conjugates

Abstract

To provide a conjugate of a formula (I) and its use in methods of treatment, and also methods for delivering an active agent into a cell.SOLUTION: A methods may be used to deliver an active agent into a nematode, a flatworm, a parasite or a bacterium. The conjugate of a formula (I) is: (formula (I)), where -D- is C1-4 alkylene or C2-4 alkenylene, preferably C2-4 alkenylene, where the alkylene or alkenylene is optionally substituted with alkyl or halo; A- is an active agent for delivery; and -RA, -RB, -RT1, -RT2, -R1, -R2, -R3, -X- and -L- are as defined herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority under GB1820626.8, filed December 18, 2018 (18 / 12 / 2018), the entire contents of which are incorporated herein by reference.

[0002] The present invention provides conjugates of pantothenic acid or a derivative thereof with an active agent for delivering the active agent to a cell or organism. The present invention also provides conjugates for use in methods of treatment and methods for preparing the conjugates. [Background technology]

[0003] Methods for treating parasitic diseases have recently focused on techniques to improve the delivery of drugs to the location of the parasite within the host, followed by subsequent uptake by the parasite. Many current methods for treating parasitic diseases rely on administering high levels of drugs to patients over a sustained period of time to ensure that the drug is provided at the target location in sufficient concentration and for sufficient duration to achieve beneficial effects. Such drug administration often results in serious side effects in treatment methods.

[0004] For this reason, it would be beneficial to provide a drug delivery strategy that allows for the administration of small amounts of drug while maintaining beneficial therapeutic effects. Recent research in this area has considered all aspects of drug delivery. Leading research has considered improving the transport of active agents to target locations within infected hosts. Exemplary strategies include adapting drug formulations and administration regimens. For example, incorporating antiparasitic agents into nanoparticles or microparticles, emulsions, or liposomes may result in more targeted delivery of the drug to pathogens (see, for example, the overview of delivery strategies for antiparasitic drugs by Kayser et al. and Date et al.).

[0005] Another line of research focuses on delivering drugs to the parasite itself, and in particular, transporting drugs across the parasite cell membrane. The use of many antibiotics is hampered by the drug's relative inability to cross the cell membrane. To address the challenge of delivering drugs to cells, researchers have resorted to linking the drug to a second agent known to cross the cell membrane. Thus, the second agent can be used to carry the drug into the parasite's cell.

[0006] As an example of this approach to treating malaria and other parasitic diseases, Sparr et al. described the preparation and use of conjugates of the antimalarial drug fosmidomycin with an octaarginine peptide. Fosmidomycin has been reported to be poorly taken up by T. gondii, M. tuberculosis, and P. berghei. The drug was covalently attached to the octaarginine peptide via a short linker, or the drug was prepared as a salt in which the counterion was a labeled octaarginine peptide. Octaarginine peptides have previously been shown to penetrate the plasma membrane of red blood cells infected with P. falciparum. The authors demonstrated that the octaarginine peptide could be used to improve the uptake of fosmidomycin into P. falciparum and other parasites, subsequently improving the drug's antiparasitic efficacy.

[0007] Landfear also reported that conjugating antiparasitic agents to functional groups associated with cellular uptake can improve uptake of antiparasitic agents into the intracellular environment. Thus, Landfear mentions the use of P2 targeting motifs, known to be substrates for P2 transporters, to increase uptake selectivity.

[0008] There is a need for additional vehicles useful for the delivery of active agents to parasites. Accordingly, the present inventors have developed conjugates useful for the delivery of active agents to intracellular and extracellular parasites, including nematodes or helminths and bacteria, that at least in some way fulfill this need and / or at least provide the public with a useful option. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent No. 0068485 [Patent Document 2] U.S. Patent No. 9,108,942 [Patent Document 3] International Publication No. 2012 064632 [Patent Document 4] International Publication No. 2012 / 097454 [Patent Document 5] International Publication No. 2019 / 060634 [Non-patent literature]

[0010] [Non-Patent Document 1] Bergen et al., Antimicrob. Agents and Chemotherapy, 2006, 50, 1953 [Non-patent document 2] Schechter et al., J. Med Chem 2002, 45(19) 4264 [Non-patent document 3] Krise and Oliyai Biotechnology: Pharmaceutical Aspects, 2007, 5(2), 101~131 [Non-patent document 4] "Protective Groups in Organic Synthesis" (T. Green and P. Wuts; 3rd ed.; John Wiley and Sons, 1999) [Non-Patent Document 5] Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990 [Non-patent document 6] Handbook of Pharmaceutical Excipients, 5th edition, 2005 [Non-Patent Document 7] PLoS Negl Trop Dis. 2011 / 9;5(9):e1260 [Non-patent document 8] Mol. Biochem. Parasitol. 164:57~65, 2009 [Non-Patent Document 9] Zhao et al. Org. Biomol. Chem. 2010, 8, 3328-3337 Summary of the Invention [Means for solving the problem]

[0011] In a general aspect, the present invention provides conjugates comprising a pantothenic acid group or a derivative thereof ("pantothenic acid group") for delivering an active agent to a cell. The pantothenic acid group is covalently linked to the active agent, either directly or via a linker.

[0012] Thus, the present invention allows for the modification of active agents with pantothenic acid groups to improve or alter the transport properties of the active agent. For example, the pantothenic acid group may improve the transport of the active agent across the cell membrane, thereby increasing the amount of the active agent in the intracellular environment.

[0013] The conjugates of the invention can be used to deliver active agents to the cells of pathogens, such as bacterial cells, or the cells of parasites, such as nematodes or helminths. The conjugates of the invention may find use in treating host subjects, such as mammalian subjects, infected with a pathogen.

[0014] The conjugates of the invention can be used to selectively deliver active agents to cells infected with a pathogen, such that the conjugate does not deliver the active agent to non-infected cells.

[0015] The conjugates of the invention can also be used to deliver active agents to pathogens that are intracellular or extracellular parasites.

[0016] In a first aspect of the present invention, there is provided a conjugate of formula (I):

[0017] [ka]

[0018] (In the formula, -R A and -R B are each independently hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkanyol, and aralkanoyl, e.g., hydrogen; or -R A and -R B together -C(R C1 )(R C2 )- to form a six-membered ring, and -R C1 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R C2 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy, such as -R C1 and -R C2 is alkyl or -R C1 and -R C2 together form oxo (=O); -R T1 and -R T2 are each independently hydrogen or alkyl, e.g., hydrogen; -R 1 and -R 2are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl, e.g., alkyl; -R 3 is hydrogen or alkyl, e.g., hydrogen; -D- is C 1~4 Alkylene or C 2~4 Alkenylene, for example C2 alkylene or C2 alkenylene, wherein the alkylene or alkenylene is optionally substituted with alkyl or halo; -X- is a covalent bond, -O-, -S-, -Se- or -N(R 4 )-, for example -N(R 4 )- and -R 4 is hydrogen or alkyl, e.g., hydrogen; -L- is a linker or a covalent bond; A- is the active agent to be delivered) and salts, solvates and protected forms thereof.

[0019] In one embodiment, the compound of formula (I) is a compound of formula (Ia-I) to (Ia-III):

[0020] [ka]

[0021] is selected from.

[0022] In one embodiment, the compound of formula (I) has formula (Ib):

[0023] [ka]

[0024] (In the formula, R A , -R B , -L- and -A are as defined for compounds of formula (I). and salts, solvates and protected forms thereof.

[0025] In one embodiment, the compound of formula (I) has formula (Ic):

[0026] [ka]

[0027] (In the formula, R A , -R B , -L- and -A are as defined for compounds of formula (I). and salts, solvates and protected forms thereof.

[0028] In one embodiment, the compound is

[0029] [ka]

[0030] isn't it.

[0031] In a second aspect of the present invention, there is provided a pharmaceutical composition comprising a conjugate of formula (I) optionally together with one or more pharmaceutically acceptable excipients.

[0032] In a third aspect of the present invention, there is provided a conjugate of formula (I) or a pharmaceutical composition comprising a conjugate of formula (I), as described in the first and second aspects of the present invention, for use in a method of treatment.

[0033] In a fourth aspect of the present invention, there is provided a conjugate of formula (I) or a pharmaceutical composition comprising a conjugate of formula (I), as described in the first and second aspects of the present invention, for use in a method of treating an infectious disease, such as a microbial infection, for example a bacterial infection; or a nematode or flatworm infection; or a parasitic infection.

[0034] In various embodiments, the infection can be caused by any one of Haemonchus contortus, Trypanosoma brucei, Theileria annulata, Plasmodium falciparum, Lotmaria passim, Babesia bovis, or Mycobacterium tuberculosis.

[0035] In various embodiments, the infection may be caused by a helminth, a kinetoplast, an apicomplexan, or a mycobacteria.

[0036] In a fifth aspect of the present invention, there is provided the use of the conjugate as a tool for drug and protein delivery to Caenorhabditis elegans, which is used as an animal model of human disease.

[0037] The present invention also provides a method for delivering a conjugate of formula (I) or a pharmaceutical composition comprising a conjugate of formula (I) as described in the first and second aspects of the present invention to a cell, the method comprising the step of contacting a cell with a conjugate of formula (I) or a composition comprising the conjugate.

[0038] In one embodiment, the method of the invention is not a method of treatment of the human or animal body. In one embodiment, the method of the invention is ex vivo.

[0039] In one aspect, there is provided a compound for use in preparing a compound of formula (I). In one embodiment, the compound is of formula (II), (III), (IV) or (V), as described in more detail below.

[0040] In a further aspect of the invention, there is provided a process for preparing a compound of formula (I), comprising reacting a compound of formula (II), (III) or (IV) with an activator, thereby obtaining a compound of formula (I).

[0041] In one embodiment, the method comprises reacting a compound of formula (V) with a compound of formula (II), (III) or (IV), thereby obtaining a compound of formula (I).

[0042] In yet another aspect of the present invention, there is provided the use of an active agent for use in a method of treatment, wherein the active agent is conjugated to a pantothenic acid group. Thus, the active agent may be a conjugate of formula (I).

[0043] These and other aspects of the invention are discussed in further detail below. [Brief explanation of the drawings]

[0044] [Figure 1] Figure 1 shows a pair of fluorescence microscopy images. The left shows an infected red blood cell containing two trophozoite-stage parasites treated with a conjugate of the invention, with the compound present throughout the parasite's cytosol. Digestive vacuoles appear as black circles, and there is no compound accumulation. The right shows several uninfected red blood cells that exhibit no fluorescence surrounding the trophozoite-stage infected cells. [Figure 2a] 1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. 2 is a fluorescence micrograph of Babesia bovis infected red blood cells treated with conjugate 5. Uninfected red blood cells do not take up the conjugate. [Figure 2b] 1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. 2 is a fluorescence micrograph of a Theileria parva infected red blood cell treated with conjugate 5. The conjugate appears to accumulate within the parasite. [Figure 2c]1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. Figure 2 is a bright field (left) and fluorescence (right) micrographs of Plasmodium falciparum infected red blood cells treated with conjugate 5. Uninfected red blood cells do not take up the conjugate. [Figure 2d] This is a series of microscopy images, including fluorescence microscopy images of organisms treated with a conjugate of the present invention. These are bright-field (left) and fluorescence (right) microscopy images of Trypanosoma brucei treated with conjugate 9 (visible as green dots in the fluorescence image). DAPI staining is also visible (blue dots in the fluorescence image). Conjugate 5 was also tested (images not shown). Conjugate 5 was taken up by T. brucei and formed small vesicles throughout the trypanosome body, but did not accumulate in lysosomes or the nucleus; rather, it accumulated in vesicles between the flagellum and lysosomes. Conjugate 9, on the other hand, was taken up much more rapidly and at much higher concentrations than compound 5. Compound 9 also spread throughout the cell, but appeared to have areas of higher concentration in the mitochondria or endoplasmic reticulum. [Figure 2e] 1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. 2 is a fluorescence micrograph of Escherichia coli treated with conjugate 5, showing uptake of the conjugate by the organism. [Figure 2f] 1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. 2 is a fluorescence micrograph of Enterococcus faecalis treated with conjugate 5, showing uptake of the conjugate by the organism. [Figure 2g] 1 is a series of micrographs including fluorescence micrographs of organisms treated with a conjugate of the invention. 2 is a fluorescence micrograph of Staphylococcus aureus treated with conjugate 5, showing uptake of the conjugate by the organism. [Figure 2h]

[0023] Figure 1 is a series of microscopy images containing fluorescence microscopy images of organisms treated with conjugates of the invention. A pair of fluorescence microscopy images of Caenorhabditis elegans treated with conjugates 1 (right image) and 5 (left image). Conjugate 5 is localized in the digestive track of C. elegans. Conjugate 1 is distributed throughout the worm. [Figure 2i] Figure 1 is a series of microscopy images including fluorescence microscopy images of organisms treated with a conjugate of the invention. Figure 2 is a pair of fluorescence microscopy images of Haemonchus contortus treated and untreated with Conjugate 1. The image on the left is a control showing autofluorescence of Haemonchus contortus. The image on the right is of Haemonchus incubated with Conjugate 1. [Figure 3] Figure 1 shows the results of an uptake study of delivery vehicles (a) Compound 2 and (b) Compound 8 in L. passim. Pictures were taken from experiments performed at 500 μM for Compound 2 and 10 μM for Compound 8, and at room temperature for 45 minutes of incubation. Size bar: 5 μm. [Figure 4] Figure 1 shows the results of evaluation of delivery vehicles and BODIPY11 in L. passim. Bar graphs represent RFU from experiments performed at 1 μM and 45 min incubation at room temperature. RFU was calculated from images using ImageJ software (units x 10). Asterisks indicate significant differences (*P<0.03) between samples and BODIPY control 1 analyzed using an unpaired Student's t-test. [Figure 5] Figure 1 shows the results of an uptake study of delivery vehicles 2 and 8 and BODIPY unit 11 in the honeybee gut. Photographs were taken from experiments performed at 100 μM and 33°C for 45 minutes. Size bar: 100 μm. Photographs show bright-field (left image) and FITC filter (right image) images. (a) BODIPY11; (b) Compound 1; (c) Compound 2; (d) Compound 8. [Figure 6]Figure 1 shows the assessment of delivery vehicle uptake in B. bovis-infected RBCs. Photographs are merged results of bright field and FITC filters. Size bar: 5 μm. a) Compound 1; b) Compound 3; c) Compound 2; d) Compound 5; e) BODIPY11. [Figure 7] Figure 1 shows the uptake assessment of the delivery vehicle (R)-P3 in RBCs infected with B. bovis. Photographs show results from experiments performed at 100 μM and 45 min of incubation at 37°C. Photographs: parasite-infected RBCs, size bar: 5 μm. (a) Bright field; (b) DAPI; (c) FITC; (d) Merged. [Figure 8] Figure 1 shows the uptake assessment of ivermectin B1a and compounds 14 and 15 and ivermectin B1a in mixed cultures of wild-type nematodes. Images are taken from experiments performed at 5 μM and 50 μM concentrations, 25°C, and 24 hours of incubation. Size bar: 100 μm. (a) Ivermectin B1a, 5 μM, bright field; (b) Compound 14, 5 μM, bright field; (c) Compound 15, 5 μM, FITC; (d) Ivermectin B1a, 50 μM, bright field. [Figure 9] Figure 9 shows uptake assessments of the bacterium, Mycobacterium tuberculosis (H37Rv strain), when incubated with 50 μg / mL of compounds 1-8, 10, and control PBS (phosphate buffered saline). Figure 9(a) shows a graph of the uptake experiment in relative fluorescence units (RFU) of compounds 1-8, 10, and control PBS after 45 minutes of incubation with bacteria. Figure 9(b) shows two bright-field images of M. tuberculosis bacteria for the uptake experiment with compounds 1 (top) and 2 (bottom). DETAILED DESCRIPTION OF THE INVENTION

[0045] The present inventors have discovered that pantothenic acid and its derivatives can be used to modify active agents to improve the delivery of the active agents in vivo.

[0046] The conjugate comprises a pantothenic acid group covalently linked to a drug, either directly or through a linker group.

[0047] Pantothenic acid is utilized in vivo to prepare coenzyme A (CoA) (see Van der Westhuyzen et al.). The first step in biosynthesis is the conversion of pantothenic acid to P-Pan, mediated by PanK. This phosphorylated compound is then converted to P-Pan-CMP, which is then converted to PPC under the action of PPCS.

[0048] Recently, analogs of pantothenic acid have been described that can disrupt coenzyme A biosynthesis. Research has shown that a range of bacteria cannot synthesize pantothenate and therefore rely on the uptake of pantothenic acid from the environment to synthesize CoA. Therefore, disrupting CoA biosynthesis is a useful strategy for treating bacterial infections.

[0049] An exemplary physiologically active derivative of pantothenic acid is CJ-15801. Structurally, CJ-15801 differs from pantothenate only in that it has a double bond in the β-alanine moiety. CJ-15801 is processed in vivo by PanK and PPCS to give P-CJ-CMP (the CJ-15801 version of P-Pan-CMP). P-CJ-CMP is a potent tight-binding inhibitor of PPCS. Thus, the processing of pantothenic acid is prevented.

[0050] Han et al. described a synthetic route to the antibiotic CJ-15801. Some intermediates are protected forms of the antibiotic, for example, the carboxylic acid terminus is protected as a benzylamide or allyloxycarbonyl group. The protected forms of the antibiotic have not been reported to have antibiotic activity, and the protecting group used at the carboxylic acid terminus is not considered an active agent.

[0051] The present inventors have established that active agents can be modified with pantothenic acid and its derivatives to improve delivery of the active agents, for example, to improve delivery of the active agents to parasites and bacteria. The present inventors have established that conjugates containing pantothenic acid groups are rapidly taken up by cells. For example, the present inventors have found that the conjugates of the present invention are taken up by Staphylococcus aureus and E. faecalis cells, among others, within 45 minutes. Thus, the conjugates of the present invention find use in Gram-positive and Gram-negative bacteria.

[0052] Furthermore, the conjugates of the present invention may be selective for certain cell types, for example, preferably taken up by bacterial cells over mammalian cells, or parasite cells over host cells. In one embodiment, the conjugates of the present invention may be preferably taken up by mammalian cells infected with a parasite over uninfected mammalian cells. In another embodiment, the conjugates of the present invention may be preferably taken up by parasite-infected cells over insect cells.

[0053] The conjugates of the present invention can be delivered to prokaryotic cells, such as bacterial cells. The conjugates of the present invention can be delivered to eukaryotic cells, including cells of eukaryotic microorganisms such as protists, including Chromalveolata microorganisms, e.g., Apicomplexan microorganisms, or Kinetoplastids, e.g., Trypanosoma and Rottomaria. The conjugates of the present invention can be delivered to helminths, e.g., C. elegans and Haemonchus contortus.

[0054] Thus, the conjugates of the present invention may find use in the delivery of agents to treat disease-associated microorganisms. Thus, the conjugates find use in the treatment of microbial infections in subjects, such as mammalian subjects.

[0055] Thus, while pantothenic acid and its derivatives are known to have uses in vivo within the CoA biosynthetic pathway (as substrates or antimetabolites), this acid and its derivatives are used in the present invention to modify, e.g., improve, the transport properties of active agents.

[0056] Clarke et al. previously described a pantothenic acid analogue to study the biosynthesis of coenzyme A (CoA). Here, pantothenic acid was covalently linked to a fluorescent dye via a diaminoalkylene linker. The pantothenic acid analogue was taken up by E. coli cells and processed intracellularly to give the coenzyme A analogue.

[0057] Pantothenic acid analogs have not been used in treatment methods, and there is no suggestion that the analogs can be delivered to mammalian cells that host the parasites. Indeed, the authors report that the analogs have negligible antibacterial activity, which they highlight as an advantage of the analogs.

[0058] The study by Clarke et al. focuses solely on studying the metabolic processing of pantothenate compounds within cells. Thus, pantothenic acid analogs are provided as substrates for natural product elucidation. There is no suggestion that pantothenic acid or its derivatives can be used as delivery vehicles to carry or deliver other active agents into cells.

[0059] In one embodiment, the conjugate of the invention is not Compound 1 from Clarke et al.. In a further embodiment, the conjugate of the invention comprises an active agent that has biological activity, e.g., antiparasitic activity, e.g., antimicrobial activity.

[0060] European Patent No. 0068485 discloses carbapenem derivatives conjugated to a pantothenic acid group for use as antibiotics. The focus of this patent is the production of these carbapenem derivatives from bacteria, such as Streptomyces sp. OA-6129. It is disclosed that these compounds have antimicrobial activity against Comamonas terrigena B-996, a β-lactam-hypersensitive microorganism, but no quantitative data are disclosed. There is no suggestion that pantothenic acid or its derivatives can be used as a delivery vehicle to carry or deliver other active agents to cells.

[0061] The present invention differs from EP 0068485 because the conjugates for use against microorganisms, e.g., bacteria, contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria than the pantothenic acid group itself (see, e.g., tests with Mycobacterium tuberculosis in the Examples).

[0062] U.S. Patent No. 9,108,942 discloses the conjugate CLX-SYN-G18-CO1 for use in treating severe pain. This conjugate contains a 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetic acid group as the active agent attached to a pantothenic acid group. Other conjugates disclosed have a wide variety of different groups attached to the active agent. There is no suggestion that the pantothenic acid group is preferred over the other groups listed in the disclosure.

[0063] The present invention is novel relative to CLX-SYN-G18-CO1 because the conjugates of the present invention contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective than the pantothenic acid group itself (see, e.g., tests with Plasmodium falciparum, Trypanosoma brucei, Theileria annulata, and Mycobacterium tuberculosis in the Examples).

[0064] WO 2012 064632 discloses 5-mercapto-1H-indazole-4,7-dione derivatives capable of inhibiting fatty acid synthase, for use in treating a wide variety of conditions, such as bacterial and protozoan infections. Some of the disclosed compounds are conjugated to a pantothenic acid group, among a wide variety of other possible groups. No biological data is provided to support the claim that the compounds have activity against bacteria or protozoa. Protozoa are not necessarily parasites.

[0065] The present invention is novel with respect to WO 2012 064632 because the conjugates for use against bacteria or parasites of the present invention contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria or parasites than the pantothenic acid group itself (see, e.g., tests with Plasmodium falciparum, Trypanosoma brucei, Theileria annulata, and Mycobacterium tuberculosis in the Examples).

[0066] WO 2012 / 097454 discloses conjugates for enhancing resistant bacterial cells, comprising an aminoglycoside moiety linked to a pantothenic acid group. These conjugates lack antibacterial activity per se, but are reported to resensitize bacteria to other antibiotics. There is no suggestion that pantothenic acid or its derivatives can be used as a delivery vehicle to deliver or carry other active agents to cells.

[0067] The present invention is novel with respect to WO 2012 / 097454 because the conjugates for use against bacteria of the present invention contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria than the pantothenic acid group itself (see, e.g., tests with Mycobacterium tuberculosis in the Examples).

[0068] WO 2019 / 060634 discloses two compounds containing cystamine derivatives conjugated to a pantothenic acid group. These compounds are intended to treat cysteamine-sensitive conditions, syndromes, and diseases, including infectious diseases such as bacterial and parasitic infections. Exemplary bacteria and parasites cited as causing cysteamine-sensitive infections include the Pseudomonas aeruginosa bacterium that causes cystic fibrosis, and the Plasmodium falciparum and Plasmodium muriatica parasites that cause malaria. Similarly, there is no suggestion that pantothenic acid or its derivatives can be used as a delivery vehicle to transport or deliver other active agents to cells.

[0069] The present invention is novel with respect to WO 2019 / 060634 because the active agent in the conjugates for use against bacteria and parasites of the present invention contains at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria and parasites than the pantothenic acid group itself (see, e.g., tests with Plasmodium falciparum, Trypanosoma brucei, Theileria annulata, and Mycobacterium tuberculosis in the Examples).

[0070] Meier et al. disclose compounds for in vitro and in vivo protein labeling containing a fluorescent dye moiety conjugated to a pantothenic acid group. These compounds were shown to be incorporated into the CoA pathway of E. coli and to bind to the carrier protein Fren. Similarly, there is no suggestion that pantothenic acid or its derivatives can be used as a delivery vehicle to carry or deliver other active agents to bacteria, such as E. coli.

[0071] The present invention is novel to Meier et al. because the conjugates for use in the delivery method to bacteria, e.g., E. coli, contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria than the pantothenic acid group itself (see, e.g., tests with Mycobacterium tuberculosis in the Examples).

[0072] Shakya et al. disclose pantothenic acid derivatives as polyketide mimetics. The mimetics are conjugated to actinorhodin acyl carrier protein (actACP) for use in studying polyketide synthases (PKS). There is no suggestion that pantothenic acid or its derivatives can be used as a delivery vehicle to carry or deliver active agents to cells.

[0073] The present invention is novel to Shakya et al. because the conjugates of the present invention contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. Furthermore, the moiety attached to the pantothenic acid group is not a dye, a small molecule drug, a polypeptide, a polynucleotide, or a polysaccharide.

[0074] Storz et al. have disclosed compounds that inhibit the PqsD enzyme. Inhibition of PqsD suppresses the production of 2-heptyl-4-hydroxyquinoline (HHQ) and Pseudomonas aeruginosa quinolone signal (PQS). The molecules HHQ and PQS are involved in regulating virulence factor production and biofilm formation in the pathogenic Gram-negative bacterium Pseudomonas aeruginosa. Therefore, PqsD is a target for the development of anti-infective drugs.

[0075] The present invention is novel to Storz et al. because the conjugates for use against bacteria of the present invention contain at least one unsaturated bond in the β-alanine portion of the pantothenic acid group, forming an enamide. This derivative of the pantothenic acid group is more selective for bacteria than the pantothenic acid group itself (see, e.g., tests with Mycobacterium tuberculosis in the Examples).

[0076] Conjugates The present invention provides a conjugate of a drug and a pantothenic acid group. Thus, in one embodiment, the conjugate has the structure:

[0077] [ka]

[0078] The group includes a group having the formula:

[0079] In this context, references to pantothenic acid groups and derivatives thereof ("pantothenic acid groups") are references to groups having the structure shown above.

[0080] In one aspect of the invention, a conjugate of formula (I):

[0081] [ka]

[0082] and salts, solvates and protected forms thereof. Substituents are discussed in detail below.

[0083] Exemplary Conjugates In one embodiment, the compound of formula (I) has formula (Ib):

[0084] [ka]

[0085] (In the formula, -R A , -R B , -L- and -A are as defined for compounds of formula (I). and salts, solvates and protected forms thereof.

[0086] In one embodiment, the compound of formula (I) has formula (Ic):

[0087] [ka]

[0088] (In the formula, -R A , -R B , -L- and -A are as defined for compounds of formula (I). and salts, solvates and protected forms thereof.

[0089] In one embodiment, the compound of formula (I) has the formula (Id):

[0090] [ka]

[0091] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 , -D- and -X- are as defined for compounds of formula (I); -L 1 - is alkylene or heteroalkylene; -L 2 - is alkylene or heteroalkylene and salts, solvates and protected forms thereof.

[0092] In one embodiment, the compound of formula (I) has the following formula:

[0093] [ka]

[0094] wherein -L- and -A are as defined for compounds of formula I. The compound is selected from the group consisting of:

[0095] In a preferred embodiment, -L- is a linker as defined for compounds of formula (Id). 1 - is C2 alkylene, and -L 2 - is a C3 alkylene or a C5 alkylene.

[0096] In one embodiment, the compound of formula I is a compound of formula (Ie) or (If). In a preferred embodiment, -L- is a linker as defined for compounds of formula (Id). In an even more preferred embodiment, -L 1 - is C2 alkylene, and -L 2 - is a C3 alkylene or a C5 alkylene.

[0097] intermediate compound In another aspect of the invention, there are provided intermediate compounds for use in the preparation of compounds of formula (I).

[0098] Thus, the compound of formula (II)

[0099] [ka]

[0100] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 , -D- and -X- are as defined for compounds of formula (I); -L 1 - is alkylene or heteroalkylene; -D 1 -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , -N3, -C=CH2, -C=C(H)(Hal) and -C≡CH, -R N and -R D are each independently alkyl, and Hal is halogen. and salts, solvates and protected forms thereof.

[0101] Compounds of formula (III):

[0102] [ka]

[0103] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 and -D- is as defined for compounds of formula (I). and salts, solvates and protected forms thereof are also provided.

[0104] The present invention also provides a compound of formula (IV):

[0105] [ka]

[0106] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 , -D- and -X- are as defined for compounds of formula (I); -L 1 - is alkylene or heteroalkylene; -L 2 - is alkylene or heteroalkylene; -D 2 -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D and Maleimidyl) and salts, solvates and protected forms thereof.

[0107] Compounds of formula (V):

[0108] [ka]

[0109] where -A is an activator; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -T is -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , -N3, -C=CH2 and -C≡CH; N and -R D are each independently selected from alkyl and salts, solvates and protected forms thereof are also provided and suitable for use in the present invention.

[0110] substituent An alkyl group refers to a monovalent hydrocarbon group. An alkyl group is fully saturated. It can be linear or branched. An alkyl group is a C 1~10 Alkyl, C 1~6 Alkyl, C 1~4 Alkyl, C 1~2 It can be alkyl or C1 alkyl (methyl).

[0111] An alkylene group refers to a divalent hydrocarbon group. An alkylene group is fully saturated. It can be linear or branched. An alkylene group is a C 1~10 Alkylene, C 1~6 Alkylene, C 1~4 Alkylene, C 1~3 Alkylene, C 2~3 Alkylene, C 1~2 It can be alkylene, C2 alkylene (ethylene) or C1 alkylene (methylene).

[0112] An alkenyl group refers to a monovalent hydrocarbon group having one or more carbon-carbon double bonds, e.g., one double bond. An alkenyl group can be fully or partially unsaturated, e.g., partially unsaturated. It can be linear or branched. An alkenyl group is a C 2~10 Alkenyl, C 2~6 Alkenyl, C 2~4 Alkenyl, C 2~3 It can be alkenyl, C3 alkenyl (allyl) or C2 alkenyl (vinyl).

[0113] An alkenylene group refers to a divalent hydrocarbon group having one or more carbon-carbon double bonds, e.g., one double bond. The alkenyl group can be fully or partially unsaturated, e.g., partially unsaturated. It can be linear or branched. An alkenylene group is a C 2~12 Alkenylene, C 2~10 Alkenylene, C 2~6 Alkenylene, C 4~6 Alkenylene, C 2~4 Alkenylene, C 2~3 It can be alkenylene, C2 alkenylene or C3 alkenylene.

[0114] An alkynyl group refers to a monovalent hydrocarbon group having one or more carbon-carbon triple bonds, e.g., one triple bond. An alkynyl group can be fully or partially unsaturated, e.g., partially unsaturated. It can be linear or branched. An alkynyl group is a C 2~10 Alkynyl, C 2~6 Alkynyl, C 2~4 Alkynyl, C 2~3 It can be alkynyl, C3 alkynyl (propargyl) or C2 alkynyl.

[0115] A cycloalkyl group refers to a monovalent cyclic hydrocarbon group. A cycloalkyl group is fully saturated. A cycloalkyl group can have one ring or two or more fused rings. A cycloalkyl group is a C 3~10 Cycloalkyl, e.g., C 3~6 Cycloalkyl, e.g., C 4~6 It can be a cycloalkyl, such as a C6 cycloalkyl (cyclohexyl).

[0116] A cycloalkylene group refers to a divalent cyclic hydrocarbon group. A cycloalkylene group is fully saturated. A cycloalkylene group can have one ring or two or more condensed rings. A cycloalkylene group is a C 3~10 Cycloalkylene, e.g., C 3~6 Cycloalkylene, e.g., C 4~6 It can be a cycloalkylene, for example a C6 cycloalkylene (cyclohexylene).

[0117] A heteroalkylene group refers to a divalent hydrocarbon group in which one or more carbon atoms are replaced by a heteroatom. A heteroalkylene group is fully saturated. It can be linear or branched. The heteroalkylene group is C 2~12 Heteroalkylene groups, such as C 3~12 Heteroalkylene, e.g., C 3~12 It may be heteroalkylene. The heteroalkylene group may be an alkylene glycol group, such as a polyalkylene glycol group. Examples include ethylene glycol groups, such as polyethylene glycol groups. The heteroalkylene group may contain one or two heteroatoms, such as one. The heteroatoms may be selected from -O-, -S- and / or -NH-.

[0118] An aryl group refers to a monovalent aromatic group. The aryl group can be a carboaryl group or a heteroaryl group. The carboaryl group is C 6~14 Carboaryl, C 6~10 Carboaryl, such as C6 carboaryl (phenyl) or C 10 It may be carboaryl (naphthyl). The heteroaryl group is C 5~10 Heteroaryl, e.g., C 5~6 It can be a heteroaryl, such as a C5 heteroaryl or a C6 heteroaryl. The heteroaryl group has one or more aromatic ring atoms selected from N, S and O. Examples of C5 heteroaryl groups include pyrrolyl and oxazolyl. Examples of C6 heteroaryl groups include pyridyl and pyrimidinyl. An aryl group can have one ring or two or more fused rings. When a heteroaryl group has two or more rings, each ring may have 5 to 7 ring atoms, of which 0 to 4 are heteroatoms (provided that at least one ring has one heteroatom).

[0119] An arylene group refers to a divalent aromatic group. The arylene group can be a carboarylene group or a heteroarylene group. The carboarylene group is C 6~14 Carboarylene, C 6~10 Carboarylene, for example C6 carboarylene (phenylene) or C 10 It may be a carboarylene (naphthylene). The heteroarylene group is C 5~10 Heteroarylene, e.g., C 5~6 Heteroarylene can be, for example, a C5 heteroarylene or a C6 heteroarylene. Heteroarylene groups have one or more aromatic ring atoms selected from N, S and O. Examples of C5 heteroarylene groups include triazolylene, pyrrolylene and oxazolylene. Examples of C6 heteroarylene groups include pyridylene and pyrimidylene. An arylene group can have one ring or two or more fused rings. When a heteroarylene group has two or more rings, each ring may have 5 to 7 ring atoms, of which 0 to 4 are heteroatoms (provided that at least one ring has one heteroatom).

[0120] A heterocyclene group refers to a divalent heterocycle. A heterocyclene group is fully saturated. Heterocyclenes are C 5~12 Heterocyclenes, such as C 5~7 Heterocyclenes, such as C 5~6 It may be a heterocyclene, for example a C6 heterocyclene. A heterocyclene can have one or two fused rings. If two rings are present, one or both rings can contain a heteroatom. Heterocyclenes can have one or more ring heteroatoms selected from O, S, and N (such as NH). Sulfur atoms can be oxides, such as SO and SO. Carbon ring atoms in a heterocyclene group may have an oxo substituent (=O). In one embodiment, an oxo substituent is provided on the carbon ring atom bearing the adjacent nitrogen ring atom, thereby providing an amide-like group in the heterocycle. If the heterocyclene has a nitrogen ring atom, the heterocyclene may be attached via the nitrogen ring atom.

[0121] An aralkyl group refers to an alkyl group having one or more, for example, one, aryl substituents. The aralkyl is connected via an alkyl group. An example of an aralkyl group is benzyl. The alkyl and aryl groups can each be as defined herein.

[0122] A cycloalkylalkyl group refers to an alkyl group having a cycloalkyl substituent. The cycloalkylalkyl group is connected via an alkyl group. The alkyl and cycloalkyl groups can each be as defined herein.

[0123] An alkanoyl group refers to an alkyl group in which the carbon of the alkyl group forming the connection is replaced with oxo (=O). An example of an alkanoyl group is acyl (C2 alkanoyl). The alkanoyl group can be based on the alkyl groups described herein.

[0124] An aralkanoyl group refers to an aralkyl group in which the carbon of the alkyl group forming the linkage is replaced with oxo (=O). An example of an aralkanoyl group is benzoyl. The aralkanoyl group can be based on the aralkyl groups described herein.

[0125] An alkoxy group refers to an alkyl ether group connected through an ether oxygen atom, where alkyl is as defined herein.

[0126] An alkenoxy group refers to an alkenyl ether connected via an ether oxygen atom. Alkenyl groups are as defined herein. In one embodiment, the ether oxygen is not provided at a carbon atom that also participates in a carbon-carbon double bond.

[0127] An alkynoxy group refers to an alkynyl ether connected via an ether oxygen atom. Alkynyl groups are as defined herein. In one embodiment, the ether oxygen is not provided at a carbon atom that also participates in a carbon-carbon triple bond.

[0128] An aralkoxy group refers to an aralkyl ether connected through an ether oxygen atom provided on the alkyl of the aralkyl, where aralkyl groups are as defined herein.

[0129] A cycloalkylalkoxy group refers to a cycloalkylalkyl ether group connected via an ether oxygen atom provided on the alkyl of the cycloalkylalkyl, where cycloalkylalkyl is as defined herein.

[0130] -R A and -R B -R A Groups and -R B groups are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, and alkanoyl, or -R A and -R B together -C(R C1 )(R C2 )-, forming a six-membered ring.

[0131] -R A and -R B together -C(R C1 )(R C2 )-group. This six-membered ring can have a -R A and -R Bare formed together with the oxygens to which they are bonded and the carbon atoms that are alpha and beta to these oxygen atoms.

[0132] [ka]

[0133] The six-membered ring is a 1,3-dioxane group. The compound may be, for example, -R C1 and -R C2 When —R is alkyl or hydrogen, it may be referred to as an acetal. C1 and -R C2 together form an oxo (=O) group. The compound can then be called a cyclic carbonate.

[0134] In one embodiment, -R A and -R B are each independently selected from hydrogen and alkyl, or -R A and -R B together -C(R C1 )(R C2 )-, forming a six-membered ring.

[0135] In one embodiment, -R A and -R B are each independently selected from hydrogen and alkyl, e.g., hydrogen.

[0136] -R A and -R B The compound where both are hydrogen is -R A and -R B together -C(R C1 )(R C2 )-. Similarly, -R A and -R B In compounds where both are not hydrogen, -R A and -R B can be formed from compounds where both are hydrogen.

[0137] The alkanoyl group is C1~6 Alkanoyl groups, e.g., C 1~4 , for example C2 alkanoyl (acyl group). Compounds bearing an alkanoyl group can be formed by reaction of an alcohol with, for example, the appropriate acid chloride or anhydride.

[0138] Typically, -R A and -R B are both hydrogen or -R A and -R B together -C(R C1 )(R C2 )-, for example -C(Me)2-.

[0139] In one embodiment, -R A is hydrogen. In one embodiment, -R B is hydrogen.

[0140] stereochemistry The conjugates of the present invention are based on pantothenic acid. Thus, the compounds of the present invention also have the stereochemical configuration of pantothenic acid, which is the (2R)-configuration. Thus, in one embodiment, the conjugate of formula (I) is

[0141] [ka]

[0142] It could be.

[0143] Thus, in one embodiment, the conjugates of the invention have (2R)-stereochemistry.

[0144] In another embodiment, the conjugate of the invention has (2S)-stereochemistry. Thus, in one embodiment, the conjugate of formula (I) is

[0145] [ka]

[0146] It could be.

[0147] -R C1 and -R C2 -R C1 The groups may be selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl. -R C2 The groups may be selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy. Or, -R C1 and -R C2 together form an oxo (=O). This can be called a carbonate protecting group for a diol functionality.

[0148] -R C2 The group may be an ether group, for example an alkoxy, and the compound may be called an orthoester.

[0149] In one embodiment, -R C1 is selected from hydrogen, alkyl and aralkyl. In one embodiment, -R C1 is selected from hydrogen and alkyl. In one embodiment, -R C1 is alkyl, for example methyl.

[0150] In one embodiment, -R C2 may be selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl. In one embodiment, -R C2 is selected from hydrogen, alkyl and aralkyl. In one embodiment, -R C2 is selected from hydrogen and alkyl. In one embodiment, -R C2 is alkyl, for example methyl.

[0151] In one embodiment, -R C1 and -RC2 are each alkyl. In one embodiment, -R C1 and -R C2 are methyl groups.

[0152] -R T1 and -R T2 -R T1 and -R T2 are each independently hydrogen or alkyl. The alkyl group is 1~8 Alkyl, e.g., C 1~4 Alkyl, e.g., C 1~2 It can be alkyl. The alkyl group can be methyl.

[0153] In one embodiment, -R T1 is hydrogen and -R T2 is hydrogen or alkyl, for example methyl. In one embodiment, -R T1 and -R T2 Each of these is hydrogen.

[0154] Pantothenic acid groups bearing alkyl substituents at the ω-position (terminal position of the pantoyl moiety, also at the β-position) are known from Bird et al. (and discussed by Spry et al.).

[0155] -R D Each-R D is independently alkyl. The alkyl group is C 1~12 Alkyl groups, such as C 1~6 Alkyl, e.g., C 1~4 Alkyl, e.g., C 1~2 The alkyl group can be a C1 alkyl (methyl). In one embodiment, each -R D is methyl.

[0156] -R N Each-R N is independently alkyl. The alkyl group is C 1~12Alkyl groups, such as C 1~6 Alkyl, e.g., C 1~4 Alkyl, e.g., C 1~2 The alkyl group can be a C1 alkyl (methyl). In one embodiment, each -R N is methyl.

[0157] -R 1 and -R 2 -R 1 and -R 2 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl. In one embodiment, -R 1 and R 2 are each independently selected from alkyl, alkenyl, aralkyl, and cycloalkylalkyl.

[0158] In one embodiment, -R 1 and R 2 are each alkyl. -R 1 Groups and -R 2 Both groups may be methyl.

[0159] -R 1 and -R 2 One of them may be methyl and the other may be other than methyl.

[0160] In one embodiment, -R 1 Ga-R 2 is the same as

[0161] Akinnusi et al. describe geminal derivatives of pantothenic amides in which the gem-dimethyl substituent of pantothenic acid is replaced with one or two alternative alkyl, alkenyl, aralkyl, and cycloalkylalkyl groups.

[0162] -R 3 In one embodiment, -R 3 is hydrogen. In one embodiment, -R3 is alkyl, for example methyl.

[0163] -D- The -D- group is an alkylene or alkenylene optionally substituted with alkyl or halo, e.g., optionally mono- or di-substituted with alkyl or halo, e.g., optionally mono- or di-substituted with alkyl, C 1~4 Alkylene or C 2~4 It is alkenylene.

[0164] Alternatively, -D- is alkenylene optionally substituted with alkyl or halo, C 2~4 It is alkenylene.

[0165] In one embodiment, -D- is alkylene or alkenylene optionally substituted with alkyl or halo, C 1~3 Alkylene or C 2~3 It is alkenylene.

[0166] Alternatively, -D- is alkenylene optionally substituted with alkyl or halo, C 2~3 It is alkenylene.

[0167] In one embodiment, -D- is a C2 alkylene or C2 alkenylene, wherein the alkylene or alkenylene is optionally substituted with alkyl or halo.

[0168] Alternatively, -D- is a C2 alkenylene, where the alkenylene is optionally substituted with alkyl or halo.

[0169] The alkylene or alkenylene may be a straight chain alkylene or a straight chain alkenylene.

[0170] In one embodiment, -D- is selected from the group consisting of -R 5 is hydrogen or alkyl, and each -R 6 is hydrogen or alkyl, -C(R 5 )2-C(R 6 )2- or -C(R5 )=C(R 6 )-.

[0171] In one embodiment, -D- is a C2 alkylene or C2 alkenylene optionally substituted with alkyl.

[0172] Alternatively, -D- is a C2 alkenylene optionally substituted with alkyl.

[0173] When -D- is a C4 alkenylene, the alkenylene can be a diene.

[0174] Preferably, -D- is C 2~3 It is alkenylene.

[0175] Even more preferably, -D- is C2 alkenylene.

[0176] double bond In one embodiment, eg a preferred embodiment, a double bond is present in the conjugate of formula (I). In one embodiment, the double bond has a trans or cis configuration.

[0177] In one embodiment, the double bond has a trans configuration, where trans refers to the configuration of the amide group across the double bond. For example, the compound of formula (Ia-I) has a trans configuration:

[0178] [ka]

[0179] In one embodiment, the double bond has a cis configuration, where cis refers to the arrangement of the amide group across the double bond. For example, the compound of formula (Ia-II) has a cis configuration:

[0180] [ka]

[0181] The present inventors have found that the geometry of the double bond can affect the selectivity of the conjugate for delivering the drug to a particular cell or to a particular organism.

[0182] When the group is a diene, both double bonds can have a trans or cis geometry, or one can be trans and the other cis.

[0183] -R 5 If the pantothenic acid group has a double bond, one -R 5 If the pantothenic acid group does not have a double bond, two -R groups may be present. 5 A group may be present, where -R 5 The groups may be the same or different.

[0184] In one embodiment, each -R 5 is hydrogen. In one embodiment, each -R 5 is alkyl, for example methyl.

[0185] -R 6 If the pantothenic acid group has a double bond, one -R 6 If the pantothenic acid group does not have a double bond, two -R groups may be present. 6 A group may be present, where -R 6 The groups may be the same or different.

[0186] In one embodiment, each -R 6 is hydrogen. In one embodiment, each -R 6 is alkyl, for example methyl.

[0187] If a double bond is present, -R 6 The group is -R 5 For example, -R 5 and -R 6 can both be hydrogen. If no double bond is present, -R6 The groups can be the same and are -R 5 For example, each -R 5 group and each -R 6 The group can be hydrogen.

[0188] -X- -X- is a covalent bond, -O-, -S-, -Se- or -N(R 4 )-, for example -N(R 4 )- and -R 4 is hydrogen or alkyl, for example hydrogen.

[0189] In one embodiment, -X- is a covalent bond, -O-, -S-, or -N(R 4 )-. In one embodiment, -X- is a covalent bond, -O-, or -N(R 4 )-. In one embodiment, -X- is a covalent bond or -N(R 4 )-. Alternatively, -X- can be a covalent bond or -O-.

[0190] In one embodiment, -X- is -N(R 4 )-. Exemplary groups include -N(H)- and -N(Me)-.

[0191] -R 4 In one embodiment, -R 4 is hydrogen. In one embodiment, -R 4 is alkyl, for example methyl.

[0192] -L- The -L- group can be a covalent bond, where the pantothenic acid group together with the -X- group directly connects the active agent-A. Alternatively, the -L- group can be a linker for indirect covalent attachment of the pantothenic acid group and the -X- group to the active agent-A.

[0193] In one embodiment, the linker -L- is * -L3 -BL 4 -GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or cycloalkylene; -L 4 - is a covalent bond, alkylene, or heteroalkylene; -G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)—, —OC(O)—, and a maleimide-derived group; —R N is hydrogen or alkyl, -L A - is a covalent bond, alkylene or heteroalkylene; -L 3 -, -B- and -L 4 When at least one of - is not a covalent bond and -B- is a covalent bond, -L 4 - is a covalent bond) is.

[0194] In one embodiment, -L 3 -and-B-and-L 4 -Not all bonds are covalent.

[0195] In one embodiment, the linker -L- is * -L 3 -GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N)C(O)—, —OC(O)—, and a maleimide-derived group; —R N is hydrogen or alkyl, -L A - is a covalent bond, alkylene, or heteroalkylene. is.

[0196] In one embodiment, -L 3 - is a C3 or C5 alkylene. In one embodiment, -G- is a maleimide-derived group.

[0197] In one embodiment, -L 3 - is a C3 or C5 alkylene and -G- is a maleimide-derived group.

[0198] In one embodiment, the linker -L- is * -L 3 -BL 5 -G- group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or heteroalkylene; -L 5 - is the formula * -(NR N C(O)-L 6 )-, the asterisk indicates the point of attachment to -B-, and -L 6 - is alkylene; G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)—, —OC(O)—, and maleimide-derived groups; -R N is hydrogen or alkyl) is.

[0199] In one embodiment, the linker comprises a maleimide-derived group:

[0200] [ka]

[0201] Includes.

[0202] A maleimide-derived group can be present at the end of the linker-L- as a -G- group, for example, for connection to an activator-A. Thus, the connection between the linker and the activator can be formed by reaction of a thiol group with a maleimide, typically when the activator has a thio functional group. For example, the activator can be a polypeptide with a cysteine ​​residue, where the sulfur atom of the activator is bonded to a carbon ring atom of the maleimide-derived group:

[0203] [ka]

[0204] Although the bond shown above is formed through a sulfur atom, the maleimide-derived group can also be attached through -O-, -Se-, and -NH-, such groups being derived, for example, from the side chain functional groups of appropriate amino acid residues (e.g., Ser, Se-Cys, and Lys, respectively).

[0205] The maleimide-derived group can be, for example, a heterocyclene in the linker -L- as the -B- group.

[0206] -L 1 - -L 1 The - group is selected from alkylene and heteroalkylene.

[0207] A heteroatom present in a heteroalkylene group cannot be attached to the -X- group, particularly if -X- is not a covalent bond. The heteroatoms present in the heteroalkylene group are -D 1 cannot bond to the group.

[0208] The heteroatoms present in the heteroalkylene group may be selected from -O-, -S-, -Se- or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O-, -S-, or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O- or -N(H)-. In one embodiment, the heteroatom present in the heteroalkylene group is —O—. In one embodiment, the heteroatom present in the heteroalkylene group is —N(H)—.

[0209] In one embodiment, -L 1 -But C 1~12 Alkylene, e.g., C 2~6 Alkylene, e.g., C 4~6 Alkylene or C 3~5 It is alkylene.

[0210] -L 2 - -L 2 The - group is selected from alkylene and heteroalkylene.

[0211] Any heteroatoms present in the heteroalkylene group cannot be attached to the nitrogen atom of the triazole. The heteroatoms present in the heteroalkylene group are -D 2 - group.

[0212] The heteroatoms present in the heteroalkylene group may be selected from -O-, -S-, -Se- or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O-, -S-, or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O- or -N(H)-. In one embodiment, the heteroatom present in the heteroalkylene group is —O—. In one embodiment, the heteroatom present in the heteroalkylene group is —N(H)—.

[0213] In one embodiment, -L 2 -But C 1~12 Alkylene, e.g., C 2~6 Alkylene, e.g., C 4~6 It is alkylene.

[0214] -L 3 - -L 3 The - group is selected from a covalent bond, alkylene, and heteroalkylene.

[0215] A heteroatom present in a heteroalkylene group cannot be attached to the -X- group, particularly if -X- is not a covalent bond.

[0216] The heteroatoms present in the heteroalkylene group may be selected from -O-, -S-, -Se- or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O-, -S-, or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O- or -N(H)-. In one embodiment, the heteroatom present in the heteroalkylene group is —O—. In one embodiment, the heteroatom present in the heteroalkylene group is —N(H)—.

[0217] In one embodiment, -L 3 -But C 1~12 Alkylene, e.g., C 2~6 Alkylene, e.g., C 4~6 It is alkylene.

[0218] -B- The -B- group is a covalent bond, arylene, heterocyclene, or cycloalkylene.

[0219] In one embodiment, -B- is a covalent bond.4 - is also a covalent bond. In one embodiment, -B- is arylene, heterocyclene, or cycloalkylene.

[0220] In one embodiment, -B- is arylene, for example, carboarylene or heteroarylene. In one embodiment, -B- is carboarylene, for example, phenylene. In one embodiment, -B- is heteroarylene, for example, triazolylene, for example, 1,2,3-triazolylene, such as 1,2,3-triazolyl-1,4-ene and 1,2,3-triazolyl-1,5-ene. In one embodiment, -B- is heterocyclene.

[0221] -L 4 - -L 4 The - group is a covalent bond, alkylene, or heteroalkylene.

[0222] The heteroatoms present in the heteroalkylene group are, in particular, -G-, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)- and -OC(O)-, they cannot be bonded to the -G- group.

[0223] The heteroatoms present in the heteroalkylene group may be selected from -O-, -S-, -Se- or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O-, -S-, or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O- or -N(H)-. In one embodiment, the heteroatom present in the heteroalkylene group is —O—. In one embodiment, the heteroatom present in the heteroalkylene group is —N(H)—.

[0224] -G- -G- group is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)—, —OC(O)—, and a maleimide-derived group; —R N is hydrogen or alkyl.

[0225] In one embodiment, -G- is a covalent bond. In one embodiment, -G- is -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)-, and maleimide-derived groups. In one embodiment, -G- is -O-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)-, and maleimide-derived groups. In one embodiment, -G- is -O-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)-, and maleimide-derived groups. In one embodiment, -G- is a covalent bond, -C(O)N(R N )-, -N(R N )C(O)— and maleimide-derived groups. In one embodiment, -G- is -C(O)N(R N )-, -N(R N )C(O)— and maleimide-derived groups. In one embodiment, -G- is a covalent bond, -C(O)N(R N )- and maleimide-derived groups. In one embodiment, -G- is -C(O)N(R N )- and maleimide-derived groups. In one embodiment, -G- is a maleimide-derived group.

[0226] -L A - -L A The - group is a covalent bond, alkylene, or heteroalkylene.

[0227] The heteroatoms present in the heteroalkylene group are, in particular, -G-, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)- and -OC(O)-, they cannot be bonded to the -G- group.

[0228] The heteroatoms present in the heteroalkylene group may be selected from -O-, -S-, -Se- or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O-, -S-, or -N(H)-. In one embodiment, the heteroatoms present in a heteroalkylene group can be selected from -O- or -N(H)-. In one embodiment, the heteroatom present in the heteroalkylene group is —O—. In one embodiment, the heteroatom present in the heteroalkylene group is —N(H)—.

[0229] In one embodiment, -L A - is a covalent bond. In one embodiment, -L A - is alkylene.

[0230] -L 5 - -L 5 -The group is of the formula * -(NR N C(O)-L 6 )-, the asterisk indicates the point of attachment to -B-, and -L 6 - is alkylene.

[0231] In one embodiment, -L 5 - is an expression *-(NHC(O)-L 6 )-. In one embodiment, -L 6 - is C 2~12 Alkylene, e.g., C 2~6 It is alkylene.

[0232] Triazole In some embodiments of the invention, the compounds contain triazole groups, particularly 1,2,3-triazoles, which may be present in the linker-L- or in precursor groups to the linker-L-.

[0233] When a 1,2,3-triazole is present, it is 1,4- or 1,5-substituted. In one embodiment, the 1,2,3-triazole is 1,4-substituted.

[0234] -D 1 -D 1 The group is -D 1 functional groups for forming covalent bonds with functionalized activators, including modified activators with functional groups for reaction with

[0235] Therefore, -D 1 -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , —N3, —C═CH2, —C═C(H)(Hal) and —C≡CH. In one embodiment, -D 1 -OH, -SH, -NH2, -NHR N , -COOH, -COH, -COOR D , —N3, —C═CH2, —C═C(H)(Hal) and —C≡CH.

[0236] In one embodiment, -D 1 -OH, -SH, -SeH, -NH2 and -NHR N is selected from. In one embodiment, -D 1 is selected from OH, —NH 2 , —COOH, —N 3 and —C≡CH. In one embodiment, -D 1 is selected from -NH2, -COOH, -N3 and -C≡CH.

[0237] -D 1 When - is -NH2, this group is suitable for forming amide, carbamate and carbamide bonds. -D 1 When is -COOH, this group is suitable for forming amide and ester bonds. -D 1 When is -N3 and -C≡CH, these groups are suitable for forming a linking 1,2,3-triazole group. -D 1 When is -C=C(H)(Hal), this group may be suitable for participating in cross-coupling reactions.

[0238] -D 2 -D 2 The group is -D 2 functional groups for forming covalent bonds with functionalized activators, including modified activators with functional groups for reaction with

[0239] Therefore, -D 2 -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR C and maleimidyl. In one embodiment, -D 2 -OH, -SH, -NH2, -NHR N , -COOH, -COH, -COOR C and maleimidyl.

[0240] In one embodiment, -D 2 -OH, -SH, -SeH, -NH2, -NHR N and maleimidyl.

[0241] In one embodiment, -D 2is selected from -OH, -NH2, -COOH and maleimidyl. In one embodiment, -D 2 is Maleimijir. In one embodiment, -D 2 is selected from —OH, —NH 2 and —COOH.

[0242] -T The -T group is a functional group for forming a covalent bond with a compound of formula (II), (III), or (IV), e.g., (III). Thus, -T can be a carboxylic acid group (e.g., present in (III)), or an -NH group (e.g., -D in a compound of formula (II)). 1 is -NH2, or in compounds of formula (IV), -D 2 is -NH2).

[0243] -T is -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , —N3, —C═CH2 and —C≡CH.

[0244] In one embodiment, -T is selected from the group consisting of -OH, -SH, -SeH, -NH2, and -NHR. N Such groups are suitable for reaction with carboxylic acid groups present in compound (III). In one embodiment, -T is selected from the group consisting of -OH, -NH and -NHR. N is selected from. In one embodiment, -T is -NH or -NHR N is.

[0245] In one embodiment, -T is selected from the group consisting of -COOH, -COH and -COOR. D Such groups are suitable for reaction with amino groups present in compounds (II) or (IV). In one embodiment, -T is -COOH.

[0246] In one embodiment, -T is -N3 or -C≡CH. Such groups are suitable for reaction with -C≡CH or -N3 present in compounds (II) or (IV), respectively.

[0247] -A The conjugate contains an active agent for delivery to a cell. The -A group is a group of the active agent that can formally (though not necessarily in practice) be derived from the active agent by removing a hydrogen group.

[0248] Active agent can be a biologically active agent, for example, a drug for use in treatment method.Active agent can be called small molecule drug, for example, when active agent is an organic small molecule and can have molecular weight of 1000Da or less, for example, 500Da or less.In some cases, active agent has molecular weight of 150Da or more, for example, 175Da or more, for example, 200Da or more.

[0249] An active agent can be biologically active even when not bound to the structure shown below:

[0250] [ka]

[0251] (In the formula, -R A , -R B , -R T1 , -R T2 , -R 1 , -R 2 , -R 3 and -D- is as defined above (i.e., the active agent is not conjugated to the pantothenic acid group).

[0252] The active agent may be a compound suitable for use in treating a microbial, eg, bacterial, infection. The active agent may be a compound suitable for use in treating a parasitic infection. The active agent may be a compound suitable for treating nematode or helminth, eg flatworm, infections. The active agent may be a compound suitable for treating a Mycobacterium infection, an Escherichia infection, a Staphylococcus infection or an Enterococcus infection. The active agent may be a compound suitable for treating a Plasmodium, Trypanosoma, Theileria or Babesia infection, and / or a Phytophthora, Crithidia or Lotmaria infection. The active agent may be a compound suitable for the treatment of a Caenorhabditis infection or a Haemonchus infection.

[0253] In the conjugates of the present invention, it is anticipated that the active agent is not also a derivative thereof with pantothenic acid. Thus, in one embodiment, -A is not a pantothenic acid group, and therefore, -A does not have the structure shown below:

[0254] [ka]

[0255] (In the formula, -R A , -R B , -R T1 , -R T2 , -R 1 , -R 2 , -R 3 and -D- is as defined above).

[0256] Thus, in one embodiment, the conjugate is not a dimer of pantothenic acid groups.

[0257] The -A group can be a dye, for example an organic dye. In one embodiment, the dye is a fluorescent dye. In one embodiment, -A is not a fluorescent dye.

[0258] The active agent may be or may include a polypeptide, eg, a protein. The active agent may be or may include a polynucleotide.

[0259] The active agent may be or may include a polysaccharide. Additionally, the polysaccharide may be a disaccharide or trisaccharide, or a polysaccharide having three or more sugar units. In one embodiment, the active agent may not be a disaccharide.

[0260] The conjugates of the invention are for use in delivering agents to a desired location, eg, a cell.

[0261] The drug is not particularly limited and can be any drug whose presence at a particular location is considered desirable.

[0262] The agent may be an active agent for use in a method of treatment or a method of diagnosis.

[0263] Generally, the activator has a functional group for forming a covalent bond with the linker. Thus, the activator may have a functional group such as -OH, -SH, -NH, -NHR. N , -COOH, -COH, -COOR C , -N3, -C=CH2, -C≡CH and maleimidyl.

[0264] For example, a thiol-containing (—SH) activator, such as a cysteine-containing polypeptide, can form a bond with a maleimide group on a linker precursor, such as compound (III) or (IV).

[0265] The active agent may be modified to incorporate specific functional groups for forming a covalent bond with the pantothenic acid group.

[0266] activity The conjugates of the invention may have activity against pathogens, such as bacteria and nematodes, where the conjugates of the invention typically comprise an active agent that has the requisite biological activity.

[0267] The compounds of the present invention may find use in the methods of treatment, for example, as described in more detail below.

[0268] In one embodiment, the conjugate may reduce parasitemia by at least 20%, at least 40%, at least 50%, at least 70, at least 80, or at least 90% compared to an untreated population or compared to a cell population treated with the active agent alone (i.e., the active agent is not conjugated to a pantothenic acid group). Parasitemia can be determined, for example, 48 hours or 72 hours after the first treatment of the parasite population.

[0269] The parasite may be a Plasmodium parasite, such as Plasmodium falciparum, or a Theileria parasite, such as Theileria annulata.

[0270] Additionally or alternatively, the parasite may be a parasite described below. The parasite may be an apicomplexan or a kinetoplastid. The parasite may be a Theileria parasite, such as T. annulata and T. parva, or a Phytophthora parasite, such as P. cinnamomi and P. agathidicida, or a Babesia parasite, such as B. bovis, or a Crithidia parasite, such as C. bombi, or a Rotomaria parasite, such as L. passim, or a Toxoplasma parasite, such as T. gondii. The parasite may also be a Plasmodium parasite, such as P. vivax, P. ovale, P. malaria, and P. knowlesi, or a Trypanosoma parasite, such as T. brucei. The parasite may also be a Plasmodium parasite, such as Plasmodium falciparum.

[0271] The conjugates of the present invention may be compounds with antimicrobial or antiparasitic activity.

[0272] In one embodiment, the conjugate may have antimicrobial activity as measured by an MIC of at most 150 μM, at most 100 μM, at most 50 μM, at most 25 μM, at most 10 μM, or at most 5 μM.

[0273] Additionally or alternatively, the conjugate may be a compound with antibacterial activity, for example against bacteria as described below. The bacterium may be a mycobacterium, such as Mycobacterium tuberculosis. The bacterium may be an Enterococcus bacterium, such as E. faecalis. The bacterium may be a coliform bacterium, such as E. coli, or a Staphylococcus bacterium, such as Staphylococcus aureus.

[0274] Antimicrobial or antiparasitic activity may be determined using the assays described herein.

[0275] In one embodiment, the conjugate has an LC of at most 10.0 μg / mL, at most 5.0 μg / mL, at most 2.0 μg / mL, at most 1.0 μg / mL, at most 0.5 μg / mL, or at most 0.1 μg / mL. 50 The compound may have anthelmintic activity as measured by Furthermore, the helminth may be a nematode or worm, such as a flatworm. The nematode or worm may be a Caenorhabditis nematode, such as C. elegans, or a Haemonchus nematode, such as Haemonchus contortus, or a Schistosoma flatworm, such as S. haematobium.

[0276] The active agent may have biological activity, and when used alone (i.e., the active agent is not conjugated to a pantothenic acid group), it may have the antiparasitic, antimicrobial, or antiparasitic activity described above for the conjugates. However, it is a feature of the present invention to provide conjugates for enhancing the biological activity of the active agent by ensuring that the active agent can be delivered to cells of a target organism. Thus, in embodiments of the present invention, conjugates containing an active agent have improved activity compared to the active agent used alone.

[0277] The conjugate of the present invention can have low toxicity. The toxicity of the conjugate measured relative to the survival rate percentage of human embryonic kidney cell lines treated with the conjugate can be, for example, 40% or less, for example, 30% or less, for example, 20% or less, for example, 10% or less. The compound can be used at a concentration of 100 μM.

[0278] Salts, solvates and other forms Examples of salts of the compounds of the present invention, such as the conjugates of formula (I), include all pharmaceutically acceptable salts, including, but not limited to, acid addition salts of strong mineral acids, such as HCl and HBr salts, and addition salts of strong organic acids, such as methanesulfonates. Further examples of salts include sulfates and acetates, such as trifluoroacetates or trichloroacetates.

[0279] Compounds of Formula (I) can also be formulated as prodrugs. Prodrugs can include antibacterial compounds described herein in which one or more amino groups are protected with a group that can be cleaved in vivo to release the biologically active compound. In one embodiment, the prodrug is an "amine prodrug." Examples of amine prodrugs include sulfomethyl, as described, for example, in Bergen et al., Antimicrob. Agents and Chemotherapy, 2006, 50, 1953, or HSO3-FMOC, as described, for example, in Schechter et al., J. Med Chem 2002, 45(19) 4264, and salts thereof. Further examples of amine prodrugs are provided by Krise and Oliyai, Biotechnology: Pharmaceutical Aspects, 2007, 5(2), 101-131.

[0280] In one embodiment, the compounds of formula (I) are provided as prodrugs.

[0281] A reference to a compound of the disclosure is also a reference to a solvate of that compound. Examples of solvates include hydrates.

[0282] The compounds of the present disclosure include compounds in which atoms are replaced by naturally occurring or non-naturally occurring isotopes. In one embodiment, the isotope is a stable isotope. Thus, the compounds described herein include, for example, deuterium-containing compounds. For example, H is 1 H, 2 H(D) and 3 H(T) can be any isotopic form; C can be 12 C. 13 C and 14 It can be any isotopic form including C; O is 16 O and 18 It can be any isotopic form containing O, etc.

[0283] Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including, but not limited to, cis- and trans-forms; E- and Z-forms; c, t, and r-forms; endo- and exo-forms; R, S, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-)-forms; keto, enol, and enolate forms; syn- and anti-forms; synclinal and anticlinal forms; α and β-forms; axial and equatorial forms; boat, chair, twist, envelope, and half-chair forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

[0284] It should be noted that, except as discussed below with respect to tautomeric forms, structural (or constitutional) isomers (i.e., isomers that differ not only in the position of their atoms in space but also in the connections between their atoms) are specifically excluded from the term "isomer" as used herein. For example, a reference to a methoxy group, -OCH3, is not considered a reference to the structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not considered a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may also include structural isomeric forms that fall within that class (e.g., C 1~6 Alkyl includes n-propyl and isopropyl; butyl includes n-, iso-, sec- and tert-butyl; methoxyphenyl includes ortho-, meta- and para-methoxyphenyl).

[0285] Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures thereof (e.g., racemic mixtures). Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by methods taught herein or by adapting known methods in a known manner.

[0286] The compounds described herein may be provided in a protected form, such that one or more functional groups within the compound may be provided with a protecting group to prevent unintended reaction, for example, during synthesis or storage.

[0287] It may be convenient or desirable to prepare, purify, and / or handle a compound in a chemically protected form. As used herein, the term "chemically protected form" refers to a compound in which one or more reactive functional groups are protected from undesired chemical reactions, i.e., in the form of a protected group or protecting group (also known as a masked group or a blocked group or a blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be carried out without affecting the protected group; the protecting group can usually be removed in a subsequent step without substantially affecting the remainder of the molecule. See, for example, "Protective Groups in Organic Synthesis" (T. Green and P. Wuts; 3rd Edition; John Wiley and Sons, 1999).

[0288] For example, amine groups can be protected as amides or urethanes, e.g., as methylamide (-NHCO-CH3); benzyloxyamide (-NHCO-OCH2C6H5, -NH-Cbz); t-butoxyamide (-NHCO-OC(CH3)3, -NH-Boc); 2-biphenyl-2-propoxyamide (-NHCO-OC(CH3)2C6H4C6H5, -NH-Bpoc), 9-fluorenylmethoxyamide (-NH-Fmoc), 6-nitroveratryloxyamide (-NH-Nvoc), 2-trimethylsilylethyloxyamide (-NH-Teoc), 2,2,2-trichloroethyloxyamide (-NH-Troc), allyloxyamide (-NH-Alloc), 2(-phenylsulfonyl)ethyloxyamide (-NH-Psec); or, where appropriate, as N-oxides (>NO·).

[0289] Carboxylic acid groups can be used as esters, e.g., C 1~7 Alkyl esters (e.g., methyl esters; t-butyl esters); C 1~7 Haloalkyl esters (e.g., C 1~7 Trihaloalkyl ester); Tri C 1~7 Alkylsilyl-C 1~7 Alkyl ester; or C 5~20 Aryl-C 1~7 It may be protected as an alkyl ester (eg, benzyl ester; nitrobenzyl ester); or as an amide, eg, as a methylamide.

[0290] Hydroxyl groups can be protected as ethers (-OR) or esters (-OC(=O)R), for example, as t-butyl ethers; benzyl, benzhydryl (diphenylmethyl) or trityl (triphenylmethyl) ethers; trimethylsilyl or t-butyldimethylsilyl ethers; or acetyl esters (-OC(=O)CH3, -OAc).

[0291] Compounds for use in the present invention include those having -R A and -R B Each of the hydroxyl groups may have a 1,3-diol functionality where each of the -R is hydrogen. Each of the hydroxyl groups may be independently protected as an ether or ester form. In the present invention, the diol may also be protected as an acetal. Thus, -R A and -R B together -C(R C1 )(R C2 )- to form a six-membered ring, and each -R C1 and -R C2 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl.

[0292] Aldehyde or ketone groups can be protected as acetals or ketals, respectively, in which the carbonyl group (>C=O) is converted to a diether (>C(OR)2), for example, by reaction with a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid. One aspect of the present invention relates to compounds in a substantially purified form and / or substantially free of contaminants.

[0293] In one embodiment the substantially purified form is at least 50% by weight, such as at least 60% by weight, for example at least 70% by weight, such as at least 80% by weight, for example at least 90% by weight, such as at least 95% by weight, for example at least 97% by weight, such as at least 98% by weight, for example at least 99% by weight.

[0294] Unless otherwise specified, a substantially pure form refers to any stereoisomeric or enantiomeric form of a compound. For example, in one embodiment, a substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, a substantially purified form refers to a single stereoisomer, e.g., an optically pure stereoisomer. In one embodiment, a substantially purified form refers to a mixture of enantiomers. In one embodiment, a substantially purified form refers to an equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, a substantially purified form refers to a single enantiomer, e.g., an optically pure enantiomer.

[0295] In one embodiment, the contaminants are 50% by weight or less, such as 40% by weight or less, for example 30% by weight or less, such as 20% by weight or less, for example 10% by weight or less, such as 5% by weight or less, for example 3% by weight or less, such as 2% by weight or less, for example 1% by weight or less.

[0296] Unless specified, contaminants refer to other compounds, i.e., other than stereoisomers or enantiomers. In one embodiment, contaminants refer to other compounds and other stereoisomers. In one embodiment, contaminants refer to other compounds and other enantiomers.

[0297] In one embodiment the substantially purified form is at least 60% optically pure (i.e. 60% of the compound on a molar basis is the desired stereoisomer or enantiomer and 40% is the undesired stereoisomer or enantiomer), such as at least 70% optically pure, such as at least 80% optically pure, such as at least 90% optically pure, for example at least 95% optically pure, such as at least 97% optically pure, for example at least 98% optically pure, such as at least 99% optically pure.

[0298] Methods for preparing conjugates The present invention also provides methods for preparing the conjugates of the present invention, including the conjugates of formula (I).

[0299] The examples herein describe methods for preparing conjugates, and these methods can be adapted to prepare other conjugates of formula (I).

[0300] Generally, the methods of the present invention use a pantothenic acid group, which is reacted with an active agent to form a conjugate. If the conjugate contains a linker, the linker can be attached to the pantothenic acid group or the active agent, and the linker can then form a connection between the two. For example, in some methods of the present invention, the pantothenic acid group is provided with a linker that terminates in a maleimide group. This maleimide group is reacted with an active agent, such as a polypeptide with a thiol functional group, to form a conjugate.

[0301] In other methods, both the pantothenic acid group and the active agent may contain a linker portion, and the completed linking group may be formed by reaction between the pantothenic acid group and the active agent. For example, in some methods of the present invention, the pantothenic acid group comprises a linker portion that terminates in an acetylene group. The active agent comprises a linker portion that terminates in an azide group. The acetylene group reacts with the azide group in a click chemistry-type reaction to form an imidazole, thereby generating a linker between the pantothenic acid group and the active agent.

[0302] The preparation of pantothenic acid derivatives has been described in the art. These methods can be adapted for use in the present invention.

[0303] The process of the present invention may also employ intermediate compounds of formula (II), (III) and (IV), and optionally compounds of formula (V).

[0304] In one aspect, there is provided a method of preparing a compound of formula (I), comprising reacting a compound of formula (II), (III) or (IV) with an activator, thereby obtaining a compound of formula (I).

[0305] Here, the activator has a functional group for reaction with a compound of formula (II), (III), or (IV). For example, a compound of formula (III) has a carboxylic acid at its terminus. This group can react with a hydroxyl, thiol, or amino functionality present on the activator to form a conjugate with an ester, thioester, or amide functionality.

[0306] In one aspect of the invention, there is provided a method of preparing a conjugate of the invention, comprising reacting a compound of formula (II) with a compound of formula (V).

[0307] Here, the active agent (V) and the pantothenic acid group (II) both comprise part of a linker, the linker parts comprising suitable functional groups to react together, thereby forming the complete linker.

[0308] The compound of formula (II)

[0309] [ka]

[0310] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 , -D- and -X- are as defined for compounds of formula (I); -L 1 - is alkylene or heteroalkylene; -D 1 -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , -N3, -C=CH2, -C=C(H)(Hal) and -C≡CH, -R N and -R D are each independently alkyl, and Hal is halogen. and salts, solvates, and protected forms thereof.

[0311] Thus, compounds of formula (II) are suitable for preparing conjugates of formula (I) in which -L- contains an alkylene or heteroalkylene group.

[0312] The compound of formula (II) is reacted with -D 1 The activators can be reacted directly with the activators described above, which are equipped with a functional group suitable for reaction with the group.

[0313] Alternatively, a compound of formula (II) can be reacted with an active agent, optionally equipped with a linker moiety, such as a compound of formula (V).

[0314] The compound of formula (V)

[0315] [ka]

[0316] where -A is an activator; -L 3 - is a covalent bond, alkylene or heteroalkylene; -T is -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D , -N3, -C=CH2 and -C≡CH; N and -R D are each independently selected from alkyl and salts, solvates and protected forms thereof.

[0317] Thus, the -T group corresponds to the -D group of the compound of formula (III) 1 The group is provided for reaction with the hydroxyl group.

[0318] The reaction of compound (II) with compound (V) produces an activator, or -T and -D 1 The present invention provides a conjugate of formula (I) having a linker containing an alkylene or heteroalkylene group connected to the alkylene or heteroalkylene group via a group formed from the reaction of

[0319] Alternatively, a compound of formula (III) can be reacted with an active agent, optionally equipped with a linker moiety, such as a compound of formula (V).

[0320] The compound of formula (III)

[0321] [ka]

[0322] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3and -D- is as defined for compounds of formula (I). and salts, solvates and protected forms thereof.

[0323] In the compound of formula (V), the -T group is a group suitable for reaction with a carboxylic acid. For example, -T can be -OH, -SH, -NH, or -NHR. N , for example -NH2 or -NHR N , for example -NH2.

[0324] Reaction of compound (III) with compound (V) provides a conjugate of formula (I) having a pantothenic acid group connected to an active agent or a pantothenic acid group connected to an alkylene or heteroalkylene group via a group formed from the reaction of -T with a carboxylic acid group.

[0325] A compound of formula (III) can be used to prepare a compound of formula (II).

[0326] Alternatively, a compound of formula (IV) can be reacted with an active agent, optionally equipped with a linker moiety, such as a compound of formula (V).

[0327] The compound of formula (IV)

[0328] [ka]

[0329] (In the formula, -R T1 , -R T2 , -R A , -R B , -R 1 , -R 2 , -R 3 , -D- and -X- are as defined for compounds of formula (I); -L 1 - is alkylene or heteroalkylene; -L 2 - is alkylene or heteroalkylene; -D2 - is -OH, -SH, -SeH, -NH2, -NHR N , -COOH, -COH, -COOR D and Maleimidyl) and salts, solvates and protected forms thereof.

[0330] The above method is based on the reaction of common functional groups to form standard bonds. Thus, the reaction of an amino functional group with a carboxyl functional group to yield an amide bond is contemplated. Similarly, the reaction of an acetylene functional group with an azide functional group to yield a triazole linking group is contemplated. The reaction conditions necessary to achieve this bond formation are well known to those skilled in the art, and exemplary conditions are provided in the examples in this case.

[0331] Treatment method The compounds of formula (I) or pharmaceutical formulations containing the compounds are suitable for use in methods of treatment and prevention. The compounds can be administered to a subject in need thereof.

[0332] The conjugates of the invention can be used in methods for treating parasites, e.g., microorganisms, including bacterial infections, protozoan infections, or helminth infections, e.g., nematodes or helminth infections, e.g., flatworm infections.

[0333] The conjugates of formula (I) are for use in methods of treatment of the human or animal body by therapy. In some aspects of the invention, compounds of formula (I) can be administered to a mammalian subject, e.g., a human, to treat a microbial infection, e.g., a bacterial infection.

[0334] Another aspect of the invention relates to the use of a compound of formula (I) in the manufacture of a medicament for use in the treatment of. In one embodiment, the medicament comprises a conjugate of formula (I). In one embodiment, the medicament is for use in the treatment of a microbial infection, for example a bacterial infection.

[0335] In one embodiment, the conjugate is suitable for use as an anthelmintic, and thus can be used to treat helminth infections, such as nematodes or helminth infections, such as schistosomiasis.

[0336] Additionally or alternatively, the conjugates can be used to treat Haemonchus infections, such as Haemonchus contortus infections. Thus, the conjugates can be used to treat Haemonchus disease. The subject to be treated can be a mammal, such as a sheep or goat.

[0337] Additionally or alternatively, the conjugates can be used to treat Schistosoma infections, such as Schistosoma haematobium infections. Thus, the conjugates can be used to treat schistosomiasis. The subject to be treated can be a mammal, such as a human.

[0338] In one embodiment, the conjugate used to treat a helminth infection has the structure Ie or II.

[0339] [ka]

[0340] In one embodiment, L is a linker as defined for compounds of formula (Id). 2 In a preferred embodiment, -L 1 - is C2 alkylene, and -L 2 - is a C3 alkylene.

[0341] In one embodiment, the linker -L- * -L 3 -GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is alkylene; -G- is a maleimide-derived group; -L A- is a covalent bond) is.

[0342] In a preferred embodiment, -L 3 - is a C3 alkylene.

[0343] In one embodiment, the conjugate is for use in treating an Escherichia infection, such as an E. coli infection, or a Staphylococcus infection, such as a Staphylococcus aureus infection.

[0344] Additionally or alternatively, the conjugate is for use in treating an Enterococcus infection, such as an E. faecalis infection.

[0345] In one embodiment, the conjugate is for use in treating an Apicomplexan infection.

[0346] For example, the conjugates can be used to treat Babesia infections, such as B. bovis. Thus, the conjugates can be used to treat babesiosis. The subject being treated can be a mammal, such as a bovine subject.

[0347] In one embodiment, the conjugate used to treat a Babesia infection has the structure Ie or Ii.

[0348] [ka]

[0349] In one embodiment, L is a linker as defined for compounds of formula (Id). 2 In a preferred embodiment, -L 1 - is C2 alkylene, and -L 2 - is a C3 alkylene.

[0350] In one embodiment, the linker -L- is * -L 3-GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is alkylene; -G- is a maleimide-derived group; -L A - is a covalent bond) is.

[0351] In a preferred embodiment, -L 3 - is a C5 alkylene.

[0352] The conjugates can be used to treat Theileria infections, such as T. annulata and T. parva. Thus, the conjugates can be used to treat tropical theileriosis and east coast fever. The subject to be treated can be a mammal, such as a bovine or ovine subject.

[0353] The conjugates can be used to treat Plasmodium infections, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malaria, and Plasmodium simianum. Thus, the conjugates can be used to treat malaria. The subject to be treated can be a mammal, for example, a human.

[0354] Additionally or alternatively, the conjugates can be used to treat Phytophthora infections, such as P. cinnamomi and P. agathidius. Thus, the conjugates can be used to treat kauri dieback. The subject to be treated can be a plant, for example, a tree.

[0355] The conjugates can be used to treat Toxoplasma infections, such as T. gondii. Thus, the conjugates can be used to treat toxoplasmosis. The subject to be treated can be a mammal, such as a human.

[0356] The conjugates can be used to treat kinetoplastid infections, such as trypanosoma infections, such as Trypanosoma brucei infections. Thus, the conjugates can be used to treat sleeping sickness.

[0357] The conjugates can be used to treat a Rotmalia infection, e.g., a Rotmalia passim infection. Additionally or alternatively, the conjugates can be used to treat a Crithidia infection, e.g., a C. bombi infection. Thus, the subject can be a bee, e.g., a bumblebee.

[0358] In one embodiment, the conjugate used to treat a Rotomaria infection has the structure If.

[0359] [ka]

[0360] The conjugates can be used to treat mycobacterial infections, such as Mycobacterium tuberculosis infections, and thus can be used to treat tuberculosis.

[0361] In one embodiment, the conjugate used to treat a mycobacterial infection has the structure If.

[0362] [ka]

[0363] The conjugate of formula (I) can be administered in conjunction with a second active agent. Administration can be simultaneous, separate or sequential.

[0364] The method and mode of administration will depend on the pharmacokinetics of the compound of the conjugate (I) and the second active agent.

[0365] By "concurrent" administration is meant that a compound of formula (I) and a second active agent are administered to a subject in a single dose by the same route of administration.

[0366] "Separate" administration means that the compound of Formula (I) and the second active agent are administered to a subject by two different routes of administration that occur simultaneously, as may occur, for example, when one agent is administered by infusion and the other is administered orally during the infusion.

[0367] "Sequential" means that two agents are administered at different times, provided that the activity of the first administered agent is present and ongoing in the subject when the second agent is administered.

[0368] Delivery method The conjugates of the invention can be used in methods of delivery, for example, methods of delivering active agents to an organism, including to cells of the organism.

[0369] The method of the invention comprises the step of exposing a conjugate of the invention, eg, a conjugate of formula (I), to an organism and allowing the conjugate to pass through the organism.

[0370] The organism may be a helminth, such as a nematode or helminth. The nematode or helminth may ingest the conjugate. The nematode or helminth may be a Caenorhabditis nematode, such as a Caenorhabditis elegans nematode, or a Haemonchus nematode, such as a Haemonchus contortus nematode, or a Schistosoma flatworm, such as a Schistosoma haematobium flatworm.

[0371] In a more preferred embodiment, the organism is a nematode or a helminth.

[0372] Additionally or alternatively, the organism may be a parasite. The parasite may ingest the conjugate. The parasite may be a Plasmodium parasite, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium simulans, or a Theileria parasite, such as T. annulata and T. parva, or a Phytophthora parasite, such as P. cinnamomi and P. agathidicida, or a Babesia parasite, such as B. bovis, or a Crithidia parasite, such as C. bombi, or a Rotomaria parasite, such as L. passim, or a Trypanosoma parasite, such as T. brucei, or a Toxoplasma parasite, such as T. gondii.

[0373] In another preferred embodiment, the organism is a parasite.

[0374] If the organism is a parasite, the parasite is preferably a Plasmodium parasite, such as Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium simulans, or a Theileria parasite, such as T. annulata and T. parva, or a Phytophthora parasite, such as P. cinnamomi and P. agathidicida, or a Babesia parasite, such as B. bovis, or a Crithidia parasite, such as C. bombyi, or a Rottomaria parasite, such as L. passim, or a Trypanosoma parasite, such as T. brucei, or a Toxoplasma parasite, such as T. gondii.

[0375] If the organism is a parasite, the parasite is more preferably a Theileria parasite, such as T. annulata and T. parva, or a Phytophthora parasite, such as P. cinnamomi and P. agathidicida, or a Babesia parasite, such as B. bovis, or a Crithidia parasite, such as C. bombyi, or a Rottomaria parasite, such as L. passim, or a Toxoplasma parasite, such as T. gondii.

[0376] The organism can be a microorganism, such as a bacterium. The microorganism, such as a bacterium, can ingest the conjugate. The microorganism, such as a bacterium, can be a Mycobacterium, such as Mycobacterium tuberculosis, or an Escherichia coli, such as E. coli, or a Staphylococcus, such as Staphylococcus aureus, or an Enterococcus, such as E. faecalis.

[0377] When the organism is a microorganism, such as a bacterium, the microorganism is preferably a Mycobacterium, such as Mycobacterium tuberculosis, or an Enterococcus, such as E. faecalis.

[0378] When the organism is a microorganism, for example a bacterium, the microorganism is more preferably a mycobacterium, for example a Mycobacterium tuberculosis bacterium.

[0379] The conjugate is capable of crossing the cell wall of the organism. Organisms can be unicellular or multicellular.

[0380] Delivery methods can be performed in vivo or ex vivo, thus the organism may be located inside a human, insect or animal, or the organism may be located outside a human, insect or animal.

[0381] treatment The term "treatment," as used herein in the context of treating a condition, generally relates to treatment and care of either humans, insects, or animals (e.g., veterinary uses), in which some desired therapeutic effect is achieved, e.g., inhibition of the progression of the condition, including slowing the rate of progression, halting the rate of progression, alleviating the symptoms of the condition, ameliorating the condition, and curing the condition. Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, the term "treatment" encompasses use in patients or subjects who have not yet developed the condition, but who are at risk of developing the condition.

[0382] The term "therapeutically effective amount," as used herein, relates to an amount of a compound, or a material, composition, or dosage form containing a compound, that is effective to produce some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio, when administered in accordance with a desired treatment regimen.

[0383] The term "treatment" includes combination treatments and therapies described herein, in which two or more treatments or therapies are combined, for example sequentially or simultaneously.

[0384] formulation In one aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) together with a pharmaceutically acceptable excipient, e.g., a carrier. The pharmaceutical composition may further comprise a second active agent. In an alternative embodiment, when a second agent is provided for use in therapy, the second agent may be formulated separately from the compound of formula (I). Accordingly, the following comments made with respect to the compound of formula (I) may also apply to a separately formulated second agent.

[0385] Although the compounds of formula (I) can be administered alone or together with a second agent, it is preferable to provide them as pharmaceutical formulations (e.g., compositions, preparations, medicaments) comprising at least one compound of formula (I) described herein together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweeteners. The formulations may further comprise other active agents, such as other therapeutic or prophylactic agents.

[0386] Thus, the present invention further provides pharmaceutical compositions as defined above, and methods for preparing pharmaceutical compositions comprising the step of admixing at least one compound of formula (I) described herein with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, such as carriers, diluents, excipients, etc. When formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound. The composition optionally further comprises a predetermined amount of a second active agent.

[0387] As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of the subject (e.g., human) in question without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0388] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical textbooks, such as Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th ed., 2005.

[0389] The formulations can be prepared by any method well known in the art of pharmacy. Such methods include the step of bringing into association a compound of formula (I) with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with the carrier (e.g., liquid carriers, finely divided solid carrier, etc.), and then, if necessary, shaping the product.

[0390] The formulations may be prepared to provide fast or slow release; immediate, delayed, timed or sustained release; or a combination thereof.

[0391] Formulations may suitably be in the form of a liquid, solution (e.g., aqueous, non-aqueous), suspension (e.g., aqueous, non-aqueous), emulsion (e.g., oil-in-water, water-in-oil), elixir, syrup, electuary, mouthwash, drops, tablets (including, e.g., coated tablets), granules, powders, lozenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mist or aerosols.

[0392] Dosage In general, the methods of the present invention can include the step of administering to a subject an effective amount of a compound of formula (I) to produce a biological effect.

[0393] Those skilled in the art will appreciate that appropriate dosages of compounds of Formula (I) and compositions comprising compounds of Formula (I) may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against risk or adverse side effects. The selected dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound of Formula (I), the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, general health, and previous medical history. The amount and route of administration of the compound of Formula (I) are ultimately at the discretion of the physician, veterinarian, beekeeper, or clinician, but generally, the dosage will be selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or deleterious side effects.

[0394] Administration can be carried out in a single dose, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective means and dosage of administration are well known to those skilled in the art and will vary depending on the formulation used for therapy, the purpose of the therapy, the target cells being treated, and the subject being treated. Single or multiple administrations can be carried out at any dose level, with the pattern being selected by the treating physician, veterinarian, or clinician.

[0395] In general, suitable doses of a compound of formula (I) range from about 10 μg to about 250 mg (more typically about 100 μg to about 25 mg) per kilogram of subject body weight per day. Where the compound of formula (I) is a salt, prodrug, or the like, the amount administered is calculated based on the parent compound, and therefore the actual mass used is increased proportionately.

[0396] Route of administration The compounds of formula (I), or pharmaceutical compositions comprising compounds of formula (I), may be administered to a subject by any convenient route of administration, whether systemically / peripherally or locally (i.e., to the desired site of action).

[0397] Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (e.g., by patch, plaster, etc.); intranasal (e.g., by nose drops); ocular (e.g., by eye drops); pulmonary (e.g., via aerosol, e.g., through the mouth or nose, e.g., by inhalation or insufflation therapy); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, e.g., by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; e.g., by implantation of a subcutaneous or intramuscular depot or reservoir.

[0398] Subjects / patients The subject / patient can be a chordate, vertebrate, mammal, placental mammal, marsupial (e.g., kangaroo, wombat), rodent (e.g., guinea pig, hamster, rat, mouse), murine (mouse), lagomorph (e.g., rabbit), avian (e.g., bird), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), porcine (e.g., pig), ovine (ovine), bovine (e.g., cow), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), insect (e.g., bee, bumblebee), or human. Additionally or alternatively, the subject / patient can be a caprine (e.g., goat).

[0399] Furthermore, the subject / patient may be in any of its forms of development, for example, a fetus. The inventors have found that the conjugates of the present invention cannot be taken up by mammalian cells, including rapidly dividing mammalian cells, for example, cells within a developing fetus.

[0400] Thus, the conjugates of the invention can be used to treat pregnant subjects and subjects contemplating pregnancy.

[0401] In one preferred embodiment, the subject / patient is a human.

[0402] It is also envisioned that the present invention may be practiced in non-human animals having microbial infections. The non-human mammal may be a rodent. Rodents include rats, mice, guinea pigs, chinchillas, and other similarly sized small rodents used in laboratory research.

[0403] Other Preferences Each and every compatible combination of the above embodiments is expressly disclosed herein as if each and every combination were individually and expressly recited.

[0404] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0405] "And / or," as used herein, is considered a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0406] The term "comprising" as used in this specification and claims means "consisting at least in part of." When interpreting statements in this specification and claims that include the term "comprising," other features may be present in addition to the feature that precedes the term in each statement. Related terms such as "comprise" and "comprises" should be interpreted similarly.

[0407] Where reference is made herein to external documents or other sources, this is generally for the purpose of providing a context for discussing features of the present invention. Unless specifically stated, reference to such external documents should not be construed as an admission that such documents or such sources are prior art or form part of the general knowledge in the art in any jurisdiction.

[0408] Unless the context dictates otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the invention but apply equally to all aspects and embodiments described. Where technically appropriate, embodiments can be combined, and thus the present disclosure covers all permutations and combinations of the embodiments provided herein.

[0409] Certain aspects and embodiments of the present invention will now be illustrated by way of example and with reference to the above-mentioned drawings. [Example]

[0410] The following examples are provided merely to illustrate the present invention and are not intended to limit the scope of the invention described herein.

[0411] General synthesis method Reactions involving air-sensitive reagents and anhydrous solvents were carried out in oven-dried glassware (130 °C). These reactions were carried out with the exclusion of air using an argon atmosphere. Acetonitrile, dichloromethane, diethyl ether, tetrahydrofuran, and toluene were purified through a Pure Solv 400-5MD solvent purification system (Innovative Technology, Inc.). All reagents were used as received unless otherwise stated. Solvents were evaporated under reduced pressure at 40 °C using a Buchi Rotavapor.

[0412] Microwave reactions were carried out using a Biotage Initiator system.

[0413] Column chromatography was performed under pressure using silica gel (FluoroChem Silica LC 60A) as the stationary phase and HPLC grade solvents as eluents. Reactions were monitored by thin layer chromatography. TLC was performed using silica gel (Merck or FluoroChem Silica 60 F). 254 The plate was then subjected to UV fluorescence (λ max The cells were visualized by extinction at 254 nm and / or by staining with KMnO4 solution or acidic ethanolic anisaldehyde immersion.

[0414] Proton magnetic resonance spectrum ( 1 H NMR) and carbon magnetic resonance spectrum ( 13C NMR was recorded at 400 MHz and 100 MHz, or 500 MHz and 125 MHz, using either a Bruker DPX Avance 400 instrument or a Bruker Avance III 500 instrument. Chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the residual solvent peak. The order of citation in parentheses is (i) the number of equivalent nuclei (by integration), (ii) the multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad, dm = double multiplet, or a combination of these terms), and (iii) the coupling constant (J) in hertz to the nearest 0.1 Hz.

[0415] IR spectra were obtained using a Golden Gate™ attachment (Shimadzu FTIR-8400) that uses a type IIa diamond as the single reflecting element so that the IR spectra of compounds (solid or liquid) can be detected directly (thin film) without any sample preparation. Only significant absorptions are reported in wavenumbers.

[0416] UV-Vis absorption spectra were recorded using a Shimadzu UV-3600 UV-Vis-NIR spectrophotometer. Brand® UV-Cuvette UV-Transparent spectrophotometric plastic cuvettes with a 10 mm path length and a 3 mL volume were used. Fluorescence emission spectra were recorded using a Shimadzu RF-5301 PC fluorescence spectrophotometer and Panorama Fluorescence 1.1 software.

[0417] High resolution mass spectra were recorded by the analytical group in the Chemistry Department at the University of Glasgow on a JEOL JMS-700 mass spectrometer with electrospray and chemical ionization or on a Bruker micro TOFq mass spectrometer with electrospray ionization.

[0418] Compound synthesis - intermediates (E)-2-(Trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate and (Z)-2-(Trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate

[0419] [ka]

[0420] 2-(Trimethylsilyl)ethyl-2-(triphenylphosphoranylidene)acetate (0.84 g, 3.9 mmol) was dissolved in benzene (60 mL) and N-formyl-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (R or S form) (0.84 g, 3.9 mmol) was added. The solution was heated to 80 °C for 18 h and then allowed to cool to room temperature. The solvent was removed in vacuo to give the crude product as a yellow oil. The crude product was purified using column chromatography (10–20% EtOAc / petroleum ether) to give the enamide product as a separable mixture of cis (0.46 g, 33%) and trans (0.75 g, 54%) isomers as a yellow oil and a white solid, respectively. NMR data obtained for this compound are consistent with literature data for this compound.

[0421] See also Sewell et al.

[0422] (E) type: 1H NMR (CDCl3, 500 MHz) δ: 8.38 (1H, d, J = 11.8 Hz), 7.97 (1H, dd, J = 14.2, 11.8 Hz), 5.59 (1H, d, J = 14.2 Hz), 4.24-4.18 (2H, m), 4.20 (1H, s), 3.72 (1H, d, J = 11.8 Hz), 3.32 (1H, d, J = 11.8 Hz), 1.52 (3H, s), 1.46 (3H, s), 1.06 (3H, s), 1.02 (2H, t, J = 8.4 Hz), 1.01 (3H, s), 0.05 (9H, s); 13 IR ν max (フィルム) / cm -1 3295, 2957, 2897, 1688, 1638, 1476; HRMS (Cl) C 17 H 32 O5NSi (M+H)+ calculated value: m / z 358.2050, measured value m / z 358.2052; melting point 126-127℃. R type: [α] D +58.9 (c=1.1, CHCl3, T=22.5℃). S-type: [α] D -56.7 (c=0.6, CHCl3, T=21.6℃).

[0423] (Z) Type: 1H NMR (CDCl3, 500 MHz) δ: 11.08 (1H, d, J = 11.6 Hz), 7.40 (1H, dd, J = 11.6, 9.0 Hz), 5.15 (1H, d, J = 8.9 Hz), 4.26-4.20 (2H, m), 4.21 (1H, s), 3.72 (1H, d, J = 11.6 Hz), 3.32 (1H, d, J = 11.6 Hz), 1.59 (3H, s), 1.47 (3H, s), 1.05 (3H, s), 1.04 (3H, s), 1.02 (2H, m), 0.05 (9H, s); 13 IR ν max (film) / cm -1 3331, 2959, 2858,1680, 1628, 1478; HRMS (Cl)C 17 H 32 Calculated for O5NSi (M+H)+: m / z 358.2050, found m / z 358.2054. R type: [α] D +40.5(c=1.0, CHCl3, T=22.5℃). S type: [α] D -38.0(c=0.8, CHCl3, T=22.5℃).

[0424] (E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid

[0425] [ka]

[0426] This compound is also known from Sewell et al. (see compound 11).

[0427] (E)-2-(Trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (R- or S-form) (110 mg, 0.310 mmol) was dissolved in THF (20 mL) and cooled to 0 °C. TBAF (0.370 mL of a 1 M solution in THF, 0.370 mmol) was added. The resulting pale yellow solution was stirred for 16 h while warming to room temperature, and then the reaction mixture was concentrated in vacuo. The crude residue was purified by flash column chromatography (0–2% MeOH / CHCl) to give the acid product as a mixture with TBAF impurity. The crude product was dissolved in EtOAc (10 mL) and water (10 mL), and 1 M aqueous NaOH was added until the solution reached pH 12. The resulting solution was stirred at room temperature for 1 h, after which the aqueous layer was collected, diluted with EtOAc (15 mL), and 1 M HCl was added until the pH reached 5. After the mixture was stirred at room temperature for an additional 1 h, the layers were separated and the aqueous phase was extracted with EtOAc (2 x 10 mL). The combined organic phases were washed with brine (20 mL), dried (NaSO), filtered, and the solvent was removed in vacuo to give the acid product (64 mg, 79%) as a white solid.

[0428] 1 H NMR (CD3OD, 400 MHz) δ: 7.95 (1H, d, J = 14.3 Hz), 5.86 (1H, J = 14.3 Hz), 4.31 (1H, s), 3.80 (1H, d, J = 11.6 Hz), 3.32 (1H, d, J = 11.6 Hz), 1.53 (3H, s), 1.45 (3H, s), 1.04 (3H, s), 1.03 (3H, s); 13 C NMR (CD3OD, 100 MHz) δ: 171.5, 170.9, 138.5, 104.0, 100.8, 78.6, 72.2, 34.3, 29.5, 22.1, 19.3, 19.0; max (film) / cm -1 3318, 3094, 2963, 2878, 1670, 1636; HRMS (Cl)C 12 H 20Calculated for ON (M+H): m / z 258.1341, found m / z 258.1346; melting point 185-186 °C. R type: [α] D +81.3(c=0.5, CHCl3, T=22.7℃). S type: [α] D -77.6(c=1.2, CHCl3, T=22.8℃).

[0429] (Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid

[0430] [ka]

[0431] (Z)-2-(Trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (R or S form) (310 mg, 0.868 mmol) was dissolved in a mixture of THF (6 mL) and water (0.150 mL) and then cooled to 0 °C. TBAF (1.30 mL of a 1 M solution in THF, 1.30 mmol) was added via syringe at 0 °C and then allowed to stir at room temperature for 16 h. The solvent was then removed in vacuo to give the crude product as a pale yellow oil. The crude residue was purified by flash column chromatography (0–2% MeOH / CHCl) to give the desired acid product (178 mg, 80%) as a colorless oil.

[0432] 1 H NMR (CDCl3, 500 MHz) δ: 11.25, (1H, d, J = 11.8 Hz), 7.54 (1H, dd, J = 11.8, 8.8 Hz), 5.22 (1H, d, J = 8.9 Hz), 4.21 (1H, s), 3.73 (1H, d, J = 11.7 Hz), 3.33 (1H, d, J = 11.7 Hz), 1.53 (3H, s), 1.46 (3H, s), 1.05 (3H, s), 1.03 (3H, s);13 C NMR (CDCl3, 125 MHz) δ: 173.6, 168.8, 138.6, 99.3, 96.6, 77.2, 71.2, 33.2, 29.2, 21.8, 19.0, 18.6; IR ν max (film) / cm -1 3302, 2993, 2940, 2870, 1678, 1601; HRMS (Cl)C 12 H 20 Calculated for O5N (M+H)+: m / z 258.1341, found m / z 258.1339. R type: [α] D +51.0(c=0.5, CHCl3, T=22.7℃). S type: [α] D -47.1(c=0.8, CHCl3, T=21.7℃).

[0433] (Z)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)acrylic acid

[0434] [ka]

[0435] (Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (R- or S-form) (40 mg, 0.160 mmol) was treated with BiCl (5 mg, 16 μmol) in MeCN (1 mL) and HO (50 μL). The crude residue was purified by flash column chromatography (0–5% MeOH / CHCl) to give the diol product (9 mg, 26%) as a colorless oil.

[0436] 1H NMR (CDCl3, 500 MHz) δ: 7.43 (1H, d, J = 8.9 Hz), 5.17 (1H, d, J = 8.9 Hz), 4.05 (1H, s), 3.49 (1H, d, J = 10.9 Hz), 3.41 (1H, d, J = 10.9 Hz), 0.94 (3H, s), 0.93 (3H, s); 13 C NMR (CDCl3, 125 MHz) δ: 174.6, 171.4, 137.3, 98.8, 76.8, 69.8, 40.8, 21.3, 20.5; IR ν max (film) / cm -1 3306, 2967, 2940, 2832, 1670; HRMS (Cl) C9H 15 Calculated for O5N (M+H)+: m / z 218.1028, found m / z 218.1029. The R type is known. S type: [α] D -30.8(c=1.4, MeOH, T=25.5℃).

[0437] (E)-N-(3-(3-hydroxypropylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0438] [ka]

[0439] (E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (50 mg, 0.195 mmol) was dissolved in CHCl (0.5 mL), and the resulting solution was treated with HBTU (110 mg, 0.290 mmol), 3-amino-propan-1-ol (22 μL, 0.290 mmol), and DIPEA (51 μL, 0.290 mmol). The resulting solution was heated at 80 °C under microwave conditions for 2.5 h, after which the solvent was removed in vacuo to give the crude product. The crude residue was purified by flash column chromatography (0–5% MeOH / EtOAc) to give the pantothenic acid product (20 mg, 33%) as a white solid.

[0440] 1 H NMR (CDCl3, 500 MHz) δ: 8.35 (1H, d, J = 10.8 Hz), 7.80 (1H, dd, J = 13.8, 10.8, Hz), 5.98 (1H, t, J = 6.0 Hz), 5.86 (1H, d, J = 13.8 Hz), 4.20 (1H, s), 3.73 (1H, d, J = 11.7 Hz), 3.66-3.63 (2H, m), 3.53-3.48 (3H, m), 3.33 (1H, d, J = 11.7 Hz), 1.74-1.69 (2H, m), 1.52 (3H, s), 1.47 (3H, s), 1.06 (3H, s), 1.01 (3H, s); 13 IR ν max (film) / cm -1 3275, 2998, 2953, 2876, 1705, 1659; HRMS (Cl)C 15 H 27 Calculated for O5N2(M+H)+: m / z 315.1920, found m / z 315.1917; mp 165-166°C.

[0441] (Z)-N-(3-(3-hydroxypropylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0442] [ka]

[0443] (l)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (50 mg, 0.195 mmol) was treated with BTFFH (92 mg, 0.290 mmol), 3-amino-propan-1-ol (22 μL, 0.290 mmol), and DIPEA (51 μL, 0.290 mmol) according to the procedure for the synthesis of the corresponding (E)-form described above. The crude residue was purified by flash column chromatography (0–5% MeOH / EtOAc) to give the pantothenic acid product (31 mg, 57%) as a white solid.

[0444] 1 H NMR ((CD3)2CO, 500 MHz) δ: 11.91 (1H, d, J = 11.1 Hz), 7.38 (1H, br s), 7.24 (1H, dd, J = 11.1, 8.9 Hz), 5.25 (1H, d, J = 8.9 Hz), 4.28 (1H, s), 3.87 (1H, br s), 3.81 (1H, d, J = 11.6 Hz), 3.58 (2H, t, J = 6.0 Hz), 3.41-3.35 (2H, m), 3.31 (1H, d, J = 11.6 Hz), 1.70-1.65 (2H, m), 1.60 (3H, s), 1.50 (3H, s), 1.04 (3H, s), 1.00 (3H, s); 13C NMR ((CD3)2CO), 125 MHz) δ: 169.3, 169.0, 133.3, 101.4, 99.7, 77.7, 71.6, 59.5, 36.3, 33.7, 33.5, 29.5, 22.0, 19.3, 19.0; IRμ max (film) / cm -1 3243, 2974, 2930, 2842, 1672, 1612; HRMS (Cl)C 15 H 26 Calculated for O5N2(M+H)+: m / z 315.1918, found m / z 315.1920; mp 80-81°C.

[0445] (E)-N-(3-(5-hydroxypentylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0446] [ka]

[0447] (E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide)acrylic acid (50 mg, 0.195 mmol) was treated with HBTU (110 mg, 0.290 mmol), 5-amino-pentan-1-ol (100 mg, 0.971 mmol), and DIPEA (51 μL, 0.290 mmol) according to the procedure for the synthesis of (E)-N-(3-(3-hydroxypropylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide. The crude residue was purified by flash column chromatography (0–5% MeOH / EtOAc) to give the pantothenic acid product (12 mg, 18%) as a colorless oil.

[0448] 1H NMR (CDCl3, 400 MHz) δ: 8.31 (1H, d, J = 11.0 Hz), 7.78 (1H, dd, J = 13.9, 11.0 Hz), 5.85 (1H, d, J = 13.9 Hz), 5.61 (1H, t, J = 5.4 Hz), 4.21 (1H, s), 3.74 (1H, d, J = 11.8 Hz), 3.67 (2H, t, J = 6.0 Hz), 3.39-3.34 (2H, m), 3.34 (1H, d, J = 11.8 Hz), 1.78 (1H, br s), 1.65-1.55 (4H, m), 1.53 (3H, s), 1.48 (3H, s), 1.48-1.41 (2H, m), 1.07 (3H, s), 1.02 (3H, s); 13 C NMR (CDCl3, 125 MHz) δ: 168.2, 166.3, 132.8, 106.3, 99.5, 77.2, 71.3, 62.6, 39.4, 33.4, 32.2, 29.5, 29.4, 23.0, 21.9, 18.8, 18.7; IR ν max (film) / cm -1 3264, 2991, 2934, 2868, 1701, 1661; HRMS (Cl)C 17 H 31 Calculated for O5N2(M+H)+: m / z 343.2238, found m / z 343.2233.

[0449] (Z)-N-(3-(5-hydroxypentylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0450] [ka]

[0451] (Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide)acrylic acid (50 mg, 0.195 mmol) was treated with BTFFH (92 mg, 0.290 mmol), 5-amino-pentan-1-ol (30 mg, 0.290 mmol), and DIPEA (51 μL, 0.290 mmol) according to the procedure for the synthesis of (E)-N-(3-(3-hydroxypropylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide. The crude residue was purified by flash column chromatography (0-5% MeOH / EtOAc) to give the pantothenic acid product (27 mg, 40%) as a colorless oil.

[0452] 1 H NMR ((CD3)2CO, 400 MHz) δ: 11.92 (1H, d, J = 11.0 Hz), 7.27 (1H, br s), 7.22 (1H, dd, J = 11.0, 8.9 Hz), 5.24 (1H, d, J = 8.9 Hz), 4.27 (1H, s), 3.80 (1H, d, J = 11.8 Hz), 3.58-3.54 (2H, m), 3.29-3.24 (2H, m), 3.31 (1H, d, J = 11.8 Hz), 1.78 (1H, br s), 1.58-1.50 (4H, m), 1.52 (3H, s), 1.50 (3H, s), 1.46-1.40 (2H, m), 1.04 (3H, s), 1.00 (3H, s); 13 C NMR ((CD3)2CO), 100 MHz) δ: 169.0, 168.6, 133.0, 101.8, 99.8, 77.8, 71.6, 62.3, 39.4, 33.7, 33.4, 30.2, 29.9, 24.1, 22.1, 19.4, 19.0; IR ν max (film) / cm -1 3293, 2992, 2937, 2868, 1652, 1609; HRMS (Cl)C 17 H 31Calculated for O5N2(M+H)+: m / z 343.2230, found m / z 343.2233.

[0453] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0454] [ka]

[0455] (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide)acrylic acid (100 mg, 0.389 mmol) was treated with BTFFH (184 mg, 0.584 mmol), 1-amino-3-butyne (46 μL, 0.584 mmol), and DIPEA (102 μL, 0.584 mmol) according to the procedure for the synthesis of (E)-N-(3-(3-hydroxypropylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide. The crude residue was purified by flash column chromatography (50-60% EtOAc / petroleum ether) to give the pantothenic acid product (118 mg, 99%) as a white solid.

[0456] 1H NMR ((CD3)2CO, 500 MHz) δ: 9.35 (1H, d, J = 11.1 Hz), 7.83 (1H, dd, J = 13.9, 11.1 Hz), 7.18 (1H, br s), 5.95 (1H, d, J = 13.9 Hz), 4.28 (1H, s), 3.78 (1H, d, J = 11.4 Hz), 3.40-3.36 (2H, m), 3.29 (1H, d, J = 10.6 Hz), 2.39 (2H, td, J = 6.9, 2.6 Hz), 2.36 (1H, t, J = 2.6 Hz), 1.47 (3H, s), 1.40 (3H, s), 1.03 (3H, s), 1.00 (3H, s). 13 C NMR ((CD3)2CO), 125 MHz) δ: 169.3, 166.9, 133.8, 106.7, 100.0, 82.7, 77.9, 71.7, 70.7, 39.1, 33.9, 29.9, 22.1, 19.9, 19.2, 19.0; IR ν max (film) / cm -1 3309, 3287, 2996, 2944, 2889, 2871, 1657, 1616; HMRS (El) C 16 H 24 Calculated for O4N2M+: m / z 308.1736, found m / z 308.1739; mp 153-154 °C; [α]D +73.9 (c = 0.5, (CH3)2CO, T = 30.0 °C).

[0457] The (S,E) form can be prepared similarly from (S,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid. [α] D -65.5(c=1.1, (CH3)2CO, T=24.4℃).

[0458] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0459] [ka]

[0460] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (110 mg, 0.357 mmol) was dissolved in a mixture of MeCN (1 mL) and HO (50 μL). BiCl (11 mg, 0.035 mmol) was then added, resulting in a milky suspension, which was stirred at room temperature for 18 h. The reaction mixture was quenched with a few drops of saturated aqueous NaHCO and subsequently filtered through a Celite® pad, which was then washed with EtOAc (10 mL). The eluent was concentrated in vacuo to give the crude product. The crude residue was purified by flash column chromatography (5–7% MeOH / CHCl) to give the diol product (49 mg, 51%) as a white solid.

[0461] 1 H NMR ((CD3)2CO, 400 MHz) δ: 9.55 (1H, d, J = 11.3 Hz), 7.87 (1H, dd, J = 14.0, 11.3 Hz), 7.27 (1H, br s), 6.00 (1H, d, J = 14.0 Hz), 4.09 (1H, d, J = 5.0 Hz), 4.03 (1H, t, J = 5.5 Hz), 3.56 (1H, br s), 3.49 (1H, dd, J = 10.5, 5.5 Hz), 3.43 (1H, dd, J = 10.6, 5.5 Hz), 3.41-3.35 (2H, m), 2.38 (2H, td, J = 6.9, 2.6 Hz), 2.37-2.35 (1H, m), 0.94 (3H, s), 0.93 (3H, s); 13IR ν max (film) / cm -1 3368, 3309, 3247, 2965, 2920, 2861, 1691, 1652; HMRS (El) C 13 H 19 Calculated for O4N2(MH)+: m / z 267.1350, found m / z 267.1344; mp 171-172°C; [α] D +78.1 (c = 1.1, (CH3)2CO, T = 30.0℃).

[0462] (Z)-N-(3-(but-3-yn-1-ylamino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0463] [ka]

[0464] (Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (R- or S-form) (125 mg, 0.49 mmol) was dissolved in CHCl (1 mL) in a 0.5-2 mL microwave vial equipped with a magnetic stir bar. BTFFH (233 mg, 0.74 mmol) was then added to DIPEA (150 μL, 0.74 mmol) and 1-amino-3-butyne (61 μL, 0.74 mmol) via micropipette. The vial was capped and heated to 80 °C under microwave conditions for 2.5 h. The solvent was then removed in vacuo to give the crude product as a pale yellow oil. This was purified by flash column chromatography (10-25% EtOAc / petroleum ether) to give the alkyne product as a white solid (110 mg, 73%).

[0465] 1 H NMR (CDCl3, 400 MHz) δ: 11.66 (1H, d, J = 10.9 Hz), 7.31 (1H, dd, J = 10.9, 8.9 Hz), 5.74 (1H, br s), 5.03 (1H, d, J = 8.9 Hz), 4.20 (1H, s), 3.72 (1H, d, J = 11.6 Hz), 3.48 (2H, m), 3.32 (1H, d, J = 11.6 Hz), 2.44 (2H, dt, J = 6.5, 2.6 Hz), 2.03 (1H, t, J = 2.6 Hz), 1.61 (3H, s), 1.47 (3H, s), 1.05 (6H, s); 13 C NMR (CDCl3, 100 MHz) δ: 168.9, 167.7, 133.7, 100.2, 99.3, 81.5, 77.3, 71.4, 70.2, 37.6, 33.3, 29.4, 21.9, 19.5, 19.1, 18.7; IR ν max (film) / cm -1 (undiluted): 3294, 2955, 1656, 1467, 1249; HRMS (ESI) C 16 H 23 Calculated m / z for N2O4(MH) 307.1663, found m / z 307.1658; mp 122-125°C. R type: [α] D +20.6(c=1.0, (CH3)2CO, T=24.4℃). S type: [α] D -23.1(c=1.4, (CH3)2CO, T=24.4℃).

[0466] (Z)-2-Methyl-3-[((R)-2,2,5,5-tetramethyl-[1,3]dioxane-4-carbonyl)-amino]-acrylic acid ethyl ester and (E)-2-methyl-3-[((R)-2,2,5,5-tetramethyl-[1,3]dioxane-4-carbonyl)-amino]-acrylic acid ethyl ester

[0467] [ka]

[0468] The formyl compound 9 (186 mg) described by Sewell et al. was added to a solution of 1-carbethoxyethylidene triphenylphosphorane in benzene to give the enamide product (175 mg, 65%) as a mixture of E and Z isomers, with an E:Z ratio of 3:1. In a general procedure, a solution of the formyl compound (1.0 mmol) in benzene (10 mL) was treated with ethoxycarbonylmethylenetriphenylphosphorane (3.0 mmol), and the resulting mixture was heated to 95 °C for 19 h. Upon completion of the reaction as indicated by TLC analysis, the solvent was removed under vacuum. The crude residue was then purified by flash column chromatography (silica gel, 10%–30% EtOAc in 40–60 petroleum ether) to give the desired enamide.

[0469] Z type: 1 H NMR (400MHz, CDCl3) δ: 10.90 (1H, bd, J = 12.4 Hz), 7.26 (1H, dd, J = 11.5, 1.3 Hz), 4.18 (2H, qd, J = 7.2, 1.4 Hz), 4.13 (1H, s), 3.66 (1H, d, J = 11.7 Hz), 3.26 (1H, d, J = 11.7 Hz), 1.79 (3H, d, J = 1.3 Hz), 1.51 (3H, s), 1.39 (3H, s), 1.25 (3H, t, J = 7.1 Hz), 0.98 (3H, s), 0.96 (3H, s). 13 IR ν max (film) / cm -1 3410, 3036, 2992, 1695, 1652. HRMS C 15 H 25Calculated O5N (M+): 299.1733. Found: 299.1735. [α] D +62.1 (c = 1.1, CHCl3). E type: 1 H NMR (400MHz, CDCl3) δ: 8.25 (1H, bd, J = 12.3 Hz), 7.86 (1H, dq, J = 12.3, 1.4 Hz), 4.14 (1H, s), 4.10 (2H, qd, J = 7.2, 1.2 Hz), 3.64 (1H, d, J = 11.8 Hz), 3.22 (1H, d, J = 11.8 Hz), 1.71 (3H, d, J = 1.4 Hz), 1.41 (3H, s), 1.39 (3H, s), 1.25 (3H, t, J = 7.1 Hz), 0.97 (3H, s), 0.91 (3H, s). 13 IR ν max (film) / cm -1 3410, 3028, 2992, 1695, 1652. HRMS C 15 H 25 Calculated O5N (M+): 299.1733. Found: 299.1735. [α] D +40.7 (c = 1.1, CHCl3).

[0470] (Z)-N-(2-Bromovinyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0471] [ka]

[0472] (Bromomethyl)triphenylphosphonium bromide (8.72 g, 20.0 mmol) and potassium tert-butoxide (2.24 g, 20.0 mmol) were dried under vacuum for 10 min, after which the flask was cooled to 0 °C and THF (40 mL) was added to give a bright yellow suspension. The mixture was stirred for 1.5 h, after which a solution of N-formyl-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (1.09 g, 5.0 mmol) in THF (5.0 mL) was added and allowed to stir at room temperature for 16 h. The reaction mixture was diluted with hexanes (30 mL) and HO (30 mL) and then filtered to remove solids. The aqueous phase was extracted with EtOAc (3 × 30 mL), and the combined organic phases were washed with brine (45 mL), dried (NaSO), filtered, and the solvent removed in vacuo to give the crude product as a brown oil. The crude residue was purified by flash column chromatography (5–10% EtOAc / petroleum ether) to give a mixture of dibromoenamide and cis-bromoenamide. The mixture was then dissolved in EtOAc (20 mL). Pd(PPh3)4 (288 mg, 0.249 mmol) was added, followed by Bu3SnH (0.670 mL, 2.49 mmol), and the mixture was stirred at room temperature for 16 h. The mixture was diluted with hexane (10 mL), filtered through Celite®, and the solvent was removed in vacuo to give the crude product as a brown oil. Column chromatography (2–4% EtOAc / petroleum ether) afforded the cis-bromoenamide (607 mg, 41% over two steps) as a pale yellow oil.

[0473] 1 H NMR (CDCl3, 400 MHz) δ: 8.61 (1H, d, J = 11.2 Hz), 7.37 (1H, dd, J = 11.2, 5.9 Hz), 5.58 (1H, d, J = 5.9 Hz), 4.20 (1H, s), 3.75 (1H, d, J = 11.8 Hz), 3.35 (1H, d, J = 11.8 Hz), 1.55 (3H, s), 1.49 (3H, s), 1.08 (3H, s), 1.06 (3H, s);13 C NMR (CDCl3, 100 MHz) δ: 167.3, 124.2, 99.3, 89.8, 77.2, 71.3, 33.2, 29.4, 21.9, 18.9, 18.7; IR ν max (film) / cm -1 3390, 2993, 2959, 2872, 1699, 1643.

[0474] (Z)-N-(2-Bromovinyl)-2,2,5,5-pentamethyl-1,3-dioxane-4-carboxamide

[0475] [ka]

[0476] (Bromomethyl)triphenylphosphonium bromide (3.27 g, 7.50 mmol) and potassium tert-butoxide (300 mg, 7.50 mmol) were dried under vacuum for 10 min, after which the flask was cooled to 0 °C and THF (10 mL) was added to give a bright yellow suspension. The mixture was stirred for 1.5 h, after which a solution of N-formyl-N,2,2,5,5-pentamethyl-1,3-dioxane-4-carboxamide (345 mg, 1.50 mmol) in THF (2.0 mL) was added and allowed to stir at room temperature for 16 h. The reaction mixture was diluted with hexane (10 mL) and HO (10 mL) and then filtered to remove solids. The aqueous phase was extracted with EtOAc (3 × 15 mL), and the combined organic phase was washed with brine (20 mL), dried (NaSO), filtered, and the solvent removed in vacuo to give the crude product as a brown oil. Column chromatography (5–10% EtOAc / petroleum ether) afforded a mixture of the dibromo compound and an unidentified impurity. The mixture was then dissolved in EtOAc (5 mL). Pd(PPh3)4 (59 mg, 0.051 mmol) was added, followed by Bu3SnH (0.164 mL, 0.612 mmol), and the mixture was stirred at room temperature for 16 h. The mixture was diluted with hexane (5 mL), filtered through Celite®, and the solvent was removed in vacuo to give the crude product as a brown oil. Column chromatography (5–20% EtOAc / petroleum ether) afforded the cis-bromoenamide (97 mg, 21% over two steps) as a pale yellow oil.

[0477] 1 H NMR (CDCl3, 400 MHz) δ: 7.14 (1H, d, J = 5.5 Hz), 6.03 (1H, d, J = 5.9 Hz), 4.32 (1H, s), 3.61 (1H, d, J = 11.4 Hz), 3.31 (1H, d, J = 11.4 Hz), 3.13 (3H, s), 1.46 (3H, s), 1.42 (3H, s), 1.26 (3H, s), 0.90 (3H, s); 13C NMR (CDCl3, 100 MHz) δ: 167.1, 134.6, 99.5, 93.5, 77.2, 72.7, 33.7, 33.5, 29.4, 21.8, 19.4, 18.4; IR ν max (film) / cm -1 3092, 2959, 2935, 2859, 1663.

[0478] (Z)-2,2,5,5-pentamethyl-N-(4-phenylbut-1-en-3-ynyl)-1,3-dioxane-4-carboxamide

[0479] [ka]

[0480] (Z)-N-(2-bromovinyl)-N,2,2,5,5-pentamethyl-1,3-dioxane-4-carboxamide (48 mg, 0.157 mmol) was dissolved in MeCN (1 mL), followed by the addition of phenylacetylene (35 μL, 0.314 mmol), EtN (87 μL, 0.628 mmol), CuI (6 mg, 31 μmol), and finally Pd(PPh) (18 mg, 16 μmol). The mixture was stirred at room temperature for 16 h and then filtered through Celite® with 20% EtOAc / hexane to give the crude product as a brown solid. The crude residue was purified by flash column chromatography (2–5% EtOAc / petroleum ether) to give the enyne product (29 mg, 59%) as a pale yellow oil.

[0481] 1H NMR (CDCl3, 400 MHz, 55℃) δ: 7.42-7.40 (2H, m), 7.33-7.30 (3H, m), 7.05 (1H, d, J = 8.7 Hz), 5.15 (1H, d, J = 8.7 Hz), 4.48 (1H, s), 3.65 (1H, d, J = 11.5 Hz), 3.55 (3H, br s), 3.35 (1H, d, J = 11.5 Hz), 1.48 (3H, s), 1.30 (3H, s), 0.94 (3H, s); 13 C NMR (CDCl3, 100 MHz, 55℃) δ: 167.8, 136.9, 131.1, 128.3, 128.2, 123.5, 99.6, 94.7, 93.2, 85.8, 76.6, 72.7, 34.2, 33.7, 29.1, 21.8, 19.2, 18.5; IR ν max (フィルム) / cm -1 2955, 2929, 2958, 2859, 1684, 1619.

[0482] Compound Synthesis-Radiolabeled Intermediates 1,2- 13 C3-2-(トリメチルシリル)エチル2-ブロモアセテート

[0483]

change

[0484] 13C2-Bromoacetic acid 284 (890 mg, 6.41 mmol) was dissolved in CHCl and cooled to 0 °C. 2-Trimethylsilylethanol (1.83 mL, 12.8 mmol), a catalytic amount of DMAP, and finally DCC (1.39 g, 6.73 mmol) were added in portions. The resulting solution was stirred for 2 h and then filtered through a Celite® bed. The Celite® pad was washed with 20% EtOAc / hexane (200 mL), followed by saturated aqueous NaHCO3 (150 mL), water (150 mL), and brine (150 mL). The solution was then dried (NaSO4), and the solvent was removed in vacuo to give the crude product as a yellow oil. The crude residue was purified by flash column chromatography (2–4% EtOAc / hexane) to give the ester product (1.05 g, 68%) as a pale yellow oil.

[0485] 1 H NMR (CDCl3, 400MHz) δ: 4.29-4.24 (2H, m), 4.00-3.61 (2H, dd, J = 152.8, 4.4 Hz), 1.07-1.00 (2H, m), 0.06 (9H, s); 13 C NMR (CDCl3, 100MHz) δ: 172.6 (d, J = 49.0 Hz), 66.3, 27.6 (d, J = 49.0 Hz), 18.7, 0.0; IR ν max (film) / cm -1 2955, 2899, 1693, 1249; HRMS (ESI) C5 13 C2H 15 Calculated for OBrNaSi (M+Na)+: m / z 262.9984, found m / z 262.9977.

[0486] 1,2- 13 C2-2-(trimethylsilylethoxycarbonylmethyl)triphenylphosphonium bromide

[0487] [ka]

[0488] 1,2- 13 C2-2-(Trimethylsilyl)ethyl 2-bromoacetate (1.05 g, 4.38 mmol) was dissolved in toluene (20 mL). Triphenylphosphine (1.15 g, 4.38 mmol) was added, and the resulting solution was stirred at room temperature for 16 hours. At this time, the reaction mixture contained a thick white precipitate. An additional 10 mL of toluene was added, followed by sonication and stirring at room temperature for an additional hour. The white precipitate was collected by filtration, washed with toluene (2 × 20 mL), and then dissolved in CHCl (50 mL). The solution was washed with brine (50 mL), dried (NaSO), and concentrated in vacuo to give the phosphonium salt (1.42 g, 65%) as a white solid.

[0489] 1 H NMR (CDCl3, 400 MHz) δ: 7.98-7.93 (6H, m), 7.85-7.80 (3H, m), 7.74-7.69 (6H, m), 5.83-5.44 (2H, ddd, J = 134.0, 17.0, 9.0 Hz), 4.12-4.07 (2H, m), 0.90-0.87 (2H, m), 0.00 (9H, s); 13 C NMR (CDCl3, 100 MHz) δ: 166.5 (d, J = 57.5 Hz), 136.7, 135.7, 131.9, 67.1, 35.0 (d, J = 57.5 Hz), 18.8, 0.00; IR ν max (film) / cm -1 3478, 3405, 2954, 2802, 1674; mp 90-91℃.

[0490] 13 C-1H-Benzotriazole-1-carboxaldehyde

[0491] [ka]

[0492] Acetic anhydride (1.34 mL, 14.2 mmol) and 13 C-formic acid (0.800 mL, 21.3 mol) was heated at 50° C. for 3 hours, at which time analysis of the crude NMR spectrum revealed 13 This allowed the calculation of the mass ratio of C1-acetic acid to formic anhydride. In this case, 9.94 mmol of mixed anhydride was formed. Benzotriazole (1.06 g, 8.92 mmol) was dissolved in THF (5 mL) at -10 °C, and the resulting solution was treated with the crude mixed anhydride mixture via syringe addition and left stirring for 1 h. The solvent was removed under vacuum, and the crude white solid that formed was azeotroped with CHCl3 to remove excess formic acid, yielding the formylated product (1.06 g, 99%) as a white solid.

[0493] 1 H NMR (500 MHz, CDCl3) δ: 10.08-9.54 (1H, d, J = 220.0 Hz), 8.28 (1H, d, J = 8.0 Hz), 8.18 (1H, d, J = 8.0 Hz), 7.73 (1H, ddd, J = 8.2, 7.2, 1.0 Hz), 7.59 (1H, ddd, J = 8.2, 7.2, 1.0, Hz); 13 C NMR (125 MHz, CDCl3) δ: 159.9, 146.5, 130.7, 129.9, 127.0, 120.4, 114.4; IR ν max (film) / cm -1 3103, 1686, 1594; HRMS (El) C6 13 Calculated for CH6ON3(M+H)+: m / z 148.0467, found m / z 147.0469; mp 93-94°C.

[0494] 13 C-(R)-N-Formyl-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0495] [ka]

[0496] (R)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (known from Han et al.) (1.33 g, 7.12 mmol) was dissolved in THF (40 mL). The solution was cooled to 0° C. and n-butyllithium (3.13 mL, 2.5 M in hexane, 7.83 mmol) was added slowly via syringe and then allowed to stir at 0° C. for 0.5 h. 13 A solution of C-1H-benzotriazole-1-carboxaldehyde (1.16 g, 7.83 mmol) in THF (10 mL) was added slowly via syringe. The reaction mixture was left stirring at RT for 4 h. The reaction mixture was diluted with isopropanol (10 mL) and then washed with saturated aqueous NaHCO3 (50 mL). The aqueous phase was separated and extracted with EtOAc (3 × 50 mL), and the combined organic phases were washed with brine (100 mL), dried (Na2SO4), filtered, and concentrated in vacuo to give the crude product as a pale yellow solid. The crude residue was purified by flash column chromatography (10–20% EtOAc / petroleum ether) to give the imide product (1.20 g, 78%) as a white solid.

[0497] 1 H NMR (CDCl3, 500 MHz) δ: 9.38-8.96 (1H, dd, J = 193.5, 10.4 Hz), 8.92 (1H, br s), 4.21 (1H, s), 3.73 (1H, d, J = 11.9 Hz), 3.35 (1H, d, J = 11.9 Hz), 1.50 (3H, s), 1.47 (3H, s), 1.08 (3H, s), 1.07 (3H, s); 13 C NMR (CDCl3, 125 MHz) δ: 170.6, 161.4, 99.7, 77.1, 71.2, 33.4, 29.4, 21.7, 18.9, 18.6; IR ν max (film) / cm -1 3264, 2994, 2960, 2882, 1724, 1657, 1457; HRMS (ESI) C913 C1H 17 Calculated for NO4Na M+: m / z 239.1083, found m / z 239.1081; mp 130-131°C.

[0498] 13 C3-(R,E)-2-(trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate and 13 C3-(R,Z)-2-(trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate

[0499] [ka]

[0500] 1,2- 13 C2-2-(Trimethylsilylethoxycarbonylmethyl)triphenylphosphonium bromide (1.42 g, 2.85 mmol) was dissolved in CHCl (50 mL) and stirred with 1 M aqueous NaOH (50 mL) at room temperature for 2 h. The organic layer was then separated and concentrated in vacuo to give crude trimethylsilyl ylide (1.24 g) as a yellow oil. The crude ylide was then dissolved in benzene (15 mL) and then 13 C-(R)-N-Formyl-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (425 mg, 1.84 mmol) was added. The resulting solution was then stirred at 80 °C for 16 h. The reaction mixture was concentrated in vacuo, and the crude residue was then purified by flash column chromatography (5-20% EtOAc / petroleum ether) to afford the cis isomer (99 mg, 15%) as a yellow oil, followed by the trans isomer (537 mg, 82%) as a white solid.

[0501] 13 C3-(R,E)-2-(trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate: 1H NMR (CDCl3, 500 MHz) δ: 8.33 (1H, d, J = 11.5 Hz), 8.13-7.71 (1H, dddd, J = 177.0, 14.5, 11.5, 5.0 Hz), 5.70-5.34 (1H, dddd, J = 163.0, 14.5, 3.0, 1.5 Hz), 4.25-4.21 (2H, m), 4.17 (1H, s), 3.67 (1H, d, J = 11.8 Hz), 3.27 (1H, d, J = 11.8 Hz), 1.52 (3H, s), 1.46 (3H, s), 1.06 (3H, s), 1.02 (2H, t, J = 8.4 Hz), 1.01 (3H, s), 0.05 (9H, s); 13 C NMR (CDCl3, 125MHz) δ: 169.4, 169.0 (dd, J = 79.5, 4.3 Hz), 137.5 (dd, J = 79.5, 4.3 Hz), 104.7 (dd, J = 79.5, 79.5 Hz), 101.0, 78.7, 72.7, 63.8, 34.9, 30.9, 23.3, 20.3, 20.1, 18.8, 0.0; IR ν max (film) / cm -1 3306, 2955, 2899, 2874, 1708, 1649, 1476; HRMS (El) C 14 13 C3H 32 O5NSi M+ calculated: m / z 360.2073, found m / z 360.2064; mp 126-127°C. 13 C3-(R,Z)-2-(trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate: 1H NMR (CDCl3, 400 MHz) δ: 11.10 (1H, d, J = 11.6 Hz), 7.62-7.09 (1H, dm, J = 175.2 Hz), 5.32-4.86 (1H, ddm, J = 168.8, 9.2 Hz), 4.23 (2H, tm, J = 8.3 Hz), 4.21 (1H, s), 3.72 (1H, d, J = 11.6 Hz), 3.32 (1H, d, J = 11.6 Hz), 1.59 (3H, s), 1.47 (3H, s), 1.05 (3H, s), 1.04 (3H, s), 1.04-1.00 (2H, m), 0.05 (9H, s); 13 C NMR (CDCl3, 100MHz) δ: 170.2, 169.8 (d, J = 75.4 Hz), 137.3 (d, J = 74.4 Hz), 100.8 (dd, J = 75.4, 74.4 Hz), 99.7, 78.7, 72.8, 63.7, 34.8, 30.8, 23.4, 20.5, 20.1, 18.9, 0.0; IR ν max (film) / cm -1 3300, 2965, 2924, 2860, 1672, 1630, 1454; HRMS (El) C 14 13 C3H 32 O5NSi M+ calculated: m / z 360.2073, found m / z 360.2064.

[0502] 13 C3-(R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid

[0503] [ka]

[0504] 13C3-(R,E)-2-(trimethylsilyl)ethyl 3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (500 mg, 1.40 mmol) was dissolved in THF (10 mL). Water (0.200 mL) was added, followed by TBAF (4.20 mL, 4.2 mmol, 1 M solution in THF) via syringe, and the resulting pale orange solution was heated at 40 °C for 18 h. The reaction mixture was concentrated in vacuo, and the crude residue was purified by flash column chromatography (0–2% MeOH / CHCl) to give an impure white solid. The crude product was dissolved in EtOAc (25 mL) and stirred with 1 M aqueous NaOH (25 mL) for 1 h. The aqueous layer was collected, diluted with EtOAc (50 mL), and then acidified to pH 5 with 1 M aqueous HCl. After stirring the mixture for 1 hour, the aqueous layer was extracted with EtOAc (3×25 mL). The combined organic layers were washed with brine (50 mL), dried (NaSO), filtered, and concentrated in vacuo to give the desired acid (287 mg, 80%) as a white solid.

[0505] 1 H NMR (CDCl3, 500 MHz) δ: 8.52 (1H, d, J = 11.8 Hz), 8.26-7.85 (1H, dddd, J = 176.5, 14.2, 11.8, 5.0 Hz), 5.76-5.40 (1H, ddm, J = 163.0, 14.2 Hz), 4.22 (1H, s), 3.71 (1H, d, J = 11.6 Hz), 3.32 (1H, d, J = 11.6 Hz), 1.55 (3H, s), 1.51 (3H, s), 1.04 (3H, s), 1.03 (3H, s); 13 C NMR (CDCl3, 125 MHz) δ: 172.6, 172.9 (dd, J = 77.3, 4.4 Hz), 138.1 (dd, J = 77.1, 4.4 Hz), 101.8 (dd, J = 77.3, 77.1 Hz), 99.6, 77.2, 71.2, 33.4, 29.4, 21.8, 18.8, 18.7; IR ν max (film) / cm-1 3315, 3085, 2995, 2880, 1695, 1668; HRMS (El) C9 13 C3H 20 O5N M+ calculated: m / z 260.1365, found m / z 260.1364; mp 187-188°C.

[0506] 13 C3-(R,E)-3-(2,4-dihydroxy-3,3-dimethylbutanamido)acrylic acid

[0507] [ka]

[0508] 13 C3-(R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (100 mg, 0.389 mmol) was dissolved in MeCN (3 mL). Water (300 μL) was added, followed by BiCl3 (12 mg, 38 μmol), resulting in a white suspension. The mixture was stirred at room temperature for 16 hours and then filtered through a small Celite® pad, which was then washed with EtOAc (20 mL). The crude residue was purified by flash column chromatography to give: 13 C3-CJ-15801 was obtained as a colorless oil (35 mg, 41%).

[0509] 1 H NMR ((CD3)2CO, 500 MHz) δ: 9.70 (1H, d, J = 10.5 Hz), 8.09-7.67 (1H, dddd, J = 174.0, 14.5, 10.5, 5.2 Hz), 5.87-5.51 (1H, ddm, J = 163.5, 14.5 Hz), 4.00 (1H, s), 3.38 (1H, d, J = 10.9 Hz), 3.31 (1H, d, J = 10.9 Hz), 0.82 (3H, s), 0.81 (3H, s); 13C NMR ((CD3)2CO, 125 MHz) δ: 173.1, 170.4 (dd, J = 76.7, 4.3 Hz), 138.5 (dd, J = 76.7, 4.3 Hz), 102.2 (dd, J = 76.7, 76.7 Hz), 77.4, 70.2, 40.3, 21.4, 20.5; IR ν max (film) / cm -1 3295, 2964, 2836, 2878, 1642, 1584; HRMS (ESI) C6 13 C3H 15 Calculated for NO5Na (M+Na)+: m / z 243.0943, found m / z 243.0940.

[0510] 13 C3-(R,E)-benzyl 3-(2,4-dihydroxy-3,3-dimethylbutanamido)acrylate

[0511] [ka]

[0512] 13A solution of C3-(R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (100 mg, 0.389 mmol) in CHCl2 (3 mL) was cooled to 0 °C, followed by the sequential addition of benzylamine (30 μL, 0.291 mmol), catalytic DMAP, and DCC (21 mg, 0.101 mmol). The resulting solution was stirred at 0 °C for 4 h, by which time a white precipitate had formed. The reaction mixture was filtered through a Celite® pad, which was then washed with EtOAc (20 mL). The organic layer was then washed with saturated aqueous NaHCO3 (15 mL), HO (15 mL), and brine (15 mL), before being dried (Na2SO4), filtered, and concentrated in vacuo to give the crude product as a white solid (71 mg, 0.203 mmol). After unsuccessful purification of the crude residue by flash column chromatography, the crude product was dissolved in a mixture of MeCN (1.5 mL) and HO (0.150 mL), followed by the addition of BiCl (6 mg, 19 μmol) to give a milky suspension, which was then stirred at room temperature for 16 h. The reaction mixture was quenched with a few drops of saturated aqueous NaHCO and subsequently filtered through a Celite® pad, which was then washed with EtOAc (10 mL). The eluent was concentrated in vacuo to give the crude product as a colorless oil. The crude residue was purified by flash column chromatography (20–70% EtOAc / petroleum ether) to give the diol product (26 mg, 41%) as a colorless oil.

[0513] 1H NMR (CDCl3, 400 MHz) δ: 9.03 (1H, d, J = 11.6 Hz), 8.29-7.77 (1H, dddd, J = 176.7, 13.8, 11.6, 4.9 Hz), 7.38-7.34 (5H, m), 5.89-5.41 (1H, ddm, J = 163.1, 13.8 Hz), 5.18 (2H, d, J = 3.3 Hz), 4.58 (1H, d, J = 4.1 Hz), 4.17 (1H, d, J = 4.1 Hz), 3.58 (1H, br d, J = 10.6 Hz), 3.53 (1H, d, J = 10.6 Hz), 3.04 (1H, br s), 1.02 (3H, s), 0.97 (3H, s); 13 C NMR (CDCl3, 100 MHz) δ: 171.4 (d, J = 3.6 Hz), 167.1 (dd, J = 80.0, 4.6 Hz), 136.8 (dd, J = 77.9, 4.6 Hz), 135.2, 128.6, 128.2, 128.1, 102.5 (dd, J = 80.0, 77.9 Hz), 77.4, 71.6, 66.2, 39.4, 20.9, 20.3; IR ν max (film) / cm -1 3316, 2963, 2934, 2875, 1682, 1649; HRMS (ESI) C 13 13 C3H 21 O5NNa M+ calculated: m / z 333.1413, found m / z 333.1407.

[0514] Compound synthesis - BODIPY amide conjugates 3-(1H-pyrrol-2-yl)propan-1-amine

[0515] [ka]

[0516] 2-(3-Azidopropyl)-1H-pyrrole (100 mg, 0.704 mmol) was dissolved in MeOH (3 mL) and placed under an argon atmosphere. Pd / C (10 mg, 10%) was added, and the argon was replaced with a hydrogen atmosphere, followed by stirring at room temperature for 1.5 hours. The crude mixture was filtered through a Celite® bed to remove Pd, washed with MeOH (10 mL), and the solvent was removed in vacuo to give the amine product (84 mg, 100%) as a pale yellow oil.

[0517] 1 H NMR (CDCl3, 400 MHz) δ: 8.73 (1H, br s), 6.68-6.66 (1H, m), 6.13-6.11 (1H, m), 5.94-5.92 (1H, m), 2.78 (2H, t, J = 6.8 Hz), 2.69 (2H, t, J = 7.4 Hz), 1.81-1.75 (2H, m), 1.57 (2H, br s); 13 C NMR (CDCl3, 100 MHz) δ: 132.1, 116.2, 108.5, 105.0, 41.7, 33.0, 25.2; IR ν max (film) / cm -1 3364, 3233, 3098, 2932, 2851; HRMS (El) C7H 13 Calculated for N2(M+H)+: m / z 125.1079, found m / z 125.1077.

[0518] (9H-Fluoren-9-yl)methyl 3-(1H-pyrrol-2-yl)propylcarbamate

[0519] [ka]

[0520] 3-(1H-Pyrrol-2-yl)propan-1-amine (719 mg, 5.75 mmol) was dissolved in CHCl (45 mL), followed by the addition of EtN (1.61 mL, 11.5 mmol) and fluorenylmethyloxycarbonyl chloride (1.64 g, 6.33 mmol). The resulting solution was then stirred at room temperature for 16 h. The reaction mixture was diluted with CHCl (25 mL) and then washed with saturated aqueous NaHCO (30 mL), water (30 mL), and brine (30 mL), dried (NaSO), filtered, and concentrated in vacuo to give the crude product as a colorless oil. Purification by flash column chromatography (10–20% EtOAc / petroleum ether) afforded the clean Fmoc-protected amine (1.15 g, 58%) as a white solid.

[0521] 1 H NMR (CDCl3, 400 MHz) δ: 8.58 (1H, br s) 7.79 (2H, d, J = 7.3 Hz), 7.61 (2H, d, J = 7.3 Hz), 7.42 (2H, t, J = 7.3 Hz), 7.32 (2H, t, J = 7.4 Hz), 6.71- 6.69 (1H, m), 6.13-6.11 (1H, m), 5.93-5.90 (1H, m) 4.79 (1H, br s), 4.47 (2H, d, J = 6.7 Hz), 4.23 (1H, t, J = 6.7 Hz), 3.30-3.27 (2H, m), 3.05 (2H, t, J = 6.8 Hz), 1.80-1.73 (2H, m); 13 C NMR (CDCl3, 125 MHz) δ: 157.2, 143.9, 141.4, 131.5, 127.7, 127.1, 125.0, 124.7, 116.6, 108.1, 105.3, 66.6, 47.3, 39.9, 31.0, 24.0; IR ν max (film) / cm -1 3403, 3341, 2982, 2928, 1690; HRMS (Cl)C 22 H 22Calculated for N2O2(M)+: m / z 346.1681, found m / z 346.1676.

[0522] 3-[(9H-Fluoren-9-ylmethoxy)carbonyl]amino-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane

[0523] [ka]

[0524] (9H-Fluoren-9-yl)methyl-3-(1H-pyrrol-2-yl)propyl carbamate (692 mg, 0.739 mmol) and 3,5-dimethyl-1H-pyrrole-2-carbaldehyde (268 mg, 2.20 mmol) were dissolved in CHCl (15 mL) and cooled to 0 °C. POCl (0.205 mL, 2.20 mmol) was then added dropwise. The mixture was stirred at room temperature for 6 h, then cooled to 0 °C again, and BF.EtO (0.987 mL, 8.00 mmol) and DIPEA (1.46 mL, 8.40 mmol) were added and the mixture was left stirring at room temperature for 12 h. The mixture was then diluted with HO (25 mL) and CHCl (10 mL), filtered through a Celite® bed, and washed with CHCl (2 × 25 mL). The combined organic layers were then dried (Na2SO4) and concentrated in vacuo to give the crude product as a dark red / green solid. The crude residue was purified by flash column chromatography (10-40% EtOAc / petroleum ether) to give the product (320 mg, 32%) as a red oil that solidified upon cooling. NMR values ​​are comparable to those of similar BODIPY compounds (see Giessler et al.).

[0525] 1H NMR (CDCl3, 400 MHz) δ: 7.77 (2H, d, J = 7.3 Hz), 7.62 (2H, d, J = 7.3 Hz), 7.40 (2H, t, J = 7.3 Hz), 7.30 (2H, t, J = 7.4 Hz), 7.08 (1H, s), 6.92 (1H, d, J = 3.9 Hz), 6.32 (1H, d, J = 3.9 Hz), 6.13 (1H, s), 5.26 (1H, br s), 4.36 (2H, d, J = 7.3 Hz), 4.22 (1H, t, J = 7.3 Hz), 3.30-3.27 (2H, m), 3.05 (2H, t, J = 7.1 Hz), 2.59 (3H, s), 2.26 (3H, s), 2.01-1.95 (2H, m); 13 C NMR (CDCl3, 125 MHz) δ: 159.7, 158.9, 156.4, 144.1, 143.5, 141.3, 134.9, 133.2, 128.5, 127.6, 127.0, 125.2, 123.7, 120.2, 119.9, 116.9, 66.6, 47.3, 40.2, 29.0, 25.6, 14.9, 11.3; IR ν max (フィルム) / cm -1 3333, 2943, 2859, 1601.

[0526] 3-(1H-ピロール-2-イル)プロパン-1-オール

[0527]

change

[0528] A solution of methyl 3-(1H-pyrrol-2-yl)propanoate (2.64 g, 17.5 mmol) in EtO (130 mL) was cooled to 0 °C, followed by the slow addition of LiAlH (996 mg, 26.3 mmol). The suspension was allowed to stir at room temperature for 16 h. The crude reaction mixture was quenched by the dropwise addition of 1 M NaOH until a neutral pH was achieved. The EtO was decanted, and the lithium / aluminum salts were washed with additional EtO (3 × 50 mL). The combined organic layers were dried (NaSO), filtered, and the solvent removed in vacuo to give the alcohol product (2.15 g, 100%) as a colorless oil.

[0529] 1 H NMR (CDCl3, 400 MHz) δ: 8.24 (1H, br s), 6.70-6.68 (1H, m), 6.15-6.13 (1H, m), 5.95-5.94 (1H, m), 3.73 (2H, t, J = 5.9 Hz), 2.75 (2H, t, J = 7.3 Hz), 1.93-1.87 (2H, m), 1.50 (1H, br s); 13 C NMR (CDCl3, 100 MHz) δ: 131.8, 116.4, 108.3, 105.2, 62.3, 32.2, 24.2; IR ν max (film) / cm -1 3365, 2940, 2976, 2850, 1012; HRMS (Cl) C7H 12 Calculated for NO (M+H)+: m / z 126.0919, found m / z 126.0917.

[0530] 2-(3-azidopropyl)-1H-pyrrole

[0531] [ka]

[0532] A solution of 3-(1H-pyrrol-2-yl)propan-1-ol (1.00 g, 8.13 mmol) in CHCl (60 mL) was cooled to 0 °C, followed by the addition of EtN (2.26 mL, 16.26 mmol) and methanesulfonyl chloride (0.755 mL, 9.76 mmol). The reaction mixture was allowed to stir at 0 °C for 1 h, then warmed to room temperature and washed with 1 M HCl (40 mL), saturated aqueous NaHCO (60 mL), and brine (60 mL), then dried (NaSO), filtered, and the solvent removed in vacuo to give the mesylate intermediate (1.52 g, 94%). The crude mesylate was then dissolved in DMF (60 mL), and the solution was treated with sodium azide (1.48 g, 22.7 mmol) and heated to 70 °C for 16 h. The reaction mixture was then cooled to room temperature before adding EtOAc (60 mL) followed by HO (60 mL). The aqueous phase was washed with EtOAc (2 x 60 mL) and then the combined organic phases were washed with brine (5 x 100 mL), dried (NaSO), filtered and the solvent removed in vacuo to give the azide product (947 mg, 88%) as a yellow oil.

[0533] 1 H NMR (CDCl3, 400 MHz) δ: 7.98 (1H, br s), 6.70-6.68 (1H, m), 6.15-6.13 (1H, m), 5.96-5.94 (1H, m), 3.34 (2H, t, J = 6.6 Hz), 2.73 (2H, t, J = 7.4 Hz), 1.92-1.87 (2H, m); 13 C NMR (CDCl3, 100 MHz) δ: 130.7, 116.5, 108.5, 105.5, 50.7, 28.9, 24.7. IR ν max (film) / cm -1 3379, 2940, 2870, 2091; HRMS (El) C7H 11 Calculated for N4(M+H)+: m / z 151.0984, found m / z 151.0987.

[0534] 3-Azido[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane, 11

[0535] [ka]

[0536] 2-(3-Azidopropyl)-1H-pyrrole (105 mg, 0.739 mmol) and 3,5-dimethyl-1H-pyrrole-2-carboxaldehyde (99 mg, 0.813 mmol) were dissolved in CHCl (5 mL) and cooled to 0 °C. POCl (76 μL, 0.813 mmol) was then added dropwise. The mixture was stirred at room temperature for 6 h, then cooled to 0 °C again, and BF.EtO (0.365 mL, 2.96 mmol) and DIPEA (0.539 mL, 3.10 mmol) were added. The mixture was then left stirring at room temperature for 12 h. The mixture was then diluted with HO (10 mL) and CHCl (5 mL), filtered through a Celite® bed, and washed with CHCl (2 × 10 mL). The combined organic layers were then dried (Na2SO4) and the solvent removed in vacuo to give the crude product as a dark red / green solid. The crude residue was purified by flash column chromatography (5% EtOAc / petroleum ether) to give the desired azide (155 mg, 57%) as a red oil that solidified upon cooling. NMR values ​​are comparable to those of similar BODIPY compounds (see Giessler et al.).

[0537] 1 H NMR (CDCl3, 500 MHz) δ: 7.09 (1H, s), 6.91 (1H, d, J = 3.9 Hz), 6.28 (1H, d, J = 3.9 Hz), 6.12 (1H, s), 3.40 (2H, t, J = 7.0 Hz), 3.05 (2H, t, J = 7.4 Hz), 2.57 (3H, s), 2.27 (3H, s), 2.04 (2H, m); 13IR ν max (film) / cm -1 2959, 2932, 2870, 2091; HRMS (ESI) C 14 H 17 Calculated for BF2N5(M+H)+: m / z 304.1545, found m / z 304.1528; mp 49-50°C.

[0538] 3-amino[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane

[0539] [ka]

[0540] A solution of 3-azido[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane (25 mg, 0.083 mmol) in THF (1 mL) was treated with polymer-bound triphenylphosphine (103 mg, 1.6 mmol g -1 ) and HO (50 μL) were added. After reaction progress by TLC, the resulting suspension was heated at 50° C. for 4 h. Upon completion, the suspension was allowed to cool to room temperature before being filtered through a bed of Celite® and the solvent removed in vacuo to give the desired amine as a red oil (61-82%). This material was used without further purification.

[0541] Amide conjugate-310

[0542] [ka]

[0543] The compound was prepared by amide coupling of 3-amino[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane and (E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid, which was prepared in situ from 3-[(9H-fluoren-9-ylmethoxy)carbonyl]amino-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane by treatment with piperidine.

[0544] Thus, 3-[(9H-fluoren-9-ylmethoxy)carbonyl]amino-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane was treated with piperidine in THF at room temperature for 3 hours. The crude product was reacted with (£)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid in the presence of HBTU, DIPEA, and CHCl under microwave conditions at 80 °C for 2.5 hours. HPLC purification afforded the crude product in less than 10% yield over two steps.

[0545] Compound synthesis - BODIPY click conjugates (R,E)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide, 1

[0546] [ka]

[0547] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (20 mg, 65 μmol) and 3-azido[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane (15 mg, 50 μmol) were dissolved in THF (2 mL). One drop of DIPEA was added via pipette, followed by a catalytic amount of CuI. The resulting mixture was heated to 70 °C for 18 h and then concentrated in vacuo to give the crude product as a brown oil. The crude residue was purified by flash column chromatography (0–2% MeOH / CHCl) to give the desired compound (23 mg, 76%) as a red oil.

[0548] 1 H NMR (CDCl3, 500 MHz) δ: 8.32 (1H, d, J = 11.4 Hz), 7.84 (1H, dd, J = 13.9, 11.4 Hz), 7.38 (1H, s), 7.09 (1H, s), 6.87 (1H, d, J = 4.1 Hz), 6.34 (1H, br s), 6.23 (1H, d, J = 4.1 Hz), 6.11 (1H, s), 5.73 (1H, d, J = 13.9 Hz), 4.40 (2H, t, J = 6.9 Hz), 4.15 (1H, s), 3.68 (1H, d, J = 11.6 Hz), 3.65-3.61 (2H, m), 3.28 (1H, d, J = 11.6 Hz), 2.97 (2H, t, J = 7.5 Hz), 2.90 (2H, t, J = 6.3 Hz), 2.53 (3H, s), 2.38-2.31 (2H, m), 2.24 (3H, s), 1.46 (3H, s), 1.42 (3H, s), 1.02 (3H, s), 0.97 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 168.0, 166.3, 160.5, 156.6, 144.2, 135.3, 133.1, 132.8, 128.2, 123.9, 122.1, 120.6, 116.6, 106.0, 99.4, 77.2, 71.3, 49.8, 38.7, 33.3, 29.4, 29.3, 25.6, 25.5, 21.9, 18.8, 18.7, 14.9, 11.3; IR ν max (film) / cm -1 3295, 2991, 2830, 2876, 1666, 1598; HRMS (ESI) C 30 H 39 Calculated for BF2N7O4(MH)+: m / z 609.3166, found m / z 609.3141; [α] D +29.6 (c = 0.6, (CH3)2CO, T = 24.4℃).

[0549] The (S,E) form of 3 can be prepared similarly from (S,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide. [α] D -21.8(c=1.2, (CH3)2CO, T=24.4℃).

[0550] (R,E)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,4-dihydroxy-3,3-dimethylbutanamide, 5

[0551] [ka]

[0552] (R,E)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (20 mg, 33 μmol) was dissolved in a mixture of THF (1 mL) and HO (50 μL), followed by the addition of BiCl (1 mg, 3 μmol) and stirring at room temperature for 16 h. The crude mixture was filtered through Celite® and then concentrated in vacuo. The crude residue was purified by flash column chromatography (5–8% MeOH / CHCl) to afford the diol product (10 mg, 56%) as a red solid.

[0553] 1 H NMR (CDCl3, 500 MHz) δ: 9.43 (1H, d, J = 11.2 Hz), 7.90-7.85 (1H, m), 7.47 (1H, s), 7.09 (2H, br s), 6.86 (1H, d, J = 3.9 Hz), 6.22 (1H, d, J = 3.9 Hz), 6.10 (1H, s), 5.81 (1H, d, J = 13.8 Hz), 5.53 (1H, br s), 4.37-4.34 (3H, m), 4.17 (1H, s), 3.56-3.50 (3H, m), 3.43 (1H, d, J = 10.7 Hz), 2.93 (2H, t, J = 7.5 Hz), 2.90 (2H, br s), 2.52 (3H, s), 2.31-2.28 (2H, m), 2.22 (3H, s), 0.93 (3H, s), 0.92 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 172.7, 167.3, 160.5, 156.6, 145.2, 144.2, 135.2, 133.8, 133.2, 128.3, 124.0, 122.1, 120.6, 116.6, 105.6, 76.8, 70.3, 49.7, 39.6, 39.1, 29.3, 25.6, 25.5, 21.0, 20.7, 14.9, 11.3; IR ν max (film) / cm -1 3295, 2970, 2829, 2873, 1661, 1597; HRMS (ESI) C 27 H 35 Calculated for BF2N7O4(MH)+: m / z 569.2853, found m / z 569.2831; mp 104-105°C; [α] D +28.1 (C = 0.5, (CH3)2CO, T = 24.4℃).

[0554] The (S,E) form (7) can be prepared similarly from (S,E)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide. [α] D -24.0(c=1.0, (CH3)2CO, T=24.4℃).

[0555] (Z)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide, (R)-2, (S)-4

[0556] [ka]

[0557] (Z)—N-(3-(but-3-yn-1-ylamino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (R or S form) (96 mg, 0.3 mmol) was dissolved in THF (1.5 mL), followed by the addition of 3-azido[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane (122 mg, 0.4 mmol) in one portion, followed by the addition of DIPEA (1 drop) and a catalytic amount of CuI. The resulting mixture was heated to 70° C. for 16 h and then concentrated in vacuo to give the crude product as a dark brown oil. The crude product was purified via column chromatography (5% MeOH / CHCl) to give the triazole product (161 mg, 85%) as a red solid.

[0558] 1 H NMR (CDCl3, 500 MHz) δ: 11.68 (1H, d, J = 11.1 Hz), 7.39 (1H, s), 7.23 (1H, dd, J = 11.1, 8.9 Hz), 7.10 (1H, s), 6.88 (1H, d, J = 3.9 Hz), 6.35 (1H, t, J = 5.5 Hz), 6.24 (1H, d, J = 3.9 Hz), 6.12 (1H, s), 5.03 (1H, d, J = 8.9 Hz), 4.42 (2H, t, J = 6.9 Hz), 4.19 (1H, s), 3.71 (1H, d, J = 11.7 Hz), 3.68-3.58 (2H, m), 3.32 (1H, d, J = 11.7 Hz), 2.97 (2H, t, J = 7.5 Hz), 2.92 (2H, t, J = 6.2 Hz), 2.54 (3H, s), 2.37 (2H, quintet, J = 7.2 Hz), 2.26 (3H, s), 1.60 (3H, s), 1.46 (3H, s), 1.04 (6H, s); 13C NMR (CDCl3, 125 MHz) δ: 168.7, 167.8, 160.6, 156.5, 145.4, 144.2, 135.3, 133.1, 133.0, 128.1, 123.8, 121.7, 120.6, 116.5, 100.8, 99.2, 77.2, 71.4, 49.6, 38.2, 33.3, 29.4, 29.2, 25.5, 25.4, 22.0, 19.0, 18.6, 14.9, 11.3; IR ν max (film) / cm -1 (undiluted): 2926, 2360, 1660, 1610, 1139; HRMS (ESI) C 30 H 40 N7BF2O4Na (M+Na) + Calculated m / z 633.3131, found m / z 633.3107; melting point range: 66-69°C. R type: [α] D +3.8(c=0.9, (CH3)2CO, T=24.4℃). S type: [α] D -1.6(c=1.0, (CH3)2CO, T=24.4℃).

[0559] (Z)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,4-dihydroxy-3,3-dimethylbutanamide, (R)-6, (S)-8

[0560] [ka]

[0561] (Z)—N-((((3-[4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (60 mg, 0.1 mmol) was dissolved in MeCN (1.2 mL) and HO (0.1 mL), followed by addition of BiCl (6 mg, 0.02 mmol). The mixture was stirred vigorously for 16 h, then filtered through Celite® and washed with MeCN (40 mL). The solvent was removed in vacuo to give the crude diol as a dark brown oil. The crude product was purified by flash column chromatography (5% MeOH / CH2Cl2) to give the diol as a red oil (52 mg, 92%).

[0562] 1 H NMR (CDCl3, 400 MHz) δ: 11.85 (1H, d, J = 11.1 Hz), 7.42 (1H, s), 7.23 (1H, dd, J = 11.1, 8.8 Hz), 7.10 (1H, s), 6.89 (1H, d, J = 4.0 Hz), 6.55 (1H, t, J = 5.5 Hz), 6.24 (1H, d, J = 4.0, Hz), 6.13 (1H, s), 5.05 (1H, d, J = 8.8 Hz), 4.42 (2H, t, J = 6.9 Hz), 4.16 (1H, s), 3.66-3.55 (2H, m), 3.53 (1H, s), 2.96 (2H, t, J = 7.5 Hz), 2.92 (2H, t, J = 6.2 Hz), 2.54 (3H, s), 2.35 (2H, quintet, J = 7.4 Hz), 2.26 (3H, s), 1.03 (3H, s), 0.98 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 172.0, 168.1, 160.6, 156.5, 145.2, 144.2, 135.3, 133.5, 133.1, 128.2, 123.9, 121.8, 120.6, 116.5, 100.6, 77.9, 71.1, 49.7, 39.4, 38.4, 29.2, 25.5, 25.4, 20.9, 20.6, 14.9, 11.3; IRν max (film) / cm -1 (undiluted): 3309, 2953, 1654, 1610, 1139; HRMS (ESI) C 27 H 36 N7BF2O4Na (M+Na) + Calculated: m / z 593.2818, Found: m / z 593.2810. R type: [α] D +15.3(c=0.6, (CH3)2CO, T=24.4℃). S type: [α] D -16.0(c=1.0, (CH3)2CO, T=24.4℃).

[0563] 7-(3-azidopropyl)-5,5-difluoro-1-(3-methoxy-3-oxopropyl)-5H-dipyrrolo[1,2-c:1',2'-F][1,3,2]diazaborin-4-ium-5-uidate

[0564] [ka]

[0565] A solution of 2-(3-azidopropyl)-1H-pyrrole (275 mg, 1.83 mmol) in CHCl (10 mL) at 0° C. was treated with methyl 3-(5-formyl-1H-pyrrol-2-yl)propanoate (365 mg, 2.01 mmol) in CHCl (10 mL), and the resulting solution was treated dropwise with POCl (0.2 mL, 2.01 mmol). The reaction mixture was stirred at room temperature for 6 hours and then cooled to 0° C. again. The mixture was then treated with BF(OEt) (1 mL, 7.3 mmol) and N,N-diisopropylethylamine (1.4 mL, 8.24 mmol), and the reaction was stirred at room temperature overnight. The mixture was then diluted with HO (15 mL) and CHCl (10 mL) before being filtered through a Celite® bed. The Celite® was washed with CHCl (2×15 mL) and the organic phases were combined. The solution was then dried over NaSO and concentrated in vacuo to give a crude dark red / green solid residue. The crude solid was purified by flash column chromatography (petroleum ether: EtOAc; 8:2) to give 271 mg (41%) of the desired ester as a red oil that solidified upon cooling.

[0566] 1 H NMR (CDCl3, 400 MHz) δ: 7.13 (1H, s), 7.03-6.95 (1H, m), 6.35 (1H, t, J = 3.7 Hz), 3.70 (1H, s), 3.40 (1H, t, J = 6.9 Hz), 3.32 (1H, t, J = 7.5 Hz), 3.07 (1H, t, J = 7.7 Hz), 2.78 (1H, t, J = 7.6 Hz), 2.11-1.96 (1H, m).

[0567] (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoic acid

[0568] [ka]

[0569] A solution of D-pantothenic acid hemicalcium salt (500 mg, 1.0 mmol) in acetone (25 mL) was treated with p-TsOH.HO (560 mg, 3.0 mmol) and 1.0 g of 4 Å molecular sieves. The reaction was stirred at room temperature until complete by TLC analysis (18 h). The suspension was then filtered through a Celite® bed, washed with acetone (2 × 15 mL), and the organic phases were combined. The combined organic washes were concentrated in vacuo, and then EtOAc (30 mL) was added to the crude residue. The resulting solution was then washed with brine (2 × 30 mL). The organic layer was dried over NaSO and concentrated in vacuo. Hexane was added dropwise to induce crystallization of the acetonide (300 mg, 55%) before complete removal of the solvent. The desired acetonide was obtained as white crystals, requiring no further purification.

[0570] 1 H NMR (CDCl3, 400 MHz) δ: 7.04 (1H, m), 4.12 (1H, s), 3.71 (1H, d, J = 11.6 Hz), 3.66-3.47 (2H, m), 3.30 (1H, d, J = 11.6 Hz), 2.65 (2H, t, J = 6.0 Hz), 1.48 (3H, s), 1.45 (3H, s), 1.06 (3H, s), 1.00 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 174.7, 170.1, 99.0, 77.2, 71.4, 34.1, 33.7, 32.9, 29.4, 22.0, 18.8, 18.7.

[0571] (R)-N-(3-(but-3-yn-1-ylamino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0572] [ka]

[0573] A solution of (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-3-carboxamido)propanoic acid (160 mg, 0.6 mmol) in CHCl (1.5 mL) was treated with HBTU (340 mg, 0.9 mmol), followed by 1-amino-3-butyne (70 μL, 0.9 mmol) and N,N-diisopropylethylamine (150 μL, 0.9 mmol). The reaction mixture was then heated in a microwave at 80 °C for 3.5 h. The reaction was then concentrated under reduced pressure. The crude residue was purified by flash column chromatography (0–5% MeOH / CHCl) to afford the desired alkyne as a red oil (175 mg, 90%).

[0574] 1 H NMR (CDCl3, 400 MHz) δ: 7.05 (1H, br s), 6.24 (1H, br s), 4.10 (1H, s), 3.71 (1H, d, J = 12.0 Hz), 3.65-3.50 (2H, m), 3.30 (1H, d, J = 12.0 Hz), 3.24-3.17 (2H, m), 2.50 (2H, t, J = 8.0 Hz), 2.42 (2H, td, J = 6.4, 2.4 Hz), 2.03 (1H, t, J = 2.4 Hz), 1.48 (3H, s), 1.46 (3H, s), 1.06 (3H, s), 0.99 (3H, s). 13 IR u max (film) / cm -1 : 3293, 2989, 1740, 1650, 1536, 1368, 1232, 1090. [α] D +9.50 (c = 0.5, CHCl3, T = 23.0℃).

[0575] (R)—N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxopropyl-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide, 10

[0576] [ka]

[0577] A solution of 3-azido[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propane (40 mg, 0.1 mmol) in THF (2.5 mL) was treated with a solution of N-(3-(but-3-yn-1-ylamino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (43 mg, 0.1 mmol) in THF (2.5 mL). The resulting mixture was treated with a catalytic amount of CuI (5 mg) followed by N,N-diisopropylethylamine (0.1 mL, 0.6 mmol). The reaction was then heated to 70 °C until complete by TLC analysis (18 h). The reaction mixture was cooled to room temperature and diluted with HO (5 mL). The mixture was extracted with EtOAc (3 × 10 mL), and the combined organic layers were washed with brine (3 × 30 mL), dried over MgSO, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (0–5% MeOH / CHCl) to afford the expected triazole 10 as a red oil (74 mg, 87%).

[0578] 1H NMR (CDCl3, 400 MHz) δ: 7.45 (1H, s), 7.13 (1H, s), 6.91 (1H, d, J = 4.0 Hz), 6.61 (1H, t, J = 5.0 Hz), 6.28 (1H, d, J = 4.0 Hz), 6.15 (1H, s), 4.44 (2H, t, J = 8.0 Hz), 4.07 (1H, s), 3.70 (1H, d, J = 12.0 Hz), 3.62-3.56 (3H, m), 3.54-3.45 (1H, m), 3.28 (1H, d, J = 12.0 Hz), 3.01 (2H, t, J = 8.0 Hz), 2.90 (2H, t, J = 6.4 Hz), 2.56 (3H, s), 2.43 (2H, t, J = 6.0 Hz), 2.39-2.33 (2H, m), 2.28 (3H, s), 1.46 (3H, s), 1.44 (3H, s), 1.03 (3H, s), 0.96 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 171.2, 169.9, 160.5, 156.5, 145.2, 135.0, 133.1, 132.0, 128.8, 123.9, 121.9, 120.6, 116.5, 99.1, 77.1, 71.4, 49.7, 38.7, 35.9, 34.8, 32.9, 29.4, 29.3, 25.8, 25.4, 22.1, 18.9, 18.6,14.9, 11.3. 19 F NMR (CDCl3, 376 MHz) δ: -70.1, -72.0. IR u max (フィルム) / cm -1 : 3357, 2922, 2850, 1740, 1650. [α] D -15.50 (c = 0.1, CHCl3, T = 23.0℃).

[0579] (R)-N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino-3-oxopropyl)-2,4-dihydroxy-3,3-dimethylbutanamide, 9

[0580] [ka]

[0581] A solution of (R)—N-((((3-[4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl]propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide 10 (40 mg, 60 μmol) in CHCN (3 mL) was treated with a catalytic amount of BiCl (5 mg) followed by HO (0.1 mL). The reaction mixture was then stirred at room temperature until complete by TLC analysis (17 h). The reaction was then quenched by the addition of a few drops of saturated aqueous NaHCO and diluted with EtOAc (5 mL). The resulting suspension was filtered through a Celite® bed, which was then thoroughly washed with EtOAc (2 × 5 mL). The combined organic washings were dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (0–10% 2 M NH in MeOH / CHCl) to afford the expected diol 9 as a red oil (10 mg, 28%).

[0582] 1H NMR (CD3OD, 400 MHz) δ: 8.57 (1H, s), 7.45 (1H, s), 7.06 (1H, s), 7.00 (1H, d, J = 4.0 Hz), 6.71 (1H, br s), 6.43 (1H, d, J = 4.0 Hz), 6.24 (1H, s), 4.51 (2H, t, J = 8.0 Hz), 4.09 (1H, s), 3.69 (1H, d, J = 12.0 Hz), 3.63-3.59 (2H, m), 3.56-3.53 (2H, m), 3.15 (1H, d, J = 12.0 Hz), 3.00 (2H, t, J = 8.0 Hz), 2.92 (2H, t, J = 6.4 Hz), 2.51 (3H, s), 2.31 (2H, t, J = 6.0 Hz), 2.20-2.12 (2H, m), 2.07 (3H, s), 1.04 (3H, s), 0.74 (3H, s). 13 C NMR (CD3OD, 100 MHz) δ: 179.0, 167.8, 160.3, 155.8, 145.4, 134.2, 132.4, 130.8, 129.6, 124.3, 122.5, 120.9, 116.2, 100.5, 77.4, 71.8, 50.1, 38.7, 35.9, 35.1, 31.6, 29.0, 25.5, 18.7, 18.4, 13.0, 11.4. 19 F NMR (CD3OD, 376 MHz) δ: -74.1, -76.0. IR u max (film) / cm -1 : 3491, 2927, 1843, 1644, 1557, 1411, 1258. [α] D -26.00 (c = 0.02, CHCl3, T = 23.0℃).

[0583] 7-(3-Azidopropyl)-3-(2-carboxyethyl)-5,5-difluoro-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium-5-uidate

[0584] [ka]

[0585] A solution of 7-(3-azidopropyl)-5,5-difluoro-1-(3-methoxy-3-oxopropyl)-5H-dipyrrolo[1,2-c:1',2'-F][1,3,2]diazaborinin-4-ium-5-ide (114 mg, 315 μmol) in THF (13 mL) was treated with HO (7 mL) and concentrated HCl (5 mL). The mixture was stirred overnight at room temperature, then diluted with HO and extracted with CHCl. ​​The organic extract was dried over NaSO and concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: EtOAc; 7:3) to give the desired acid as a red solid in 68% yield.

[0586] 1 H NMR (CDCl3, 400 MHz) δ: 7.14 (1H, s), 7.01 (2H, dd, J = 11.9, 4.1 Hz), 6.36 (2H, d, J = 4.1 Hz), 3.40 (2H, t, J = 6.9 Hz), 3.32 (2H, t, J = 7.5 Hz), 3.07 (2H, t, J = 7.7 Hz), 2.84 (2H, t, J = 7.5 Hz), 2.08-2.00 (2H, m). 13 C NMR: (CDCl3, 100 MHz) δ: 161.56, 160.42, 134.85, 130.79, 130.63, 127.97, 118.56, 51.02, 33.05, 29.84, 28.14, 26.17, 24.06, 14.25. 19 F NMR: (377 MHz, CDCl3) δ -143.75, -143.84, -143.93, -144.02. IR ν max (film) / cm -1 2954, 2925, 2854, 2097, 1709, 1604, 1439, 1114.

[0587] (rac)-Praziquanamine-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one

[0588] [ka]

[0589] A 1N HCl solution (7.5 mL) was added to a 25 mL round-bottom flask containing praziquantel (250 mg, 0.8 mmol) in 2 mL of EtOH. The mixture was refluxed for 60 h. After this time, the reaction was cooled to room temperature, washed with 5 mL of EtOAc, and cooled in an ice bath. 5 M NaOH was then added to adjust the pH to 12-14 (using pH paper). The aqueous layer was extracted with CHCl, and the organic layer was dried over NaSO and concentrated in vacuo. The pale yellow solid was purified by column chromatography (CHCl:MeOH; 9:1) to give the pure product, praziquantel, in 83% yield (135 mg). The obtained NMR data are consistent with those previously reported (PLoS Negl Trop Dis. 2011 Sep;5(9):e1260).

[0590] 1 H NMR (CDCl3, 400 MHz) δ: 7.25-7.09 (4H, m), 4.90-4.83 (1H, m), 4.80 (1H, dd, J = 10.0, 4.5 Hz), 3.73 (1H, dd, J = 13.0, 4.7 Hz), 3.60 (2H, dd, J = 56.8, 17.4 Hz), 3.05-2.68 (4H, m), 2.00 (1H, s).

[0591] 5,5-Difluoro-1,3-dimethyl-7-(3-oxo-3-(4-oxo-3,4,6,7-tetrahydro-l-pyrazino[2,1-a]isoquinoline 2(11bH)-yl)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium-5-uido, 12

[0592] [ka]

[0593] A solution of BODIPY acid (53 mg, 0.18 mmol) and PZQ amine (35 mg, 0.18 mmol) in 1 mL of CHCl was treated with EDC (80 mg, 0.36 mmol), and the reaction was stirred overnight at room temperature. Upon completion of the reaction, as indicated by TLC (CHCl:MeOH; 95:5), the mixture was concentrated in vacuo. The remaining red oil was dissolved in EtOAc and washed with saturated aqueous NaCO and brine. The organic layer was dried over NaSO and concentrated under reduced pressure. The crude residue was purified by column chromatography (CHCl:MeOH; 98:2) to give a red solid in 90% yield (80 mg). The obtained NMR data are consistent with those previously reported. Mol. Biochem. Parasitol. 164:57-65, 2009.

[0594] 1 H NMR (CDCl3, 400 MHz) δ: 7.28-7.00 (4H, m), 6.88 (1H, t, J = 3.7 Hz), 6.38 6.09 (1H, m), 5.19-5.12 (1H, m), 4.95-4.62 (2H, m), 4.54-4.35 (1H, m), 3.90 (1H, dd, J = 86.2, 18.0 Hz), 3.34 (1H, t, J = 7.5 Hz), 3.15-2.70 (4H, m), 2.59 (2H, d, J = 8.3 Hz), 2.26 (2H, s), 1.28 (2H, s).

[0595] 3-(3-Azidopropyl)-5,5-difluoro-7-(3-oxo-3-(4-oxo-3,4,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium-5-ui

[0596] [ka]

[0597] A solution of the bis-functionalized BODIPY acid (40 mg, 0.11 mmol) and PZQ amine (23 mg, 0.11 mmol) in 1 mL of CHCl was treated with EDC (55 mg, 0.23 mmol), and the resulting mixture was stirred at room temperature overnight. Upon completion of the reaction, as determined by TLC (CHCl:MeOH; 95:5), the mixture was concentrated in vacuo. The remaining red oil was dissolved in EtOAc and washed with saturated aqueous NaCO and brine. The organic layer was dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (CHCl:MeOH; 97:3) to afford the desired product as a red solid in 90% yield (80 mg).

[0598] 1 H NMR (CDCl3, 400 MHz) δ: 7.32-6.96 (4H, m), 6.39 (1H, td, J = 11.0, 4.1 Hz), 5.15 (1H, dd, J = 13.4, 2.8 Hz), 4.87-4.67 (1H, m), 4.46-4.34 (1H, m), 3.92 (1H, dd, J = 83.6, 18.0 Hz), 3.44-3.30 (2H, m), 3.18-2.69 (4H, m), 2.11-1.96 (1H, m), 1.60 (1H, s), 1.30-1.20 (1H, m), 1.25 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 170.33, 164.18, 135.42, 134.90, 132.83, 132.37, 129.45, 127.63, 127.12, 126.84, 125.69, 119.98, 76.84, 55.65, 55.03, 51.04, 48.97, 45.39, 39.16, 29.84, 28.87, 28.17, 26.21, 24.46. IR ν max (film) / cm -12924, 2854, 2096, 1649, 1259, 1114. HRMS (ESI) C 27 H 28 BF2N7O2[M+Na] + Calculated value: 553.2294 m / z, observed value: 553.2283 m / z.

[0599] 5,5-Difluoro-7-(3-oxo-3-(4-oxo-3,4,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-2(11bH)-yl)propyl)-3-(3-(4-(2-((E)-3-((R)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylamido)ethyl)-1H-1,2,3-triazol-1-yl)propyl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-4-ium-5-ui de, 13

[0600] [ka]

[0601] A solution of PZQ-BODIPY-azide (37 mg, 70 μmol) and pantothenate-derived alkyne (22 mg, 70 μmol) in dry THF (1.5 mL) was treated with catalytic amounts of CuI and DIPEA. The resulting mixture was heated at 40 °C and stirred overnight. When TLC indicated consumption of the starting material (CHCl:MeOH; 96:4), the reaction was cooled to room temperature, and the crude product was purified by column chromatography. The compound was obtained as a dark red solid in 20% yield (11 mg) as a mixture of triazoles.

[0602] 1H NMR (CDCl3, 400 MHz) δ 8.30 (1H, d, J = 11.3 Hz), 7.81 (1H, dt, J = 20.3, 10.2 Hz), 7.44 (1H, s), 7.35 (1H, s), 7.26-6.92 (8H, m), 6.46-6.28 (2H, m), 5.75 (1H, dd, J = 13.8, 4.5 Hz), 5.15 (1H, dd, J = 13.3, 3.0 Hz), 4.88-4.68 (2H, m), 4.47-4.32 (3H, m), 4.15 (1H, dd, J = 6.8, 5.2 Hz), 4.04 (1H, d, J = 17.5 Hz), 3.91-3.57 (4H, m), 3.39-3.24 (3H, m), 3.07-2.71 (8H, m), 2.37 (2H, dq, J = 15.0, 7.5 Hz), 2.17 (2H, s), 1.46 (3H, s), 1.38 (3H, s) 0.97 (3H, s), 0.87 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ 168.1, 164.2, 134.9, 129.4, 127.1, 99.5, 76.8, 71.4, 50.9, 33.4, 29.5, 22.0, 18.9, 18.8. 19 HRMS (ESI) C 43 H 52 BF2N9O6[M+Na] + The calculated value is 861.4030 m / z, and the measured value is 861.4004 m / z.

[0603] Compound synthesis - クマリンコンジュゲート エチル2-(7-(ジメチルアミノ)-2-オキソ-2H-クロメン-4-イル)アセテート

[0604]

change

[0605] The ZnCl2 was stirred under vacuum at 140 °C for 18 h, finally dried over a flame, then cooled and weighed. A solution of 3-(dimethylamino)phenol (2.92 mL, 16.0 mmol) in EtOH (10 mL) was heated with ethyl acetone dicarboxylate (2.00 g, 14.6 mmol) followed by ZnCl2 (2.38 g, 17.5 mmol). The resulting suspension was then heated at reflux for 16 h, cooled to room temperature, and poured onto ice (50 g). The aqueous phase was extracted with CHCl2 (3 × 30 mL), and the combined organic phases were washed with brine (50 mL), dried (NaSO4), filtered, and concentrated in vacuo to give the crude product as a dark purple oil. The crude residue was purified by flash column chromatography (20–50% EtOAc / petroleum ether) to give the coumarin product as a pale yellow solid (1.94 g, 48%). The NMR data are consistent with the literature (see Ma et al.).

[0606] 1 H NMR (CDCl3, 400 MHz) δ: 7.40 (1H, d, J = 9.0 Hz), 6.61 (1H, dd, J = 9.0, 2.6 Hz), 6.51 (1H, d, J = 2.6 Hz), 6.05 (1H, br s), 4.18 (2H, q, J = 7.1 Hz), 3.67 (2H, d, J = 0.9 Hz), 3.05 (6H, s), 1.25 (3H, t, J = 7.1 Hz); 13 C NMR (CDCl3, 100 MHz) δ: 169.1, 161.7, 156.0, 153.0, 148.4, 125.3, 110.7, 109.0, 108.5, 98.4, 61.5, 40.1, 38.2, 14.1.

[0607] 2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetic acid

[0608] [ka]

[0609] To a solution of ethyl 2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetate (1.92 g, 6.97 mmol) in THF (42 mL) was added a solution of LiOH (338 mg, 14.1 mmol) in HO (85 mL). The resulting solution was stirred at room temperature for 4 h, at which time the reaction mixture was washed with EtO (2 × 100 mL). The aqueous layer was then acidified to pH 2 with 1 M HCl, and the resulting yellow precipitate was filtered off to give the product acid (1.24 g, 72%) as a yellow solid. NMR data are consistent with the literature (see Ma et al.).

[0610] 1 H NMR ((CD3)2CO, 400 MHz) δ: 7.56 (1H, d, J = 8.9 Hz), 6.76 (1H, dd, J = 8.9, 2.6 Hz), 6.54 (1H, d, J = 2.6 Hz), 6.08 (1H, br s), 3.85 (2H, d, J = 0.7 Hz), 3.10 (6H, s), 2.88 (1H, br s); 13 C NMR ((CD3)2CO, 100 M Hz) δ: 170.8, 161.4, 157.0, 154.1, 150.3, 126.7, 111.2, 109.8, 109.4, 98.7, 40.2, 37.9.

[0611] N-(3-azidopropyl)-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetamide

[0612] [ka]

[0613] A solution of 3-azidopropan-1-amine (50 mg, 0.500 mmol) (known from Zabrodski et al.) in CHCl (1.5 mL) was cooled to 0 °C, followed by the addition of 2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetic acid (49 mg, 0.200 mmol), EDC (46 mg, 0.240 mmol), DIPEA (70 μL, 0.400 mmol), and a catalytic amount of DMAP. After stirring at 0 °C for 15 min, the reaction mixture became cloudy with the formation of a white precipitate. Additional CHCl (3 mL) was added, and the mixture was sonicated to aid stirring. The slurry was allowed to warm to room temperature with stirring for 16 h, after which time the suspension had become a yellow solution. The reaction mixture was concentrated in vacuo to give the crude product as a yellow oil. The crude residue was purified by flash column chromatography (0-2% MeOH / CH2Cl2) to afford the azide product (40 mg, 84%) as a yellow solid.

[0614] 1 H NMR ((CD3)2CO, 400 MHz) δ: 7.58 (1H, d, J = 9.0 Hz), 7.45 (1H, br s), 6.71 (1H, dd, J = 9.0, 2.6 Hz), 6.49 (1H, d, J = 2.6 Hz), 6.02 (1H, br s), 3.66 (2H, d, J = 0.7 Hz), 3.34 (2H, t, J = 6.9 Hz), 3.30-3.25 (2H, m), 3.07 (6H, s), 1.77-1.70 (2H, m); 13 C NMR ((CD3)2CO, 100 MHz) δ: 168.7, 161.5, 156.9, 154.1, 151.3, 126.9, 111.1, 109.7, 109.5, 98.6, 49.7, 40.5, 40.2, 37.5, 29.6; IR ν max (film) / cm -1 3283, 2920, 2881, 2807, 2085, 1705, 1604; HRMS (El) C 16 H 18Calculated for O3N5(MH)+: m / z 328.1415, found m / z 328.1404.

[0615] (R,E)-N-(3-(2-(1-(3-(2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetamido)propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0616] [ka]

[0617] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (59 mg, 0.192 mmol) and N-(3-azidopropyl)-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetamide (46 mg, 0.192 mmol) were dissolved in THF (2 mL). One drop of DIPEA was added via pipette, followed by a catalytic amount of CuI. The resulting mixture was heated to 40 °C for 18 h and then reduced in vacuo to remove the solvent. The crude residue was purified by flash column chromatography (0–5% MeOH / CHCl) to give the desired compound (78 mg, 74%) as a yellow oil.

[0618] 1H NMR ((CD3)2CO, 500 MHz) δ: 9.42 (1H, d, J = 11.0 Hz), 7.81 (1H, dd, J = 14.0, 11.0, Hz), 7.69 (1H, s), 7.60 (1H, d, J = 9.0 Hz), 7.59 (1H, t, J = 5.7 Hz), 7.21 (1H, t, J = 5.7 Hz), 6.72 (1H, dd, J = 9.0, 2.6 Hz), 6.48 (1H, d, J = 2.6 Hz), 6.02 (1H, br s), 5.95 (1H, d, J = 14.0 Hz), 4.35 (2H, t, J = 6.9 Hz), 4.26 (1H, s), 3.75 (1H, d, J = 11.6 Hz), 3.66 (2H, s), 3.53-3.50 (2H, m), 3.26 (1H, d, J = 11.6 Hz), 3.22-3.19 (2H, m), 3.06 (6H, s), 2.39 (2H, t, J = 6.9 Hz), 2.09-2.02 (2H, m), 1.44 (3H, s), 1.37 (3H, s), 1.00 (3H, s), 0.98 (3H, s); 13 C NMR ((CD3)2CO, 125 MHz) δ: 169.3, 168.7, 167.0, 161.6, 156.9, 154.1, 151.4, 145.8, 133.8, 126.9, 123.0, 111.1, 109.8, HRMS (ESI) C 32 H 42 Calculated value of N7O7(MH)+: m / z 636.3151, measured value m / z 636.3133; mp 141-142℃.

[0619] (R,E)-N-(3-(2-(1-(3-(2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetamido)propyl)-1H-1,2,3-triazol-4-yl)ethylamino)-2-oxoprop-1-enyl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0620] [ka]

[0621] (R,E)-N-(3-(but-3-ynylamino)-3-oxoprop-1-enyl)-2,4-dihydroxy-3,3-dimethylbutanamide (49 mg, 0.183 mmol) and N-(3-azidopropyl)-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acetamide (44 mg, 0.183 mmol) were dissolved in THF (2 mL). One drop of DIPEA was added via pipette, followed by a catalytic amount of CuI. The resulting mixture was heated to 70 °C for 18 h and then concentrated in vacuo to remove the solvent to give the crude product as a yellow oil. The crude residue was purified by flash column chromatography (0–10% 7 M NH in MeOH / CHCl) to give the desired compound (33 mg, 30%) as a yellow oil.

[0622] 1H NMR ((CD3)2SO, 500 MHz) δ: 10.20 (1H, d, J = 10.9 Hz), 8.31 (1H, t, J = 5.5 Hz), 7.95 (1H, t, J = 5.7 Hz), 7.87 (1H, s), 7.62 (1H, dd, J = 11.0, 10.9 Hz), 7.55 (1H, d, J = 9.0 Hz), 6.73 (1H, dd, J = 9.0, 2.6 Hz), 6.55 (1H, d, J = 2.6 Hz), 6.01 (1H, br s), 5.86 (1H, d, J = 14.0 Hz), 5.68 (1H, br s), 5.51 (1H, br s), 4.31 (2H, t, J = 6.9 Hz), 3.87 (1H, s), 3.62 (2H, s), 3.38-3.32 (3H, m), 3.16 (1H, d, J = 10.4 Hz), 3.09-3.05 (2H, m), 3.02 (6H, s), 2.76 (2H, t, J = 7.3 Hz), 1.96-1.91 (2H, m), 0.83 (3H, s), 0.80 (3H, s); 13 C NMR ((CD3)2SO, 125 MHz) δ: 170.5, 168.0, 166.1, 160.7, 155.4, 152.8, 151.2, 145.4, 133.0, 125.9, 122.2, 109.4, 109.0, 108.2, 105.1, 97.4, 74.9, 67.4, 46.9, 39.7, 39.6, 39.2, 38.8, 38.5, 29.7, 25.7, 21.1, 19.9; IR ν max (フィルム) / cm -1 3295, 2945, 2929, 2876, 1711, 1653, 1615, 1598; HRMS (ESI) C 29 H 38 Calculated value of N7O7(MH)+: m / z 596.2838, measured value m / z 596.2822; mp 146-147℃.

[0623] Compound Synthesis-Fusun Chemical 5-(phenylthio)-1H-benzo[d]imidazol-2-amine

[0624] [ka]

[0625] To a suspension of fenbendazole (1.00 g, 3.34 mmol) in DMSO (12 mL) and water (4 mL) was added potassium hydroxide (750 mg, 13.4 mmol). The resulting mixture was heated to 80° C. to dissolve the material. After heating for 72 h, the mixture was allowed to cool to room temperature and then diluted with EtOAc (100 mL) and water (100 mL). The layers were separated and the aqueous layer was washed with additional EtOAc (2×75 mL), followed by washing the combined organic layers with brine (100 mL), drying over sodium sulfate, and concentration in vacuo to give the desired guanidine (796 mg, 99%) as a pale purple solid.

[0626] 1 H NMR ((CD3)2CO, 400 MHz) δ: 10.17 (1H, br s), 7.35 (1H, dd, J = 1.7, 0.5 Hz), 7.27-7.22 (3H, m), 7.14 - 7.10 (4H, m), 5.97 (2H, br s).

[0627] tert-Butyl 3-oxo-3-(5-(phenylthio)-1H-benzo[d]imidazol-2-ylamino)propylcarbamate

[0628] [ka]

[0629] 5-(Phenylthio)-1H-benzo[d]imidazol-2-amine (116 mg, 0.482 mmol) and N-(tert-butoxycarbonyl)-L-alanine (137 mg, 0.723 mmol) were dissolved in DMF (4.5 mL) to give a purple solution. After cooling the mixture to 0 °C, HBTU (292 mg, 0.771 mmol) and DIPEA (134 μL, 0.771 mmol) were added and allowed to stir for 16 h. The reaction mixture was diluted with EtOAc (30 mL) and then washed with 1 M NaOH (25 mL), water (25 mL), and brine (4 × 20 mL), dried (NaSO), and reduced in vacuo to give the crude product as a yellow oil. Purification by silica gel column chromatography (0–2% MeOH / CHCl) afforded the desired product (185 mg, 93%) as a white solid.

[0630] 1 H NMR ((CD3)2CO, 400 MHz) δ: 11.87 (2H, br s), 7.80 (1H, s), 7.70 (1H, d, J = 8.1 Hz), 7.40-7.36 (3H, m), 7.31-7.26 (3H, m), 6.25 (1H, br apparent t), 3.60-3.56 (2H, m), 2.93 (2H, t, J = 6.7 Hz), 1.47 (9H, s). HRMS (ESI) C 21 H 24 N4NaO3S (M+Na) + Calculated: m / z 435.1461, Found: m / z 435.1443.

[0631] (R,E)-2,2,5,5-tetramethyl-N-(3-oxo-3-(3-oxo-3-(5-(phenylthio)-1H-benzo[d]imidazol-2-ylamino)propylamino)prop-1-enyl)-1,3-dioxane-4-carboxamide

[0632] [ka]

[0633] tert-Butyl 3-oxo-3-(5-(phenylthio)-1H-benzo[d]imidazol-2-ylamino)propylcarbamate (60 mg, 0.145 mmol) was dissolved in a mixture of CHCl (1 mL) and TFA (1 mL) and stirred at room temperature for 2 h. The reaction mixture was then reduced in vacuo to give the crude TFA salt as a pink oil. The crude salt was dissolved in DMF (1.5 mL) and cooled to 0 °C. Then, (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (27 mg, 0.106 mmol), HBTU (60 mg, 0.159 mmol), and DIPEA (56 μL, 0.318 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 16 hours, then diluted with EtOAc (15 mL), washed with saturated NaHCO (15 mL), water (15 mL), and brine (4 × 10 mL), dried (NaSO), and reduced in vacuo to give the crude product as a yellow oil. Purification by silica gel column chromatography (0–5% MeOH / CHCl) afforded the desired product (31 mg, 53%) as a cream-colored solid.

[0634] 1 H NMR ((CD3)2CO, 500 MHz) δ: 11.33 (1H, br s), 11.30 (1H, br s), 9.41 (1H, d, J = 10.8 Hz), 7.85 (1H, dd, J = 14.0, 10.8 Hz), 7.69 (1H, s), 7.58 (1H, d, J = 8.2 Hz), 7.33-7.18 (7H, m), 5.96 (1H, d, J = 14.0 Hz), 4.23 (1H, s), 3.78 (1H, d, J = 11.7 Hz), 3.68-3.65 (2H, m), 3.29 (1H, d, J = 11.7 Hz), 2.86 (2H, t, J = 6.5 Hz), 1.47 (3H, s), 1.39 (3H, s), 1.03 (3H, s), 1.00 (3H, s).

[0635] Compound synthesis - albendazole conjugates 5-(Propyl)-1H-benzo[d]imidazol-2-amine

[0636] [ka]

[0637] To a suspension of albendazole (886 mg, 3.34 mmol) in MeOH (25 mL) and water (6.5 mL) was added potassium hydroxide (375 mg, 6.68 mmol). The resulting mixture was heated to reflux, giving a yellow solution. After heating for 72 h, TLC analysis indicated the presence of starting material. Additional potassium hydroxide (375 mg, 6.68 mmol) was added, and the mixture was stirred for an additional 24 h. After the mixture was allowed to cool to room temperature, the MeOH was removed in vacuo. The remaining aqueous layer was then diluted with CHCl (3 × 20 mL), and the combined organic layers were washed with brine (50 mL), dried over sodium sulfate, and concentrated in vacuo to give the desired guanidine (501 mg, 72%) as a pale gray solid.

[0638] 1 H NMR ((CD3)2SO, 400 MHz) δ: 10.67 (1H, br s), 7.14 (1H, d, J = 1.5 Hz), 7.03 (1H, dd, J = 8.1, 0.5 Hz), 6.92 (1H, br), 6.20 (2H, s), 2.79 (2H, t, J = 7.3 Hz), 1.56-1.47 (2H, m), 0.94 (3H, t, J = 7.3 Hz). Reference: Zhao et al. Org. Biomol. Chem. 2010, 8, 3328~3337.

[0639] (R,E)-2,2,5,5-tetramethyl-N-[3-oxo-3-[(5-propylsulfanyl-1H-benzimidazol-2-yl)amino]prop-1-enyl]-1,3-dioxane-4-carboxamide

[0640] [ka]

[0641] 5-(Propyl)-1H-benzo[d]imidazol-2-amine (33 mg, 0.159 mmol) and (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (27 mg, 0.106 mmol) were dissolved in DMF (1 mL) at 0 °C. HBTU (60 mg, 0.159 mmol) and DIPEA (28 μL, 0.159 mmol) were added and the resulting mixture was allowed to stir for 16 h while warming to room temperature. The reaction was diluted with EtOAc (15 mL) and washed with saturated NaHCO (15 mL), water (15 mL), and brine (4 × 10 mL), then dried (NaSO) and reduced in vacuo to give the crude product as a yellow oil. Multiple purification attempts by silica gel column chromatography (0-5% MeOH / EtOAc) afforded the desired product (9 mg, 17%) as a white solid.

[0642] 1 H NMR (CDCl3, 500 MHz) δ: 11.65 (1H, br), 7.82 (1H, dd, J = 12.0, 8.9 Hz), 7.40 (1H, d, J = 1.7 Hz), 7.37 (1H, d, J = 8.4 Hz), 7.11 (1H, dd, J = 8.4, 1.7 Hz), 6.33 (2H, br), 6.08 (1H, d, J = 12.0 Hz), 4.31 (1H, s), 3.78 (1H, d, J = 11.8 Hz), 3.39 (1H, d, J = 11.8 Hz), 2.92 (2H, t, J = 7.3 Hz), 1.72-1.65 (2H, m), 1.64 (3H, s), 1.52 (3H, s), 1.10 (6H, s), 1.03 (3H, t, J = 7.3 Hz). 13C NMR (CDCl3, 125 MHz) δ: 169.2, 167.9, 154.5, 140.7, 132.7, 124.3, 122.8, 118.4, 112.8, 99.4, 97.2, 77.4, 71.2, 36.8, 33.4, 29.7, 29.3, 22.6, 21.8, 19.1, 18.7, 13.4. HRMS (ESI) C 22 H 31 N4O4S (M+H) + Calculated m / z 447.2066, found m / z 447.2037.

[0643] tert-Butyl 3-oxo-3-(5-(propyl)-1H-benzo[d]imidazol-2-ylamino)propylcarbamate

[0644] [ka]

[0645] 5-(Propyl)-1H-benzo[d]imidazol-2-amine (100 mg, 0.482 mmol) and N-(tert-butoxycarbonyl)-L-alanine (137 mg, 0.732 mmol) were dissolved in DMF (4.5 mL) to give a purple solution. After cooling the mixture to 0 °C, HBTU (292 mg, 0.771 mmol) and DIPEA (134 μL, 0.771 mmol) were added and allowed to stir for 16 h. The reaction mixture was diluted with EtOAc (30 mL) and then washed with 1 M NaOH (25 mL), water (25 mL), and brine (4 × 20 mL), dried (NaSO), and reduced in vacuo to give the crude product as a yellow oil. Purification by silica gel column chromatography (0–2% MeOH / CHCl) afforded the desired product (125 mg, 69%) as a white solid.

[0646] 1H NMR ((CD3)2SO, 500 MHz) δ: 12.06 (1H, br), 11.54 (1H, s), 7.50-7.36 (2H, m), 7.11 (1H, d, J = 7.9 Hz), 6.91 (1H, t, J = 5.6 Hz), HRMS (ESI) C 18 H 26 N4NaO3S (M+Na) + Calculated m / z 401.1618, found m / z 401.1596.

[0647] (R,E)-2,2,5,5-tetramethyl-N-(3-oxo-3-(3-oxo-3-(5-(propyl)-1H-benzo[d]imidazol-2-ylamino)propylamino)prop-1-enyl)-1,3-dioxane-4-carboxamide

[0648] [ka]

[0649] tert-Butyl 3-oxo-3-(5-(propyl)-1H-benzo[d]imidazol-2-ylamino)propylcarbamate (60 mg, 0.159 mmol) was dissolved in a mixture of CHCl (1 mL) and TFA (1 mL) and stirred at room temperature for 2 h. The reaction mixture was then reduced in vacuo to give the crude TFA salt as a pink oil. The crude salt was dissolved in DMF (1.5 mL) and cooled to 0 °C. Then, (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (27 mg, 0.106 mmol), HBTU (60 mg, 0.159 mmol), and DIPEA (56 μL, 0.318 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 16 hours, then diluted with EtOAc (15 mL), washed with saturated NaHCO (15 mL), water (15 mL), and brine (4 × 10 mL), dried (NaSO), and reduced in vacuo to give the crude product as an off-white solid. Purification by silica gel column chromatography (0–10% MeOH / EtOAc) afforded the desired product (32 mg, 58%) as a white solid.

[0650] 1 H NMR ((CD3)2CO, 500 MHz) δ: 11.52 (2H, br), 9.38 (1H, d, J = 10.8 Hz), 7.83 (1H, dd, J = 14.0, 10.8 Hz), 7.59 (1H, s), 7.45 (1H, d, J = 8.4 Hz), 7.28 (1H, t, J = 5.7 Hz), 7.20 (1H, dd, J = 8.4, 1.7 Hz), 5.93 (1H, d, J = 14.0 Hz), 4.26 (1H, s), 3.75 (1H, d, J = 11.8 Hz), 3.66-3.62 (2H, m), 3.26 (1H, d, J = 11.8 Hz), 2.88 (2H, t, J = 7.3 Hz), 2.83 (2H, t, J = 6.5 Hz), 1.64-1.56 (2H, m), 1.44 (3H, s), 1.36 (3H, s), 1.00-0.97 (9H, m); HRMS (ESI) C25 H 36 N5O5S (M+H) + Calculated value: 518.2437; measured value: 518.2406.

[0651] Compound Synthesis - Protein Conjugates tert-Butyl (5-hydroxypentyl)carbamate

[0652] [ka]

[0653] To a 100 mL flask were added 5-aminopentanol (500 mg, 4.84 mmol) and CHCl (10 mL). The mixture was stirred for 5 min, followed by the addition of EtN (1.3 mL, 9.6 mmol), followed by the dropwise addition of a solution of BocO (1.16 g, 5.3 mmol) in CHCl (10 mL). The reaction mixture was then stirred at room temperature for 16 h, after which it was diluted with water (20 mL). The phases were separated, and the aqueous phase was extracted with CHCl (2 × 20 mL). The combined organic phases were washed with brine (60 mL), dried over NaSO, and filtered. The solvent was removed in vacuo to give a yellow oil. The crude product was purified by chromatography (CHOH / CHCl (1–5%)) to give the desired alcohol as a yellow oil (730 mg, 74%).

[0654] 1 H NMR (CDCl3, 400 MHz) δ: 4.5 (1H, br s), 3.67 (2H, t, J = 6.4 Hz), 3.15 (2H, dd, J = 12.8, 6.3 Hz), 1.6-1.49 (4H, m), 1.45 (9H, s), 1.44-1.39 (2H, m). 13 C NMR (CDCl3, 100 MHz) δ: 156.0, 79.1, 62.7, 40.4, 32.2, 29.8, 28.4, 22.9.

[0655] tert-Butyl (5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamate

[0656] [ka]

[0657] To a 25 mL flask was added tert-butyl (5-hydroxypentyl)carbamate (203 mg, 1 mmol), maleimide (107 mg, 1.1 mmol), triphenylphosphine (283 mg, 1.08 mmol), and THF (5 mL). The mixture was stirred at room temperature for 10 min, after which DIAD (0.23 mL, 1.2 mmol) was added dropwise. The reaction mixture was stirred at room temperature for an additional 16 h, at which point the solvent was reduced in vacuo to give a crude yellow oil. The crude residue was purified by flash chromatography (CHOH / CHCl (0–1%)) to give the desired compound (248 mg, 87%).

[0658] 1 H NMR (CDCl3, 400 MHz) δ: 6.70 (2H, s), 4.5 (1H, br s), 3.53 (2H, t, J = 7.2 Hz), 3.12 (2H, dd, J = 12.6, 6.2 Hz), 1.67-1.57 (2H, m), 1.56-1.47 (2H, m), 1.46 (9H, s), 1.36-1.30 (2H, m). 13 C NMR (CDCl3, 100 MHz) δ: 170.8, 156.3, 134.0, 79.1, 70.1, 40.3, 37.6, 29.8, 28.4, 23.9.

[0659] tert-Butyl (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)carbamate

[0660] [ka]

[0661] To a 25 mL flask was added tert-butyl (3-hydroxypropyl)carbamate (0.25 mL, 1.5 mmol), maleimide (175 mg, 1.8 mmol), PPh3 (472 mg, 1.8 mmol), and THF (7 mL). The mixture was stirred at room temperature for 10 min, after which DIAD (0.35 mL, 1.8 mmol) was added dropwise. The reaction was then stirred at room temperature for an additional 18 h, and the solvent was reduced in vacuo to give a crude yellow oil. This crude oil was purified by column chromatography (ethyl acetate / petroleum ether (0–30%)) to give a colorless oil (257 mg, 69%).

[0662] 1 H NMR (CDCl3, 400 MHz) δ: 6.63 (2H, s), 5.08 (1H, br s), 3.55-3.42 (2H, t, J = 6.7 Hz), 3.06-2.9 (2H, dd, J = 11.9, 5.8 Hz), 1.73-1.63 (2H, q, J = 6.7 Hz), 1.38 (9H, s). 13 C NMR (CDCl3, 100 MHz) δ: 170.9, 170.3, 135.1, 79.3, 70.1, 37.3, 34.9, 28.4.

[0663] 1-(5-aminopentyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate salt

[0664] [ka]

[0665] To a 10 mL flask was added tert-butyl (5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)carbamate (140 mg, 0.49 mmol), CHCl (0.6 mL), and trifluoroacetic acid (0.6 mL). The resulting mixture was then stirred at room temperature for 2.5 h, after which the reaction mixture was diluted with CHCl (2 mL) and quenched with water (5 mL). The phases were separated, and the organic phase was washed with water (5 × 3 mL). The combined organic phase was then extracted with CHCl (3 × 5 mL), after which the water was removed under high vacuum for 36 h. The product was obtained as white crystals (107 mg, 73%).

[0666] 1 H NMR (MeOD, 400 MHz) δ: 6.71 (2H, s), 3.40 (2H, t, J = 7.2 Hz), 2.80 (2H, t, J = 7.2 Hz), 1.62-1.49 (4H, m), 1.30-1.23 (2H, m). 13 C NMR (MeOD, 125 MHz) δ: 171.2, 133.9, 39.1, 36.6, 27.6, 26.5, 23.1. 19 F NMR (MeOD, 470 MHz) δ: -76.8.

[0667] 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propan-1-aminium 2,2,2-trifluoroacetate salt

[0668] [ka]

[0669] To a 10 mL flask was added tert-butyl (3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)carbamate (250 mg, 0.98 mmol), CHCl (1.5 mL), and trifluoroacetic acid (1.5 mL). The mixture was stirred at room temperature for 3 h. After this time, the reaction mixture was diluted with CHCl (2 mL), followed by the addition of water (5 mL). The organic phase was washed with water (5 × 3 mL). The combined aqueous phases were then washed with CHCl (3 × 5 mL), after which the water was removed under high vacuum in vacuo for 36 h. The product was obtained as a white solid (250 mg, 95%).

[0670] 1 H NMR (MeOD, 500 MHz) δ: 6.86 (2H, s), 3.63 (2H, t, J = 7.0 Hz), 2.95 (2H, t, J = 7.5 Hz), 1.97-1.91 (2H, m). 13 C NMR (MeOD, 125 MHz) δ: 171.1, 134.1, 37.0, 34.0, 26.5. 19 F NMR (MeOD, 470 MHz) δ: -76.8 (3F).

[0671] Perfluorophenyl (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido) acrylate

[0672] [ka]

[0673] To a 10 mL flask was added (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (100 mg, 0.38 mmol), 2,3,4,5,6-pentafluorophenol (90 mg, 0.48 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (88 mg, 0.45 mmol), 4-(dimethylamino)pyridine (2 mg, 0.016 mmol), and CHCl (3 mL). The resulting mixture was then stirred at room temperature for 24 h. At this point, the solvent was reduced in vacuo, and the crude product was purified by flash column chromatography (CHCl (100%)). After purification, the starting acrylic acid (10 mg, 10%) was recovered along with the expected product (60 mg, 40%) as a yellow oil and the corresponding Z isomer (60 mg, 40%) also as a yellow oil.

[0674] 1 H NMR (CDCl3, 500 MHz) δ: 8.61 (1H, d, J = 12.5 Hz), 8.1 (1H, dd, J = 12.5,12.5 Hz), 5.76 (1H, d, J = 14 Hz), 4.18 (1H, s), 3.66 (1H, d, J = 12.0 Hz), 3.27 (1H, d, J = 12.0 Hz), 1.45 (3H, s), 1.40 (3H, s), 0.99 (3H, s), 0.96 (3H, s). 13 C NMR (CDCl3, 125 MHz) δ: 168.2, 162.9, 140.2, 140.0, 138.8, 136.8, 99.7, 98.8, 77.0, 71.2, 33.5, 29.4, 21.7, 18.9, 18.8. 19 F NMR (CDCl3, 470 MHz) δ: -152.0, -158.0, -163.0. HRMS (ESI) C 18 H 18 Calculated for F5NO5M+: m / z 423.1105, found m / z 423.1081.

[0675] Perfluorophenyl (R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido) acrylate

[0676] [ka]

[0677] To a 10 mL flask was added (R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (35 mg, 0.13 mmol), 2,3,4,5,6-pentafluorophenol (38 mg, 0.20 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (38 mg, 0.19 mmol), 4-(dimethylamino)pyridine (3 mg, 0.02 mmol), and CHCl (2 mL). The reaction mixture was stirred at room temperature for 18 h, and then the solvent was reduced in vacuo. The crude residue was then purified by flash column chromatography (petroleum ether:ethyl acetate (0–20%)). After purification, the desired ester was obtained as a yellow oil (40 mg, 64%) along with the corresponding E isomer (22 mg, 35%) as a yellow oil.

[0678] 1 H NMR (CDCl3, 500 MHz) δ: 10.80 (1H, d, J = 11.5 Hz), 7.63-7.59 (1H, dd, J = 9.0, 8.5 Hz), 5.39 (1H, d, J = 8.5 Hz), 4.16 (1H, s), 3.64 (1H, d, J = 12.0 Hz), 3.25 (1H, d, J = 12.0 Hz), 1.42 (3H, s), 1.39 (3H, s), 0.99 (3H, s), 0.97 (3H, s). 13 C NMR (CDCl3, 125 MHz) δ: 168.9, 163.6, 142.4, 140.3, 138.9, 136.8, 99.4, 93.8, 77.2, 71.1, 33.3, 31.9, 21.7, 18.9, 18.5. 19F NMR (CDCl3, 470 MHz) δ: -151.6, -158.6, -162.7. HRMS (ESI) C 18 H 18 Calculated for F5NO5M+: m / z 423.1105, found m / z 423.1080.

[0679] Perfluorophenyl (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoate

[0680] [ka]

[0681] To a 10 mL flask was added (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoic acid (150 mg, 0.57 mmol), 2,3,4,5,6-pentafluorophenol (141 mg, 0.76 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (132 mg, 0.68 mmol), 4-(dimethylamino)pyridine (3 mg, 0.02 mmol), and CHCl (4 mL). The resulting mixture was stirred at room temperature for 18 h, after which the solvent was reduced in vacuo. The crude residue was purified by flash column chromatography (petroleum ether-ethyl acetate (0-40%)) to afford the desired ester as a white solid (200 mg, 81%).

[0682] 1 H NMR (CDCl3, 500 MHz) δ: 7.01 (1H, br s), 4.13 (1H, s), 3.78-3.60 (2H, m), 3.71 (1H, d, J = 12.5 Hz), 3.31 (1H, d, J = 11.5 Hz), 2.98 (2H, t, J = 6.5 Hz), 1.47 (3H, s), 1.45 (3H, s), 1.08 (3H, s), 1.00 (3H, s). 13C NMR (CDCl3, 100 MHz) δ: 170.1, 168.1, 99.1, 77.2, 71.4, 34.1, 33.5, 33.0, 29.3, 22.0, 18.7, 18.6. 19 F NMR (CDCl3, 470 MHz) δ: -152.6, -157.6, -162.0. HRMS (ESI) C 18 H 20 Calculated for F5NO5M+: m / z 425.1262, found m / z 425.1237.

[0683] (R,E)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0684] [ka]

[0685] To a 10 mL flask was added perfluorophenyl (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (25 mg, 0.06 mmol), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propan-1-aminium 2,2,2-trifluoroacetate salt (14.5 mg, 0.05 mmol), and CHCl (1.5 mL). The mixture was stirred at room temperature for 10 min, followed by the addition of DIPEA (10 μL, 0.06 mmol). Stirring was continued at room temperature for 20 h, after which the mixture was reduced in vacuo and the residue purified by flash column chromatography (CHCl:CHOH (0–4%)) to give the desired product as a pink solid (8 mg, 48%) (combined with recovered starting material (5 mg)).

[0686] 1H NMR (CDCl3, 400 MHz) δ: 8.23 ​​(1H, d, J = 10.8 Hz), 7.77 (1H, dd, J = 11.2, 11.2 Hz), 6.65 (2H, s), 5.95 (1H, br s), 5.75 (1H, d, J = 14 Hz), 4.12 (1H, s), 3.64 (1H, d, J = 11.6 Hz), 3.52 (2H, t, J = 6 Hz), 3.24 (1H, d, J = 11.6 Hz), 3.22-3.17 (2H, m), 1.76-1.70 (2H, m), 1.44 (3H, s), 1.38 (3H, s), 0.98 (3H, s), 0.93 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 171.1, 168.0, 162.9, 134.2, 123.0, 105.9, 99.4, 77.2, 71.3, 35.9, 34.7, 33.4, 29.4, 28.3, 21.9, 18.8, 18.7.IR ν max (undiluted) / cm -1 : 3323, 3050, 2926, 1705, 1664, 1600, 1097. HRMS (El + G) C 19 H 27 Calculated value for N3O6M+: m / z 393.1900, found m / z 393.1903. [α] D +54.667 (c = 0.3, CHCl3, T = 23.9℃).

[0687] (R,E)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0688] [ka]

[0689] To a 10 mL flask was added perfluorophenyl (R,E)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (60 mg, 0.14 mmol), 1-(5-aminopentyl)-1H-pyrrole-2,5-dione trifluoroacetate salt (38 mg, 0.12 mmol), and CHCl (2.2 mL). The mixture was stirred at room temperature for 10 min, followed by the addition of DIPEA (20 μL, 0.12 mmol). Stirring was continued at room temperature for an additional 72 h, after which the mixture was reduced in vacuo and the concentrate purified by flash column chromatography (CHCl:CHOH (0–4%)) to give the desired product as a colorless oil (32 mg, 60%).

[0690] 1 H NMR (CDCl3, 400 MHz) δ: 8.21 (1H, d, J = 10.4 Hz), 7.69 (1H, dd, J = 10.8,10.8 Hz), 6.63 (2H, s), 5.73 (1H, d, J = 14 Hz), 5.47 (1H, br s), 4.11 (1H, s), 3.63 (1H, d, J = 11.6 Hz), 3.45 (2H, t, J = 6.8 Hz), 3.25-3.17 (2H, m), 3.2 (1H, d, J = 11.6 Hz), 3.22-3.17 (2H, m), 1.76-1.70 (2H, m), 1.57-1.47 (4H, m), 1.43 (3H, s), 1.38 (3H, s), 1.30-1.27 (2H, m), 0.98 (3H, s), 0.93 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 170.9, 168.0, 166.2, 134.0, 132.7, 106.2, 99.4, 77.2, 71.3, 39.3, 37.4, 33.3, 29.4, 28.9, 28.2, 23.8, 21.9, 18.8, 18.7.IR ν max (undiluted) / cm -1: 3329, 3050, 2933, 1701, 1662, 1097, 720. HRMS (ESI) C 21 H 31 Calculated value for N3O6M+: m / z 421.2213, found m / z 421.2207. [α] D +25.09 (c = 2.2, CHCl3, T = 23.0℃).

[0691] (R,Z)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0692] [ka]

[0693] To a 10 mL flask was added perfluorophenyl(R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (25 mg, 0.05 mmol), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propan-1-aminium 2,2,2-trifluoroacetate salt (14.5 mg, 0.05 mmol), and CHCl (1.4 mL). The resulting mixture was stirred at room temperature for 10 min, after which DIPEA (10 μL, 0.06 mmol) was added. The reaction was stirred at room temperature for 72 h, after which the mixture was reduced in vacuo, and the crude residue was purified by flash column chromatography (CHCl:CHOH (0–3%)) to give the desired product as a yellow oil (4 mg, 17%).

[0694] 1H NMR (CDCl3, 400 MHz) δ: 11.58 (1H, d, J = 11.2 Hz), 7.25-7.20 (1H, dd, J = 9.2, 9.2 Hz), 6.65 (2H, s), 5.89 (1H, t, J = 6 Hz), 4.98 (1H, d, J = 8.0 Hz), 4.12 (1H, s), 3.65 (1H, d, J = 11.6 Hz), 3.53 (2H, t, J = 6.4 Hz), 3.25 (1H, d, J = 11.6 Hz), 3.23-3.13 (2H, m), 1.77-1.70 (2H, m), 1.53 (3H, s), 1.39 (3H, s), 0.98 (6H, s). 13 C NMR (CDCl3, 100 MHz) δ: 171.0, 168.8, 167.8, 134.2, 133.4, 100.5, 99.2, 77.2, 71.4, 33.5, 34.8, 33.1, 29.3, 28.3, 22.0, 19.0, 18.6.IR ν max (undiluted) / cm -1 : 3311, 3050, 2928, 1705, 1654, 1097, 696. HRMS (El) C 19 H 27 Calculated value for N3O6M+: m / z 393.1900, Found m / z 393.1898. [α] D +24.00 (c = 0.5, CHCl3, T = 23.8℃).

[0695] (R,Z)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0696] [ka]

[0697] To a 10 mL flask was added perfluorophenyl (R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate (30 mg, 0.07 mmol), 1-(5-aminopentyl)-1H-pyrrole-2,5-dione trifluoroacetate salt (15 mg, 0.05 mmol), and CHCl (1.2 mL). The reaction mixture was stirred at room temperature for 10 min, followed by the addition of DIPEA (10 μL, 0.06 mmol). Stirring was continued at room temperature for 72 h, after which the mixture was reduced in vacuo, and the crude concentrate was purified by flash column chromatography (CHCl:CHOH (0–3%)) to give the desired product as a colorless oil (12 mg, 40%).

[0698] 1 H NMR (CDCl3, 400 MHz) δ: 11.59 (1H, d, J = 11.2 Hz), 7.23-7.18 (1H, dd, J = 4.0, 2.0 Hz), 6.63 (2H, s), 5.37 (1H, t, J = 5.2 Hz), 4.92 (1H, d, J = 9.2 Hz), 4.12 (1H, s), 3.64 (1H, d, J = 11.6 Hz), 3.46 (2H, t, J = 6.8 Hz), 3.25 (1H, d, J = 11.6 Hz), 3.22-3.19 (2H, m), 1.58-1.43 (4H, m), 1.53 (3H, s), 1.37 (3H, s), 1.32-1.21 (2H, m), 1.18 (3H, s), 0.99 (3H, s). 13 C NMR (CDCl3, 100 MHz) δ: 170.9, 169.4, 168.2, 134.1, 133.2, 100.9, 99.2, 77.2, 71.4, 38.9, 37.3, 33.3, 29.7, 28.7, 28.1, 23.8, 22.0, 19.0, 18.6. HRMS (ESI) C 21 H 31 Calculated for N3O6M+: m / z 421.2213, Found m / z 421.2189. IR ν max (undiluted) / cm-1 : 3323, 2926, 2854, 1703, 1656, 1465, 1097, 720. [α] D +12.5 (c = 0.8, CHCl3, T = 23.9℃).

[0699] (R)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0700] [ka]

[0701] To a 25 mL flask was added perfluorophenyl (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoate (95 mg, 0.22 mmol), 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propan-1-aminium 2,2,2-trifluoroacetate salt (80 mg, 0.29 mmol), and CHCl (8 mL). The mixture was then stirred at room temperature for 10 min, after which DIPEA (70 μL, 0.42 mmol) was added. Stirring was continued at room temperature for an additional 72 h, after which the mixture was reduced in vacuo. The crude concentrate was then purified by flash column chromatography (CHCl:methanol (0–3%)) to give the desired product as a colorless oil (52 mg, 59%).

[0702] 1H NMR (CDCl3, 500 MHz) δ: 7.00 (1H, t, J = 7 Hz), 6.65 (2H, s), 6.32 (1H, t, J = 7 Hz), 4.01 (1H, s), 3.67 (1H, d, J = 11.6 Hz), 3.55-3.42 (2H, m), 3.50 (2H, t, J = 8 Hz), 3.21 (1H, d, J = 15 Hz), 3.16-3.11 (2H, m), 2.39 (2H, t, J = 7.5 Hz), 1.74-1.68 (2H, m), 1.38 (3H, s), 1.34 (3H, s), 0.96 (3H, s), 0.90 (3H, s). 13 C NMR (CDCl3, 125 MHz) δ: 171.1, 170.9, 170.0, 134.2, 99.0, 77.2, 71.4, 36.1, 36.0, 34.8, 32.9, 30.9, 29.4, 28.2, 22.1, 18.8, 18.6.IR ν max (undiluted) / cm -1 : 3310, 3098, 2945, 1706, 1647, 1533, 1097, 696. HRMS (ESI) C 19 H 29 Calculated value for N3O6M+: m / z 395.2056, Found m / z 395.2033. [α] D +21.231 (c = 1.3, CHCl3, T = 23.7℃).

[0703] (R)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide

[0704] [ka]

[0705] To a 25 mL flask was added perfluorophenyl (R)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)propanoate (95 mg, 0.22 mmol), 1-(5-aminopentyl)-1H-pyrrole-2,5-dione trifluoroacetate salt (60 mg, 0.20 mmol), and CHCl (5 mL). The mixture was stirred at room temperature for 10 min, after which DIPEA (60 μL, 0.36 mmol) was added. Stirring was continued at room temperature for an additional 72 h, after which the mixture was reduced in vacuo. The crude residue was purified by flash column chromatography (CHCl:CHOH, (0–3%)) to give the desired adduct as a colorless oil (90 mg, 95%).

[0706] 1 H NMR (CDCl3, 500 MHz) δ: 6.97 (1H, t, J = 5.5 Hz), 6.63 (2H, s), 5.98 (1H, br s), 4.00 (1H, s), 3.61 (1H, d, J = 11.5 Hz), 3.58-3.46 (2H, m), 3.44 (2H, t, J = 7.0 Hz), 3.21 (1H, d, J = 11.5 Hz), 3.17-3.12 (2H, m), 2.36 (2H, t, J = 6.0 Hz), 1.56-1.42 (4H, m), 1.39 (3H, s), 1.34 (3H, s), 1.26-1.18 (2H, m), 1.03 (3H, s), 0.80 (3H, s). 13 C NMR (CDCl3, 125 MHz) δ: 170.9, 170.8, 170.2, 134.2, 99.0, 77.2, 71.4, 39.3, 37.4, 36.1, 34.9, 32.9, 29.4, 29.2, 28.1, 23.9, 22.1, 18.8, 18.6.IR ν max (undiluted) / cm -1 : 3330, 2930, 1706, 1656, 1521, 1097, 696. HRMS (ESI) C 21 H 33Calculated value for N3O6M+: m / z 423.2369, found m / z 423.2364. [α] D +24.267 (c = 1.5, CHCl3, T = 23.7℃).

[0707] (R,E)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0708] [ka]

[0709] To a 5 mL flask was added (R,E)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (12 mg, 0.03 mmol), CHCN (0.2 mL), bismuth chloride (1 mg, 0.003 mmol), and HO (10 μL). The reaction mixture was stirred at room temperature for 24 h and then quenched with saturated aqueous NaHCO. The mixture was then diluted with ethyl acetate and filtered through Celite®. The solvent was removed in vacuo, and the crude residue was purified by flash column chromatography (CHCl:methanol:NH (0–5%)) to give the desired product as a colorless oil (10 mg, 92%).

[0710] 1H NMR ((CD3)2CO), 400 MHz) δ: 9.56 (1H, br s), 7.86 (1H, dd, J = 12.0, 11.2 Hz), 7.08 (1H, br s), 6.87 (2H, s), 6.00 (1H, d, J = 13.6 Hz), 5.17 (1H, d, J = 7.0 Hz), 3.95 (2H, d, J = 5.2 Hz), 3.38 (2H, t, J = 6.8 Hz), 3.24 (1H, d, J = 11.6 Hz), 3.12-3.11 (2H, m), 1.64-1.61 (2H, m), 1.16 (6H, s). 13 C NMR ((CD3)2CO), 100 MHz) δ: 170.8, 134.2, 133.0, 105.4, 76.5, 69.4, 39.3, 36.4, 35.2, 28.9, 20.4, 19.7. IR ν max (undiluted) / cm -1 : 3323, 2924, 1701, 1654, 1329, 1188. HRMS (ESI) C 16 H 23 Calculated value for N3O6M+: m / z 353.1587, found m / z 353.1562. [α] D +16.8 (c = 0.5, acetone, T = 24.0°C).

[0711] (R,E)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0712] [ka]

[0713] In a 5 mL flask, (R,E)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (10 mg, 0.02 mmol), CHCN (0.2 mL), bismuth chloride (1 mg, 0.003 mmol), and HO (10 μL) were combined. The reaction mixture was then stirred at room temperature for 24 h and then quenched with saturated aqueous NaHCO. The mixture was diluted with ethyl acetate and filtered through Celite®. The solvent was removed in vacuo, and the crude residue was purified by flash column chromatography (CHCl:methanol:NH (0–5%)) to give the desired product as a colorless oil (3 mg, 33%).

[0714] HRMS(ESI) C 18 H 27 Calculated for N3O6M+: m / z 381.1900, found m / z 381.1880.

[0715] (R,Z)-N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0716] [ka]

[0717] To a 5 mL flask was added (R,Z)—N-(3-((3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (18 mg, 0.05 mmol), CHCN (0.3 mL), bismuth chloride (3 mg, 0.009 mmol), and HO (30 μL). The reaction was stirred at room temperature for 24 h and then treated with a few drops of saturated aqueous NaHCO solution and then diluted with ethyl acetate. The solution was filtered through Celite® and the solvent was removed in vacuo. The crude residue was purified by flash column chromatography (CHCl:methanol:NH (0–5%)) to afford the desired adduct (5 mg, 30%) as a colorless oil.

[0718] 1 H NMR ((CD3)2CO, 400 MHz) δ: 11.85 (1H, d, J = 8.4 Hz), 7.12-7.07 (1H, dd, J = 9.2, 8.8 Hz), 6.73 (2H, s), 5.07 (1H, d, J = 6.0 Hz), 3.98 (1H, s), 3.42-3.30 (3H, m), 3.29 (1H, d, J = 10.8 Hz), 3.09-3.04 (2H, m), 1.75-1.66 (2H, m), 1.16 (6H, s). 13 C NMR ((CD3)2CO), 100 MHz) δ: 170.8, 134.2, 133.0, 100.1, 76.5, 69.4, 39.3, 36.1, 35.1, 28.9, 20.4, 19.7. HRMS (ESI) C 16 H 23 Calculated for N3O6M+: m / z 353.1587, found m / z 353.1567.

[0719] (R,Z)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0720] [ka]

[0721] To a 5 mL flask was added (R,Z)—N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxoprop-1-en-1-yl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (19 mg, 0.04 mmol), CHCN (0.3 mL), BiCl (3 mg, 0.009 mmol), and HO (30 μL). The mixture was stirred at room temperature for 24 h and treated with a few drops of saturated aqueous NaHCO. The mixture was diluted with ethyl acetate and then filtered through Celite®. The solvent was removed in vacuo, and the crude residue was purified by flash column chromatography (CHCl:methanol:NH (0–5%)) to give the desired product as a colorless oil (5 mg, 29%).

[0722] HRMS(ESI) C 18 H 27 Calculated for N3O6M+: m / z 381.1900, found m / z 381.1877.

[0723] (R)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxopropyl)-2,4-dihydroxy-3,3-dimethylbutanamide

[0724] [ka]

[0725] To a 5 mL flask was added (R)-N-(3-((5-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentyl)amino)-3-oxopropyl)-2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamide (100 mg, 0.23 mmol), CHCN (1.6 mL), BiCl (8 mg, 0.02 mmol), and HO (80 μL). The mixture was stirred at room temperature for 24 h and then treated with a few drops of saturated aqueous NaHCO solution. The mixture was then diluted with ethyl acetate and filtered through Celite®. The solvent was removed in vacuo, and the crude residue was purified by flash column chromatography (CHCl:methanol:NH (0–5%)) to give the desired product as a yellow oil (43 mg, 47%).

[0726] 1 H NMR ((CD3)2CO), 500 MHz) δ: 7.52 (1H, t, J = 6.0 Hz), 7.22 (1H, br s),6.73 (2H, s), 3.80 (1H, s), 3.41-3.24 (6H, m), 3.06-3.02 (2H, m), 2.28 (2H, t, J = 5.5 Hz), 1.46-1.34 (4H, m), 1.20-1.18 (2H, m), 0.86 (3H, s), 0.74 (3H, s). 13 C NMR (CDCl3, 125 MHz) δ: 173.6, 170.9, 170.8, 134.2, 76.3, 69.6, 63.3, 39.2, 38.8, 37.1, 35.2, 29.4, 28.0, 23.8, 21.1, 19.8.IR u max (undiluted) / cm -1 : 3310, 2931, 1700, 1646, 1539, 1410, 695. HRMS (ESI) C 18 H 29 Calculated value for N3O6M+: m / z 383.2056, Found m / z 383.2051. [α] D +13.023 (c = 4.3, acetone, T = 23.9°C).

[0727] Compound Synthesis - Ivermectin Conjugates 5-O-(tert-butyldimethylsilyl) avermectin B1a

[0728] [ka]

[0729] A solution of ivermectin B1a (980 mg, 1.1 mmol) in anhydrous DMF (8.0 mL) was treated with imidazole (495 mg, 7.3 mmol), followed by a solution of TBDMSCI (560 mg, 3.28 mmol) in anhydrous DMF (2.0 mL). The reaction mixture was stirred at room temperature for 2.5 h and then diluted with EtO (25 mL) followed by HO (15 mL). The resulting emulsion was then stirred for 0.5 h, the organic layer was separated, and the aqueous phase was extracted with EtO (3 × 25 mL). The combined organic layers were washed with HO (5 × 100 mL) and brine (2 × 100 mL). The combined organic washes were dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, elution gradient 0–30% EtOAc in petroleum ether) to afford the expected product as a white solid (638 mg, 58%).

[0730] 11H NMR (CDCl3, 400 MHz) δ: 5.83 - 5.78 (1H, br m), 5.73 - 5.69 (2H, br m), 5.38 (1H, d, J = 3.2 Hz), 5.32 (1H, br s), 5.29 - 5.28 (1H, br m), 4.97 (1H, d, J = 9.6 Hz), 4.76 (1H, d, J = 3.2 Hz), 4.67 (1H, d, J = 14.4 Hz), 4.56 (1H, d, J = 14.8 Hz), 4.42 (1H, br s), 3.92(1H, br s), 3.85 - 3.78 (2H, br m), 3.77 - 3.72 (1H, br m,), 3.68 - 3.57 (2H, br m), 3.50 - 3.45 (1H, br m), 3.41 (3H, s), 3.40 (3H, s), 3.36 (1H, br s), 3.25 - 3.19 (2H, br m), 3.14 (1H, t, J = 8.8 Hz), 2.53 (1H, apparently t, J = 6.8 Hz), 2.35 - 2.29 (2H, br m), 2.28 - 2.25 (1H, br m), 2.21 (1H, dd, J = 13.2, 5.0 Hz), 1.98 (1H, dd, J = 12.0, 4.4 Hz), 1.77 (3H, s), 1.74 - 1.71 (1H, br m), 1.64 (1H, d, J = 11.1 Hz), 1.59 - 1.52 (7H, br m), 1.49 (3H, s), 1.47 - 1.39 (2H, br m), 1.33 (1H, t, J = 11.6 Hz), 1.26 (3H, br s), 1.25 (3H, br s), 1.14 (3H, d, J = 6.8 Hz), 0.96 - 0.92 (3H, br m), 0.91 (9H, s), 0.87 - 0.81 (4H, br m), 0.76 (3H, d, J = 4.4 Hz), 0.12 (6H, s). 13C NMR (CDCl3, 100 MHz) δ: 174.0, 140.2, 137.5, 137.4, 135.0, 124.8, 119.3, 118.3, 117.3, 98.4, 97.4, 94.7, 81.8, 80.3, 80.2, 80.0, 79.3, 78.1, 76.5, 76.0, 69.4, 68.6, 68.1, 67.9, 67.2, 56.4, 56.3, 45.7, 41.1, 39.6, 36.8, 35.7, 35.4, 34.5, 34.1, 31.2, 27.3, 26.8, 25.8, 20.2, 20.0, 18.4, 17.6, 17.4, 15.1, 12.4, 12.0, -4.6, -4.8.

[0731] 4''-O-[(R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate]-5-O-(tert-butyldimethylsilyl)avermectin B1a

[0732] [ka]

[0733] 5-O-(tert-butyldimethylsilyl) avermectin B 1aA solution of (45 mg, 40 μmol) in CHCl (1 mL) was treated sequentially with DCC (19 mg, 90 μmol), followed by DMAP (10 mg, 80 μmol) and a solution of (R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylic acid (10 mg, 40 μmol) in CHCl (1.5 mL). The reaction mixture was stirred at room temperature until completion (18 h), as indicated by TLC analysis. The reaction was then diluted with CHCl (5 mL). The organic phase was washed with 1 M HCl (7 mL), followed by saturated aqueous NaHCO (7 mL) and brine (7 mL). The organic layer was dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, elution gradient 0-10% EtOAc in petroleum ether) to give TBS-protected ivermectin as a colorless oil (30 mg, 61%).

[0734] 11H NMR (CDCl3, 400 MHz) δ: 11.12 (1H, d, J = 11.6 Hz), 7.52 - 7.43 (1H, m), 5.87 - 5.82 (1H, br m), 5.79 - 5.71 (2H, br m), 5.42 (1H, br s), 5.36 - 5.31 (2H, br m), 5.19 (1H, d, J = 8.9 Hz), 5.00 (1H, d, J = 9.9 Hz), 4.79 (1H, br s), 4.77 (1H, apparently t, J = 9.6 Hz), 4.70 (1H, d, J = 14.6 Hz), 4.60 (1H, d, J = i4.5 Hz), 4.46 (1H, br s), 4.22 (1H, s), 3.96 (1H, br s), 3.92 - 3.83 (3H, m), 3.73 (1H, d, J = 11.7 Hz), 3.70 - 3.60 (3H, m), 3.45 (3H, s), 3.42 - 3.38 (4H, m), 3.34 (1H, d, J = 11.7 Hz), 3.28 - 3.20 (2H, m), 2.53 (1H, br s), 2.38 - 2.32 (2H, m), 2.31 - 2.22 (2H, m), 2.00 (1H, dd, J = 11.7, 3.8 Hz), 1.81 (3H, s), 1.79 - 1.62 (9H, m), 1.59 (3H, s), 1.47 (3H, s), 1.44 - 1.41 (5H, m), 1.37 (1H, t, J = 12.4 Hz), 1.28 (3H, br s), 1.27 (3H, br s), 1.18 (3H, d, J = 6.3 Hz), 1.07 (3H, s), 1.05 (3H, s), 0.99 - 0.96 (3H, m), 0.94 (9H, s), 0.88 - 0.85 (4H, m), 0.81 - 0.79 (1H, m), 0.15 (6H, s). 13 It should be noted that there is an unclear "見かけ上t" in the original text which is translated as "apparently t" tentatively, and "i4.5 Hz" might be a typo in the original which is kept as is for translation.C NMR (CDCl3, 100 MHz) δ: 174.0, 168.9, 167.2, 140.3, 137.5, 137.4, 136.6, 135.0, 124.8, 119.3, 118.3, 117.2, 99.3, 98.4, 98.2, 97.5, 94.8, 81.9, 80.8, 80.2, 80.0, 79.2, 77.2, 77.1, 76.6, 75.7, 75.6, 71.3, 69.5, 68.7, 67.9, 67.1, 57.2, 56.5, 45.7, 41.1, 39.6, 36.8, 35.7, 35.4, 35.2, 34.5, 34.1, 33.3, 31.2, 29.3, 27.2, 26.9, 25.8, 21.9, 20.3, 20.0, 18.9, 18.6, 18.4, 17.4 (2C), 15.2, 12.4, 12.1, -4.5, -4.8. HRMS (ESI) C 68 H 109 NO 18 Si [M-CO2] + Calculated m / z 1211.7516, Found m / z 1211.6699. IR u max (film) / cm -1 : 3420, 2945, 2879, 1775, 1670, 1550, 1392, 1128. [α] D +12.21 (c = 0.7, CHCl3, T = 23.1℃).

[0735] 4''-O-[(R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate]avermectin B1a, 14

[0736] [ka]

[0737] A solution of 4"-O-[(R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate]-5-O-(tert-butyldimethylsilyl)avermectin B1a (30 mg, 20 μmol) in MeOH (2.5 mL) was treated with a catalytic amount of p-TsOH (3 mg). The reaction mixture was stirred at 18 °C for 30 min. The reaction was then diluted with HO (17 mL) followed by EtOAc (20 mL). The organic layer was washed with HO (3 × 20 mL) and brine (20 mL). The organic phase was dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, elution gradient 0–3% MeOH / CH2Cl2) to give 4″-O-[(R,Z)-3-(2,2,5,5-tetramethyl-1,3-dioxane-4-carboxamido)acrylate]avermectin B1a as a yellow oil (16 mg, 58%).

[0738] 1H NMR (CDCl3, 400 MHz) δ: 11.27 (1H, d, J = 11.6 Hz), 7.52 (1H, dd, J = 11.6 Hz, 9.6 Hz), 5.89 (1H, d, J = 9.2 Hz), 5.82-5.69 (2H, m), 5.44 (1H, br s), 5.42-5.32 (2H, m), 5.23 (1H, d, J = 9.2 Hz), 5.00 (1H, d, J = 10.0 Hz), 4.80 (1H, br s), 4.75 (1H, d, J = 9.5 Hz), 4.72-4.65 (2H, m), 4.32 (1H, d, J = 6.0 Hz), 4.23 (1H, s), 4.13 (1H, br s), 3.99 (2H, m), 3.92-3.84 (2H, m), 3.73-3.62 (4H, m), 3.58 (1H, d, J = 6.8 Hz), 3.45 (3H, br s), 3.38 (3H, br s), 3.30 (1H, br s), 3.28-3.21 (3H, m), 2.54 (1H, br s), 2.39-2.32 (2H, m), 2.32-2.22 (2H, m), 2.00 (1H, dd, J = 12.0, 4.4 Hz), 1.89 (3H, s), 1.77 (1H, d, J = 11.6 Hz), 1.67 (1H, d, J = 9.6 Hz), 1.62-1.47 (16H, m), 1.47-1.34 (3H, m), 1.29-1.25 (3H, m), 1.20-1.15 (6H, m), 1.07 (3H, s), 1.00 (3H, s), 0.95 (3H, t, J = 7.2 Hz), 0.87 (3H, d, J = 6.6 Hz), 0.83-0.78 (4H, m). 13C NMR (CDCl3, 100 MHz) δ: 174.0, 168.9, 167.2, 140.3, 138.0, 137.9, 137.2, 135.0, 124.7, 120.4, 118.3, 118.0, 107.0, 98.5, 98.3, 97.4, 94.8, 81.8, 80.7, 80.4, 79.2, 79.0, 77.9, 77.2, 76.3, 75.8, 75.7, 71.3, 68.6, 68.5, 67.7, 67.2, 67.1, 57.1, 56.5, 45.7, 41.1, 39.7, 36.9, 35.7, 35.4, 35.1, 34.5, 34.1 (2C), 31.2, 28.0, 27.2, 22.9, 20.8, 20.5, 20.2, 20.0, 18.8, 18.4, 17.4 (2C), 15.1, 12.4, 12.1. HRMS (ESI) C 62 H 95 NO 18 [M-CO2] + Calculated m / z 1097.6651, Found m / z 1097.1176. IR u max (film) / cm -1 : 3680, 2947, 2879, 1783, 1645, 1498, 1288. [α]D +4.50 (c = 0.2, CHCl3, T = 23.0℃).

[0739] Compound synthesis - BODIPY-ivermectin conjugate 5-(3-Azidopropyl)-1H-pyrrole-2-carbaldehyde

[0740] [ka]

[0741] Anhydrous DMF (20 mL) was cooled to 0 °C and then treated with POCl (0.6 mL, 6.6 mmol). The resulting solution was then stirred at 0 °C for 5 minutes and then at room temperature for an additional 30 minutes. The reaction was then cooled to 0 °C again and treated with a solution of 2-(3-azidopropyl)-1H-pyrrole (805 mg, 5.3 mmol) in anhydrous DMF (2 mL). The mixture was then heated to 40 °C until complete by TLC analysis (18 h). The reaction was once again cooled to room temperature, diluted with EtOAc (5 mL), and treated with 4 M aqueous NaOH (5 mL). The phases were separated, and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with HO (5 × 20 mL), brine (2 × 20 mL), and dried over NaSO. The resulting solution was concentrated under reduced pressure to give the desired aldehyde as a brown oil (490 mg, 51%). The product was used without further purification.

[0742] 1 H NMR (CDCl3, 400 MHz) δ: 9.95 (1H, br s), 9.41 (1H, s), 6.93-6.91 (1H, m), 6.13-6.12 (1H, m), 3.01 (2H, t, J = 8.0 Hz), 2.72 (2H, t, J = 7.0 Hz), 1.8- 1.6 (2H, m). 13 C NMR (CDCl3, 100 MHz) δ: 178.3, 129.7, 128.5, 127.8, 109.6, 60.4, 28.0, 24.4. HRMS (ESI) C8H 10 Calculated for NO [M]: m / z 178.0855, found m / z 178.0851. IR u max (film) / cm -1 : 3233, 2831, 2099, 1735, 1653, 1376.

[0743] tert-Butyl (E)-3-(1H-pyrrol-2-yl)acrylate

[0744] [ka]

[0745] A solution of pyrrole-2-carboxaldehyde (1.5 g, 15.2 mmol) in benzene (110 mL) was treated with (tert-butoxycarbonylmethylene)triphenylphosphorane (10.0 g, 26.5 mmol). The reaction mixture was heated to 80 °C until complete by TLC analysis (22 h). The reaction was then cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (0–20% EtOAc / PE) to afford the expected ester as an orange oil (2.45 g, 83%).

[0746] 1 H NMR (CDCl3, 400 MHz) δ: 9.00 (1H, br s), 7.49 (1H, d, J = 16.0 Hz), 6.92 (1H, apparent s), 6.55 (1H, apparent s), 6.29 (1H, apparent s), 6.01 (1H, d, J = 16.0 Hz), 1.55 (9H, s). 13 C NMR (CDCl3, 100 MHz) δ: 167.1, 133.3, 128.5, 122.0, 113.7, 113.3, 110.8, 80.2, 28.2. HRMS (ESI) C 11 H 15 NO2[M+Na] + Calculated value: m / z 216.0995, Found value: m / z 216.0966. IR u max (film) / cm -1 : 3358, 2919, 2848, 1713, 1632, 1150.

[0747] tert-Butyl 3-(1H-pyrrol-2-yl)propanoate

[0748] [ka]

[0749] A solution of (E)-3-(1H-pyrrol-2-yl)acrylate (2.4 g, 12.4 mmol) in anhydrous MeOH (95 mL) was stirred under an argon atmosphere for 5 minutes. Pd / C (10%, 160 mg, 7 mol%) was added to the solution, and the reaction was placed under a hydrogen atmosphere and stirred at room temperature until complete (18 hours) by TLC analysis. The reaction was filtered through a Celite® bed and washed with MeOH (2 × 25 mL). The organic phases were combined and concentrated in vacuo to give the desired ester as a brown oil (2.23 g, 92%). The resulting product required no further purification.

[0750] 1 H NMR (CDCl3, 400 MHz) δ: 8.62 (1H, br s), 6.69 (1H, apparent s), 6.12 (1H, apparent d, J = 2.4 Hz), 5.93 (1H, apparent s), 2.89 (2H, t, J = 6.4 Hz), 2.57 (2H, t, J = 6.4 Hz), 1.47 (9H, s). 13 C NMR (CDCl3, 100 MHz) δ: 173.6, 131.3, 116.7, 107.8, 105.4, 80.7, 35.7, 28.1, 22.6. HRMS (ESI) C 11 H 17 NO2[M+Na] + Calculated m / z 218.1151, Found m / z 218.1168. IR u max (film) / cm -1 : 3394, 2919, 2848, 1644.

[0751] tert-Butyl 3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate

[0752] [ka]

[0753] A solution of 5-(3-azidopropyl)-1H-pyrrole-2-carbaldehyde (330 mg, 1.9 mmol) in CHCl (8 mL) at 0° C. was treated with a solution of tert-butyl 3-(1H-pyrrol-2-yl)propanoate (346 mg, 1.8 mmol) in CHCl (2 mL). The resulting mixture was then treated dropwise with POCl (150 μL, 1.5 mmol) and stirred at room temperature for 6.5 h before being cooled to 0° C. again. The reaction mixture was then treated sequentially with BF.EtO (0.9 mL, 7.2 mmol), followed by N,N-diisopropylethylamine (1.5 mL, 8.5 mmol). The mixture was then stirred at room temperature until complete by TLC analysis (18 h). The reaction was quenched with HO (10 mL), diluted with CHCl (5 mL), and filtered through a Celite® bed. The Celite® was washed with CHCl (2 × 10 mL), and the organic phases were combined. The combined organic washes were dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, elution gradient 0–15% EtOAc in petroleum ether) to afford the expected ester as a red oil (71 mg, 9%).

[0754] 1 H NMR (CDCl3, 400 MHz) δ: 7.13 (1H, s), 7.00-6.92 (2H, m), 6.37- 6.34 (2H, m), 3.35-3.27 (4H, m), 2.80 (2H, t, J = 8.0 Hz), 2.67 (2H, t, J = 8.0 Hz), 1.46 (9H, s), 1.27 (2H, apparently t, J = 7.2 Hz). 13 C NMR (CDCl3, 100 MHz) δ: 172.7, 161.6, 161.1, 134.7, 134.6, 130.6, 130.2, 127.8, 118.7, 118.1, 80.7, 51.8, 34.2, 32.8, 29.7, 28.2, 24.2. 19F NMR (CDCl3, 376 MHz) δ: -144.17, -144.26, -144.35, -144.44. HRMS (ESI) C 19 H 24 BF2N5O2[M-N3] + Calculated m / z 361.1899, Measured m / z 361.3293. IR u max (film) / cm -1 : 3172, 2978, 2150, 1733, 1609, 1490, 1439, 1155. λ exc =467nm. (510nm emission, c=0.2nM, MeOH). λ emis =516nm, (315nm excitation, c=0.2nM, MeOH).

[0755] Methyl 3-(5-formyl-1H-pyrrol-2-yl)propanoate

[0756] [ka]

[0757] Anhydrous DMF (10 mL) was cooled to 0 °C and treated with POCl (0.6 mL, 6.6 mmol). The resulting solution was then stirred at 0 °C for 5 minutes and then at room temperature for an additional 30 minutes. The reaction was once again cooled to 0 °C and treated with a solution of methyl 3-(1H-pyrrol-2-yl)propanoate (800 mg, 5.2 mmol) in anhydrous DMF (2 mL). The mixture was then heated to 40 °C until complete by TLC analysis (14 h). The reaction was once again cooled to room temperature, diluted with EtOAc (18 mL), and treated with 4 M aqueous NaOH (5 mL). The phases were separated, and the aqueous layer was extracted with EtOAc (3 × 18 mL). The combined organic layers were washed with HO (5 × 30 mL), brine (2 × 30 mL), and dried over NaSO. The resulting solution was concentrated under reduced pressure to give the desired aldehyde as a brown oil (630 mg, 66%). The product was used without further purification.

[0758] 1 H NMR (CDCl3, 400 MHz) δ: 10.01 (1H, br s), 9.42 (1H, s), 6.89 (1H, apparent t, J = 4.0 Hz), 6.1-6.0 (1H, apparent t, J = 4.0 Hz), 3.73 (3H, s), 3.02 (2H, t, J = 7.0 Hz), 2.72 (2H, t, J = 7.1 Hz). 13 C NMR (CDCl3, 100 MHz) δ: 178.4, 173.3, 116.8, 132.3, 122.1, 109.6, 52.0, 33.3, 22.8. HRMS (ESI) C9H 11 NO3[M+H] + Calculated value: m / z 182.0739, Found value: m / z 182.0828. IR u max (film) / cm -1 : 3248, 2924, 2853, 1735, 1647.

[0759] Methyl 3-[3-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-5-yl]propanoate

[0760] [ka]

[0761] A solution of 2-(3-azidopropyl)-1H-pyrrole (100 mg, 0.7 mmol) in CHCl (5 mL) at 0° C. was treated with a solution of 3-(5-formyl-1H-pyrrol-2-yl)propanoate (60 mg, 0.3 mmol) in CHCl (5 mL). The resulting mixture was then treated dropwise with POCl (100 μL, 1.0 mmol) and stirred at room temperature for 6.5 hours before being cooled to 0° C. again. The reaction mixture was then treated sequentially with BF.EtO (0.2 mL, 1.6 mmol), followed by N,N-diisopropylethylamine (0.3 mL, 1.7 mmol). The reaction was stirred at room temperature until complete by TLC analysis (18 hours). The reaction mixture was then quenched with H₂O (10 mL), diluted with CHCl (5 mL), and filtered through a bed of Celite®. The Celite® was washed with CH2Cl2 (2 × 10 mL) and the organic phases were combined. The combined organic washes were dried over Na2SO4 and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (silica gel, elution gradient 0 to 30% EtOAc in petroleum ether) to afford the expected ester as a red oil (31 mg, 26%).

[0762] 1 H NMR (CDCl3, 400 MHz) δ: 7.15 (1H, s), 7.04 (1H, d, J = 4.0 Hz), 7.01 (1H, d, J = 4.4 Hz), 6.3 (2H, apparently t, J = 4.0 Hz), 3.72 (3H, s), 3.42 (2H, t, J = 6.8 Hz), 3.34 (2H, t, J = 7.6 Hz), 3.09 (2H, t, J = 7.6 Hz), 2.81 (2H, t, J = 7.6 Hz), 2.09-2.02 (2H, m). 13 C NMR (CDCl3, 100 MHz) δ: 164.6, 159.9, 156.5, 146.8, 140.1, 134.9, 128.3, 127.9, 120.9, 114.7, 51.8, 50.8, 34.7, 28.0, 25.4, 21.4.19 F NMR (CDCl3, 376 MHz) δ: -143.7, -143.8, -143.9, -144.0. HRMS (ESI) C 16 H 18 BF2N5O2[MH] + Calculated m / z 360.1522, Measured m / z 360.3250. IR u max (film) / cm -1 : 3171, 2936, 2098, 1734, 1609, 1497, 1175. λ exc =467nm (510nm emission, c=0.2nM, MeOH). λ emis =516nm (315nm excitation, c=0.2nM, MeOH).

[0763] 3-[3-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-5-yl]propionic acid

[0764] [ka]

[0765] A solution of methyl 3-[3-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-5-yl]propanoate (30 mg, 80 μmol) in THF (4 mL) at 0° C. was treated with HO (2.6 mL) followed by concentrated HCl (1.6 mL). The reaction mixture was then stirred at room temperature until complete by TLC analysis (18 h). The reaction mixture was then diluted with CHCl (20 mL) and stirred at room temperature for 1 h. The organic layer was separated and washed with HO (2 × 20 mL) followed by brine (2 × 25 mL). The resulting solution was dried over NaSO and concentrated under reduced pressure to give the expected carboxylic acid as a red oil (15 mg, 52%). The product was used without further purification.

[0766] 1H NMR (CDCl3, 400 MHz) δ: 7.16 (1H, s), 7.05 (1H, d, J = 4.0 Hz), 7.01 (1H, d, J = 3.2 Hz), 6.39-6.38 (2H, m), 3.42 (2H, t, J = 6.8 Hz), 3.35 (2H, t, J = 7.6 Hz), 3.10 (2H, t, J = 7.6 Hz), 2.86 (2H, t, J = 7.6 Hz), 2.10-2.02 (2H, m). 13 C NMR (CDCl3, 100 MHz) δ: 166.2, 161.6, 159.9, 153.1, 149.0, 134.9, 130.8, 128.8, 127.9, 118.5, 50.9, 31.9, 30.5, 26.0, 23.8. 19 F NMR (CDCl3, 376 MHz) δ:-143.75, -143.84, -143.92, -144.01. HRMS (ESI) C 15 H 16 BF2N5O2[MH] + Calculated m / z 346.1365, Measured m / z 346.3293. IR u max (film) / cm -1 : 3423, 2915, 2850, 1644. λ exc =467nm (510nm emission, c=5nM, MeOH). λ emis =510nm (315nm excitation, c=5nM, MeOH).

[0767] 4''-O-[3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate]-5-O-(tert-butyldimethylsilyl) avermectin B1a

[0768] [ka]

[0769] A solution of 5-O-(tert-butyldimethylsilyl)avermectin B1a (40 mg, 40 μmol) in CHCl (2.5 mL) was treated sequentially with a solution of 3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoic acid (15 mg, 40 μmol) in CHCl (2.5 mL), followed by DCC (16 mg, 70 μmol) and DMAP (3 mg, 20 μmol). The reaction mixture was then stirred at room temperature until complete by TLC analysis (18 h). The reaction was then concentrated under reduced pressure, and the crude residue was purified by flash column chromatography (silica gel, elution gradient 0–20% EtOAc in petroleum ether) to give 4″-O-[3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate]-5-O-(tert-butyldimethylsilyl)avermectin B1a as a red oil (23 mg, 39%).

[0770] 11H NMR (CDCl3, 400 MHz) δ: 7.14 (1H, s), 7.00 (2H, d, J = 4.4 Hz), 6.43 (1H, d, J = 4.0 Hz,), 6.37 (1H, d, J = 4.0 Hz), 5.85 - 5.82 (1H, m), 5.77 - 5.72 (2H, m), 5.41 (1H, br s,), 5.36 - 5.31 (2H, m), 5.00 (1H, d, J = 9.6 Hz), 4.79 (1H, br s), 4.74 (1H, apparently t, J = 10.0 Hz), 4.71 - 4.68 (1H, m), 4.60 (1H, d, J = 14.4 Hz), (原文本此处的“見かけ上t”推测为“apparently t”,“見かけ上d”推测为“apparently d”)4.45 (1H, br s), 3.96 (1H, br s), 3.88 - 3.83 (3H, m), 3.70 - 3.60 (3H, m), 3.45 (6H, br s), 3.41 - 3.37 (4H, m), 3.34 - 3.31 (1H, m), 3.28 - 3.20 (2H, m), 2.86 - 2.79 (4H, m), 2.53 (1H, br s), 2.38 - 2.32 (2H, m), 2.29 (1H, apparently d, = 11.6 Hz), 2.24 (1H, dd, J = 12.0, 4.0 Hz), 2.00 (1H, dd, J = 12.8, 4.4 Hz), 1.98 - 1.93 (2H, m), 1.81 (3H, s), 1.79 - 1.62 (9H, m), 1.53 (3H, s), 1.41 - 1.36 (3H, m), 1.29 - 1.27 (6H, br s), 1.17 (1H, d, J = 6.8 Hz), 0.96 - 0.92 (12H, m), 0.89 - 0.86 (4H, m), 0.81 - 0.79 (1H, m), 0.15 (6H, s). 13C NMR (CDCl3, 100 MHz) δ: 174.0, 172.7, 162.1, 160.5, 140.2, 137.5 (2C), 135.0 (2C), 134.8, 130.4, 127.9, 124.8, 119.3, 118.7, 9, 67. 9, 67. 2, 67. 1, 56.8, 56.5, 51.8, 45.7, 41.1, 39.6, 36.8, 35.7, 35.4, 34.9, 34.5, 34.1, 33.2, 33.0, 31.2, 29.7, 28.1, 27.3, 25.8, 25.4, 20.3, 20.0, 18.4, 17.7, 17.4, 15.2, 12.4, 12.1, -4.5, -4.8. 19 F NMR (CDCl3, 376 MHz) δ: - 144. 21, -144.30, -144.39, -144.48. HRMS (ESI) C 11 H 106 BF2N5O 15 Calculated value for Si [MH]+: m / z 1344.7516, Found m / z 1344.3033. IR u max (film) / cm -1 : 3360, 2961, 2921, 2851, 1632.

[0771] 4''-O-[3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate]avermectin B1a, 15

[0772] [ka]

[0773] A solution of 4"-O-[3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate]-5-O-(tert-butyldimethylsilyl)avermectin B1a (6 mg, 4 μmol) in MeOH (2.0 mL) was treated with a catalytic amount of p-TsOH (3 mg). The reaction mixture was stirred at 18 °C for 30 min. The reaction was then diluted with HO (15 mL) followed by EtOAc (20 mL). The organic layer was washed with HO (3 × 20 mL) and brine (20 mL). The organic phase was dried over NaSO and concentrated under reduced pressure. The crude residue was purified by flash column chromatography (0–40% EtOAc / petroleum ether) to afford 4″-O-[3-(5-(3-azidopropyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacen-3-yl)propanoate]avermectin B1a, 15, as a red oil (4 mg, 72%). MS(ESI) C 65 H 92 BF2N5O 15 [MH] + Calculated value for m / z 1230.6651, observed value m / z 1230.1914.

[0774] General method for reacting proteins with conjugate precursors To a 10 mL flask, the maleimide compound (12 mmol), iLOV protein (10 mg, 0.7 mmol), and 1 mL of pH 7 phosphate buffer were added with stirring at room temperature for 1 hour. The reaction mixture was then loaded onto a column loaded with 1 mL of Strep-Tactin Superflow resin (IBA Lifescience) and 1 mL of pH 8 buffer (150 mM TRIS, 100 mM NaCl). The column was sealed and incubated on a rotor for 15 minutes at 4°C. Unreacted compounds were eluted with 4 mL of pH 8 buffer, after which 15 μL of SUMO protein and pH 8 buffer (100 mM TRIS, 150 mM NaCl) were added. The column was sealed and incubated on a rotor for 18 hours at 4°C. After incubation, the solution (now green) was collected in a tube and concentrated in a centrifuge at 14,000 rpm for 7 minutes. The concentration was calculated by Bradford protein assay and the conjugate solution was stored at -80°C until required.

[0775] Compounds P1 to P11 were prepared in this manner (see Table 1 below).

[0776] Conjugate uptake test Uptake studies using the conjugates of the present invention have been carried out in several organisms, with a particular focus on uptake by C. elegans.

[0777] The conjugates tested are shown in the table below, along with similarly tested reference compounds for comparison.

[0778] [Table 1A]

[0779] [Table 1B]

[0780] [Table 1C]

[0781] [Table 1D]

[0782] C. elegans test The nematode rearing medium (NGM) plate containing C. elegans was washed with 1.0-1.5 mL of standard M9 buffer and the wash solution was collected in an Eppendorf tube. The liquid containing C. elegans was then spun at 10,000 rpm for 1 minute at room temperature. The supernatant was removed and fresh M9 buffer (500 µL) was added to create a new stock solution of C. elegans in M9 buffer.

[0783] The C. elegans suspension (100 μL) was added to a specified amount of M9 buffer (determined by the final desired concentration of the compound to be tested: 399 μL for the BODIPY-tagged derivative) contained in a separate Eppendorf tube. To this was then added a 25 mM solution of the compound in DMSO (1 μL). The resulting suspension was mixed using a vortex machine. Once mixing was complete, the Eppendorf tube was placed on its side to ensure that the worms had not settled to the bottom of the tube and that the test compound was evenly dispersed.

[0784] The resulting mixture was incubated in the dark (aluminum foil was used to shield the tubes, which were also kept in a cabinet) at room temperature for 2-4 hours.

[0785] Other samples were incubated overnight, where OP50 bacteria (3 μL) were added to the worms for feeding purposes and the worms were kept at 20°C in the dark.

[0786] After incubation, the tube contents were gently mixed by hand (typically by simple rotation of the tube) to achieve a homogenous suspension. A suspension sample (250 μL) was taken and mixed with M9 buffer (500 μL). The resulting suspension was placed in a centrifuge and spun at 10,000 rpm for 1 minute at room temperature. The supernatant was removed, and the washing process was repeated two more times, each time using an additional amount of fresh M9 buffer (500 μL). After the final wash, sufficient supernatant was removed to leave a final sample volume of approximately 30 μL.

[0787] A 1.5% agarose solution in water was preheated in a standard household microwave (power setting 4) for 60-90 seconds. Two drops of this solution were removed and placed on a glass slide. The slide was covered with a second glass slide. After 1 minute, the slides were carefully slid apart, with the agarose remaining on one side. An aliquot (15-20 μL) of the final C. elegans solution was placed on this slide. To this, 5 μL of 20 mM sodium azide solution was added to inhibit nematode mobility. Petrolatum was then placed around the agarose area before the second slide was placed back on. The slide was not pressed down.

[0788] The worms were then analyzed using a Zeiss Axiskop 2 Plus microscope with a Hamamatsu Photonics Orca-ER (CA 742-95) camera. Images were resolved using OpenLab on a Mac operating system.

[0789] Uptake studies demonstrate that changes to the structure of the conjugate can optimize uptake into C. elegans and alter the location of accumulation, as illustrated using the compounds described below as examples.

[0790] Dye uptake in C. elegans Compounds 1-11 were evaluated at 50 μM concentrations after 3 h and overnight incubation using the general uptake protocol described for C. elegans.

[0791] Compound 1- (R)-t-kCJ-BODIPY After 3 hours of incubation, compound 1 could be observed in the pharynx, intestinal lumen, and enterocytes of both adult and larval stages. This was the greatest uptake observed after a 3-hour incubation period compared to compounds 2 to 8. After 20 hours of incubation, strong fluorescence was localized in the pharynx, intestinal lumen, and enterocytes of both adult and larval stages.

[0792] Compound 2- (R)-c-kCJ BODIPY After 3 hours of incubation, trace amounts of Compound 2 were observed in the pharynx, intestinal cells, and intestinal lumen of larval stages. No adult worms were present on the slides. After 20 hours of incubation, fluorescence was localized to the intestinal cells, intestinal lumen, and to a lesser extent, in the pharynx of larvae.

[0793] Compound 3- (S)-t-kCJ BODIPY After 3 hours of incubation, compound 3 was present in the pharynx, enterocytes, and intestinal lumen of larval stages. Uptake was much less than that of the enantiomeric analog compound 1, consistent with active uptake. In adult worms, fluorescence was detected in the pharynx, enterocytes, and intestinal lumen. Fluorescence was also present in early stages of egg development. After overnight incubation, fluorescence was visible in the pharynx, enterocytes, and intestinal lumen of larvae.

[0794] Compound 4- (S)-c-kCJ BODIPY After 3 hours of incubation, trace amounts of compound 4 were present in the intestinal cells of the larval stage. After 20 hours of incubation, fluorescence was detected in the pharynx and intestinal cells of the larval stage and in the early stages of egg development.

[0795] Compound 5- (R)-t-dCJ BODIPY After 3 hours of incubation, small amounts of compound 5 were present in the intestinal cells and intestinal lumen of larval stages and in the pharynx, intestinal lumen, and intestinal cells of adult nematodes. Compound 5 was not detected in embryos. After 20 hours of incubation, fluorescence remained in the pharynx, intestinal cells, and intestinal lumen of both larval and adult stages. Fluorescence was also detected in early stages of egg development, but not in embryos.

[0796] Compound 6- (R)-c-dCJ BODIPY After 3 hours of incubation, compound 6 was visible in the intestinal cells and lumen of the larval stage, and to a lesser extent in the pharynx. In adult worms, fluorescence was located in the pharynx, intestinal lumen, and intestinal cells. Fluorescence was not observed in embryos. Uptake was much less than that of the enantiomeric analog compound 8, consistent with active uptake. After 20 hours of incubation, fluorescence was present in the intestinal lumen and intestinal cells of larvae, and to a lesser extent in the pharynx. Fluorescence was also present in the pharynx, intestinal cells, and lumen of adult worms, as well as in early stages of egg development. Fluorescence was not observed in embryos.

[0797] Compound 7- (S)-t-dCJ BODIPY After 3 hours of incubation, small amounts of compound 7 were visible in the intestinal cells and intestinal lumen of larvae, but not in the pharynx. In adult worms, fluorescence was located in the intestinal lumen and intestinal cells, and to a lesser extent, in the pharynx. After 20 hours of incubation, fluorescence was present in the intestinal cells and intestinal lumen of larval stages, but not in the pharynx. Fluorescence was also detected in the intestinal lumen, intestinal cells, and to a lesser extent, in the pharynx of adult worms. Fluorescence was also located in early stages of egg development.

[0798] Compound 8- (S)-c-dCJ BODIPY After 3 hours of incubation, compound 8 was visible in the intestinal cells and intestinal lumen of larvae, and to a lesser extent in the pharynx. This was the second-highest uptake observed compared to compounds 1-7 after a 3-hour incubation period. In adult worms, fluorescence was observed in the pharynx, intestinal lumen, intestinal cells, and eggs. After 20 hours of incubation, fluorescence was present in the pharynx, intestinal lumen, and intestinal cells of larvae. In adult worms, fluorescence was detected in the pharynx, intestinal lumen, and intestinal cells, but not in embryos.

[0799] Compound 9 - BODIPY Pantothenate After 3 hours of incubation, trace amounts of compound 9 were visible in the intestinal cells of both larval and adult nematodes, and to a lesser extent in the pharynx. After 20 hours of incubation, small amounts of fluorescence could be detected in the intestinal cells of both larval and adult stages. In both cases, the amount of fluorescence was lower than that of the enamide derivatives, compounds 1–8.

[0800] Compound 10-ketal pantothenic acid BODIPY After 3 hours of incubation, trace amounts of compound 10 were detected in the intestinal cells and intestinal lumen of larvae. Compound 10 was also visible to a lesser extent in the pharynx of larvae. No adult worms were present on the slides. After 20 hours of incubation, fluorescence was visible in the intestinal cells of larval stages and, to a lesser extent, in the pharynx. The amount of fluorescence present after 20 hours was comparable to that of the enamide compound, which exhibited the lowest uptake.

[0801] Compound 11 - BODIPY azide Worms exposed to BODIPY11 showed faint fluorescence localized in the intestinal lumen and enterocytes after 3 hours, with no significant increase in fluorescence even after 20 hours of incubation.

[0802] Praziquantel uptake in C. elegans To compare the ability of conjugates to improve uptake of the drug praziquantel, two praziquantel derivatives were prepared. Praziquantel was tagged with a BODIPY fluorescent tag (compound 12) and incorporated into the conjugate of the present invention (compound 13). Compounds 12 and 13 were evaluated at a concentration of 50 μM using the C. elegans uptake protocol described above for compounds 1-11. Microscopic readings were taken after 3, 24, and 72 hours.

[0803] Compound 12 - Praziquantel BODIPY After 3 hours of incubation, compound 12 was weakly taken up by the pharynx and intestinal cells of C. elegans larvae and by adult nematodes, primarily in the intestinal cells and to a lesser extent in the pharynx. After 24 hours of incubation, larvae showed no increased uptake, and only small amounts of compound 12 were located in the pharynx and intestinal cells. Adult nematodes were not present in the 24-hour samples collected. After 72 hours of incubation, only the intestinal cells of larvae showed the presence of small amounts of fluorescence. In the adult stage, fluorescence was visible in the intestinal lumen, intestinal cells, and pharynx. The nematodes remained viable without any apparent adverse effects.

[0804] Compound 13 - Praziquantel-BODIPY conjugate After 3 hours of incubation, compound 13 was strongly localized in the pharynx, intestinal cells, and intestinal lumen of larval stages. In adult nematodes, compound 13 was strongly localized in the pharynx, intestinal cells, and intestinal lumen, but was not visible in embryos. After 20 hours of incubation, compound 13 uptake was three-fold increased compared to compound 12, despite its larger molecular weight. After 24 hours of incubation, compound 13 was strongly observed in the pharynx, intestinal cells, and intestinal lumen of larvae. Adult nematodes were not present on the slides at this time point. After 72 hours, compound 13 was still strongly visible in the pharynx, intestinal lumen, and intestinal cells of larvae. In adult nematodes, compound 13 was strongly visible in the pharynx and intestinal lumen, as well as in embryos and eggs. The nematodes remained viable without any apparent adverse effects.

[0805] Protein uptake in C. elegans Conjugates containing PhiLOV proteins were tested against C. elegans.

[0806] Chemical ligation and purification experiments yielded effective PhiLOV protein conjugates in M9 buffer. The amount of M9 buffer added was determined based on the required final concentration. C. elegans (30–40 per protein sample analyzed) were selected and added to the protein solution. The resulting suspension was treated with OP50 E. coli solution for nematode maintenance. The nematode suspension was incubated at 20°C in the dark and covered with aluminum foil.

[0807] Aliquots (10-25 μL) were taken at 24, 48, and 72 hours, transferred to new Eppendorf tubes, and diluted with M9 buffer (200 μL). The new suspension was placed on ice for 1-2 minutes and then centrifuged at 10,000 rpm for 1 minute. The supernatant was removed, and the concentrated nematodes were washed again (2 x 200 μL). After the final wash, a residual volume of nematodes (approximately 30 μL) was left.

[0808] For microscopy, a 1.5% agarose solution in water was heated in a standard household microwave oven (power 4) for 1-1.5 minutes. Two drops of the agarose solution were placed on a glass slide and covered with another glass slide. After 1 minute, the two slides were carefully separated. 15-20 μL of the final C. elegans solution was placed on the slide with the remaining agarose. To this, 5 μL of 20 mM sodium azide solution was added to inhibit nematode mobility.

[0809] After placing petroleum jelly around the agarose area, a top coverslip was placed back on. The coverslip was not pressed down. C. elegans were then analyzed using a Zeiss Axiskop 2 Plus microscope with a Hamamatsu Photonics Orca-ER (CA 742-95) camera. Images were analyzed using OpenLab on a Mac operating system.

[0810] Feeding was repeated 48 hours later by adding 2 μL of OP50 bacteria for feeding purposes, and worm incubation continued at 2°C in the dark.

[0811] Using the above approach, 11 protein conjugates were tested for their ability to deliver phiLOV proteins to C. elegans. Results indicate that the conjugates can deliver large biological molecules to different parts of the nematode. The location and efficacy of delivery can be regulated by small structural modifications to the delivery vehicle, as demonstrated by compounds P1–P11 (including a negative control, a positive control using compound 1, and an untagged phiLOV protein control).

[0812] Negative control - M9 buffer After 24 hours of incubation with M9 buffer alone, both larval and adult stages of C. elegans exhibited small amounts of cellular autofluorescence in the intestinal cells. After 48 and 72 hours, there was no change in the autofluorescence level. At the 72 hour time point, the adult nematodes were dead.

[0813] Positive control - Compound 1-(R)-t-kCJ BODIPY After 24 hours of incubation with Compound 1, fluorescence could be observed in the pharynx, intestinal lumen, and enterocytes of both adult and larval stages of C. elegans. After 48 and 72 hours of incubation, fluorescence was still localized in the pharynx, intestinal lumen, and enterocytes of both adult and larval worms. At 72 hours, adult nematodes were dead.

[0814] Protein control - phiLOV protein After 24 hours of incubation with untagged phiLOV protein (a 13 kDa protein used at a concentration of 236 μM), a small amount of fluorescence was visible in the pharynx of adult worms, but no fluorescence was present in the larval stages. After 48 and 72 hours, there was no change (i.e., no fluorescence) in the larval stages. The small amount of fluorescence in the pharynx of adult worms also showed no discernible change.

[0815] Compound P1 After 24 hours of incubation with protein conjugate P1 (305 μM), only autofluorescence could be detected in the intestinal cells of the larval stage. In adult worms, a small amount of protein was located in the pharynx. After 48 hours of incubation with protein conjugate P1, fluorescence could be observed in the pharynx and intestinal cells of the larval stage. A small amount of fluorescence was also visible in the intestinal lumen. No adult worms were present on the slides. After 72 hours, the fluorescence level remained unchanged.

[0816] Compound P2 After 24 hours of incubation with conjugate P2 (305 μM), fluorescence was observed in the pharynx and intestinal lumen of larval stages. A small amount of fluorescence was also visible in intestinal cells. Protein conjugate P2 did not show significant improvement in uptake compared to the untagged protein control. Adult stages were not present on the slide. After 48 hours of incubation, larval stages showed increased amounts of fluorescence in the pharynx, intestinal cells, and intestinal lumen. Adult stages showed localization of fluorescence in the pharynx and intestinal lumen. After 72 hours, fluorescence remained localized in the larval pharynx, intestinal lumen, and intestinal cells. In adult worms, fluorescence was observed in the pharynx and intestinal lumen.

[0817] Compound P3 After 24 hours of incubation with protein conjugate P3 (305 μM), a small amount of fluorescence was visible in the pharynx and intestinal lumen of larval stages. In adult stages, fluorescence was visible in the pharynx, intestinal lumen, and intestinal cells. After 48 hours of incubation, a significant increase in the amount of fluorescence was detected in the pharynx, intestinal cells, and to a lesser extent, in the intestinal lumen of larvae. In adult worms, an increase in fluorescence was seen in the pharynx and intestinal lumen. After 72 hours of incubation, fluorescence was visible in the pharynx, intestinal lumen, and intestinal cells of older larval stages. Adult stages showed fluorescence in the intestinal lumen and intestinal cells.

[0818] Compound P4 After 24 hours of incubation with protein conjugate P4 (305 μM), fluorescence was visible in the pharynx, intestinal lumen, and, to a lesser extent, intestinal cells of larvae. In adult worms, fluorescence was observed in the pharynx, intestinal lumen, and intestinal cells. Protein conjugate P4 did not show significant improvement in uptake compared to the untagged protein control. After 48 hours of incubation, fluorescence was present in the pharynx and intestinal lumen of larvae. In adult worms, fluorescence was present in the pharynx and intestinal lumen. After 72 hours of incubation, there was a significant amount of fluorescence in the pharynx and intestinal lumen of larval stages, and a lesser amount in intestinal cells. In adult worms, fluorescence was clearly visible in the intestinal cells, intestinal lumen, and pharynx.

[0819] Compound P5 After 24 hours of incubation with protein conjugate P5 (305 μM), fluorescence was present in the intestinal lumen and enterocytes of larvae. In adult worms, fluorescence could be observed in the intestinal lumen and, to a lesser extent, in the pharynx. After 48 hours of incubation, fluorescence could be seen in the intestinal cells and intestinal lumen of larvae, and to a lesser extent, in the pharynx. No adult worms were present on the slide. After 72 hours, variable amounts of fluorescence could be observed in the intestinal lumen and enterocytes of larvae. No protein could be detected in the larval pharynx. No adult worms were present on the slide.

[0820] Compound P6 After 24 hours of incubation with the protein conjugate P6 (305 μM), only intrinsic autofluorescence could be observed in either adult or larval stages. After 48 hours of incubation, fluorescence was present in the intestinal lumen and enterocytes of younger larvae, as well as in the pharynx. In adult worms, fluorescence was observed in the intestinal lumen. After 72 hours, fluorescence was present in the intestinal lumen of larval stages. No adult worms were present on the slides.

[0821] At a higher concentration of protein conjugate P6 (435 μM), low fluorescence was observed in the pharynx and intestinal lumen of larvae after 24 hours. After 24 hours, only intrinsic autofluorescence was visible in adult worms. After 48 hours, larvae still showed small amounts of fluorescence in the pharynx, intestinal lumen, and intestinal cells. No adult worms were present on the slide. After 72 hours, larvae showed small amounts of fluorescence in intestinal cells and intestinal lumen. No adult worms were present on the slide.

[0822] Compound P7 After 24 hours of incubation with protein conjugate P7 (305 μM), fluorescence was present in the intestinal lumen of larvae. In adult stages, fluorescence was present in the intestinal cells and, to a lesser extent, in the pharynx. After 48 hours of incubation, fluorescence could be observed in the intestinal lumen and intestinal cells of larvae and in the pharynx of younger larval stages. No adult worms were present on the slide. After 72 hours of incubation, fluorescence was visible in the intestinal lumen and intestinal cells of larval stages. No adult worms were present on the slide.

[0823] At a higher concentration of protein conjugate P7 (435 μM), fluorescence was visible in the gut lumen of larvae and, to a lesser extent, in the pharynx after 24 hours of incubation. In adult worms, fluorescence was detected in the gut lumen and, to a lesser extent, in the pharynx after 24 hours. After 48 hours of incubation, larvae showed high levels of fluorescence in the gut lumen and, to a lesser extent, in the pharynx. No adult worms were present on the slide. After 72 hours, high levels of fluorescence were present in the gut lumen of the larval stage. A small amount of fluorescence was visible in the pharynx of larvae. No adult worms were present on the slide.

[0824] Compound P8 After 24 hours of incubation with protein conjugate P8 (305 μM), a small amount of fluorescence was observed in the intestinal cells of larvae. A small amount of fluorescence was also present in the pharynx of adult worms. Protein conjugate P8 showed significantly reduced uptake compared to protein conjugate P11. After 48 hours, a small amount of fluorescence could be detected in the intestinal lumen and intestinal cells of larval stages. No adult worms were present on the slide. After 72 hours, low levels of fluorescence remained in the intestinal lumen and intestinal cells of larval stages. No adult worms were present on the slide.

[0825] At a higher concentration of protein conjugate P8 (435 μM), fluorescence was visible in the intestinal lumen of larvae and, to a lesser extent, in the pharynx after 24 hours. After 24 hours of incubation, fluorescence was observed in the intestinal lumen of adult worms and, to a lesser extent, in the pharynx. Protein conjugate P8 showed significantly reduced uptake compared to protein conjugate P11. After 48 hours of incubation, fluorescence was visible in the intestinal lumen of larvae, with variable amounts in enterocytes and a low distribution in the pharynx. In adult worms, fluorescence was visible in the pharynx and intestinal lumen. After 72 hours, larvae showed the presence of fluorescence in the intestinal lumen and, to a lesser extent, in the pharynx. No adult worms were present on the slides.

[0826] Compound P9 After 24 hours of incubation with the protein conjugate P9 (305 μM), small amounts of fluorescence were visible in the intestinal lumen of larvae and the pharynx of adult worms. After 48 hours of incubation, fluorescence could be observed in the intestinal lumen and enterocytes of both larval and adult stages. After 72 hours of incubation, fluorescence was present in the intestinal lumen and enterocytes of young larvae. Fluorescence was also visible in the pharynx, enterocytes, and intestinal lumen of adult worms.

[0827] After 24 hours of incubation with a high concentration of the protein conjugate P9 (435 μM), fluorescence was visible in the intestinal lumen of the larvae. No adult worms were present on the slide. After 48 hours of incubation, increased fluorescence was observed in the intestinal lumen of the larval stage. No adult worms were present on the slide. After 72 hours of incubation, larvae showed protein uptake in the intestinal lumen and to a lesser extent in the pharynx and enterocytes. No adult worms were present on the slide.

[0828] Compound P10 After 24 hours of incubation with protein conjugate P10 (305 μM), fluorescence was present in the intestinal lumen of larvae. Low fluorescence was also detected in the pharynx of adult worms. After 48 hours, larvae showed high levels of fluorescence in the intestinal lumen and enterocytes. Fluorescence was also visible in the intestinal lumen of adult worms. After 72 hours, fluorescence was visible in the enterocytes, intestinal lumen, and to a lesser extent, the pharynx of larvae. No adult worms were present on the slides.

[0829] At a high concentration of protein conjugate P10 (435 μM), fluorescence was visible in the intestinal cells and intestinal lumen of larval stages after 24 hours. In adult worms, low fluorescence was visible in the pharynx. After 48 hours of incubation, larvae showed fluorescence in the intestinal cells and intestinal lumen, and to a lesser extent in the pharynx. In adult worms, fluorescence was visible in the intestinal lumen. After 72 hours, fluorescence was present in the intestinal cells, intestinal lumen, and pharynx of larvae. No adult worms were present on the slides.

[0830] Compound P11 After 24 hours of incubation with protein conjugate P11 (267 μM), fluorescence was visible in the intestinal lumen of larvae and, to a lesser extent, in the pharynx. Protein conjugate P11 showed significantly improved uptake compared to the untagged control and compound P8. This supports the hypothesis that protein conjugate P11 is actively transported into the worms and not simply ingested by them. In adult worms, fluorescence was observed in the pharynx and, to a lesser extent, in the intestinal lumen. After 48 hours of incubation, larvae showed fluorescence in the intestinal lumen and enterocytes, and to a lesser extent in the pharynx. Fluorescence in adult worms was located in the intestinal lumen and, to a lesser extent, in the pharynx. After 72 hours, larval stages showed fluorescence in enterocytes, the intestinal lumen, and, to a lesser extent, the pharynx. No adult worms were present on the slides.

[0831] Plasmodium falciparum test Plasmodium falciparum parasites were cultured in complete RPMI 1640 medium (Thermo Fisher) containing 10% Albumax II serum (Thermo Fisher). The compounds used for the test were prepared as 100 mM solutions in DMSO and diluted to the appropriate concentrations using complete medium. For testing, a 96-well plate with an appropriate number of wells, each containing 50 μL of blood and 200 μL of serum, was used. The parasitemia range was approximately 3-5%. For each well, the serum was removed and replaced with an equal volume of serum containing the compound to be tested. The 96-well plate was then incubated for 45 minutes at 37°C in an atmosphere of 96% N2, 3% CO2, and 1% O2. After this, the cells were concentrated via centrifugation at 2209 rpm. The medium was removed, and the cells were resuspended in another 200 μL of fresh serum. The cells were centrifuged and washed in this manner two more times. After the final wash, approximately 5-10 μL of blood was applied to the slide and a coverslip was placed on top to smear the blood. The edges of the slide were then sealed with nail polish to ensure cells were contained before imaging. Slides were analyzed using a Zeiss Axioplan 2 microscope system, and images were captured using Volocity 3D Image Analysis software with a FITC fluorescent filter.

[0832] Compounds 1, 5, 9, 10, and 11 were tested for uptake at 25 μM in both synchronized and mixed cultures. As expected, compound 11 diffused into all erythrocytes (both infected and non-infected) through what appeared to be a diffusion-controlled mechanism. Compound 1 was not taken up by either infected or non-infected erythrocytes in all cultures. However, compound 5 was selectively taken up by infected erythrocytes, and the fluorescent compound accumulated within the parasite itself (i.e., the compound was internalized by the parasite in infected erythrocytes). Compound 10 was not appreciably taken up, whereas compound 9 was taken up very rapidly by both infected and non-infected erythrocytes, with only minor differences in uptake between the two sets of erythrocytes.

[0833] Further Plasmodium falciparum testing The BODIPY analog-amide conjugate 310 shown below was partially purified via HPLC.

[0834] [ka]

[0835] Compounds were then incubated at a concentration of 25 μM with 50 μL aliquots of trophozoite-infected erythrocytes (trophozoite-stage Plasmodium falciparum) for 45–60 minutes. Due to their size, trophozoite-stage parasites provided the best results when imaged under a fluorescence microscope. After incubation, samples were centrifuged and the supernatant removed. Cells were washed with an appropriate buffer to remove background fluorescence from the compounds in solution. Slides were then prepared from the washed live cells and examined using fluorescence microscopy on an Applied Precision DV Elite microscope system. An inverted microscope and a 100x oil-immersion objective were used. Images were captured using a CoolSNAP_HQ2 / HQ2-ICX285 camera and acquired using WoRx version 5.5 software.

[0836] The images show that the compound accumulates in the parasite in infected red blood cells. For healthy red blood cells, fluorescence is absent, so selectivity exists between healthy and infected cells, likely due to uptake via a novel permeability pathway (NPP) (see Saliba et al. and Kirk et al.) that is created when healthy cells are infected. Fluorescence is absent in the parasite's digestive vacuole, but is present throughout the cytosol.

[0837] It can also be seen that within the cytosol, spots of high fluorescence can be observed, indicating areas of high conjugate concentration, an example of which is shown in Figure 1.

[0838] Trypanosoma brucei test T. brucei was cultured in complete RPMI 1640 medium (Thermo Fisher) containing 10% Albumax II (Thermo Fisher) serum. Test compounds were prepared as 100 mM solutions in DMSO and diluted to the appropriate concentrations using complete medium. For testing, a 96-well plate with the appropriate number of wells, each containing 200 μL of serum and trypanosomes, was used. For each well, the serum was removed and replaced with an equal volume of serum containing the test compound (10–25 μM). The 96-well plate was then incubated for 45 minutes at 37°C in an atmosphere of 96% N2, 3% CO2, and 1% O2. After this, the trypanosomes were concentrated via centrifugation at 2209 rpm. The medium was removed, and the cells were resuspended in another 200 μL of fresh serum. The trypanosomes were centrifuged and washed two more times in this manner. After the final wash, approximately 5–10 μL of serum was applied to a slide and a coverslip was placed on top. The edges of the slides were then sealed with nail polish to ensure the cells were contained before imaging. The slides were analyzed using a Deltavision deconvolution microscope system.

[0839] The uptake of compounds 5 and 9 in T. brucei was examined.

[0840] The images suggest that compound 5 is taken up by T. brucei and forms small vesicles throughout the trypanosome body, but does not accumulate in lysosomes or the nucleus, but rather in vesicles between the flagellum and lysosomes.

[0841] On the other hand, compound 9 is taken up much more rapidly and at much higher concentrations than compound 5. Compound 9 is also distributed throughout the cell, but appears to have regions of higher concentration in the mitochondria or endoplasmic reticulum.

[0842] Theileria annulata test T. annulata was cultured in RPMI 1640 containing 10% Albumax II (Thermo Fisher) serum. The cell concentration in the parent culture was calculated using a standard hemocytometer. Cultures were grown to a concentration of 2 × 10 cells / ml in RPMI 1640. 5 The cells were diluted to 1000 cells / mL. For each compound and concentration tested, 2 mL of culture was centrifuged at 1000 rpm for 5 minutes. After removing the medium, the cells were resuspended in 950 μL of complete medium containing the desired concentration of the compound to be tested, which was prepared from a 100 mM stock solution of the compound in DMSO. Once resuspended, the cells were transferred to a 5-well plate (1 mL volume / well). They were then incubated for 1 hour at 37°C in an atmosphere of 5% CO2 in air. After the incubation was complete, the contents of each well were transferred to a 10 mL centrifuge tube. Cold RPMI 1640 (Thermofisher) or HBSS was added to bring the volume to 5 mL. The suspension was then centrifuged at 1000 rpm and 4°C for 5 minutes. The supernatant was then removed, and the cells were washed twice more in this manner. After the final wash, the cell pellet was resuspended in a minimal amount of RPMI 1640 and then spotted onto a glass slide. The slides were then cover slipped and analyzed using an Olympus BX60 UV microscope system equipped with a Spot RT3 camera and a FITC fluorescent filter and Spot Image software.

[0843] The uptake of compounds 1, 5, 9, 10, and 11 in T. annulata was examined. Images suggest that compounds 5 and 9 are taken up by T. annulata. Compound 9 is taken up more rapidly, and its fluorescence can be detected at low concentrations, even though it is present in infected lymphocytes. Compound 5, on the other hand, selectively accumulates in intracellular parasites. Compound 11 diffuses indiscriminately, whereas compounds 1 and 10 show very limited uptake.

[0844] Bacterial testing The bacteria were cultured by inoculating LB medium (Thermo-Fisher) with the desired bacteria and incubating overnight at 37°C in an air atmosphere.600 The culture was then diluted appropriately to a concentration of 2 × 10 5 The cells were then concentrated to a concentration of 1000 cells / mL. 1 mL of this culture was placed in a 5 mL centrifuge tube and centrifuged at 13,000 rpm for 5 minutes. The supernatant was removed and replaced with 1 mL of a 25 μM solution of compound in medium. The culture was incubated at 37°C for 45 minutes. The culture was centrifuged as before, the supernatant removed, and the pellet resuspended in 1 mL of PBS. The cells were washed in this manner two more times. After the final wash, the pellet was resuspended in 35 μL of water and spotted onto a slide. The slide was then protected with a cover slide and analyzed using an Olympus UV microscope system with a FITC fluorescence filter and Leica imaging analysis software.

[0845] In the case of bacterial cultures, compound 5 was tested in all cases, and uptake was observed in both Gram-negative and Gram-positive bacteria.

[0846] cytotoxicity The conjugates of the present invention are based on pantothenate and CJ-15801, the former being an essential vitamin and well tolerated in vivo in human subjects.

[0847] The cytotoxicity of the conjugates can be measured against HEK cells using the methods described below.

[0848] Test compounds were incubated at 100 μM with human embryonic kidney cells (HEK293) in Greiner 384 cell star plates for 24 hours before the addition of CellTitre-Glo® (Promega), a luminescent indicator of cell viability. Compounds showing greater than 40% cytotoxicity across two tests performed at 100 μM were considered cytotoxic in this assay.

[0849] Interestingly, the representative compound shown below was inactive in the screen at 3.7±0.6% at 100 μM concentration.

[0850] [ka]

[0851] The above compound can be prepared as shown below using hexafluorophosphate benzotriazole tetramethyluronium (HBTU), N,N-diisopropylethylamine (DIPEA), and CHCl at 80° C. for 2.5 hours. The yield was 58%.

[0852] [ka]

[0853] The parent carrier was tested for intrinsic clearance in female CD1 mouse microsomes and showed a liver clearance of 1.0 mL / min / g, where the carrier is a CJ-15801 framework with amide bonds.

[0854] Loto Maria Passim Exam In recent years, colony collapse disorder has become one of the major challenges for honeybee survival. The European honeybee (Apis mellifera) has played an important role in agriculture and as a biological model for identifying pathogens and symbionts. Lotomaria passim (L. passim), a member of the Kinetoplastidae class, has emerged as a major parasite of honeybees worldwide (Schwarz et al., Ravoet et al., and Paxton et al.).

[0855] Parasite uptake test (CJ-15801 coupled with BODIPY FL and pantothenic acid derivatives) A 100 mM stock solution of the BODIPY derivatives was prepared in dimethyl sulfoxide (DMSO). L. passim strain PRA-403 (ATCC) was cultured in modified C. bombyi medium (Tognazzo et al.). Parasite cultures were centrifuged, the supernatant removed, and 500 μL of L. passim medium containing the compound (final concentrations of 500, 100, 10, or 1 μM for the BODIPY derivatives) or DMSO (0.1%) control was added. The mixture was incubated at room temperature for 45 min. After incubation, the samples were centrifuged, and the pellets were washed with PBS (3 × 500 μL). The samples were transferred to slides and observed under a fluorescence microscope (NIKON ECLIPSE Ni), or 100 μL was added to a 96-well microplate and the fluorescence intensity was measured using a Varioskan LUX or BioTek microscope reader.

[0856] Honeybee intestinal uptake test (CJ-15801 coupled with BODIPY and pantothenic acid derivatives) Honeybee (A. mellifera) hives were obtained and allowed to feed ad libitum from spring to autumn. To provide sufficient food during the winter, bee hives were fed pollen mixed with 50% (v / v) sucrose. Honeybees were dissected, and the digestive tract, including the crop, midgut, and rectum, was removed and immediately added to 500 μL of the delivery vehicle: test sample diluted in PBS to a final concentration of 100 μM or DMSO (0.1%) control. The mixture was incubated at 33°C for 45 min. After incubation, the samples were centrifuged, and the intestines were washed with PBS buffer (3 x 500 μL). The samples were transferred to slides and observed under a fluorescence microscope (NIKON ECLIPSE Ni).

[0857] Results - Parasite Uptake Test A set of CJ-BODIPY derivatives was tested on L. passim strain PRA-403 at 10 μM and 500 μM and incubated for 45 minutes at room temperature. After incubation, samples were analyzed by fluorescence microscopy. Parasites treated with 500 μM (R)-trans-ketal 2 exhibited fluorescence in the parasite cytosol, with the derivative forming fluorescent dots. On the other hand, parasites incubated with 10 μM of the diol derivative (S)-cis-diol 8 exhibited fluorescence surrounding the cell membrane but not in the cytoplasm (Figure 3).

[0858] Uptake studies were performed with the CJ-BODIPY derivatives at final concentrations of 1 μM and 100 μM. After 45 minutes of incubation at room temperature, fluorescence intensity was assessed using a microplate reader. Quantitative results confirmed the fluorescence microscopy findings and suggested preferential uptake of the ketal derivatives. Derivative 2 showed the highest readings among all the compounds tested, while derivative 4 showed one of the lowest amounts of uptake. Compound 1 exhibited approximately one-quarter the fluorescence of derivative 2 at 1 μM. Compounds 5 and 8 had relatively low uptake (Figure 4).

[0859] Results - Intestinal uptake test The digestive tract (crop, midgut, and rectum) of adult honeybees was removed and incubated with 100 μM derivatives 1, 2, 8, and 11 at 33°C for 45 min. Derivatives 2 and 8 showed slight fluorescence in the midgut and rectum, but none in the crop. Compound 2 was localized on the surface of the gut, but not inside. Intestines treated with BODIPY11 showed only autofluorescence. Incubation with derivative 1 resulted in high fluorescence in the midgut and rectum, but not in the crop (Figure 5).

[0860] Without wishing to be bound by theory, the results suggest that using concentrations of derivatives below 100 μM reduces the likelihood of distribution of the test compound outside the wasp's digestive system, maintaining the compound in close proximity to the parasite.

[0861] Thus, these derivatives appear to be suitable tools for delivering molecules to Lotomaria passim with minimal permeability through the honeybee's digestive system.

[0862] Babesia bovis test B. bovis is a protozoan parasite that shares a taxonomic group with the genus Theileria and is placed in the order Piroplasmida due to its pear-shaped appearance within infected erythrocytes. Babesia reproduces by binary fission, causing the characteristic presence of diploids or tetrads in stained infected erythrocytes. Two species, B. bovis and B. bigemina, cause considerable economic impact in the livestock industry (Yamagishi et al.).

[0863] Short-term study in B. bovis (CJ-15801 and pantothenic acid derivatives coupled with BODIPY FL and phiLOV (R475G, K476C)) A 100 mM stock solution of the delivery vehicle was prepared using DMSO. B. bovis Texas strain (provided by Nagasaki University) was cultured in GIT medium (Kohjin-bio Co.) containing 10% bovine red blood cells (RBCs) using a microaerophilic stationary culture system (Bork et al.). Parasite cultures were centrifuged and washed once with GIT medium. RBCs were pelleted, and 20 μl of infected RBC mixture (10 μl of concentrated infected RBCs + 10 μl of GIT) was prepared. Then, 180 μl of GIT medium containing the compound to be evaluated (final concentrations of 25 μM for BODIPY derivatives and 100 μM for phiLOV derivatives) or DMSO (0.1%) / PBS control was added. The sample was incubated at 37°C for 45 minutes. (Samples were stained with Hoechst 33342 diluted at 1 μg / mL in GIT medium.) After incubation, samples were centrifuged and pellets were washed 3x with GIT medium. The final solution of 50% hematocrit infected RBC mixture was transferred to a slide and observed by confocal laser scanning microscopy (Nikon A1). Parasitemia levels were monitored by staining thin blood smears with Giemsa solution.

[0864] FACS experiments on B. bovis (CJ-15801 and pantothenic acid derivatives coupled with phiLOV (R475G, K476C)) B. bovis Texas strain was cultured in GIT medium (Kohjin-bio Co.) containing 10% bovine red blood cells (RBCs) using the microaerophilic stationary culture system (Bork et al.). Parasite cultures were centrifuged and washed once with GIT medium. RBCs were pelleted, and 20 μl of infected RBC mixture (10 μl of concentrated infected RBCs + 10 μl of GIT) was prepared. 180 μl of GIT medium containing test compound (final concentration 150–250 μM) or DMSO (0.1%) / PBS control was then added. Samples were incubated at 37°C for 45 min. (Samples were stained using DRAQ5 at a final concentration of 20 μM in GIT medium.) After incubation, samples were centrifuged, and the pellets were washed 3x with GIT medium, transferred to FACS tubes, and analyzed using BD FACSVerse.

[0865] Long-term study in B. bovis (CJ-15801 and pantothenic acid derivatives coupled with phiLOV (R475G, K476C)) B. bovis Texas strain was cultured in GIT medium (Kohjin-bio Co.) containing 10% bovine red blood cells (RBCs) using the microaerophilic stationary culture system (Bork et al.). Parasite cultures were centrifuged and washed once with GIT medium. RBCs were pelleted, and 20 μl of infected RBC mixture (10 μl of concentrated infected RBCs + 10 μl of GIT) was prepared. 180 μl of GIT medium containing test compounds (final concentrations of 100 or 5 μM for phiLOV derivatives) or DMSO (0.1%) / PBS control was then added. The mixture was incubated at 37°C for 45 min. After incubation, the samples were centrifuged, and the pellets were washed 3x with GIT medium. 180 μL of GIT was then added, and the samples were incubated at 37°C. After 5 and 48 hours of incubation, samples were stained with 1:200 diluted Hoechst 33342 (Thermofisher) in GIT medium, and a 50% hematocrit infected RBC mixture was transferred to a slide and observed by confocal laser scanning microscopy (Nikon A1). After 24 hours, fresh GIT medium was added to the sample. Parasitemia levels were monitored by staining thin blood smears with Giemsa solution.

[0866] result Uptake experiments were performed by incubating parasites (approximately 5% parasitized erythrocytes (PPE)) with 25 μM of each derivative for 45 min at 37 °C. After incubation, fluorescence intensity was calculated from confocal microscopic images. These images were analyzed with ImageJ 1.50b software, following the same procedure as applied to C. elegans (Miller et al. and Fricker et al.) (Figure 6).

[0867] Compound 1 showed the highest uptake, significantly higher than derivative 3 (just under 50%). Analog 2 and the untagged BODIPY control 11 (just under 90%) showed much lower values ​​compared to compound 1. On the other hand, compound 5 also showed lower uptake (just under 30%) compared to compound 1.

[0868] The phiLOV double mutant (R475G, K476C) was used to generate derivatives P11, P1, P8, and P3, which were tested for uptake in the Texas strain of B. bovis, using phiLOV as a control. Derivatives and controls were incubated at 100 μM and 37°C for 45 min. After incubation, samples were analyzed by confocal microscopy using Hoeschst 33342 (Thermofisher) as a costai. Only samples treated with derivative P3 showed fluorescent uptake in the binary pattern characteristic of B. bovis (Figure 7).

[0869] In a separate experiment, derivatives P11, P1, P8, and P3, as well as untagged phiLOV as a control, were incubated in approximately 9% parasitized erythrocytes (PPE) for 45 min at 37°C. After incubation, samples were sorted using DRAQ5 (Thermofisher) as a costai. FACS results were consistent with the confocal findings, with derivative P3 demonstrating significant uptake and localization within the parasite.

[0870] Parasite growth effect assays were performed by incubating B. bovis-infected RBCs for 45 minutes in the presence of 5 μM and 100 μM phiLOV-functionalized derivatives. Initial PPE was approximately 1% and 3%, respectively. Samples were collected 48 hours after exposure, and PPE was calculated using Giemsa solution before microscopic analysis. All derivatives showed similar cell growth and were not significantly different from the control. Parasite growth reached comparable levels in all cultures, including those treated with derivative P3, indicating the non-toxicity of the derivatives to the parasite in long-term experiments.

[0871] Mycobacterium test Uptake test in Mycobacterium tuberculosis (CJ-15801 coupled with BODIPY FL and pantothenic acid derivatives) A 20 mg / mL stock solution of BODIPY derivative compounds 1–8 and 10 was prepared in DMSO. Mycobacterium tuberculosis strain H37Rv (provided by the University of Queensland) was centrifuged, the medium removed, and the pellet resuspended in PBS / 0.02% tyloxapol. A 1 mL aliquot was transferred to an Eppendorf tube, centrifuged, the supernatant removed, and the pellet resuspended in 1 mL of BODIPY derivative (final concentration 50 μg / mL). The mixture was incubated for 45 minutes at room temperature with stirring and protected from light. After incubation, the samples were centrifuged, the pellet washed with PBS (3 × 100 μL), and transferred to a 96-well microplate for evaluation of fluorescence intensity using a microplate reader (Agilent). 25 μL of the solution was spotted onto a glass slide and then dried on a heating block at 90°C for 10 minutes. Cells were fixed by immersion in 10% formalin for 30 minutes. The slides were then air-dried and mounted with DAKO mounting medium (Agilent) before being observed under a fluorescent microscope.

[0872] result The set of compounds was tested using the M. tuberculosis strain H37Rv, which has been widely studied for biomedical purposes due to its ease of genetic manipulation, but more importantly, the cells retain full virulence and drug susceptibility in animal models of tuberculosis.

[0873] Uptake experiments were performed as described above by incubating bacteria with 50 μg / mL (approximately 80 μM) of compounds 1 to 8, 10 and control PBS (phosphate buffered saline) for 45 min at room temperature.

[0874] The results are shown in Figure 9.

[0875] The results showed that the enamide ketal derivatives (compounds 1-4) showed higher intensity compared to the enamide diols (compounds 5-8), and that the R enantiomers of the enamide ketal derivatives (compounds 1 and 2) were preferred over their S counterparts (compounds 3 and 4). Compounds 1 and 2 showed the highest fluorescence signals, with compound 2 showing approximately 2.5-fold higher intensity compared to compound 1. Interestingly, as shown in the image in Figure 9(b), the bacterial shape was clearer in the case of compound 2, with fluorescence localized to the poles. The pantothenic acid derivative 10 showed the lowest uptake.

[0876] Nematode test Uptake studies in nematodes (ivermectin B1a, pantothenic acid derivatives coupled with ivermectin, and BODIPY-ivermectin conjugates) Mixed cultures of wild-type nematodes (roundworms, cestodes, and trematodes) obtained from Massey University's sheep farm were incubated for 20 hours with compound 14, compound 15, and commercial ivermectin B1a as a control. The protocol followed was the same as the previously used C. elegans protocol.

[0877] result Compound 14 showed enhanced activity against nematodes compared to commercially available ivermectin B1a. Nematodes treated with compound 14 showed paralysis at 5 μM compared to 50 μM for ivermectin B1a. Compound 15 showed an activity profile similar to that of ivermectin B1a (FIG. 8).

[0878] References

Claims

1. A conjugate of formula (I) or a pharmaceutical composition comprising a conjugate of formula (I) for use in a method of treating a nematode or flatworm infection, the conjugate of formula (I) being 【Chemistry 1】 (In the formula, -R A and -R B are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkanoyl, and aralkanoyl; or -R A and -R B Together we can -C(R C1 )(R C2 )- to form a six-membered ring, and -R C1 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R C2 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy, or -R C1 and -R C2 together are oxo (=O); -R T1 and -R T2 are each independently hydrogen or alkyl; -R 1 and -R 2 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R 3 is hydrogen or alkyl; -D- is C 2～4 Alkenylene or C 1～4 alkylene, said alkenylene or alkylene optionally substituted with alkyl or halo; -X- is a covalent bond, -N(R 4 )-, -O-, -S- or -Se-, -R 4 is hydrogen or alkyl; -L- is a linker or a covalent bond; A- is an active agent to be delivered, including dyes, small molecule drugs, polypeptides, polynucleotides, or polysaccharides. and salts, solvates and protected forms thereof.

2. A method of delivering an active agent to a nematode or flatworm, comprising the steps of contacting the nematode or flatworm with a conjugate of formula (I) or a pharmaceutical composition comprising a conjugate of formula (I); A method which is not a method of treatment of the human or animal body, wherein the conjugates of formula (I) and pharmaceutical compositions are as described in claim 1.

3. 3. The conjugate for use according to claim 1 or the method of delivery according to claim 2, wherein the nematode or flatworm is selected from the group consisting of Caenorhabditis nematodes, such as C. elegans; Haemonchus nematodes, such as H. contortus; and Schistosoma flatworms, such as S. haematobium.

4. 4. The conjugate for use according to claim 1 or 3, wherein A- is a small molecule drug, a polypeptide or a polysaccharide.

5. 5. The conjugate for use according to any one of claims 1, 3 or 4, wherein A- is a small molecule drug having a molecular weight of 1000 Da or less.

6. 6. The conjugate for use according to any one of claims 1 or 3 to 5, wherein A- is a small molecule drug having a molecular weight of 150 Da or more.

7. 4. A conjugate for use in the method of delivery according to claim 2 or 3, wherein A- is a dye, a polypeptide or a polysaccharide.

8. -D- is C 2 Alkenylene or C 2 8. The conjugate for use or method of delivery according to any one of claims 1 to 7, wherein said alkenylene or alkylene is optionally substituted with alkyl or halo.

9. -D- is C optionally substituted with alkyl or halo 2 9. The conjugate for use or method of delivery according to claim 8, which is alkenylene.

10. 10. The conjugate for use or method of delivering according to any one of claims 1 to 9, wherein the alkenylene group has a trans configuration.

11. 11. The conjugate for use or method of delivering according to any one of claims 1 to 10, wherein the alkenylene group has a cis configuration.

12. -D- is C 2 9. The conjugate for use or method of delivery according to claim 8, wherein said conjugate is an alkylene.

13. -R T1 is hydrogen, -R T2 is hydrogen or alkyl, e.g., -R T1 is hydrogen, -R T2 13. The conjugate for use or method of delivery according to any one of claims 1 to 12, wherein is hydrogen.

14. -R 1 and -R 2 14. The conjugate for use or method of delivery according to any one of claims 1 to 13, wherein each is independently selected from alkyl, alkenyl, aralkyl and cycloalkylalkyl.

15. -R 1 and -R 2 15. The conjugate for use or method of delivery according to claim 14, wherein each is alkyl, e.g. methyl.

16. -R A and -R B are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkanoyl, and aralkanoyl, e.g., -R A and -R B 16. The conjugate for use or method of delivery according to any one of claims 1 to 15, wherein each of is hydrogen.

17. -R A and -R B Together, C(R C1 )(R C2 )- to form a six-membered ring, and -R C1 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R C2 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy, or -R C1 and -R C2 17. The conjugate for use or method of delivery according to any one of claims 1 to 16, wherein taken together is oxo (=O).

18. -R C1 and -R C2 18. The conjugate for use or method of delivery according to claim 17, wherein each is alkyl, e.g. methyl.

19. -R 3 19. The conjugate for use or method of delivery according to any one of claims 1 to 18, wherein is hydrogen.

20. -X- is a covalent bond or -N(R 4 )-, for example, -X- is -N(R 4 20. The conjugate for use or method of delivery according to any one of claims 1 to 19, wherein

21. -R 4 21. The conjugate for use or method of delivery according to any one of claims 1 to 20, wherein is hydrogen.

22. 22. A conjugate for use or method of delivery according to any one of claims 1 to 21, wherein -L- is a linker.

23. -L-, * -L 3 -BL 4 -GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or cycloalkylene; -L 4 - is a covalent bond, alkylene, or heteroalkylene; -G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)- and maleimide derived groups; -R N is hydrogen or alkyl; -L A - is a covalent bond, alkylene or heteroalkylene; -L 3 -, -B- and -L 4 When at least one of - is not a covalent bond and -B- is a covalent bond, -L 4 - is a covalent bond) 23. The conjugate for use or method of delivery according to claim 22,

24. -L-, * -L 3 -BL 5 -G- group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or heteroalkylene; -L 5 - is the formula * -(NR N C(O)-L 6 )-, the asterisk indicates the point of attachment to -B-, and -L 6 - is alkylene; G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)- and maleimide derived groups; -R N is hydrogen or alkyl) 23. The conjugate for use or method of delivery according to claim 22,

25. 25. The conjugate for use or method of delivery according to any one of claims 22 to 24, wherein -L- comprises a 1,2,3-triazole.

26. 25. The conjugate for use or method of delivery according to any one of claims 22 to 24, wherein -L- contains a maleimide derivative group.

27. Conjugates of formula (I): 【Chemistry 2】 (In the formula, -R A and -R B are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkanoyl, and aralkanoyl; or -R A and -R B Together we can -C(R C1 )(R C2 )- to form a six-membered ring, and -R C1 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R C2 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy, or -R C1 and -R C2 together are oxo (=O); -R T1 and -R T2 are each independently hydrogen or alkyl; -R 1 and -R 2 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R 3 is hydrogen or alkyl; -D- is C 2～4 alkenylene, said alkenylene optionally substituted with alkyl or halo; -X- is a covalent bond, -N(R 4 )-, -O-, -S- or -Se-, -R 4 is hydrogen or alkyl; -L- is a linker or a covalent bond; A- is an active agent to be delivered, including dyes, small molecule drugs, polypeptides, polynucleotides, or polysaccharides. and salts, solvates and protected forms thereof.

28. 28. The conjugate of claim 27, wherein A- is a dye.

29. 28. The conjugate of claim 27, wherein A- is a small molecule drug having a molecular weight of 1000 Da or less.

30. 30. The conjugate of claim 27 or 29, wherein A- is a small molecule drug having a molecular weight of 150 Da or more.

31. 28. The conjugate of claim 27, wherein A- is or comprises a polypeptide.

32. 28. The conjugate of claim 27, wherein A- is or comprises a polynucleotide.

33. 28. The conjugate of claim 27, wherein A- is or comprises a polysaccharide.

34. 34. The conjugate of claim 27 or 33, wherein A- is or comprises a disaccharide.

35. 34. The conjugate of claim 27 or 33, wherein A- is not and does not contain a disaccharide.

36. -D- is C 2 36. The conjugate of any one of claims 27 to 35, which is alkenylene, said alkenylene optionally substituted with alkyl or halo.

37. 37. The conjugate of any one of claims 27 to 36, wherein the alkenylene group has a trans configuration.

38. 37. The conjugate of any one of claims 27 to 36, wherein the alkenylene group has a cis configuration.

39. -R T1 is hydrogen, -R T2 is hydrogen or alkyl, e.g., -R T1 is hydrogen, -R T2 The conjugate of any one of claims 27 to 38, wherein is hydrogen.

40. -R 1 and -R 2 40. The conjugate of any one of claims 27 to 39, wherein each is independently selected from alkyl, alkenyl, aralkyl, and cycloalkylalkyl.

41. -R 1 and -R 2 41. The conjugate of any one of claims 27 to 40, wherein each is alkyl, e.g. methyl.

42. -R A and -R B are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkanoyl, and aralkanoyl, e.g., -R A and -R B 42. The conjugate of any one of claims 27 to 41, wherein each is hydrogen.

43. -R A and -R B Together, C(R C1 )(R C2 )- to form a six-membered ring, and -R C1 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkylalkyl; -R C2 is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aralkyl, cycloalkylalkyl, alkoxy, alkenoxy, alkynoxy, aralkoxy, and cycloalkylalkoxy, or -R C1 and -R C2 The conjugate of any one of claims 27 to 42, wherein taken together is oxo (=O).

44. -R C1 and -R C2 44. The conjugate of any one of claims 27 to 41 and 43, wherein each is alkyl, e.g. methyl.

45. -R 3 45. The conjugate of any one of claims 27 to 44, wherein is hydrogen.

46. -X- is a covalent bond or -N(R 4 46. ​​The conjugate of any one of claims 27 to 45, wherein

47. -X- is -N(R 4 47. The conjugate of any one of claims 27 to 46, wherein

48. -R 4 48. The conjugate of any one of claims 27 to 47, wherein is hydrogen.

49. 49. The conjugate of any one of claims 27 to 48, wherein -L- is a linker.

50. -L-, * -L 3 -BL 4 -GL A -group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or cycloalkylene; -L 4 - is a covalent bond, alkylene, or heteroalkylene; -G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)- and maleimide derived groups; -R N is hydrogen or alkyl; -L A - is a covalent bond, alkylene or heteroalkylene; -L 3 -, -B- and -L 4 When at least one of - is not a covalent bond and -B- is a covalent bond, -L 4 - is a covalent bond) 50. The conjugate of any one of claims 27 to 49, wherein

51. -L-, * -L 3 -BL 5 -G- group (wherein the asterisk indicates the point of attachment to -X-; -L 3 - is a covalent bond, alkylene, or heteroalkylene; -B- is a covalent bond, arylene, heterocyclene, or heteroalkylene; -L 5 - is the formula * -(NR N C(O)-L 6 )-, the asterisk indicates the point of attachment to -B-, and -L 6 - is alkylene; G- is a covalent bond, -O-, -S-, -N(R N )-, -C(O)-, -C(O)N(R N )-, -C(O)O-, -N(R N )C(O)-, -OC(O)- and maleimide derived groups; -R N is hydrogen or alkyl) 50. The conjugate of any one of claims 27 to 49, wherein

52. 52. The conjugate of any one of claims 27 to 51, wherein -L- contains a 1,2,3-triazole.

53. 52. The conjugate of any one of claims 27 to 51, wherein -L- contains a maleimide derivative group.

54. 54. A pharmaceutical composition comprising a conjugate of formula (I) according to any one of claims 27 to 53, optionally together with one or more pharma- ceutically acceptable excipients.

55. 55. A conjugate of formula (I) according to any one of claims 27 to 54, or a pharmaceutical composition comprising a conjugate of formula (I), for use in a method of treatment.

56. 55. A conjugate of formula (I) according to any one of claims 27 to 54, or a pharmaceutical composition comprising a conjugate of formula (I), for use in a method for treating parasitic infections of the human and animal body.

57. 55. A method for delivering an active agent to a parasite, comprising contacting the parasite with a conjugate of formula (I) as defined in any one of claims 27 to 54, or a pharmaceutical composition comprising a conjugate of formula (I); the method is not a method for treating the human or animal body.

58. Parasites include Plasmodium parasites, such as P. falciparum, P. vivax, P. ovale, P. malaria and P. knowlesi; Theileria parasites, such as T. annulata and T. parva; Phytophthora parasites, such as P. cinnamomi and P. agathidicida; 58. The conjugate for use according to claim 56, or the method of delivery according to claim 57, selected from Babesia parasites, such as B. bovis; Crithidia parasites, such as C. bombi; Lotmaria parasites, such as L. passim; Trypanposoma parasites, such as T. brucei; or Toxoplasma parasites, such as T. gondii.

59. 55. A conjugate of formula (I) as defined in any one of claims 27 to 54, or a pharmaceutical composition comprising a conjugate of formula (I), for use in a method for treating microbial infections, such as bacterial infections, of the human and animal body.

60. 55. A method for delivering an active agent to a microorganism, such as a bacterium, comprising contacting the microorganism, such as a bacterium, with a conjugate of formula (I) as defined in any one of claims 27 to 54, or a pharmaceutical composition comprising a conjugate of formula (I); the method is not a method for treating the human or animal body.

61. 61. The conjugate for use according to claim 59, or the method of delivery according to claim 60, wherein the microorganism, e.g. bacterium, is selected from Mycobacteria bacteria, e.g. M. tuberculosis; Escherichia bacteria, e.g. E. coli; Staphylococcus bacteria, e.g. S. aureus; or Enterococcus bacteria, e.g. E. faecalis.

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