub moiety and the TPP moiety is appropriate, the compound will bind to the CBS in TRAP1. These results confirm that a compound structure with an appropriate length is essential for binding to TRAP1 (see Figures 1 and 2).

[0313] Example 4. Binding strength analysis FP (Fluorescence polarization) analysis Example 4-1. Use of SB-TM2 probe Human full-length TRAP1 protein (400 nM) and SB-TM2 probe (100 nM) were added to FP buffer (35 mM NaCl, 2.7 mM KCl, 4.3 mM NaHPO, 1.4 mM KHPO (pH 7.3), 1 mM DTT, 2 mM MgCl, 0.1 mg / mL BSA) in a final volume of 100 μl. The mixture was incubated at room temperature for 1 hour in a 96-well plate. FP was measured using a SYNERGY NEO microplate reader with an excitation wavelength of 440 nm and an emission wavelength of 500 nm.

[0314] As a result, we confirmed that TPP-8 can competitively bind with SB-TM2, and that if the alkyl chain length is C8 or longer, it can bind to the CBS of TRAP1. We also confirmed that the binding strength increases as the alkyl chain length increases. Furthermore, when comparing the binding strength with SMX (MitoQ), we confirmed that TPP-12, TPP-14, and TPP-16 have superior binding strength compared to SMX (MitoQ). In particular, TPP-16, which has the longest alkyl chain length, exhibited twice the binding strength to SMX (see Figure 4).

[0315] Furthermore, it was confirmed that among the antioxidants bound to TPP, those with a sufficient bond distance between TPP and the antioxidant bind to the CBS of TRAP1 (see Figure 6).

[0316] Furthermore, we confirmed that other synthetic substances linked between TPP and hydrocarbons have the ability to bind to the CBS of TRAP1 (see Figure 8).

[0317] Example 4-2. Use of PU-H71-FITC For FP analysis, purified recombinant TRAP1 (400 nM) was incubated with the fluorescent probe PU-H71-FITC (10 nM), synthesized as described in Taldone et al., 2013, in the presence of increasing inhibitor concentrations for 2 h. Fluorescence polarization was measured using a microplate reader (Synergy NEO, BioTek) at room temperature.

[0318] As a result, it was confirmed that alkyl-TPP did not compete with PU-H71-FITC (an Hsp90 / TRAP1 inhibitor that targets the ATP binding site) and did not bind to the ATP binding site (see Figure 9b).

[0319] Furthermore, we confirmed that TPP-antioxidant conjugates with long linkers did not compete with PU-H71-FITC (an Hsp90 / TRAP1 inhibitor that targets the ATP binding site) and did not bind to the ATP binding site (see Figure 11b).

[0320] Example 5. ATPase activity analysis The present inventors performed an analysis to confirm that the compounds disclosed herein bind to the ATP binding site in TRAP1 and affect the activity of the ATPase.

[0321] TRAP1 ATPase activity was measured using the PiColorLock Gold Phosphate Detection Kit (Abcam). 0.5 μM TRAP1 (wild-type or mutant) was preincubated with various concentrations of inhibitor for 30 min, followed by incubation with 0.2 mM ATP for 3 h at 37°C in ATPase activity assay buffer containing 50 mM Tris-HCl, 20 mM KCl, and 6 mM MgCl2 (pH 7.4). Next, 20 μL of PiColorLock Gold reagent and accelerator mixture (100:1) was added to each sample (100 μL). After a 5-min incubation, color development was stopped by adding 10 μL of stabilizer. Absorbance was measured at 620 nm using a microplate reader (Synergy NEO, BioTek). Background signals were normalized by subtracting the absorbance of unreacted samples.

[0322] As a result, in the case of alkyl-TPP, ATPase activity decreased with increasing concentration of PU-H71, but did not show a concentration-dependent decrease in ATPase activity (see Figure 9a), suggesting that alkyl-TPP acts in a different manner from PU-H71, which inhibits ATPase activity by binding to the ATP binding site.

[0323] Furthermore, the TPP-antioxidant conjugate and other synthetic substances (SB-U011,014,015) did not show any tendency to decrease ATPase activity in a concentration-dependent manner (see FIGS. 11a and 14).

[0324] Example 6. Protein Expression Analysis The inventors performed protein expression analysis to determine whether the compounds disclosed herein inhibit TRAP1 or cytoplasmic Hsp90.

[0325] Cell incubation and treatment The human cancer cell line 22Rv1 was purchased from the American Type Culture Collection (ATCC) and maintained according to the supplier's recommendations. Briefly, cancer cells were incubated in DMEM or RPMI medium (GIBCO) containing 10% fetal bovine serum (FBS; ATCC) and 1% penicillin / streptomycin (GIBCO) at 37°C in a humidified atmosphere of 5% CO. Cells were not incubated for more than 6 months.

[0326] 22Rv1 cells were treated with 5 μM of each compound, incubated for 2 hours, and then analyzed by Western blot.

[0327] antibody Anti-phospho-AMPKα, anti-Cdk4, anti-CHOP, and anti-SIRT3 antibodies were purchased from Cell Signaling Technology. Anti-Akt and anti-AMPK were purchased from Santa Cruz Biotechnology. Anti-Hsp70 was purchased from BD Biosciences. Anti-β-actin antibody was purchased from MP Biomedicals. Anti-SDHB was purchased from Abcam.

[0328] Western blot Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 10% nonfat milk in TBST (TBS with 0.05% Tween-20) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The membranes were washed three times with TBST for 1 hour and then incubated with secondary antibodies (1:5000) diluted in 10% nonfat milk in TBST for 1 hour. The membranes were washed three times with TBST and visualized using an enhanced chemiluminescence detection kit (BioRad).

[0329] The experimental results showed that TPP-10-16 inhibited TRAP1 and reduced SDHB and SIRT3, with TPP-14 and 16 showing particularly potent effects. However, none of the alkyl-TPPs caused any changes in the expression of Akt or Cdk4 proteins, nor did they cause any changes in Hsp70, which is used as a marker for Hsp90 inhibition (see Figure 10). This confirms that alkyl-TPPs of a certain length inhibit TRAP1 without affecting cytoplasmic Hsp90.

[0330] Furthermore, in the case of TPP-antioxidant conjugates, conjugates with long linkers showed a decrease in SDHB and SIRT3 upon TRAP1 inhibition without inhibiting Hsp90, while short linkers had no effect on protein expression (see Figure 12). This confirms that only TPP-conjugates with the appropriate length inhibit TRAP1. Furthermore, unlike compounds with short linkers, compounds with the appropriate length activated AMPK (p-AMPK) and induced the mitochondrial unfolded protein response (CHOP), markers of TRAP1 inhibition (see Figure 13).

[0331] Furthermore, Western blot results also confirmed that other synthetic compounds inhibited TRAP1 (see Figure 15).

[0332] Example 7. In vivo experiments to confirm the effects of compounds on cancer In vivo mouse xenografting Immunodeficient athymic nude mice (male, 8 weeks old) were purchased from OrientBio. Mice were maintained in a pathogen-free facility (12-h dark / light cycle) at the UNIST In Vivo Research Center and provided with standard chow and water. All animal experiments were approved by UNIST (UNISTIACUC-19-11). 22Rv1 cells (1 × 10 7) was injected subcutaneously into both flanks of nude mice. 3 Once tumor size reached 100%, vehicle (DMSO) and 3 mg / kg of drug (MitoQ, SB-U014, and SBU015) dissolved in 20% cremophor EL (Sigma) in PBS were administered intraperitoneally daily. Tumor volume was measured daily using electronic calipers and calculated using the following formula: V = 1 / 2 × (width) × length. Upon completion of the experiment, animals were euthanized, and tumors were collected for histology and Western blot analysis. Band intensity was quantified using ImageJ software (National Institutes of Health, USA). In this case, Western blot analysis was performed similarly to the method described above.

[0333] The experimental results showed that tumor size and weight were reduced when treated with SB-U014,015 compared to when treated with DMSO (see Figure 16). Additionally, Western blot analysis showed that TRAP1 inhibition reduced SDHB and SIRT3, activated AMPK (p-AMPK), and induced the mitochondrial unfolded protein response (CHOP), while no changes were observed in Akt, Cdk4, or Hsp70 (see Figure 17).

[0334] This confirmed that the compounds disclosed herein inhibit the growth of cancer cells and reduce their size through TRAP1 inhibition.

[0335] Example 8. In vivo experiments to confirm the effects of compounds on ophthalmic diseases 8-1. Experimental induction of oxygen-induced retinopathy (OIR) in TRAP1 knockout mice.

[0336] Black6 J strain mice were used for the TRAP1+ / + (wild-type), TRAP1+ / - (heterozygous), and TRAP1- / - (knockout) experimental mice. On day 7 after birth, the pups and their mothers were placed in an in vivo chamber (Coy-lab) and supplied with hyperoxia (75% O2). On day 12 after birth, the pups were removed from the in vivo chamber and placed in a surrogate mother, after which they were supplied with normal oxygen (21% O2). On day 17 after birth, the pups' eyes were enucleated and used for the experiments.

[0337] The TRAP1 knockout mice were created by mating heterozygous female mice with male mice, and the offspring were compared. Genotype analysis was performed on the day of eye enucleation.

[0338] 8-2. Eye drop treatment To create an in vivo oxygen-induced retinopathy (OIR) model, pups were treated with eye drops for six days, from the day they were removed from a hyperoxia-enriched environment to the day their eyes were harvested (days 12-17 after birth). Eye drops were administered three times daily at four-hour intervals. Mito Q was diluted to 1 mM liposomally and 10 μl of the drug was then dropped into the pups' eyes, after which they were asked to blink for one minute. Any remaining drug on the outside of the eyeball was wiped with a cotton swab, and the mice were then returned to their mother's care.

[0339] 8-3. Intravitreal injection of drugs To create an in vivo oxygen-induced retinopathy (OIR) model, pups were given an intraocular injection on the day they were released to normal oxygen after hyperoxia (day 12 after birth). The pups were anesthetized with 50 μl of anesthetic (2.5% Avertin, solvent: 1X phosphate buffered saline (PBS)) intraperitoneally. A hole was made in the pup's eye with a pinholder. A 1 μl volume of the drug was injected into the hole at a rate of 0.1 μl / sec using a fine glass capillary. MitoQ was diluted to a concentration of 0.1 mg / ml in triple-distilled water (0.1% DMSO + 99.9% triple-distilled water).

[0340] 8-4. Whole mount staining and microscopic imaging of tissue An in vivo oxygen-induced retinopathy model was established, and eyes were harvested on day 17 of birth. As a negative control, eyes raised in a normal oxygen environment were harvested on day 17 of birth. Mice were anesthetized via intraperitoneal injection of 2.5% Avertin in 1X phosphate-buffered saline, and 10 ml of 1X PBS was injected into the left ventricle to remove all blood. The eyes were enucleated and fixed in 4% paraformalin for 24 hours at 4°C. The retinas were isolated and then cut radially into four sections. After blocking (1X PBS + 0.5% BSA + 0.1% Tryton-X-100) for 1 hour at room temperature, they were treated with primary antibody (CD31.1:100) for 24 hours at 4°C. The next day, the tissues were washed four times for 20 minutes each with washing solution (1X PBS + 0.1% Tryton X-100) and then treated with secondary antibody (Alexa fluor-594, 1:500, Invitrogen) diluted in blocking solution at 4°C for 24 hours. The next day, the tissues were washed five times for 20 minutes each with washing solution (1X PBS + 0.1% Tryton X-100) and then mounted on slides with mounting solution (Vector Lab. H-1700) and fixed.

[0341] Images of the entire tissue were observed using a stereofluorescence microscope (Axion xoom, Zeiss), and enlarged photographs of specific areas were observed using a confocal scanning microscope (Multi-photon confocal microscopy, LSM 780, Zeiss).

[0342] 8-5. In vivo streptozotocin-induced diabetic retinopathy model Experiments were performed using TRAP1 wild-type, heterozygous, and knockout mice. Eight-week-old male mice were intraperitoneally injected with streptozotocin (Sigma) diluted in 0.1 M sodium citrate solution (pH 5.0) at a concentration of 75 mg / kg once daily for five days. One week later, blood glucose was measured to confirm the induction of glycosuria by measuring levels above 350 mg / dL. From week 8 to week 16 after STZ injection, the mice were provided with weaning food supplemented with water three times a week. At 16 weeks of age after STZ injection, the eyes were enucleated and analyzed.

[0343] 8-6. Immunofluorescence staining The enucleated eyeballs were fixed in 4% paraformalin for 24 hours at 4°C. After tissue processing, paraffin blocks were made, and the tissue was cut into 10 μm thick pieces, which were then attached to slides to prepare tissue sections.

[0344] Paraffin was dissolved with xylan, and the xylan was removed with 100%, 80%, 70%, and 50% ethanol. After rehydration with triple-distilled water, the slides were placed in 10 mM sodium citrate (pH 6.0) and heated in a pressure cooker for 10 minutes to warm to room temperature. Permeabilization was achieved with 1X PBS + 1% Tryton X-100 solution at room temperature for 1 hour, followed by blocking with 1X PBS + 5% BSA + 5% FBS + 0.3% Tryton X-100 for 1 hour at room temperature. The slides were then incubated with primary antibodies (TRAP1, Thermo, 1:100; Glutamine synthase, Millipore, 1:100; GFAP, Abcam, 1:50; HIF1α, Novus, 1:20) at 4°C for 24 hours. The following day, the slides were washed three times with 1X PBS + 0.3% Tryton X-100 for 5 minutes each. The slides were treated with secondary antibodies (Alelx fluor-488, 546, 633, 1:500) at room temperature for 1 hour, washed, and then stained with DAPI (thremo. 300 nM) for nuclei. The slides were fixed with mounting solution.

[0345] Images were observed using a confocal scanning microscope (Multi-photon confocal microscopy, LSM 780, Zeiss).

[0346] 8-7. Western blot analysis Mouse eyes were enucleated and retinas were isolated. Whole cell lysates were prepared using RIPA lysis buffer and subjected to electrophoresis. After transfer to a PVDF membrane, the membrane was blocked with 10% nonfat dry milk at room temperature for 1 hour, followed by treatment with primary antibodies (TRAP1, Abcam; HIF1α, Novus; VEGF-A, Abcam; GFAP, Millipore; and β-actin, Millipore; all diluted 1:500) for 18 hours at 4°C. The following day, secondary antibodies (anti-mouse or rabbit-HRP, KPL) were applied for 1 hour at room temperature, and protein expression was analyzed using Western blotting detection reagents (Bio-Rad).

[0347] For quantitative analysis, area fraction was calculated using Image J and normalized using β-actin.

[0348] 8-8. Real-time polymerase chain reaction (real-time PCR) After enucleation of mouse eyes, the RNA layer was isolated using Trizol and chloroform, and RNA was extracted using an RNA extraction kit (Qiagen). cDNA was synthesized using a cDNA synthesis kit (NEB), and quantitative analysis of the cDNA was performed by real-time polymerase chain reaction using SYBR Real-Time Polymerase Chain Reaction Master Mix (Enzynomics). Amplification reactions were performed using a LightCycler (Roche). Analysis was performed using comparative CT analysis. Normalization was performed using β-actin, and the increase or decrease in the expression level of target genes (TRAP1, VEGF-A, ANGPTL4) in the experimental group was analyzed compared to the mean expression level in the control group.

[0349] 8-9. Experimental results 18 and 19, TRAP1 expression is increased in both the oxygen-induced retinopathy model and the STZ (streptozotocin)-induced diabetic retinopathy model. Furthermore, it can be seen that the hypoxia marker HIF1α and downstream angiogenic factor VEGF-A increase along with the increase in TRAP1 in these models. Furthermore, the staining results confirm that TRAP1 is colocalized with the staining of GS (glutamine synthase), a marker of Müller cells responsible for the production of various angiogenic factors during the progression of retinal disease.

[0350] Referring to FIG. 20, it can be seen that in the OIR model in which TRAP1 was knocked out, the neovascular area and avascular area were reduced.

[0351] Furthermore, referring to the results of HIF1α staining of the retina in FIG. 21, it can be seen that HIF1α was reduced when TRAP1 was knocked out in the STZ or OIR model.

[0352] Referring to the VEGF-A and ANGPTL4 expression results in FIG. 22a, the mRNA levels of VEGF-A and ANGPTL4 were significantly increased by Con TRAP1. + / + Compared to STZ TRAP1 + / + STZ-TRAP1 - / - Furthermore, referring to FIG. 22b, TRAP1 expression was not elevated in the OIR model retina. + / + The mRNA levels of VEGF-A and ANGPTL4 were significantly higher than those of TRAP1 + / - and TRAP1 - / - It decreased by.

[0353] Finally, referring to the results in Figures 23 and 24, it can be confirmed that MitoQ treatment significantly reduces both the avascular and neovascular areas in the retina of the OIR model.

[0354] Example 9. Drug activity analysis using MIO-M1 HRE cell line MIO-M1 Müller cells were transfected (using the Jetprime kit) with the 5HRE / GFP plasmid (addgene. #46926) and selected for plasmid transfection using the selectable marker G418 (Neomycin) at 1 mg / ml. Stable cell lines were generated by selecting single-cell colony-forming cells. The resulting MIO-M1-HRE / GFP stable cell line was then aliquoted into 96-well plates and treated with various concentrations of drugs the following day. After 24 hours of exposure to a hypoxic environment (1% O2), GFP (Ex / Em: 488 / 507) fluorescence signals were measured using a SYNERGY NEO microplate reader (BioTek Instrument). DMSO, the solvent used to dissolve the drugs, was used as a negative control, and relative fluorescence values ​​were calculated relative to the negative control (100%).

[0355] As a result, referring to Figures 25, 26, and 27, it was confirmed that alkyl-TPP, TPP-antioxidant conjugates (with linkers of appropriate lengths), and other synthetic compounds that were confirmed to inhibit TRAP1 all have HIF1α inhibitory activity.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical formula 1] In this case, L is (CH ２ ) ｎ Including, The n is an integer of 7 or more and 40 or less, A is, unsubstituted cyclohexyl, or a pharmaceutically acceptable salt thereof.

2. The compound is 2. The compound of claim 1 selected from the group consisting of: or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which binds to TRAP1 and competes with SB-TM2.

4. A TRAP1 inhibitor comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

5. 10. An inhibitor of the binding between TRAP1 and a client protein, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition for treating an ophthalmic disease, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

7. 10. A method for treating a rheumatoid arthritis comprising administering to a subject in need thereof a compound of claim 1 or a pharmaceutically acceptable salt thereof; The method for treating an ophthalmic disease, wherein the subject is a mammal other than a human.

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