Fluorescence systems for biological imaging and their applications

Abstract

The present invention relates to compounds of Formula I, in which Y, Ar1, Ar2, X, R1, and R2 are defined herein, and to their use in various biological imaging techniques and therapeutic methods. The present invention relates, in aspects, to conjugates comprising compounds of Formula I and their related uses and therapeutic uses.

Description

Technology Field

[0001] The present invention relates to Y, Ar1, Ar2, X, and R 1 and R 2 Compound of Formula I as defined herein:

[0002]

[0003] The invention relates to the use thereof in various biological imaging techniques and therapeutic methods. Furthermore, the invention relates to a conjugate comprising a compound of Formula I and related uses and therapeutic uses. Background Technology

[0004] Fluorescence imaging has rapidly become a powerful tool for investigating biological processes, particularly in living cells, allowing cellular phenomena to be observed from a biological perspective. The development of single-molecule visualization techniques has significantly improved the utility of fluorescence microscopy in these applications, enabling the tracking of proteins and small molecules in their endogenous environments. From probes capable of detecting specific molecules to compounds located in specific intracellular organelles, the field of biological imaging is becoming a rapidly emerging area.

[0005] Fluorescent synthetic retinoids as described in WO 2016 / 055800 A are used as research tools in the field of fluorescence imaging, providing useful insights into retinoid activity and metabolism in natural environments by tracking cellular uptake and localization. However, extensive retinoid signaling in biology makes targeting with retinoids difficult, limiting their widespread use as fluorescent probes and therapeutic agents.

[0006] The development of reliable markers for non-mammalian cell types also remains a challenge. For example, while some commercially available fluorescent probes targeting specific organelles within mammalian cells can be used in plants, their signal quality and specificity are generally poor, and the relatively high molecular weight of fluorescent compounds affects labeling efficiency. Furthermore, known fluorescent probes often have excitation ranges similar to chlorophyll, leading to signal interference in plant cell imaging.

[0007] Consequently, it would be beneficial to provide fluorescent compounds that address one or more of these drawbacks and can be utilized as versatile fluorophores in a wide variety of imaging and bio-targeting techniques. In terms of functionality, compounds with improved flexibility—specifically, ease of attachment of various targeting or reactive groups or manipulation and amplification of the chromophore—would be beneficial, as would excellent physical properties such as good water solubility. Excellent photoresponsive characteristics, such as the ability to act as a photosensitizer when activated by light of an appropriate wavelength, are also beneficial, making them useful for photodynamic therapy (PDT) and various ROS-mediated applications in various cell types.

[0008] Accordingly, the present invention generally relates to fluorescent compounds and their use in various biological imaging and targeting techniques.

[0009] The present invention relates, in aspects, to a new compound itself and its use as a biological probe, in particular as a fluorescent probe.

[0010] The present invention relates, in aspects, to the use of compounds in Raman imaging and fluoRaman imaging techniques and related imaging methods.

[0011] The present invention relates, in aspects, to a method for deprotecting a compound to form a deprotected compound for bonding, as well as to a deprotected compound formed by such method.

[0012] The present invention relates, in aspects, to a method for controlling the properties of a compound of Formula I to incorporate a function of targeting cell-localization.

[0013] The present invention relates, in aspects, to conjugates comprising compounds, and to the use of these conjugates in imaging, therapeutic, and non-therapeutic applications. The conjugate may comprise, for example, a compound of the present invention that is directly conjugated to a targeting agent or an active agent, or conjugated through a linker or spacer group.

[0014] The present invention relates, in aspects, to pharmaceutical compositions comprising such compounds and conjugates, and to the use of such compounds, conjugates, and compositions in the treatment of various pathological conditions or diseases. The present invention, in aspects, includes the use of compounds for controlling the generation of reactive oxygen species (ROS) in therapeutic applications.

[0015] The present invention relates, in aspects, to formulations comprising such compounds and conjugates, and to the use of such compounds, conjugates, and formulations in applications for controlling the generation of ROS in plant, fungal, and bacterial cells.

[0016] Additional aspects and embodiments of the present invention are defined as defined in the claims and are described in more detail below.

[0017] The present invention provides a compound of Formula I in a free form or a salt form and a diastereomer thereof:

[0018]

[0019] In the above formula,

[0020] R 1 is a C1-10 alkyl group that is H or optionally substituted with one or more N atoms; R 2-(CH2) is a C1-10 alkyl group optionally substituted with one or more N atoms n R 3 , -(CH2) n NHR 3 and -(CH2)2(COCH2) n R 3 Selected from, where n is an integer between 1 and 10, and R 3 is -NH2, -OH, -SO2PhCH3 or -COOH, or R 2 -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2) n CH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R 8 or -(CH2) n PPh3 + Br - but, R 8 is -OH or -NHOH, n is an integer from 1 to 8, and m is an integer from 1 to 4; or

[0021] R 1 and R 2 It forms part of the heterocyclic group Y having 3-12 ring atoms;

[0022] Ar1 and Ar2 are each independently aromatic groups; and

[0023] X is selected from unsaturated esters, ketones, carboxylic acids, imidazolone, pyridine, oxazolone, oxazolidinone, barbituric acid, and thiobarbituric acid, wherein

[0024] However, if Ar1 is phenyl and R1 and R2 form part of a heterocyclic group Y having 3-12 ring atoms, N of the heterocyclic group is located at the para position relative to the acetylene group of the compound of Formula I.

[0025] In general terms, the compound of Formula I has a basic structure of diarylacetylene, generally exemplified by the diphenylacetylene structure, and at one end para - Amino (electron donor group) and other ends para - It has an electron-conjugating group and implements a dipole system through electron conjugation.

[0026] The inventors have fortunately discovered that the compound of Formula I possesses remarkable utility in biological imaging techniques. For example, this compound has been proven to penetrate mammalian, bacterial, fungal, and plant cells, demonstrating its widespread applicability to hosts for imaging applications. The unique structure of this compound provides utility in terms of functional groups for the system, namely, in particular Y and R 1 or R 2It allows for the attachment of targeting or reactive groups through reactions with the amine group of the moiety as well as reactions at other positions, such as the X group. This enables the attachment of targeting functional groups, such as photoaffinity labels, by allowing the conjugation of reactive functional groups, such as targeting motifs for cellular localization, and / or the conjugation or attachment of biomolecules and other small molecule drugs, such as peptides and antibodies. Compared to previously known fluorescent probes, the low molecular weight of this compound facilitates cell penetration, allowing for targeting without fluctuation when a moiety, such as an anticancer drug, is conjugated to the compound, as demonstrated using the model drug vorinostat. The ability of this compound to act as a photosensitizer, when combined with selectively conjugated drug molecules, provides various useful applications through the control of ROS in plant, fungal, and bacterial cells, for example, in the production of targeted herbicides or in seed enhancement applications, such as in photodynamic therapy (PDT). The structural flexibility of this molecule in terms of modularity also provides the opportunity to incorporate a second fluorophore capable of excitation at different wavelengths, leading to a host of additional potential applications. Furthermore, this structure of the compound enables its use in Raman imaging and fluoRaman imaging techniques. Surprisingly, the inventors found that Ar1 is phenyl and R 1 and R 2 In embodiments of the present invention in which a heterocyclic group Y forms part of the heterocyclic group, it has been demonstrated that the nitrogen arrangement of the heterocyclic group at the para-position relative to the central acetylene group of the compound is significantly more efficient in terms of photophysical properties compared to the ortho-position equivalent. This has significant advantages in terms of application in imaging techniques.

[0027] The compound of the present invention has a general structural formula represented by the aforementioned formula I.

[0028] In this document, the term "diastereomer" refers to isomers that have the same composition but differ in the spatial arrangement of atoms. The term "diastereomer" is intended to encompass alkene diastereomers.

[0029] In this document, the term “heterocyclic group” means a monocyclic or bicyclic ring group containing 3 to 12 ring atoms and having 1 to 3 functional groups or heteroatoms selected from the group consisting of N, S, SO, SO2, O2, and O, in addition to a nitrogen atom of Formula I. In this document, the term “heterocyclic group” encompasses aromatic, partially unsaturated, and saturated ring systems. Examples of non-aromatic groups include, but are not limited to, piperidinyl, piperazinyl, morpholinyl, thiomophorinyl, dioxydothiomophorinyl, pyrrolidine-1-yl, pyrrolidine-3-yl, azetidin-1-yl, azetidin-3-yl, aziridine-1-yl, azepan-1-yl, azepan-3-yl, azepan-4-yl, etc. Examples of aromatic (heteroaryl) groups include, but are not limited to, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, indolyl, and benzothiadiazolyl groups. In one embodiment, the heterocyclic group is a saturated ring system. The heterocyclic group may be optionally substituted. In one embodiment, the heterocyclic group is an alkyl group, -COCH3, -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2) n CH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R8 or -(CH2) n PPh3 + Br - It can be substituted as, where R 8 is -OH or -NHOH, n is an integer from 1 to 8, and m is an integer from 1 to 4.

[0030] "The fact that N of the heterocyclic group is in the para position relative to the acetylene group means that N constituting part of the heterocyclic group Y is a para-substituted donor group in the compound; and in the embodiment where Ar1 is phenyl, it is located in the para position relative to the central acetylene moiety of the compound of Formula I. To avoid doubt, if the heterocyclic group contains two or more nitrogen atoms, one of the N atoms is in the para position.

[0031] In this document, the term "aromatic group" encompasses carbocyclic and heterocyclic unsaturated rings comprising 5 to 19 ring atoms, preferably 5 to 13 ring atoms. The aromatic group may be monocyclic or polycyclic, preferably monocyclic, dicyclic, or tricyclic, and more preferably monocyclic or dicyclic. In heterocyclic aromatic groups, the ring group may comprise one or more of N, O, or S atoms. Examples of suitable aromatic groups include pyrrole, furan, benzofuran, thiophene, phenyl, imidazole, pyrazole, oxazole, thiazole, oxathiazole, pyridine, pyrimidine, pyrazine, pyridazine, and triazine. The aromatic group may be optionally substituted, for example, groups such as fluoride, chloride, bromide, and iodide, alkyl groups, alkenyl groups, and amine groups (-CH2-(CH2) n -NH2), hydroxyl group (-CH2-(CH2) n -OH) and carboxyl groups (-CH2-(CH2) n -COOH)(n can be 0-10), or can be substituted with aromatic or PEG-derived groups.

[0032] In one embodiment, Ar2 is selected from the following:

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] In one embodiment, Ar1 is selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, thiazole, and oxathiazole groups.

[0040] In one embodiment, Ar1 and Ar2 may each be independently selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, thiazole, and oxathiazole groups.

[0041] In one embodiment, Ar1 and Ar2 can each be independently selected from phenyl, thiophene, furan, benzofuran, thiazole, and oxathiazole groups.

[0042] In one embodiment, Ar1 is a phenyl group.

[0043] In one embodiment, Ar1 is a phenyl group, and Ar2 is selected from phenyl, thiophene, furan, thiazole, and oxathiazole groups.

[0044] X is an electron-deficient group. As described herein, the term "electron-deficient group" refers to a functional group having a reduced electron density compared to the rest of the chemical structure of the molecule of Formula I. As will be obvious to those skilled in the art, the electron-deficient group must not only have a lower electron density compared to the rest of the chemical structure of the molecule of Formula I but also be non-toxic. This implies that, for example, nitro and nitrile groups are generally unsuitable.

[0045] In the present invention, X is an unsaturated ester, ketone, carboxylic acid, imidazolone, pyridine, oxazolone, oxazolidinone, barbituric acid, thiobarbituric acid, -CH=CH-C(=O)OR 4 (R 4 is C2-C 10 alkyl, or alkenyl, aryl, or glycol group); -CH=CH-C(=O)R 5 (R 5 is C2-C 10 Alkyl, or alkenyl or aryl group, -CF3 or -NH2); -(OCH2CH2OH) n (n = 1 - 6), or selected from nitrogen-containing heterocycles, optionally nitrogen-containing heterocycles containing 5-6 ring atoms.

[0046] As used herein, the term “alkyl” refers to a fully saturated, branched, unbranched, or cyclic hydrocarbon moiety, i.e., a primary, secondary, or tertiary alkyl, or, where appropriate, a cycloalkyl or an alkyl substituted with a cycloalkyl. Unless otherwise noted, the alkyl group comprises 6 to 10 carbon atoms, preferably 1 to 6, or more preferably 1 to 4. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n -profile, iso -profile, n -butyl, sec -butyl, iso -butyl, tert -butyl, n -Pentyl, isopentyl, neopentyl, n -hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n -Heptyl, n -Octyl, n - Nonil and n There are decil, etc.

[0047] The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds.

[0048] In this document, the term "halogen" or "halo" means F, Cl, Br, or I.

[0049] The term "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system composed solely of hydrogen and carbon, containing 6 to 19 carbon atoms, preferably 6 to 10, wherein the ring system may be partially saturated. Alkyl groups include, but are not limited to, fluorophenyl, phenyl, indenyl, and naphthyl groups. The term "aryl" encompasses an aryl radical optionally substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, halo, haloalkyl, cyano, nitro, amino, amidine, aryl, aralkyl, cycloalkyl, heterocyclyl, heteroaryl, or heteroarylalkyl. A preferred alkyl group is an optionally substituted phenyl or naphthyl group.

[0050] In one embodiment of the present invention, R 1 and R 2 constitutes a part of the heterocyclic group Y. In this embodiment, the heterocyclic group Y may be selected from, for example, the following:

[0051]

[0052] R 7 C1-C 10 Alkyl group, -COCH3, -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2)nCH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R 8 or -(CH2) n PPh3 + Br - It could be, but, R 8is -OH or -NHOH, n is an integer from 1 to 8, and m is an integer from 1 to 4.

[0053] Alternatively, R 1 ≠ H or can be a C1-10 alkyl group optionally substituted with one or more N atoms, and R 2 is a carbon-10 alkyl group optionally substituted with one or more N atoms, -(CH2) n R 3 , -(CH2) n NHR 3 and (CH2)2(COCH2) n R 3 Selected from, where n is an integer between 1 and 10, and R 3 is -NH2, -OH, -SO2PhCH3 or -COOH, or R 2 -COCH3, -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2) n CH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R 8 or -(CH2) n PPh3 + Br - It could be, but, R 8 is -OH or -NHOH, n is an integer from 1 to 8, and m is an integer from 1 to 4. In this embodiment, preferably, R 1 is H or a C1-10 alkyl group, and R 2 is (CH2) n R 3 , -(CH2) n NHR 3 or (CH2)2(COCH2) n R3 and, where n is an integer from 1 to 10, and R 3 is -NH2, -OH, -SO2PhCH3, or -COOH.

[0054] In one embodiment, X is selected from the following: -CH=CH-C(=O)OR 4 (R 4 = C2-C 10 Alkyl, alkenyl, aryl, or glycol group); -CH=CH-C(=O)R 5 (R 5 = C2-C 10 Alkyl, alkenyl, or aryl group, -CF3 or NH2); -(OCH2CH2OH) n (n = 1 - 6), or optionally a nitrogen-containing heterocycle containing 5-6 ring atoms.

[0055] If X is an N-containing heterocycle, it can be selected from the following:

[0056]

[0057] In the above equation, R 4 and R 5 is defined as above, and R 6 is H or alkyl.

[0058] In one embodiment, X is selected from the following:

[0059]

[0060] In the compound of Formula I, Ar1 is phenyl and R 1 and R 2 In embodiments where ga forms part of the heterocyclic group Y, N of the heterocyclic group is located at the para position relative to the acetylene group of the compound of Formula I. Ar1 is phenyl; R 1 and R 2In one embodiment in which ga forms part of the heterocyclic group Y, N of the heterocyclic group attached to Ar1 is not in an ortho position with respect to the acetylene group of the compound of Formula I. This means that the compound of Formula I is not, for example, as follows:

[0061]

[0062] In one embodiment, the compound of Formula I is selected from the following:

[0063] In one embodiment, the compound of Formula I is compound 6, compound 7, compound 43, compound 51, compound 55, compound 57, compound 59, compound 64, compound 69, or compound 71.

[0064] In one embodiment, the compound of Formula I is compound 6, compound 7, compound 43, or compound 69.

[0065] The compound according to the present invention is essentially fluorescent. In one aspect, the present invention provides a compound of Formula I for use in fluorescent imaging.

[0066] The flexible chemical properties of the compound of Formula I advantageously allow for selective cell targeting and / or cell localization, making the compound of Formula I a powerful tool in biological imaging. For example, the compound of the present invention can readily conjugate various targeting biomolecules to provide useful information regarding cell uptake and localization through fluorescence imaging techniques.

[0067] Due to the modular nature of the compound structure, the compound structure of Formula I can expand the chromophore by modifying it with various functional groups to approach or reach the near-infrared (NIR) region. Fluorescence in the near-infrared region (1,000–1,700 nm) is particularly useful for biological and biomedical imaging due to its deep penetration, spatial high resolution, and weak biofluorescence (Stolik et al. J. Photochem. Photobiol. B. 57 (20000), 90-93).

[0068] In one aspect, the present invention provides a compound of Formula I for use in Raman imaging.

[0069] The aspects of the present invention relate to the use of the compound of Formula I in Raman imaging.

[0070] Specifically, the internal acetylene functional group of the compound of Formula I is in the 'cell-silencing' Raman region (1800–2600 cm⁻¹). -1 ), that is, since it generates unique vibrational frequencies in the region where endogenous molecules do not vibrate, this compound can be used to image specific target molecules in a biological environment using Raman-based techniques.

[0071] In terms of aspects, the compound is a dual-mode imaging agent.

[0072] Aspects of the present invention relate to the use of compounds in a combination of fluorescence imaging techniques and Raman imaging techniques by constructing a powerful tool for imaging complex biological systems, for example, by superimposing fluorescence to provide environmental information with Raman to provide quantitative mapping.

[0073] Furthermore, the present invention relates to a method for monitoring cell development, such as cell differentiation or apoptosis. In embodiments, such a method may include the steps of administering an effective amount of a compound of Formula I and detecting emitted fluorescence. Alternatively, a method for monitoring cell development, such as cell differentiation or apoptosis, may, without limitation, include imaging the distribution of a compound of Formula I by detecting CARS (coherent anti-Stokes Raman scattering) and SRS (stimulated Raman scattering).

[0074] Accordingly, the present invention provides a probe comprising a compound of Formula I in its aspects.

[0075] The flexible chemical properties of the compound of Formula I advantageously allow for selective targeting and / or localization to cell types, making the compound of Formula I a powerful tool for biological imaging.

[0076] The present invention relates, in aspects, to a method for modifying the properties of a compound to incorporate a function of targeting cell-localization. For example, a reactive amine group of a compound can undergo a one-step acylation, alkylation, or sulfonylation reaction to introduce a targeting motif for intracellular localization, such as a triphenylphosphonium cation (localization to the mitochondrial matrix) and a tosylsulfonamide group (localization to the endoplasmic reticulum (ER)).

[0077] The present method also relates to inactivated derivatives of compounds.

[0078] As understood by those skilled in the art, compounds incorporating reactive functional groups, such as amine, hydroxy, or carboxylic acid groups, are usually protected as inactivated derivatives for storage, namely as amides, ethers, or esters. Activation of these compounds for further reaction or conjugation involves removing the protecting group by, for example, treatment with a strong acid solution (amide → amine), a strong Lewis acid (ether → hydroxy), and a strong basic aqueous solution (ester → carboxylic acid). Alternatively, for example, the reactive amine group can be further derivatized to utilize a functional group that provides orthogonal reactivity for conjugation reactions inaccessible to the parent compound, such as, for example, conversion from an amine to an acrylamide for reaction with a thiol, provision of a carboxylic acid for reaction with another amine or hydroxyl through reaction of an amine with a cyclic anhydroide, or conversion of an amine to azidoacetamide for a cycloaddition reaction of an azide / alkyne.

[0079] The present invention relates, in aspects, to protected and unprotected compounds of Formula I.

[0080] The present invention, in aspects, provides a conjugate comprising a compound of Formula I and a targeting substance or an active substance. The targeting substance or active substance may be, for example, a reactive group such as a photoaffinity labeling substance, a small molecule drug such as an anticancer agent including vorinostat, methotrexate, and fulvestrant, a biomolecule such as a protein or peptide containing a cell adhesion sequence such as RGD (tripeptide Arg-Gly-Asp), a carbohydrate such as glucose or the polysaccharide sucrose, or a biologic agent such as an aptamer, affimer, or antibody.

[0081] For example, the targeting substance or active substance may contain a photo-reactive functional group that operates at a different wavelength than the fluorescent compound of Formula I, so that the compound can be dissociated through a photoreactive linker, or a photoaffinity label can be activated to tag a target protein / receptor or enzyme. Suitable photoaffinity labels include diaziridine (diazirin), which can easily attach to the amine group of the compound of Formula I.

[0082] The targeting substance or active substance may be covalently coupled to the compound of Formula I, for example, through amide, ester, or ether linkages. 'Click-chemistry,' that is, techniques for linking substances to biomolecules, may also be used to prepare the conjugate of the present invention. The targeting substance or active substance may be attached to the compound of Formula I using a linker such as asymmetric (bifunctional) PEG or other spacer groups. Suitable functional group compounds that may be available include carboxyl groups for amide formation, alcohols and carboxylic acid groups for ester formation, alkyl electrophiles and alcohols for ether formation, and alkylazides and acetylenes for click-reaction.

[0083] In one embodiment, the conjugate comprises a compound of the following formula:

[0084] The targeting substance or active substance can be a small molecule drug, such as an anticancer drug.

[0085] In one embodiment, the conjugate comprises the compound of Formula 6, the compound of Formula 7, the compound of Formula 43, the compound of Formula 51, the compound of Formula 55, the compound of Formula 57, the compound of Formula 59, the compound of Formula 64, the compound of Formula 69, or the compound of Formula 71.

[0086] In one embodiment, the conjugate comprises a compound of Formula 6, a compound of Formula 7, a compound of Formula 43, or a compound of Formula 69:

[0087]

[0088]

[0089]

[0090]

[0091] In one embodiment, the conjugate comprises a compound of Formula 6 and a small molecule drug. In one embodiment, the conjugate comprises a compound of Formula 6 and an anticancer drug. In one embodiment, the conjugate comprises a compound of Formula 6 and vorinostat or an analog thereof.

[0092] In one embodiment, the conjugate comprises a compound of Formula 7 and a small molecule drug. In one embodiment, the conjugate comprises a compound of Formula 7 and an anticancer drug. In one embodiment, the conjugate comprises a compound of Formula 7 and vorinostat or an analog thereof.

[0093] In addition, the present invention relates to the use of these conjugates in imaging, therapeutic, and non-therapeutic applications.

[0094] The present invention relates, in aspects, to the use of a compound of Formula I in the generation of reactive oxygen species (ROS) upon activation of the compound by light.

[0095] Triplet-state photosensitizers (PS) typically contain a light-harvesting region that performs the dual functions of light-harvesting and intersystem crossing, where single-state electrons pass non-radiatively to the triplet state. Quenching of the triplet-excited state results in the generation of reactive oxygen species (ROS), which can arise from radicals derived from ground-state molecular oxygen or from direct chemical reactions with surrounding molecules. Local ROS generation is an immune defense strategy adopted in response to pathogen attacks in animal and plant systems. In animal, plant, fungal, and bacterial cells, ROS exert various regulatory effects depending on their production rate and extent; apoptosis is observed at high concentrations, while irritant responses are commonly observed at low concentrations (Guo et al. Stem Cells Dev. 2010, 19, 1321-1331).

[0096] Photodynamic therapy (PDT) utilizes the ability of photosensitizers to generate ROS, typically destroying cancer cells, pathogenic microorganisms, and / or unwanted tissues through apoptosis. Typically, photosensitive compounds are excited near or within specific target tissues or conditions (e.g., microbial infections, neoplasms, tumors, etc.), inducing the generation of large amounts of ROS and subsequent tissue destruction. Since ROS trigger cell proliferation at low concentrations, they can be utilized in wound healing or, more generally, tissue regeneration therapies.

[0097] Therefore, PDT relies on targeting photosensitive compounds to accumulate at desired locations, such as diseased tissues, and localizing light delivery to activate ROS generation. Although compounds for use in PDT are known, they generally have various disadvantages, such as small absorption peaks that cause problems with photoactivation, particularly in the case of bulky tumors where achieving photopenetration can be difficult; long biological half-lives that induce prolonged skin photosensitivity after treatment; poor pharmacological properties such as poor water solubility; and poor targeting ability (i.e., lack of ability to target and accumulate in specific tissues or cells that cause significant off-target damage).

[0098] In a beneficial way, the compound of the present invention is biologically inert in an unactivated state, but generates ROS when exposed to short-wavelength visible light irradiation of low to medium energy.

[0099] Therefore, the compound of Formula I can be utilized to generate reactive oxygen species (ROS) to control cell development—specifically, cell proliferation, differentiation, and apoptosis—thereby achieving various therapeutic and non-therapeutic applications. The compound of Formula I exhibits efficient targeting and thus may have low off-target effects, making it particularly beneficial for applications mediated by ROS control. Furthermore, this compound can be adapted to various cell types, enabling selective targeting effects.

[0100] Accordingly, in some aspects, the present invention relates to the use of the compounds or conjugates of the present invention in photodynamic therapy (PDT).

[0101] ROS generation can be controlled according to therapeutic needs to achieve, for example, the induction of apoptosis to remove cells, the induction of growth in wound healing, or a combination thereof. In an exemplary embodiment, for example in wound healing, high levels of ROS generation may be triggered first to induce apoptosis of bacterial and / or fungal cells, and then low levels of ROS generation may be used to assist skin regeneration.

[0102] In one aspect, the present invention provides a method for treating a patient using photodynamic therapy (PDT), comprising administering a compound of Formula I or a conjugate thereof, and activating the compound of Formula I to generate ROS.

[0103] In another aspect, the present invention provides the use of a compound of Formula I or a conjugate thereof in the manufacture of a drug for use in treating diseases or pathological conditions in which the control of cell proliferation, differentiation, or apoptosis is beneficial.

[0104] In another aspect, the present invention provides a method for treating a patient with a disease or pathological condition in which control of cell proliferation, differentiation, or apoptosis is beneficial, comprising administering a compound of Formula I or a conjugate thereof to the patient in a therapeutically effective amount.

[0105] Diseases or conditions where the control of cell proliferation, differentiation, or apoptosis is beneficial include, for example, cancer, for example, neuroneoplasms, skin disorders such as acne, and skin injuries such as burns, diabetic foot ulcers, UV damage, and skin aging.

[0106] The compound of Formula I can act as a chemotherapy agent or chemopreventive agent due to its ability to control cell development, that is, its ability to control proliferation, differentiation, and apoptosis in normal and tumor cells. In particular, the compound of Formula I can regulate proliferation, differentiation, and apoptosis in normal cells, premature cells, and malignant cells in vitro and in vivo.

[0107] In an embodiment of the present invention, the compound may act as a chemotherapy agent or chemopreventive agent in treating or preventing precancerous or cancerous conditions including the skin, oral cavity, larynx, lungs, bladder, vulva, breasts, kidneys, liver, prostate, eyes, or digestive tract.

[0108] This compound can act as a chemotherapy agent or chemopreventive agent in treating or preventing basal cell carcinomas, squamous cell carcinomas, such as head and neck tumors and bladder tumors.

[0109] This compound can act as a chemotherapy agent or chemopreventive agent in treating or preventing leukemia, particularly acute promyelocytic leukemia.

[0110] The compound of Formula I can act to promote cell proliferation, for example, the proliferation of skin or nerve cells, and to assist in wound healing. The compound of Formula I can be used to promote tissue health and development, particularly to promote the health and development of the skin, bones, nerves, teeth, body hair, and / or mucous membranes of the human or animal body. The compound of the present invention can be used to prevent or treat skin conditions such as signs of aging (particularly wrinkles and age spots), acne (particularly severe and / or intractable acne), psoriasis, stretch marks, keratosis pilaris, emphysema, and alopecia.

[0111] In an embodiment of the present invention, a conjugate of Formula I can be utilized in PDT. For example, an embodiment of the present invention relates to a conjugate of Formula I containing a small molecule therapeutic agent, e.g., an anticancer drug. Due to the relatively small size of the compound of Formula I compared to conventional fluorescent dyes, the anticancer drug can be targeted without alteration; that is, as demonstrated with vorinostat, the bioconjugate behaves as if the compound of Formula I is not attached, thereby maintaining the cytotoxic effect. Thus, the conjugate can be delivered to a target site, where the drug performs its normal function before the conjugate is exposed to UV light, thereby inducing control of ROS generation. In the context of anticancer drugs, for example, the apoptotic effect of the drug can be complemented by ROS-mediated apoptosis; that is, the anticancer drug can induce initial death of cancer cells and subsequently trigger apoptosis to kill the remaining cells.

[0112] Another aspect provides a pharmaceutical composition for use in treating or alleviating a disease or condition in which control of cell differentiation or apoptosis is beneficial, comprising a compound of Formula I as defined herein or a conjugate thereof, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, or carriers. Such compositions may optionally comprise one or more additional therapeutic substances.

[0113] In embodiments, the pharmaceutical composition may include a compound of Formula I conjugated with a therapeutic substance, such as a small molecule drug like an anticancer drug.

[0114] In embodiments, the pharmaceutical composition may comprise a compound of Formula I conjugated with borinostat or an analog thereof.

[0115] A conjugate comprising the compound of Formula I and vorinostat or an analog thereof exerts the inherent cytotoxic activity derived from the hydroxyamic acid of vorinostat, which can be complemented and enhanced by the application of UV, 405 nm, or 2-photon 800 nm light to induce an additional photoactivated cell-death effect.

[0116] The term "therapeutically effective" amount or "effective amount" refers to an amount of the compound or composition of the present invention effective for producing a desired therapeutic, improving, inhibiting, or preventive effect.

[0117] The dosage of a compound or conjugate administered to a human or animal body will vary depending on factors such as the intended use and the method of administration, as understood by those skilled in the art.

[0118] The term "pharmaceutical composition" refers to a composition suitable for administration to a patient. That is, the term "pharmaceutical composition" refers to a composition comprising a compound of the present invention, or a conjugate or mixture thereof, or a salt, solvate, prodrug, isomer, or tautomeric isomer, optionally together with one or more pharmaceutically acceptable excipients, carriers, or diluents. The term "pharmaceutical composition" is also intended to encompass both bulk compositions (i.e., forms not yet made into individual dosage units) and individual dosage units. Such individual dosage units include tablets, pills, caplets, ampoules, etc.

[0119] Those skilled in the art will know that the compounds of the present invention can be converted into prodrugs and / or solvates. The term “prodrug” refers to a compound (e.g., a drug precursor) that is converted in vivo to provide the compound of the present invention or a pharmaceutically acceptable salt, hydrate, or solvate of the compound. The conversion may occur through various mechanisms (e.g., metabolic or chemical processes), such as, for example, through hydrolysis in the blood.

[0120] The compounds of the present invention may or may not be solvated with pharmaceutically acceptable solvents such as water, ethanol, etc. For example, a solvate will be understood as being isolable when, for example, one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. The term "solvate" encompasses both solution-phase solvates and isolable solvates. Suitable solvates include, but are not limited to, ethanolates, methanolates, hydrates, etc.

[0121] The compound to be used in the present invention includes a salt thereof, and references to the compound of the present invention are intended to encompass references to its salt unless otherwise noted. Suitable salts include, for example, acidic salts formed with inorganic acids and / or organic acids, basic salts formed with inorganic bases and / or organic bases, as well as salts formed with cations that may be formed and are included in the term "salt(s)" herein ("internal salts"). While other salts may be useful in some cases, pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred. Exemplary acid addition salts that may be available include acetate, ascorbate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, campersulfonate, fumarate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate, naphthalenesulfonate, nitrate, oxalate, phosphate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, Examples include toluenesulfonate (also known as tosylate). Exemplary basic salts that may be available include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, dicyclohexylamine, tThere are salts with organic bases such as butylamine (e.g., organic amines), and salts with amino acids such as arginine, lysine, etc. Basic nitrogen-containing groups can be quaternized using substances such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long-chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides, and iodides), and arylalkyl halides (e.g., benzyl and phenethyl bromides).

[0122] Compounds for use in the present invention include pharmaceutically acceptable esters thereof, wherein the non-carbonyl moiety of the carboxylic acid region of the ester group is a straight-chain or branched-chain alkyl (e.g., acetyl, n-propyl, t -butyl or n-butyl), alkoxyalkyl (e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g., phenoxymethyl), aryl (e.g., halogen, C 1-4 alkyl or C 1-4 There may be (2) sulfonate esters such as alkylsulfonyl or aralkylsulfonyl (e.g., methanesulfonyl); (3) amino acid esters (e.g., L-valyl or L-isolucyl); (4) phosphonate esters; and (5) carboxylic acid esters obtained by esterification of a hydroxyl group, selected from mono-, di-, or triphosphate esters.

[0123] Polymorphic forms of the compounds of the present invention and the salts, solvates, esters, and prodrugs of the compounds of the present invention are also intended to be included in the present invention.

[0124] A suitable dosage of the compound of the present invention for administration to a patient may be determined by a person skilled in the art, for example, by an attending physician, pharmacist, or other person skilled in the art, and may vary depending on factors such as the patient's weight, health, age, frequency of administration, method of administration, presence of any other active ingredient, and symptoms for which the compound is administered.

[0125] Examples of excipients, diluents, and carriers include buffers, as well as fillers and extenders, such as starch, cellulose, sugar, mannitol, and silicic acid derivatives. Binders may also be included. Reinforcing agents may also be included.

[0126] Optionally, the compound of Formula I may be administered in combination with one or more additional therapeutic substances. When used in combination with one or more additional therapeutic substances, the compound of the present invention may be administered together or sequentially.

[0127] The composition can be administered via various routes, such as oral, parenteral (subcutaneous, intravenous, intramuscular, and peritoneal, etc.), rectal, dermal, transdermal, intrathoracic, intrapulmonary, mucosal, intraocular, and nasal routes.

[0128] Suitable forms of administration will be recognized by those skilled in the art and include, in particular, tablets, capsules, solutions, suspensions, powders, aerosols, ampoules, pre-filled syringes, small infusion containers or multi-dose containers, creams, milks, gels, dispersants, microemulsions, lotions, impregnated pads, ointments, eye drops, nose drops, lozenges, etc.

[0129] The generation of ROS can be controlled in non-therapeutic applications using compounds of Formula I and their conjugates. Interestingly, compounds of Formula I have been proven to penetrate other cell types, such as plant cells, leading to various other uses including targeted herbicides, seed reinforcement, and proliferation enhancement applications.

[0130] Accordingly, the present invention relates to a formulation comprising, in some aspects, a compound of Formula I or a conjugate thereof, optionally together with one or more formulation components. Such formulation components include, but are not limited to, preservatives, thickening agents, antifoaming agents, etc. Such formulation components may optionally contain additional active ingredients, e.g., herbicides, etc.

[0131] The present invention relates, in aspects, to a formulation comprising the said compound and conjugate, and to the use of said compound, conjugate and formulation in controlling the generation of ROS in plants, fungi and bacteria.

[0132] The present invention relates, in aspects, to a compound of Formula I in the form of a glass or salt and stereoisomers thereof:

[0133]

[0134] In the above formula,

[0135] R 1 is a C1-10 alkyl group that is H or optionally substituted with one or more N atoms, and R 2 -(CH2) is a C1-10 alkyl group optionally substituted with one or more N atoms n R 3 , -(CH2) n NHR 3 and -(CH2)2(COCH2) n R 3 Selected from, where n is an integer between 1 and 10, and R 3 is -NH2, -OH, -SO2PhCH3 or -COOH; or

[0136] R 1 and R 2 forms part of a heterocyclic group Y having 3-12 ring atoms, provided that R 1 and R 2 If ga forms part of a heterocyclic group Y having 3-12 ring atoms, N of the heterocyclic group is in the para position relative to the acetylene group of the compound of Formula I;

[0137] Ar1 and Ar2 are each independently aromatic groups; and

[0138] X is an electron-deficient group.

[0139] The present invention relates, in aspects, to a compound of Formula I in the form of a glass or salt and stereoisomers thereof:

[0140]

[0141] In the above formula,

[0142] R 1 is a C1-10 alkyl group that is H or optionally substituted with one or more N atoms, and R 2 -(CH2) is a C1-10 alkyl group optionally substituted with one or more N atoms n R 3 and -(CH2)2(COCH2) n R 3 Selected from, where n is an integer between 1 and 10, and R 3 is -NH2, -OH, or -COOH; or

[0143] R 1 and R 2 It forms part of the heterocyclic group Y having 3-12 ring atoms;

[0144] Ar1 and Ar2 are each independently aromatic groups; and

[0145] X is an electron-deficient group.

[0146] Examples :

[0147] The present invention will now be described by simple examples with reference to the attached drawings. Brief explanation of the drawing

[0148] Figure 1 illustrates the synthesis of a coupling partner and reference compound 77. Figure 2 illustrates the synthesis of a compound of the exemplary formula I. Figure 3 illustrates the absorption and emission spectra of the compound of the present invention and a reference compound. FIG. 4 illustrates the synthesis of (a) a THP-protected analog of borinostat, compound 37; (b) a form in which a THP-protected analog of borinostat is conjugated to compound 6, compound 38; and (c) a form in which an unprotected borinostat analog is conjugated to compound 6, compound 39. Figure 5 illustrates cell viability using CellTitreGlow analysis for primary, HPV-negative oral squamous cell carcinoma cells (a) cell line SJG-26; and (b) cell line SJG-41. Figure 6 illustrates the results of MTT viability analysis in (a) non-investigation and (b) investigation-analysis. Figure 7 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 7 and various organelle markers. Figure 8 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 13 and various organelle markers. Figure 9 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 14 and various organelle markers. Figure 10 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 12 and various organelle markers. Figure 11 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 15 and various organelle markers. Figure 12 shows a tiled image of co-stained HaCaT keratinocytes treated with compound 6 and various organelle markers. Figure 13 is a tiled arrangement of fluorescence images of the intracellular localization of compounds 7 (Column A), 14 (Column B), 12 (Column C), and 15 (Column D) in black-grass cells. Figure 14 illustrates the cell viability of black-grass cells treated with compounds 7, 15, 12, and 14 upon UV treatment. FIG. 15(i) shows the optical density over time of the cell suspension of compound 12 Microbacterium smegmatis treated with (1-100 μM) M. smegmatis This shows the overnight growth curve of ). For half of the sample, approximately 15 mW / cm² for 5 minutes as shown in Fig. 15(ii). 2 Light with a wavelength of 405 nm was irradiated. Fig. 16 shows compounds co-stained with propidium iodide (visualization of non-viable cells) and Syto 9 (visualization of both viable and non-viable cells). 6 Staphylococcus epidermidis treated with (1 μM) S. epidermidis This shows the cases of ) irradiated with light and ) not irradiated. Photographs were obtained using a wide-field microscope in the blue (Compound 6), green (Syto 9), and red (Propidium Iodide) channels shown in columns 1-3, respectively. Fig. 17 shows the optical density over time of the cell suspension of compound 6 Staphylococcus epidermidis treated with (1-100 μM) S. epidermidisThis shows the overnight growth curve of ). Half of the sample was at approximately 15 mW / cm² for 5 minutes. 2 Light (R) with a wavelength of 405 nm was irradiated. Fig. 18 shows compounds co-stained with propidium iodide (visualization of non-viable cells) and Syto 9 (visualization of both viable and non-viable cells). 12 Bacillus subtilis treated with (1 μM) B. subtilis Figure 18(a) and Figure 18(b) illustrate the case of cells not irradiated with light. Photographs were obtained using a wide-field microscope in the blue (Compound 6), green (Syto 9), and red (Propidium Iodide) channels (shown in columns 1-3, respectively). Fig. 19 shows the compound under light irradiation (R) and light non-irradiation (NR). 12 Bacillus subtilis treated with (1-100 μM) B. subtilis This plots the overnight growth curve of ). Approximately 15 mW / cm² for 5 minutes on the sample. 2 Light (R) with a wavelength of 405 nm was irradiated. FIG. 20 shows the optical density of the cell suspension over time, under light irradiation and light non-irradiation. 6 (10, 5, 1 μM) treated Bacillus subtilis ( B. subtilis This plots the overnight growth curve of ). Approximately 15 mW / cm² for 5 minutes on half of the sample. 2 Light (R) with a wavelength of 405 nm was irradiated. Fig. 21 shows a compound captured by a confocal microscope at laser excitation of 405 nm. 12 (10 μM) treated Bacillus subtilis ( B. subtilisThis is a diagram of a cell. The emission spectrum at 500 / 50 nm was image-captured. Post-processing was performed using the 'Find edges' function in ImageJ to identify the location of the intracellular compound. Specific details for implementing the invention

[0149] Examples 1: Synthesis of the compound of exemplary formula I:

[0150] 1.1 Coupling Partner Synthesis

[0151] 1.1.1. tert -butyl (2 E )-3-(4- Ethinylphenyl ) Prof -2- Enoate , 3 synthesis

[0152] tert -butyl (2 E The synthesis of )-3-(4-ethynylphenyl)prop-2-enoate (3) is exemplified in FIG. 1(i). Triethylamine (Et3N) (250 mL) was degassed by injecting Ar for 1 hour. 4-bromobenzaldehyde (18.5 g, 100.0 mmol), Pd(PPh3)2Cl2 (1.4 g, 2.00 mmol), CuI (0.38 g, 2.00 mmol), and trimethylsilylacetylene (15.2 mL, 110.0 mmol) were added under Ar, and the resulting suspension was stirred at room temperature (RT) for 16 hours (h). The suspension was diluted with heptane, passed through a short Celite / SiO2 plug, and the extract was evaporated to obtain a dark solid crude product (24 g). This is purified by Kugelrohr distillation (130-150°C, 9.0 Torr) to obtain the compound 1 It was obtained as an off-white solid (21.5 g, >100%), which was passed to the next step without further purification. Tert- Butyl diethylphosphonoacetate (14.4 mL, 61.5 mmol) and LiCl (2.54 g, 60.0 mmol) were added to anhydrous tetrahydrofuran (THF) (100 mL), the resulting solution was stirred for 15 minutes, and the compound therein 1 (10.1 g, 50.0 mmol) was added. 1,8-diazabicyclo[5.4.0]undek-7-ene (DBU) (8.2 mL, 55.0 mmol) was slowly added to this solution, and the formed slurry was stirred at RT for 16 hours. This was poured over crushed ice and extracted with ethyl acetate (EtOAc). The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain a white solid crude product (18 g). This was purified by recrystallization from heptane to obtain the compound 2 was obtained as a colorless crystalline solid (10.99 g, 73%): 1 H NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 1.53 (s, 9H), 6.36 (d, J = 16.0 Hz, 1H), 7.40 - 7.49 (m, 4H), 7.54 (d, J = 16.0 Hz, 1H). Compound 2 (10.95 g, 36.4 mmol) and K2CO3 (7.55 g, 54.6 mmol) were added to methanol (MeOH) / dichloromethane (DCM) (200 mL, 1:3), and the resulting solution was stirred at RT for 3 hours. The solution was diluted with DCM, the organic phase was rinsed with a saturated NH4Cl solution and H2O, and then dried (MgSO4) and evaporated to obtain a crude product solid (8 g). This was purified by recrystallization from heptane to obtain the compound 3 was obtained as a colorless crystalline solid (5.96 g, 72%): 1 H NMR (600 MHz, CDCl3) δ 1.53 (s, 9H), 3.17 (s, 1H), 6.36 (d, J= 16.0 Hz, 1H), 7.43 - 7.49 (m, 4H), 7.54 (d, J = 16.0 Hz, 1H); 13 C NMR (151 MHz, cdcl3) δ 28.1, 79.0, 80.6, 83.2, 121.2, 123.5, 127.7, 132.5, 135.0, 142.4, 166.0; IR (ATR) v max / cm -1 3281m, 3064w, 3000w, 2980w, 2936w, 1691s, 1641m, 1370m, 1296s, 1153s, 1002m, 980m, 832s; MS(ASAP): m / z = 228.1 [M+H] + ; C 15 H 16 O2[M+H] + HRMS (ASAP) 계산치: 228.1150, 실측치 228.1161.

[0153] 1.1.2 1-(4- iodophenyl ) Piperazine, synthesis of 4

[0154] The synthesis of 1-(4-iodophenyl)piperazine (4) is illustrated in FIG. 1(ii). A solution of 1-phenylpiperazine (20.5 mL, 134.0 mmol) in acetic acid (AcOH) / H2O (3:1, 84 mL) was mechanically stirred at 55°C, to which a solution of ICl (24.0 g, 148.0 mmol) in AcOH / H2O (3:1, 84 mL) was added dropwise. The resulting slurry was stirred for an additional hour, then cooled to RT and stirred again for an hour. The slurry was poured onto crushed ice, and a 20% aqueous NaOH solution was added to the pH to 13. Then, the solution was extracted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a dark solid crude product. This was purified by SiO2 chromatography (9:1, DCM / MeOH, 1% Et3N) to obtain a pale yellow solid, and this was recrystallized from MeOH / H2O (1:1) to obtain Compound 4 as a beige solid (18.5 g, 48%): 1 H NMR (600 MHz, CDCl3) δ 2.97 - 3.03 (m, 4H), 3.07 - 3.14 (m, 4H), 6.65 - 6.69 (m, 2H), 7.48 - 7.52 (m, 2H); 13 C NMR (151 MHz, CDCl3) δ 45.9, 49.9, 81.4, 118.0, 137.7, 151.3; IR (ATR) v max / cm -1 3032w, 2955w, 2829m, 1582m, 1489m, 1243s, 914m, 803s; MS(ASAP): m / z = 289.0 [M+H] + ; C 10 H 13 N2I [M] + HRMS (ASAP) Calculated: 288.0124, Measured: 288.0114.

[0155] 1.1.3 2- Chloro- N -(4- iodophenyl )- N - Methylacetamide , synthesis of 8

[0156] 2-chloro- N -(4-iodophenyl)- N The synthesis of -methylacetamide (8) is exemplified in FIG. 1 (iii). 4-iodo- N methylaniline (13.9 g, 59.7 mmol) was dissolved in DCM (100 mL), and a mixture prepared by adding chloroacetyl chloride (5.2 mL, 65.7 mmol) and Et3N (9.2 mL, 65.7 mmol) was stirred at room temperature (RT) for 16 hours. Subsequently, the solution was diluted with DCM, rinsed with a saturated NH4Cl solution and H2O, and then dried (MgSO4) and evaporated to obtain the crude product solid. This was purified by SiO2 chromatography (8:2, heptane / EtOAc) to obtain the compound 8 was obtained as an off-white solid (8.26 g, 45%): 1 H NMR (600 MHz, CDCl3) δ 3.28 (s, 3H), 3.83 (s, 2H), 6.95 - 7.06 (m, 2H), 7.78 (d, J = 8.1 Hz, 2H); 13 C NMR (151 MHz, CDCl3) δ 37.9, 41.2, 93.9, 129.0, 139.3, 142.4, 166.1; IR (ATR) v max / cm -1 2996w, 2947w, 1664s, 1480m, 1371m, 1260m, 1009m, 824m, 552s; MS (ASAP) m / z = 310.0 [M+H] + ; C9H 10 ONICl [M+H] +HRMS (ASAP) Calculated value: 309.9496, Measured value 309.9494.

[0157] 1.1.4 2-amino- N -(4- iodophenyl )- N - methylacetamide , synthesis of 10

[0158] 2-amino- N -(4-iodophenyl)- N The synthesis of -methylacetamide (10) is exemplified in FIG. 1(iv). Compound 8 (8.23 g, 26.6 mmol) and potassium phthalimide (7.39 g, 39.9 mmol) were dissolved in dimethylformamide (DMF) (40 mL), and the prepared mixture was heated to 120°C and stirred for 5 hours. The solution was cooled and diluted with H2O. The formed precipitate was isolated by filtration, washed with H2O, and then recrystallized from ethanol (EtOH) to obtain the compound 9 was obtained as a white solid (9.26 g, 83%). Compound 9 (9.2 g, 11.51 mmol) was dissolved in EtOH (50 mL), and the prepared mixture was heated under reflux. Hydrazine hydrate (64%, 1.22 mL, 24.09 mmol) was added to the mixture, and the mixture was stirred under reflux for 3 hours. Afterward, the suspension was cooled, and the formed precipitate was filtered. The filtrate was evaporated to obtain a crude oily solid (7 g), which was purified by SiO2 chromatography (9:1, DCM / MeOH + 1% Et3N) to obtain the compound 10 was obtained as a crystalline white solid (5.97 g, 94%): 1 H NMR (600 MHz, CDCl3) δ 3.13 (s, 2H), 3.25 (s, 3H), 6.92 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H);13 C NMR (151 MHz, CDCl3) δ 37.3, 44.1, 93.3, 129.1, 139.1, 142.4, 172.6; IR (ATR) v max / cm -1 3365m, 3301w, 3055w, 2947w, 2885w, 1649s, 1570m, 1486m, 1423m, 1345m, 1109m, 1013m, 892s; MS(ES): m / z = 291.1 [M+H] + ; C9H 12 N2OI [M+H] + HRMS (ES) Calculated value: 290.9994, Measured value 291.0012.

[0159] 1.1.5 N-(2- aminoethyl )-4- Iodine -N- Methylaniline , 11's synthesis

[0160] N -(2-aminoethyl)-4-iodo- N The synthesis of -methylaniline, (11) is exemplified in FIG. 1(v). Compound 10 (5.72 g, 19.72 mmol) was dissolved in anhydrous toluene (50 mL) under N2, and BH3.Me2S (2.0 M, 10.35 mL, 20.70 mmol) was added to this solution. The resulting solution was refluxed and stirred for 16 hours. The solution was cooled, 10% Na2CO3 was added, and the solution was vigorously stirred for 10 minutes. Subsequently, the solution was diluted with EtOAc, rinsed with H2O and brine, dried (MgSO4), and evaporated to obtain a crude yellow oil product (4.4 g). This was purified by SiO2 chromatography (9:1, DCM:MeOH, 0.5% Et3N), and the compound 11 It was obtained as a yellow oil (3.46 g, 64%), and this was immediately transferred to the next step: 1H NMR (400 MHz, CDCl3) δ 2.90 (t, J = 6.6 Hz, 2H), 2.93 (s, 3H), 3.36 (t, J = 665 Hz, 2H), 6.47 - 6.57 (m, 2H), 7.41 - 7.49 (m, 2H).

[0161] 1.1. 6 (4 Z )-2- methyl -4-({4-[2-( trimethylsilyl ) Ethinyl ]phenyl} Methylidene )-4,5- die Synthesis of Hydro-1,3-Oxazole-5-one, 16

[0162] (4 Z )-2-methyl-4-({4-[2-(trimethylsilyl)ethynyl]phenyl}methylidene)-4,5-dihydro-1,3-oxazole-5-one ( 16 The synthesis of ) is exemplified in FIG. 1(vi). Compound 1 (5.0 g, 24.7 mmol), N Acetylglycine (3.46 g, 29.6 mmol) and sodium acetate (NaOAc) (2.43 g, 29.6 mmol) were dissolved in acetic anhydride (25 mL), and the resulting solution was stirred at 80°C for 16 hours. The solution was cooled, and ice water was added to obtain an orange precipitate. This was filtered, rinsed with H2O, and dried to obtain the compound 16 It was obtained as an orange / brown solid (6.92 g, 91%) and was passed directly to the next step without further purification: 1 H NMR (400 MHz, CDCl3) δ 0.27 (s, 9H), 2.42 (s, 3H), 7.09 (s, 1H), 7.47 - 7.53 (m, 2H), 7.98 - 8.04 (m, 2H).

[0163] 1.1.7 4 Z )-1-(2- methoxyethyl )-2- methyl -4-({4-[2-( trimethylsilyl ) Ethinyl ]phenyl} Methylidene Synthesis of )-4,5-dihydro-1H-imidazole-5-one, 17

[0164] (4 Z The synthesis of )-1-(2-methoxyethyl)-2-methyl-4-({4-[2-(trimethylsilyl)ethynyl]phenyl}methylidene)-4,5-dihydro-1H-imidazole-5-one (17) is exemplified in FIG. 1(vii). 16 (5.50 g, 19.4 mmol) and 2-methoxyethylamine (1.68 mL, 19.4 mmol) were dissolved in pyridine (40 mL), and the resulting solution was stirred at RT for 0.5 hours. N , O Bistrimethylsilylacetamide (9.49 mL, 38.8 mmol) was added, and the solution was stirred at 110°C for 16 hours. Subsequently, the solution was cooled and diluted with EtOAc; the organic phase was then rinsed with a saturated NH4Cl solution, H2O, and brine, followed by drying (MgSO4) and evaporation to obtain a dark oil crude product (7.7 g). This was purified by SiO2 chromatography (Et2O), and the compound 17 was obtained as a light brown solid (4.03 g, 61%): 1 H NMR (400 MHz, CDCl3) δ 0.26 (s, 9H), 2.42 (s, 3H), 3.30 (s, 3H), 3.53 (t, J = 5.1 Hz, 2H), 3.77 (t, J = 5.1 Hz, 2H), 7.02 (s, 1H), 7.43 - 7.51 (m, 2H), 8.02 - 8.11 (m, 2H); 13C NMR (101 MHz, CDCl3) δ -0.1, 16.0, 41.0, 59.0, 70.5, 96.8, 105.0, 124.5, 125.8, 131.8, 132.1, 134.3, 139.0, 163.9, 170.6; IR (ATR) v max / cm -1 2957w, 2896w, 2833w, 2154m, 1710s, 1645s, 1599m, 1562s, 1405s, 1357s, 1249s, 1126m, 862s, 841s; MS(ES): m / z = 341.2 [M+H] + ; C 19 H 24 N2O2Si [M+H] + HRMS (ES) Calculated value: 341.1685, Measured value 341.1681.

[0165] 1.1. 8 (4 Z )-4-[(4- Ethinylphenyl ) Methylidene ]-1-(2- methoxyethyl )-2- methyl -4,5- die Synthesis of Hydro-1H-Imidazole-5-one, 18

[0166] (4 Z The synthesis of )-4-[(4-ethynylphenyl)methylidene]-1-(2-methoxyethyl)-2-methyl-4,5-dihydro-1H-imidazole-5-one (18) is exemplified in FIG. 1(viii). Compound 17(3.6 g, 10.57 mmol) and K2CO3 (2.92 g, 21.14 mmol) were added to DCM / MeOH (9:1, 50 mL), and the formed suspension was high-speed stirred for 20 hours. This suspension was diluted with DCM and H2O, and the organic phase was rinsed with a saturated NH4Cl solution and H2O, followed by drying (MgSO4) and evaporation to obtain a brown oil crude product (3.2 g). This was purified by SiO2 chromatography (1:1, PE / EtOAc) to obtain the compound 18 was obtained as a yellow solid (1.99 g, 70%): 1 H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 3.20 (s, 1H), 3.31 (s, 3H), 3.53 (t, J = 5.1 Hz, 2H), 3.78 (t, J = 5.1 Hz, 2H), 7.03 (s, 1H), 7.49 - 7.54 (m, 2H), 8.07 - 8.12 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 16.0, 41.0, 59.0, 70.5, 79.2, 83.6, 123.4, 125.6, 131.8, 132.3, 134.7, 139.2, 164.1, 170.6; IR (ATR) v max / cm -1 3285m, 3241m, 2986w, 2933w, 2891w, 2831w, 2104w, 1704s, 1643s, 1600m, 1592s, 1404s, 1356s, 1125s, 838m; MS(ES): m / z = 269.1 [M+H] + ; C 16 H 17 N2O2[M+H] + HRMS (ES) Calculated value: 269.1290, Measured value 269.1290.

[0167] 1.1.9. (4 Z )-2-phenyl-4-({4-[2-( trimethylsilyl) Ethinyl ]phenyl} Methylidene )-4,5- all Synthesis of dihydro-1,3-oxazole-5-one, 20

[0168] (4 Z The synthesis of )-2-phenyl-4-({4-[2-(trimethylsilyl)ethynyl]phenyl}methylidene)-4,5-dihydro-1,3-oxazole-5-one (20) is exemplified in FIG. 1(ix). 1 (12.5 g, 61.7 mmol), benzoylaminoethanolic acid (hypuric acid) (13.3 g, 74.0 mmol), and NaOAc (6.07 g, 74.0 mmol) were dissolved in acetic anhydride (80 mL), and the resulting solution was heated at 100°C for 18 hours. The solution was cooled and diluted with water, at which point a yellow precipitate was formed. This was filtered and dried to obtain a crude yellow solid, which was purified by SiO2 chromatography (95:5, PE / EtOAc) to obtain the compound 20 was obtained as a bright yellow solid (23.25 g, >100%): 1 H NMR (400 MHz, CDCl3) δ 0.28 (s, 9H), 7.20 (s, 1H), 7.50 - 7.58 (m, 4H), 7.63 (ddt, J = 8.4, 6.7, 1.4 Hz, 1H), 8.11 - 8.17 (m, 2H), 8.16 - 8.21 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ -0.1, 97.9, 104.7, 125.5, 125.8, 128.4, 129.0, 130.5, 132.1, 132.3, 133.4, 133.5, 133.7, 163.8, 167.4; IR (ATR) v max / cm -1 3063w, 2959w, 2898w, 2155m, 1768s, 1654s, 1598m, 859s; MS(ES): m / z = 346.1 [M+H]+ ; C 21 H 20 NO2Si [M+H] + HRMS (ES) Calculated value: 346.1263, Measured value 346.1266.

[0169] 1.1.10 (4 Z )-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4-({4-[2-( trimethylsilyl Synthesis of )ethynyl]phenyl}methylidene)-4,5-dihydro-1H-imidazole-5-one, 21

[0170] (4 Z The synthesis of )-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4-({4-[2-(trimethylsilyl)ethynyl]phenyl}methylidene)-4,5-dihydro-1H-imidazole-5-one, (21) is exemplified in FIG. 1(x). 20 (10.36 g, 30.0 mmol) and 4-(2-aminoethyl)morpholine (3.93 mL, 30.0 mmol) were dissolved in pyridine (65 mL), and the resulting solution was stirred at RT for 0.5 hours. N , O Bistrimethylsilylacetamide (14.67 mL, 60.0 mmol) was added, and the solution was stirred at 110°C for 18 hours. Subsequently, the solution was cooled and diluted with DCM; the organic phase was rinsed with a saturated NH4Cl solution, H2O, and brine, followed by drying (MgSO4) and evaporation to obtain a dark solid crude product. This was purified by SiO2 chromatography (1:9, PE / EtOAc) to obtain the compound 21 It was obtained as a viscous red oil and then slowly crystallized (12.91 g, 94%), and passed directly to the next step without further purification: 1 H NMR (400 MHz, CDCl3) δ 0.26 (s, 9H), 2.24 - 2.31 (m, 4H), 2.45 (t, J = 6.3 Hz, 2H), 3.47 - 3.56 (m, 4H), 3.91 (t, J= 6.3 Hz, 2H), 7.18 (s, 1H), 7.46 - 7.51 (m, 2H), 7.51 - 7.58 (m, 3H), 7.79 - 7.87 (m, 2H), 8.13 - 8.19 (m, 2H).

[0171] 1.1. 11 (4 Z )-4-[(4- Ethinylphenyl ) Methylidene ]-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4,5-dihydro-1H-imidazole-5-one, 22 Synthesis of

[0172] (4 Z )-4-[(4-ethynylphenyl)methylidene]-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4,5-dihydro-1H-imidazole-5-one, 22 The synthesis of is exemplified in Fig. 1(xi). Compound 21 (12.91 g, 28.2 mmol) and K2CO3 (7.8 g, 56.42 mmol) were added to DCM / MeOH (4:1, 100 mL), and the formed suspension was high-speed stirred for 20 hours. This suspension was diluted with DCM and H2O, the organic phase was rinsed with a saturated NH4Cl solution and H2O, and then dried (MgSO4) and evaporated to obtain the crude product solid. This was purified by SiO2 chromatography (100% EtOAc) to obtain the compound 22 was obtained as a yellow solid (7.69 g, 71%): 1 H NMR (400 MHz, CDCl3) δ 2.24 - 2.30 (m, 4H), 2.44 (t, J = 6.3 Hz, 2H), 3.21 (s, 1H), 3.43 - 3.57 (m, 4H), 3.91 (t, J = 6.3 Hz, 2H), 7.18 (s, 1H), 7.49 - 7.59 (m, 5H), 7.78 - 7.85 (m, 2H), 8.14 - 8.21 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 39.0, 53.6, 56.6, 66.7, 79.5, 83.6, 123.6, 127.2, 128.4, 128.8, 129.9, 131.3, 132.2, 132.3, 134.7, 139.5, 163.4, 171.6; IR (ATR) v max / cm -1 3290w, 3238w, 2956w, 2854w, 2811w, 1705s, 1640s, 1597m, 1491s, 1446m, 1391s, 1351s, 1314m, 1115s, 868m; MS(ES): m / z = 386.2 [M+H] + ; C 24 H 24 N3O2[M+H] + HRMS (ES) Calculated: 386.1869, Measured: 386.1858.

[0173] 1.1.12 5- iodothiophene -2- Carbualdehyde , 24 Synthesis of

[0174] 5-iodothiophene-2-carbaldehyde, 24 The synthesis of is exemplified in FIG. 1(xii). In a solution of 2-thiophenecarboxaldehyde (9.34 mL, 100.0 mmol) in EtOH (50 mL) at 50 °C N - Iodosuccinimide (24.75 g, 110.0 mmol) and p - Toluenesulfonic acid monohydrate (1.90 g, 10.0 mmol) was added, and the resulting solution was stirred at 50°C for 20 minutes. 1 M HCl (80 mL) was added, and the mixture was extracted with EtOAc; the resulting solution was rinsed with a saturated Na2S2O3 solution, H2O, and brine, then dried (MgSO4) and evaporated to obtain the compound 24 was obtained as a yellow oil and slowly crystallized (25.34 g, >100%): 1H NMR (300 MHz, CDCl3) δ 7.39 (s, 2H), 9.77 (s, 1H).

[0175] 1.1.13 tert -butyl (2 E )-3-(5- iodothiophene -2-days) Prof -2- Enoate , 25 Synthesis of

[0176] tert -butyl (2 E )-3-(5-iodothiophene-2-yl)prop-2-enoate, 25 The synthesis of is exemplified in Fig. 1(xiii). Tert - Butyl diethylphosphonoacetate (8.5 mL, 36.0 mmol) and LiCl (1.49 g, 35.2 mmol) were added to anhydrous THF (100 mL), the resulting solution was stirred for 15 minutes, and the compound therein 24 (6.97 g, 29.3 mmol) was added. DBU (4.82 mL, 32.2 mmol) was slowly added to this solution, and the resulting slurry was stirred at RT for 16 hours. This was poured onto crushed ice and extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain a brown oil crude product (12 g). This was purified by SiO2 chromatography (9:1, heptane / EtOAc) to obtain the compound 25 was obtained as an orange oil (10.99 g, 73%): 1 H NMR (700 MHz, CDCl3) δ 1.51 (s, 9H), 6.07 (d, J = 15.7 Hz, 1H), 6.85 (d, J = 3.8 Hz, 1H), 7.18 (d, J = 3.8 Hz, 1H), 7.58 (dd, J = 15.7, 0.6 Hz, 1H);13 C NMR (176 MHz, CDCl3) δ 28.2, 80.7, 119.8, 131.6, 134.7, 137.9, 145.7, 165.8; IR (ATR) v max / cm -1 2976w, 2931w, 1698s, 1622s, 1417m, 1367m, 1256m, 1140s, 964m, 793m; MS(ES): m / z = 359.2 [M+H] + .

[0177] 1.1.14 tert -butyl (2 E )-3-(5- Ethinylthiophene -2-days) Prof -2- Enoate, 26 Synthesis of

[0178] tert -butyl (2 E )-3-(5-ethynylthiophene-2-yl)prop-2-enoate, 26 The synthesis of is exemplified in Fig. 1(xiv). Et3N (150 mL) was degassed by injecting Ar for 1 hour. Compound 25 (8.4 g, 24.98 mmol), Pd(PPh3)2Cl2 (0.175 g, 0.25 mmol), CuI (48 mg, 0.25 mmol), and trimethylsilylacetylene (4.15 mL, 30.0 mmol) were added under Ar, and the formed suspension was stirred at RT for 16 hours. The suspension was diluted with methyl tert-butyl ether (MTBE), passed through a short Celite / SiO2 plug, and the extract was evaporated to obtain a brown oil crude product (8.8 g). This was purified by SiO2 chromatography (95:5, heptane / EtOAc), tert -butyl (2 E)-3-{5-[2-(trimethylsilyl)ethinyl]thiophene-2-yl}prop-2-enoate was obtained as an orange oil (8.51 g, >100%), which was transferred to the next step without further purification: 1 H NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 1.51 (s, 9H), 6.12 (d, J = 15.7 Hz, 1H), 7.05 (d, J = 3.8 Hz, 1H), 7.12 (d, J = 3.8 Hz, 1H), 7.57 (dd, J = 15.7, 0.6 Hz, 1H). In MeOH / DCM solution (1:10, 110 mL) tert -butyl (2 E )-3-{5-[2-(trimethylsilyl)ethynyl]thiophene-2-yl}prop-2-enoate (8.51 g, 27.76 mmol) and K2CO3 (7.67 g, 55.55 mmol) were added, and the prepared mixture was stirred at RT under N2 for 16 hours. Subsequently, the solution was diluted with DCM, rinsed with a saturated NH4Cl solution, H2O, and brine, then dried (MgSO4) and evaporated to obtain the crude product solid (3.6 g). This was purified by SiO2 chromatography (97:3, heptane / EtOAc) to obtain the compound 26 It was obtained as a bright yellow oil (3.50 g, 54%), and this was transferred to the next step: 1 H NMR (400 MHz, CDCl3) δ 1.53 (s, 9H), 3.45 (s, 1H), 6.16 (d, J = 15.7 Hz, 1H), 7.08 (d, J = 3.8 Hz, 1H), 7.18 (d, J = 3.8 Hz, 1H), 7.59 (dd, J = 15.8, 0.6 Hz, 1H).

[0179] 1.1.15 4-( Azetidine -1-day) Benzaldehyde , 28 Synthesis of

[0180] 4-(azetidine-1-yl)benzaldehyde ( 28 The synthesis of ) is exemplified in Fig. 1(xv). Azetidin.HCl (1.81 g, 19.4 mmol) and K2CO3 (5.89 g, 42.6 mmol) were added to a solution of 4-fluorobenzaldehyde (1.52 mL, 14.2 mmol) in dimethyl sulfoxide (DMSO) (50 mL), and the prepared solution was stirred at 110°C for 40 hours. The solution was cooled, diluted with H2O, and extracted with EtOAc (x3). The organic phase was rinsed with H2O and brine, then dried with MgSO4 and evaporated to obtain a yellow solid crude product. This was purified by SiO2 chromatography (7:3, PE / EtOAc) to obtain the compound 28 was obtained as a yellow crystalline solid (2.04 g, 89%): 1 H NMR (400 MHz, CDCl3) δ 2.44 (pent, J = 7.4 Hz, 2H), 3.98 - 4.06 (t, J = 7.4 Hz, 4H), 6.32 - 6.43 (m, 2H), 7.65 - 7.75 (m, 2H), 9.71 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 16.4, 51.4, 109.7, 125.7, 131.9, 155.0, 190.3; IR (ATR) v max / cm -1 3040w, 3002w, 2921m, 2856m, 2730w, 1672s, 1586s, 1551s, 1523s, 1476m, 1435m, 1382s, 1301s, 1221s, 1154s, 818s, 683s; MS(ES): m / z = 162.1 [M+H] + ; C 10 H 12 NO [M+H]+ HRMS (ES) Calculated value: 162.0919, Measured value 162.0922.

[0181] 1.1.16 1-(4- Ethinylphenyl ) Azetidine , 29 Synthesis of

[0182] 1-(4-ethynylphenyl)azetidine ( 29 The synthesis of ) is illustrated in FIG. 1(xv). Compound in anhydrous MeOH (30 mL) 28 K2CO3 (1.71 g, 12.4 mmol) and dimethyl-1-diazo-2-oxopropylphosphonate (1.12 mL, 7.44 mmol) were added to a solution (1.0 g, 6.2 mmol) under Ar, and the formed suspension was stirred at RT for 72 hours. The solution was diluted with EtOAc, rinsed with 5% NaHCO3, H2O, and brine, then dried (MgSO4) and evaporated to obtain a brown oil crude product (1.16 g). This was purified by SiO2 chromatography (9:1, PE:EtOAc) to obtain the compound 29 was obtained as a white solid (0.199 g, 20%): 1 H NMR (300 MHz, CDCl3) δ 2.37 (pent, J = 7.4 Hz, 2H), 2.97 (s, 1H), 3.90 (t, J = 7.4 Hz, 4H), 6.31 - 6.36 (m, 2H), 7.31 - 7.37 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 16.7, 52.0, 74.7, 84.8, 109.6, 110.6, 133.0, 151.8; IR (ATR) v max / cm -1 3287w, 2963w, 2918w, 2855w, 2099w, 1609s, 1514s, 1355m, 1171m, 1123m, 824m; MS(ES): m / z= 158.1 [M+H] + ; C 11 H 12 N [M+H] + HRMS (ES) Calculated value: 158.0970, Measured value 158.0971.

[0183] 1.1. 17 (4 Z )-4-[(4- Bromophenyl ) Methylidene ]-2-phenyl-4,5- Dihydro Synthesis of -1,3-oxazole-5-one, 31

[0184] (4 Z )-4-[(4-bromophenyl)methylidene]-2-phenyl-4,5-dihydro-1,3-oxazole-5-one ( 31 The synthesis of ) is illustrated in Fig. 1(xvi). 4-bromobenzaldehyde (28.46 g, 153.8 mmol), hyfuric acid (35.83 g, 200.0 mmol), and NaOAc (16.4 g, 200.0 mmol) were dissolved in acetic anhydride (150 mL), and the resulting solution was heated at 100°C for 18 hours. The solution was cooled and diluted with water, at which point a yellow precipitate was formed. This was dissolved in DCM, the organic phase was rinsed with water, and then dried (MgSO4) and evaporated to obtain a yellow solid crude product. This was suspended in DCM / EtOAc (1:1), and the resulting suspension was stirred for 0.5 hours. The precipitate was collected by filtration, rinsed with cold EtOAc, and then dried to obtain the compound 31 was obtained as a bright yellow solid (40.5 g, 80%): 1 H NMR (400 MHz, CDCl3) δ 7.17 (s, 1H), 7.51 - 7.58 (m, 2H), 7.59 - 7.67 (m, 3H), 8.05 - 8.11 (m, 2H), 8.15 - 8.22 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 167.3, 163.9, 133.8, 133.6, 133.6, 132.4, 132.2, 130.1, 129.0, 128.5, 125.9, 125.4; IR (ATR) v max / cm -1 3088w, 3061w, 3044w, 1651s, 1580s, 1553m, 1483m, 1323s, 1298s, 1159m, 980m, 820s; MS(ES): m / z = 328.0, 330.0 [M+H] + ; C 16 H 11 NO2Br [M+H] + HRMS (ES) Calculated value: 327.9973, Measured value 327.9974.

[0185] 1.1. 18 tert -butyl N -{2-[(4 Z )-4-[(4- Bromophenyl ) Methylidene ]-5-oxo-2-phenyl-4,5-dihydro-1H-imidazole-1-yl]ethyl}carbamate, 32 of synthesis

[0186] tert -butyl N -{2-[(4 Z )-4-[(4-bromophenyl)methylidene]-5-oxo-2-phenyl-4,5-dihydro-1H-imidazole-1-yl]ethyl}carbamate ( 32 The synthesis of ) is illustrated in Fig. 1(xvi). Compound 31 (15.0 g, 45.7 mmol) and tert -butyl N (2-aminoethyl)carbamate (7.24 mL, 45.7 mmol) was dissolved in pyridine (80 mL), and the resulting solution was stirred at RT for 0.5 hours. N , OBistrimethylsilylacetamide (22.35 mL, 91.4 mmol) was added, and the solution was stirred at 110°C for 18 hours. Subsequently, the solution was cooled and diluted with EtOAc; the organic phase was then rinsed with 5% HCl, H2O, and brine, followed by drying (MgSO4) and evaporation to obtain the crude red oil product. This was purified by SiO2 chromatography (7:3, PE / EtOAc) to obtain the compound 32 It was obtained as an orange / red solid (18.69 g, 87%) and was passed directly to the next step without further purification: 1 H NMR (400 MHz, CDCl3) δ 1.37 (s, 9H), 3.40 (q, J = 6.0 Hz, 2H), 3.90 (t, J = 6.0 Hz, 2H), 4.81 - 4.88 (m, 1H), 7.16 (s, 1H), 7.50 - 7.62 (m, 5H), 7.76 - 7.88 (m, 2H), 8.01 - 8.14 (m, 2H).

[0187] 1.1.19 (4 Z )-1-(2- aminoethyl )-4-[(4- Bromophenyl ) Methylidene ]-2-phenyl-4,5- Dihydro -1H-imidazole-5-one, 33 Synthesis of

[0188] (4 Z )-1-(2-aminoethyl)-4-[(4-bromophenyl)methylidene]-2-phenyl-4,5-dihydro-1H-imidazole-5-one ( 33 The synthesis of ) is illustrated in Fig. 1(xvi). Compound 32 (7.0 g, 14.88 mmol) was dissolved in trifluoroacetic acid (TFA) / DCM (1:3, 80 mL), and the resulting solution was stirred at RT for 16 hours. The solution was evaporated to obtain crude product oil (16 g). This was SiO2 Compound 33 was obtained as a red solid mixed with impurities (8.89 g, >100%) by purification by chromatography (95:5, DCM / MeOH, 1% Et3N). This was suspended in EtOAc and stirred for 0.5 hours, then the formed precipitate was filtered and rinsed with cold EtOAc, and the compound 33 was obtained as a bright yellow solid (2.39 g, 43%): 1 1H NMR (300 MHz, DMSO- d 6) δ 2.98 (t, J = 6.7 Hz, 2H), 3.95 (t, J = 6.7 Hz, 2H), 7.20 (s, 1H), 7.58 - 7.71 (m, 5H), 7.60 - 7.80 (br, 2H), 7.83 - 7.88 (m, 2H), 8.20 - 8.29 (m, 2H).

[0189] 1.1.20 5-[2-( trimethylsilyl ) Ethinyl ]Pyridine-2- Carbualdehyde , 40 Synthesis of

[0190] 5-[2-(trimethylsilyl)ethinyl]pyridine-2-carbaldehyde ( 40 The synthesis of ) is illustrated in Fig. 1(xvii). Et3N (400 mL) was degassed by injecting Ar for 1 hour. 5-bromopyridine-2-carboxaldehyde (20.0 g, 108 mmol), trimethylsilylacetylene (16.5 mL, 119 mmol), Pd(PPh3)2Cl2 (700 mg, 1.00 mmol), and CuI (190 mg, 1.00 mmol) were added under Ar, and the resulting suspension was stirred at RT for 18 hours. The mixture was diluted in Et2O and then passed through Celite / SiO2 to form the compound 40 was obtained as an orange solid (23.0 g, >100%): 1H NMR (400 MHz, CDCl3) δ 0.28 (s, 9H), 7.90 (d, J = 1.2 Hz, 2H), 8.81 (t, J = 1.2 Hz, 1H), 10.06 (s, 1H); 13 C NMR (176 MHz, CDCl3) δ -0.3, 100.6, 102.7, 120.8, 124.6, 139.8, 151.0, 152.8, 192.5; IR (ATR) v max / cm -1 3039w, 2961w, 2835w, 2158w, 1710s, 1575m, 1468w, 1425w, 1233s, 1217s, 839s; M.S. (ES) m / z = 204.0 [M+H] + ; C 11 H 13 NOSi [M+H] + HRMS (ES) Calculated value: 204.0839, Measured value 204.0839.

[0191] 1.1. 21-methyl (2 E )-3-{5-[2-( trimethylsilyl ) Ethinyl ]pyridine-2-il} Prof -2-Enoate, 41 Synthesis of

[0192] Methyl (2 E )-3-{5-[2-(trimethylsilyl)ethinyl]pyridine-2-yl}prop-2-enoate ( 41 The synthesis of ) is illustrated in Fig. 1(xviii). Trimethylphosphonoacetate (21.0 mL, 129.8 mmol) and LiCl (5.5 g, 129.8 mmol) were added to anhydrous THF (300 mL) at 0°C, the formed solution was stirred for 15 minutes, and the compound therein 40(22.0 g, 108.2 mmol) was added. DBU (19.4 mL, 129.8 mmol) was slowly added to this solution, and the resulting slurry was stirred at RT for 16 hours. This was poured onto crushed ice and extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain a brown solid as the crude product (31.5 g). This was purified by SiO2 chromatography to obtain the compound 41 was obtained as a white solid (16.2 g, 58%): 1 H NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 3.79 (s, 3H), 6.90 (d, J = 15.7 Hz, 1H), 7.32 (dd, J = 8.1, 0.9 Hz, 1H), 7.62 (d, J = 15.7 Hz, 1H), 7.72 (dd, J = 8.0, 2.1 Hz, 1H), 8.66 (d, J = 2.1 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ -0.3, 51.8, 100.1, 101.3, 120.7, 122.6, 123.2, 139.4, 142.6, 151.6, 152.8, 166.9; IR (ATR) v max / cm -1 3020w, 2955w, 2901w, 2160w, 1717s, 1644m, 1582m, 1547m, 1473m, 1318s, 1204s, 842s; M.S. (ES) m / z = 260.1 [M+H] + ; C 14 H 17 NO2Si [M+H] + HRMS (ES) Calculated value: 260.1101, Measured value 260.1101.

[0193] 1.1.22 methyl (2 E )-3-(5- Ethinylpyridine -2-days) Prof -2- Enoate , 42 Synthesis of

[0194] Methyl (2 E )-3-(5-ethynylpyridine-2-yl)prop-2-enoate ( 42 The synthesis of ) is illustrated in FIG. 1(xix). Compound 41 (5.0 g, 19.2 mmol) was dissolved in a mixture of DCM (80 mL) and MeOH (10 mL), and K2CO3 (5.3 g, 38.4 mmol) was added. The formed suspension was stirred at RT for 16 hours and then diluted with DCM and H2O. The organic phase was rinsed with a saturated NH4Cl solution and H2O and then dried (MgSO4) to obtain a white solid crude product (3.4 g). This was purified by recrystallization from petroleum ether to obtain the compound 42 was obtained as a white solid (3.06 g, 85%): 1 H NMR (400 MHz, CDCl3) δ 3.31 (s, 1H), 3.81 (s, 3H), 6.93 (d, J = 15.7 Hz, 1H), 7.36 (dd, J = 8.1, 0.9 Hz, 1H), 7.65 (d, J = 15.7 Hz, 1H), 7.77 (dd, J = 8.0, 2.1 Hz, 1H), 8.71 (d, J = 1.7 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 51.9, 80.3, 82.1, 119.7, 123.0, 123.3, 139.7, 142.5, 152.1, 153.0, 166.9; IR (ATR) v max / cm -13245m, 3015w, 2970w, 2951w, 2104w, 1738m, 1609s, 1632w, 1443m, 1368m, 1293m, 1272s, 869m; M.S. (ES) m / z = 188.1 [M+H] + ; C 11 H 10 NO2[M+H] + HRMS (ES) Calculated value: 188.0706, Measured value 188.0706.

[0195] 1.1.23 (2 E )-3-(5- Ethinylpyridine -2-days) Prof -2- Enoyiksan , 44 Synthesis of

[0196] (2 E The synthesis of )-3-(5-ethynylpyridine-2-yl)prop-2-enoic acid (44) is illustrated in FIG. 1(xx). Compound 41 Dissolve (5.41 g, 20.9 mmol) in THF (40 mL), and 20% aq. w / v After adding NaOH (10 mL), the mixture was refluxed and stirred for 18 hours. The formed suspension was cooled, diluted with H2O and EtOAc, and then titrated to pH 1 using 20% ​​HCl. The organic phase was rinsed with H2O and Bryn, then dried (MgSO4) and evaporated to obtain the compound. 44 was obtained as an off-white solid (4.14 g, >100%): 1 H NMR (400 MHz, CDCl3) δ 3.33 (s, 1H), 6.93 (d, J = 15.1 Hz, 1H), 7.41 (d, J = 6.8 Hz, 1H), 7.73 (d, J = 15.1 Hz, 1H), 7.81 (dd, J = 6.8, 2.0 Hz, 1H), 8.75 (s, 1H).

[0197] 1.1.24 2- Methylpropyl (2 E )-3-(5- Ethinylpyridine -2-days) Prof -2- Enoate , 45 Synthesis of

[0198] 2-methylpropyl (2 E )-3-(5-ethynylpyridine-2-yl)prop-2-enoate ( 45 The synthesis of ) is illustrated in Fig. 1(xx). Compound 44 (4.14 g, 23.9 mmol) was dissolved in DMF (60 mL), at which point K2CO3 (6.6 g, 47.8 mmol) and 1-bromo-2-methylpropane (5.2 mL, 47.8 mmol) were added, and the resulting suspension was stirred at RT for 18 hours. This was diluted with DCM and H2O, the organic phase was rinsed with a saturated NH4Cl solution and H2O, and then dried (MgSO4) and evaporated to obtain a brown oil crude product (5.23 g). This was purified by SiO2 chromatography (9:1, PE / EtOAc) to obtain the compound 45 was obtained as a white solid (1.03 g, 19%): 1 H NMR (700 MHz, CDCl3δ 0.97 (d, J = 6.8 Hz, 6H), 1.96 - 2.05 (hept, J = 6.8 Hz, 1H), 3.30 (s, 1H), 4.00 (d, J = 6.6 Hz, 2H), 6.94 (d, J = 15.7 Hz, 1H), 7.38 (dd, J = 8.0, 0.8 Hz, 1H), 7.65 (d, J = 15.7 Hz, 1H), 7.78 (dd, J = 8.0, 2.1 Hz, 1H), 8.72 (d, J = 2.1 Hz, 1H);13 C NMR (176 MHz, CDCl3) δ 19.09, 27.78, 70.87, 80.29, 82.06, 119.61, 123.25, 123.50, 139.71, 142.20, 152.28, 152.97, 166.58; IR (ATR) v max / cm -1 3238m, 2966w, 2953w, 2876w, 2108w, 1695s, 1640s, 1550m, 1313s, 1292s, 1160s; M.S. (ES) m / z = 230.1 [M+H] + ; C 14 H 16 NO2[M+H] + HRMS (ES) Calculated value: 230.1176, Measured value 230.1176.

[0199] 1.1.25 8- Methoxy -8- Okso-oktano-iksan , 47 Synthesis of

[0200] 8-Methoxy-8-Oxo-octanoic acid ( 47 The synthesis of ) is illustrated in Fig. 1(xxi). Dimethyl suberate salt (112.5 g, 556 mmol) was dissolved in MeOH (400 mL) and the solution was cooled to 0°C. At this point, KOH (31.2 g, 556 mmol) was added, and the resulting solution was stirred at RT for 4 hours. Diethyl ether (400 mL) and H2O were added, and the organic layer was separated and obtained. The aqueous layer was acidified to pH 3 and extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain a waxy solid as a crude product. This was suspended in hexane, filtered, and vigorously stirred for 0.5 hours. The filtrate was evaporated to obtain the compound 47 was obtained as a clear oil (60.51 g, 58%): 1H NMR (400 MHz, CDCl3) δ 1.27 - 1.42 (m, 4H), 1.57 - 1.69 (m, 4H), 2.30 (t, J = 7.5 Hz, 2H), 2.34 (t, J = 7.5 Hz, 2H), 3.66 (s, 3H), 10.25 (s, 1H).

[0201] 1.1.26 methyl 7-[( Oksan -2- Iloxy ) Carbamoil ] heptanoate , 48 Synthesis of

[0202] Methyl 7-[(oxane-2-yloxy)carbamoyl]heptanoate ( 48 The synthesis of ) is illustrated in Fig. 1(xxi). Compound 47 (4.0 mL, 22.3 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (4.88 g, 27.8 mmol) were dissolved in DCM (70 mL), and the solution was cooled to 0°C. 4-methylmorpholine (3.06 mL, 27.8 mmol) was added dropwise over 5 minutes, and the resulting solution was stirred at 0°C for 72 hours, to O -(Tetrahydropyran-2-yl)hydroxylamine (2.48 g, 21.2 mmol) and 4-methylmorpholine (2.77 mL, 26.0 mmol) were added, and the solution was stirred for an additional 16 hours. The solution was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow oil as a crude product (9.5 g). This Purified by SiO2 chromatography (1:1, PE / EtOAc), the compound 48 was obtained as a clear oil (5.26 g, 86%): 1H NMR (400 MHz, CDCl3) δ 1.27-1.32 (m, 4H), 1.54 - 1.70 (m, 7H), 1.71 - 1.87 (m, 3H), 2.09 (br, 2H), 2.28 (t, J = 7.5 Hz, 2H), 3.57 - 3.63 (m, 1H), 3.64 (s, 3H), 3.86 - 3.98 (m, 1H), 4.92 (br, 1H), 8.59 (br, 1H); 13 C NMR (101 MHz, CDCl3) δ 24.6, 25.0, 28.6, 33.9, 51.4, 62.6, 77.3, 102.4, 170.4, 174.2; IR (ATR) v max / cm -1 3202br, 2940m, 2858w, 1736s, 1656s, 1455m, 1204m, 1064s. 1 H NMR (400 MHz, CDCl3) δ 1.27-1.32 (m, 4H), 1.54 - 1.70 (m, 7H), 1.71 - 1.87 (m, 3H), 2.09 (br, 2H), 2.28 (t, J = 7.5 Hz, 2H), 3.57 - 3.63 (m, 1H), 3.64 (s, 3H), 3.86 - 3.98 (m, 1H), 4.92 (br, 1H), 8.59 (br, 1H); 13 C NMR (101 MHz, CDCl3) δ 24.6, 25.0, 28.6, 33.9, 51.4, 62.6, 77.3, 102.4, 170.4, 174.2; IR (ATR) v max / cm -1 3202br, 2940m, 2858w, 1736s, 1656s, 1455m, 1204m, 1064s; MS(ES): m / z = 288.2 [M+H] + ; C 14 H 26 NO5[M+H] +: HRMS (ES) Calculated value 288.1805, Measured value 288.1805.

[0203] 1.1.27 7-[(Oksan-2- Iloxy ) Carbamoil ] heptanoic acid , 49 Synthesis of

[0204] 7-[(oxane-2-yloxy)carbamoyl]heptanoic acid ( 49 The synthesis of ) is illustrated in Fig. 1(xxi). Compound 48 (5.0 g, 17.4 mmol) was dissolved in MeOH (60 mL) and H2O (20 mL), to which NaOH (2.78 g, 69.6 mmol) was added, and the resulting solution was stirred at 50°C for 18 hours. The solution was evaporated, and the residue was suspended in H2O. 5% HCl was added to carefully titrate the pH to 3 / 4, and the solution was extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain the compound 49 was obtained as a clear oil (4.27 g, 90%): 1 H NMR (400 MHz, CDCl3) δ 1.28-1.40 (m, 4H), 1.52-1.69 (m, 7H), 1.74-1.84 (m, 3H), 2.11 (br, 2H), 2.32 (t, J = 7.4 Hz, 2H), 3.58-3.66 (m, 1H), 3.88-4.00 (m, 1H), 4.93 (br, 1H), 8.96 (br, 1H), 10.12 (br, 1H); IR (ATR) v max / cm -1 3200br, 2938, 2860w, 1707s, 1644s, 1455m, 1357m, 1204s, 1035s, 871s; MS(ES): m / z = 296.1 [M+H] + ; C 13 H 23NO5Na [M+H] + HRMS (ES) Calculated value: 296.1468, Measured value 296.1466.

[0205] 1.1.28 methyl (2 E )-3-(5-{2-[4-(4-{7-[(Oksan-2- Iloxy ) Carbamoil ] heptanoyl } piperazine-1-yl)phenyl]ethinyl}pyridine-2-yl)prop-2-enoate, 50 Synthesis of

[0206] Methyl (2 E )-3-(5-{2-[4-(4-{7-[(oxane-2-yloxy)carbamoyl]heptanoyl}piperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate ( 50 The synthesis of ) is illustrated in FIG. 1(xxii). Compound 49 (0.88 g, 3.23 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.71 g, 4.03 mmol) were dissolved in DCM (60 mL) at 0°C, and 4-methylmorpholine (0.44 mL, 4.03 mmol) was added dropwise over 5 minutes. The resulting mixture was stirred at 0°C for 2 hours, and the compound therein 43 (1.07 g, 3.08 mmol) and 4-methylmorpholine (0.41 mL, 3.63 mmol) were added, and the mixture was stirred at RT for 16 hours. The mixture was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (1.31 g). This was purified by SiO2 chromatography (98:2, DCM / MeOH) to obtain the compound 50 was obtained as a yellow solid (1.25 g, 67%): 1H NMR (700 MHz, CDCl3) δ 1.29 - 1.42 (m, 4H), 1.55 - 1.67 (m, 7H), 1.70 - 1.87 (m, 3H), 2.01 - 2.19 (m, 2H), 2.35 (t, J = 7.6 Hz, 2H), 3.22 (t, J = 5.3 Hz, 2H), 3.26 (t, J = 5.3 Hz, 2H), 3.57 - 3.64 (m, 3H), 3.76 (t, J = 5.3 Hz, 2H), 3.80 (s, 3H), 3.91 - 3.98 (m, 1H), 4.94 (s, 1H), 6.81 - 6.88 (m, 2H), 6.90 (d, J = 15.7 Hz, 1H), 7.36 (dd, J = 8.0, 0.8 Hz, 1H), 7.41 - 7.46 (m, 2H), 7.65 (d, J = 15.7 Hz, 1H), 7.75 (dd, J = 8.0, 2.2 Hz, 1H), 8.66 - 8.74 (m, 1H), 8.75 - 8.94 (m, 1H); 13 C NMR (176 MHz, CDCl3) δ 18.7, 25.0, 25.2, 28.1, 28.7, 28.9, 33.1, 33.2, 41.3, 45.4, 48.3, 48.6, 52.0, 62.6, 85.2, 95.2, 102.5, 113.0, 115.5, 121.6, 122.3, 123.7, 133.1, 138.7, 143.0, 151.0, 151.1, 152.4, 167.3, 171.8; IR (ATR) v max / cm -1 3217br, 3000w, 2945m, 2856w 2211w, 1738s, 1640s, 1605s, 1577m, 1516s, 1437s, 1366s, 1231s, 820s; MS(ES): m / z = 603.2 [M+H] + ; C34 H 42 N4O6[M+H] + HRMS (ES) Calculated value: 603.3177, Measured value 603.3178.

[0207] 1.1.29 2- Methylpropyl (2 E )-3-(5-{2-[4-(4-{7-[(Oksan-2- Iloxy ) Carbamoil ] heptanoyl }Piperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate, 54 Synthesis of

[0208] 2-methylpropyl (2 E )-3-(5-{2-[4-(4-{7-[(oxane-2-yloxy)carbamoyl]heptanoyl} piperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate ( 54 The synthesis of ) is illustrated in FIG. 1(xxiii). Compound 49 (0.54 g, 1.97 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.45 g, 2.58 mmol) were dissolved in DCM (50 mL) at 0°C, and 4-methylmorpholine (0.32 mL, 2.97 mmol) was added dropwise over 5 minutes. The resulting mixture was stirred at 0°C for 2 hours, and the compound therein 46 (0.56 g, 1.44 mmol) and 4-methylmorpholine (0.32 mL, 2.97 mmol) were added, and the mixture was stirred at RT for 16 hours. The mixture was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (1.7 g). This was purified by SiO2 chromatography (98:2, DCM / MeOH) to obtain the compound 54 was obtained as a yellow solid (0.55 g, 59%): 1 H NMR (700 MHz, CDCl3) δ 0.98 (d, J= 6.8 Hz, 6H), 1.35 - 1.40 (m, 4H), 1.50 - 1.61 (m, 3H), 1.63 - 1.67 (m, 4H), 1.74 - 1.86 (m, 3H), 2.01 (hept, J = 6.8 Hz, 1H), 2.07 - 2.20 (m, 2H), 2.37 (t, J = 7.5 Hz, 2H), 3.24 (t, J = 5.3 Hz, 2H), 3.27 (t, J = 5.3 Hz, 2H), 3.61 - 3.64 (m, 3H), 3.78 (t, J = 5.3 Hz, 2H), 3.92 - 3.97 (m, 1H), 4.00 (d, J = 6.6 Hz, 2H), 4.95 (s, 1H), 6.85 - 6.89 (m, 2H), 6.93 (d, J = 15.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.44 - 7.48 (m, 2H), 7.66 (d, J = 15.8 Hz, 1H), 7.77 (dd, J = 8.0, 2.1 Hz, 1H), 8.57 (s, 1H), 8.73 (d, J = 2.1 Hz, 1H); 13 C NMR (176 MHz, CDCl3) δ 19.1, 24.9, 25.0, 27.8, 28.0, 28.5, 28.7, 32.9, 41.2, 45.2, 48.2, 48.5, 62.5, 70.8, 85.0, 95.0, 102.4, 112.9, 115.4, 121.4, 122.7, 123.4, 133.0, 138.6, 142.5, 150.8, 151.1, 152.2, 166.7, 171.6; (ATR) v max / cm -13191br, 2940m, 2857w, 2209w, 1708s, 1641s, 1605s, 1517s, 1234s, 1204s, 1021s, 753m; MS(ES): m / z = 645.3 [M+H] + ; C 37 H 49 N4O6[M+H] + HRMS (ES) Calculated value: 645.3647, Measured value 645.3647.

[0209] 1.1. 30 tert -butyl (2 E )-3-(4-{2-[4-(4-{7-[( Oksan -2- Iloxy ) Carbamoil ] Heptano piperazine-1-yl)phenyl]ethynyl}phenyl)prop-2-enoate, 56 of synthesis

[0210] tert -butyl (2 E )-3-(4-{2-[4-(4-{7-[(oxane-2-yloxy)carbamoyl]heptanoyl} piperazine-1-yl)phenyl]ethynyl}phenyl)prop-2-enoate ( 56 The synthesis of ) is illustrated in Fig. 1(xxiv). Compound 49 (0.22 g, 0.80 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.18 g, 1.00 mmol) were dissolved in DCM (30 mL), and the solution was cooled to 0°C. 4-methylmorpholine (0.11 mL, 1.00 mmol) was added dropwise over 5 minutes, and the resulting solution was stirred at 0°C for 2 hours, to which the compound 6(0.3 g, 0.77 mmol) and 4-methylmorpholine (0.1 mL, 0.90 mmol) were added, and the solution was stirred for an additional 18 hours. The solution was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (0.62 g). This was purified by SiO2 chromatography (97:3 -> 95:5, DCM / MeOH) to obtain the compound 56 was obtained as a yellow solid (0.30 g, 61%): 1 H NMR (400 MHz, CDCl3) δ 1.31 - 1.43 (m, 4H), 1.53 (s, 9H), 1.55 - 1.72 (m, 7H), 1.74 - 1.89 (m, 3H), 2.13 (s, 2H), 2.37 (t, J = 7.5 Hz, 2H), 3.19 - 3.32 (m, 4H), 3.56 - 3.70 (m, 3H), 3.79 (t, J = 5.1 Hz, 3H), 3.87 - 4.01 (m, 1H), 4.95 (s, 1H), 6.37 (d, J = 16.0 Hz, 1H), 6.89 (d, J = 8.5 Hz, 2H), 7.39 - 7.53 (m, 6H), 7.56 (d, J = 16.0 Hz, 1H), 8.48 (s, 1H); 13 C NMR (176 MHz, CDCl3) δ 18.5, 24.9, 25.0, 28.0, 28.2, 28.5, 28.7, 32.9, 33.1, 41.2, 45.3, 48.4, 48.7, 62.5, 80.6, 88.0, 91.9, 102.4, 113.8, 115.5, 120.6, 125.3, 127.8, 129.1, 130.4, 131.7, 132.8, 134.0, 142.7, 150.6, 166.2, 170.5, 171.6; IR (ATR) v max / cm -13218br, 2933m, 2855w, 2209w, 1700s, 1633s, 1596s, 1520s, 1518m, 1440m, 1325m, 1234s, 1207s, 1153s, 1159m, 1128m, 1036s, 820s; MS(ES): m / z = 644.4 [M+H] + ; C 38 H 50 N3O6[M+H] + HRMS (ES) Calculated value: 644.3700, Measured value 644.3675.

[0211] 1.1. 31 tert -butyl (2 E )-3-(5-{2-[4-(4-{7-[( Oksan -2- Iloxy ) Carbamoil ] Heptano piperazine-1-yl)phenyl]ethynyl}thiophene-2-yl)prop-2-enoate, 5 8 Synthesis of

[0212] tert -butyl (2 E )-3-(5-{2-[4-(4-{7-[(oxane-2-yloxy)carbamoyl]heptanoyl} piperazine-1-yl)phenyl]ethynyl}thiophene-2-yl)prop-2-enoate ( 58 The synthesis of ) is illustrated in Fig. 1(xxv). Compound 49 (0.22 g, 0.80 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.18 g, 1.00 mmol) were dissolved in DCM (30 mL), and the solution was cooled to 0°C. 4-methylmorpholine (0.11 mL, 1.00 mmol) was added dropwise over 5 minutes, and the resulting solution was stirred at 0°C for 2 hours, to which the compound 27(0.3 g, 0.76 mmol) and 4-methylmorpholine (0.1 mL, 0.90 mmol) were added, and the solution was stirred for an additional 20 hours. The solution was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain an orange oil as a crude product (0.6 g). This was purified by SiO2 chromatography (97:3 -> 95:5, DCM / MeOH) to obtain the compound 58 was obtained as a yellow oil (0.32 g, 65%): 1 H NMR (400 MHz, CDCl3) δ 1.34 - 1.40 (m, 4H), 1.51 (s, 9H), 1.59 - 1.69 (m, 6H), 1.75 - 1.84 (m, 4H), 2.12 (s, 2H), 2.32 - 2.41 (m, 2H), 3.20 - 3.29 (m, 4H), 3.59 - 3.65 (m, 3H), 3.77 (t, J = 5.2 Hz, 2H), 3.87 - 4.00 (m, 1H), 4.94 (s, 1H), 6.12 (d, J = 15.6 Hz, 1H), 6.79 - 6.91 (m, 2H), 7.06 - 7.14 (m, 2H), 7.38 - 7.45 (m, 2H), 7.59 (d, J = 15.6 Hz, 1H), 8.70 (s, 1H); 13 C NMR (176 MHz, CDCl3) δ 18.6, 24.9, 25.0, 25.2, 28.0, 28.1, 28.2, 28.2, 28.5, 28.7, 32.9, 33.0, 41.2, 45.2, 48.2, 48.5, 51.5, 56.0, 62.5, 63.8, 80.6, 81.4, 96.0, 102.4, 113.0, 115.4, 119.3, 126.2, 130.6, 132.0, 132.7, 135.5, 140.3, 150.7, 165.9, 170.5, 171.7; IR (ATR) v max / cm -13233br, 2934m, 2860w, 2203w, 1700s, 1674s, 1620s, 1604s, 1513m, 1442m, 1368s, 1232s, 1150s, 1036m, 655s; MS(ES): m / z = 650.3 [M+H] + ; C 36 H 48 N3O6S [M+H] + HRMS (ES) Calculated value: 650.3264, Measured value 650.3262.

[0213] 1.1.32 methyl (2 E )-3-4-[2-( trimethylsilyl ) Ethinyl ] Phenylprop -2- Enoate , 60 Synthesis of

[0214] Methyl (2 E )-3-4-[2-(trimethylsilyl)ethynyl]phenylprop-2-enoate ( 60 The synthesis of ) is illustrated in Fig. 1(xxvi). Anhydrous THF (10 mL) was placed in a round-bottom Schrenk flask, and then methyl 2-(diethoxyphosphoryl)acetate (1.4 mL, 6 mmol) and LiCl (0.25 g, 5.9 mmol) were added. The prepared reaction mixture was stirred at 0°C for 15 minutes. Compound 1 (1 g, 4.9 mmol) was subsequently added, followed by the slow addition of DBU (0.81 mL, 5.4 mm). The reaction mixture was heated to RT and stirred for an additional 16 hours. The reaction mixture was poured onto crushed ice and extracted with EtOAc; the organic extract was then rinsed with H2O and brine, followed by MgSO4 The product was dried and evaporated over a medium to obtain a light brown solid as a crude product (1.4 g). The crude product was purified by SiO2 column chromatography (using Pet. Et:EtOAc, 9:1 as the eluent) to obtain the compound 60 was obtained as a white solid (87.2 mg, 69%): 1 H NMR (CDCl3, 400 MHz) δ 0.25 (s, 9H), 3.81 (s, 3H), 6.43 (d, J 16 Hz, 1H), 7.43-7.49 (m, 4H), 7.65 (d, J 16 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 167.38, 144.03, 134.45, 132.54, 127.99, 125.16, 118.69, 104.61, 96.87, 51.93, 0.32, 0.04.

[0215] 1.1.33 methyl (2 E )-3-(4- Ethinylphenyl ) Prof -2- Enoate , 5 Synthesis of

[0216] Methyl (2 E )-3-(4-ethynylphenyl)prop-2-enoate ( 5 The synthesis of ) is illustrated in Fig. 1(xxvi). MeOH:DCM (1:3, 2 mL) was added to a round-bottom flask, and then the compound 60 (0.87 g, 3.4 mmol) and K2CO3 (0.7 g, 5.06 mmol) were added. The reaction mixture was stirred at RT for 3 hours. Subsequently, the prepared solution was diluted in DCM, the organic phase was rinsed with NH4Cl (saturated) and H2O, and then dried and evaporated over an MgSO4 phase to obtain a white solid crude product. The crude product was then purified by recrystallization from heptane to obtain the compound5 was obtained as a white crystalline solid (0.5 g, 77%): 1 H NMR δ 3.18 (s, 1H), 3.81 (s, 3H), 6.42 - 6.46 (d, J 16.02 Hz, 1H), 7.48-7.50 (m, 4H), 7.64 - 7.68 (d, J 16.02 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 167.32, 143.89, 134.84, 132.73, 128.05, 124.09, 118.97, 83.28, 79.35, 51.96.

[0217] 1.1.34 2-(2- Methoxyethoxy )ethyl (2 E )-3-(4- Ethinylphenyl ) Prof -2- Enoate , 61 Synthesis of

[0218] 2-(2-methoxyethoxy)ethyl (2 E The synthesis of )-3-(4-ethynylphenyl)prop-2-enoate (61) is illustrated in FIG. 1(xxvi). Compound 5 (22.5 mg, 0.12 mmol) was dissolved in diethylene glycol monomethyl ether (2 mL), then K2CO3 (1 mg, 0.007 mmol) was added, and the reaction mixture was stirred at RT for 24 hours. The prepared reaction mixture was diluted in H2O and extracted with DCM; the organic extract was rinsed with H2O and then dried and evaporated over MgSO4 to obtain a yellow oil as a crude product (157.8 mg). Subsequently, the crude product was purified by Kugelrohr distillation (70-80°C, 9 Torr) to obtain the compound 61 It was obtained as a yellow oil (25.9 mg, 62%). 1H NMR (CDCl3, 400 MHz) δ 3.18 (s, 1H), 3.40 (s, 3H), 3.56 - 3.59 (m, 2H), 3.67 - 3.70 (m, 2H), 3.77 - 3.80 (m, 2H), 4.37 - 4.40 (m, 2H), 6.48 (d, J = 16 Hz, 1H), 7.45 - 7.51 (m, 4H), 7.67 (d, J = 16 Hz, 1H); 13 C NMR (CDCl3, 101 MHz) δ 166.84, 144.06, 134.84, 132.73, 128.07, 124.08, 119.08, 83.28, 79.36, 72.05, 70.69, 69.42, 63.90, 59.27; MS (ESI) m / z = 275.1 [M+H] + ; C 16 H 19 O4[M+H] + HRMS (ESI) Calculated value: 275.1283, Measured value 275.1286.

[0219] 1.1.35 2-(2- Methoxyethoxy )ethyl (2 E )-3-(4-{2-[4-(4-{8-[(Oksan-2- Iloxy )amino]octanoyl}piperazine-1-yl)phenyl] Ethinyl }phenyl) Prof -2- Enoate , 63 Synthesis of

[0220] 2-(2-methoxyethoxy)ethyl (2E)-3-(4-{2-[4-(4-{8-[(oxane-2-yloxy)amino]octanoyl}piperazine-1-yl)phenyl]ethynyl}phenyl)prop-2-enoate ( 63 The synthesis of ) is illustrated in FIG. 1(xxvii). Compound 492-chloro-4,6-dimethoxy-1,3,5-triazine (328 mg, 1.20 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (270 mg, 1.51 mmol) were added to a round-bottom flask containing 40 mL of DCM. After cooling the resulting solution to 0°C, 4-methylmorpholine (156 µL, 1.44 mmol) was added dropwise. The reaction mixture was stirred at 0°C until the 2-chloro-4,6-dimethoxy-1,3,5-triazine was completely consumed. Compound 62 (500 mg, 1.15 mmol) and 4-methylmorpholine (156 µL, 1.44 mmol) were added, and the reaction mixture was stirred at RT for 16 hours. The prepared reaction mixture was diluted in DCM, rinsed with H2O, and then dried and evaporated over MgSO4 to obtain the crude product as an orange solid, which was purified by SiO2 chromatography (9:1, DCM / MeOH) to obtain the compound 62 It was obtained as an orange solid (0.5 g, 65%). 1 H NMR (CDCl3, 400 MHz) δ 1.41-1.34 (m, 6H), 1.70-1.63 (m, 6H), 3.21-3.28 (m, 4H), 3.57-3.59 (m, 2H), 3.61-3.66 (m, 4H), 3.68-3.70 (m, 2H), 3.71-3.73 (m, 1H), 3.77 -3.81 (m, 4H), 3.83-3.86 (m, 2H), 3.95 (s, 3H), 4.36-4.41 (m, 2H), 4.95 (s, br, 1H), 6.48 (d) J 15.9 Hz, 1H), 6.88 (d J 8.8 Hz, 2H), 7.44-7.46 (m, 2H), 7.47-7.51 (m, 4H), 7.68 (d) J 15.9 Hz, 1H).

[0221] 1.1.36 6-[2-( trimethylsilyl ) Ethinyl ]Pyridine-3- Carbualdehyde, 65 Synthesis of

[0222] 6-[2-(trimethylsilyl)ethinyl]pyridine-3-carbaldehyde ( 65 The synthesis of ) is illustrated in Fig. 1(xxviii). 2-chloropyridine-3-carboxaldehyde (10 g, 70.6 mmol), trimethylsilylacetylene (13.7 mL, 99.5 mmol), Na2PdCl4 (0.41 g, 1.4 mmol), CuI (0.2 g, 1.06 mmol), PtBu3HBF4 (0.81 g, 2.8 mmol), and Na2CO3 (11.13 g, 105 mmol) were added to a round-bottom flask containing toluene (150 mL) that had been degassed with Ar. The reaction mixture was stirred at 100°C for 20 hours. After evaporation, the crude reaction product mixture was purified by SiO2 column chromatography (petroleum ether:EtOAc, 7:3 as eluent) to obtain the compound 65 It was obtained as a brown solid (4.4 g, 31%). 1 H NMR (400 MHz, CDCl3) d 0.30 (s, 9H), 7.60 (d J 7.5 Hz, 1H), 8.12 (dd J 8.1, 2.1 Hz, 1H), 9.0 (dd J 2.1, 0.8 Hz, 1H), 10.1(s, 1H).

[0223] 1.1.37 6- Ethinylpyridine -3- Carbualdehyde , 66 Synthesis of

[0224] 6-ethynylpyridine-3-carbaldehyde ( 66 The synthesis of ) is illustrated in Fig. 1(xxviii). Compound 65(4.4 g, 21.64 mmol) was dissolved in MeOH:DCM (1:3, 180 mL), and then K2CO3 (3.23 g, 23.4 mmol) was added. The reaction mixture was stirred at RT for 2 hours. The crude reaction product was subsequently dissolved in DCM, rinsed with NH4Cl and H2O, and then dried and evaporated over MgSO4. After Kugelrohr distillation at 150°C (9 Torr), the pure compound 66 It was obtained as an off-white solid (1.4 g, 45%). 1 H NMR (400 MHz, CDCl3) d 3.41 (s, 1H), 7.64 (d J 8.0 Hz, 1H), 8.15 (dd J 8.0, 2.1 Hz, 1H), 9.05 (dd J 2.1, 0.8 Hz, 1H), 10.12 (s, 1H).

[0225] 1.1.38 diethyl ((Iso- Butoxycarbonyl ) methyl ) Phosphonate , 67 Synthesis of

[0226] Diethyl ((iso-butoxycarbonyl)methyl) phosphonate ( 67The synthesis of ) is illustrated in Fig. 1 (xxix). 2-methyl-1-propanol (0.74 mL, 8.0 mmol) was added under Ar to a round-bottom Schlenk flask containing toluene anhydride (40 mL), followed by the addition of diethylphosphonoacetic acid (1.35 mL, 8.4 mmol), DIPEA (3.62 mL, 20.8 mmol), and propylphosphonic anhydride (6.62 mL, 10.4 mmol). The prepared reaction mixture was stirred at RT for 4 hours. Subsequently, the reaction product mixture was diluted with H2O, and the organic phase was extracted with EtOAc. The combined organic extract was rinsed with HCl (10% aq.), NaHCO3(sat.), and brine, and then dried and evaporated over MgSO4. The compound 67 (1.92 g, 95%) was used in the next step without purification. 1 H NMR (400 MHz, CDCl3) d 0.94 (d J 6.7 Hz, 6H), 1.34 (t J 14.1, 7.0 Hz, 6H), 1.90 - 2.00 (m, 1H), 2.97 (d) J 21.6 Hz, 2H), 3.92 (dd J 6.7, 0.5 Hz, 2H), 4.13 - 4.21 (m, 4H).

[0227] 1.1.39 2- Methylpropyl (2E)-3-(6- Ethinylpyridine -3-days) Prof -2- Enoate , 68 Synthesis of

[0228] 2-methylpropyl(2E)-3-(6-ethynylpyridine-3-yl)prop-2-enoate ( 68 The synthesis of ) is illustrated in Fig. 1(xxx). Compound 67(1.92 g, 7.6 mmol) and LiCl (0.314 g, 7.41 mmol) were added to a round-bottom Schlenk flask containing anhydrous THF (10 mL) under Ar, and the prepared reaction mixture was cooled to 0°C and stirred for 15 minutes. Compound 66 (0.810 g, 6.18 mmol) was added, followed by dropwise addition of DBU (1.01 mL, 6.8 mmol). The reaction mixture was heated to RT and stirred continuously for an additional 16 hours. The crude reaction product was poured onto crushed ice and extracted with EtOAc; the organic extract was rinsed with Bryn and then dried and evaporated over MgSO4. The compound was purified by SiO2 column chromatography. 68 It was obtained as a bright yellow solid (1.3 g, 92%). 1 H NMR (400 MHz, CDCl3) d 0.99 (d J 6.7 Hz, 6H), 1.97 - 2.07 (m, 1H), 3.27 (s, 1H), 4.01 (d) J 6.7 Hz, 2H), 6.54 (d J 16.1 Hz, 1H), 7.50 (d J 8.2 Hz, 1H), 7.65 (d J 16.1 Hz, 1H), 7.82 (dd J 8.2, 2.2 Hz, 1H), 8.72 (d J 2.2 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 166.31, 149.86, 143.29, 139.98, 134.56, 130.11, 127.61, 121.54, 82.51, 79.33, 71.19, 27.95, 19.28); C 14 H 16 NO2[M+H] + HRMS (ESI) Calculated value: 230.1181, Measured value 230.1181.

[0229] 1.1. 40 2 - Methylpropyl (2 E )-3-(6-{2-[4-(4-{7-[( Oksan -2- Iloxy ) Carbamoil ] Hepta Noyl} piperazine-1-yl) phenyl] ethinyl} pyridine-3-yl)prop-2-enoate, 70 of synthesis

[0230] 2-methylpropyl (2 E )-3-(6-{2-[4-(4-{7-[(oxane-2-yloxy)carbamoyl]heptanoyl} piperazine-1-yl) phenyl] ethinyl} pyridine-3-yl)prop-2-enoate ( 70 The synthesis of ) is illustrated in FIG. 1(xxxi). Compound 49 (370 mg, 1.34 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (300 mg, 1.7 mmol) were dissolved in DCM, and after cooling the obtained solution to 0°C, 4-methylmorpholine (250 mL, 2.27 mmol) was added dropwise, and the reaction mixture was continuously stirred at 0°C for 4 hours. Compound 69 (500 mg, 1.28 mmol) and 4-methylmorpholine (102 mL, 0.92 mmol) were added, and the prepared reaction mixture was heated to RT and stirred continuously overnight. The crude product of the prepared reaction mixture was diluted in DCM, rinsed with H2O, and then MgSO4 A yellow solid crude product was obtained by drying and evaporating on a surface (1 g). Subsequently, this was purified by SiO2 column chromatography (DCM:MeOH, 9:1) to obtain the compound 70 was obtained as a bright yellow solid (0.6 g, 72%): 1 H NMR (400 MHz, CDCl3) δ 0.99 (d J6.7 Hz, 6H), 1.33 - 1.42 (m, 6H), 1.64 - 1.71 (m, 6H), 1.76 - 1.87 (m, 4H), 1.99 - 2.06 (m, 1H), 2.10 - 2.17 (m, 2H), 3.24 - 3.32 (m, 4H), 3.60 - 3.67 (m, 4H), 3.71 - 3.74 (m, 1H), 3.84 - 3.87 (m, 1H), 4.01 (d) J 6.7 Hz, 2H), 4.95 (s, 1H), 6.53 (d) J 16 Hz, 1H), 7.66 (d J 16 Hz, 1H), 7.52 - 7.56 (m, 1H), 7.84 (d J 8.3 Hz, 1H), 8.72 (d J 2.1 Hz, 1H), 6.89 (d J 8.8 Hz, 2H), 7.50 - 7.54 (m, 2H).

[0231] 1.1.41 1-(4- iodophenyl )-4- Methylpiperazine , 72 Synthesis of

[0232] 1-(4-iodophenyl)-4-methylpiperazine ( 72 The synthesis of ) is illustrated in FIG. 1(xxxii). Compound 4 (2.88 g, 10.0 mmol) was dissolved in DMF (20 mL) under Ar, at which point iodomethane (0.93 mL, 15.0 mmol) and Et3N (2.09 mL, 15.0 mmol) were added, and the solution was stirred at RT for 72 hours. The formed precipitate was then filtered to obtain the crude product as a solid (6.4 g). This was purified by SiO2 chromatography (DCM / MeOH, 9:1) to obtain the compound 72 was obtained as an off-white solid (1.22 g, 40%): 1H NMR (400 MHz, CDCl3) δ 2.34 (s, 3H), 2.51 - 2.58 (m, 4H), 3.13 - 3.21 (m, 4H), 6.64 - 6.71 (m, 2H), 7.46 - 7.55 (m, 2H); 13 C NMR (101 MHz, CDCl3) δ 46.1, 48.6, 54.9, 81.3, 118.0, 137.7, 150.8; IR (ATR) v max / cm -1 2959w, 2832m, 2793m, 1672m, 1490s, 1447m, 1390m, 1292s, 1235s, 1144s, 1009m, 908s, 811s; MS(ES): m / z = 303.0 [M+H] + ; C 11 H 15 N2I [M+H] + HRMS (ES) Calculated value: 303.0353, Measured value 303.0351.

[0233] 1.1.42 1- methyl -4-(2- nitrophenyl )piperazine, 74 Synthesis of

[0234] 1-Methyl-4-(2-nitrophenyl)piperazine ( 74 The synthesis of ) is illustrated in Fig. 1(xxxiii). 1-fluoro-2-nitrobenzene (9 mL, 85.0 mmol) was added to DMSO (60 mL), at which time N methylpiperazine (18.9 mL, 170.0 mmol) and K2CO3 (23.4 g, 170 mmol) were added. The prepared red solution was stirred at 110°C for 24 hours, then cooled and diluted with H2O. The mixture was extracted with DCM (3 x), rinsed with saturated NH4Cl and H2O, dried (MgSO4), and evaporated to obtain the compound 74 was obtained as a red oil and transferred directly to the next step (21.0 g, >100%):1 H NMR (300 MHz, CDCl3) δ 2.35 (s, 3H), 2.52 - 2.60 (m, 4H), 3.03 - 3.14 (m, 4H), 6.98 - 7.06 (m, 1H), 7.14 (dd, J = 8.2, 1.7 Hz, 1H), 7.40 - 7.53 (m, 1H), 7.75 (dd, J = 8.2, 1.7 Hz, 1H).

[0235] 1.1.43 2-(4- Methylpiperazine -1-day)aniline, 75 Synthesis of

[0236] 2-(4-methylpiperazine-1-yl)aniline ( 75 The synthesis of ) is illustrated in Fig. 1(xxxiii). Compound 74 (21.0 g, 85.0 mmol) was dissolved in EtOH (200 mL), to which concentrated hydrochloric acid (c. HCl) (20 mL) and Sn(II)Cl2 (48.4 g, 255.0 mmol) were added, and the prepared mixture was refluxed and stirred for 18 hours. The mixture was cooled, and the solvent was evaporated to obtain the crude residue, which was then dissolved in DCM. The organic phase was rinsed with 5% NaOH and H2O, then dried (MgSO4) and evaporated to obtain a yellow solid crude product (4.7 g). This was purified by SiO2 chromatography (9:1, DCM / MeOH) to obtain the compound 75 was obtained as a yellow solid (3.08 g, 19%): 1 H NMR (400 MHz, CDCl3) δ 2.36 (s, 3H), 2.45 - 2.65 (m, 4H), 2.95 (t, J = 4.9 Hz, 4H), 3.96 (br, 2H), 6.68 - 6.77 (m, 2H), 6.93 (td, J = 7.7, 1.2 Hz, 1H), 7.02 (dd, J = 7.7, 1.2 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 46.2, 50.9, 55.9, 115.0, 118.5, 119.8, 124.5, 139.1, 141.4; IR (ATR) v max / cm -1 3389m, 3294w, 2939w, 2980w, 1619s, 1503s, 1449s, 1283s, 1139s, 1011s, 927m.

[0237] 1.1.44 1-(2- iodophenyl )-4- Methylpiperazine , 76 Synthesis of

[0238] 1-(2-iodophenyl)-4-methylpiperazine ( 76 The synthesis of ) is illustrated in Fig. 1(xxxiii). Compound 75 (2.0 g, 10.4 mmol) was dissolved in c. HCl (3 mL) and H2O (12 mL), and the resulting solution was cooled to 0°C. NaNO2 (0.86 g, 12.5 mmol, solution in 3 mL H2O) was slowly added over 2 minutes, and the resulting suspension was stirred at 0°C for 2 hours, during which time KI (3.45 g, 20.8 mmol) was added in a fractional manner, and the suspension was subsequently stirred at RT for 72 hours. The suspension was extracted with DCM, rinsed with saturated NaHCO3 and water, dried with (MgSO4), and evaporated to obtain a solid as a crude product. This was purified by SiO2 chromatography (9:1, DCM / MeOH) to obtain the compound 76 was obtained as a dark solid (2.64 g, 84%): 1 H NMR (300 MHz, CDCl3) δ 2.54 (s, 3H), 2.90 (s, 4H), 3.18 (t, J = 4.9 Hz, 4H), 6.81 (td, J = 7.8, 1.5 Hz, 1H), 7.06 (dd, J= 8.0, 1.5 Hz, 1H), 7.31 (ddd, J = 8.0, 7.3, 1.5 Hz, 1H), 7.83 (dd, J = 7.8, 1.5 Hz, 1H); 13 C NMR (176 MHz, CDCl3) δ 45.2, 51.0, 54.9, 98.0, 121.2, 125.9, 129.3, 139.9, 152.4; IR (ATR) v max / cm -1 3006w, 2879m, 2833m, 1738w, 1579w, 1468s, 1461s, 1371s, 1289m, 1230s, 1145s, 1012s, 972m, 762m.

[0239] 1.1.45 (3- Chloro -2- oxopropyl ) triphenylphosphonium chloride, 78 Synthesis of

[0240] (3-chloro-2-oxopropyl)triphenylphosphonium chloride ( 78 The synthesis of ) is illustrated in Fig. 1 (xxxiv). 1,3-dichloroacetone (15.0 g, 118 mmol) and triphenylphosphine (31.0 g, 118 mmol) were dissolved in toluene (60 mL), and the suspension was stirred at RT for 72 hours. The formed suspension was filtered, and the isolated solid was rinsed with toluene and Et2O to obtain the compound 78 was obtained as a white solid (43.1 g, 94%): 1 H NMR (400 MHz, DMSO) δ 4.88 (s, 2H), 5.88 (d, J = 12.8 Hz, 2H), 7.72 - 7.87 (m, 15H); all other data were consistent with the literature (doi:10.1016 / j.poly.2014.11.029).

[0241] 1.1.46 1- Chloro-3-( Triphenylphosphanylidene )propane-2-one, 79 Synthesis of

[0242] 1-chloro-3-(triphenylphosphanylidene)propan-2-one ( 79 The synthesis of ) is illustrated in Fig. 1(xxxiv). Compound 78 (43.1 g, 110.7 mmol) was dissolved in MeOH (60 mL), at which point Na2CO3 (5.87 g, 55.4 mmol, solution in 60 mL H2O) was added, and the formed suspension was high-speed stirred for 0.5 hours. The suspension was diluted with approximately 300 mL of H2O, and the mixture was filtered. The isolated solid was subsequently dissolved in DCM, dried (MgSO4), and evaporated to obtain the compound 79 was obtained as a white solid (32.1 g, 82%): 1 H NMR (400 MHz, CDCl3) δ 4.01 (s, 2H), 4.29 (d, J = 24.0 Hz, 1H), 7.44 - 7.51 (m, 6H), 7.54 - 7.60 (m, 3H), 7.61 - 7.69 (m, 6H); all other data were consistent with the literature ( https: / / doi.org / 10.1021 / jo101864n ).

[0243] 1.1. 47 (3 E )-1- Chloro -4-{5-[2-( trimethylsilyl ) Ethinyl ]pyridin-2-il}but-3-en-2-on, 80 Synthesis of

[0244] (3 E )-1-chloro-4-{5-[2-(trimethylsilyl)ethinyl]pyridine-2-yl}but-3-en-2-one ( 80 The synthesis of ) is illustrated in Fig. 1(xxxiv). Compound 40 (7.5 g, 36.9 mmol) and compound 79(13.0 g, 36.9 mmol) was dissolved in DCM (60 mL), and the solution was stirred at RT for 48 hours. The dark reaction solution was evaporated, and the crude product solid was purified by SiO2 chromatography to obtain the compound 80 was obtained as a white solid (7.67 g, 75%): 1 H NMR (400 MHz, CDCl3) δ 0.27 (s, 9H), 4.32 (s, 2H), 7.40 (dd, J = 8.1, 0.9 Hz, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.65 (d, J = 15.6 Hz, 1H), 7.77 (dd, J = 8.1, 2.1 Hz, 1H), 8.69 (d, J = 2.1 Hz, 1H); 13 C NMR (75 MHz, CDCl3) δ -0.3, 47.8, 100.9, 101.2, 121.4, 124.4, 125.6, 139.5, 142.4, 151.1, 152.9, 191.2; IR (ATR) v max / cm -1 3033w, 2959w, 2920w, 2157w, 1709s, 1622m, 1473w, 1399w, 1248m, 981m, 867s, 841s; MS(ES): m / z = 278.1 [M+H] + ; C 14 H 17 NOCl [M+H] + HRMS (ES) Calculated value: 278.0768, Measured value 278.0769.

[0245] 1.1. 48 4 -[( E )-2-{5-[2-( trimethylsilyl ) Ethinyl ]pyridine-2-il} Ethenil ]-1,3-thiazole-2-amine , 81 of synthesis

[0246] 4-[( E )-2-{5-[2-(trimethylsilyl)ethynyl]pyridine-2-yl}ethenyl]-1,3-thiazole-2-amine ( 81 The synthesis of ) is illustrated in Fig. 1 (xxxiv). Compound 80 (8.5 g, 30.6 mmol) and thiourea (2.8 g, 36.7 mmol) were dissolved in EtOH (70 mL), and the solution was refluxed and stirred for 18 hours. The mixture was cooled and evaporated to obtain the crude residue, which was purified by SiO2 chromatography (1:1, cyclohexane / EtOAc) to obtain the compound 81 was obtained as an off-white solid (4.24 g, 46%): 1 H NMR (400 MHz, CDCl3) δ 0.25 (s, 9H), 6.83 (s, 1H), 7.08 (d, J = 15.4 Hz, 1H), 7.12 (s, 2H), 7.41 (d, J = 15.4 Hz, 1H), 7.46 (dd, J = 8.1, 0.8 Hz, 1H), 7.80 (dd, J = 8.1, 2.2 Hz, 1H), 8.58 (dd, J = 2.2, 0.8 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 0.2, 89.2, 91.1, 98.1, 102.5, 109.6, 116.8, 121.7, 127.2, 127.4, 139.2, 149.2, 154.8, 168.1; IR (ATR) v max / cm -1 3305br, 3117br, 2959w, 2899w, 2157m, 1724m, 1628m, 1582m, 1536m, 1504m, 1471m, 1367m, 1249s, 860s, 842s, 758s; MS(ES): m / z = 300.1 [M+H] + ; C 15 H18 N3SSi [M+H] + HRMS (ES) Calculated value: 300.0985, Measured value 300.0985.

[0247] 1.1.49 4-[( E )-2-(5- Ethinylpyridine -2-days) Ethenil ]-1,3-thiazol-2- amine, 82 Synthesis of

[0248] 4-[( E )-2-(5-ethynylpyridine-2-yl)ethenyl]-1,3-thiazole-2-amine ( 82 The synthesis of ) is illustrated in Fig. 1(xxxiv). Compound 81 5.0 g (16.7 mmol) was dissolved in 80 mL of THF, and the solution was cooled to -40°C. Tetrabutylammonium fluoride (TBAF) (18.3 mL, 18.3 mmol, 1.0 M in THF) was added dropwise, and the resulting solution was stirred at -40°C for 1 hour and then allowed to reach RT. The solution was diluted with H2O and extracted with DCM. The organic phase was rinsed with H2O, dried (MgSO4), and evaporated to obtain a dark solid crude product. This was purified by SiO2 chromatography (cyclohexane / EtOAc, 1:1) to obtain the compound 82 was obtained as a yellow solid (2.68 g, 71%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 4.45 (s, 1H), 6.83 (s, 1H), 7.09 (d, J = 15.4 Hz, 1H), 7.12 (s, 2H), 7.40 (d, J = 15.4 Hz, 1H), 7.49 (dd, J = 8.3, 0.9 Hz, 1H), 7.83 (dd, J = 8.3, 2.2 Hz, 1H), 8.61 (dd, J= 2.2, 0.9 Hz, 1H); 13 13C NMR (101 MHz, DMSO- d 6) δ 80.9, 84.3, 109.5, 116.4, 121.6, 127.3, 139.4, 149.2, 152.0, 154.9, 168.1; IR (ATR) v max / cm -1 3284br, 3113br, 3016w, 2105w, 1738s, 1626s, 1581s, 1528m, 1468w, 1366s, 1217s, 917m; MS(ES): m / z = 228.1 [M+H] + ; C 12 H 10 N3S [M+H] + HRMS (ES) Calculated value: 228.0590, Measured value 228.0588.

[0249] 1.1.50 4-(4- iodophenyl )morpholine, 83 Synthesis of

[0250] 4-(4-iodophenyl)morpholine ( 83 The synthesis of ) is illustrated in Fig. 1 (xxxv). 4-phenylmorpholine (12.5 g, 76.6 mmol) and NaHCO3 (10.3 g, 122.6 mmol) were suspended in H2O (100 mL), and the mixture was cooled to ca. 12°C. Iodine (20.4 g, 80.4 mmol) was slowly added, and the formed suspension was high-speed stirred at RT for 4 hours. A saturated aqueous solution of Na2S2O3 was added, and the precipitated solid was isolated by filtration to obtain a dark gray solid as a crude product (27 g). This was purified by recrystallization from EtOH to obtain the compound 83 was obtained as a gray solid (16.3 g, 74%): 1H NMR (300 MHz, CDCl3) δ 3.07 - 3.16 (m, 4H), 3.80 - 3.89 (m, 4H), 6.61 - 6.72 (m, 2H), 7.47 - 7.58 (m, 2H); 13 C NMR (176 MHz, CDCl3) δ 48.8, 66.6, 81.7, 117.6, 137.8, 150.8; IR (ATR) v max / cm -1 2966w, 2890w, 2856w, 2829w, 1583m, 1490m, 1258, 1234s, 1118s, 922s, 811s; MS(ES): m / z = 290.0 [M+H] + ; C 10 H 13 NOI [M+H] + HRMS (ES) Calculated value: 290.0044, Measured value 290.0037.

[0251] 1.2 Preparation of Reference Compounds

[0252] 1.2. 1 methyl (2 E )-3-(5-{2-[2-(4- Methylpiperazine -1-1)phenyl] Ethinyl }pyridine-2-yl)prop-2-enoate, 77 of synthesis

[0253] Methyl (2 E )-3-(5-{2-[2-(4-methylpiperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate ( 77 The synthesis of ) is illustrated in Fig. 1 (xxxiii). Et3N (20 mL) was degassed by injecting Ar for 1 hour. Compound 76 (175 mg, 0.58 mmol), compound 42(120 mg, 0.64 mmol), Pd(PPh3)2Cl2 (21 mg, 0.03 mmol), and CuI (6 mg, 0.03 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 18 hours. Subsequently, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (95:5, DCM / MeOH) to obtain the compound 77 was obtained as a yellow oil (105 mg, 50%): 1 H NMR (400 MHz, CDCl3) δ 2.39 (br, 3H), 2.68 (br, 4H), 3.29 (br, 4H), 3.82 (s, 3H), 6.94 (d, J = 15.7 Hz, 1H), 6.96 - 7.00 (m, 2H), 7.28 - 7.35 (m, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.51 (dd, J = 7.8, 1.6 Hz, 1H), 7.68 (d, J = 15.7 Hz, 1H), 7.79 (dd, J = 8.0, 2.1 Hz, 1H), 8.76 (d, J = 1.6 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 51.3, 51.9, 55.5, 91.2, 93.4, 115.7, 118.0, 121.4, 121.8, 122.4, 123.6, 130.3, 134.1, 138.6, 142.7, 151.3, 152.2, 154.3, 167.1; IR (ATR) v max / cm -1 3006w, 2879m, 2833m, 1738w, 1579w, 1468s, 1461s, 1371s, 1289m, 1230s, 1145s, 1012s, 972m, 762m.

[0254] 1.3 Preparation of Exemplary Compounds

[0255] 1.3.1 tert -butyl (2 E )-3-(4-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }phenyl)prop-2-enoate, 6 of synthesis

[0256] The synthesis of exemplary compound 6 is illustrated in FIG. 2(i). Et3N (80 mL) was degassed by injecting Ar for 1 hour. Compound 4 (2.16 g, 7.5 mmol), compound 3 (1.80 g, 7.88 mmol), Pd(PPh3)2Cl2 (260 mg, 0.39 mmol) and CuI (71 mg, 0.39 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 24 hours. Afterward, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (9:1, DCM / MeOH, 1% Et3N) and recrystallization from MeOH to obtain the compound 6 was obtained as a yellow solid (2.11 g, 72%): 1 H NMR (400 MHz, CDCl3) δ 1.53 (s, 9H), 3.22-3.28 (m, 4H), 3.38-3.45 (m, 4H), 6.37 (d, J = 15.9 Hz, 1H), 6.77 - 6.95 (m, 2H), 7.33 - 7.53 (m, 6H), 7.56 (d, J = 15.9 Hz, 1H) ; IR (ATR) v max / cm -1 2967w, 2916w, 2830w, 2212w, 1687s, 1629m, 1595m, 1518m, 1326m, 1241m, 1159m, 1128m, 986m, 831s, 819s; MS(ASAP): m / z = 389.2 [M+H] + ; C 25 H 29N2O2[M+H] + HRMS (ASAP) Calculated: 389.2229, Measured: 389.2231.

[0257] 1.3. 2-methyl (2 E )-3-(4-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }phenyl) Prof -2-Enoate, 7 of synthesis

[0258] Exemplary compounds 7 The synthesis of is exemplified in Fig. 2(i). Et3N (150 mL) was degassed by injecting Ar for 1 hour. Compound 4 (4.50 g, 15.6 mmol), compound 5 (3.05 g, 16.4 mmol), Pd(PPh3)2Cl2 (550 mg, 0.78 mmol) and CuI (150 mg, 0.78 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 24 hours. Subsequently, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (9:1, DCM / MeOH, 1% Et3N) and subsequent recrystallization from MeOH to obtain the compound 7 was obtained as a yellow solid (2.74 g, 51%): 1 1H NMR (600 MHz, DMSO- d 6) δ 2.82-2.94 (m, 4H), 3.14-3.24 (m, 4H), 3.73 (s, 3H), 6.67 (d, J = 16.0 Hz, 1H), 6.94 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 16.0 Hz, 1H), 7.74 (d, J= 8.0 Hz, 2H); 13 13C NMR (151 MHz, DMSO- d 6) δ 44.9, 47.5, 51.5, 87.6, 92.7, 110.7, 114.5, 118.3, 124.9, 128.6, 131.3, 132.5, 133.5, 143.6, 151.2, 166.6; IR (ATR) v max / cm -1 3039w, 2952w, 2909w, 2830w, 2204w, 2173w, 1698s, 1630s, 1593m, 1518m, 1312m, 1243s, 1168s, 987m, 831s, 817s; MS(ASAP): m / z = 347.2 [M+H] + ; C 22 H 23 N2O2[M+H] + HRMS (ASAP) Calculated value: 347.1760, Measured value 347.1736.

[0259] 1.3. 3-methyl (2 E )-3-[4-(2-{4-[(2- aminoethyl )( methyl )amino]phenyl} Ethinyl ) phenyl]prop-2-enoate, 12 of synthesis

[0260] Methyl (2 E )-3-[4-(2-{4-[(2-aminoethyl)(methyl)amino]phenyl} ethynyl) phenyl]prop-2-enoate, 12 The synthesis of the compound is illustrated in FIG. 2(ii). 11 (3.46 g, 12.53 mmol) was dissolved in Et3N (120 mL), and the solution was degassed by injecting Ar for 1 hour. Compound 5(2.57 g, 13.8 mmol), Pd(PPh3)2Cl2 (440 mg, 0.63 mmol), and CuI (120 mg, 0.63 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 72 hours. Afterward, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (9:1, DCM / MeOH, 0.5% Et3N) to obtain the compound 12 was obtained as a yellow solid (2.44 g, 58%): 1 1H NMR (600 MHz, DMSO- d 6) δ 2.94 (t, J = 7.0 Hz, 2H), 2.97 (s, 3H), 3.56 (t, J = 7.0 Hz, 2H), 3.73 (s, 3H), 6.67 (d, J = 16.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 7.40 (d, J = 8.9 Hz, 2H), 7.47 - 7.54 (m, 2H), 7.67 (d, J = 16.0 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H); 13 13C NMR (151 MHz, DMSO- d 6) δ 36.3, 38.1, 49.6, 51.5, 78.7, 79.0, 79.2, 87.4, 93.1, 108.6, 111.9, 118.2, 118.2, 125.1, 128.6, 131.2, 132.7, 133.3, 143.6, 148.9, 166.6; IR (ATR) v max / cm -1 3403br, 3042w, 2952w, 2888w, 2208m, 1698s, 1632m, 1608m, 1594s, 1522s, 1313s, 1169s, 1134s, 817s; MS(ASAP): m / z = 335.2 [M+H] + ; C21 H 23 N2O2[M+H] + HRMS (ASAP) Calculated value: 335.1760, Measured value 335.1743.

[0261] 1.3. 4-methyl (2 E )-3-(4-{2-[4-(4- Acetylpiperazine -1-1)phenyl] Ethinyl }phenyl) print Rope-2-Enoate, 13 Synthesis of

[0262] Methyl (2 E )-3-(4-{2-[4-(4-acetylpiperazine-1-yl)phenyl]ethinyl}phenyl) prop-2-enoate, 13 The synthesis of is illustrated in FIG. 2(iii). Compound 7 (0.35 g, 1.01 mmol) was dissolved in DCM (10 mL), at which point acetyl chloride (86 µL, 1.21 mmol) and pyridine (98 µL, 1.21 mmol) were added, and the resulting solution was stirred at RT for 16 hours. The solution was diluted with DCM, rinsed with a saturated NH4Cl solution and H2O, dried (MgSO4), and evaporated to obtain a yellow solid as a crude product (0.4 g). This was purified by SiO2 chromatography (97.5:2.5, DCM / MeOH) to obtain the compound 13 was obtained as a yellow solid (0.38 g, 97%): 1 H NMR (600 MHz, CDCl3) δ 2.15 (s, 3H), 3.24 (t, J = 5.3 Hz, 2H), 3.27 (t, J = 5.3 Hz, 2H), 3.63 (t, J = 5.2 Hz, 2H), 3.78 (t, J = 5.3 Hz, 2H), 3.81 (s, 3H), 6.44 (d, J = 16.0 Hz, 1H), 6.88 (d,J = 8.4 Hz, 2H), 7.41 - 7.47 (m, 2H), 7.46 - 7.54 (m, 4H), 7.67 (d, J = 16.0 Hz, 1H); 13 C NMR (151 MHz, CDCl3) δ 21.3, 41.1, 45.9, 48.3, 48.6, 51.7, 88.0, 92.1, 113.8, 115.6, 118.1, 125.7, 128.0, 131.8, 132.9, 133.7, 144.0, 150.5, 167.3, 169.0; IR (ATR) v max / cm -1 3039w, 2947w, 2836w, 2205w, 2173w, 1699m, 1627s, 1594m, 1521m, 1446m, 1425m, 1311m, 1236s, 1164s, 994s, 835s, 822s; MS(ASAP): m / z = 388.2 [M+H] + ; C 24 H 24 N2O3[M+H] + HRMS (ASAP) Calculated: 388.1787, Measured: 388.1793.

[0263] 1.3. 5 (3-{4-[4-(2-{4-[(1 E )-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}ethinyl)phenyl]piperazine-1-yl}propyl)triphenylphosphonium bromide, 14 Synthesis of

[0264] (3-{4-[4-(2-{4-[(1 E )-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}ethinyl)phenyl]piperazine-1-yl}propyl)triphenylphosphonium bromide, 14 The synthesis of the compound is illustrated in FIG. 2(iv). 7(0.35 g, 1.01 mmol) was dissolved in anhydrous DMF (10 mL) under Ar, at which point K2CO3 (0.167 g, 1.2 mmol) and (3-bromopropyl)triphenylphosphonium bromide (0.47 g, 1.01 mmol) were added, and the resulting solution was stirred at 80°C for 16 hours. The solution was cooled, diluted with H2O, and extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried (MgSO4) and evaporated to obtain a yellow solid crude product (0.5 g). This was purified by SiO2 chromatography (95:5, DCM / MeOH) and recrystallization from a DCM / heptane solution, thereby obtaining the compound 14 was obtained as a yellow solid (0.44 g, 60%): 1 H NMR (600 MHz, CDCl3) δ 1.82-1.91 (m, 2H), 2.52-2.58 (m, 4H), 2.74 (t, J = 6.3 Hz, 2H), 3.16-3.23 (m, 4H), 3.79 (s, 3H), 3.91-3.99 (m, 2H), 6.41 (d, J = 16.0 Hz, 1H), 6.77 - 6.84 (m, 2H), 7.32 - 7.42 (m, 2H), 7.39 - 7.52 (m, 4H), 7.64 (d, J = 16.0 Hz, 1H), 7.66-7.73 (m, 6H), 7.75-7.81 (m, 3H), 7.81 - 7.90 (m, 6H); 13 C NMR (151 MHz, CDCl3) δ 19.8 (d, J = 3.2 Hz), 20.1 (d, J = 51.8 Hz), 47.9, 51.7, 52.7, 57.1 (d, J = 16.5 Hz), 87.6, 92.5, 112.7, 114.9, 117.9, 118.2, 118.7, 125.8, 127.9, 130.4 (d, J= 12.5 Hz), 131.7, 132.7, 133.4, 133.6 (d, J = 10.0 Hz), 135.0 (d, J = 3.1 Hz), 144.0, 150.8, 167.3; IR (ATR) v max / cm -1 3362br, 2952w, 2876w, 2826w, 2206w, 1703m, 1630m, 1595s, 1519s, 1437s, 1425m, 1324m, 1240s, 1169s, 1111s, 996s, 823s; MS(ES): m / z = 649.4 [M] + ; C 43 H 42 N2O2P [M] + HRMS (ES) Calculated value: 649.2984, Measured value 649.2991.

[0265] 1.3. 6-methyl (2 E )-3-{4-[2-(4-{methyl[2-(4-methylbenzenesulfonamido) ethyl]amino}phenyl)ethynyl]phenyl}prop-2-enoate, 15 of synthesis

[0266] Methyl (2 E )-3-{4-[2-(4-{methyl[2-(4-methylbenzenesulfonamido) ethyl]amino}phenyl)ethynyl]phenyl}prop-2-enoate, 15 The synthesis of is illustrated in Fig. 2(v). Compound 12 (0.35 g, 1.05 mmol) was dissolved in DCM (30 mL), at this time p -Toluenesulfonyl chloride (0.24 g, 1.26 mmol) and Et3N (0.18 mL, 1.26 mmol) were added, and the resulting solution was stirred at RT for 16 hours. The solution was diluted with DCM, rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (0.5 g). This was purified by SiO2 chromatography (99:1, DCM / MeOH) to obtain the compound 15was obtained as a yellow solid (0.47 g, 92%): 1 H NMR (600 MHz, CDCl3) δ 2.42 (s, 3H), 2.92 (s, 3H), 3.15 (q, J = 6.4 Hz, 2H), 3.48 (t, J = 6.4 Hz, 2H), 3.81 (s, 3H), 4.78 (t, J = 6.4 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 6.57 - 6.62 (m, 2H), 7.29 (d, J = 8.1 Hz, 2H), 7.34 - 7.39 (m, 2H), 7.45 - 7.52 (m, 4H), 7.66 (d, J = 16.0 Hz, 1H), 7.70 - 7.74 (m, 2H); 13 C NMR (151 MHz, CDCl3) δ 21.5, 38.6, 40.3, 51.7, 52.2, 87.5, 92.8, 110.5, 112.0, 117.9, 126.0, 127.0, 128.0, 129.8, 131.6, 133.0, 133.3, 136.7, 143.6, 144.1, 148.8, 167.4; IR (ATR) v max / cm -1 3241br, 2949w, 2921w, 2857w, 2210m, 1711m, 1632w, 1595s, 1524s, 1320m, 1156s, 1145s, 819s; MS(ASAP): m / z = 489.2 [M+H] + ; C 28 H 29 N2O4S [M+H] + HRMS (ASAP) Calculated: 489.1848, Measured: 489.1866.

[0267] 1.3. 7 (4 Z )-1-(2- methoxyethyl )-2- methyl -4-[(4-{2-[4-(piperazine-1-yl)phenyl] to [Tinyl}phenyl)methylidene]-4,5-dihydro-1H-imidazole-5-one, 19 Synthesis of

[0268] (4 Z )-1-(2-methoxyethyl)-2-methyl-4-[(4-{2-[4-(piperazine-1-yl)phenyl] ethinyl}phenyl)methylidene]-4,5-dihydro-1H-imidazole-5-one, 19 The synthesis of is illustrated in Fig. 2(vi). Et3N (90 mL) was degassed by injecting Ar for 1 hour. Compound 4 (1.43 g, 4.97 mmol), compound 18 (1.60 g, 5.96 mmol), Pd(PPh3)2Cl2 (175 mg, 0.25 mmol), and CuI (48 mg, 0.25 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 18 hours. The suspension was diluted with CHCl3, the organic phase was rinsed with a saturated NaHCO3 solution, H2O, and brine, then dried (MgSO4) and evaporated to obtain an orange oil as a crude product. This was purified by SiO2 chromatography (92.5:7.5, DCM / MeOH, 1% Et3N), and the compound 19 was obtained as a bright orange solid (1.61 g, 76%): 1 H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 2.95 - 3.10 (m, 4H), 3.15 - 3.27 (m, 4H), 3.31 (s, 3H), 3.53 (t, J = 5.1 Hz, 2H), 3.78 (t, J = 5.1 Hz, 2H), 6.81 - 6.91 (m, 2H), 7.05 (s, 1H), 7.37 - 7.48 (m, 2H), 7.48 - 7.56 (m, 2H), 8.06 - 8.17 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 16.0, 41.0, 45.8, 49.2, 59.0, 70.5, 88.3, ​​92.7, 113.0, 115.0, 125.4, 126.1, 131.5, 131.9, 132.8, 133.5, 138.7, 151.4, 163.5, 170.6; IR (ATR) v max / cm -1 2943w, 2929w, 2206m, 1700s, 1639s, 1592s, 1561m, 1538m, 1519m, 1403m, 1357m, 1262s, 1136m, 835m; MS(ES): m / z = 429.2 [M+H] + ; C 26 H 29 N4O2[M+H] + HRMS (ES) Calculated value: 429.2291, Measured value 429.2279.

[0269] 1.3. 8 (4 Z )-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4-[(4-{2-[4-(piperazine-1-yl)phenyl]ethynyl}phenyl)methylidene]-4,5-dihydro-1H-imidazole-5-one, 23 Synthesis of

[0270] (4 Z )-1-[2-(morpholine-4-yl)ethyl]-2-phenyl-4-[(4-{2-[4-(piperazine-1-yl)phenyl]ethynyl}phenyl)methylidene]-4,5-dihydro-1H-imidazole-5-one, 23 The synthesis of is illustrated in Fig. 2(vii). Et3N (90 mL) was degassed by injecting Ar for 1 hour. Compound 4 (2.00 g, 6.94 mmol), compound 22(3.21 g, 8.33 mmol), Pd(PPh3)2Cl2 (250 mg, 0.35 mmol), and CuI (67 mg, 0.35 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 40 hours. The suspension was diluted with DCM, the organic phase was rinsed with a saturated NaHCO3 solution, H2O, and brine, then dried with (MgSO4) and evaporated to obtain an orange solid as the crude product. This was purified by SiO2 chromatography (95:5, DCM / MeOH, 1% Et3N) to obtain the compound 23 was obtained as a bright red solid (2.80 g, 74%): 1 ¹H NMR (400 MHz, CDCl₃) δ 1 H NMR (400 MHz, CDCl3) δ 2.23 - 2.32 (m, 4H), 2.45 (t, J = 6.3 Hz, 2H), 3.02 (s, 4H), 3.21 (s, 4H), 3.44 - 3.58 (m, 4H), 3.91 (t, J = 6.3 Hz, 2H), 6.80 - 6.91 (m, 2H), 7.20 (s, 1H), 7.40 - 7.47 (m, 2H), 7.48 - 7.65 (m, 5H), 7.75 - 7.89 (m, 2H), 8.13 - 8.23 ​​(m, 2H); 13 C NMR (101 MHz, CDCl3) δ 39.0, 53.6, 56.6, 66.8, 88.3, ​​93.1, 112.7, 114.9, 125.8, 127.8, 128.4, 128.8, 130.0, 131.2, 131.5, 132.3, 132.8, 133.5, 139.0, 151.5, 162.9, 171.6; MS(ES): m / z = 546.3 [M+H] + ; C 34 H 36 N5O2[M+H] + HRMS (ES) Calculated value: 546.2869, Measured value 546.2824.

[0271] 1.3. 9 tert -butyl (2 E )-3-(5-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }Thiophen-2-yl)prop-2-enoate, 27 of synthesis

[0272] tert -butyl (2 E )-3-(5-{2-[4-(piperazine-1-yl)phenyl]ethinyl}thiophene-2-yl) prop-2-enoate, 27 The synthesis of is illustrated in Fig. 2(viii). Et3N (75 mL) was degassed by injecting Ar for 1 hour. Compound 4 (2.31 g, 8.00 mmol), compound 26 (2.11 g, 9.01 mmol), Pd(PPh3)2Cl2 (280 mg, 0.4 mmol), and CuI (76 mg, 0.4 mmol) were added under Ar, and the formed suspension was stirred at 65°C for 72 hours. The suspension was diluted with DCM, rinsed with H2O and brine, and then dried (MgSO4) and evaporated to obtain an orange solid as the crude product. This was purified by SiO2 chromatography (92:8, DCM:MeOH) to obtain the compound 27 was obtained as a bright yellow / orange solid (1.4 g, 44%): 1 H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 3.35 - 3.43 (m, 4H), 3.53 - 3.61 (m, 4H), 6.13 (d, J = 15.7 Hz, 1H), 6.87 (d, J = 8.9 Hz, 2H), 7.10 (d, J = 3.9 Hz, 1H), 7.13 (d, J = 3.9 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 15.7 Hz, 1H); 13C NMR (151 MHz, CDCl3) δ 28.2, 44.9, 47.9, 80.6, 81.4, 96.0, 113.1, 115.4, 119.3, 126.2, 130.6, 132.0, 132.7, 135.5, 140.3, 150.8, 165.9; IR (ATR) v max / cm -1 2977w, 2929w, 2820w, 2194w, 1698s, 1617m, 1602m, 1526w, 1323m, 1141s, 812w; MS(ES): m / z = 395.3 [M+H] + ; C 23 H 27 N2O2S [M+H] + HRMS (ES) Calculated value: 395.1793, Measured value 395.1792.

[0273] 1.3. 10 methyl (2 E )-3-(4-{2-[4-( Azetidine -1-1)phenyl] Ethinyl }phenyl) Prof -2-Enoate, 30 Synthesis of

[0274] Methyl (2 E )-3-(4-{2-[4-(azetidine-1-yl)phenyl]ethynyl}phenyl)prop-2-enoate ( 30 The synthesis of ) is illustrated in FIG. 2(ix). Compound 29 (0.182 g, 1.16 mmol) was dissolved in Et3N (30 mL), and the solution was degassed by injecting Ar for 1 hour. Methyl (2 E)-3-(4-iodophenyl)prop-2-enoate (0.288 g, 1.0 mmol), Pd(PPh3)2Cl2 (35 mg, 0.05 mmol), and CuI (10 mg, 0.05 mmol) were added under Ar, and the resulting suspension was stirred at 60°C for 16 hours. The suspension was diluted with diethyl ether (Et2O), passed through Celite / SiO2, and evaporated to obtain a yellow solid crude product. This was purified by SiO2 chromatography (8:2, PE / EtOAc) and subsequent recrystallization from acetonitrile (MeCN) to obtain the compound 30 was obtained as a bright yellow crystalline solid (0.204 g, 64%): 1 H NMR (400 MHz, CDCl3) δ 2.38 (pent, J = 7.2 Hz, 2H), 3.81 (s, 3H), 3.90 - 3.97 (m, 4H), 6.35 - 6.40 (m, 2H), 6.43 (d, J = 16.0 Hz, 1H), 7.36 - 7.40 (m, 2H), 7.44 - 7.51 (m, 4H), 7.66 (d, J = 7.2 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 16.7, 51.7, 52.0, 87.2, 93.2, 110.4, 110.7, 117.8, 126.2, 127.9, 131.6, 132.7, 133.2, 144.1, 151.6, 167.3; IR (ATR) v max / cm -1 2963w, 2922w, 2855w, 2207m, 1713s, 1632m, 1595m, 1522m, 1366m, 1325m, 1314m, 1173s, 820s, 731s; MS(ES): m / z = 318.1 [M+H] + ; C 21 H 20 NO2[M+H] +HRMS (ES) Calculated: 318.1494, Measured: 318.1494.

[0275] 1.3.11 (4 Z )-1-(2- aminoethyl )-4-[(4-{2-[4-( Azetidine -1-1)phenyl] Ethinyl } phenyl) Methylidene ]-2-phenyl-4,5- Dihydro -1H-imidazole-5-one, 34 Synthesis of

[0276] (4 Z )-1-(2-aminoethyl)-4-[(4-{2-[4-(azetidine-1-yl)phenyl]ethynyl} phenyl) methylidene]-2-phenyl-4,5-dihydro-1H-imidazole-5-one ( 34 The synthesis of ) is exemplified in Fig. 2(x). Et3N (50 mL) was degassed by injecting Ar for 1 hour. Compound 33 (0.52 g, 1.4 mmol), compound 29 (0.25 g, 1.59 mmol), Pd(PPh3)2Cl2 (56 mg, 0.08 mmol), and CuI (15 mg, 0.08 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 20 hours. The solution was evaporated to obtain the crude residue, which was purified by SiO2 chromatography (97:3, DCM / MeOH, 1% Et3N) to obtain the compound 34 was obtained as a red solid (0.52 g, 83%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 2.33 (p, J = 7.3 Hz, 2H), 2.66 (t, J = 6.7 Hz, 2H), 3.73 (t, J = 6.7 Hz, 2H), 3.87 (t, J= 7.3 Hz, 4H), 6.36 - 6.44 (m, 2H), 7.17 (s, 1H), 7.34 - 7.38 (m, 2H), 7.51 - 7.57 (m, 2H), 7.58 - 7.66 (m, 3H), 7.89 - 7.94 (m, 2H), 8.24 - 8.33 (m, 2H).

[0277] 1.3. 12 methyl (2 E )-3-(5-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }pyridine-2-yl)prop-2-enoate, 43 of synthesis

[0278] Methyl (2 E The synthesis of )-3-(5-{2-[4-(piperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate (43) is illustrated in FIG. 2(xi). Et3N (125 mL) was degassed by injecting Ar for 1 hour. Compound 4 (2.88 g, 10.0 mmol), compound 42 (2.05 g, 11.0 mmol), Pd(PPh3)2Cl2 (350 mg, 0.5 mmol), and CuI (95 mg, 0.5 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 72 hours. Afterward, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (95:5 -> 9:1, DCM / MeOH, 1% Et3N) to obtain the compound 43 was obtained as a bright yellow solid (3.12 g, 90%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 3.08 - 3.40 (m, 4H), 6.91 (d, J = 15.7 Hz, 3H), 7.41 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 15.7 Hz, 1H), 7.78 (dd, J= 8.2, 0.8 Hz, 1H), 7.96 (dd, J = 8.1, 2.2 Hz, 1H), 8.73 (d, J = 2.1 Hz, 1H); 13 C NMR (101 MHz, DMSO) δ 51.8, 84.8, 95.7, 109.8, 114.3, 121.0, 121.5, 124.4, 132.7, 138.8, 143.0, 150.5, 151.6, 166.3; IR (ATR) v max / cm -1 2950m, 2835w, 2209m, 1711s, 1639m, 1605s, 1577m, 1516s, 1319s, 821s; M.S. (ES) m / z = 348.2 [M+H] + ; C 21 H 22 N3O2[M+H] + HRMS (ES) Calculated value: 348.1707, Measured value 348.1707.

[0279] 1.3. 13-methylpropyl (2 E )-3-(5-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }pyridine-2-yl)prop-2-enoate, 46 of synthesis

[0280] Methylpropyl (2 E )-3-(5-{2-[4-(piperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate ( 46 The synthesis of ) is illustrated in Fig. 2(xii). Et3N (60 mL) was degassed by injecting Ar for 1 hour. Compound 4 (0.74 g, 2.58 mmol), compound 45(0.65 g, 2.83 mmol), Pd(PPh3)2Cl2 (91 mg, 0.13 mmol), and CuI (25 mg, 0.13 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 72 hours. Afterward, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (95:5 -> 9:1, DCM / MeOH, 1% Et3N) to obtain the compound 46 was obtained as a bright yellow solid (0.62 g, 62%): 1 H NMR (400 MHz, CDCl3) δ 0.98 (d, J = 6.7 Hz, 6H), 2.01 (hept, J = 6.7 Hz, 1H), 2.93 - 3.07 (m, 4H), 3.17 - 3.28 (m, 4H), 4.01 (d, J = 6.7 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 15.7 Hz, 1H), 7.39 (dd, J = 8.1, 0.9 Hz, 1H), 7.45 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 15.7 Hz, 1H), 7.77 (dd, J = 8.0, 2.1 Hz, 1H), 8.73 (dd, J = 2.1, 0.8 Hz, 1H); IR (ATR) v max / cm -1 2959m, 2874w, 2834w, 2209m, 1709s, 1640m, 1605s, 1515s, 1203s, 1146s, 821s; M.S. (ES) m / z = 390.2 [M+H] + ; C 24 H 28 N3O2[M+H] + HRMS (ES) Calculated value: 390.2177, Measured value 390.2176.

[0281] 1.3. 14-methyl (2 E )-3-{5-[2-(4-{4-[7-( Hydroxycarbamoyl ) heptanoyl ]piperazine-1-yl}phenyl)ethinyl]pyridine-2-yl}prop-2-enoate, 51 of synthesis

[0282] Methyl (2 E The synthesis of )-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazine-1-yl}phenyl)ethynyl]pyridine-2-yl}prop-2-enoate (51) is illustrated in FIG. 2(xiii). 50 (0.78 g, 1.29 mmol) was dissolved in DCM / MeOH (1:2, 60 mL) and cooled to 0°C, at this time p TSA·H2O (0.32 g, 1.68 mmol) was added. The resulting solution was stirred at 0°C for 2 hours and further stirred at RT for 3.5 hours. It was then diluted with DCM, rinsed with a saturated NaHCO3 solution and H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (0.7 g). This was purified by SiO2 chromatography (95:5 -> 9:1 DCM / MeOH) to obtain the compound 51 was obtained as a bright yellow solid (280 mg, 42%): 1 1H NMR (700 MHz, DMSO- d 6) δ 1.23 - 1.28 (m, 4H), 1.44 - 1.49 (m, 4H), 1.92 (t, J = 7.4 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 3.23 (t, J = 5.4 Hz, 2H), 3.26 - 3.29 (m, 2H), 3.58 (t, J = 5.4 Hz, 4H), 3.74 (s, 3H), 6.90 (d, J= 15.7 Hz, 1H), 6.96 - 7.00 (m, 2H), 7.42 - 7.45 (m, 2H), 7.68 (d, J = 15.7 Hz, 1H), 7.75 - 7.83 (m, 1H), 7.96 (dd, J = 8.1, 2.2 Hz, 1H), 8.63 (s, 1H), 8.73 (d, J = 2.1 Hz, 1H), 10.31 (s, 1H); 13 C NMR (176 MHz, DMSO- d 6) δ 24.6, 25.0, 28.4, 28.5, 32.2, 32.2, 40.5, 44.4, 46.9, 47.2, 51.7, 84.9, 95.4, 110.4, 114.7, 120.9, 121.5, 124.4, 132.7, 138.8, 142.9, 150.5, 150.8, 151.6, 166.3, 169.1, 170.7; IR (ATR) v max / cm -1 3241br, 2933w, 2910w, 2846w, 2212w, 1723m, 1650s, 1601s, 1514m, 1231m, 1207m, 1033m, 830m; MS(ES): m / z = 519.3 [M+H] + ; C 29 H 35 N4O5[M+H] + HRMS (ES) 계산치: 519.2603, 실측치 519.2602.

[0283] 1.3. 15 2 - Methylpropyl (2 E )-3-{5-[2-(4-{4-[7-( Hydroxycarbamoyl ) heptanoyl ] piperazine-1-yl}phenyl)ethinyl]pyridine-2-yl}prop-2-enoate, 55 of synthesis

[0284] 2-메틸프로필 (2 E)-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazine-1-yl}phenyl)ethynyl]pyridine-2-yl}prop-2-enoate ( 55 The synthesis of ) is illustrated in Fig. 2(xiv). Compound 54 (0.55 g, 0.85 mmol) was dissolved in DCM / MeOH (1:2, 60 mL) and cooled to 0°C, to p TSA·H2O (0.21 g, 1.11 mmol) was added. The resulting solution was stirred at 0°C for 2 hours and further stirred at RT for 3.5 hours. It was then diluted with DCM, rinsed with a saturated NaHCO3 solution and H2O, dried (MgSO4), and evaporated to obtain a yellow solid crude product (0.7 g). This was purified by SiO2 chromatography (9:1, DCM / MeOH) to obtain the compound 55 was obtained as a bright yellow solid (340 mg, 71%): 1 1H NMR (700 MHz, DMSO- d 6) δ 0.94 (d, J = 6.7 Hz, 6H), 1.23 - 1.30 (m, 4H), 1.46 - 1.51 (m, 4H), 1.90 - 2.01 (m, 3H), 2.33 (t, J = 7.5 Hz, 2H), 3.23 (t, J = 5.5 Hz, 2H), 3.29 (t, J = 5.5 Hz, 2H), 3.59 (t, J = 5.3 Hz, 4H), 3.97 (d, J = 6.6 Hz, 2H), 6.92 (d, J = 15.8 Hz, 1H), 6.96 - 7.01 (m, 2H), 7.40 - 7.48 (m, 2H), 7.68 (d, J = 15.8 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.96 (dd, J = 8.1, 2.2 Hz, 1H), 8.64 (d,J = 1.5 Hz, 1H), 8.74 (d, J = 2.2 Hz, 1H), 10.32 (s, 1H); 13 13C NMR (176 MHz, DMSO- d 6) δ 18.9, 24.6, 25.0, 27.3, 28.4, 28.5, 32.2, 32.2, 40.5, 44.4, 46.9, 47.2, 70.1, 84.9, 95.4, 110.4, 114.7, 120.8, 121.9, 124.3, 132.6, 132.8, 138.7, 138.9, 142.7, 142.8, 150.6, 150.8, 151.6, 151.6, 165.8, 169.1, 170.7; IR (ATR) v max / cm -1 3245br, 2933m, 2846m, 2212w, 1710m, 1649s, 1601s, 1544m, 1369m, 1231s, 1031m, 971m; MS(ES): m / z = 561.3 [M+H] + ; C 32 H 41 N4O5[M+H] + HRMS (ES) Calculated value: 561.3071, Measured value 561.3071.

[0285] 1.3. 16 tert -butyl (2 E )-3-{4-[2-(4-{4-[7-( Hydroxycarbamoyl ) heptanoyl ]piperazine-1-yl}phenyl)ethinyl]phenyl}prop-2-enoate, 57 of synthesis

[0286] tert -butyl (2 E )-3-{4-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazine-1-yl}phenyl)ethynyl]phenyl}prop-2-enoate ( 57 The synthesis of ) is illustrated in Fig. 2(xv). Compound 56Dissolve (0.14 g, 0.22 mmol) in DCM / MeOH (1:4, 12.5 mL) It was cooled to 0℃, and at this time p TSA·H2O (12.7 mg, 0.067 mmol) was added, and the resulting solution was stirred at 0°C for 2 hours, followed by stirring at RT for 2 hours. The solution was evaporated to obtain the crude product solid, which was then purified by SiO2 chromatography (95:5 -> 9:1, DCM / MeOH) to obtain the compound 57 was obtained as a yellow solid (67.5 mg, 55%): 1 1H NMR (600 MHz, DMSO- d 6) δ 1.23 - 1.30 (m, 4H), 1.46 - 1.50 (m, 12H), 1.93 (t, J = 7.4 Hz, 2H), 2.33 (t, J = 7.4Hz, 2H), 3.19 - 3.24 (m, 2H), 3.24 - 3.29 (m, 2H), 3.58 (t, J = 4.9 Hz, 4H), 6.56 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 16.0 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 8.66 (s, 1H), 10.33 (s, 1H); 13 13C NMR (176 MHz, DMSO- d6) δ 24.6, 25.0, 27.8, 28.4, 28.5, 32.2, 32.2, 40.6, 44.4, 47.0, 47.4, 80.0, 87.6, 92.4, 111.1, 114.8, 120.5, 124.6, 128.5, 131.4, 132.5, 133.7, 142.6, 150.6, 165.4, 169.1, 170.7; IR (ATR) v max / cm -1 3231br, 2929w, 2854w, 2206w, 1704m, 1653s, 1632m, 1598s, 1540m, 1324m, 1234s, 1154s, 1054m, 968m, 826s; MS(ES): m / z = 560.3 [M+H] + ; C 33 H 42 N3O5[M+H] + HRMS (ES) Calculated value: 560.3119, Measured value 560.3119.

[0287] 1.3. 17 tert -butyl (2 E )-3-{5-[2-(4-{4-[7-( Hydroxycarbamoyl ) heptanoyl ]piperazine-1-yl}phenyl)ethinyl]thiophene-2-yl}prop-2-enoate, 59 of synthesis

[0288] tert -butyl (2 E )-3-{5-[2-(4-{4-[7-(hydroxycarbamoyl)heptanoyl]piperazine-1-yl}phenyl)ethinyl]thiophene-2-yl}prop-2-enoate ( 59 The synthesis of ) is illustrated in Fig. 2(xvi). Compound 58 (0.3 g, 0.46 mmol) was dissolved in DCM / MeOH (1:4, 12.5 mL) and cooled to 0°C, at this time pTSA·H2O (27 mg, 0.14 mmol) was added. The resulting solution was stirred at 0°C for 2 hours, then stirred again at RT for 2 hours, and evaporated to obtain a yellow oil as a crude product. This was purified by SiO2 chromatography (DCM / MeOH, 95:5 -> 9:1) to obtain the compound 59 was obtained as a bright yellow solid (49 mg, 19%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 1.21 - 1.30 (m, 4H), 1.43 - 1.56 (m, 13H), 1.93 (t, J = 7.3 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H), 3.18 - 3.26 (m, 2H), 3.26 - 3.31 (m, 2H), 3.54 - 3.64 (m, 4H), 6.18 (d, J = 15.7 Hz, 1H), 6.97 (d, J = 9.0 Hz, 2H), 7.32 (d, J = 3.8 Hz, 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.49 (d, J = 3.8 Hz, 1H), 7.66 (dd, J = 15.7, 0.6 Hz, 1H), 8.65 (s, 1H), 10.32 (s, 1H); 13 C NMR (176 MHz, DMSO) δ 24.6, 25.0, 27.8, 28.4, 28.5, 32.2, 32.2, 40.5, 44.4, 46.8, 47.2, 80.2, 80.9, 96.6, 110.2, 114.7, 118.9, 125.3, 132.2, 132.5, 132.7, 135.6, 139.6, 150.8, 165.1, 169.1, 170.7; IR (ATR) v max / cm -13235br, 2978w, 2928w, 2855w, 2832w, 2188w, 1704m, 1654s, 1603s, 1525m, 1249s, 1145s; M.S. (ES) m / z = 566.2 [M+H] + ; C 31 H 30 N3O5S [M+H] + HRMS (ES) Calculated value: 566.2689, Measured value 566.

[0289] 1.3.18 2-(2- Methoxyethoxy )ethyl-(2 E )-3-(4-{2-[4-(piperazine-1 yl)phenyl] Ethinyl }phenyl) Prof -2- Enoate , 62 Synthesis of

[0290] 2-(2-methoxyethoxy)ethyl-(2 E )-3-(4-{2-[4-(piperazine-1-yl)phenyl]ethynyl}phenyl) prop-2-enoate ( 62 The synthesis of ) is illustrated in FIG. 2(xvii). Compound 4 (788 mg, 2.73 mmol), compound 61 (788.3 mg, 2.87 mmol), Pd(PPh3)2Cl2 (91.24 mg, 0.13 mmol), and CuI (24.75 mg, 0.13 mmol) were added to a Schlenk flask under Ar. Degassed Et3N (10 mL) was added, and the resulting suspension was stirred at 60°C for 24 hours. Afterward, the solvent was evaporated to obtain an orange solid as a crude product, which was purified by SiO2 chromatography (9:1, DCM / MeOH) to obtain the compound 62 It was obtained as an orange solid (794 mg, 67%). 1H NMR (CDCl3, 400 MHz) δ 3.16-3.24 (m, 2H), 3.4 (s, 3H), 3.46-3.51 (m, 4H), 3.56-3.59 (m, 2H), 3.63-3.70 (m, 6H), 3.77-3.80 (m, 2H), 4.36-4.40 (m, 2H), 6.48 (d, J = 16 Hz, 1H), 6.88 (dt, J 8.9, 2 Hz, 2H), 7.46-7.52 (m, 6H), 7.68 (d, J = 16 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 166.95, 144.31, 133.17, 132.01, 128.18, 116.68, 72.06, 70.69, 69.45, 63.87, 59.27, 46.51, 46.00, 43.47, 8.80; C 26 H 31 N2O4[M+H] + HRMS (ESI) calculated value 435.2284, measured value 435.2283.

[0291] 1.3. 19 2 -(2- Methoxyethoxy )ethyl(2E)-3-{4-[2-(4-{4-[8-( Hydroxyamino ) Octanoyl]piperazine-1-yl}phenyl) ethinyl]phenyl}prop-2-enoate, 64 of synthesis

[0292] 2-(2-methoxyethoxy)ethyl (2 E )-3-{4-[2-(4-{4-[8-(hydroxyamino)octanoyl]piperazine-1-yl}phenyl)ethynyl]phenyl}prop-2-enoate ( 64 The synthesis of ) is illustrated in FIG. 2(xviii). Compound 63(384 mg, 0.55 mmol) was dissolved in DCM:MeOH (1:2), and after cooling the resulting solution to 0°C, para-toluenesulfonic acid monohydrate (pTsOH·H2O) (56.3 mg, 0.28 mmol) was added. Then, the reaction mixture was stirred at RT for 5 hours. Additionally p TsOH·H2O (56.3 mg, 0.28 mmol) was added, and the reaction mixture was stirred at RT for an additional 16 hours. Afterward, the crude reaction product was diluted in DCM, rinsed with a saturated NaHCO3 solution and brine, and then MgSO4 An orange solid crude product was obtained by drying and evaporating on a surface. The crude product was purified by SiO2 column chromatography (DCM:MeOH, 9:1 as eluent) to obtain the compound 64 was obtained as an orange solid (60.3 mg, 18%): 1 ¹H NMR (DMSO-d6, 400 MHz) δ 1.22-1.32 (m, 6H), 1.44-1.52 (m, 6H), 1.93 (t J 14.7 Hz, 7.3 Hz, 2H), 2.33 (t J 14.7 Hz, 7.3 Hz, 3H), 3.19-3.23 (m, 4H), 3.24 (s, 3H), 3.43-3.46 (m, 3H), 3.54-3.60 (m, 8H), 3.65-3.69 (m, 2H), 4.23-4.29 (m, 3H), 6.72 (d J 16 Hz, 1H), 6.97 (d J 8.9 Hz, 2H), 7.42 (d J 8.9 Hz, 2H), 7.52 (d J 8.4 Hz, 2H), 7.67 (d J 16 Hz, 1H), 7.7 (d J 8.4 Hz, 1H), 8.64-8.67 (m, 1H), 10.33 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ 132.39, 128.49, 114.60, 71.04, 69.39, 57.88, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89, 38.69, 32.03, 28.23, 24.83; C 34 H 44 N3O7[M+H] + HRMS (ESI) Calculated value: 606.3179, Measured value 606.3193.

[0293] 1.3. 20 2 - Methylpropyl (2E)-3-(6-{2-[4-(piperazine-1-yl)phenyl] Ethinyl }pyridine-3-yl)prop-2-enoate, 69 of synthesis

[0294] 2-methylpropyl (2E)-3-(6-{2-[4-(piperazine-1-yl)phenyl]ethynyl}pyridine-3-yl)prop-2-enoate ( 69 The synthesis of ) is illustrated in FIG. 2(xix). Compound 4 (1.21 g, 4.2 mmol), compound 68 (1.0 g, 4.4 mmol), Pd(PPh3)2Cl2 (147 mg, 0.21 mmol), and CuI (39 mg, 0.21 mmol) were placed in a round-bottom Schlenk flask under Ar, and then 50 mL of Et3N, which had been degassed with N2 for 1 hour, was added. The prepared reaction mixture was stirred at 60°C for 24 hours. After purification by SiO2 column chromatography (DCM:MeOH, 9:1), the compound 69 It was obtained as a bright yellow solid (1.1 g, 67%). 1 H NMR (400 MHz, CDCl3) d 0.99 (d J 6.7 Hz, 6H), 1.98 - 2.05 (m, 1H), 3.20 - 3.26 (m, 4H), 3.40 - 3.44 (m, 4H), 4.01 (d) J6.7 Hz, 2H), 6.52 (d J 16.0 Hz, 1H), 6.88 (d J 9.0Hz, 2H), 7.48 - 7.55 (m, 3H), 7.65 (d J 16.0 Hz, 1H), 7.81 (dd J 8.45, 2.2 Hz, 1H), 8.72 (d J 2.2 Hz, 1H); 13 C NMR (101 MHz, CDCl3) δ 166.51, 151.05, 150.13, 144.99, 140.39, 138.20, 134.32, 133.67, 126.91, 120.65, 119.00, 115.71, 92.36, 88.06, 71.10, 44.61, 27.97, 19.29; C 24 H 28 N3O2[M+H] + HRMS (ESI) Calculated value: 390.2182, Measured value 390.2181.

[0295] 1.3. 21 2 - Methylpropyl (2 E )-3-{6-[2-(4-{4-[7-( Hydroxycarbamoyl ) heptanoyl ] piperazine-1-yl}phenyl) ethinyl]pyridine-3-yl}prop-2-enoate, 71 of synthesis

[0296] 2-methylpropyl (2 E )-3-{6-[2-(4-{4-[7-(hydroxycarbamoyl) heptanoyl]piperazine-1-yl}phenyl) ethinyl]pyridine-3-yl}prop-2-enoate ( 71 The synthesis of ) is illustrated in FIG. 2(xx). Compound 70 (500 mg, 0.76 mmol) was dissolved in DCM:MeOH (1:2), and the obtained solution was cooled to 0°C. pTsOH·H2O (197.6 mg, 0.988 mmol) was added, and the reaction mixture was heated to RT and stirred continuously for 6 hours. The crude product reaction mixture was diluted in DCM, rinsed with a saturated NaHCO3 solution and brine, and then dried and evaporated over MgSO4 to obtain a bright yellow solid as the crude product (0.3 g). Subsequently, this was purified by SiO2 column chromatography (DCM:MeOH, 9:1) to obtain the compound 71 was obtained as a bright yellow solid (90.4 mg, 21%): 1 H NMR (400 MHz, DMSO-d6) d 0.95 (d J 6.7 Hz, 6H), 1.22-1.31 (m, 6H), 1.44-1.53 ​​(m, 6H), 1.91-1.95 (m, 2H), 1.96-2.00 (m, 1H), 3.55-3.62 (m, 4H), 3.97 (d) J 6.6 Hz, 2H), 6.85 (d J 16.0 Hz, 1H), 7.01 (d J 9.0Hz, 2H), 7.44-7.52 (m, 3H), 7.72 (d J 16.0 Hz, 1H), 8.23 ​​(dd J 8.4 Hz, 2.3 Hz, 1H), 8.88-8.91 (m, 1H), 10.34 (s, 1H); C 32 H 41 N4O5[M+H] + HRMS (ESI) Calculated value: 561.3077, Measured value 561.3087.

[0297] 1.3. 22-methyl (2 E )-3-(5-{2-[4-(4- Methylpiperazine -1-1)phenyl] Ethinyl }pyridine-2-yl)prop-2-enoate, 73 of synthesis

[0298] Methyl (2 E)-3-(5-{2-[4-(4-methylpiperazine-1-yl)phenyl]ethynyl}pyridine-2-yl)prop-2-enoate ( 73 The synthesis of ) is illustrated in Fig. 2(xxi). Et3N (60 mL) was degassed by injecting Ar for 1 hour. Compound 72 (1.11 g, 3.66 mmol), compound 42 (0.75 g, 4.02 mmol), Pd(PPh3)2Cl2 (128 mg, 0.18 mmol), and CuI (34 mg, 0.18 mmol) were added under Ar, and the formed suspension was stirred at 60°C for 72 hours. Subsequently, the solvent was evaporated to obtain the crude product solid, which was purified by SiO2 chromatography (95:5 -> 9:1, DCM / MeOH, 1% Et3N) and subsequent recrystallization from MeCN, thereby obtaining the compound 73 was obtained as a bright yellow solid (1.02 g, 77%): 1 H NMR (700 MHz, CDCl3) δ 2.35 (s, 3H), 2.56 (t, J = 5.0 Hz, 4H), 3.26 - 3.30 (m, 4H), 3.82 (s, 3H), 6.87 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 15.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.41 - 7.46 (m, 2H), 7.66 (d, J = 15.6 Hz, 1H), 7.76 (dd, J = 8.0, 2.1 Hz, 1H), 8.72 (d, J = 2.1 Hz, 1H); 13C NMR (176 MHz, CDCl3) δ 46.1, 47.9, 51.8, 54.8, 84.8, 95.4, 111.9, 114.9, 121.6, 122.0, 123.5, 132.9, 138.5, 142.9, 150.8, 151.3, 152.2, 167.2; IR (ATR) v max / cm -1 3066w, 3036w, 2878w, 2797w, 2212m, 1714s, 1640m, 1603m, 1543m, 1515s, 1305s, 1241s, 1190s, 1161s, 1006m; M.S. (ES) m / z = 362.2 [M+H] + ; C 22 H 24 N3O2[M+H] + HRMS (ES) Calculated: 362.1863, Measured: 362.1863.

[0299] 1.3. 23 4 -[( E )-2-(5-{2-[4-(morpholine-4-yl)phenyl] Ethinyl }pyridine-2-il) Ethe [Nyl]-1,3-thiazole-2-amine, 84 of synthesis

[0300] 4-[( E )-2-(5-{2-[4-(morpholine-4-yl)phenyl]ethynyl}pyridine-2-yl)ethenyl]-1,3-thiazole-2-amine ( 84 The synthesis of ) is illustrated in Fig. 2(xxii). A mixture of Et3N (30 mL) and DMF (60 mL) was degassed by injecting Ar for 1 hour. Compound 83 (2.3 g, 8.0 mmol), compound 82(2.0 g, 8.8 mmol), Pd(PPh3)2Cl2 (281 mg, 0.4 mmol), and CuI (76 mg, 0.4 mmol) were added under Ar, and the resulting solution was stirred at 60°C for 72 hours. The suspension was cooled, H2O was added, and the mixture was filtered to obtain a brown solid as the crude product. This was suspended in a DCM / EtOAc / acetone (1:1:1) mixture, stirred for 0.5 hours, and then filtered to obtain the compound 84 was obtained as a bright yellow solid (3.03 g, >100%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 3.18 - 3.23 (m, 4H), 3.73 (t, J = 5.1 Hz, 4H), 6.82 (s, 1H), 6.97 (d, J = 8.3 Hz, 3H), 7.06 - 7.17 (m, 3H), 7.36 - 7.45 (m, 3H), 7.49 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 8.64 (dd, J = 0.8 Hz, 1H).

[0301] Examples 2: of example compounds light absorption and fluorescence emission measurement

[0302] The peak absorption and fluorescence emission wavelengths of compounds 6, 7, 12, 13, 14, 15, 19, 23, 27, 30, and 34 were measured in various solvents, and the results are shown in Table 1. Absorption measurements were recorded at a concentration of 10 μM, and emission measurements were recorded at a concentration of 100 nM. The emission spectrum was recorded by excitation at the absorption peak (S0->S1).

[0303] compound menstruum λ abs (max) / nm λ em (max) / nm 6 toluene 358 482 DCM 368 550 7 toluene 361 504 DCM 362 563 12 toluene 380 482 DCM 371 551 13 toluene 358 464 DCM 361 547 14 toluene 367 506 DCM 361 531 15 toluene 381 473 DCM 377 545 19 toluene 403 515 chloroform 403 584 MeOH 395 - 23 chloroform 424 616 27 toluene 380 493 DCM 371 524 30 chloroform 374 535 34 chloroform 432 628

[0304] Table 1: Peak absorption and emission wavelengths of compounds 6, 7, 12, 13, 14, 15, 19, 23, 27, 30, and 34 in various solvents.

[0305] Examples 3: of para-substituted compounds and ortho-substituted compounds Mineraphysical Comparison of characteristics

[0306] To compare the photophysical behavior of the para-substituted compound of the present invention with that of the ortho-substituted compound, compound 73 and reference compound 77 were synthesized according to Example 1:

[0307]

[0308] Solutions of Compound 73 and Compound 77 were prepared in chloroform at concentrations of 10 μM and 100 nM, respectively. The absorption spectra of each compound (10 μM) were recorded from 200 to 800 nm using a CARY100 UV-Visible spectrometer and are shown in Fig. 3a after limiting the solvent background. Fig. 3a shows a decrease in the absorption coefficient and a substantial hypochromic shift as a result of the donor moiety shifting from the para-position of Compound 73 to the ortho-position of Compound 77. Additionally, Fig. 3a shows the approximate bandwidth of a 405 nm violet-excited laser light source, which is typical in fluorescence microscopy used for cell imaging studies. Compound 73 While silver was efficiently excited by a light source, 77 It absorbed very weakly at this wavelength.

[0309] Evaluate these effects and 73 and 77 To compare the fluorescence emission characteristics of these compounds, chloroform (100 nM) solutions were excited at both 360 nm and 405 nm. Upon excitation at 360 nm, this wavelength approached the maximum absorption of these compounds, 73 and 77This was excited very efficiently. Fig. 3b shows that although these two types of compounds can be excited at this wavelength, the compound 73 This shows that it emits substantially stronger fluorescence as a result of the improvement in quantum yield. In addition, the compound 73 silver compounds 77 Compared to, it exhibited a significant long-wavelength shift (bathochromic shift), which means that charge transfer is more efficient in para-substituted compounds, which translates to a more significant dipole moment across the entire molecule, i.e., a larger Stokes shift.

[0310] Both of these compounds were excited at 405 nm to compare whether they were suitable for imaging using a typical fluorescence microscope. Figure 3c shows the compound when excited at 405 nm. 73 While the emission intensity from is similar to that at 360 nm excitation, the compound 77 It shows that it does not absorb efficiently at 405 nm and emits very weak fluorescence at 405 nm. Therefore, 77 It is not a suitable fluorophor for cell imaging experiments using a 405 nm excitation source.

[0311] In conclusion, the para-substituted diphenylacetylene fluorophores exhibit improved photophysical properties compared to the corresponding ortho-substituted compounds, due to stronger and longer wavelength light absorption and more efficient fluorescence emission with increased charge transfer behavior.

[0312] Examples 4: Conjugate Synthesis

[0313] 4.1 Conjugation to anticancer drug molecules

[0314] Vorinostat, an approved anticancer drug, was conjugated to Compound 6. To evaluate the effect of the conjugate on the activity of vorinostat, three compounds were prepared: THP-protected analogs of vorinostat (Compound 37); a form in which a THP-protected analog of vorinostat is conjugated to compound 6 (compound 38 ); a form of compound 6 conjugated with an unprotected borinostat analog (compound 39 ).

[0315] 4.1.1 Borinostat's THP -Protected analogue (compound) 37 )of synthesis

[0316] The synthesis of a protected analog of vorinostat is illustrated in Fig. 4(a). Ethyl 4-aminobenzoate (16.87 g, 102 mmol) was dissolved in anhydrous THF under N2. Oxanone-2,9-dione (anhydrous cerberic acid) (15.95 g, 102 mmol) was added, and the resulting solution was stirred at RT for 16 hours. The suspension was diluted with H2O, and the precipitate was filtered and rinsed with H2O. This was purified by SiO2 chromatography (7:3 -> 1:1, heptane / EtOAc) to obtain the compound 35 was obtained as a white solid (6.62 g, 20%), and this was immediately transferred to the next step: 1 ¹H NMR (400 MHz, DMSO- d 6) δ 1.22 - 1.34 (m, 7H), 1.42 - 1.53 (m, 2H), 1.53 - 1.64 (m, 2H), 2.15 - 2.22 (m, 2H), 2.33 (t, J = 7.4 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 7.70 - 7.74 (m, 2H), 7.86 - 7.91 (m, 2H), 10.20 (s, 1H), 11.94 (br, 1H). Compound 351-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).HCl (1.28 g, 6.70 mmol) and hydroxybenzothiazole (HOBt) (hydrate, 0.91 g, 6.7 mmol) were added to this, and the formed suspension was stirred at RT for 0.5 hours. O -(tetrahydro-2 H -pyran-2-yl)hydroxylamine (0.78 g, 6.70 mmol) and N , N Diisopropylethylamine (DIPEA) (1.46 mL, 8.40 mmol) was added, and the solution was stirred at RT for 16 hours. The solution was diluted with H2O and extracted with DCM. The organic phase was rinsed with H2O, dried (MgSO4), and evaporated to obtain a bright yellow oil as the crude product. This was purified by SiO2 chromatography (7:3, heptane / acetone) to obtain the compound 36 ...was obtained as an off-white solid (0.81 g, 34%) and was passed directly to the next step without further purification. Compound 36 (0.62 g, 1.47 mmol) and NaOH (0.13 g, 3.13 mmol) were dissolved in MeOH / H2O (18 mL, 2:1), and the resulting solution was stirred at 50°C for 16 hours. The solution was cooled, diluted with H2O, acidified to pH 4, and extracted with EtOAc. The organic phase was rinsed with H2O and brine, then dried with (MgSO4) and evaporated to obtain the compound 37 was obtained as a white solid (0.44 g, 76%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 1.20 - 1.34 (m, 4H), 1.44 - 1.69 (m, 10H), 1.97 (t, J = 7.3 Hz, 2H), 2.33 (t, J= 7.4 Hz, 2H), 3.45 - 3.52 (m, 1H), 3.87 - 3.94 (m, 1H), 4.79 (br, 1H), 7.67 - 7.72 (m, 2H), 7.84 - 7.89 (m, 2H), 10.17 (s, 1H), 10.90 (s, 1H), 12.68 (br, 1H); 13 13C NMR (101 MHz, DMSO- d 6) δ 18.3, 24.7, 27.8, 28.3, 28.4, 32.1, 36.4, 61.3, 100.8, 118.2, 124.8, 130.4, 143.4, 166.9, 169.0, 171.8; IR (ATR) v max / cm -1 3301w, 2972w, 2944w, 2855w, 1662s, 1593m, 1523m, 1405m, 1295m, 913m, 734s; MS(ES): m / z = 393.4 [M+H] + ; C 20 H 29 N2O4[M+H] + HRMS (ES) Calculated: 393.2026, Measured: 393.2027.

[0317] 4.1.2 to Compound 6 Borinostat's THP -Protected analogue bonded Form (compound) 38 Synthesis of )

[0318] compound 37 (0.36 g, 0.9 mmol) was dissolved in anhydrous DMF (10 mL) under N2, EDC.HCl (0.18 g, 1.17 mmol) and HOBt (hydrate, 0.12 g, 0.9 mmol) were added to this, and the formed suspension was stirred at RT for 0.5 hours. Compound 6(0.35 g, 0.9 mmol) and DIPEA (0.24 mL, 1.35 mmol) were added, and the solution was stirred at RT for 40 hours. The solution was diluted with H2O and extracted with DCM. The organic phase was rinsed with H2O, dried (MgSO4), and evaporated to obtain a yellow oil as a crude product (0.69 g). This was purified by SiO2 chromatography (97:3, DCM / MeOH) to obtain the compound 38 was obtained as a yellow solid (0.54 g, 79%): 1 H NMR (400 MHz, CDCl3) δ 1.20 - 1.35 (m, 4H), 1.52 (s, 9H), 1.53 - 1.70 (m, 7H), 1.72 - 1.82 (m, 3H), 2.02 - 2.12 (m, 2H), 2.31 (t, J = 7.4 Hz, 2H), 3.25 (br, 4H), 3.57 - 4.00 (m, 6H), 4.95 (s, 1H), 6.36 (d, J = 16.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.41 - 7.50 (m, 6H), 7.54 (d, J = 16.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H), 8.67 (s, 1H), 9.36 (s, 1H); 13 C NMR (101 MHz, CDCl3) δ 18.5, 24.9, 25.0, 25.2, 28.0, 28.1, 28.3, 28.5, 32.9, 37.1, 48.6, 62.4, 80.6, 88.0, 91.8, 102.3, 114.0, 115.7, 119.5, 120.5, 125.2, 127.8, 128.1, 130.0, 131.7, 132.8, 133.9, 140.3, 142.7, 150.3, 166.2, 170.3, 170.7, 172.4; IR (ATR) vmax / cm -1 3252br, 2933w, 2858w, 2251w, 2210w, 1698m, 1666m, 1630m, 1596s, 1519s, 1436m,1235m, 1152s, 1136s, 731s; MS(ES): m / z = 763.5 [M+H] + ; C 45 H 55 N4O7[M+H] + HRMS (ES) Calculated value: 763.4071, Measured value 763.4086.

[0319] 4.1.3 Unprotected in Compound 6 Borinostart analogue bonded form (compound 39 Synthesis of )

[0320] compound 38 (0.36 g, 0.47 mmol) was dissolved in MeOH / DCM (20 mL, 3:1) and cooled to 0°C. p -Toluenesulfonic acid ( p After adding TSA).H2O (29 mg, 0.15 mmol), the formed solution was high-speed stirred at RT for 3 hours. p TSA·H2O (14 mg, 0.075 mmol) was additionally added, and the solution was stirred for 1 hour. The solution was evaporated to obtain a yellow solid as a crude product, which was purified by SiO2 chromatography (95:5, DCM / EtOH -> 9:1, DCM / MeOH) to obtain a bright yellow solid, which was further recrystallized from EtOH to form a compound 39 was obtained as a pale yellow solid (131 mg, 41%): 1 ¹H NMR (400 MHz, DMSO- d 6) δ 1.21-1.35 (m, 4H), 1.48 (s, 9H), 1.51-1.64 (m, 4H), 1.94 (t, J = 7.4 Hz, 2H), 2.31 (t,J = 7.4 Hz, 2H), 3.25-3.42 (m, 4H), 3.62 (br, 4H), 6.54 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 7.40-7.43 (m, 4H), 7.51 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 16.0 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 8.66 (s, 1H), 10.06 (s, 1H), 10.33 (s, 1H); 13 C NMR (101 MHz, DMSO- d 6) δ 25.0, 25.0, 27.9, 28.4, 32.3, 36.4, 47.3, 80.1, 87.7, 92.5, 111.3, 114.9, 118.4, 120.5, 124.7, 128.2, 128.5, 129.8, 131.4, 132.6, 133.7, 140.7, 142.7, 150.6, 165.5, 169.0, 169.1, 171.6; IR (ATR) v max / cm -1 3285br, 2975w, 2931w, 2851w, 2822w, 2208w, 2167w, 1706m, 1655m, 1626m, 1596s, 1520s, 1391m, 1234m, 1154s, 1136s, 976m, 825s, 736s; MS(ES): m / z = 679.6 [M+H] + ; C 40 H 47 N4O6[M+H] + HRMS (ES) 계산치: 679.3496, 실측치 679.3510.

[0321] Examples 5: Conjugate Analysis

[0322] 5.1 Cells Survivability analyze

[0323] Cell viability was measured using the CellTitreGlo® assay according to the manufacturer's instructions. Two types of HPV-negative primary oral squamous cell carcinoma cells (SJG-26 and SJG-41) were treated with Compounds 37, 38, and 39 for 72 hours, followed by the assay. The cells were not irradiated with light. IC50 of vorinostat alone (not shown). 50 was confirmed to be 1.6 μM; the IC of compound 39 50 The values ​​were nearly identical (1.3 μM for SJG-26 and 1.4 μM for SJG-41). The analysis results are shown in Figures 5a (cell line SJG-26) and 5b (cell line SJG-41).

[0324] 5.2 MTT cell Survivability analyze

[0325] MTT assay was performed according to the following procedure: cells were treated with compounds 37 / 38 / 39 at various concentrations for 1 hour at 37°C / 5% CO2, and 56 Jmm -2 The cells were irradiated for 5 minutes. Afterward, the cells were incubated for 24 hours at 37°C / 5% CO2. The culture medium was removed, and the cells were rinsed with PBS. Phenol-free medium was added, followed by the addition of 12 mM MTT stock solution, and the cells were incubated at 37°C for 2 hours. DMSO was additionally added, and the cells were incubated in a humid chamber at 37°C. Subsequently, the absorbance was measured at 540 nm to determine the degree of cell viability. The results are shown in Figure 6.

[0326] MTT cell viability analysis in SJG-41 cells treated with Compound 37, Compound 38, Compound 39, and vorinostat for 24 hours prior to analysis. Note that analysis measurements were standardized against DMSO-treated cells (dotted line). While non-light-irradiated Compound 38 did not affect cell viability, Compound 39 induced apoptosis with an efficacy similar to that of vorinostat alone, suggesting that the conjugation of vorinostat to the fluorescent compounds of the present invention does not negatively affect the cytotoxicity of vorinostat. However, after light irradiation, Compound 39 and Compound 38 resulted in significant apoptosis. The efficacy of Compound 39 was approximately 10 times higher compared to non-modified vorinostat. Therefore, compound 39 exerts inherent cytotoxic activity derived from hydroxylic acid, which can be complemented and enhanced upon application of UV, 405 nm, or 2-photon 800 nm, thereby inducing additional photoactivated cell-death effects.

[0327] Examples 6: Mammals intracellular localization of compounds

[0328] To investigate the localization of compounds in biological cells, biological cells were co-stained with the compounds of Formula I and specific organelle markers (fluorescent dyes and antibodies). The following compounds were investigated: compounds 6, 7, 12, 13, 14, and 15.

[0329] experiment:

[0330] 6.1 Cell Lines and Media

[0331] HaCaT keratinocyte cell lines were used in the following experimental procedure. Cells were incubated in cell culture medium (94% Dulbetto's Modified Eagle Medium (DMEM), 5% fetal bovine serum (FBS), and 1% penicillin streptomycin solution (Pen-Strep)).

[0332] 6.2 Staining using organelle dyes

[0333] Cells were seeded into an 8-well plate at 25,000 cells / mL. 200 µL of cell suspension was added to each well, and the cells were incubated for 2 days, after which staining and imaging were performed.

[0334] To visualize mitochondria, cells were probed with the mitochondrial dye MitoTracker® Deep Red. Cells to be stained were incubated for 30 minutes with 200 µl of MitoTracker® Deep Red solution per well (N=3) (200 nM MitoTracker® and 1 μM Compound I in cell culture medium).

[0335] Nile Red was used to identify intracellular lipids. 200 µl of Nile Red lipophilic dye (10 µg / ml Nile Red and 1 µM compound of Formula I in cell culture medium) was added to each well (N=3) and incubated for 30 minutes.

[0336] LysoTracker® Red DND-99 dye was used to detect intracellular lysosomes. 200 µl of LysoTracker® Red DND-99 (a solution of 50 nM LysoTracker® and 1 μM of Formula I compound in cell culture medium) was added to each well (N=3) and incubated for 30 minutes.

[0337] To visualize the endoplasmic reticulum (ER), cells were stained with BODIPY® ER-Tracker® Red. 200 µl of BODIPY ER-Tracker® Red (solution of 1 μM BODIPY® and 1 μM Formula I compound in cell culture medium) was added to each well (N=3) and incubated for 30 minutes.

[0338] After incubation, the dye-containing cell culture medium was removed, and the cells were washed twice with 200 µl of phosphate-buffered saline (PBS). After washing, 200 µl of PBS was added to each well for imaging.

[0339] Paragraph 6.3 Ramin Staining using A / C antibodies

[0340] To visualize the nuclear lamina, cells were probed with anti-lamin A / C antibodies. Cells were plated onto 22 x 22 mm coverslips (10,000 cells / ml) and incubated for 2 days prior to staining. Excess medium was removed by rinsing the cells with PBS. Cells were fixed in 4% paraformaldehyde (PFA) at room temperature for 10 minutes, followed by two washes with PBS for 5 minutes each. After washing, cells were permeated in 0.4% Triton X-100 in PBS for 10 minutes. Subsequently, cells were washed three times with PBS for 5 minutes each and incubated in blocking buffer (1% BSA, 0.1% fish gelatin, and 0.1% Triton X-100 in PBS) at room temperature for 15 minutes. Cells were incubated with the primary antibody (mouse anti-lamin A / C IgG in block buffer) for 1 hour at room temperature. Then, the cells were washed twice in block buffer and incubated with the secondary antibody (anti-mouse Alexa-594 IgG in block buffer) for 30 minutes at room temperature. The cells were rinsed twice in PBS for 10 minutes at room temperature.

[0341] 6.4 Dyeing using the compound of Formula I

[0342] To stain cells with the compound of Formula I, a 5 μM solution of the compound of Formula I in PBS was added to the cells and left at room temperature for 30 minutes. Then, the cells were washed five times in PBS for 5 minutes each. After washing, the cells were mounted on uncharged microscope slides using 6 μL of Mowiol® per cover slip as the mounting medium.

[0343] 6.5 Imaging

[0344] A Zeiss 880 confocal microscope was used for all imaging tasks.

[0345] compound Here (nm) Emission range (nm) Compound of Formula I 405 450 - 550 MitoTracker® Deep Red 633 640 - 680 Nile Red 594 600 - 640 LysoTracker® Red DND-99 594 600 - 640 BODIPY® ER-Tracker Red 594 600 - 640 Alexa-594 anti-mouse IgG 594 600 - 640

[0346] Table 2: Imaging conditions

[0347] 6.6 Analysis

[0348] Statistics on co-locality between the compound of Equation I and organelle marker images were calculated using ImageJ Coloc2 software. Backgrounds were removed from each image, and the region of interest (ROI) was used as the analysis target. The point diffusion function (PSF) for each image was calculated as 2.0, and Costes' iterations were set to 100. Statistics were quantified using Pearson's correlation coefficient (PCC). PCCs are presented as numbers ranging from +1 to -1: 1 = Perfect co-localization; 0 = No correlation; -1 = Perfect anti-co-localization.

[0349] 6.7 Results

[0350] Individual images of each compound and organelle marker were captured and are shown in FIGS. 7-12. The green image on the left (column 1) is the compound of Formula I, the red image in the center (column 2) is the organelle marker, and the image on the right (column 3) is a superposition of the two images.

[0351] Figure 7 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 7 and various organelle markers. Column 1 visualizes Compound 7 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 7 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 7. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 7. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 7. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 7. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 7.

[0352] Figure 8 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 13 and various organelle markers. Column 1 visualizes Compound 13 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 13 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 13. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 13. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 13. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 13. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 13.

[0353] Figure 9 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 14 and various organelle markers. Column 1 visualizes Compound 14 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 14 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 14. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 14. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 14. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 14. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 14.

[0354] Figure 10 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 12 and various organelle markers. Column 1 visualizes Compound 12 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 12 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 12. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 12. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 12. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 12. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 12.

[0355] Figure 11 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 15 and various organelle markers. Column 1 visualizes Compound 15 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 15 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 15. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 15. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 15. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 15. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 15.

[0356] Figure 12 is a tiled arrangement of co-stained images of HaCaT keratinocytes probed with Compound 6 and various organelle markers. Column 1 visualizes Compound 6 in green, Column 2 visualizes various organelle markers in red, and Column 3 superimposes images of Compound 6 (green) and organelle markers (red). Column A is stained with MitoTracker (red) to investigate the mitochondrial localization of Compound 6. Column B is stained with Nile Red (red) to investigate the lipophilic localization of Compound 6. Column C is stained with LysoTracker® Red DND-99 (red) to investigate the lysosome localization of Compound 6. Column D is stained with BODIPY® ER-Tracker Red (red) to investigate the endoplasmic reticulum (ER) localization of Compound 6. Column E was stained (red) with anti-lamin A / C antibody to investigate the subnuclear membrane localization of compound 6.

[0357] Table 3-8 below shows the average PCC values ​​for each organelle marker indicating the degree of co-localization with compounds 7, 13, 14, 12, 15, and 6, respectively. PPC values ​​for the anti-lamin A / C antibody could not be calculated because there were not enough pixels per image to generate reliable data.

[0358] small agency Marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value 0.12 0.39 0.75 0.32 Joint-locality

[0359] Table 3: Mean correlation coefficient (PCC) for locality between Compound 7 and various organelle markers in HaCaT keratinocytes.

[0360] small agency Marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value -0.35 0.00 0.22 -0.18 No joint-locality

[0361] Table 4: Mean correlation coefficient (PCC) for locality between compound 13 and various organelle markers in HaCaT keratinocytes.

[0362] organelle marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value 0.65 0.51 0.11 0.68 No joint-locality

[0363] Table 5: Mean correlation coefficient (PCC) for locality between compound 14 and various organelle markers in HaCaT keratinocytes.

[0364] organelle marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value 0.14 0.37 0.73 0.34 No joint-locality

[0365] Table 6: Mean correlation coefficient (PCC) for locality between compound 12 and various organelle markers in HaCaT keratinocytes.

[0366] organelle marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value 0.16 0.82 0.21 0.30 No joint-locality

[0367] Table 7: Mean correlation coefficient (PCC) for locality between compound 15 and various organelle markers in HaCaT keratinocytes.

[0368] organelle marker MitoTracker ® Nile Red LysoTracker ® BODIPY ® ER-Tracker port- Ramin A / C PCC value 0.08 0.42 0.81 0.48 Joint-locality

[0369] Table 8: Mean correlation coefficient (PCC) for locality between Compound 6 and various organelle markers in HaCaT keratinocytes.

[0370] In summary, Compound 7 was primarily located in lysosomes, with some located in the ER and Golgi region, and also exhibited some lipophilic staining. Compound 13 appeared to stain the peripheral regions of the cell, but co-localization with the organelle markers used was observed to be undetectable. Compound 14 exhibited mitochondrial and ER localization, and some lipophilic staining was also observed. Compound 12 exhibited primarily lysosome localization, with some ER localization and lipophilic staining present. Compound 15 was observed to exhibit primarily lipophilic localization. Compound 6 exhibited primarily lysosome localization, with some ER localization and lipophilic staining also observed.

[0371] Examples 7: Localization of compounds in plant cells

[0372] 7.1 Black- Grass cell suspension Preparation of culture

[0373] Black-grass cell suspension cultures were initiated from embryogenic calli. Suspension cultures were subcultured every 10 days. Logarithmic cells (5 days after subculture) were used for all experiments.

[0374] 7.2 Cover

[0375] Compounds 7, 14, 12, and 15 were resuspended in DMSO (5 mM). 10 mL of black-grass cell suspension culture was labeled with the compounds (final concentration 1 μM) at room temperature for 1 hour. The cell culture was rinsed twice with culture medium to remove excess compounds. Cells were observed using a conchimous microscope (Leica SP8) with an HP PL APO 63x objective lens. Images were acquired at excitation / emission of 405 / 460–540 nm. The acquired images were processed using LasX software (Leica).

[0376] 7.3 Cytotoxicity Analysis

[0377] Compounds 7, 14, 12, and 15 were treated to 5 mL of black-grass cell suspension culture at concentrations of 0.1, 1, 5, and 10 μM for 1 hour at room temperature. Cells were treated with 0.1% DMSO as a control. Cells were irradiated with light (~365 nm) for 5 minutes and then incubated for 24 hours at 25°C at 150 rpm. Additionally, the cytotoxicity of the compounds was analyzed in the absence of light irradiation. Cell viability in 5 biological replicates for each concentration was confirmed by fluorescence analysis (FDA / PI). Cell viability % was calculated using the following formula:

[0378] Viability % = {(Living cells (FDA) / (Living cells + Dead cells)} x 100

[0379] Statistical analysis of cell viability % using one-way analysis of variance (ANOVA) and subsequent Tukey HSD posthocThe test was performed using SPSS 23 (IBM, Chicago, IL, USA).

[0380] 7.4 Results

[0381] The results are shown in Figures 13 and 14.

[0382] 7.4.1 Compound 7

[0383] Compound 7 generated an acceptable signal in black-grass cell suspension cultures. As can be seen in Figure 13, the compound was observed to label the inner cell membrane, but Compound 7 showed a stronger signal in cell vesicles (possibly lipid vesicles).

[0384] 7.4.2 Compound 14

[0385] Compound 14, which possesses a triphenylphosphonium moiety, has been shown to target mitochondria in mammalian cells. However, this compound has been observed to label not only the inner cell membrane but also small vesicles. Given that mitochondria are very abundant organelles in living organisms, compound 14 did not appear to label mitochondria in Black-Grass cells.

[0386] 7.4.3 Compound 12

[0387] Compound 12 generated a strong signal in black-grass cells. It was observed to specifically label the plasma membrane and cell plate.

[0388] 7.4.4 Compound 15

[0389] Compound 15, which has a tosyl sulfonamide moiety, has been shown to label endoplasmic reticulum in mammalian cells. However, this compound has been observed to label small vesicles in black-grass cells. The inventors hypothesized that the small vesicles labeled by this compound could be peroxisomes.

[0390] 7.4.5 Black- Grass Cytotoxicity of compounds to cell cultures

[0391] The above results demonstrate that the compounds of Formula I appear to target various organelles in black-grass cultures. Next, tests were performed to determine whether these compounds could be observed to have a negative effect on cell viability after light irradiation. To ensure that light irradiation was necessary to trigger cytotoxicity, the percentage of cell viability of black-grass cells treated with the compounds was evaluated without light irradiation.

[0392] Compounds 7 and 15 did not reduce black-grass viability regardless of concentration or light irradiation treatment. In contrast, treatment with 1 μM of compound 14 significantly reduced black-grass cell viability. At this concentration, the cytotoxic effect of compound 14 appears to be independent of light irradiation, as a significant decrease in cell viability was observed in non-irradiation treatment. Treatment with compound 12 at 5 μM and 10 μM significantly reduced black-grass cell viability. Furthermore, only the cytotoxic effect of compound 12 was observed after light irradiation.

[0393] Imaging and cytotoxicity analyses suggested that Compound 12 specifically targets the plasma membrane in black-grass cell cultures. Furthermore, Compound 12 was able to kill black-grass cells when treated at high concentrations (5 μM and 10 μM). In short, Compound 12 is likely a reliable marker for plasma membrane localization in plant cells and thus has the potential to be used as a photosensitizer for ROS production in plant cells.

[0394] Examples 8: Localization of compounds in bacterial cells

[0395] 8.1 Preparation of Bacterial Cell Cultures

[0396] Microbacterium smegmatis, Staphylococcus epidermis, and Bacillus subtilis were used in the following experimental procedures:

[0397] Staphylococcus epidermidis samples were taken from plate cultures, inoculated into LB medium, and cultured overnight at 30°C for about 16 hours.

[0398] Bacillus subtilis samples were taken from plate cultures, inoculated into LB medium, and cultured overnight at 37°C for about 16 hours.

[0399] Microbacterium smegmatis samples were taken from plate cultures and inoculated into Middlebrook 7H9 broth supplemented with Middlebrook ADC proliferation supplement and cultured overnight at 37°C for about 16 hours.

[0400] 8.2 Cytotoxicity Analysis

[0401] Cultures of Microbacterium smegmatis, Staphylococcus epidermis, and Bacillus subtilis were prepared as follows:

[0402] bacterial strains Sample preparation ( 5 ml of Amount of overnight culture added to fresh medium, μl) Compound of the present invention Sample processing (each preparations Amount of compound added to, μM) Microbacterium smegmatis 50 Compound 12 0, 1, 10, 100 Staphylococcus epidermis 50 Compound 6 0, 1, 10, 100 Bacillus subtilis 50 Compound 12 0, 1, 10, 100 Bacillus subtilis 50 Compound 6 0, 1, 10, 100

[0403] Table 9: Preparation of bacterial cultures

[0404] The samples were incubated under dark conditions at room temperature for approximately 2 hours. The black, transparent-bottomed Costar TM 200 µl of sample was added to each well of a 96-well plate.

[0405] Approximately 15 mW / cm² for 5 minutes at cells 2 The compound was irradiated with light. The cytotoxicity of the compound was also evaluated without light irradiation.

[0406] A 96-well plate was placed in a plate reader, and the proliferation curve protocol was established to be performed at the following parameters:

[0407] · Incubation temperature: 37℃

[0408] · OD reading wavelength 600 nm

[0409] · 250 cycles, read every 5 minutes

[0410] · Stir for 5 seconds before reading

[0411] This was carried out overnight to obtain a kinetic proliferation curve based on optical density readings.

[0412] 8.3 Dyeing using Compound 6 and Compound 12

[0413] Microbacterium smegmatis, Staphylococcus epidermis, and Bacillus subtilis were stained with compound 6. Bacillus subtilis was stained with compound 12.

[0414] Samples prepared according to Table 9 were treated with the compound by diluting a 10 mM compound stock solution in the medium to a concentration of 100 μM. Then, this solution was further diluted in the medium at a 1:10 ratio to prepare 10 μM and 1 μM medium solutions containing the compound. Subsequently, 50 μL of cell culture was added to the 100 μM, 10 μM, and 1 μM compound-containing medium preparations.

[0415] 8.4 propidium Iodide and Syto TM Dyeing using 9

[0416] After treatment as described in Table 9, each of the three bacterial strains was Syto TM Baclight equipped with solutions of 9 and propidium iodide, respectively TM Staining was performed using a staining kit. One spare sample was treated with each compound at 0.1 μM and included in this analysis.

[0417] Microbacterium smegmatis, Staphylococcus epidermis, and Bacillus subtilis were stained with propidium iodide to identify non-viable cells and with Syto 9 to identify all cells.

[0418] The following dyeing procedure was applied:

[0419] 1. 1 ml of each sample was placed into the wells of a 12-well plate;

[0420] 2. Half of the 12-well plate is approximately 15 mW / cm² 2 Light was irradiated for 5 minutes;

[0421] 3. The contents of each well were placed into individual Eppendorfers and centrifuged at 10,000 rpm for 3 minutes to obtain culture pellets;

[0422] 4. The medium was removed, and each pellet was resuspended in 200 µl of 1X PBS and centrifuged at 10,000 rpm for 3 minutes;

[0423] 5. Baclight TM The staining solution preparation consists of 1 ml 1X PBS, 3 µl propidium iodide, and 3 µl Syto TM Made using 9;

[0424] 6. Each pellet was resuspended in 200 µl of staining solution and incubated at room temperature for 15 minutes;

[0425] 7. Then, the sample was centrifuged at 10,000 rpm for 3 minutes and resuspended in 1X PBS. This process was repeated 3 times to remove any excess staining solution;

[0426] 8. 20 µl of each sample was dropped onto a poly-L-lysine coated coverslip, left for 15 minutes, then excess sample was removed and finally washed with 1X PBS;

[0427] 9. Replace the Baclight cover slip provided in the kit. TM It was mounted on the slide using mounting oil.

[0428] 8.5 Imaging

[0429] 8.5.1 wide field of view fluorescence Imaging

[0430] Images were acquired using 63x and 100x oil immersion lenses on a Zeiss Cell observer wide-field microscope. Sets of blue, green, and red filters were used to acquire fluorescence images of the irradiated compounds, Syto 9 and propidium iodide, respectively (see Table 10).

[0431] Channel color compound Max here (nm) Maximum emission (nm) blue Compound 6 / 12 365 397 green Syto 9 450 515 red propidium iodide 546 590

[0432] Table 10: Wide-field imaging conditions

[0433] 8.5.2 Confocal Imaging

[0434] High-resolution images of Bacillus subtilis were acquired using a Leica SP5 laser scanning confocal microscope. A 100x oil-immersed objective lens was used for additional digital magnification. A 405 nm excitation and 450 nm–600 nm emission range were applied for fluorescence image acquisition.

[0435] 8.6 Results

[0436] The results are listed in Figures 15-21.

[0437] 8.6.1 Microbacterium In Smegmatis Cytotoxicity of Compound 12

[0438] Figure 15(i) shows the overnight growth curve of Microbacterium smegmatis after treatment with Compound 12, and Figure 15(ii) shows the overnight growth curve of Microbacterium smegmatis after treatment with Compound 12 and light irradiation.

[0439] No significant difference was observed between the treatment control and the untreated control in the non-photoactivated samples. However, the light-irradiated samples began to show some cytotoxicity at a concentration of 100 μM.

[0440] 8.6.2 Staphylococcus In Epidermis Cytotoxicity of Compound 6

[0441] Figure 16 shows Staphylococcus epidermis treated with compound 6 before and after light irradiation. Control cells untreated with compound 6 are also shown. Compound 6 is observed in blue (Column 1), Syto 9 is observed in green (Column 2) by visualizing all viable and non-viable cells, and propidium iodide is observed in red (Column 3) by visualizing non-viable cells.

[0442] In the image, red fluorescent cells increased after treatment with Compound 6 compared to the untreated control group. Curves were constructed by averaging the 8-microwell OD measurements for each sample type. Error bars represent the standard error of the 8-well measurements. At concentrations of 100 and 10 μM, no proliferation was observed regardless of photoactivation. The non-photoactivated 1 μM sample showed some effect, with an extended lag phase (time before proliferation begins) compared to the untreated control group. When the 1 μM sample was photoactivated, the proliferation lag phase was significantly extended to a maximum of approximately 15 hours, whereas the lag phase of the untreated sample was extended by only about 2 hours.

[0443] 8.6.3 Bacillus In Subtilis Cytotoxicity of Compounds 6 and 12

[0444] Figure 18 shows Bacillus subtilis treated with compound 12 before and after light irradiation (Figure 18(a) and 18(b), respectively). Compound 12 is observed as blue (i). Cells were co-stained with Syto 9, which is observed as green (2), to visualize all cells. Cells were also stained with propidium iodide, which is observed as red (3), to visualize non-viable cells.

[0445] Compound 12 was observed in the blue channel in both light-irradiated and light-non-irradiated images, indicating cell adhesion / absorption. After light irradiation, the proportion of non-viable (red) cells increased compared to the light-non-irradiated sample. Therefore, it appears that the cytotoxicity of Compound 12 is present in Bacillus subtilis.

[0446] Figure 19 shows the overnight proliferation curves of Bacillus subtilis cells treated with compound 12 before and after light irradiation. At treatment concentrations of 100 μM and 10 μM, no proliferation was observed regardless of light irradiation. Both untreated control samples exhibited similar levels of proliferation. The light-non-irradiated 1 μM samples showed slightly lower proliferation than the untreated samples, and the lag phase was prolonged.

[0447] Figure 20 shows the overnight proliferation curves of Bacillus subtilis cells treated with compound 6 before and after light irradiation. At concentrations of 0, 5, and 1 μM, the non-light-irradiated samples exhibited similar proliferation levels. When light-irradiated, these samples showed slight inhibition of proliferation. At a treatment concentration of 10 μM, proliferation decreased and the induced phase was prolonged, and these effects were much more pronounced in the light-irradiated samples.

[0448] Compound 12 was more cytotoxic than compound 6 at both 10 and 1 μM concentrations.

[0449] 8.6.4 Bacillus In Subtilis Locality of Compound 12

[0450] Figure 21 shows Bacillus subtilis cells treated with compound 12. Compound 12 was strongly localized at the peptidoglycan site of Bacillus subtilis cells.

[0451] The detailed experiments described above demonstrate the cytotoxicity of compounds 6 and 12 against Gram-positive cells Staphylococcus epidermis and Bacillus subtilis. Depending on the concentration, this can be present even without photoactivation. As such, these small molecules offer a promising alternative to traditional antibiotics to which many organisms have become resistant. The response to photoactivation may also be beneficial in the treatment of skin diseases or potentially usable as an insecticide in the context of plant pathogens.

[0452] Attachment to the endospores of Bacillus subtilis cells demonstrates intracellular uptake, which is often problematic for macromolecular drugs. The spore-forming cycle of these bacteria provides innate protection from harmful environments and chemical treatments, making it difficult to eradicate pathogens that may undergo this process. Actively killing endospores will be a novel method of cell death against spore-forming pathogens.

[0453] All features disclosed herein (including the appended claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined in any combination except that at least some of these features and / or steps are mutually exclusive. Each feature disclosed herein (including the appended claims, abstract, and drawings) may be substituted with alternative features providing the same, equivalent, or similar purpose unless otherwise stated. Accordingly, unless otherwise stated, each disclosed feature is merely one example of a comprehensive set of equivalent or similar features. The invention is not limited to the details of the embodiments(s) described above. The invention extends to any new one or any new combination of features disclosed herein (including the appended claims, abstract, and drawings), or to any new one or any new combination of steps of any disclosed method or process.

Claims

Claim 1 Compounds of Formula I in the form of glass or salts, or diastereomers thereof: The above formula I is a compound of formula 19, 23, or 34, or: ; or in the above formula I, R 1 and R 2 forms part of a heterocyclic group Y having 3-12 ring atoms, wherein Y is selected from the following groups: R 7 alkyl group, -COCH3, -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2) n CH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R 8 or -(CH2) n PPh3 + Br - but, R 8 is -OH or -NHOH, n is an integer from 1 to 8, and m is an integer from 1 to 4; Ar1 ​​is selected from phenyl, pyridine, pyrimidine, thiophene, furan, benzofuran, or thiazole groups; and Ar2 is selected from the following groups: R 1 is a C1-10 alkyl group that is H or optionally substituted with one or more N atoms; R 2 -(CH2) is a C1-10 alkyl group optionally substituted with one or more N atoms n R 3 , -(CH2) n NHR 3 and -(CH2)2(COCH2) n R 3 Selected from, where n is an integer between 1 and 10, and R 3 is -NH2, -OH, -SO2PhCH3 or -COOH, or R 2 -C(O)(CH2) n C(O)R 8 , -C(O)(CH2) m O(CH2) m C(O)R 8 , -C(O)(CH2) n CH(CH3)C(O)R 8 , -S(O)2(CH2) n C(=O)R 8 , -S + (O - )(CH2) n C(=O)R 8 or -(CH2) n PPh3 + Br - but, R 8 is -OH or -NHOH, n is an integer from 1 to 8, m is an integer from 1 to 4; and X is the expression -CH=CH-C(=O)OR 4 is R 4 methyl, C2-C 10 Selected from unsaturated esters of alkyl, alkenyl, or aryl origin; carboxylic acids; and imidazolones, provided that Ar1 is phenyl and R 1 and R 2 When is formed as part of a heterocyclic group Y having 3-12 ring atoms, N of the heterocyclic group is located at the para position relative to the acetylene group of the compound of Formula I. Claim 2 A composition for use in fluorescence imaging comprising a compound of Formula I according to claim 1. Claim 3 A composition for use in Raman imaging or fluoRaman imaging, comprising a compound of Formula I according to claim 1. Claim 4 A probe comprising a compound of Formula I according to claim 1. Claim 5 A compound of Formula I according to claim 1; and a conjugate comprising a targeting substance or an active substance. Claim 6 In paragraph 5, the targeting substance or active substance is a conjugate selected from small molecule drugs, peptides or proteins, saccharides or polysaccharides, aptamers or affimers, or antibodies. Claim 7 A composition for use in controlling cell development, comprising a compound of Formula I according to claim 1; or a conjugate comprising a compound of Formula I according to claim 1 and a targeting substance or an active substance. Claim 8 A composition for use in photodynamic therapy comprising a compound of Formula I according to claim 1; or a conjugate comprising a compound of Formula I according to claim 1 and a targeting substance or an active substance. Claim 9 A formulation comprising a compound of Formula I according to claim 1; or a conjugate comprising a compound of Formula I according to claim 1 and a targeting substance or an active substance, optionally combined with one or more co-formulants. Claim 10 A composition for treating a patient having a disease or pathological condition in which control of cell proliferation, differentiation, or apoptosis is useful, comprising: a compound of Formula I according to claim 1; or a conjugate comprising a compound of Formula I according to claim 1 and a targeting substance or active substance, wherein the disease or pathological condition in which control of cell proliferation, differentiation, or apoptosis is useful is cancer, a skin disorder, or a skin injury, and said cancer includes neuroneoplasm; a precancerous or cancerous pathological condition of the skin, oral cavity, larynx, lung, bladder, vulva, breast, kidney, liver, prostate, eye, or digestive tract; basal cell carcinoma; squamous cell carcinoma; or leukemia, and said skin disorder includes acne, psoriasis, stretch marks, keratosis pilaris, emphysema, or alopecia, and said skin injury includes burns, diabetic foot ulcers, UV damage, or skin aging. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete

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