Photoswitchable chemical induced dimerization systems for control of cellular activities with light

Abstract

The present application refers to photoswitchable chemical induced dimerization systems for reversible and repetitive control of cellular activities with light. Compounds, test systems, methods and uses are disclosed how the invention can be applied in the investigation of cellular processes. A photoswitchable compound consisting of: ligand A – linker A – photoswitch moiety – linker B – ligand B or ligand (photoswitch moiety) A – linker B – ligand B, wherein ligand A, ligand (photoswitch moiety) A and ligand B are ligands capable of binding a ligand binding domain of a first and a second protein. Linker A and linker B are as specified in the specification. The photoswitch moiety is of formula (I), formula (II), the cis-isomer thereof or of formula (II) wherein R1, R2, R3, R4 and A are as defined in the specification.

Description

[0001] PHOTOSWITCHABLE CHEMICAL INDUCED DIMERIZATION SYSTEMS FOR CONTROL OF CELLULAR ACTIVITIES WITH LIGHT

[0002] FIELD OF THE INVENTION

[0003] The invention relates to photoswitchable chemical induced dimerization systems for control of cellular activities with light.

[0004] BACKGROUND OF THE INVENTION

[0005] Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. Optogenetic systems based on natural photoreceptors such as rhodopsins, light-oxygen-voltage (LOV) proteins, cryptochromes and phytochromes, mediate intra- or intermolecular interactions in response to light. However, the photoreceptors are limited in the scope of modification. Whereas chemo-optogenetic systems use photoactivatable and / or photocleavable small-molecule dimerizers that are readily subject to chemical modification.

[0006] Chemo-optogenetic systems represent next-generation optogenetic approaches. First, many photosensitive proteins suffer from basal activity in the dark state and relatively low affinity (Kd = 0.1-10 pM) in the on state. In theory, improvement of binding affinity results in a kinetically slower system and also enhances the binding of optogenetic modules in the dark. In contrast, there is no binding between the caged dimerizer and the dimerization module, while the uncaged dimerizer usually binds in high affinity (Kd in the nanomolar range). Hence, the dynamic range of chemo-optogenetic systems is significantly improved. Second, chemo-optogenetic systems are operated by a single short pulse of light, while optogenetic systems usually require constant pulses of illumination to keep them in the on state, as photosensitive proteins undergo relaxation in the dark. WO2013 / 178593 A1 discloses bioorthogonal chemical inducers of reversible dimerization for control of protein interactions in cells. WO 2019 / 008175 A1 discloses photo-activatable / cleavable chemical inducers of dimerization for control of protein interactions in cells by light. However, in contrast to optogenetic systems that can be controlled with repetitive cycles, current chemo-optogenetic dimerization systems are subjected to only one-time or one-round control by UV / blue light, as the dimerizer is disrupted by light illumination. This limits the scope of application in research that requires cycling or gating and studies in the whole organism.

[0007] SUMMARY OF THE INVENTION

[0008] One object of the invention is to alleviate, mitigate or eliminate one or more drawbacks of the prior art.

[0009] One object of the invention is to provide a compound that can induce dimerization and subsequent dedimerization between two proteins, without being consumed during the process.

[0010] One object of the invention is to provide a compound that can repeatedly induce dimerization and subsequent dedimerization between two proteins, the same two protein or different.

[0011] One object of the invention is to provide a chemo-optogenetic dimerization system that can undergo multiple rounds of dimerization controlled by light.

[0012] According to one aspect of the invention, there is a photoswitchable compound consisting of: ligand A - linker A - photoswitch moiety - linker B - ligand B, wherein ligand A is capable of binding a ligand binding domain of a first protein; the photoswitch moiety is of formula (I), formula (II), or of formula (III), or any isomer of formula (I), formula (II) or formula (III);

[0013]

[0014] wherein each of R1, R2, R3and R4is independently selected from hydro, fluoro, chloro, bromo, methyl, methoxy and ethylthio; X is selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, -OCH2-, -CH2O-, -NHCH2-, -CH2NH-, -CH2S-, -SCH2-, -NR5CH2- and - CH2NR5-; R5is selected from hydro, methyl, ethyl and acetyl; and ligand B is capable of binding a ligand binding domain of a second protein. In the prior art, chemo-optogenetic systems may undergo dimerization and sometimes also dedimerization. Those processes, however, consume the dimerizer, typically by bond cleavage, meaning that the processes may not be repeated. The compounds according to the invention are based on photo-switchable moieties, which repeatedly may change between trans and cis configurations (see Examples 1-13). This property may be utilized to induce dimerization and dedimerization, without being consumed during the process. This is shown in Examples 14-17.

[0015] Ligands A and B may be the same ligand in the case of homodimerization.

[0016] Photoswitch moiety could also be part of ligand A or ligand B. A photoswitchable compound could consist of: ligand (photoswitch moiety) A - linker B - ligand B, wherein ligand (photoswitch moiety) A is capable of binding a ligand binding domain of a first protein; the photoswitch moiety is of formula (I), or formula (II), the c / s-isomer thereof or of formula (III), and ligand B is capable of binding a ligand binding domain of a second protein.

[0017] The photoswitch moiety can switch between the well-defined cis and trans (i.e. E and Z) configurations upon light irradiation. The photoswitch moiety adopts either cis or trans configuration in the dark state and undergoes configurational change to the excited state upon light irradiation. The photoswitch moiety can undergo thermal relaxation to the dark state in the dark or undergo configurational change to the dark state upon irradiation with light of another wavelength. In that regard the chemical inducers of dimerization of the present invention are photo-switchable which means that under distinct irradiation conditions the dimerization can be induced and reversed with light independently. Herein, the chemical inducers of dimerization of the present invention are also denoted as CIDs, sCID, or dimerizers. However, dimerization and reversal of dimerization (dedimerization) cannot be induced under the same irradiation conditions. In other words, irradiation conditions for dimerization and dedimerization are orthogonal, i.e. while the dimerization is induced by irradiation with light or in the dark under irradiation condition A. The dimerization of the two proteins via the chemical inducers of dimerization of the present invention is reversed by irradiation with light or in the dark under irradiation condition B. Irradiation condition A and B may differ in the used wavelength of light or in the dark.

[0018] According to one embodiment of the invention, linker A is an C2-C12 alkylene moiety optionally interrupted by at least one group selected from an amide, an ether, an ester, a sulfonamide, a triazole, a carbon-carbon single bond, a carbon-carbon double bond and a carbon-carbon triple bond.

[0019] According to one embodiment of the invention, linker B is an C2-C12 alkylene moiety optionally interrupted by at least one group selected from an amide, an ether, an ester, a sulfonamide, a triazole, a carbon-carbon single bond, a carbon-carbon double bond and a carbon-carbon triple bond.

[0020] According to one embodiment of the invention, linkers A and B are selected from the group comprising or consisting of polyethylene glycol (PEG), triethylene glycol, tetraethylene glycol (TEG), alkyl chain with up to 30 carbon atoms, phosphodiesters, glycosides, amides, esters, diesters, thioesters, aldol products, acetate moieties, polyethylenes, isoprenoids, phenyl groups, aromatic groups, heterocyclic groups, ethers, thioethers, and imines.

[0021] Ligand A is a ligand that is capable of binding a ligand binding domain (LBD-A). Ligand B is another ligand that is capable of binding a second ligand binding domain (LBD-B).

[0022] According to one embodiment of the invention, ligands A and B are selected from trimethoprim (TMP) and HaloTag ligands, such as chlorohexane derivatives. HaloTag refers to a modified haloalkane dehalogenase. HaloTag ligands can bind with HaloTag in a rapid and covalent fashion. On the other hand, TMP (5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine) is a well-known bacteriostatic antibiotic which is used above all in the prophylaxis and treatment of urinary tract infections. It belongs to the group of dihydrofolate reductase (DHFR) inhibitors that confer non-covalent binding with DHFR with high affinity (nM range) and specificity. The action of TMP could also be replaced by other DHFR binding ligands, such as methotrexate (MTX). TMP has the following structure:

[0023]

[0024] MTX has the following structure:

[0025]

[0026] The current ligand-ligand binding domain (LBD) pairs, TMP-DHFR and chlorohexane-HaloTag, could be replaced by other ligand-LBD pairs, which can be organized in a similar way as did in the current invention. For example, chlorohexane-HaloTag could be replaced by other protein / peptide tags and respective ligands, such as SNAP-tag (benzylguanine), CLIP-tag (benzylcytosine), His-tag (Ni-NTA), tetracysteine-tag (FIAsH or ReAsH), D4-tag (Zn-DpaTyrs), HyRe-tag (hydrazide), SpyCatcher protein domain (SpyTag peptide), E3-tag (K3 peptide), coiled-coil peptide (coiled-coil peptide), PYP-tag (thioester derivative of cinnamic acid / coumarin), BL-tag (p-lactam compounds), Cutinase-tag (p-nitrophenyl phosphonate), ACP-tag (coenzyme A derivatives), AP-tag (ketobiotin), Q-tag (cadaverine derivatives), LAP-tag (lipoic acid), formylglycine generating enzyme substrate-tag (hydrazide or aminooxy), Sortase A substrate-tag (poly-glycine peptide), CAAX-tag (prenyl diphosphate), Split intein-tag (another half of split intein peptide fragment), AnkX substrate-tag (CDP-choline), Tubtag (tyrosine derivatives), unnatural amino acids containing a biorthogonal chemistry handle (the reagent that reacts with the biorthogonal chemistry handle), and others. The TMP-DHFR pair could be replaced by a selective inhibitor and its cognate protein, such as synthetic ligand of FKBP (SLF) and FKBP protein, SLF analog (SLF’) and FKBP(F36V) protein. For further reading, consult Klewer, L. and Wu, Y-W (Chem. Eur. J. 2019, 25, 12452 - 12463). SLF has the following structure:

[0027]

[0028] SLF’ has the following structure:

[0029]

[0030] Thus, according to one embodiment of the invention, ligands A and B are independently selected from selected from C2-C10haloalkyl, benzyl guanine, benzyl cytosine, maleimide, glutamic acid, methotrexate, SLF, SLF’ and trimethoprim.

[0031] According to one embodiment of the invention, ligand A is selected from C2-C10haloalkyl, benzyl guanine, benzyl cytosine, maleimide, glutamic acid, methotrexate, SLF, SLF’ and trimethoprim.

[0032] According to one embodiment of the invention, ligand A is selected from C2-C haloalkyl, benzyl guanine, benzyl cytosine, methotrexate, SLF, SLF’ and trimethoprim. According to one embodiment of the invention, ligand A is

[0033]

[0034] According to one embodiment of the invention, ligand A is

[0035]

[0036] According to one embodiment of the invention, ligand B is selected from C2-C haloalkyl, benzyl guanine, benzyl cytosine, maleimide, glutamic acid, methotrexate, SLF, SLF’ and trimethoprim.

[0037] According to one embodiment of the invention, ligand B is selected from C2-C haloalkyl, benzyl guanine, benzyl cytosine, maleimide, glutamic acid and trimethoprim.

[0038] According to one embodiment of the invention, ligand B is C2-C10 haloalkyl, such as C4-C6haloalkyl, such as chlorohexyl.

[0039] According to one embodiment of the invention, linker A is an C2-C12 alkylene moiety optionally interrupted by at least one group selected from an amide, an ether, an ester and a sulfonamide. According to one embodiment of the invention, linker A is an alkylene group, wherein 1 to 3 of the methylene groups in said alkylene group may be replaced by an -O- group, wherein said alkylene group binds to ligand A by a group selected from an amide, an ether, an ester, a sulfonamide, a triazole, a carbon-carbon single bond, a carboncarbon double bond and carbon-carbon triple bond, and wherein said alkylene group binds to the photoswitchable moiety by a group selected from an amide, an ether, an ester, a sulfonamide, a triazole, a carbon-carbon single bond, a carbon-carbon double bond and carbon-carbon triple bond.

[0040] According to one embodiment of the invention, linker A is an alkylene group, wherein 1 to 3 of the methylene groups in said alkylene group may be replaced by an -O- group, wherein said alkylene group binds to ligand A by a group selected from an amide, an ether, an ester and a sulfonamide, and wherein said alkylene group binds to the photoswitchable moiety by a group selected from an amide, an ether, an ester and a sulfonamide.

[0041] According to one embodiment of the invention, linker B is an C2-C12 alkylene moiety optionally interrupted by at least one group selected from an amide, an ether, an ester and a sulfonamide.

[0042] According to one embodiment of the invention, linker B is an alkylene group, wherein 1 to 3 methylene groups in said alkylene group may be replaced by an -O- group, wherein said alkylene group binds to ligand B by a group selected from an amide, an ether, an ester a sulfonamide, a triazole, a carbon-carbon single bond, a carboncarbon double bond and a carbon-carbon triple bond, and wherein said alkylene group binds to the photoswitchable moiety by a group selected from an amide, an ether, an ester, a sulfonamide, a triazole, a carbon-carbon single bond, a carbon-carbon double bond and a carbon-carbon triple bond.

[0043] According to one embodiment of the invention, linker B is an alkylene group, wherein 1 to 3 methylene groups in said alkylene group may be replaced by an -O- group, wherein said alkylene group binds to ligand B by a group selected from an amide, an ether, an ester and a sulfonamide, and wherein said alkylene group binds to the photoswitchable moiety by a group selected from an amide, an ether, an ester and a sulfonamide.

[0044] According to one embodiment of the invention, linker A is selected from

[0045]

[0046] According to one embodiment of the invention, linker B is selected from

[0047]

[0048] According to one embodiment of the invention, X is selected from -CH2-, -CH2CH2-, -OCH2-, -CH2O-, -NHCH2-, -CH2NH-, -CH2S- and -SCH2-.

[0049] According to one embodiment of the invention, each of R1, R2, R3and R4is independently selected from hydro, methyl, chloro and fluoro.

[0050] According to one embodiment of the invention, the photoswitch moiety

[0051]

[0052] Examples 1, 2, 14 and 17, it is shown that photoswitchable compounds having such photoswitch moiety, such as TAC and TAC-3, were able to reversibly control cellular processes, by repeatedly induce dimerization and dedimerization (Figures 4 and 6). According to one embodiment of the invention, the photoswitch moiety

[0053]

[0054] In Examples 3, 13, 15 and 17, it is shown that a photoswitchable compounds having such photoswitch moiety, such as TMC and TSMC, were able to reversibly control cellular processes, by repeatedly induce dimerization and dedimerization (Figures 7D, 18B).

[0055] According to one embodiment of the invention, the photoswitch moiety

[0056]

[0057] Examples 10 and 17, it is shown that a photoswitchable compound having such photoswitch moiety, such as TPC, was able to reversibly control cellular processes, by repeatedly induce dimerization and dedimerization (Figure 8D).

[0058] According to one embodiment of the invention, linkers A and B are selected from

[0059]

[0060] According to one embodiment of the invention, linker A is H and linker B is

[0061]

[0062] According to one embodiment of the invention, linker A is

[0063]

[0064] and linker B is

[0065] mbodiment of the invention, linker A is

[0066]

[0067] and linker B is

[0068]

[0069] According to one embodiment of the invention, the photoswitch moiety is

[0070]

[0071] , wherein X is selected from -CH2-, -CH2CH2-, -CH2CH2CH2-, - OCH2-, -CH2O-, -NR5CH2-, -CH2NR5-, -CH2S- and -SCH2-,

[0072] wherein R5is selected from hydro, methyl, ethyl and acetyl. TDC dimerizers using diazocine as the photo-switchable show excellent photostability, which enables many photoswitching cycles (Examples 11, 12, and 14).

[0073] According to one embodiment of the invention, the photoswitch moiety is

[0074]

[0075] , wherein X is selected from -CH2-, -CH2CH2-, -OCH2-, -CH2O-, - NHCH2-, -CH2NH-, -CH2S-, and -SCH2-.

[0076] According to one embodiment of the invention, X is ethylene.

[0077] According to one embodiment of the invention, the photoswitch moiety is

[0078]

[0079] According to one embodiment of the invention, ligand A is selected from

[0080]

[0081] the photoswitchable moiety selected from

[0082]

[0083] each of R1, R2, R3and R4is independently selected from hydro, methyl, chloro and fluoro;

[0084] linker B is selected from

[0085]

[0086] ; and ligand B is chlorohexyl.

[0087] According to one embodiment of the invention, the compound is selected from

[0088]

[0089] According to one aspect of the invention, there is provided a chemo-optogenetic system for reversibly and repetitively testing intracellular test compound interactions in cells in vitro and / or in vivo, comprising:

[0090] a) the photoswitchable compound according to the invention;

[0091] b) conjugate 1 comprising a test compound 1 and at least a HaloTag; and c) conjugate 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or the SLF’ binding domain of FKBP (F36V).

[0092] According to another aspect of the invention said conjugate 1 and / or conjugate 2 of the chemo-optogenetic system comprises further a component for identification and / or purification of the conjugate and / or a first or second peptide or protein.

[0093] According to another aspect of the invention test compound 1 and test compound 2 of said chemo-optogenetic system are selected independently of each other among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

[0094] In one aspect of the invention the bacterial DHFR of the chemo-optogenetic system is eDHFR.

[0095] In another aspect of the invention is a there is provided a chemo-optogenetic system for reversibly and repetitively testing intracellular protein interactions in cells in vitro and / or in vivo, comprising:

[0096] a) the photoswitchable compound according to the invention;

[0097] b) fusion protein 1 comprising a test compound 1 and at least a HaloTag; and c) fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or the SLF’ binding domain of FKBP (F36V).

[0098] According to another aspect of the invention said fusion protein 1 and / or fusion protein 2 of the chemo-optogenetic system comprises further a component for identification and / or purification of the conjugate and / or a first or second peptide or protein.

[0099] According to another aspect of the invention test compound 1 and test compound 2 of said chemo-optogenetic system are selected independently of each other among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

[0100] Another aspect of the invention is the use of the chemo-optogenetic system as presented herein for reversibly and repetitively testing the interactions of a test compound 1 with a test compound 2.

[0101] One aspect of the invention is a method for reversibly and repetitively testing intracellular test compound interactions in cells in vitro and / or in vivo, comprising the following steps: a) providing, transfecting and expressing the DNA sequence of a conjugate 1, comprising a test compound 1 and at least a HaloTag, in a cell / s;

[0102] b) providing, transfecting and expressing the DNA sequence of a conjugate 2, comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or SLF’ binding domain of FKBP (F36V), in said cell / s;

[0103] c) adding a photoswitchable compound according to the invention to said cell / s and admitting them to pass the plasma membrane;

[0104] d) activating and / or deactivating said photoswitchable compound with light under irradiation condition A for activation and under irradiation condition B for deactivation;

[0105] e) determining the change in a selected test parameter system; and

[0106] f) optionally repeating step d) and e) one or more times.

[0107] In one aspect of the method the test compound 1 and test compound 2 are selected independently from one another among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

[0108] In one aspect of the invention the conjugate 1 and 2 as mentioned herein is a fusion protein 1 and 2 respectively.

[0109] According to one aspect of the invention, there is provided a dimerization system for reversibly and repetitively testing intracellular protein interaction in eukaryotic cells, comprising

[0110] a) a photoswitchable compound according to the invention;

[0111] b) fusion protein 1 generated from a test compound 1 (a plasmid with a DNA sequence encoding fusion protein 1) and at least HaloTag; and

[0112] c) fusion protein 2 generated from a test compound 2 (a plasmid with a DNA sequence encoding fusion protein 2) and at least the TMP binding domain of a bacterial DHFR. The photoswitchable compound according to the invention refers to the compound consisting of ligand A - linker A - photoswitch moiety - linker B - ligand B, as defined herein.

[0113] Fusion proteins or chimeric proteins are proteins created through the joining of two or more DNA-sequences, mostly created through genetic engineering, which originally coded for separate proteins or protein domains. The fusion proteins according to the invention may be a recombinant fusion protein created through genetic engineering of a fusion gene. This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame followed by a stop codon. The resulting DNA sequence will then be expressed by a cell as a single protein. The fusion protein may be engineered to include the full sequence of both original proteins (HaloTag or bacterial DHFR and protein of interest) or only a portion of either. Fusion proteins of at least the HaloTag and a bacterial DHFR, in particular E. coli DHFR (eDHFR), respectively, with the two proteins the interactions of which shall be tested can be generated according to conventional methods. Thus, preferred fusion proteins according to the invention may comprise the binding domain of HaloTag or respectively the binding domain of a bacterial DHFR, in particular eDHFR, and a protein or protein domain of interest (POI, for example Bicaudal D2 (1-594) and Rab5). Furthermore, the fusion proteins may also comprise some additional amino acids for example as a linking moiety or spacer between two proteins or protein domains. Additional amino acids may also be caused by strategy of genetic engineering. The fusion protein preferably comprises in addition a component for identification and / or purification of the fusion proteins, like a GST protein, FLAG peptide, a Myc-Peptide, a human influenza hemagglutinin (HA)-peptide, a hexa-his peptide (6xHis-tag), a fluorescent protein (variants of GFP, CFP, YFP, BFP, RFP, far-red FP), or a protein tag that can be labeled by a fluorescent dye (e.g. SNAP-tag, etc). The DNA constructs coding for the fusion proteins are going to be transfected to the test cell culture by conventional methods.

[0114] According to one embodiment of the invention, these DNA constructs are going to be expressed under the control of a suitable promoter gene sequence. For example, the cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. Ideally, the promoter can be easily regulated by the experimenter. Suitable promoter systems include for example tetracycline controlled promoter systems. It is therefore preferred if the DNA constructs include inducible promoter systems that are regulated by particular chemical or physical factors.

[0115] According to one aspect of the invention, there is provided a dimerization system for reversibly and repetitively testing intracellular protein interaction in eukaryotic cells, comprising

[0116] a) a photoswitchable compound according to the invention;

[0117] b) fusion protein 3 generated from a test compound 3 (a plasmid with a DNA sequence encoding fusion protein 3), at least HaloTag and at least the TMP binding domain of a bacterial DHFR.

[0118] According to one aspect of the invention, there is provided a dimerization system with the above-mentioned components, wherein test compound 1, test compound 2 or test compound 3 expresses a fluorescent protein.

[0119] Suitable fluorescent proteins can be selected from various variants of blue fluorescent protein (e.g., BFP, TagBFP, etc), cyan fluorescent proteins (e.g., mTurquoise2, ECFP, etc.), yellow fluorescent proteins (e.g., YFP, Citrine), green fluorescent proteins (e.g., EGFP), red fluorescence protein (e.g., mCherry), and far-red fluorescent proteins (e.g, mCardinal).

[0120] According to one aspect of the invention, there is provided a dimerization system with the above-mentioned components, wherein test compound 1, test compound 2 or test compound 3 expresses a protein tag that can be specifically labeled by a fluorescent dye. Suitable protein tags include SNAP-tag, CLIP-tag, His-tag, tetracysteine-tag, D4-tag, HyRe-tag, SpyTag, E3-tag, FKBP(F36V)-tag, PYP-tag, BL-tag, Cutinase-tag, ACP-tag, AP-tag, Q-tag, LAP-tag, formylglycine generating enzyme substrate-tag, Sortase A substrate-tag, CAAX-tag, Split intein-tag, AnkX substrate-tag, Tub-tag, unnatural amino acids (UAAs), and others.

[0121] Here, we term the dimerization system for reversibly and repetitively testing intracellular protein interaction in cells in vitro and / or in vivo according to the invention a chemo-optogenetic system since the system combines engineered fusion proteins with their stimulation or proteins by light by the dimerizer, in other words the photoswitchable compound according to the invention.

[0122] According to one aspect of the invention, there is provided a method for reversibly and repetitively testing intracellular protein interaction in cell / s in vitro and / or in vivo, comprising the steps of:

[0123] a) Providing, transfecting and expressing the DNA sequence of a fusion protein 1, comprising a test compound 1 and at least HaloTag in a cell / s

[0124] b) providing, transfecting and expressing the DNA sequence of a fusion protein 2, comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or SLF’ binding domain of FKBP (F36V), in said cell / s;

[0125] c) adding a photoswitchable compound according to the invention to said cell / s, preferably eukaryotic cells, and admitting them to pass the plasma membrane; d) activating and / or deactivating the photoswitchable compound according to the invention with light under irradiation condition A;

[0126] e) measuring the physiological effect by determining the change in a selected test parameter system;

[0127] f) deactivating the photoswitchable compound according to the invention with light under irradiation condition B; and

[0128] g) optionally repeating step d), e) and f) one or more times

[0129] According to one aspect of the invention, there is provided a method for reversibly and repetitively testing intracellular protein interactions in cells in vitro and / or in vivo, comprising the following steps:

[0130] a) providing, transfecting and expressing the DNA sequence of a fusion protein 1, comprising a test compound 1 and at least a HaloTag, in a cell / s;

[0131] b) providing, transfecting and expressing the DNA sequence of a fusion protein 2, comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or SLF’ binding domain of FKBP (F36V), in said cell / s;

[0132] c) adding a photoswitchable compound according to the invention to said cell / s and admitting said compound to pass the plasma membrane; d) activating and / or deactivating said photoswitchable compound with light under irradiation condition A for activation and under irradiation condition B for deactivation;

[0133] e) determining the change in a selected test parameter system; and

[0134] f) optionally repeating step d) and e) one or more times.

[0135] According to one aspect of the method of the invention said test compound 1 and test compound 2 are selected independently from one another among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

[0136] According to one aspect of the invention, there is provided a method for reversibly and repetitively testing intracellular protein interaction in cells in vitro and / or in vivo comprising the following steps;

[0137] a) Providing, transfecting and expressing the DNA sequence of a fusion protein 1, comprising a test compound 1 and at least HaloTag in a cell / s;

[0138] b) providing, transfecting and expressing the DNA sequence of a fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or SLF’ binding domain of FKBP (F36V), in said cell / s;

[0139] c) adding a photoswitchable compound according to the invention to said cell / s, preferably eukaryotic cells, and admitting said compound to pass the plasma membrane;

[0140] d) controlling the physiological effect by photoactivation and photodeactivation of the protein interaction process using either different wavelengths of light or in the dark; and

[0141] e) determining the change in a selected test parameter system; and

[0142] f) optionally repeating step d) and e) one or more times. According to one aspect of the invention, there is provided a method for reversibly and repetitively testing intracellular protein interaction in cells in vitro and / or in vivo comprises the following steps

[0143] a) Providing, transfecting and expressing the DNA sequence of a fusion protein 1 comprising a test compound 1 and at least HaloTag in a cell / s;

[0144] b) providing, transfecting and expressing the DNA sequence of a fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR to said cell / s;

[0145] c) adding a photoswitchable compound according to the invention to said cell / s and admitting said compound to pass the plasma membrane;

[0146] d) activating and / or deactivating the photoswitchable compound of general formula (I, II, III) with light under irradiation condition A for activation and under irradiation condition B for deactivation; and

[0147] e) determining the change in a selected test parameter system upon activation and / or deactivation of the photoswitchable compound according to the invention; and

[0148] f) optionally repeating step d) and e) one or more times.

[0149] The method for reversibly and repetitively testing intracellular protein interaction in cells may comprise the step a') instead of step a) and step b):

[0150] a') Providing, transfecting and expressing the DNA sequence of a fusion protein 3 comprising a test compound 3, at least HaloTag and at least the TMP binding domain of a bacterial DHFR.

[0151] The method for reversibly and repetitively testing intracellular protein interaction in cells may further comprise the step c') between step c) and step d):

[0152] c') after incubation, briefly wash the cell to remove the unreacted compound.

[0153] The cell / s as specified in this invention can be prokaryotic or eukaryotic cell / s.

[0154] In one aspect of the invention the cell / s specified herein are prokaryotic cells.

[0155] In another aspect of the invention the cell / s specified herein are eukaryotic cell / s.

[0156] As said before, the interaction introduced by a photoswitchable compound according to the invention after activation with light under irradiation condition A or in the dark is reversible by deactivation under irradiation condition B or in the dark. Therefore, the present invention refers also to above said method, wherein the generated effect is reversed by irradiating the cells, preferably eukaryotic cells, with light which causes the photoswitchable moiety of the photoswitchable compound according to the invention to undergo configurational change.

[0157] In this method test compound 1 and test compound 2 are selected independently from each other among proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, oligonucleic acids like DNA or RNA.

[0158] According to one embodiment of the invention, the bacterial DHFR in this method is eDHFR. Thus the method for reversibly and repetitively testing intracellular protein interaction in cells in vitro and / or in vivo, comprises the following steps:

[0159] a) Providing, transfecting and expressing the DNA sequence of a fusion protein 1 comprising a test compound 1 and at least HaloTag;

[0160] b) providing, transfecting and expressing the DNA sequence of a fusion protein 2 comprising a test compound 2 and at least the TMP binding domain of eDHFR; c) adding photoswitchable compound according to the invention to cells and letting them pass the plasma membrane;

[0161] d) activating and / or deactivating the photoswitchable compound according to the invention with light under irradiation condition A for activation and under irradiation condition B for deactivation; and

[0162] e) determining the change in a selected test parameter system, and

[0163] f) optionally repeating step d) and e) one or more times.

[0164] In this method activation of the photoswitchable compound according to the invention is (photo-)activated by irradiation with light or in the dark under irradiation condition A and (photo-)deactivated by irradiation with light or in the dark under irradiation condition B. Preferably, the irradiation condition A corresponds to the dark. It is also preferred that the irradiation condition B corresponds to irradiation with a laser diode at a wavelength of 405 nm. The present application is also directed to this method, wherein test compound 1 and test compound 2 is a physiological peptidic compound, respectively.

[0165] Suitable test parameter systems may be selected depending on the test compounds. Test parameter systems may be selected from the group comprising or consisting of: measurement of an enzymatic reaction like phosphorylation or dephosphorylation, proteolysis, ubiquitination or deubiquitination, glycosylation, acetylation or deacetylation, determining the transcription or translation of a gene of interest or a reporter gene, determining the activation of a signaling pathway, microscopic measurement of protein or peptide localization in cells, dimerization (binding) assay by fluorescent resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), protein compliment assay (PCA), fluorescence anisotropy, fluorescence correlation spectroscopy (FCS) I fluorescence cross-correlation spectroscopy (FCCS), yeast two-hybrid (Y2H), size exclusion chromatography, cosedimentation, pull-down, microarray, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), microscale thermophoresis, and chemical field-effect transistors. The transcription of a transporter gene may be determined by measurement of the corresponding mRNA or of the protein the gene encodes for. Alternatively, the result of an enzymatic reaction may be determined if the gene of interest or the reporter gene encodes for an enzyme.

[0166] A likewise interesting application is to regulate gene expression. Thus, the inventive method can be carried out in such a way that test compound 1 is a transcriptional modulator or epigenetic modifier and test compound 2 is a DNA binding protein such as TALE and CRISPR with catalytically dead Cas9 (dCas9), or test compound 1 is a DNA binding protein and test compound 2 is a transcriptional modulator or epigenetic modifier.

[0167] A likewise interesting application is to regulate genome editing and cell therapy. Thus the inventive method can be carried out in such a way that test compound 1 is fragment A and test compound 2 is fragment B of a protein / enzyme such as Cas9, caspase and chimeric antigen receptors (CARs), or test compound 1 is fragment B and test compound 2 is fragment A. Dimerization induced by the photoswitchable compound leads to reconstitution of a fully functional protein / enzyme by bringing both fragments in close proximity. Alternatively, the inventive method can be used in an arrangement, wherein test compound 3 is a protein / enzyme such as Cas9, caspase and chimeric antigen receptors (CARs). Dimerization induced by the photoswitchable compound may lead to inhibition of the function of a protein / enzyme, which could be restored by dedimerization.

[0168] A likewise interesting application is to test the interactions between a pharmaceutical drug (a pharmaceutical drug authorized for medical use in humans and / or animals, a corresponding prodrug or a drug candidate under investigation) and a physiological peptidic compound. Thus the inventive method can be used in an arrangement, wherein test compound 1 is a pharmaceutical drug and test compound 2 is a physiological peptidic compound, or test compound 1 is a physiological peptidic compound and test compound 2 is a pharmaceutical drug.

[0169] This test arrangement can be varied by investigating a gene regulating protein and a pharmaceutical drug. Thus the inventive method can be carried out in such a way that test compound 1 is a gene regulating protein and test compound 2 is a pharmaceutical drug, or test compound 1 is a pharmaceutical drug and test compound 2 is a gene regulating protein.

[0170] In molecular biology the use of commercially available kits is becoming more popular. They include all components needed for performing a specific test and / or analysis arrangement in reasonable amounts. Often some or all of the components are already dissolved in a suitable test solution or buffer in useful concentrations.

[0171] Therefore, this invention also applies to a kit, comprising

[0172] a) a photoswitchable compound;

[0173] b) the nucleotide sequences or the vectors including the nucleotide sequences coding for at least the binding domains of HaloTag and a bacterial DHFR.

[0174] Optionally, the kit according to the invention can also comprise

[0175] c) chemical means for promoting transfection. These means depend on the transfection method to be used. Possible chemical transfection methods include calcium phosphate method, dendrimer method, lipofection, and polycation-based methods.

[0176] BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Figure 1: Photoswitchable dimerization system. (A) Photostationary states of azobenzene. (B) TACs switch between trans and cis states. (C, E) Working principle of the photoswitchable dimerization systems. (D) Photostationary states of diazocine. (F) TDCs switch between cis and trans states.

[0178] Figure 2: Photoisomerization of TAC. (A) UV-Vis absorption spectra of TAC (50 pM) in DMSO in the dark (solid line) and after illumination at 405 nm (dash line) or 530 nm (dot line). (B) Consecutive cycles of trans-to-cis (Aex = 405 nm) and cis-to-trans photoisomerization (Aex= 530 nm) of TAC (50 pM) in DMSO at 37°C. (C) UV-Vis absorption spectra for the c / s-to- trans thermal relaxation of TAC (50 pM) in DMSO at 37°C in the dark. (D) Time course of the thermal relaxation of TAC. The solid line was resulted from a single exponential fit to obtain the rate constant and the half-life.

[0179] Figure 3: Native-PAGE of a mixture of HaloTag and eDHFR in the absence or presence of TAC with and without illumination at 405 nm. (A) trans-T AC (without illumination at 405 nm) induces heterodimerization of HaloTag and eDHFR in vitro. (B) c / s-TAC (with illumination at 405 nm) does not induce dimerization.

[0180] Figure 4: TAC induces repeated dimerization with light, while CONC induces one round dimerization with light in cells. (A) Light-induced reversible protein translocation in HeLa cells co-expressing mCherry-eDHFR and EGFP- HaloTag-ActA (located at mitochondria). Dimerization was induced by adding 1pM TAC. 405-nm illumination (+405) led to dedimerization. Thermal relaxation in the dark for 5 min (A) led to redimerization. Scale bar: 20 pm. (B) Quantification of mCherry and EGFP colocalization over multiple dimerization cycles in HeLa or Cos7 cells co-expressing mCherry-eDHFR and EGFP-HaloTag-ActA. Grey boxes indicated periods of 405 nm illumination. Light grey lines represent individual cells from at least 4 biologically independent experiments. Black lines represent the mean of all replicates. Co-localization was quantified as Pearson correlation coefficient (PCC). (C) HeLa cells co-expressing mCherry-eDHFR and HaloTag-EGFP-ActA (located at mitochondria) were treated with CONC (1.1 pM, 30min) and were illuminated with 458- nm light to induce protein dimerization. The dimerization was reversed by illumination at 405 nm. Scale bar, 10 pm.

[0181] Figure 5: (A) Schematic of how TAC can be applied to reversibly control endosomal positioning with light. (B) Control of endosomal positioning in HeLa cells co-expressing HaloTag-EGFP-Rab5a and KIF5B-mCherry- eDHFR. Dimerization was induced by adding 1pM TAC resulting in anterograde endosomal transport. 405-nm illumination (+405) led to dedimerization and endosome dispersal. Thermal relaxation in the dark for 5 min (A) led to redimerization. Scale bar: 10 pm.

[0182] Figure 6: (A) Absorption spectra of TAC-3 (50 pM) in DMSO in the dark, upon illumination at 405 nm or 530 nm. (B) Thermal relaxation of c / s-TAC-3 (50 pM) in DMSO at 37°C in the dark. (C) Time course of the cis-to-trans thermal relaxation of TAC-3 (50 pM in DMSO) in the dark at 37°C. Solid lines showed single exponential fits to obtain rate constants and halflives. (D) Kinetics of thermal relaxation in the dark leading to redimerization after dedimerization induced by 405-nm illumination in HeLa cells. (E) Light-induced reversible protein translocation in HeLa cells co-expressing mCherry-eDHFR and EGFP-HaloTag-ActA (located at mitochondria). Dimerization was induced by adding 1 pM TAC-3. 405- nm illumination (+405) led to dedimerization

[0183] Figure 7: Characterization of TMC. (A) UV-Vis absorption spectra of TMC (50 pM in DMSO) in the dark (solid line) and after irradiation at 405 nm (dash line) for trans-to-c / s photoisomerization or at 530 nm (dot line) for c / s-to- trans photoisomerization. (B) Time course of the cis-to-trans thermal relaxation of TMC (50 pM in DMSO) in the dark at 37°C. Solid lines showed single exponential fits to obtain rate constants and half-lives. (C) Native-PAGE of a mixture of HaloTag and eDHFR in the absence or presence of TMC with and without illumination at 405 nm. (left panel) trans-TMC (without illumination at 405 nm) induces heterodimerization of HaloTag and eDHFR in vitro, (right panel) c / s-TMC (with illumination at 405 nm) does not induce dimerization. (D) Photoswitchable dimerization in cells. Light-induced reversible protein translocation in HeLa cells coexpressing mCherry-eDHFR and EGFP-HaloTag-ActA (located at mitochondria). Dimerization was induced by adding 1pM TMC compound. 405-nm illumination (+405) led to dedimerization. Thermal relaxation in the dark for 5 min (A) led to redimerization.

[0184] Figure 8: (A) Absorption spectra of TPC (50 pM) in DMSO in the dark, upon illumination at 405 nm or 530 nm. (B) Consecutive cycles of trans-to-cis (lex = 405 nm) and cis-to-trans photoisomerization (lex = 530 nm) of TPC (50 pM) in DMSO at 37°C. (C) UV / Vis spectral scans with an interval of 2 h / 2 h on trans-TPC and c / s-TPC incubated with 10 mM GSH in DMSO: PBS= 1:1. (D) Light-induced reversible protein translocation in HeLa cells co-expressing mCherry-eDHFR and EGFP-HaloTag-ActA (located at mitochondria). Dimerization was induced by adding 1 pM TPC. 405-nm illumination (+405) led to dedimerization. Thermal relaxation in the dark for 5 min (A) led to redimerization.

[0185] Figure 9: UV-Vis absorption spectra of ABF4, ABF4-DD, ABF4-DW, ABF4-WW,

[0186] ABF4-AW and ABF4-AA in PBS: DMSO = 4:1 in the dark and after irradiation at 530 nm for trans-to-cis photoisomerization or at 405 nm for cis-to-trans photoisomerization.

[0187] Figure 10: The cis-to-trans thermal relaxation of ABF4-DD (A) and ABF4-DW (B) in the dark at 37°C in PBS: DMSO 4:1. Solid lines showed single exponential fits to obtain rate constants and half-lives. (C-E) Time course of thermal relaxation of ABF4-WW (C), ABF4-AW (D) and ABF4-AA (E). Fraction of c / s-form is calculated as (Adark -At) / (Adark - A530). A stands for absorption at 380 nm.

[0188] Figure 11: Photoisomerization of TFC-1, TFC-2, TFC-3 and TFC-4. (A) UV-Vis absorption spectra in DMSO in the dark (solid line) and after illumination at 530 nm (dash line) or 405 nm (dot line). (B) UV-Vis absorption spectra for the cis-to-trans thermal relaxation in PBS: DMSO = 1:1 at 37°C in the dark.

[0189] Figure 12: (A) Native-PAGE of a mixture of HaloTag and eDHFR in the absence or the presence of TFCs with and without illumination at 530 nm. (B) Protein-fragment complementation, treated with 100 nM TFCs in the dark, in the 530-nm light, or initially in the 530-nm light followed by the 405-nm light. Lower panel: quantification of relative bioluminescence intensity. (C) Complementation of split NanoLuc in HeLa cells, treated with 1 pM TFC-4 and left in the dark or subjected to 530-nm illumination, untr: untreated vehicle. Lower panel: quantification of relative bioluminescence intensity.

[0190] Figure 13: (A) Absorption spectra of TCC (50 pM) in DMSO in the dark, upon irradiation at 405 nm, 530 nm or 627 nm. (B) Consecutive cycles of trans- to-c / s (Aex = 627 nm) and cis-to-trans photoisomerization (Aex= 405 nm) of TCC in DMSO. (C) Time-course of the cis-to-trans thermal relaxation of TCC in PBS: DMSO = 1:1 at 37°C in the dark. The solid line was resulted from a single exponential fit to obtain the half-life. (D) Proteinfragment complementation, treated with 100 nM TCC in the dark, in the 627-nm light or initially in the 627-nm light followed by 405-nm light. Lower panel: quantification of relative bioluminescence intensity.

[0191] Figure 14: Photoisomerization of TDCs. (A) UV-Vis absorption spectra of TDC-1,

[0192] TDC-2, and TDC-3 in PBS: DMSO = 1:1 in the dark (solid line) and after illumination at 405 nm (dash line) or 530 nm (dot line). (B) Consecutive cycles of cis-to-trans ( ex = 405 nm) and trans-to-cis photoisomerization (Aex = 530 nm) of TDC-1 or TDC-3 in PBS: DMSO = 1:1. (C) Time course of the trans-to-cis thermal relaxation of TDC-1, TDC-2 and TDC-3 in PBS: DMSO = 1:1 at 37°C in the dark. The solid line showed a single exponential fit to obtain the rate constant and the half-life.

[0193] Figure 15: TDCs induce repeated dimerization with light in cells. (A)

[0194] Complementation of split NanoLuc in HeLa cells expressing HaloTag- LgBiT and SmBiT-eDHFR, treated with 5 pM TDC-1, TDC-2 or TDC-3 in the dark or with 405-nm illumination, untr: untreated vehicle. (B) Quantification of bioluminescence in (A). Mean intensities were determined using Imaged. Data are shown as mean ± SD of 3 biological replicates. Statistical significance was determined using One-way ANOVA. (C) Light-induced reversible protein translocation in HeLa cells co-expressing mCherry-eDHFR and EGFP-HaloTag-ActA (located at mitochondria). Dimerization was induced by 405 nm illumination (405). Thermal relaxation in the dark (A) led to dedimerization. (D) Quantification of mCherry and EGFP colocalization over multiple dimerization cycles in HeLa cells. Data are shown as mean ± SD of 3 biologically independent experiments.

[0195] Figure 16: (A) Schematic of TPC-mediated complementation of the split NanoLuc system consisting of HaloTag-LgBiT and SmBiT-eDHFR. (B) Schematic of illumination using a photomask for patterning. (C) HeLa cells transiently transfected with HaloTag-LgBiT and SmBiT-eDHFR were treated with 1 pM TPC and illuminated using a photomask. Detection of NanoLuc complementation by bioluminescence was done using NanoGio Live Cell Assay. (D) Quantification of bioluminescence in (C). Statistical significance was determined using unpaired t-test. (E) Complementation of split NanoLuc in subpopulations of HeLa expressing HaloTag-LgBiT and SmBiT-eDHFR, treated with TPC and exposed to 405-nm light using different photomasks.

[0196] Figure 17: (A) Schematic of TPC-mediated control of protein stability in cells. In cells expressing eDHFRDD-POI and HaloTag-mCherry, the POI is continuously degraded. Upon treatment with TPC, eDHFRDDis stabilized in a dimerization-dependent manner. Illumination of cells with 405nm light results in dedimerization, dissociation of eDHFRDDfrom HaloTag and subsequent degradation of the fusion protein. (B) Representative western blot of parental Hek293FT or Hek293FT stably expressing eDHFRDD-HA-KRASG12Vand HaloTag-mCherry treated with vehicle or 1 pM TPC for 30min. Cells were washed and incubated for 16h and subsequently illuminated at 405 nm for 6 h or in the dark.

[0197] Figure 18: (A) Absorption spectra of TSMC (30 pM) in PBS: DMSO 1:1 in the dark, upon irradiation at 405 nm or 530 nm (B) Light-induced reversible protein translocation in HeLa cells co-expressing mCherry-eDHFR and EGFP- HaloTag-ActA (located at mitochondria). Dimerization was induced by adding 1 pM TSMC. 405-nm illumination (+405) led to dedimerization. Thermal relaxation in the dark for 5 min (A) led to redimerization. Scale bar: 10 pm.

[0198] EXAMPLES

[0199] Below follows a number of non-limiting examples of the invention.

[0200] The following table lists the abbreviations used in this section.

[0201] Abbreviations: n-Bu4NBr: Tetra-n-butylammonium bromide; TFA: trifluoroacetic acid; DCM: dichloromethane; RT: room temperature, DMSO: Dimethyl sulfoxide; EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; HOBt: Hydroxybenzotriazole; DIPEA: A / , A / -diisopropylethylamine; DMF: A / , A / -dimethylformamide; HATU: 1-[Bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DMAP: 4-Dimethylaminopyridine; NBS: A / -bromosuccinimide, NMP: A / -Methyl-2-pyrrolidone, Oxone: potassium peroxymonosulfate, Pd2dba3: tris(dibenzylideneacetone)dipalladium(0) Pd(dppf)Cl2: [1,1 ' - Bis(diphenylphosphino)ferrocene]dichloropalladium(ll). Pd(PPh3)4: Tetrakis(triphenylphosphine)palladium(0). TCFH: Ch loro- A / , A / , A / ’, A / ’ -tetra methylformamidinium hexafluorophosphate. NMI: A / -Methylimidazole. XantPhos: 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene. PyBOP: (Benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate, THF: tetrahydrofuran. EA: ethyl acetate. PE: petroleum ether.

[0202] Preparation of compounds

[0203] Example 1:

[0204] Synthesis of compound TAC:

[0205]

[0206] Compound 3: tert-Butyl 3-((6-chlorohexyl)oxy)propanoate. To a solution of compounds 1 (2.0 g, 14.64 mmol) and 2 (5.6 g, 43.92 mmol) in DCM (30 mL) was added tetrabutylammonium bromide (944 mg, 2.93 mmol) and 50% NaOH (3.5 mL). The reaction mixture was stirred at room temperature (RT) for overnight. Then, H2O (20 mL) was added and the resulting mixture was extracted with DCM (3 x 30 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (EA / PE) to furnish compound 3 (3.1g, 80% yield) as a colorless oil.

[0207] 1H NMR (400 MHz, CDCI3) 53.65 (t, J = 6.5 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H), 3.43 (t, J = 6.5 Hz, 2H), 2.48 (t, J = 6.5 Hz, 2H), 1.80-1.73 (m, 2H), 1.60 - 1.53 (m, 2H), 1.49 - 1.36 (m, 13H);13C NMR (100 MHz, CDCI3) 5 171.0, 80.4, 70.8, 66.4, 45.0, 36.3, 32.5, 29.4, 28.0, 26.6, 25.4; HRMS(ESI-TOF): m / z = 265.0635 (calcd. for [M + H]+, 265.1565).

[0208] Compound 5: 4-((2,4-Diaminopyrimidin-5-yl)methyl)-2,6-dimethoxy-phenol. 5g of trimethoprim (TMP) 4 was dissolved in 60 mL 48% HBr pre-heated to ca. 100°C. Reaction was stirred for ca. 20 min. and then quenched by slow addition of 12 mL 50% NaOH. Reaction was allowed to cool to RT, and placed at 4°C overnight, allowing crystals to form. Crystals were filtered and washed with ice-cold water. Crystals were dissolved in ca. 25 mL of boiling water, and the solution was neutralized with NH4OH, leading to recrystalization. Crystals were filtered and dried under vacuum, yielding the desired product 5 (3 g, 60 %).1H NMR (400 MHz, CD3OD) 5 7.18 (s, 1H), 6.54 (bs, 2H), 3.82 (s, 6H), 3.63 (s, 2H); HRMS(ESI-TOF): m / z = 277.1295 (calcd. for [M + H]+, 277.1295).

[0209] Compound 6: tert-Butyl 2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)acetate. To a solution of the compound 5 (1.0 g, 3.6 mmol) in anhydrous DMSO (12 mL) was addedfBuOK (449 mg, 4.0 mmol) and stirred under Ar atmosphere at RT for 20 min. Then tert-butyl bromoacetate (0.59 mL, 4.0 mmol) was added to the solution and stirred at RT. When completed (within 2.0 h), the reaction mixture was diluted with EA (30 mL) and washed by water (3 x 20 mL ). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Then the residual brown oil was perified by column chromatography (MeOH / DCM) to afford the desired product 6 (342 mg, 25% yield) as a white solid.1H NMR (500 MHz, CD3OD) 5 7.23 (s, 1H), 6.58 (s, 2H), 4.42 (s, 2H), 3.81 (s, 6H), 3.67 (d, J = 1.0 Hz, 2H), 1.49 (s, 9H);13C NMR (126 MHz, CD3OD) 5 170.6, 166.3, 156.2, 154.4, 140.4, 136.4, 134.1, 111.1, 107.3, 83.0, 70.9, 56.7, 33.9, 28.3; HRMS(ESI-TOF): m / z = 391.1972 (calcd. for [M + H]+, 391.1976).

[0210] Compound 8: (E)-N-(4-((4-aminophenyl)diazenyl)phenyl)-2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)acetamide. To a solution of 6 (500 mg, 1.28 mmol) in DCM (3 mL) was added TFA (3 mL) and stirred at RT for 1 h, then solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. The residual oil was dissolved in anhydrous DMF (4 mL), then EDCI (307 mg, 1.6 mmol), HOBt (216 mg, 1.6 mmol), and DIPEA (207 pL, 1.6 mmol) were added and the solution was stirred at RT for 30 min. A solution of 7 (340 mg, 1.6 mmol) in anhydrous DMF (1 mL) was added and stirred at RT for 16 h. Then the solvent was removed under reduced pressure and the residual oil was purified by column chromatography (MeOH / DCM) to obtain the desired product 8 (302 mg, 44% yield) as a yellowish oil.1H NMR (500 MHz, CD3OD) 57.90 -7.84 (m, 2H), 7.83 - 7.77 (m, 4H), 7.25 (t, J = 1.1 Hz, 1H), 6.98 - 6.91 (m, 2H), 6.67 (s, 2H), 4.59 (s, 2H), 3.92 (s, 6H), 3.70 (s, 2H);13C NMR (126 MHz, CD3OD) 6 170.6, 166.3, 161.7, 156.2, 153.9, 150.5, 147.3, 140.5, 140.4, 137.0, 134.7, 126.2, 124.2, 121.3, 117.6, 110.8, 107.3, 73.8, 56.9, 33.9; HRMS(ESI-TOF): m / z = 529.2311 (calcd. for [M + H]+, 529.2307).

[0211] Compound TAC: (E)-3-((6-chlorohexyl)oxy)-N-(4-((4-(2-(4-((2,4-diamino pyrimidin-5-yl)methyl)-2,6 dimethoxyphenoxy) acetamido) phenyl) diazenyl) phenyl)propanamide. To a stirred solution of compound 3 (56.1 mg, 0.212 mmol) in DCM (1 mL) was added TFA (1 mL). The reaction solution was stirred at RT for 1 h. The solvent was removed under reduced pressure, and further removed by coevaporation with toluene (2 x 1 mL). To a stirred solution of this residue in dry DMF (4 mL) was added compound 8 (56.0 mg, 0.106 mmol), HATU (161.2 mg, 0.424 mmol), DMAP (2.6 mg, 0.021 mmol), and DIPEA (54 pL, 0.318 mmol). The mixture was stirred at RT for 18 h. The mixture was diluted with EA (20 mL), washed with H2O (20 mL), and brine (3 x 20 mL), dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash colomn chromatography (MeOH / DCM) to give the final product TAC (6.2 mg, 8% yield) as a solid.1H NMR (850 MHz, DMSO-d6) 6 10.28 (s, 1H), 10.11 (s, 1H), 7.96 - 7.78 (m, 8H), 7.53 (s, 1H), 6.65 (s, 2H), 6.14 (s, 2H), 5.72 (s, 2H), 4.51 (s, 2H), 3.80 (s, 6H), 3.68 (t, J= 6.2 Hz, 2H), 3.57 (t, J= 6.7 Hz, 2H), 3.55 (s, 2H), 3.39 (t, J = 6.5 Hz, 2H), 2.59 (t, J = 6.2 Hz, 2H), 1.67 - 1.64 (m, 2H), 1.50 -1.46 (m, 2H), 1.37 - 1.33 (m, 2H), 1.30 - 1.27 (m, 2H);13C NMR (214 MHz, DMSO-d6) 5 169.8, 167.9, 162.2, 155.4, 151.8, 148.0, 147.5, 142.0, 140.6, 136.7, 134.3, 123.44, 123.42, 119.7, 119.2, 105.9, 105.7, 72.3, 70.0, 66.2, 56.1, 45.3, 37.4, 33.0, 32.0, 29.0, 26.1, 24.9; HRMS (ESI-TOF): m / z = 719.3071 (calcd. for [M + H]+, 719.3067).

[0212] Example 2:

[0213] Synthesis of compound TAC-3

[0214]

[0215] Compound 11: 5-(4-bromo-3,5-dimethoxybenzyl)pyrimidine-2,4-diamine. To a solution of compound 9 (2.45 g, 10.0 mmol) in dry 25 mL DMSO were added anilinopro pionitrile (1.61 g, 11.0 mmol) andfBuOK (1.29 g, 11.5 mmol) subsequently. The reaction mixture was stirred at RT for 1 h and then partitioned between EA and H2O (100 mL each). The aqueous layer was extracted with EA (2 x 100 mL) and the combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure to obtain the crude intermediate compound 10, which was used directly for the next step. A guanidine solution was prepared separately by dissolving guanidine hydrochloride (2.87 g, 30.0 mmol) in 30 mL dry EtOH and treating it withfBuOK (3.37 g, 30.0 mmol). This mixture was allowed to stir for 30 min at RT before it was filtered and the filter cake was washed with another 30 mL of EtOH. The obtained filtrate was then added to the crude intermediate of the first step and stirred overnight at 75°C. Upon cooling, the product crystallized from the reaction mixture. It was filtered off, washed successively with ice-cooled H2O and H2O / EtOH (1:1) and dried in vacuo, yielding the desired product 11 (1.9 g, 56% yield for 2 steps) as a yellow solid.1H NMR (400 MHz, CDCI3) 57.78 (s, 1 H), 6.39 (s, 2H), 4.85 (br, NH2), 4.59 (br, NH2), 3.85 (s, 6H), 3.68 (s, 2H). HRMS (ESI-TOF): m / z = 339.0441 (calcd. for [M + H]+, 339.0451).

[0216] Compound 13: (E)-4-((4-bromophenyl)diazenyl)phenol. Compound 12 (1.72 g, 10.0 mmol) was dissolved in 13 mL 2 M aqueous HCI and cooled to 0 °C before a solution of NaNO2 (690 mg, 10.0 mmol) in 10 mL water was added dropwise. The mixture was stirred at 0°C for 30 min. In a separate flask, phenol (941 mg, 10.0 mmol) dissolved in 20 mL of water was treated with NaOH (400 mg, 10.0 mmol) and Na2CO3(2.12 g, 20.0 mmol) and the resulting solution was cooled to 0°C. To this, the diazonium solution was carefully added dropwise and upon complete addition the mixture was stirred at 0°C for 30 min. After acidifying the reaction mixture with 2 M aqueous HCI, the precipitate was filtered off, washed with water and dried in vacuo to yield the orange solid 13 (2.3 g, 83% yield).1H NMR (400 MHz, DMSO-d6) 6 10.38 (s, OH), 7.81 (d, J = 8.4 Hz, 2H), 7.76 (s, 4H), 6.95 (d, J = 8,7 Hz, 2H).

[0217] Compound 14: tert-butyl (E)-2-(4-((4-bromophenyl)diazenyl)phenoxy)acetate.

[0218] Compound 13 (1.5 g, 5.4 mmol) was dissolved in 20 mL acetone, and tert-butyl 2-bromoacetate (1.6g, 8.2 mmol) and K2CO3 (896 mg, 6.5 mmol) were added. The reaction mixture was stirred at 50°C overnight before the solvent was removed under reduced pressure and the residue was partitioned between EA and H2O (50 mL each). The aqueous layer was extracted with EA (2 x 50 mL), the combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure and the residual oil was purified by column chromatography (EA / PE) to obtain the desired product 14 (1.56 g, 74% yield) as a brown solid.1H NMR (400 MHz, CDCh) 5 7.84 (d, J = 9.0 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.53 (s, 2H), 1.47 (s, 9H).

[0219] Compound 15: ferf-butyl (E)-2-(4-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)diazenyl)phenoxy)acetate. Compound 14 (1.36g, 3.47 mmol) and Pin2B2 (1.06 g, 4.17 mmol) were dissolved in 20 mL DMSO, then Pd(dppf)Cl2 (254 mg, 0.35 mmol) and KOAc (1.02 g, 10.41 mmol) were added. The reaction mixture was stirred at 80°C overnight under N2. The solution was diluted with 50 mL H2O and the aqueous layer was extracted with EA (2 x 50 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure and the residual oil was purified by column chromatography (EA / PE) to obtain the desired product 15 (1.1 g, 73% yield) as an orange solid.1H NMR (400 MHz, CDCI3) 57.95 -7.92 (m, 4H), 7.86 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 9.1 Hz, 2H), 4.60 (s, 2H), 1.50 (s, 9H), 1.37 (s, 12H).13C NMR (100 MHz, CDCI3) 5 167.5, 160.3, 154.4, 147.5, 135.6, 124.8, 121.7, 114.8, 84.0, 82.7, 65.8, 28.0, 24.9. HRMS(ESI-TOF): m / z = 439.2386 (calcd. for [M + H]+, 439.2399).

[0220] Compound 16: ferf-butyl (E)-2-(4-((4'-((2,4-diaminopyrimidin-5-yl)methyl)-2',6'-dimethoxy-[1,1 '-biphenyl]-4-yl)diazenyl)phenoxy)acetate. To a solution of Compound 11 (100 mg, 0.29 mmol) and compound 15 (142 mg, 0.32 mmol) in 2 mL DME were added Pd(PPhs)4(34 mg, 0.03 mmol) and 1.25 mL 2M K2CO3. The reaction mixture was stirred at 85°C for 1 h under microwave irradiation. The solution was diluted with 5 mL H2O and the aqueous layer was extracted with EA (2 x 5 mL). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated under reduced pressure, and the residual oil was purified by column chromatography (MeOH / DCM) to obtain the desired product 16 (146 mg, 87% yield) as a yellow solid.1H NMR (400 MHz, CDCI3) 57.92 - 7.89 (m, 4H), 7.83 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 9.1 Hz, 2H), 6.48 (s, 2H), 4.85 (s, NH2), 4.70 (s, NH2), 4.60 (s, 2H), 3.75 (s, 2H), 3.70 (s, 6H), 1.50 (s, 9H).13C NMR (100 MHz, CDCI3) 5 167.6, 162.8, 161.9, 160.0, 158.0, 156.2, 151.3, 147.7, 139.5, 136.5, 131.8, 124.6, 122.0, 117.5, 114.8, 106.3, 104.1, 82.7, 77.3, 65.8, 56.0, 35.1, 28.1. HRMS(ESI-TOF): m / z = 571.2645 (calcd. for [M + H]+, 571.2663).

[0221] Compound 20: ferf-Butyl (2-((6-chlorohexyl)oxy)ethyl)carbamate. To a solution of Compound 18 (1.47 g, 9.13 mmol) in dry THF (30 mL) was added NaH (365 mg, 9.13 mmol) dropwise at 0°C. The reaction mixture was stirred at 0°C for 30 min, then compound 19 (1.5 g, 6.08 mmol) in dry THF (5 mL) was added slowly. The reaction mixture was stirred at RT overnight and quenched with H2O and extracted with EA, the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to give the desired product 20 (177 mg, 11 % yield) as a colorless oil.1H NMR (600 MHz, CDCI3) 53.53 (t, J = 6.6 Hz, 2H), 3.45 (t, J = 5.0 Hz, 2H), 3.42 (t, J = 6.6 Hz, 2H), 3.29 - 3.28 (m, 2H), 1.79 - 1.75 (m, 2H), 1.60 - 1.55 (m, 2H), 1.47 - 1.44 (m, 11 H), 1.42 -1.34 (m, 2H);13C NMR (150 MHz, CDCI3) 8 155.9, 79.19, 70.9, 69.7, 45.0, 40.4, 32.5, 29.4, 28.4, 26.7, 25.4; HRMS (ESI-TOF): m / z = 280.1845 (calcd. for [M + H]+, 280.1674).

[0222] Compound TAC-3: (E)-N-(2-((6-chlorohexyl)oxy)ethyl)-2-(4-((4'-((2,4-diamino pyrimidin-5-yl)methyl)-2',6'-dimethoxy-[1, T-biphenyl]-4-yl) diazenyl) phenoxy) acetamide. Compound 16 (40 mg, 0.07 mmol) was dissolved in DCM (2 mL) at 0°C and TFA (1 mL) was added. The mixture was stirred at RT for 1 h. The solvent was concentrated under reduced pressure to give the intermediate 17, which was used directly for next step without further purification. Compound 20 (20 mg, 0.07 mmol) was dissolved in DCM (2 mL) at 0 °C and TFA (1 mL) was added. The mixture was stirred at RT for 1 h. The solvent was concentrated under reduced pressure to give the intermediate 21, which was used directly for next step without further purification. The intermediate 17 and 21 were dissolved in DCM (5 mL), and EDCI (15 mg, 0.08 mmol), HOBt (12 mg, 0.08 mmol) and DIPEA (97 pL, 0.56 mmol) were added. The mixture was stirred at RT overnight. The solvent was concentrated under reduced pressure and the resulting mixture was purified by column chromatography (MeOH / DCM) to yield the desired product TAC-3 (27 mg, 57% yield for 2 steps) as a yellow solid.1H NMR (400 MHz, CDCI3) 67.88 - 7.84 (m, 4H), 7.69 (s, 1 H), 7.40 (d, J = 7.6 Hz, 2H), 6.97 (d, J= 8.6 Hz, 2H), 6.87 (s, NH), 6.40 (s, 2H), 5.19 (br. NH2), 4.85 (br. NH2), 4.52 (s, 2H), 3.69 - 3.64 (m, 8H), 3.48 - 3.42 (m, 6H), 3.36 - 3.33 (m, 2H), 1.72 - 1.65 (m, 2H), 1.52 - 1.46 (m, 2H), 1.41 - 1.28 (m, 4H). HRMS (ESI-TOF): m / z = 676.2981 (calcd. for [M + H]+, 676.3009).

[0223] Example 3:

[0224] Synthesis of compound TMC

[0225]

[0226] Compound 24: (E)-4-((4-aminophenyl)diazenyl)-3,5-dimethylaniline. To a solution of AcOH (10 mL), Propionic acid (6 mL), H2SO4 (1.5 mL) at 0°C was added Compound 22 (1.38 g, 10 mmol), then NaNCh (828 mg, 12 mmol) in H2O (2 mL) was added dropwise. The reaction mixture was stirred for 1 h. Then Urea (0.2 g) in H2O (1 mL) was added and the mixture was stirred for 5 min. Compound 23 (1.21 g, 10 mmol) dissolved in HCO2H (20 mL) was added dropwise and the reaction was stirred for 30 min to form precipitate, filtered, and used for the next step without further purification. The intermediate (2.7 g, 10 mmol) was dissolved in dioxane (50 mL) and Na2S 9H2O (7.2 g, 30 mmol) dissolved in H2O (10 mL) was added, the reaction mixture was stirred 4 h at 90 °C. The mixture was diluted with EA and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to give the target product 24 as a red solid (1.34 g, 50% yield for two steps).1H NMR (400 MHz, CDCI3) 5 7.74 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz, 2H), 6.41 (s, 2H), 3.85 (br. 4H), 2.43 (s, 6H);13C NMR (100 MHz, CDCI3) 5 148.4, 146.2, 146.2, 143.0, 134.3, 123.9, 115.3, 114.6, 20.0; HRMS(ESI-TOF): m / z = 241.1446 (calcd. for [M + H]+, 241.1448).

[0227] Compound 25: (E)-N-(4-((4-aminophenyl)diazenyl)-3,5-dimethylphenyl)-3-((6 chlorohexyl)oxy)propanamide. To a solution of 3 (165 mg, 0.62 mmol) in DCM (3 mL) was added TFA (3 mL) and stirred at RT for 1 h, then solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. The oil was dissolved in anhydrous DMF (4 mL), HATU (262 mg, 0.69 mmol) and DIPEA (318 pL, 1.88 mmol) was added and stirred at RT for 30 min. Then a solution of compound 24 (150 mg, 0.62 mmol) in anhydrous DMF (1 mL) was added and the reaction mixture was stirred at RT overnight. The solvent was removed under reduced pressure and the residual brown oil was purified by column chromatography (EA / PE) to give the target product 25 (160 mg, 62% yield) as a red solid.1H NMR (400 MHz, CDCI3) 58.34 (s, 1 H), 7.75 (d, J = 8.7 Hz, 2H), 7.27 (s, 2H), 6.73 (d, J = 8.7 Hz, 2H), 3.76 (t, J = 5.6 Hz, 2H), 3.56 - 3.51 (m, 4H), 2.63 (t, J = 5.6 Hz, 2H), 2.35 (s, 6H), 1.81 - 1.74 (m, 2H), 1.70 - 1.63 (m, 2H), 1.51 -1.42 (m, 4H);13C NMR (100 MHz, CDCI3) 5 169.9, 149.3, 147.6, 145.8, 137.1, 132.2, 124.5, 119.8, 114.5, 71.2, 66.7, 44.9, 38.1, 32.5, 29.5, 26.7, 25.6, 19.3; HRMS(ESI-TOF): m / z = 431.2208 (calcd. for [M + H]+, 431.2208). Compound TMC: (E)-3-((6-chlorohexyl)oxy)-N-(4-((4-(2-(4-((2,4-diamino pyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)acetamido)phenyl)diazen yl)-3,5-dimethylphenyl)propanamide. To a solution of 6 (32 mg, 0.08 mmol) in DCM (1 mL) was added TFA (1 mL) and stirred at RT for 1 h, then solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. The residual oil was dissolved in anhydrous DMF (2 mL), PyBOP (52 mg, 0.1 mmol) and DIPEA (68 pL, 0.4 mmol) was added and stirred at RT for 30 min. Then a solution of compound 12 (43 mg, 0.1 mmol) in anhydrous DMF (1 mL) was added and the reaction mixture was stirred at RT overnight. The solvent was removed under reduced pressure and the residual red oil was purified by column chromatography (MeOH / DCM) to give the target molecule TMC (19 mg, 32% yield) as a red solid.1H NMR (600 MHz, DMSO-d6) 6 10.09 (s, NH), 10.02 (s, NH), 7.89 - 7.82 (m, 4H), 7.53 (s, 1H), 7.44 (s, 2H), 6.65 (s, 2H), 6.16 (s, NH2), 5.74 (s, NH2), 4.51 (s, 2H), 3.81 (s, 6H), 3.66 (t, J = 6.1 Hz, 2H), 3.59 - 3.55 (m, 4H), 3.39 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 6.1 Hz, 2H), 2.38 (s, 6H), 1.69 - 1.64 (m, 2H), 1.50 - 1.46 (m, 2H), 1.38 - 1.27 (m, 4H);

[0228] 13C NMR (150 MHz, DMSO-d6) 6 169.6, 167.8, 162.2, 162.0, 151.8, 148.6, 145.3, 140.5, 139.5, 136.7, 134.3, 132.6, 132.4, 123.0, 119.8, 119.7, 119.3, 105.9, 105.7, 72.3, 69.9, 66.2, 56.1, 45.3, 37.3, 33.0, 32.0, 29.0, 26.1, 24.9, 19.7; HRMS(ESI-TOF): m / z = 747.3364 (calcd. for [M + H]+, 747.3380).

[0229] Example 4:

[0230] Synthesis of ort / ?o-tetrafluoroazobenzenes:

[0231]

[0232] Compound ABF4: (E)-1,2-bis(2,6-difluorophenyl)diazene. 2,6-difluoroaniline (26) (500 mg, 3.87 mmol) and a freshly ground mixture of KMnC (2 g) and FeSO4‘7H2O (2 g) were dissolved in DCM (40 mL). The solution was refluxed overnight, filtered through celite, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM / PE) to give the target product ABF4(121 mg, 25% yield) as an orange / red solid.1H NMR (400 MHz, CDCh) 5 7.41 - 7.34 (m, 2H), 7.10 - 7.04 (m, 4H); HRMS (ESI-TOF): m / z = 255.1227 (calcd. for [M + H]+, 255.0540).

[0233] Compound 27: 4-Bromo-2,6-difluoroaniline. To a solution of 2,6-difluoroaniline (20 g, 154.91 mmol) in CHCI3 (100 mL) was added NBS (30.3 g, 170.40 mmol) dropwise. The mixture was stirred at RT overnight, and then diluted with water. The two phases were separated and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM / PE) to give 27 (24.6 g, 87%) as a purple solid.1H NMR (400 MHz, CDCh) 67.00 (dd, J = 6.5, 1.5 Hz, 2H), 3.59 (br, NH2); HRMS (ESI-TOF): m / z = 207.1746 (calcd. for [M + H]+, 207.9568).

[0234] Compound 28: (E)-1,2-bis(4-bromo-2,6-difluorophenyl)diazene. To a solution of 27 (5.0 g, 24.04 mmol) in DCM (100 mL) was added a freshly ground mixture of KMnCU (10 g) and FeSO4‘7H2O (10 g). The solution was refluxed overnight, filtered through celite, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM / PE) to give 28 (1.76 g, 36%) as an orange / red solid.1H NMR (400 MHz, CDCI3) 57.29 - 7.26 (m, 4H); HRMS (ESI-TOF): m / z = 410.8749 (calcd. for [M + H]+, 410.8750).

[0235] Compound ABF4-DD: E)-N, N '-(diazene-1,2-diylbis(3,5-difluoro-4,1 -phenylene))diacetamide. Compound 28 (100 mg, 0.24 mmol), acetamide (86 mg, 1.45 mmol), CS2CO3 (274 mg, 0.42 mmol), Pd2dba3 (10 mg, 0.012 mmol) together with 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (28 mg, 0.048 mmol) were dissolved in 10 mL of dry dioxane. The reaction mixture under ishwas heated at 90°C for 2 h. The mixture was diluted with EA and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to give the target product ABF4-DD (72 mg, 81% yield) as an orange- red solid.1H NMR (400 MHz, DMSO-de) 6 10.63 (s, 2NH), 7.49 (d, J= 12.0 Hz, 4H), 2.12 (s, 6H); HRMS (ESI-TOF): m / z = 369.0968 (calcd. for [M + H]+, 369.0969).

[0236] Compound 29: 4-Amino-3,5-difluorobenzonitrile. A mixture of copper(l) cyanide (11.8 g, 0.13 mol) and 27 (13.6 g, 0,065 mol) in dry A / -methylpyrolidone (60 mL) was refluxed for 3 h. The mixture was then poured into a NH3 12% aqueous solution and extracted with EA. The organic phase was dried over Na2SC, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM / PE) to give 29 (8.7 g, 87%) as a white solid.1H NMR (400 MHz, CDCI3) 5 7.14 (dd, J = 6.1, 2.2 Hz, 2H), 4.28 (br, NH2); HRMS (ESI-TOF): m / z = 155.0414 (calcd. for [M + H]+, 155.0415).

[0237] Compound 30: 4-Amino-3,5-difluorobenzoic acid. Compound 29 (8.7 g, 56.45 mmol) was suspended in 50% aq. H2SO4 (50 mL). The reaction mixture was heated to 80°C and vigorously stirred overnight. After cooling to RT, the mixture was carefully neutralized with 30% aq. NaOH and the white precipitate was filtered off. Recrystallization from ethanol yielded 30 (9.6 g, 97 %) as a white solid.1H NMR (400 MHz, DMSO-de) 67.39 (dd, J = 7.2, 2.4 Hz, 2H), 6.06 (br, NH2); HRMS (ESI-TOF): m / z = 174.0361 (calcd. for [M + H]+, 174.0361).

[0238] Compound 31: (E)-4-((4-bromo-2, 6-difluorophenyl)diazenyl)-3,5-difluorobenzoic acid. To a solution of 30 (259 mg, 1.5 mmol) in DCM (10 mL) and acetone (2 mL) was added Oxone® (1,84g, 3.0 mmol) dissolved in H2O (5 mL). The solution was stirred at RT for 2 h. After separation of the layers, the aqueous organic layer was washed with H2O and concentrated under reduced pressure. Without any further purification the crude product was suspended in 40 mL of acetic acid / toluene / TFA 6:6:1 and the corresponding aniline compound 27 (210 mg, 1.0 mmol) was added. The mixture was stirred at RT for 3 days. The solution was diluted with H2O and extracted with EA, the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product 31 as a light yellow solid (210 mg, 55% yield for 2 steps).1H NMR (400 MHz, Acetone-d6) 67.80 (d, J = 9.1 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H);13C NMR (100 MHz, Acetone-de) 6 163.9, 156.7, 156.0, 154.1, 153.4, 134.1, 125.2, 117.0, 113.9; HRMS (ESI-TOF): m / z = 377.1228 (calcd. for [M + H]+, 376.9543). Compound ABF4-DW: (E)-4-((4-acetamido-2,6-difluorophenyl)diazenyl)-3,5-difluoro-N-methyl benzamide. To a solution of Compound 31 (70 mg, 0.19 mmol) in dry DMF (5 mL) was added PyBOP (118 mg, 0.23 mmol) and DIPEA (161 pL, 0.95 mmol) and the mixture was stirred at RT for 30 min. Then a solution of methylamine hydrochloride (19 mg, 0.28 mmol) in dry DMF (1 mL) was added and stirred at RT for 16 h. The solvent was removed under reduced pressure and the residual oil was purified by column chromatography (EA / PE) to give the desired intermediate 45 mg as a red solid. Then the intermediate, acetamide (20 mg, 0.35 mmol), CS2CO3 (150 mg, 0.46 mmol), Pd2 ba3 (6 mg) and Xantphos (14 mg) were dissolved in 5 mL of dry dioxane. The solution was heated under N2 at 90 °C for 2 h. The mixture was diluted with EA and washed with brine. The two phases were separated, and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to give the target product ABF4-DW (21 mg, 31% yield for 2 steps) as a red solid.1H NMR (600 MHz, Acetone-cfe) 67.69 (d, J = 9.5 Hz, 2H), 7.56 (d, J = 12.0 Hz, 2H), 2.93 (d, J = 4.6 Hz, 3H), 2.17 (s, 3H); HRMS (ESI-TOF): m / z = 369.0967 (calcd. for [M + H]+, 369.0969).

[0239] Compound 32: Ethyl 4-amino-3,5-difluorobenzoate. Compound 30 (5.0 g, 28.89 mmol) was dissolved in EtOH (50 mL) and H2SO4 (1 mL), and refluxed for 5 h. The mixture was neutralized with saturated NaHCOs, extracted with DCM, the two phases were separated and the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give 32 ( 5.1 g, 88% yield )as a pale brown solid.1H NMR (400 MHz, CDCI3) 57.53 (dd, J =7.1, 2.1 Hz, 2H) 4.33 (q, J = 7.1 Hz, 2H), 4.12 (br, NH2), 1.37 (t, J= 7.1 Hz, 3H); HRMS (ESI-TOF): m / z = 202.0674 (calcd. for [M + H]+, 202.0674).

[0240] Compound 33: Diethyl 4,4'-(diazene-1,2-diyl)(E)-bis(3,5-difluorobenzoate ). To a solution of 32 (2.9 g, 14.5 mmol) in DCM (50 mL) was added a freshly ground mixture of KMnO4 (6 g) and FeSO4‘7H2O (6 g). The solution was refluxed overnight, filtered through celite, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (DCM / PE) to give 33 ( 0.89 g, 32%) as an orange / red solid.1H NMR (400 MHz, CDCI3) 67.75 (d, J = 8.9 Hz, 4H), 4.43 (q, J = 7.1 Hz, 4H), 1.43 (t, J = 7.1 Hz, 6H); HRMS (ESI-TOF): m / z = 399.0977 (calcd. for [M + H]+, 399.0962).

[0241] Compound 34: (E)-4,4'-(diazene-1,2-diyl)bis(3,5-difluorobenzoic acid). A solution of compound 33 (484 mg, 1.22 mmol) and KOH (274 mg, 4.88 mmol) in H2O / THF 2:1 (15 mL) was heated at 70°C for 2 h. The mixture was diluted with H2O and EA, the aqueous layer was washed twice with EA, and subsequently acidified with 1 M HCI. The orange precipitate was filtered to give 34 (415 mg, quant.) as an orange solid.1H NMR (400 MHz, DMSO-d6) 5 7.83 (d, J = 9.8 Hz, 4H); HRMS (ESI-TOF): m / z = 343.0343 (calcd. for [M + H]+, 343.0336).

[0242] Compound ABF4-WW: (E)-4,4'-(diazene-1,2-diyl)bis(3,5-difluoro-N-methyl benzamide). Compound 34 (90 mg, 0.26 mmol) was dissolved in anhydrous DMF (5 mL), then PyBOP (274 mg, 0.52 mmol) and DIPEA (353 pL, 2.08 mmol) were added and stirred at room temperature for 30 min. Then a solution of methylamine hydrochloride (35 mg, 0.52 mmol) in anhydrous DMF (1 mL) was added and the mixture was stirred at room temperature for overnight. Then the solvent was removed under reduced pressure and the residual oil was purified by column chromatography (ethyl acetate I hexane mixtures) to give the desired product ABF4-WW (76 mg, 79% yield), as a red solid1H NMR (600 MHz, DMSO-d6) 68.90 (s, NH), 7.82 (d, J = 6.5 Hz, 4H), 2.83 (s, 6H);13C NMR (150 MHz, DMSO-d6) 6 163.7, 154.8, 138.9, 132.3, 112.5, 26.9; HRMS (ESI-TOF): m / z = 369.1808 (calcd. for [M + H]+, 369.0969).

[0243] Compound ABF4-AW: ethyl (E)-4-((2,6-difluoro-4- (hydroxymethyl)phenyl)diazenyl)-3,5-difluorobenzoate. To a solution of 8 (450 mg, 0.12 mmol) in dry THF (20 mL) at 0°C was added DIBAL-H (2.25 mL, 2.25 mmol) dropwise. The reaction mixture was stirred at 0°C for 2 h and then quenched with 1N HCI, diluted with H2O and EA. The organic layer was dried over Na2SO4 and concentrated under reduced pressure, the residual oil was purified by column chromatography (MeOH / DCM) to give the desired product ABF4-AW (239 mg, 60% yield) as a red solid.1H NMR (400 MHz, CDCI3) 57.72 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 10.0 Hz, 2H), 4.77 (s, 2H), 4.42 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) 6 164.0, 156.1, 155.0, 147.5, 134.7, 132.9, 130.4, 113.9, 110.3, 63.8, 62.2, 14.3; HRMS (ESI-TOF): m / z = 357.0856 (calcd. for [M + H]+, 357.0857).

[0244] Compound ABF4-AA: (E)-(diazene-1,2-diylbis(3,5-difluoro-4,1-phenylene))dimethanol. Compound 33 (50 mg, 0.12 mmol) was dissolved in dry THF (10 mL) and stirred at 0°C, then DIBAL-H (627 pL, 0.62 mmol) was added dropwise. The reaction mixture was stirred at 0°C for 2 h, then quenched with 1N HCI, then diluted with H2O and EA. The organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure. The residual oil was purified by column chromatography (EA / PE) to give the desired product ABF4-AA (35 mg, 86% yield) as an orange / red solid.1H NMR (400 MHz, CD3OD) 57.16 (d, J = 10.1 Hz, 4H), 4.68 (s, 4H);13C NMR (100 MHz, CD3OD) 5 155.4, 148.0, 129.9, 109.6, 62.3; HRMS (ESI-TOF): m / z = 315.0746 (calcd. for [M + H]+, 315.0751). Example 5:

[0245] Synthesis of compound TFC-1:

[0246]

[0247] Compound 35: (E)-4-((4-((2-((6-chlorohexyl)oxy)ethyl)carbamoyl)-2,6 difluorophenyl) diazenyl) -3,5-difluorobenzoic acid. Compound 20 (82 mg, 0.29 mmol) was dissolved in DCM (3 mL) at 0°C and 3 mL of TFA was added slowly, and then the reaction mixture was stirred at RT for 1 h. The solvent was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. To a solution of 34 (100 mg, 0.29 mmol) in dry DMF (5 mL) was added EDCI (56 mg, 0.29 mmol), HOBt (45 mg, 0.29 mmol), and DIPEA (300 L, 1.75 mmol) and the solution was stirred at RT for 30 min. Then the intermediate in dry DMF (1 mL) was added and the mixture was stirred at RT overnight. The solvent was removed under reduced pressure and the residual oil was purified by column chromatography (MeOH / DCM)) to obtain the desired product 35 as a red solid (37 mg, 85% yield).1H NMR (400 MHz, CD3OD) 57.82 - 7.78 (m, 2H), 7.70 - 7.67 (m, 2H), 3.64 - 3.60 (m, 4H), 3. 56 - 3.51 (m, 4H), 1.79 - 1.72 (m, 2H), 1.66 - 1.59 (m, 2H), 1.50 - 1.42 (m, 4H);13C NMR (100 MHz, CD3OD) 5 165.0, 156.2, 153.6, 138.3, 134.6, 133.8, 132.7, 113.67, 113.6, 111.6, 70.5, 68.5, 48.2, 44.2, 39.4, 32.4, 29.2, 26.3, 25.1;

[0248] HRMS (ESI-TOF): m / z = 504.1313 (calcd. for [M + H]+, 504.1308).

[0249] Compound 37: ferf-butyl (2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxy phenoxy) ethylcarbamate. To a solution of Compound 6 (184 mg, 0.66 mmol) and 36 (179 mg, 0.80 mmol) in DMF (10 mL) was added CS2CO3 (433 mg, 1.33 mmol), and then the reaction mixture was stirred at 70°C overnight. The solution was diluted with H2O and extracted with EA, the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to give the desired product 37 (162 mg, 58% yield) as a white solid.1H NMR (400 MHz, CDCI3) 57.79 (s, 1H), 6.40 (s, 2H), 5.92 (s, NH), 4.85 (s, 2NH), 4.65 (s, 2NH), 4.06 (t, J = 4.7 Hz, 2H), 3.83 (s, 6H), 3.67 (s, 2H), 3.40 -3.37 (m, 2H), 1.47 (s, 9H);13C NMR (100 MHz, CDCI3) 5 162.7, 162.0, 156.4, 156.2, 153.6, 135.3, 134.2, 106.3, 104.7, 78.9, 73.2, 56.0, 40.6, 34.7, 28.5; HRMS (ESI-TOF): m / z = 420.2244 (calcd. for [M + H]+, 420.2241).

[0250] Compound TFC-1: (E)-N-(2-((6-chlorohexyl)oxy)ethyl)-4-((4-((2-(4-((2,4-diaminopyrimidin-5-yl) methyl)-2,6-dimethoxyphenoxy)ethyl)carbamoyl)-2,6-difluorophenyl) diazeny l)-3,5-difluorobenzamide. Compound 37 (25 mg, 0.06 mmol) was dissolved in DCM (1 mL) at 0°C and 1 mL of TFA was added slowly, and then the reaction mixture was stirred at RT for 1 h. The solution was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. To a solution of Compound 35 (30 mg, 0.06 mmol) in dry DMF (3 mL) was added HATU (25 mg, 0.065 mmol) and DIPEA (52 pL, 0.30 mmol), and the solution was stirred at RT for 30 min. Then the intermediate in dry DMF (1 mL) was added and the reaction mixture was stirred at RT overnight. The solvent was removed under reduced pressure and the residual red oil was purified by column chromatography (MeOH / DCM) to afford the desired product TFC-1 (31 mg, 68% yield) as an orange-red solid.1H NMR (400 MHz, CDCI3) 57.78 (s, 1H), 7.59 - 7.51 (m, 4H), 6.45 (s, 2H), 4.86 (s, 2NH), 4.64 (s, 2NH), 4.22 (t, J = 4.7 Hz, 2H), 3.84 (s, 6H), 3.77 -3.62 (m, 8H), 3.58 - 3.50 (m, 4H), 1.84 - 1.77 (m, 2H), 1.67 - 1.61 (m, 2H), 1.52 - 1.40 (m, 4H);13C NMR (100 MHz, CDCI3) 5164.1, 163.9, 162.7, 161.9, 156.7, 156.1, 154.0, 153.4, 153.2, 138.3, 138.0, 135.0, 134.5, 133.1, 111.7, 111.5, 106.2, 105.2, 72.1, 71.2, 68.9, 56.3, 45.0, 40.2, 34.7, 32.5, 29.7, 29.4, 26.7, 25.5; HRMS (ESI-TOF): m / z = 805.2844 (calcd. for [M + H]+, 805.2846).

[0251] Example 6:

[0252] Synthesis of compound TFC-2:

[0253]

[0254] Compound 38: tert-butyl (E)-3-(4-amino-3,5-difluorophenyl)acrylate. Compound 27 (4.0 g, 19.23 mmol), ferf-butyl acrylate (5.7 mL, 38.41 mmol), Pd(OAc)2 (430 mg, 1.92 mmol) and TEA (8.02 mL, 57.69 mmol) were dissolved in DMF (30 mL), and the reaction mixture was stirred at 90°C under N2 overnight. H2O (60 mL) was added slowly and the resulting mixture was extracted with EA (3 x 40 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (EA / PE) to give 38 (4.8 g, 98% yield) as a red-brown oil.1H NMR (400 MHz, CDCI3) 6 7.37 (d, J = 15.9 Hz, 1H), 6.97 (dd, J = 7.4, 1.9 Hz, 2H), 6.15 (d, J = 15.9 HZ, 1H), 3.94 (br, NH2) 1.50 (s, 9H);13C NMR (100 MHz, CDCI3) 5 166.3, 151.6, 142.0, 126.0, 123.8, 118.7, 110.7, 80.6, 28.3; HRMS (ESI-TOF): m / z = 256.1138 (calcd. for [M + H]+, 256.1144).

[0255] Compound 39: ferf-butyl 3-(4-amino-3,5-difluorophenyl)propanoate. To a solution of compound 38 (4.8 g, 18.67 mmol) in MeOH (50 mL) was added Pd / C (230 mg), and then the solution was stirred at RT overnight, filtered through celite, dried over Na2SC, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography (EA / PE) to give compound 39 (4.25 g, 99% yield) as a lightyellow oil.1H NMR (400 MHz, CDCI3) 56.65 (dd, J = 7.4, 1.6 Hz, 2H), 3.61 (br, NH2), 2.76 (t, J = 7.6 Hz, 2H), 2.45 (t, J = 7.6 Hz, 2H), 1.40 (s, 9H);13C NMR (100 MHz, CDCI3) 5 172.0, 152.1, 130.2, 121.9, 110.9, 80.6, 37.0, 30.2, 28.1; HRMS (ESI-TOF): m / z = 258.1302 (calcd. for [M + H]+, 258.1300).

[0256] Compound 40: 3-(4-amino-3,5-difluorophenyl)propan-1-ol. To a solution of Compound 39 (950 mg, 3.69 mmol) in THF (20 mL) at 0°C was added DIBAL-H in Toluene (9.23 mL, 9.23 mmol) dropwise. The solution was stirred for 4 h, and Then, H2O (10 mL) was added slowly and extracted with EA (3 x 30 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (EA / PE) to give 40 (548 mg, 82% yield) as a brown solid.1H NMR (400 MHz, CDCI3) 66.65 (dd, J = 7.6, 1.6 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.86 (br, NH2), 2.58 (t, J = 7.5 Hz, 2H), 1.84 - 1.77 (m, 2H);13C NMR (100 MHz, CDCI3) 5 152.2, 131.4, 121.5, 111.05, 110.9, 62.0, 34.0, 31.2; HRMS (ESI-TOF): m / z = 188.0888 (calcd. for [M + H]+, 188.0881).

[0257] Compound 41: 4-(3-bromopropyl)-2,6-difluoroaniline. Compound 40 (370 mg, 2.02 mmol) and PPhs (795 mg, 3.03 mmol) were dissolved in DCM (20 mL) at 0°C, then CBu (1.0 g, 3.03 mmol) was added dropwise. The solution was stirred at RT overnight and concentrated under vacuum. The resulting residue was purified by column chromatography (EA / PE) to give 41 (386 mg, 73% yield) as a colurless oil.1H NMR (400 MHz, CDCI3) 56.67 (dd, J = 7.5, 1.8 Hz, 2H), 3.56 (br, NH2), 3.36 (t, J = 6.5 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 2.12 - 2.05 (m, 2H);13C NMR (100 MHz, CDCI3) 5 152.0, 129.8, 121.8111.11, 110.9, 33.9, 32.9, 32.7; HRMS (ESI-TOF): m / z = 250.0042 (calcd. for [M + H]+, 250.0037).

[0258] Compound 42: (E)-4-((4-(3-bromopropyl)-2,6-difluorophenyl)diazenyl)-3,5-difluorobenzoic acid. Compound 30 (173 mg, 1 mmol) was dissolved in DCM (20 mL) and acetone ( 4 mL), then Oxone (1.23 g, 2 mmol) in H2O ( 10 mL) was added. The solution was stirred at RT for 2 h. After separation of the layers, the aqueous organic layer was washed with water and concentrated under reduced pressure. Without any further purification the crude product was suspended in 26 mL of acetic acid / toluene / TFA 6:6:1 and the corresponding aniline compound 41 (200 mg, 1.0 mmol) was added. The resulting mixture was stirred at RT for 3 days. The solution was diluted with H2O and extracted with EA, the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product 42 (142 mg, 44% yield for 2 steps) as a light yellow solid.1H NMR (400 MHz, CDCI3) 57.79 (d, J = 8.5 Hz, 2H), 6.96 (d, J= 10.0 Hz, 2H), 3.43 (t, J= 6.3 Hz, 2H), 2.87 (t, J= 7.2 Hz, 2H), 2.24 - 2.18 (m, 2H); HRMS (ESI-TOF): m / z = 419.0015 (calcd. for [M + H]+, 419.0013).

[0259] Compound 43: (E)-4-((4-(3-bromopropyl)-2,6-difluorophenyl)diazenyl)-N-(2-((6-chlorohexyl)oxy)ethyl)-3,5-difluorobenzamide. Compound 20 (72 mg, 0.26 mmol) was dissolved in DCM (2 mL) at 0°C and 2 mL of TFA was added slowly and then the reaction mixture was stirred at RT for 1 h. The solvent was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. To a solution of 42(109 mg, 0.26 mmol) was added EDCI (60 mg, 0.31 mmol), HOBt (48 mg, 0.31 mmol) and DIPEA (220 uL, 1.3 mmol). The solution was stirred at RT for 30 min. Then the intermediate in DCM (1 mL) was added. The mixture was stirred at RT overnight, diluted with H2O, and extracted with EA. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to yield the target product 43 (60 mg, 40 % yield) as a red solid.1H NMR (400 MHz, CDCI3) 67.48 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 10.0 Hz, 2H), 3.68 - 3.60 (m, 4H), 3.55 - 3.48 (m, 4H), 3.42 (t, J = 6.4 Hz, 2H), 2.85 (t, J = 7.4 Hz, 2H), 2.23 - 2.17 (m, 2H), 1.81 - 1.74 (m, 2H), 1.66 - 1.59 (m, 2H), 1.51 - 1.35 (m, 4H);13C NMR (100 MHz, CDCI3) 5 164.4, 156.0, 155.3, 147.2, 137.0, 133.7, 129.9, 112.9, 111.6, 71.3, 69.0, 45.1, 40.3, 34.1, 33.2, 32.6, 32.3, 29.5, 26.8, 25.6; HRMS (ESI-TOF): m / z = 580.2195 (calcd. for [M + H]+, 580.0984).

[0260] Compound TFC-2: (E)-N-(2-((6-chlorohexyl)oxy)ethyl)-4-((4-(3-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)propyl)-2,6-difluor ophenyl)diazenyl)-3,5-difluorobenzamide. To asolution of 5 (23 mg, 0.08 mmol) in DMF (3 mL) was added K2CO3 (37 mg, 0.27 mmol) and the mixture was stirred at RT for 30 min. Then compound 43 (16 mg, 0.027 mmol) in DMF (1 mL) was added. The solution was stirred at RT overnight and concentrated under reduced pressure, and the resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product TFC-2 (2.8 mg, 15% yield) as a red solid.1H NMR (400 MHz, CDCI3) 5 7.63 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 10.4 Hz, 2H), 6.38 (s, 2H), 5.55 (br, NH2), 5.06 (br, NH2), 3.95 (t, J = 5.9 Hz, 2H), 3.81 - 3.78 (m, 8H), 3.66 - 3.61 (m, 4H), 3.54 - 3.47 (m, 4H), 2.94 (t, J = 7.5 Hz, 2H), 2.06 (t, J = 6.6 Hz, 2H), 1.81 - 1.73 (m, 2H), 1.64 - 1.58 (m, 2H), 1.48 - 1.37 (m, 4H); HRMS (ESI-TOF): m / z = 776.2949 (calcd. for [M + H]+, 776.2945).

[0261] Example 7:

[0262] Synthesis of compound TFC-3:

[0263]

[0264] Compound 44: ethyl (E)-4-((2,6-difluoro-4-(hydroxymethyl)phenyl)diazen yl)-3,5-difluorobenzoate. To a solution of 33 (450 mg, 0.12 mmol) in dry THF (20 mL) at 0°C was added DIBAL-H (2.25 mL, 2.25 mmol) dropwise. The reaction mixture was stirred at 0°C for 2 h and then quenched with 1N HCI, diluted with H2O and EA. The organic layer was dried over Na2SC and concentrated under reduced pressure, the residual oil was purified by column chromatography (MeOH / DCM) to give the desired product 44 (239 mg, 60% yield) as a red solid.1H NMR (400 MHz, CDCI3) 57.72 (d, J = 8.8 Hz, 2H), 7.09 (d, J= 10.0 Hz, 2H), 4.77 (s, 2H), 4.42 (q, J= 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) 5164.0, 156.1, 155.0, 147.5, 134.7, 132.9, 130.4, 113.9, 110.3, 63.8, 62.2, 14.3; HRMS (ESI-TOF): m / z = 357.0856 (calcd. for [M + H]+, 357.0857). Compound 45: (E)-4-((2,6-difluoro-4-(hydroxymethyl)phenyl)diazenyl)-3,5-difluorobenzoic acid. Compound 44 (240 mg, 0.674 mmol) was dissolved in THF (5 mL) and KOH (76 mg, 1.34 mmol) in H2O (10 mL) was added, and the solution was stirred at 70°C for 4 h. The solution was diluted with H2O and extracted with EA. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the target product 45 (210 mg, 95% yield) as an orange solid, which was used for the next step directly without further purification.

[0265] Compound 46: (E)-N-(2-((6-chlorohexyl)oxy)ethyl)-4-((2,6-difluoro-4- (hydroxymethyl)phenyl)diazenyl)-3,5-difluorobenzamide. Compound 20 (123 mg, 0.44 mmol) was dissolved in DCM (4 mL) at 0°C and 4 mL of TFA was added slowly and then the reaction mixture was stirred at RT for 1 h. The solution was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. Compound 45 (120 mg, 0.365 mmol), EDCI (77 mg, 0.40 mmol), HOBt (62 mg, 0.40 mmol) were dissolved in DCM (10 mL) and DIPEA (248 uL, 1.46 mmol) was added. The solution was stirred at RT for 30 min and then the intermediate in DCM (1 mL) was added. The mixture was stirred at RT overnight. The solution was diluted with H2O and extracted with DCM, the organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to yield the target product 46 (98 mg, 55% yield) as a red solid.1H NMR (400 MHz, CDCI3) 57.39 (d, J = 8.9 Hz, 2H), 7.02 (d, J = 10.1 Hz, 2H), 4.70 (s, 2H), 3.61 - 3.53 (m, 4H), 3.48 - 3.41 (m, 4H), 1.74 - 1.67 (m, 2H), 1.59 - 1.52 (m, 2H), 1.44 - 1.28 (m, 4H);13C NMR (100 MHz, CDCI3) 5 164.3, 155.9, 155.2, 147.3, 137.0, 133.5, 130.3, 111.5, 110.1, 71.2, 68.9, 63.7, 45.0, 40.2, 32.5, 29.4, 26.7, 25.5; HRMS (ESI-TOF): m / z = 490.1512 (calcd. for [M + H]+, 490.1515).

[0266] Compound 47: (E)-4-((4-(bromomethyl)-2,6-difluorophenyl)diazenyl)-N-(2-((6-chlorohexyl)oxy)ethyl)-3,5-difluorobenzamide. Compound 46 (41 mg, 0.084 mmol) and PPh3(27 mg, 0.1 mmol) were dissolved in DCM (5 mL) at 0°C, then CBu (34 mg, 0.1 mmol) was added dropwise. The solution was stirred at RT overnight and concentrated under vacuum. The resulting residue was purified by column chromatography (MeOH / DCM) to obtain the product 47 (37 mg, 81% yield) as a red solid.1H NMR (400 MHz, CDCI3) 67.41 (d, J = 8.9 Hz, 2H), 7.05 (d, J = 9.3 Hz, 2H), 4.37 (s, 2H), 3.61 - 3.53 (m, 4H), 3.48 - 3.39 (m, 4H), 1.74 - 1.67 (m, 2H), 1.59 - 1.52 (m, 2H), 1.44- 1.30 (m, 4H);13C NMR (100 MHz, CDCI3) 5164.2, 155.6, 155.3, 142.8, 137.4, 133.3, 131.1, 113.4, 111.5, 71.2, 68.9, 45.0, 40.2, 32.4, 30.7, 29.4, 26.6, 25.5;

[0267] HRMS (ESI-TOF): m / z = 552.0677 (calcd. for [M + H]+, 552.0671).

[0268] Compound TFC-3: (E)-N-(2-((6-chlorohexyl)oxy)ethyl)-4-((4-((4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)methyl)-2,6-difluo rophenyl)diazenyl)-3,5-difluorobenzamide. To a solution of 5 (56 mg, 0.20 mmol) in DMF (3 mL) was added K2CO3 (37 mg, 0.27 mmol). The mixture was stirred at RT for 30 min and then compound 47 (37 mg, 0.07 mmol) in DMF (1 mL) was added. The solution was stirred at RT overnight and then concentrated under reduced pressure. The resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product TFC-3 (8.6 mg, 18 % yield) as a red solid.1H NMR (400 MHz, CDCI3) 5 7.41 (d, J= 9.2 Hz, 2H), 7.24 (d, J= 9.3 Hz, 2H), 6.91 (s, 1H), 6.52 (br, NH), 6.32 (s, 2H), 5.54 (br, NH2), 5.05 (br, NH2), 4.99 (s, 2H), 3.75 (s, 6H), 3.59 - 3.41 (m, 10 H), 1.73 - 1.68 (m, 2H), 1.57 - 1.52 (m, 2H), 1.43 - 1.30 (m, 4H); HRMS (ESI-TOF): m / z = 748.2631 (calcd. for [M + H]+, 748.2632).

[0269] Example 8:

[0270] Synthesis of compound TFC-4:

[0271]

[0272] Compound 49: 6-chlorohexan-1 -amine hydrochloride. To a solution of 48 (2.0 g, 17.07 mmol) in CHCI3 (25 mL) at 0°C was added SOCI2 (1.86 mL, 25.62 mmol). The solution was refluxed for 3 h and concentrated to give the target molecule 49 (2.79 g, 95% yield) as a light-yellow solid.1H NMR (400 MHz, CDCI3) 58.28 (br, NH2), 3.54 (t, J = 6.3 Hz, 2H), 3.01 (br, 2H), 1.82 - 1.75 (m, 4H), 1.53 - 1.39 (m, 4H); HRMS (ESI-TOF): m / z = 136.0887 (calcd. for [M + H]+, 136.0888).

[0273] Compound 50: (E)-4-((4-((6-chlorohexyl)carbamoyl)-2,6-difluoro phenyl) diazenyl)-3,5-difluorobenzoic acid. Compound 34 (90 mg, 0.26 mmol), EDCI (56 mg, 0.29 mmol), HOBt (45 mg, 0.29 mmol) were dissolved in DMF (4 mL) and DIPEA (223 uL, 1.32 mmol) was added. The solution was stirred at RT for 30 min, and then compound 49 (46 mg, 0.26 mmol) in DMF (1 mL) was added. The mixture was stirred at RT overnight. The solution was diluted with H2O and extracted with EA, the combined organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product 50 (75 mg, 63% yield) as a red solid.1H NMR (400 MHz, CD3OD) 67.77 (d, J = 9.2 Hz, 2H), 7.65 (d, J = 10.9 Hz, 2H), 3.56 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 7.2 Hz, 2H), 1.82 - 1.75 (m, 2H), 1.69 - 1.62 (m, 2H), 1.55 -1.40 (m, 4H);13C NMR (151 MHz, CD3OD) 6 165.1, 164.9, 155.1, 154.8, 138.4, 134.9, 133.8, 132.7, 113.4, 111.5, 44.3, 39.8, 32.3, 28.8, 26.3, 25.9; HRMS (ESI-TOF): m / z = 460.1025 (calcd. for [M + H]+, 460.1046). Compound TFC-4: (E)-N-(6-chlorohexyl)-4-((4-((2-(4-((2,4-diamino pyrimi din-5-yl)methyl)-2,6-dimethoxyphenoxy)ethyl)carbamoyl)-2,6-difluoroph enyl)diazenyl)-3,5-difluorobenzamide. Compound 37 (33 mg, 0.05 mmol) was dissolved in DCM (2 mL) at 0°C and 2 mL of TFA was added slowly and then the reaction mixture was stirred at RT for 1 h. The solution was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. To a solution of 50 (20 mg, 0.04 mmol) in DMF (2 mL) was added HATLI (20 mg, 0.05 mmol) and DIPEA (30 uL, 0.17 mmol). The solution was stirred at RT for 30 min. Then the intermediate in DMF (1 mL) was added and the mixture was stirred at RT overnight. The solution was concentrated under reduced pressure and the resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product TFC-4 (26 mg, 79% yield as a red solid.1H NMR (400 MHz, DMSO-d6) 68.89 (t, J = 5.4 Hz, 1 H), 8.78 (t, J = 5.4 Hz, 1 H), 7.81 (dd, J= 10.1, 4.2 Hz, 4H), 7.49 (s, 1H), 6.58 (s, 2H), 6.50 (br, NH2), 6.07 (br, NH2), 3.96 (t, J = 5.8 Hz, 2H), 3.69 - 3.54 (m, 12 H), 3.31 - 3.26 (m, 2H), 1.76 - 1.69 (m, 2H), 1.59 -1.52 (m, 2H), 1.46- 1.31 (m, 4H);13C NMR (150 MHz, DMSO-d6) 6163.4, 163.2, 163.0, 155.7, 154.0, 153.2, 139.0, 135.9, 135.0, 132.3, 112.6, 106.9, 106.3, 71.1, 56.3, 45.8, 40.5, 33.3, 32.4, 29.1, 26.5, 26.2; HRMS (ESI-TOF): m / z = 761.2578 (calcd. for [M + H]+, 761.2584).

[0274] Example 9:

[0275] Synthesis of compound TCC

[0276]

[0277] Compound 52: methyl 4-amino-3,5-dichlorobenzoate. To a solution of compound 51 (4.0 g, 19.42 mmol) in MeOH (60 mL) was added concentrated H2SO4 (4 mL). The mixture was stirred under reflux overnight. The solution was concentrated under reduced pressure. The resulting mixture was dissolved in EA (60 mL) and washed with H2O (2 x 40 mL). The organic phase was dried over Na2SC, filtered, and concentrated under reduced pressure to give compound 52 (4.1 g, 96% yield) as a white solid.1H NMR (400 MHz, CDCI3) 57.88 (s, 2H), 3.87 (s, 3H).

[0278] Compound 53: dimethyl 4,4'-(diazene-1,2-diyl)(E)-bis(3,5-dichlorobenzoate). To a solution of compound 52 (500 mg, 2.27 mmol) in DCM (10 mL) was added DBU (692 mg, 4.54 mmol). The solution was stirred at RT for 5 min. before being cooled down to -78°C. Then NCS (606 mg, 4.54 mmol) was added. The reaction was stirred at -78°C for 10 min. the solution was washed with saturated NaHCCh, H2O, and 1 M HCI. The organic phase was concentrated under reduced pressure to give compound 53 (356 mg, 72% yield) as an orange solid.1H NMR (400 MHz, DMSO-d6) 58.17 (s, 4H), 3.93 (s, 6H). Compound 54: (E)-4,4'-(diazene-1,2-diyl)bis(3,5-dichlorobenzoic acid).

[0279] Compound 53 (436 mg, 1 mmol) was dissolved in MeOH (20 mL) and KOH (560 mg, 10 mmol) in H2O (5 mL) was added. The mixture was stirred at 60°C for 3 h. The solution was concentrated under reduced pressure and the resulting residual was acidic by 1 M HCI. The precipitate was filtered off to obtain the product 54 (376 mg, 92% yield) as an orange solid.1H NMR (400 MHz, DMSO-d6) 68.11 (s, 4H), 3.62 (br, COOH).13C NMR (100 MHz, DMSO-d6) 6 164.4, 148.9, 133.4, 130.3, 126.2.

[0280] Compound 55: (E)-3,5-dichloro-4-((2,6-dichloro-4-((6-chlorohexyl) carbamoyl) phenyl)diazenyl)benzoic acid. Compound 54 (204 mg, 0.5 mmol), EDCI (105 mg, 0.55 mmol) and HOBt (84 mg, 0.55 mmol) were dissolved in DMF (4 mL) and DIPEA (340 uL, 2 mmol) was added. The solution was stirred at RT for 30 min, and then compound 49 (86 mg, 0.5 mmol) in DMF (1 mL) was added. The mixture was stirred at RT overnight. The solution was diluted with H2O and extracted with EA. the combined organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product 55(102 mg, 40% yield) as a red solid.1H NMR (400 MHz, CD3OD) 58.13 (s, 2H), 8.02 (s, 2H), 3.57 (t, J = 6.6 Hz, 2H), 3.39 (t, J = 7.2 Hz, 2H), 1.83 - 1.74 (m, 2H), 1.69 - 1.61 (m, 2H), 1.54 - 1.43 (m, 4H).

[0281] Compound TCC: (E)-3,5-dichloro-N-(6-chlorohexyl)-4-((2,6-dichloro-4-((2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy) ethyl)carbamoyl) phenyl)diazenyl)benzamide. Compound 37 (24 mg, 0.057 mmol) was dissolved in DCM (2 mL) at 0 °C and 2 mL of TFA was added slowly and then the reaction mixture was stirred at RT for 1 h. The solvent was removed under vacuum to give the intermediate as a colorless oil, which was used for the next step directly without further purification. To a solution of compound 55 (25 mg, 0.048 mmol) in DMF (5 mL) were added HATU (22 mg, 0.057 mmol) and DIPEA (33 uL, 0.19 mmol). The solution was stirred at RT for 30 min. Then the intermediate in DMF (1 mL) was added and the mixture was stirred at RT overnight. The solution was concentrated under reduced pressure and the resulting mixture was purified by column chromatography (MeOH / DCM) to yield the target product TCC (31 mg, 80% yield) as a red solid.1H NMR (600 MHz, DMSO-d6) 68.92 (t, J = 5.4 Hz, CONH), 8.81 (t, J = 5.4 Hz, CONH), 8.14 (d, J = 2.3 Hz, 5H), 6.58 (s, 2H), 6.11 (s, NH2), 5.71 (s, NH2), 3.97 (t, J = 5.9 Hz, 2H), 3.70 (s, 8H), 3.65 (t, J = 6.6 Hz, 2H), 3.57 (t, J = 5.5 Hz, 2H), 3.53 (s, 2H), 1.77 - 1.72 (m, 2H), 1.59 - 1.54 (m, 2H), 1.46 - 1.41 (m, 2H), 1.39 - 1.34 (m, 2H).13C NMR (151 MHz, DMSO-de) 6 162.9, 162.6, 162.2, 152.6, 147.8, 147.8, 136.7, 136.6, 136.0, 134.4, 128.6, 128.6, 126.1, 105.8, 70.6, 55.8, 45.4, 40.0, 32.9, 31.9, 28.6, 26.0, 25.7.

[0282] Example 10:

[0283] Synthesis of compound TPC:

[0284]

[0285] Compound 56: 5-(4-(2-bromoethoxy)-3,5-dimethoxybenzyl)pyrimidine-2,4-diamine. To a solution of compound 5 (277 mg, 1 mmol) in 4 mL DMF was added K2CO3 (276 mg, 2 mmol), the reaction mixture was stirred at RT for 10 min. Then dibromoethane (431 iL, 5 mmol) was added dropwise. The reaction mixture was stirred at RT overnight. The solvent was concentrated under vacuum and the resulting residue was purified by column chromatography (MeOH / DCM) to furnish compound 56 (115 mg, 30% yield) as a white solid.1H NMR (600 MHz, CD3OD) 67.50 (s, 1 H), 6.52 (s, 2H), 4.15 (t, J = 6.5 Hz, 2H), 3.78 (s, 6H), 3.63 (s, 2H), 3.57 (t, J = 6.5 Hz, 2H).

[0286] 13C NMR (151 MHz, CD3OD) 6 163.1, 161.7, 154.4, 153.2, 135.4, 134.7, 106.6, 105.4, 72.6, 55.2, 33.1, 29.3.

[0287] Compound 58: (E)-2-(4-((4-hydroxyphenyl)diazenyl)-1H-pyrazol-1-yl)acetic acid.

[0288] To a solution of compound 57 (300 mg, 1.4 mmol) in 5 mL H2O was added concentrated HCI (584 pL, 7.0 mmol) at 0°C. Then NaNCh (145 mg, 2.1 mmol) dissolved in 7 mL H2O was added dropwise. The solution was stirred at 0°C for 15 min. In a separate flask, phenol (158 mg, 1.7 mmol) dissolved in 10 mL of water was treated with NaOH (151 mg, 3.8 mmol) and Na2CO3(401 g, 3.8 mmol) and the resulting solution was cooled to 0°C. To the diazonium solution, the phenol solution was carefully added dropwise and upon complete addition the mixture was stirred at 0°C for 30 min. After acidifying the reaction mixture with 2 M aqueous HCI, the precipitate was filtered off, washed with water and dried in vacuo to obtain the target product 58 (244 mg, 71% yield for 2 steps) as a yellow solid.1H NMR (400 MHz, DMSO-de) 6 10.14 (s, 1H), 8.19 (s, 1H), 7.77 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 4.40 (s, 2H). HRMS (ESI-TOF): m / z = 247.1052 (calcd. for [M + H]+, 247.0826).

[0289] Compound 59: (E)-N-(6-chlorohexyl)-2-(4-((4-hydroxyphenyl)diazenyl)-1 H-pyrazol-1-yl)acetamide. Compound 58 (100 mg, 0.41 mmol) and compound 49 (140 mg, 0.81 mmol) were dissolved in 5 mL DMF. Then EDCI (118 mg, 0.61 mmol), HOBt (94 mg, 0.61 mmol) and DIPEA (348 pL, 2.05 mmol) were added, subsequently. The reaction mixture was stirred at RT overnight. The solution was diluted with 20 mL H2O and extracted with EA (2 x 30 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Then the residual brown oil was purified by column chromatography (EA / PE) to afford the desired product 59 (120 mg, 81% yield) as a yellow solid.1H NMR (600 MHz, CD3OD) 68.24 (s, 1H), 7.96 (s, 1H), 7.68 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H) 4.89 (s, 2H), 3.54 (t, J = 6.7 Hz, 2H), 3.24 - 3.21 (m, 2H), 1.77 - 1.73 (m, 2H), 1.56 - 1.51 (m, 2H), 1.48 - 1.43 (m, 2H), 1.38 - 1.33 (m, 2H).13C NMR (151 MHz, CD3OD) 5 167.4, 160.1, 146.2, 141.7, 132.5, 128.4, 123.8, 115.2, 54.3, 44.2, 39.1, 32.3, 28.7, 26.2, 25.8.

[0290] HRMS (ESI-TOF): m / z = 364.1848 (calcd. for [M + H]+, 364.1535).

[0291] Compound TPC: (E)-N-(6-chlorohexyl)-2-(4-((4-(2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)ethoxy)phenyl)diazenyl)-1H-pyrazol-1-yl)acetamide. To a solution of compound 59 (25 mg, 0.068mmol) and compound 56 (22 mg, 0.057 mmol) was added K2CO3 (8.6 mg, 0.06 mmol). The reaction mixture was stirred at 70°C overnight. Then the solution was concentrated under vacuum and the resulting residue was purified by column chromatography (MeOH / DCM) to yield the target molecule TPC (11 mg, 30% yield) as a yellow solid.1H NMR (600 MHz, CDCI3) 5 8.13 (s, 1H), 8.06 (s, 1H), 7.80 (d, J = 8.9 Hz, 2H), 7.70 (s, 1H), 7.01 (d, J = 8.9 Hz, 2H), 6.39 (s, 2H), 5.33 (br, NH2), 4.89 (br, NH2), 4.86 (s, 2H), 4.40 - 4.38 (m, 2H), 4.36 - 4.35 (m, 2H), 3.78 (s, 6H), 3.67 (s, 2H), 3.52 (t, J = 6.7 Hz, 2H), 3.30 - 3.26 (m, 2H), 1.78 - 1.74 (m, 2H), 1.53 - 1.48 (m, 2H), 1.47 - 1.42 (m, 2H),, 1.33 - 1.29 (m, 2H).

[0292] HRMS (ESI-TOF): m / z = 666.2878 (calcd. for [M + H]+, 666.2914).

[0293] Example 11:

[0294] Synthesis of TDC-1 and TDC-3:

[0295]

[0296] Compound 61: 4,4'-(ethane-1,2-diyl)bis(3-nitroaniline). To a solution of 60 (4.0 g, 18.84 mmol) in H2SO4 (30 mL) at 60°C was added a solution of NaNCh (3.5 g, 41.45 mmol) in H2SO4 (40 mL) dropwise. The mixture was stirred at 60°C for 4 h and afterwards poured into 50 mL of ice-water. The resulting suspension was neutralised by the addition of anaqueous ammonia solution (32%). The red precipitate was filtered off, washed with H2O and dried in vacuum to give the product 61 (5.7 g, 99% yield).1H NMR (400 MHz, DMSO-d6) 67.06 (d, J = 2.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 6.78 (dd, J = 8.2, 2.2 Hz, 2H), 5.59 (s, 2NH2), 2.86 (s, 4H); HRMS (ESI-TOF): m / z = 303.1085 (calcd. for [M + H]+, 303.1088).

[0297] Compound 62: (Z)-11,12-dihydrodibenzo[c,g][1,2]diazocine-3,8-diamine. A suspension of 61 (1.0 g, 3.3 mmol) in a mixture of EtOH (140 mL) and a solution of NaOH (7.94 g, 198.0 mmol) in H2O (35 mL) was heated to 70 °C. A solution of glucose (5.96 g, 33.0 mmol) in H2O (20 mL) was added and the reaction mixture was stirred overnight. After cooling to room temperature, 500 mL water was added, and the resulting mixture was extracted with EA. The combined organic phase was dried over Na2SO4, and the solvent was concentrated under reduced pressure. The resulting mixture was purified by column chromatography (EA / PE) to yield the target product 62 (160 mg, 21% yield) as a brown solid.1H NMR (400 MHz, DMSO-d6) 66.65 (d, J= 8.1 Hz, 2H), 6.22 (dd, J = 8.1, 2.3 Hz, 2H), 5.95 (d, J = 2.3 Hz, 2H), 5.03 (s, 2NH2), 2.57 -2.50 (m, 4H); HRMS (ESI-TOF): m / z = 239.1272 (calcd. for [M + H]+, 239.1291).

[0298] Compound 63: (Z)-N-(8-amino-11,12-dihydrodibenzo[c,g][1,2]diazocin-3-yl)-2-(4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6 dimethoxy phenoxy) acet amide. To a solution of 6 (141 mg, 0.36 mmol) in DCM (2 mL) was added TFA (2 mL) and stirred at RT for 1 h, then solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. The residual oil was dissolved in in dry DMF (5 mL), HATU (148 mg, 0.40 mmol) and DIPEA (310 pL, 1.80 mmol) were added. The reaction mixture was stirred at RT for 30 min, then 62 (94 mg, 0.40 mmol) in dry DMF (2 mL) was added. The reaction mixture was stirred at RT overnight. Then, H2O (10 mL) was added and the resulting mixture was extracted with EA (3 x 20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (MeOH / DCM) to give 63 (47 mg, 24% yield) as a yellow solid.1H NMR (600 MHz, CD3OD) 57.45 (s, 1H), 7.22 - 7.19 (m, 2H), 7.00 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1 H), 6.58 (s, 1 H), 6.39 (dd, J = 8.2, 2.2 Hz, 1 H), 6.17 (d, J = 2.2 Hz, 1H), 4.49 (s, 2H), 3.82 (s, 6H), 3.66 (s, 2H), 2.83 - 2.66 (m, 4H);13C NMR (150 MHz, CD3OD) 5 170.3, 164.6, 161.3, 157.1, 156.7, 153.5, 152.2, 147.6, 137.1, 136.5, 136.4, 131.4, 131.3, 126.4, 119.3, 118.4, 115.5, 110.7, 108.3, 106.6, 105.5, 73.50, 56.6, 34.1, 32.1, 31.3; HRMS (ESI-TOF): m / z = 555.2452 (calcd. for [M + H]+, 555.2463 ).

[0299] Compound 65: ferf-butyl 2-((6-chlorohexyl)oxy)acetate. To a solution of compound 1 (1.36 g, 0.01 mol) in toluene (50 mL) was addednBu4NBr (644 mg, 2 mmol) and compound 64 (5.85 g, 0.03 mol) as well as 50% NaOH (50 mL). The reaction mixture was stirred at RT overnight. The mixture was extracted with EA (2 x 50 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (EA / PE) to give 65 (2.2 g, 88% yield) as a colorless oil.1H NMR (400 MHz, CDCI3) 53.94 (s, 2H), 3.55 - 3.49 (m, 4H), 1.81 - 1.74 (m, 2H), 1.66 - 1.59 (m, 2H), 1.47 - 1.38 (m, 13H);13C NMR (100 MHz, CDCI3) 5 169.8, 81.5, 71.5, 68.8, 45.0, 32.5, 29.5, 28.1, 26.7, 25.4; HRMS (ESI-TOF): m / z = 251.1406 (calcd. for [M + H]+, 251.1408 ).

[0300] Compound TDC-1: (Z)-3-((6-chlorohexyl)oxy)-N-(8-(2-(4-((2,4-diamino pyrimidin-5-yl)methyl)-2,6 dimethoxyphenoxy)acetamido)-11,12-dihydro dibenzo[c,g][1,2]diazocin-3-yl)propanamide. To a solution of 3 (21 mg, 0.08 mmol) in DCM (2 mL) was added TFA (2 mL) and stirred at RT for 1 h, then the solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. To a solution of the intermediate in dry DMF (3 mL) was added PyBOP (44 mg, 0.09 mmol) and DIPEA (66 pL, 0.39 mmol). The reaction mixture was stirred at RT for 30 min, then 63 (47 mg, 0.08 mmol) in dry DMF (1 mL) was added. The reaction mixture was stirred at RT overnight. Then, H2O (10 mL) was added and the resulting mixture was extracted with EA (3 x 20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (MeOH / DCM) to give TDC-1 (12 mg, 21% yield) as a light-yellow solid.1H NMR (600 MHz, DMSO-d6) 59.97 (s, NH), 9.84 (s, NH), 7.42 (s, 1 H), 7.27 - 7.23 (m, 3H), 7.17 (dd, J = 7.3, 0.9 Hz, 1 H), 7.05 (d, J = 8.3 Hz, 1 H), 7.00 (d, J = 8.3 Hz, 1 H), 6.66 (s, 2H), 4.42 (d, J = 6.1 Hz, 2H), 3.75 (s, 6H), 3.61 - 3.59 (m, 4H), 3.53 (t, J = 6.6 Hz, 2H), 3.33 (t, J = 6.6 Hz, 2H), 2.77 - 2.73 (m, 4H), 1.63 - 1.58 (m, 2H), 1.45 - 1.40 (m, 2H), 1.33 - 1.28 (m, 2H), 1.26 - 1.21 (m, 2H);13C NMR (151 MHz, DMSO-d6) 6 169.4, 167.4, 164.0, 155.1, 155.0, 154.2, 152.0, 139.9, 137.8, 136.6, 134.6, 133.7, 130.3, 130.2, 123.4, 122.5, 117.9, 117.5, 108.8, 108.8, 108.3, 106.3, 72.1, 69.9, 66.1, 56.1, 45.3, 37.2, 32.1, 32.0, 30.4, 30.4, 28.9, 26.0, 24.9. HRMS (ESI-TOF): m / z = 745.3221 (calcd. for [M + H]+, 745.3229).

[0301] Compound TDC-3: (Z)-2-((6-chlorohexyl)oxy)-N-(8-(2-(4-((2,4-diamino pyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy)acetamido)-11,12-dihydro dibenzo[c,g][1,2]diazocin-3-yl)acetamide. The same procedure as TDC-1 was applied starting with compound 65 (24 mg, 0.09 mmol) and compound 63 (53.0 mg, 0.09 mmol). The crude product was purified by column chromatography (MeOH / DCM) to give the desired product TDC-3 (12 mg, 18% yield) as a light-yellow solid.1H NMR (400 MHz, CD3OD) 57.18 - 7.08 (m, 5H), 6.95 - 6.89 (m, 2H), 6.52 (s, 2H), 4.41 (d, J = 1.7 Hz, 2H), 3.91 (s, 2H), 3.74 (s, 6H), 3.57 (s, 2H), 3.46 - 3.41 (m, 4H), 2.80 - 2.70 (m, 4H), 1.68 - 1.61 (m, 2H), 1.58 - 1.51 (m, 2H), 1.39 - 1.28 (m, 2H), 1.23 - 1.17 (m, 2H);13C NMR (100 MHz, CD3OD) 5 169.6, 169.0, 155.5, 155.4, 154.7, 152.5, 139.0, 136.3, 136.2, 135.5, 133.2, 130.2, 130.0, 124.6, 118.8, 118.3, 109.9, 109.5, 105.9, 72.2, 71.4, 69.8, 55.5, 44.3, 32.5, 32.3, 30.5, 29.3, 28.9, 26.3, 25.0; HRMS (ESI-TOF): m / z = 731.3065 (calcd. for [M + H]+, 731.3072).

[0302] Example 12:

[0303] Synthesis of compound TDC-2:

[0304]

[0305] Compound 66: (Z)-N-(8-amino-11,12-dihydrodibenzo[c,g][1,2]diazocin-3-yl)-3-((6-chlorohexyl)oxy)propanamide. To a solution of 3 (111 mg, 0.42 mmol) in DCM (4mL) was added TFA (4 mL) and stirred at RT for 1 h, then the solvent was removed under reduced pressure. The residual pale yellow oil was used in the next step without further purification. Then the intermediate was dissolved in dry DMF (4 mL), and PyBOP (262 mg, 0.50 mmol) and DIPEA (217 pL, 1.26 mmol) were added. The reaction mixture was stirred at RT for 30 min, then compound 62 (100 mg, 0.42 mmol) in dry DMF (1 mL) was added. The solution was stirred at RT overnight. Then, H2O (10 mL) was added and the resulting mixture was extracted with EA (3 x 20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (MeOH / DCM) to give 66 (85 mg, 47% yield) as a light-yellow solid.1H NMR (400 MHz, CDCI3) 58.40 (br, NH), 7.14 (d, J = 8.3 Hz, 1 H), 7.05 (s, 1 H), 6.91 (d, J = 8.0 Hz, 1 H), 6.75 (d, J = 7.6 Hz, 1 H), 6.39 (d, J = 8.1 Hz, 1 H), 6.22 (s, 1 H), 3.71 (t, J = 5.0 Hz, 2H), 3.54 - 3.49 (m, 4H), 2.91 - 2.83 (m, 2H), 2.73 - 2.58 (m, 4H), 1.80 - 1.73 (m, 2H), 1.67 -1.60 (m, 2H), 1.51 - 1.35 (m, 4H);13C NMR (100 MHz, CDCI3) 5 170.0, 156.2, 155.8, 136.7, 130.7, 130.3, 124.2, 118.4, 118.2, 114.5, 109.9, 105.6, 71.4, 66.7, 45.2, 38.1, 32.6, 31.5, 31.0, 29.6, 26.8, 25.7; HRMS (ESI-TOF): m / z = 429.2037 (calcd. for [M + H]+, 429.2052). Compound 68: ferf-butyl (2-amino-5-(4-hydroxy-3,5 dimethoxybenzyl) pyrimidin-4-yl)carbamate. To a solution of 5 (400 mg, 1.46 mmol), TEA (304 uL, 2.19 mmol) and 4-DMAP (36 mg, 0.29 mmol) in THF / DMF (5 / 5 mL) was added (Boc)2O (317 mg, 1.46 mmol) dropwise. The solution was stirred at RT overnight. Then H2O (30 mL) was added and the resulting mixture was extracted with EA (3 x 30 mL). The combined organic extracts were dried over anhydrous Na2SC, filtered and concentrated under vacuum. The resulting residue was purified by column chromatography (MeOH / DCM) to give 68 (156 mg, 29% yield) as a off-white solid.1H NMR (400 MHz, DMSO-cfe) 6 9.18 (s, 1H), 8.14 (s, 1H), 7.70 (s, 1H), 6.52 (s, 4H), 3.70 (s, 6H), 3.56 (s, 2H), 1.42 (s, 9H);13C NMR (100 MHz, DMSO-d6) 5162.3, 156.1, 154.7, 147.9, 133.9, 128.9, 111.7, 106.2, 78.6, 56.0, 32.7, 28.0. HRMS (ESI-TOF): m / z = 377.1830 (calcd. for [M + H]+, 377.1819).

[0306] Compound TDC-2: 4-((2,4-diaminopyrimidin-5-yl)methyl)-2,6-dimethoxy phenyl (Z)-(8-(3-((6-chlorohexyl)oxy)propanamido)-11,12-dihydrodibenzo

[0307] [c,g][1,2]diazocin-3-yl)carbamate. To a solution of 66 (57 mg, 0.13 mmol) in dry DCM (5 mL) was added triphosgene (20 mg, 0.07 mmol) and NaHCOa (33 mg, 0.40 mmol). Then the mixture was stirred at RT for 2 h. The solvent was concentrated under vacuum and the intermediate 67 was used for the next step without furthor perification. To a solution of 68 (50 mg, 0.13 mmol) and TEA (20 uL, 0.15 mmol) in dry DCM (5 mL) was added a solution of the intermediate in dry DCM (1 mL) dropwise. The reaction mixture was stirrd at RT for 4 h. Then, H2O (10 mL) was added and the resulting mixture was extracted with DCM (3 x 20 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The crude product was dissolved in dry DCM (6 mL) at 0°C and TFA (3 mL) was added slowly. The reaction mixture was stirred at RT for 2 h. Then the solvent was concentrated under vacuum. The resulting residue was purified by preparation HPLC to give the target molecule TDC-2 (9.8 mg, 11% yield for three steps) as a light-yellow solid.1H NMR (400 MHz, DMSO-d6) 67.80 (br, NH2), 7.55(br, NH2), 7.48 (s, 1 H), 7.23 (d, J = 2.0 Hz, 1H), 7.18 - 7.12 (m, 2H),, 7.03 - 7.00 (m, 3H), 6.70 (s, 2H), 3.78 - 3.59 (m, 10 H), 3.54 (t, J = 6.6 Hz, 2H), 2.73 (s, 4H), 1.66 - 1.59 (m, 2H), 1.48 - 1.42 (m, 2H), 1.35 - 1.23 (m, 4H);13C NMR (151 MHz, DMSO-d6) 6 169.8, 164.5, 155.6, 155.4, 154.6, 152.8, 151.3, 138.6, 138.2, 137.9, 136.3, 130.9, 130.6, 128.1, 126.8, 122.9, 122.7, 118.0, 109.2, 108.8, 106.1, 70.4, 66.6, 56.4, 45.8, 42.6, 37.6, 32.4, 30.9, 30.8, 29.4, 26.5, 25.3. HRMS (ESI-TOF): m / z = 731.3053 (calcd. for [M + H]+, 731.3067).

[0308] Example 13:

[0309] Synthesis of compound TSMC

[0310]

[0311] Compound 77: (E)-3-((E)-(4-bromophenyl)diazenyl)-4-hydroxypent-3-en-2-one. ):

[0312] The solution of sodium nitrite (828 mg, 12 mmol) in water (5.0 mL) was dropped into the solution of 76 (1.72 g, 10 mmol) in acetic acid (15 mL), concentrated hydrochloric acid (2.3 mL), and water (10 mL) at 0 °C. After stirring at 0 °C for 1.5 h, the solution was transferred to the mixture of pentane- 2,4-dione (1.33 mL, 12 mmol) and sodium acetate trihydrate (4.08 g, 30 mmol) in water (5.0 mL) and ethanol (5.0 mL). After stirring at RT for 2 h, the mixture was filtered to collect precipitation. The precipitation was washed with water, 50% ethanol / water, and hexane to give 77 (2.79 g, 98% yield) as a yellow solid.1H NMR (400 MHz, DMSO-d6) 6 13.81 (s, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 2.47 (s, 3H), 2.40 (s, 3H).13C NMR (100 MHz, DMSO-d6) 5 197.3, 196.8, 141.8, 134.4, 132.8, 118.7, 117.5, 31.7, 26.8.

[0313] Compound 78: ferf-butyl (E)-2-(4-((4-bromophenyl)diazenyl)-3,5-dimethyl-1H-pyrazol-1-yl)acetate. The suspension of 77 (1.0 g, 3.53 mmol) and hydrazine monohydrate (173 pL, 3.56 mmol) in ethanol (20 mL) was stirred overnight at reflux temperature. After cooling, the solvent was removed in vacuo to give the crude pyrazole as a yellow solid. Then the suspension of crude pyrazole, tert-butyl bromoacetate (673 pL, 4.59 mmol) and cesium carbonate (1.72 g, 5.29 mmol) in dry DMF (10 mL) was stirred at 50 °C for 15 h. After cooled, the solvent was removed by evaporation and the crude material was purified by normal column chromatography (DCM / MeOH) to give 78 (0.98 g, 71% yield for 2 steps) as a yellow solid.1H NMR (400 MHz, CDCI3) 57.65 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.7 Hz, 2H), 4.74 (s, 2H), 2.54 (s, 3H), 2.49 (s, 3H), 1.48 (s, 9H).13C NMR (100 MHz, CDCI3) 5 166.3, 152.3, 143.1, 140.3, 135.4, 132.0, 123.4, 123.3, 83.2, 51.4, 28.0, 14.0, 9.8.

[0314] Compound 79: ferf-butyl (E)-2-(4-((4-((2-hydroxyethyl)thio) phenyl) dia zenyl)-3,5-dimethyl-1H-pyrazol-1-yl)acetate. To a solution of compound 78 (392 mg, 1 mmol) in 1,4-dioxane (10 mL) were added 2-Mercaptoethanol (85 pL, 1.2 mmol), Xantphos-PdG3 (19 mg, 0.02 mmol) and TEA (140 pL, 1 mmol). The mixture was stirred under N2 at 70°C overnight. After cooled, the solvent was removed by evaporation and the crude material was purified by normal column chromatography (DCM / MeOH) to give 79 (304 mg, 78% yield) as a yellow solid.1H NMR (400 MHz, CDCI3) 57.71 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 4.73 (s, 2H), 3.79 (t, J = 6.0 Hz, 2H), 3.17 (t, J= 6.0 Hz, 2H), 2.53 (s, 3H), 2.49 (s, 3H), 1.48 (s, 9H).13C NMR (100 MHz, CDCI3) 5166.3, 151.9, 143.1, 139.9, 136.7, 135.4, 129.6, 122.4, 83.1, 60.3, 51.3, 36.8, 27.9, 13.9, 9.8.

[0315] Compound 80: (E)-N-(6-chlorohexyl)-2-(4-((4-((2-hydroxyethyl)thio) phenyl) dia zenyl)-3,5-dimethyl-1H-pyrazol-1-yl)acetamide. To a stirred solution of compound 79 (134 mg, 0.34 mmol) in DCM (5 mL) was added TFA (5 mL) at RT. The reaction solution was stirred at RT for 1 h and removed under reduced pressure, and further removed by co-evaporation with toluene (1 mL x 2) to give the residue, which was directly used in the next step without further purification. To a stirred solution of this residue in dry DCM (10 mL) was added compound 49 (119 mg, 0.686 mmol), EDCI (99 mg, 0.51 mmol), HOBt (79 mg, 0.51 mmol) and DIPEA (174 pL, 1.03 mmol). The reaction mixture was stirred at RT for 18 h. Then the mixture was concentrated under reduced pressure and the crude material was purified by normal column chromatography (DCM / MeOH) to give 80 (86 mg, 55% yield for 2 steps) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 7.73 (d, J =8.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 6.18 (s, NH) 4.72 (s, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.49 (t, J = 6.6 Hz, 2H), 3.27 - 3.18 (m, 4H), 2.58 (s, 3H), 2.52 (s, 3H), 1.76 - 1.69 (m, 2H), 1.52 -1.38 (m, 4H), 1.31- 1.25 (m, 2H).13C NMR (100 MHz, CDCl3) δ 166.6, 151.7, 144.1, 140.2, 137.6, 135.4, 129.5, 122.5, 60.4, 52.2, 44.8, 39.4, 36.7, 32.4, 29.2, 26.4, 26.0, 14.0, 9.9.

[0316] Compound 81: (E)-2-((4-((1-(2-((6-chlorohexyl)amino)-2-oxoethyl)-3,5-dimethyl-1 H-pyrazol-4-yl)diazenyl)phenyl)thio)ethyl 4-methylbenzene sulfonate. To a stirred solution of compound 80 (40 mg, 0.088 mmol) in DCM (5 mL) was added TsCI (25 mg, 0.132 mmol) and TEA (19 pL, 0.132 mmol). The reaction mixture was stirred at RT overnight. Then the mixture was concentrated under reduced pressure and the crude material was purified by normal column chromatography (DCM / MeOH) to give 81 (31 mg, 58% yield) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.34 - 7.31 (m, 4H), 6.16 (s, NH), 4.72 (s, 2H), 4.16 (t, J =7.3 Hz, 2H), 3.49 (t, J = 6.7 Hz, 2H), 3.28 -3.18 (m, 4H), 2.59 (s, 3H), 2.52 (s, 3H), 2.44 (s, 3H), 1.77 - 1.69 (m, 2H), 1.52 - 1.32 (m, 4H), 1.30 - 1.24 (m, 2H).

[0317] 13C NMR (100 MHz, CDCl3) δ 166.6, 151.9, 145.0, 144.2, 140.3, 136.4, 135.4, 132.8, 129.9, 129.6, 127.9, 122.6, 122.6, 67.7, 52.3, 44.9, 39.4, 32.4, 31.9, 29.2, 26.4, 26.0, 21.7, 14.1, 9.9.

[0318] Compound TSMC: (E)-N-(6-chlorohexyl)-2-(4-((4-((2-(4-((2,4-diamino pyrimidin-5-yl)methyl)-2,6-dimethoxyphenoxy) ethyl)thio)phenyl) diazenyl)-3,5-dimethyl-1 H-pyrazol-1-yl)acetamide. To a solution of compound 81 (20 mg, 0.033 mmol) and compound 5 (18 mg, 0.066 mmol) in 5 mL dry DMF was added Cs2CO3(22 mg, 0.066 mmol). The reaction mixture was stirred at 70°C overnight. Then the solution was concentrated under vacuum and the resulting residue was purified by column chromatography (MeOH / DCM) to yield the target molecule TSMC (6.6 mg, 28% yield) as a yellow solid.1H NMR (600 MHz, CDCI3) 57.71 (d, J = 8.4 Hz, 2H), 7.45 - 7.42 (m, 3H), 6.35 (s, 2H), 6.13 (s, NH), 4.71 (s, 2H), 4.17 (t, J = 7.3 Hz, 2H), 3.81- 3.78 (m, 8H), 3.64 (s, 2H), 3.36 (t, J = 7.3 Hz, 2H), 3.27 - 3.23 (m, 2H), 2.58 (s, 3H), 2.52 (s, 3H), 1.74 -1.72 (m, 2H) 1.51 - 1.46 (m, 2H), 1.43 - 1.39 (m, 2H), 1.31 - 1.27 (m. 2H).

[0319] 13C NMR (151 MHz, CDCl3) δ 166.9, 154.2, 151.5, 144.3, 140.1, 138.6, 136.2, 135.6, 129.6, 128.7, 124.8, 122.5, 121.2, 107.8, 105.3, 105.2, 71.5, 56.3, 52.4, 51.1, 45.0, 39.5, 34.5, 32.5, 29.4, 26.5, 26.2, 14.2, 10.1.

[0320] Example 14:

[0321] Absorption spectra and photoisomerization

[0322] Absorption spectra measurement: UV-Vis absorption spectra were recorded using Cary 5000 spectrophotometer with a 10 mm width quartz cuvette. The sample was irradiated with LEDs emitting at 405 nm or 530 nm (405 nm, Luxeon SZ-01-U7; 530 nm, Luxeon SP-03-G3; 627 nm, Luxeon MR-D0040-20T).

[0323] Half-life measurement: UV-Vis absorption spectra of compounds in DMSO or PBS / DMSO were recorded at 37°C at different time points. The maximum absorbance were plotted versus time. Data were fitted to a single exponential function to give the rate constants k. The corresponding half-lives were obtained using the equation t1 / 2= ln2 / k.

[0324] The UV-vis absorption spectra of TAC and TAC-3 in the dark-adapted state trans form) showed a strong TT-TT* band at 370 nm and a weak n-rr* band near 460 nm, while c / s-TAC showed a strong n-rr* band at 460 nm, which resembled that of azobenzene (Figure 2A, 6A). Illumination with 405 nm or 530-nm light led to trans-to-cis or c / s-to-trans isomerization of TAC and TAC-3 to the photostationary state (PSS) with the cis form or trans form, respectively (Figures 2A, 6A). Remarkably, multiple rounds of photoisomerization did not lead to any noticeable photobleaching or photo-oxidation, suggesting the robustness of the chemical photoswitch (Figure 2B). The cis forms of TAC and TAC-3 are thermally stable in DMSO, with a relaxation half-life of ca. 72 min and 1.9 h at 37°C in the dark, respectively (Figure 2C, D, Figure 6B, C).

[0325] The modular design makes it facile to incorporate different photoswitches to the dimerizer. The spectrum of azo-dimerizers could be expanded by using different azophotoswitches. The absorption wavelength for photoisomerization and the rate of thermal isomerization of the cis form can be tuned by substitutions on azobenzenes.

[0326] 3,4-dimethyl azobenzene and arylazopyrazole were incorporated into the dimerizer, resulting in TMC, TPC and TSMC, respectively (see the synthesis in Example 3, 10 and 13). The UV-vis absorption spectra of TMC, TPC and TSMC in trans and cis states are similar to those of TACs (Figures 7A, 8A and 18A). In contrast, the cis form of TMC and TPC are more thermally stable in DMSO than that of TAC. c / s-TMC has a relaxation half-life of ca. 22 h at 37°C in the dark (Figure 6B), while c / s-TPC is stable within 24 h at 37°C in the dark (Figure 8C). Moreover, TPC is resistant to the reduction by GSH, the major reductant in cells, suggesting that TPC is stable and can be used in cells in long term (Figure 8C).

[0327] or / ?o-tetrafluoroazobenzenes (ABF4s) displays n-n* and n- it* absorptions in the UV and blue region, respectively (Figure 9, Table 1). Illumination with 530-nm (green) or 405-nm light led to trans-to-cis or cis-to-trans isomerization of ABF4s, respectively (Figure 9). Moreover, ABF4s have much slower thermal relaxation rate (hours to days) (Figure 10, Table 1). The ABF4-WW, ABF4-AW and ABF4-AA photoswitches were chosen to incorporate into the dimerizer, resulting in TFC dimerizers (see the synthesis in Examples 5-8). TFCs underwent trans-to-cis photoisomerization upon illumination at 530 nm, which was reverted by illumination at 405 nm (Figure 11). The cis form of TFCs displayed relaxation half-life of »> 48 h at 37°C in PBS: DMSO = 1:1 in the dark (Figure 11, Table 2).

[0328] The orf / io-tetrachloroazobenzene undergoes trans-to-cis isomerization with red light.

[0329] TCC dimerizer based on tetra-orf / io-chloro azobenzene was prepared (see the synthesis in Example 9). TCC was switched to the cis form upon illumination at 627 nm (Figure 13A, B), The cis form of TCC displayed relaxation half-life of 2.8 h at 37°C in PBS: DMSO = 1:1 in the dark (Figure 13C, Table 2).

[0330] As opposed to azobenzenes, 5,6-dihydrodibenzo[c,g][1,2]diazocine is thermodynamically more stable in the cis (Z) form and undergoes reversible photoisomerization from the cis (Z) to trans (E) form. TDC dimerizers using diazocine as the photo-switchable linker were prepared (see the synthesis in Examples 11-12). The absorptions of Z and E isomers in the visible region are well separated (Figure 14A, Table 3). The E isomer was quantitatively switched back to the Z isomer upon illumination at 530 nm (Figure 14A). The excellent photostability enables many photoswitching cycles (Figure 14B). The half-lifes of thermal E— > Z relaxation are 1.7-3.4 h at 37°C in PBS: DMSO = 1:1 (Figure 14C, Table 3). Table 1. Absorption, photostationary state (PSS) and half-lives of thermal relaxation in the dark for o-tetrafluoroazobenzenes at 37°C in PBS: DMSO = 4:1.

[0331]

[0332] n.d. not determined, a: not separated by HPLC.

[0333] Table 2. Spectral features, PSSs and thermal relaxation rates of TFCs and TCC at 37°C in PBS: DMSO (1:1).

[0334]

[0335] Table 3. Absorption and half-lives of thermal relaxation in the dark forTDC-1, TDC-2 and TDC-3 at 37°C in PBS: DMSO = 1:1.

[0336]

[0337]

[0338] Example 15:

[0339] Protein dimerization in vitro

[0340] 5 pM HaloTag protein and 6 pM TAC orTMC in 100 pL HEPES buffer (20 mM HEPES and 40 mM NaCI) were incubated at 37°C for 30 min, then 10 pM eDHFR protein was added followed by incubating at 37°C for 30 min. Then the reaction mixture was divided two parts: one of them was irradiated with 405 nm for 10 min followed by native polyacrylamide gel electrophoresis (PAGE) under 405 nm light; the other was subjected to native PAGE without 405 nm light. frans-TAC or trans-TMC induced dimerization, while c / s-TAC or c / s-TMC did not (Figure 3, Figure 7C). 10 pM Halotag protein and 12 pM frans-TFCs in 100 pL HEPES buffer (20 mM HEPES and 40 mM NaCI) were incubated at 37°C for 30 min. Then the reaction mixture was divided two parts: one was added 20 pM eDHFR protein and then incubated at 37°C for 30 min; the other was firstly irradiated with 530 nm for 10 min to induce trans-to-cis isomerization, followed by adding 20 pM eDHFR protein and then incubated at 37°C for 30 min. trans-TFCs induced dimerization, while c / s-TFCs induced dimerization with different efficiencies (Figure 12A).

[0341] TFCs / TCC-mediated control of enzymatic activity in vitro

[0342] To demonstrate the ability of TFCs and TCC to reversibly control protein function, we employed a complementation assay using split NanoLuc luciferase. We fused eDHFR to the C-terminal fragment of NanoLuc (SmBiT-eDHFR) and HaloTag to the N-terminal fragment of NanoLuc (HaloTag-LgBiT). We purified recombinant SmBiT-eDHFR and HaloTag-LgBiT fusion proteins.

[0343] 200 nM HaloTag-LgBiT protein was incubated with 200 nM TFCs or TCC at 37°C for 10 min, followed by adding 240 nM eDHFR-SmBiT protein and incubated for 20 min. The reaction mixture was subjected to 530 nm illumination (94 mW / cm2) for 1 min or 530 nm illumination (94 mW / cm2) for 1 min then 405 nm illumination (13 mW / cm2) for 5 min, or 627 nm illumination (56 mW / cm2) for 5 min or 627 nm illumination (56 mW / cm2) for 5 min then 405 nm illumination (13 mW / cm2) for 5 min. Detection of NanoLuc complementation was done using Nano-Gio Luciferase Assay (Promega) according to the manufacturer’s instructions. Images were acquired using a ChemiDoc imaging system (Bio-Rad).

[0344] Addition of TFCs in the dark efficiently reconstituted NanoLuc activity, generating bioluminescent signal. Under 530-nm illumination, no change in signal was observed for TFC-1, TFC-2, and TFC-3, whereas a 3-fold reduction in signal was observed for TFC-4 (Figure 12B), in line with the native PAGE results (Figure 12A). Subsequent illumination at 405 nm led to full signal recovery with TFC-4 (Figure 12B). Similarly, TCC enabled rapid and reversible optical control of protein-fragment reconstitution (Figure 13D)

[0345] Example 16:

[0346] Linker strategy in the design of photoswitchable dimerizers

[0347] Guided by the molecular orbital (MO) theory, we expected that substituting the para position of azobenzene affects its photophysical properties. Therefore, it is important to explore the chemistry of the linkers between the ligand and the photoswitch. To this end, we synthesized ort / ?o-tetrafluoroazobenzene (ABF4) and its derivatives with different combinations of electron-donating and electron-withdrawing groups (EDGs and EWGs) at the para position (ABF4-DD, ABF4-DW, ABF4-WW, ABF4-AW, and ABF4-AA) (Figure 9, see synthesis in Example 4).

[0348] Three key features were evaluated: (1) A(n-n*)> the separation of the n-rr* bands of trans and cis isomers, (2) the thermal relaxation half-lives of c / s-isomers, and (3) the percentage of trans / cis photostationary states (PSSs) (Table 1). A larger Δ(λn-π*) indicates better separation of the n-n* bands, leading to more efficient trans-cis photoconversion. A longer thermal relaxation half-life of the cis isomer is beneficial for bistable photoswitches.

[0349] The n-n* bands of trans and cis isomers of unsubstituted ABF4are well separated with A(Xn-TT‘) of 22 nm. Illumination with visible light produced PSSs with 87% cis isomer (530 nm) and 76% trans isomer (405 nm) (Figure 9, Table 1). However, when substituting ABF4at the para position with EDGs or a combination of EDG and EWG (ABF4-DD or ABF4-DW), the n-n* bands of trans and cis isomers largely overlap, reducing Δ(λn-π*) of ABF4-DD to 11 nm. Consequently, 530-nm and 405-nm illumination of ABF4-DD resulted in only 50% trans-to-cis and 58% cis-to-trans photoconversions, respectively (Figure 9, Table 1).

[0350] In contrast, when substituting ABF4with EWGs, weak EDGs (such as alkyl groups) or a combination of both (ABF4-WW, ABF4-AW, and ABF4-AA), the n-rr* bands are well separated, with Δ(λn-π*) increased to 43 nm, 39 nm, and 40 nm, respectively. This allows for more selective excitation of either isomer with visible light. 530-nm light produced 83% (ABF4-WW) and 84% (ABF4-AA) cis isomers, while 405-nm light generated 92% (ABF4-WW) and 91% (ABF4-AA) trans isomers (Figure 9, Table 1). Importantly, ABF4-WW, ABF4-AW and ABF4-AA exhibit strong bistable characters, i.e. very slow thermal relaxation of the cis isomer (ti / 2 »> 48 h at 37 °C in aqueous solution, compared to 23.3 h for ABF4-DD and 8.0 h for ABF4-DW under the same conditions) (Figure 10, Table 1).

[0351] Given the superior photophysical properties of ABF4-WW, ABF4-AW and ABF4-AA, we first synthesized TFC-1 based on ABF4-WW (see the synthesis in Example 5). Native PAGE analysis showed that frans-TFC-1 (in the dark) effectively induces dimerization of HaloTag and eDHFR. However, c / s-TFC-1 (with 530-nm illumination) did not cause dedimerization (Figure 12A). This suggested that while TFC-1 undergoes efficient reversible photoisomerization, it fails to promote dedimerization upon trans-to-cis photoconversion. We speculated that the linkers in TFC-1 may not facilitate dedimerization.

[0352] Next, we optimized the linker A (between TMP and ABF4) by introducing alkyl linkers, as alkyl groups at the para position of ABF4are also beneficial for its photophysical properties, as demonstrated with ABF4-AW and ABF4-AA. We synthesized TFC-2 and TFC-3 containing propyl and methyl groups in the linker A, respectively (see the synthesis in Examples 6 and 7). Native PAGE analysis revealed that trans isomers of both compounds efficiently induce HaloTag and eDHFR dimerization, while c / s-TFC-3 (with 530 nm illumination) partially induced dedimerization (Figure 12A).

[0353] Inspired by the limited success of modifying linker A, we proceeded to change linker B (between ABF4and chloroalkyl). TFC-4 was synthesized with modified linker B compared to TFC-1 while keeping the same ABF4-WW photoswitch (see the synthesis in Example 8). Native PAGE analysis demonstrated that the trans and cis isomers of TFC-4 successfully induce dimerization and dedimerization, respectively (Figure 12A). These results suggested that the linker strategy plays a crucial role in controlling light-switchable dimerization. Optimization of linker strategy for TDCs

[0354] As the linker strategy affects the photoswitchable dimerization efficiency, three TDCs (TDC-1, TDC-2 and TDC-3) were synthesized (see the synthesis in Examples 11 and 12). To test whether TDCs can induce protein dimerization in living cells, we used a complementation assay of split NanoLuc luciferase. NanoLuc could then be reversibly reconstituted under control of the TDC systems. TDC-controlled protein dimerization was switched on with 405-nm light and off in the dark. However, the efficiency of protein complementation using TDC-1 after 405-nm illumination was relatively poor, resulting in a 1.5-fold increase in bioluminescence, due to the relatively high level of basal dimerization in the dark (Figure 15A, B). While changing linker A but keeping the original linker B in TDC-2 resulted in decreased dimerization in the dark (basal activity), TDC-2 failed to induce further dimerization in the light (Figure 15A, B). In contrast, changing linker B while keeping the original linker A resulted in TDC-3 with minimal basal activity in the dark, yielding a 3-fold increase of bioluminescence signal in the light (Figure 15A, B).

[0355] Altogether, both photoswitch nature and linker strategy play crucial roles in the development of photoswitchable dimerizers. The nature of photoswitch largely determines the wavelength of photoswitching and the thermal stability of the photoisomer in the dark (its bistable character). We have shown that linker chemistry also affects the photophysical properties of the photoswitch by influencing its molecular orbital (MO) energy. Rational design guided by MO theory could aid in optimizing linker chemistry. Importantly, the linker strategy is essential for controlling the dynamic range between dimerization on and off states (as seen with TFCs and TDCs). Moreover, the photophysical properties of the photoswitch also influence the dynamic range since the extent of photoisomerization determines the ratio of trans and cis configurations. For instance, TCC (with o-tetrachloroazobenzene photoswitch) achieves only 50% trans-to-c / s photoisomerization under red light, compared to 83-87% for o-tetrafluoroazobenzenes under green light (Table 2). Consequently, the TCC-controlled system has a higher off-state signal under red light compared to the TFC-4 system under green light, resulting in a lower dynamic range (Figures 12B and 13D), despite both photoswitchable dimerizers utilizing the same linker strategy. Example 17:

[0356] Protein dimerization in cells

[0357] Dimerizer was dissolved in DMSO at 10 mM and stored in amber plastic microcentrifuge tubes at -80°C as original stocks. For short-term storage, a dimerizer DMSO stock was diluted to a final working concentration of 1 mM and stored in amber plastic microcentrifuge tubes at -20°C. Working under normal room lighting did not cause any detectable premature trans-cis or cis-trans isomerization of dimerizer. For live cell imaging, a fresh compound stock was diluted in DMEM to a working concentration of 5 pM, cells were treated with 1 pM or 5 pM dimerizer. After incubation with dimerizer, cells were washed twice by pre-warmed phosphate buffered saline (PBS) before imaging.

[0358] Cell culture and transfection

[0359] HeLa cells were maintained at 37°C under 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1 % nonessential amino acid (NEAA, 100 x) and 1 % penicillinstreptamycin (100 x). For transient transfection, DNA (1 pg) was dissolved in 100 pl gibco opti-MEM and incubated with 1 pl X-treme GENE HP DNA transfection reagent for 15 min at room temperature. Then, 30 pl DNA was added into each imaging well (SARSTEDT 8-well slide) seeding 1-2 x 104cells in 300 pl complete DMEM.

[0360] Confocal laser scanning microscopy

[0361] For confocal microscopy, the cells were imaged on a Leica SP8 or SP5 inverted confocal system (Leica Microsystems) equipped with a HC PL APO 63x / 1.40 oil immersion lens and a temperature-controlled hood maintained at 37°C and 5% CO2. Imaging was carried out ~24h post transfection using 8-well imaging chambers (SARSTEDT x-well slide) in neutral DMEM (phenol red free) supplemented with additional 10 % FBS, 1 % NEAA and 1 % penicillin streptomycin at 37°C under 5 % CO2. Routinely, images were recorded at 1 % to 5% laser power with a scanning speed of 100 Hz, a line average of one, a frame average of two and a size of 512 x 512 or 1024 x 1024 pixel. The zoom, gain and pinhole were adjusted for each experiment individually. Light-control was performed on a Leica SP8 system using a 4.2 mW 405 nm diode laser at 1% laser power (42 pW), a scanning speed of 200 Hz, and a pixel density of 1024 x 1024 (final illumination intensity = 0.21 J / cm2). Alternatively, lightcontrol was performed on a Leica SP5 system using an illumination intensity of 0.94 J / cm2(93.75 pW laser power of 405 nm diode, a scanning speed of 100 Hz and a pixel density of 1024 x 1024).

[0362] Protein-fragment complementation in Hela cells

[0363] HeLa cells were seeded into black p-Plate 24 Well plates (Ibidi) and transfected with constructs encoding HaloTag-HA-LgBiT and eDHFR-HA-SmBiT. After 24 h, cells were treated with 1 pM or 5 pM dimerizer for 30 min, prior to wash-off and replacement of media with Opti-MEM reduced serum media. Cells were subjected to 405 nm illumination (55 mW / cm2) for 7 min or 530 nm illumination (78 mW / cm2) for 2 min prior to detection of NanoLuc complementation using Nano-Gio Live Cell Assay (Promega) according to the manufacturer’s instructions. Images were acquired using a ChemiDoc imaging system (Bio-Rad).

[0364] In HeLa cells co-expressing mCherry-eDHFR and EGFP-HaloTag-ActA (ActA represents a mitochondria targeting sequence from Listeria monocytogenes protein ActA), addition of TAC led to rapid recruitment of mCherry-eDHFR from the cytosol to mitochondria (Figure 4A). A short pulse of illumination at 405 nm led to rapid dissociation. Thermal relaxation to the trans state in the dark led to association. The process was repeated over several consecutive cycles without a detectable loss in dimerization efficacy and speed (Figure 4A, B). Similarly, TAC-3-, TMC-, TPC- or TSMC-mediated reversible dimerization was controlled by light in multiple rounds in live cells (Figures 6E, 7D, 8D, 18B).

[0365] In contrast, photo-activatable / cleavable chemical inducers of dimerization, CONC, which is described in WO 2019 / 008175 A1, induces only a single cycle of dimerization with light. Photoactivation by a short pulse of illumination at 458 nm leads to recruitment of mCherry-eDHFR from the cytosol to mitochondria, while photodeactivation results in disruption of the dimerizer and consequently of mCherry-eDHFR from mitochondria. However, redimerization is not possible, as the dimerizer is disrupted by light (Figure. 4C).

[0366] To demonstrate the ability of TAC to reversibly control cellular processes, two constructs, KIF5B-mCherry-eDHFR that contains the N-terminal motor domain (1-560) of the kinesin KIF5B, and Halo-EGFP-Rab5a that contains the early endosome resident Rab5a GTPase, were co-expressed in HeLa cells. Addition of 1 pM TAC recruited Rab5 positive endosomes to the kinesin forcing anterograde endosomal transport and accumulation at the cell periphery (Figure 5). 405-nm illumination released the endosome from the kinesin resulting in dispersal away from the periphery. This process could be repeated several times by cycling on and off 405 nm illumination (Figure 5B).

[0367] Similarly, dimerizers with different photoswitches and linkers (TFC, TCC and TDC) also induced reversible protein dimerization in cells (Figures 12C, 13A, 15A, 15C). These results suggest that the modular design of photoswitchable dimerizers is versatile. By incorporating different photoswitches or ligands to the dimerizer, the spectrum of dimerizers could be expanded.

[0368] Patterning of enzymatic activity in cells

[0369] To demonstrate the spatial resolution of light-controlled dimerization in cell populations, a patterning approach using split NanoLuc was employed. In HeLa cells co-expressing HaloTag-LgBiT and SmBiT-eDHFR fusion proteins, addition of TPC led to complementation of the functional NanoLuc, resulting in bioluminescent signal upon addition of the cell-permeable NanoLuc substrate. 405-nm (530-nm) illumination of a subpopulation of cells using a pattern mask (7 minutes; 1.5 mW / cm2) resulted in localized dedimerization and the appearance of a distinct pattern after addition of the NanoLuc substrate (Figure 16).

[0370] TPC-mediated control of protein stability in cells

[0371] The high stability of TPC in mammalian cells further allows use of this molecule for extended periods of time, while retaining its pohotoswitchable properties. Combining this system with a degron-based protein degradation tool consisting of a destabilization domain (DD, an eDHFR mutant) that is continuously degraded, therefore enables TPC-mediated, dimerization-dependent and light-controllable manipulation of protein levels in cells. In cells expressing HaloTag and a protein-of-interest (POI) such as oncogenic KRASG12Vfused to DD, addition of 1 M TPC for 16 h results in stabilization of the otherwise constitutively degraded POI. Subsequent PD by illumination of the cells with 405nm light (0.15 mW / cm2) triggers dedimerization of DD from HaloTag, resulting in efficient degradation of the target POI within hours. In contrast, non-illuminated samples retain stabilized levels of the POI (Figure 17).

Claims

1. CLAIMS1. A photoswitchable compound consisting of:3.ligand A - linker A - photoswitch moiety - linker B - ligand B, ligand A-photoswitch moiety- linker B - ligand B or ligand A - linker A - photoswitch moiety-ligand B; wherein4.ligand A or ligand A-photoswitch moiety is capable of binding a ligand binding domain of a first peptide or protein;5.the photoswitch moiety is of formula (I), formula (II), or of formula (III) or any isomer of formula (I), formula (II) or formula (III)7.

8. wherein each of R1, R2, R3and R4is independently selected from hydro, fluoro, chloro, bromo, methyl, methoxy and ethylthio;9.X is selected from -CH2-, -CH2CH2-, - CH2CH2CH2-, -OCH2-, -CH2O-, -NHCH2-, -CH2NH-, -CH2S-, -SCH2-, -NR5CH2- and -CH2NR5-; R5is selected from hydro, methyl, ethyl and acetyl;10.and ligand B or photoswitch moiety -ligand B is capable of binding a ligand binding domain of a second peptide or protein; wherein11.ligand A and ligand B are selected from C2-C10haloalkyl, benzyl guanine, benzyl cytosine, maleimide, glutamic acid, methotrexate, SLF, SLF’ and trimethoprim;12.and wherein13.linker A is any of; a bond, -O-CH2-NH-CO-, -O-CH2-CO-NH-, -O-CH2-CH2-NH-CO-, -O-CO-NH-, -O-CH2-CH2-CH2-, -O-CH2-, -O-CH2-CH2-O-, or -O-CH2-CH2-S- and wherein said linker B is any of a bond, -NH-CO-CH2-CH2-O-, -O-CH2-CO-NH-CH2- CH2-O-, -CO-NH-CH2-CH2-O-, -CO-NH-,-CH2-CO-NH-,-CO-NH-CH2-CH2-O-, -NH- CO-CH2-O- or -CH2-CH2-CO-NH-.14.2 The compound according to any of the previous claims, said compound being selected from16.

3. Chemo-optogenetic system for reversibly and repetitively testing intracellular test compound interactions in cells in vitro and / or in vivo, comprising:19.a) the photoswitchable compound according to any of the previous claims; b) conjugate 1 comprising a test compound 1 and at least a HaloTag; and c) conjugate 2 comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or the SLF’ binding domain of FKBP (F36V).

4. Chemo-optogenetic system according to claim 3, wherein conjugate 1 and / or conjugate 2 comprise further a component for identification and / or purification of the conjugate and / or a first or second peptide or protein.

5. Chemo-optogenetic system according to claim 3 or 4, wherein test compound 1 and test compound 2 are selected independently of each other among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

6. Chemo-optogenetic system according to any of claims 3 to 5, wherein the bacterial DHFR is eDHFR.

7. Use of the chemo-optogenetic system as defined in any of claims 3 to 6 for reversibly and repetitively testing the interactions of a test compound 1 with a test compound 2.

8. Method for reversibly and repetitively testing intracellular test compound interactions in cells in vitro and / or in vivo, comprising the following steps:24.a) providing, transfecting and expressing the DNA sequence of a conjugate 1, comprising a test compound 1 and at least a HaloTag, in a cell / s;25.b) providing, transfecting and expressing the DNA sequence of a conjugate 2, comprising a test compound 2 and at least the TMP binding domain of a bacterial DHFR or SLF’ binding domain of FKBP (F36V), in said cell / s;26.c) adding a photoswitchable compound according to any of claims 1-2 to said cell / s and admitting them to pass the plasma membrane;27.d) activating and / or deactivating said photoswitchable compound with light under irradiation condition A for activation and under irradiation condition B for deactivation;28.e) determining the change in a selected test parameter system; and29.f) optionally repeating step d) and e) one or more times.

9. Method according to claim 8, wherein test compound 1 and test compound 2 are selected independently from one another among gene products, proteins, protein domains, peptides, polypeptides, glycopeptides, proteins with secondarily modified amino acids, peptides or proteins with protecting groups, saccharides, small molecules, lipids, polynucleotides, oligonucleic acids, DNA and RNA.

10. Kit, comprising31.a) a compound according to any of claims 1 -2 having a ligand A and a ligand B that is selected from C2-C10haloalkyl, SLF’ and / or trimethoprim;32.b) a nucleotide sequences or a vector / s and / or plasmid / s comprising nucleotide sequences coding for at least HaloTag and the test compound 1 and a bacterial DHFR or eDHFR or FKBP (F36V) and test compound 2.

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