Vinyl isocyanide compounds as antibacterial agents

Abstract

Disclosed is a compound of formula (I) or (II) 1 , Y 2 and Y 3 CR 1 or N]. Such compounds are used as antibiotics and antifungals.

Description

[Technical Field]

[0001] The present invention relates to compounds used in treating infectious diseases, more particularly as antibiotics and antifungal substances, methods for producing such compounds, and reagents used in such methods. [Background technology]

[0002] The emergence of antimicrobial drug-resistant (AMR) bacteria represents a serious threat to human health. Currently, there is a shortage of new antibiotics under development, particularly those capable of targeting Gram-positive bacterial biofilms (Non-patent Literature 1: MJ Renwick, DM Brogan, E. Mossialos, A systematic review and critical assessment of incentive strategies for discovery and development of novel antibiotics. Journal of Antibiotics 69, 73-88 (2016)). In addition, there is an urgent need for new drugs to treat multidrug resistance in bacteria and fungi.

[0003] International Publication No. 2008 / 124836 (Patent Document 1) discloses methods and compounds for controlling bacterial virulence, methods for identifying further compounds for controlling bacterial virulence, and methods, compounds, and compositions for treating subjects with bacterial infections to reduce bacterial virulence in said subjects. European Patent No. 0440887 (Patent Document 2) discloses a process for preparing erbustatin and erbustatin analogs. International Publication No. 2021 / 145729 (Patent Document 3) discloses pharmaceutical compositions for preventing or treating cancer, inflammatory diseases, or metabolic diseases.

[0004] Microbial biofilms are communities of microbial cells that are protected by being immersed in an extracellular polymer matrix and are difficult to disrupt. Biofilms can form on biological and abiotic surfaces ranging from heart valves to medical devices such as implanted catheters and prostheses. It is estimated that 80% of endobacterial infections are associated with biofilms (Non-patent Literature 2: D. Davies, Understanding biofilm resistance to antibacterial agents. Nature Reviews Drug Discovery 2, 114-122 (2003)). Biofilm-associated infections can be refractory even to antibiotic concentrations up to 1000 times higher than the minimum inhibitory concentration (MIC) for plankton (Non-patent Literature 3: I. Olsen, Biofilm-specific antibiotic tolerance and resistance. European Journal of Clinical Microbiology & Infectious Diseases 34, 877-886 (2015)). Therefore, there is a need for antibiotics that are useful in reducing, preventing, and / or eradicating bacterial biofilms. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2008 / 124836 Brochure [Patent Document 2] European Patent No. 0440887 [Patent Document 3] International Publication No. 2021 / 145729 brochure [Non-patent literature]

[0006] [Non-Patent Document 1] MJ Renwick, DM Brogan, E. Mossialos, A systematic review and critical assessment of incentive strategies for discovery and development of novel antibiotics. Journal of Antibiotics 69, 73-88 (2016) [Non-Patent Document 2] D. Davies, Understanding biofilm resistance to antibacterial agents. Nature Reviews Drug Discovery 2, 114-122 (2003) [Non-Patent Document 3] I. Olsen, Biofilm-specific antibiotic tolerance and resistance. European Journal of Clinical Microbiology & Infectious Diseases 34, 877-886 (2015) [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need to address the problems associated with conventional technologies and to provide antibiotics and antifungal substances that can prevent or treat bacterial and other infections, and are particularly effective against biofilms.

[0008] Addressing this need is the objective of this invention. [Means for solving the problem]

[0009] Therefore, in the first embodiment, a compound of formula (I) or formula (II): [ka] The present invention provides a salt thereof, a solvate (or diastereomer), or a tautomer thereof. [In the formula, Y1, Y2, and Y3 are independently selected from C-R1 or N; Each R1 is independently selected from H, C1-C6 alkyl, OH, OR, NHCOR, NHSO2R, CONHR, CONHSO2R, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or R7; each R is independently selected from H or C1-C6 alkyl; R2 is selected from substituted or unsubstituted aryls, substituted or unsubstituted heteroaryls, or R7; R3 and R4 are independently selected from H or C1-C6 alkyl groups; R7 is based on the following equation: [ka] and; R a and R b is independently selected from H or C1-C6 alkyl groups; or R a and R b They form a substituted or unsubstituted 5- or 6-membered ring together with the atoms to which they are bonded; R5 is selected from substituted, unsubstituted, or heteroaryl aryls; R6 is selected from H or C1-C6 alkyl groups.

[0010] Substituted or unsubstituted 5 or 6-membered rings are substituted or unsubstituted cyclyl or heterocyclyl rings, appropriately C 5-20 Cyclyl or C 5-10 It may contain heterocyclines.

[0011] A substituted or unsubstituted 5 or 6-membered ring may include rings forming one of a substituted or unsubstituted aryl or heteroaryl ring or a fused ring structure, and appropriately, a substituted or unsubstituted 5 or 6-membered ring may include a substituted or unsubstituted C 5-20 Aryl or C 5-10 Contains heteroaryl compounds.

[0012] Substituted or unsubstituted 5 or 6-membered rings include pyrrolidine, pyrrole, pyridine, furan, thiophene, oxazole, isoxazole, isoxazine, oxadiazole (e.g., 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl), oxatriazole, thiazole, isothiazole, imidazole, pyrazole, pyridazine, pyrimidine, pyrazine, triazole (e.g., 1,2,4-triazole), triazine (e.g., 1,2,4-triazine), tetrazole, azaindole (e.g., 5-azaindole or 7-azaindole), azaindazole (e.g., 7 The following can be selected: azaindoleazole, azabenzimidazole (e.g., 5-azabenzimidazole), benzofuran, isobenzofuran, indole, quinoline, quinazoline, isoindole, indidine, isoindoline, benzothiofuran, benzoxazole, benzoisoxazole, benzothiazole, benzimidazole, indazole, benzodioxol, benzoflazan, benzothiadiazole, benzotriazole, purines (e.g., adenine, guanine), pyrrolo[1,2-a]pyrazine, pyrazolo[1,5-a]pyridine, 1H-pyrazolo[3,4-d]pyrimidine, pyrazolo[1,5-b]pyridazine, and pteridine.

[0013] The aforementioned compound is a compound of formula (III): [ka] It is possible. [In the formula, R1 is selected from H, C1-C6 alkyl, OH, OR, NHCOR, NHSO2R, CONHR, CONHSO2R, or substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl; Y1, Y2, and Y3 are independently selected from CR or N; Each R is independently selected from H or C1-C6 alkyl groups.

[0014] The compound is a compound of formula (III):

Chemical formula

[0015] The compound is a compound of formula (V):

Chemical formula

[0016] The compound may be a compound of formula (VI):

[0017]

Chemical formula

[0018] Suitably, R5 may be substituted or unsubstituted aryl, pyridyl, pyrazyl, pyridazyl or pyrimidyl.

[0019] Suitably, R3 and R4 may each be H.

[0020] Suitably, each of Y1, Y2 and Y3 may each be C-R1.

[0021] Suitablely, at least one R1 is selected from OH, OR, NHCOR, NHSO2R, CONHR, CONHSO2R, and each R is independently selected from H or C1-C6 alkyl. More preferably, each of Y1, Y2, and Y3 is a CR 1a This is possible, and each R 1a R7 is independently selected from H, C1-C6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or R7.

[0022] Appropriately, the compound may be selected from the following compounds: [ka] JPEG0007876210000008.jpg105120

[0023] In a second embodiment, a method for producing the vinyl isocyanide compound of the first embodiment, a) Phosphonate of formula (X): [ka] [In the formula, R 11 and R 12 The step of providing [which is independently selected from C3-C5 alkyl groups and optionally independently selected from isopropyl, isobutyl, and t-butyl], b) The step of reacting the phosphonate with a carbonyl compound in the presence of a base. This provides a method that includes this.

[0024] Carbonyl compounds are compounds of formula (XI) or (XII): [ka] It is possible. [In the formula, Y1, Y2, and Y3 are independently selected from C-R1 or N; Each R1 is independently selected from H, C1-C6 alkyl, OH, OR, NHCOR, NHSO2R, CONHR, CONHSO2R, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or R7; each R is independently selected from H or C1-C6 alkyl; R2 is selected from substituted or unsubstituted aryls, substituted or unsubstituted heteroaryls, or R7; R3 is selected from H or C1-C6 alkyl groups; R7 is based on the following equation: [ka] and; R a and R b is independently selected from H or C1-C6 alkyl groups; or R a and R b They form a substituted or unsubstituted 5- or 6-membered ring together with the atoms to which they are bonded; R5 is selected from substituted or unsubstituted aryls or heteroaryls; R6 is selected from H or C1-C6 alkyl groups.

[0025] The base may include a non-nucleophilic base, optionally a Li base, and optionally a base selected from lithium bis(trimethylsilyl)amide (LHMDS), lithium tetramethylpiperidide (LiTMP), and lithium diisopropylamide (LDA).

[0026] Appropriately, R3 could be H.

[0027] The phosphonate can be appropriately reacted with a carbonyl in the presence of THF as a base and solvent.

[0028] In the third aspect, a reagent for use in the method of the third aspect, comprising a compound of formula (X): [ka] The reagent contains [wherein R 11 and R 12 The element is independently selected from C3-C5 alkyl groups, and optionally independently selected from isopropyl, isobutyl, and t-butyl groups.

[0029] Compounds of formula (I) or (II), as well as their salts and solvates, may be used as pharmaceuticals, particularly for use in the treatment of infectious diseases, and more specifically for use in the treatment of bacterial, fungal, or protozoal diseases. When the compound is for the treatment of a bacterial disease, the disease may be caused by Gram-negative bacteria or Gram-positive bacteria.

[0030] Accordingly, the fourth embodiment provides a pharmaceutical composition comprising a compound of formula (I) or (II), a salt or solvate thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0031] Further specific preferred embodiments are presented in the attached independent and dependent claims. The features of the dependent claims may be combined with the features of the independent claims as appropriate and in combinations other than those explicitly presented in the claims, as supported by the description.

[0032] definition "Substituting" (or "substituted"), when used with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of that substituent or moiety are replaced by one or more non-hydrogen atoms or groups, provided that the valence requirements are met and the resulting compound is chemically stable.

[0033] "May be substituted" refers to a parent group that may be unsubstituted or substituted with one or more substituents. Appropriately, unless otherwise specified, if any substituents are present, an optional parent group includes one to three optional substituents. When a group can be an "optional group substituted with one, two, or three substituents," this means that the group may be substituted with zero, one, two, or three of any substituents. Appropriately, the group is substituted with one, two, or three of any substituents. When a group can be an "optional group substituted with one or two substituents," this means that the group may be substituted with zero, one, or two of any substituents. Appropriately, the group may be substituted with zero or one of any substituents. In some embodiments, the group may not be substituted. In other embodiments, the group may be substituted with one of any substituents.

[0034] Any substituent is C 1-8 Alkyl, C 2-7 Alkenil, C 2-7 Alkinyl, C 1-12 Alkoxy, C 5-20 Ariel, C 3-10 Cycloalkyl, C 3-10 Cycloalkenyl, C 3-10 Cycloalkynyl, C 3-20 Heterocyclyl, C 3-20 The following groups may be selected: heteroaryl, acetal, acyl, acylamide, acyloxy, amidino, amide, amino, aminocarbonyloxy, azide, carboxy, cyano, ether, formyl, guanidino, halo, hemiacetal, hemiketal, hydroxamic acid, hydroxyl, imido acid, imino, ketal, nitro, nitroso, oxo, oxycarbonyl, oxycarbonyloxy, sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamide, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, and ureido groups. In some embodiments, any substituent may be OH, C 1-8 Alkyl, OC 1-12One, two, or three arbitrary substituents independently selected from alkyl and halogens. More preferably, any substituents are OH, C 1-8 Alkyl and OC 1-12 Selected from alkyl groups; more preferably, any substituent is C 1-8 Alkyl and OC 1-12 Selected from alkyl groups.

[0035] "Independently" or "independently selected" means, for example, "each R 16 , R 17 H and C are independent of each other. 1-8 Alkyl, ... is used in the context of a description, and refers to a functional group, for example, R 16 Each example is R in the compound. 16 or R 17 This means that, independently of any other example, the choice is made from the enumerated options. For example, if H is R in the compound 16 For the first example, it may be selected; methyl is R in the compound. 16 The following example may be selected: ethyl is R in the compound. 17 The first example can be selected.

[0036] C 1-8 Alkyl groups are generally linear and branched saturated hydrocarbon groups having 1 to 8 carbon atoms; appropriately, C 1-7 Alkyl; appropriately, C 1-6 Alkyl; appropriately, C 1-5 Alkyl; more appropriately, C 1-4 Alkyl; more appropriately, C 1-3 This refers to alkyl groups. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, penta-1-yl, penta-2-yl, penta-3-yl, 3-methylbuta-1-yl, 3-methylbuta-2-yl, 2-methylbuta-2-yl, 2,2,2-trimethylethi-1-yl, n-hexyl, n-heptyl, n-octyl, and similar groups.

[0037] "Alkylene" refers to a divalent radical derived from alkanes, which may be linear or branched, as exemplified by -CH2CH2CH2CH2-. Alkylenes may have the number of carbon atoms of the alkyl group as discussed above.

[0038] "C 6-26 "Aralkyl" refers to an arylalkyl group that has 6 to 26 carbon atoms and is substituted with an aryl group. More precisely, the alkyl group is C 1-6 It is an alkyl group, and the aryl group is phenyl. 6-26 Examples of aralkyl include benzyl and phenethyl. In some cases, C 6-26 The aralkyl group may be substituted, and may be substituted C 6-26 An example of an aralkyl group is 4-methoxyrubenzyl.

[0039] "C 5-20 "Aryl" refers to fully unsaturated monocyclic, bicyclic, and polycyclic aromatic hydrocarbons having at least one aromatic ring and a specified number of carbon atoms constituting the members of that ring (e.g., C 5-20 An aryl group refers to an aryl group having 5 to 20 carbon atoms as ring members. The aryl group may be bonded to the parent group and to the substrate by any of the ring atoms, as long as such bonding or substitution does not violate valence requirements, and may include one or more nonhydrogen substituents. Appropriately, C 5-20 Ariel is C 6-14 Aryl, or C 6-12 Ayl, or more accurately, C 6-10 The group is selected from the aryl group. Examples of aryl groups include phenyl.

[0040] "Arylene" refers to a divalent radical derived from an aryl group, such as -C6H4-, which is an arylene derived from phenyl.

[0041] A halogen or halo refers to a group selected from F, Cl, Br, and I. Preferably, the halogen or halo is F or Cl. In some embodiments, the halogen is preferably F. In other embodiments, the halogen is preferably Cl.

[0042] "C 5-10 A "heteroaryl" or "5-10 membered heteroaryl" refers to an unsaturated monocyclic or bicyclic aromatic group containing 5-10 ring atoms, whether carbon or heteroatoms, of which 1-5 are ring heteroatoms. Preferably, any monocyclic heteroaryl ring has 5-6 ring atoms and 1-3 ring heteroatoms. Preferably, each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. Bicyclic rings include fused ring systems, particularly bicyclic groups in which a monocyclic heterocycle containing 5 ring atoms is fused to a benzene ring. Heteroaryl groups may be bonded to a parent group or to a substrate at any of their ring atoms, provided that such bonding or substitution does not violate valency requirements and does not result in a chemically unstable compound, and may contain one or more non-hydrogen substituents.

[0043] Examples of monocyclic heteroaryl groups include, but are not limited to, N1: Pyrrole, pyridine; O1:Fran; S1: Thiofen; N1O1: Oxazole, isoxazole, isoxazine; N2O1: Oxadiazole (e.g., 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl); N3O1: Oxatriaazole; N1S1: Thiazole, Isothiazole; N2: Imidazole, pyrazole, pyridazine, pyrimidine, pyrazine; N3: Triazoles, triazines; and, N4: Tetrazole It encompasses what is derived from it.

[0044] Examples of heteroaryls containing fused rings include, but are not limited to, O1: Benzofuran, isobenzofuran; N1: Indole, isoindole, indoridine, isoindoline; S1: Benzothiofuran; N1O1: Benzoxazole, Benzoisoxazole; N1S1: Benzothiazole; N2: Benzimidazole, indazole; O2: Benzodioxol; N2O1: Benzofurazan; N2S1: Benzothiadiazole; N3: Benzotriazole; and N4: Purines (e.g., adenine, guanine), pteridines; It encompasses what is derived from it.

[0045] "Heteroarylene" refers to a divalent radical derived from a heteroaryl group (such as the one mentioned above), as exemplified by pyridinyl-[C5H3N]-. Heteroarylenes can be monocyclic, bicyclic, or tricyclic systems. Typical heteroarylenes, though not limited to these, can be selected from triazolylene, tetrazolylen, oxadiazolylen, pyridylene, furylene, benzofuranylene, thiophenylene, benzothiophenylene, quinolinylene, pyrrolylene, indolylene, oxazolylene, benzooxazolylene, imidazolylen, benzimimidazolylene, thiazolylen, benzothiazolylen, isoxazolylene, pyrazolylene, isothiazolylene, pyridadinylene, pyrimidinylene, pyradinylene, triazinylene, cinolinylene, phthalazine, quinazolinylene, pyrimidinylene, azepinylene, oxepinylene, and quinoxalinylene. Heteroarylenes may be substituted.

[0046] "C 6-16 A "heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group. More precisely, as defined above, alkyl is C 1-6It is an alkyl group, and the heteroaryl group is C 5-10 It is a heteroaryl compound. 6-16 Examples of heteroarylalkyl groups include pyrrole-2-ylmethyl, pyrrole-3-ylmethyl, pyrrole-4-ylmethyl, pyrrole-3-ylethyl, pyrrole-4-ylethyl, imidazole-2-ylmethyl, imidazole-4-ylmethyl, imidazole-4-ylethyl, thiophene-3-ylmethyl, furan-3-ylmethyl, pyridine-2-ylmethyl, pyridine-2-ylethyl, thiazole-2-ylmethyl, thiazole-4-ylmethyl, thiazole-2-ylethyl, pyrimidine-2-ylpropyl, and similar groups.

[0047] "C 3-20 A "heterocyclyl" refers to a saturated or partially unsaturated monocyclic, bicyclic, or polycyclic group having 3 to 20 ring atoms, of which 0 to 10 are ring heteroatoms, regardless of whether they are carbon atoms or heteroatoms. Appropriately, each ring has 3 to 8 ring atoms and 1 to 4 ring heteroatoms (for example, appropriately, C 3-5 A heterocyclyl refers to a heterocyclyl group having 3 to 5 ring atoms and 1 to 4 heteroatoms as ring members. The ring heteroatoms can be independently selected from nitrogen, oxygen, and sulfur.

[0048] Similar to bicyclic cycloalkyl groups, bicyclic heterocyclyl groups may include isolated rings, spiro rings, fused rings, and bridging rings. Heterocyclyl groups may be bonded to the parent group or to the substrate at any of the ring atoms, provided that such bonding or substitution does not violate valence requirements and does not result in a chemically unstable compound, and may include one or more nonhydrogen substituents.

[0049] Examples of monocyclic heterocyclyl groups include, but are not limited to, N1: Aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine; O1: Oxirane, oxetane, tetrahydrofuran, dihydrofuran, tetrahydropyran, dihydropyran, pyran, oxepin; S1: Thiran, Thietan, Tetrahydrothiophene, Tetrahydrothiopyran, Thiepan; O2: dioxoiane, dioxane, and dioxepane; O3: Trioxane; N2: imidazoiidine, pyrazolidine, imidazoline, pyrazoline, piperazine: N1O1: Tetrahydroxazole, dihydroxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine; N1S1: Thiazolin, thiazolidinedione, thiomorpholin; N2O1: Oxadiazine; O1S1: Oxatiol and oxathian (thioxane); and N1O1S1: Oxathiazine It encompasses what is derived from it.

[0050] Examples of substituted monocyclic heterocyclyl groups include those derived from sugars, in cyclic form, for example, furanoses such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses such as ariopyranose, altropyranose, glucopyranose, mannopyranose, globyranose, idopyranose, galactopyranose, and talopyranose.

[0051] "Drugs," "active pharmaceutical ingredients," "active active ingredients," and similar terms refer to compounds that can be used to treat conditions requiring treatment (e.g., compounds of formula (I) and the compounds specifically named above).

[0052] An "excipient" refers to any substance that may affect the bioavailability of a drug but is otherwise pharmacologically inactive.

[0053] The term "or its pharmaceutically acceptable salt, solvate, tautomer, stereoisomer or mixture" means that it also includes pharmaceutically acceptable salts, solvates, tautomers, and stereoisomer forms of the indicated structure. The term "mixture" means that mixtures of these forms may exist, for example, that the compound of the present invention may include both tautomers and pharmaceutically acceptable salts.

[0054] A "pharmaceutically acceptable" substance is one that is appropriate for use in contact with the target tissue, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, and similar consequences, and that is effective for its intended use and corresponds to a reasonable benefit-risk ratio.

[0055] "Pharmaceutical composition" refers to a combination of one or more active pharmaceutical ingredients and one or more excipients.

[0056] As used herein, “solvate” refers to a variable stoichiometric complex formed by a solute and a solvent. Pharmaceutically acceptable solvates may be formed from crystalline compounds in which solvent molecules are incorporated into the crystal lattice during crystallization. The incorporated solvent molecules may be water molecules or, but are not limited to, non-aqueous molecules such as ethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethanolamine, and ethyl acetate molecules.

[0057] As used herein, the term “subject” refers to human or non-human mammals. Examples of non-human mammals include domesticated animals such as sheep, horses, cattle, pigs, goats, rabbits, and deer; as well as companion animals such as cats, dogs, rodents, and horses.

[0058] The "therapeutic dose" of a drug refers to the amount of the drug or composition that is effective in treating the subject and therefore produces the desired therapeutic, remission, inhibitory, or preventive effect. The therapeutic dose may depend, in particular, on the subject's weight and age, as well as the route of administration.

[0059] "To treat" means to reverse, alleviate, inhibit the progression of, or prevent a disorder, disease or medical condition to which such term is applicable, or to reverse, alleviate, inhibit the progression of, or prevent one or more symptoms of such disorder, disease or medical condition.

[0060] "Treatment" refers to the act of "treating" as defined immediately above.

[0061] As used herein, the term "comprising" means "including at least a portion of" and is meant to be inclusive or open-ended. When interpreting each description in this specification that includes the term "comprising", there may be features, elements and / or steps other than those that begin with that term. Related terms such as "comprise" and "comprises" should be interpreted similarly.

[0062] The term "consisting essentially of" limits the claim to the specified materials or steps of the claimed invention and those that do not "substantially affect the basic and novel characteristics". When the phrase "consisting essentially of" appears in an item in the characterizing part of the claim rather than immediately following the preamble, it limits only the elements described in that item.

[0063] The term "consists of" excludes any element, step, or component not specified in the claim; "consists of" is defined as "closing the claim to the inclusion of substances other than those enumerated, excluding impurities that normally accompany them." If the phrase "consists of" appears in a feature section of the claim rather than immediately following the premise, it limits only the elements described in that section; other elements are not excluded from the claim as a whole. While various embodiments in this specification are shown using the phrase "includes" in various contexts, it should be understood that relevant embodiments may also be described using the phrases "essentially consists of" or "consists of." [Brief explanation of the drawing]

[0064] Embodiments of the present invention will be further described here with reference to the accompanying drawings. [Figure 1] Mean increase in mass of tobacco hawk moth (Manduca sexta) injected with 10 μL of PBS(+) and 1% NaN3(-) controls at 1 mg / mL according to the present invention. The mass of tobacco hawk moth larvae (Manduca) was recorded 24 and 72 hours after inoculation (n=5). [Figure 2] Prevention (left) and reduction (right) of MRSA252 biofilm by selected compounds according to the present invention. The graph shows (from left to right) non-bacterial control; MRSA252 without antimicrobial agent; compounds 2; 13; 14; 22; 36; and 41. [Figure 3(a)] ~ [Figure 3(c)] Prevention of highly biofilm-forming Staphylococcus aureus (S. aureus) strains at 50 μg / mL. This includes Staphylococcus aureus AS68(a), AS140(b), and AS68(c) using compounds at various concentrations. [Figure 4] Removal of mature AS68 biofilm at 50 μg / mL (top) with the selected compound according to the present invention; and at various concentrations (bottom) with compound 41. [Figure 5]Time-kill assay for compound 41 in late logarithmic phase MRSA252. [Figure 6] SEM images of MRSA252 bacteria (a) and MRSA252 cells (b and c) 8 hours after the addition of compound 41. [Figure 7] When MRSA252 cells were treated with compound 41, changes in the bacterial cell membrane were demonstrated by fluorescence microscopy (right). Control MRSA252 cells without antibiotic treatment (left). [Figure 8] Percentage of membrane potential of MRSA cells exposed to a wide range of compounds for 180 minutes. [Figure 9] The average percentage of K+ ions remaining in MRSA cells exposed to CTAB and compound 41 for 60 minutes. [Figure 10] MIC values ​​of MRSA252 after continuous passage in the presence of compound 36 and ofloxacin. [Figure 11a] ~ [Figure 11b] a) NMR data for compound 41 and b) NMR data for compound 41 after storage in an organic solvent for 6 months. [Figure 12] NMR data for compound 39 in the presence of glutathione at various interval settings. [Figure 13] NMR data for glutathione in DMSO-D6. [Figure 14] NMR data for compound 39 (30 minutes after glutathione addition). [Figure 15] NMR data for compound 39 (5 ​​hours after glutathione addition). [Figure 16] NMR data for compound 39 (24 hours after glutathione addition). [Figure 17] MIC data plotted for compound 41 before and after cysteine ​​addition (Origin Lab). [Figure 18a] ~ [Figure 18b] NMR data for compound 41 before (a) and after (b) 4 hours of exposure to 1M acetic acid. [Modes for carrying out the invention]

[0065] The library of vinyl isocyanide compounds synthesized in this work demonstrated good activity against planktonic Staphylococcus aureus. Further biological studies revealed their excellent antibiofilm properties, completely preventing biofilm growth at sub-MIC concentrations (even as low as 1 μg / mL, the lowest concentration tested). These compounds also demonstrated no systemic toxicity issues even at concentrations exceeding 500 μg / mL using both Galleria mellonella and tobacco hawk moth systemic infection models. Despite the identification of the cell membrane as the target site of action for these compounds, the selected “lead compounds” showed remarkable selectivity, exhibiting no significant cytotoxicity against human embryonic kidney cells (HEK293 cells), and no hemolysis was detected at 32 μg / mL (the highest concentration tested). Unlike typical organic-based antibiotics, further in vitro studies indicate that bacteria may face difficulties in conferring resistance to these novel vinyl isocyanide compounds. After 18 consecutive passages, no detectable resistance was identified, further suggesting their high potential as novel antibiotics.

[0066] Novel diisopropyl isocyanomethylphosphonate reagents have been shown to undergo highly diastereoselective Horner-Wadsworth-Emmons (HWE) reactions with cyclic and acyclic aldehydes to yield vinyl isocyanides with excellent (E)-selectivity. A series of experimental studies have been conducted to explain how the isopropylphosphonate reagents yield significantly higher levels of (E)-diastereocontrol than their corresponding ethyl derivatives. The stereoselectivity in these HWE reactions is determined in the first irreversible addition step, in the absence / presence of non-classical alkoxide-CH interactions, by the transition state of the isopropyl HWE reagent responsible for its high (E)-selectivity. This novel isopropyl HWE reagent has been used as a key reactant in the first seven-step synthesis of byelyankacin and its aglycone, vinyl isocyanide natural products produced by Gram-negative bacterial species residing in the intestines of entomopathogenic nematodes that prey on insect larvae in soil. This access to vinyl cyanides allowed the inventors to demonstrate that byeliancasin is a versatile natural product that acts both as an inhibitor of insect phenol oxidase, which produces melanin as part of the insect immune response, and as an antibiotic that prevents competing Gram-positive bacteria from consuming insect carcasses. Byeliancasin and its aglycone have been shown to demonstrate good antiviral activity against clinically relevant methicillin-resistant Staphylococcus aureus strains, thus confirming the potential of vinyl isocyanides as antibiotics for treating clinically relevant bacterial infections.

[0067] The synthesis and testing of 4-phenolvinyl isocyanide (2) demonstrated its moderate activity against the planktonic bacteria of Staphylococcus aureus (MRSA252: 90 μg / mL and MSSA476: 100 μg / mL). Subsequently, in order to identify the moieties essential for its biological activity, it was decided to conduct a small structure-activity relationship (SAR) study on this specific compound (2). The synthesis, SAR study and biological performance of the synthesized compound library are detailed herein.

[0068] To investigate whether these compounds exhibit broad antibacterial activity, all synthesized compounds were tested against three clinically relevant Gram-positive pathogen Staphylococcus aureus strains (MRSA252, MSSA476 and MSSA15981) and two Gram-negative species: Pseudomonas aeruginosa PAO1 and Escherichia coli DH5α using a semi-quantitative disk diffusion assay at a set compound concentration of 500 μg / mL (Table 3). The results showed that most of these compounds were specifically active against Staphylococcus aureus, and a broad inhibition zone was observed. Generally, only a small inhibition zone or no inhibition zone was observed for both Gram-negative species with the said compounds. Therefore, the minimum inhibitory concentration (MIC) of the biologically active compounds against the Staphylococcus aureus strains; MRSA252 and MSSA47-6 was further evaluated using the liquid dilution method (Table 1). Based on the results of both assays, the important structural elements and functional groups required for the compounds to retain their antibacterial activity could be identified.

[0069] Structure-activity relationship (SAR) Isocyanide group Disk diffusion assays revealed that substitution of the isocyanide group in its cyanide derivative (compound 3) resulted in a complete loss of biological activity. The absence of any significant inhibitory sites in all five strains tested (Table 3) demonstrates that compound 3 lacks anti-vitality. Therefore, it can be justified that the isocyanide moiety must play an essential role in binding to the target site of action within the bacterium that confers anti-vitality.

[0070] vinyl group Next, the significance of the double bond in 4-phenol vinyl isocyanide (2) was evaluated. Saturating this double bond (4) resulted in complete loss of activity for all five strains, demonstrating the necessity of this functional group. During this part of the SAR study, 4-isocyanophenol (5) was also prepared.

[0071] Other compounds Other compounds (36, 37, and 38) (Figure 2) retained their anti-vitality in disk diffusion assays, and a broad inhibitory range was observed (Table 3).

[0072] [ka] Scheme 1. Synthesis

[0073] [ka] Scheme 2: Library of novel vinyl isocyanide compounds and their synthesis

[0074] Evaluation of these compounds by MIC demonstrated their increased efficacy against Staphylococcus aureus, as evidenced by a significant decrease in the MIC for each compound (36 and 38). Comparing compounds 36 and 37, it can be concluded that the ortho-positioned fragment improves antimicrobial performance (Table 1). Surprisingly, compound 38, which does not contain a hydroxyl group at the para position, was highly active against MRSA252: 5 μg / mL and MSSA476: 3 μg / mL.

[0075] However, as demonstrated by the partial insolubility of compounds 36-38 in 2% DMSO / water, the addition of styrene reduces the water solubility of the compounds. To improve water solubility, compounds 39 and 40 were synthesized, in which the ortho-position styrene analogs were replaced with 4-vinylpyridine and 2-vinylpyrazine, respectively. These two compounds could also contain the essential functional groups required to maintain their antimicrobial activity, but were considered to have improved water solubility, which is important for their possible in vivo applications. Compound 39 showed excellent activity against both MRSA252 (12 μg / mL) and MSSA476 (15 μg / mL) while maintaining its activity against Gram-negative Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) (Table 3). Antiviral activity was maintained in compound 40, but its activity against both MRSA252 (32 μg / mL) and MSSA476 (26 μg / mL) was slightly reduced (Table 1).

[0076] Compound 41 was synthesized by replacing the hydroxyl functional group in compound 36 with an amide functional group. Compound 41 showed excellent activity against all three Staphylococcus aureus strains, as well as against Pseudomonas aeruginosa PAO1, in disk diffusion assays: MIC values ​​(MRSA252: 6 μg / mL and MSSA476: 8 μg / mL). In the synthesis of compound 41, it was possible to separate the E and Z isomer forms by column chromatography. Independent MIC results obtained for each isomer revealed that the E isomer (41) was considerably more potent than its Z counterpart (42), suggesting that the E isomer in these particular "second-generation" compounds is the preferred orientation for maximizing interaction with the target site of action. To demonstrate the importance of the second vinyl bond in these compounds, compound 43 (lacking the vinyl moiety) was synthesized. Not to the inventors' surprise, the resulting MIC increased against both MRSA252 and MSSA476, demonstrating the importance of this moiety. Finally, to further improve the solubility of these second-generation compounds, compound 44 containing a more polar and potentially protonated pyridine fragment was synthesized. Despite demonstrating antivitality against four strains in disk diffusion assays, the MIC of 44 was shown to be elevated against both Staphylococcus aureus strains (MRSA252: 35 μg / mL and MSSA476: 46 μg / mL).

[0077] 2. Systemic toxicity The identification of novel compounds with antibacterial properties is the starting point in the long process of antibiotic discovery (LL Silver, Challenges of Antibacterial Discovery. Clinical Microbiology Reviews 24, 71-+ (2011)). In this process, many compounds fail to selectively target pathogenic bacterial cells rather than eukaryotic cells and are therefore unsuitable for use as antibiotics. Broadly speaking, due to their isomeric properties with respect to cyanides, isocyanide functional groups are often thought to have the same level of toxicity, and therefore, the inventors were aware of the problems associated with this essential part (I. Ugi, U. Fetzer, U. Eholzer, H. Knupfer, Offerman.K, ISONITRILE SYNTHESES. Angewandte Chemie-International Edition 4, 472-& (1965)). Therefore, in vivo systemic toxicity studies were conducted using wax moths to determine whether they exhibit systemic toxicity to living organisms. The evaluation of the toxicity of novel compounds in mammalian models such as mice and rats is costly, time-consuming, and requires careful ethical considerations (AP Desbois, PJ Coote, in Advances in Applied Microbiology, Vol 78, AI Laskin, S. Sariaslani, GM Gadd, Eds. (Elsevier Academic Press Inc, San Diego, 2012), vol. 78, pp. 25-53).Furthermore, the use of invertebrate models of infection to determine toxicity has increased dramatically recently, as many of these invertebrates share many common features with those of the mammalian innate immune system (AP Desbois, PJ Coote, in Advances in Applied Microbiology, Vol 78, AI Laskin, S. Sariaslani, GM Gadd, Eds. (Elsevier Academic Press Inc, San Diego, 2012), vol. 78, pp. 25-53). Promisingly, this study shows that all compounds (2, 6-44), with the exception of compound 5, are considered non-toxic, with survival rates in Galleria moths comparable to those obtained with phosphate-buffered saline (PBS) controls. Evidence of high survival rates, as shown in Table 4 (comparison with controls), shows that injection of 10 μL of compounds at concentrations above 500 μg / mL did not negatively affect Galleria moth survival, with the exception of compound 5. This would indicate that these novel compounds do not exhibit harmful systemic toxicity in vivo, and therefore we will further elaborate on their potential use as antibiotics.

[0078] [Table 1] JPEG0007876210000016.jpg99146

[0079] At this stage of the new drug discovery program, it was decided to select a small number of compounds for further microbiological and toxicological evaluation (Scheme 3). The compounds selected for further analysis were based on their relative ease of synthesis, disk diffusion assay data, obtained MICs, and their low systemic toxicity. The compounds selected for further analysis were as follows: [ka] Scheme 3. Six compounds selected for further research.

[0080] The use of wax moths to evaluate systemic toxicity studies provided a direct method for screening for any acute toxicity issues associated with lead compounds. One problem encountered with this model is the complexity of obtaining quantitative results, as only viability / death outcomes can be recorded. As an alternative and quantitative approach to systemic toxicity, it was decided to use a novel model using the tobacco hawk moth as the target, instead of the considerably smaller and more difficult-to-handle wax moth. The tobacco hawk moth assay allows for the measurement of larval mass changes over several hours / days, providing information not only about survival but also about the adaptability of the organism (CP Silva et al., Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cellular Microbiology 4, 329-339 (2002)). Promisingly, a 72-hour assay performed at a concentration of 1 mg / mL with the selected lead compound yielded identical results to those obtained using the wax moth infection model, showing no apparent toxicity issues for the compound. The mass increase in hawk moths injected with the lead compound was comparable to that of those injected with the positive control (PBS) (Figure 1). As a negative control, hawk moths were injected with a 1% NaN3 solution. After 24 hours, their mass was significantly decreased compared to the mass increase demonstrated by the larvae injected with the lead antibiotic compound. The hawk moths injected with NaN3 appeared extremely unhealthy, with signs of induced vomiting and diarrhea, and by 48 hours, all negative control hawk moths had died. The hawk moths injected with the lead antibiotic remained healthy, as demonstrated by further weight gain.

[0081] 3. Research on Staphylococcus aureus strains The disk diffusion assay demonstrated that the library of synthesized compounds could be selective in inhibiting Gram-positive bacteria, specifically Staphylococcus aureus. At this point in the study, it was decided to perform disk diffusion assay studies on 50 strains of Staphylococcus aureus. This was undertaken to demonstrate that the lead compounds could inhibit a large number of Staphylococcus aureus strains, both methicillin-resistant and methicillin-sensitive. Furthermore, it was thought that using a wide range of Staphylococcus aureus mutants with specific gene deletions could provide insight into possible protein targets of lead antibiotics if certain strains were resistant to the lead compounds. However, all six compounds used in this study were able to inhibit the growth of all 50 strains of Staphylococcus aureus assayed at a selected concentration of 500 μg / mL (Table 5).

[0082] 4. Biofilm Research Next, we investigated the potential of lead compounds to inhibit the growth of Staphylococcus aureus biofilms and further disrupt established biofilms. Since previous studies had determined the MICs of each compound against plankton MRSA bacteria, we assumed that a compound concentration of 50 μg / mL (above the MIC of all lead compounds except for 2) was a reasonable concentration to start the study.

[0083] The biofilm biomass was estimated by crystal violet staining. Surprisingly, the growth of Staphylococcus aureus MRSA252 biofilms was completely slowed by all lead compounds, suggesting that they have good biofilm-preventive properties at 50 μg / mL. Typically, the MICs that prevent and / or eradicate bacterial biofilms can be up to 1000 times higher than plankton MICs (I. Olsen, Biofilm-specific antibiotic tolerance and resistance. European Journal of Clinical Microbiology & Infectious Diseases 34, 877-886 (2015)). Therefore, it was encouraging that the lead compounds prevented biofilm formation at concentrations similar to their plankton MICs. Surprisingly, compound 2 also completely prevented biofilm formation at approximately half its plankton MIC (Figure 2). Subsequently, we wanted to investigate whether the selected compounds could remove mature Staphylococcus aureus biofilms. With each compound, biofilm biomass was significantly attenuated, as measured by crystal violet staining, and the biofilm was completely removed at 50 μg / mL.

[0084] Based on these studies, biofilm prevention and eradication assays were repeated using strains of Staphylococcus aureus with a high biofilm-forming nature. The Asarm(AS)68 and AS140 strains selected for this study have mutations in their agar quorum-sensing system, which is known to enhance biofilm formation (BR Boles, AR Horswill, agr-mediated dispersal of Staphylococcus aureus biofilms. Plos Pathogens 4, 13 (2008)). In this study, the lead compounds demonstrated complete prevention of Staphylococcus aureus biofilms (Figure 3). All lead compounds were able to completely inhibit biofilm growth at 50 μg / mL. Subsequently, it was established that the MIC required to completely prevent biofilm formation of the AS68MRSA strain was less than 1 μg / mL for all lead compounds against Staphylococcus aureus AS68 (Figure 3).

[0085] The attenuation of biomass by lead compounds in 24-hour-old biofilms was measured by a crystal violet staining assay. All compounds, most notably compound 41, showed significant attenuation of the biomass of Staphylococcus aureus AS68 biofilms, even if not 100% eradication (Figure 4). Further assays were performed to study the biofilm attenuation properties of compound 41. Even at a low concentration of 2 μg / ml (the lowest concentration tested), the biofilm depth was reduced by 86% (Figure 4).

[0086] 5. Cytotoxicity Cytotoxicity studies were conducted by CO-ADD (The Community for Open Antimicrobial Drug Discovery), established by the Wellcome Trust (UK) and the University of Queensland (Australia) (C. f. OAD Discovery, "Hit Confirmation of Antibiotics," (2016)). In this study, compounds were screened against the human fetal kidney cell line, HEK293, at a set concentration of 32 μg / mL. At this specific concentration (in some cases shown to prevent 32× biofilm formation), the lead compounds (2, 13, 14, 22, 36, 41) showed no toxicity to the HEK293 cell line, further demonstrating the potential of these lead compounds as antibiotics (Table 6).

[0087] A common problem encountered with novel drugs is hemolysis, also known as drug-induced immunolytic hemolytic anemia. This can result in the premature destruction of healthy red blood cells, causing a number of side effects ranging from shortness of breath and dizziness to blood clots and heart failure (H. Bilgin, A. Eren, S. Kara, Hemolytic Anemia and Heart Failure Caused by Anti-C and Anti-E Immunization. Jcpsp-Journal of the College of Physicians and Surgeons Pakistan 26, 539-540 (2016)). A hemolysis assay performed by the CO-ADD team showed that HC 10 The values ​​exceeded 32 μg / mL (the maximum concentration tested) (Table 6), suggesting that the lead compound did not exhibit significant hemolytic activity at concentrations below 32 μg / mL.

[0088] 6. Stability assay Compounds containing isocyanide groups are generally known to be highly reactive and can react with electrophiles, nucleophiles, and radicals (R. Ramozzi, N. Cheron, B. Braida, PC Hiberty, P. Fleurat-Lessard, A valence bond view of isocyanides' electronic structure. New Journal of Chemistry 36, 1137-1140 (2012)). Therefore, it was important to identify any stability issues that the lead compounds might encounter under storage conditions and in the presence of glutathione and cysteine. The compounds are stable in organic solvents for more than six months, as demonstrated by NMR (Figure 11). Another common feature of the lead compounds is the presence of vinyl fragments. It was hypothesized that the presence of glutathione and cysteine, found in eukaryotic cells, could interact with the electrophilic alkene groups in the compounds and thus block their anti-vitality. To investigate this potentially harmful in vivo process, compound 39 was exposed to glutathione for up to 24 hours, while compound 41 was added to cysteine ​​for the same amount of time. NMR studies performed after the elapsed time confirmed that compound 39 underwent no structural changes after exposure to glutathione. Furthermore, the MIC value of 41 remained unaffected in the presence of cysteine, confirming the stability of our compounds in the presence of both cysteine ​​and glutathione (Figures 12-16 and 17).

[0089] Furthermore, the hydrolysis of isocyanide compounds to their corresponding formamides under acidic conditions is well documented in the literature. It was important to identify whether the lead compounds would hydrolyze in vivo to their corresponding formamide compounds under the acidic conditions present in the stomach. To determine this, compound 41 was stirred in 1M acetic acid (pH 2.37) for 6 hours, mimicking the time an oral drug would be present in the stomach. The NMR data obtained after 6 hours was identical to that of compound 41 before the assay (Figure 18). In addition to NMR, IR analysis was performed to further analyze the data at approximately 2100 cm⁻¹. -1As indicated by the sharp signal, the presence of isocyanide groups after acid treatment was confirmed.

[0090] 7. Research on the mechanism of action The following studies investigated the precise mechanisms of action of these compounds, with time-kill assays, resistance studies, live-dead staining, and morphological analysis of bacteria exposed to the compounds providing insights into specific mechanisms of action. Time-kill assays were used to assess whether the compounds were bacteriostatic or bactericidal. Compound 41 showed bactericidal activity against late logarithmic phase MRSA252 (resuspended in TSB) (defined as a minimum 3-log decrease in bacterial titer), and a 4-log decrease in bacterial density was measured at a concentration of 2×MIC after 20 hours of exposure (Figure 5) (LL Ling et al., A new antibiotic kills pathogens without detectable resistance (vol 517, pg 455, 2015). Nature 520, (2015)).

[0091] Scanning electron microscopy (SEM) is a powerful technique typically used in drug discovery for antimicrobial agents to reveal the morphological characteristics of bacterial cells when exposed to novel compounds. In this study, SEM images revealed characteristic Staphylococcus aureus cells without the addition of an isocyanide compound (Figure 6). The cells are typical of healthy Staphylococcus aureus. In contrast, with exposure to compound 41 (4×MIC), after overnight incubation of the bacteria, SEM images showed a high degree of disruption of cell morphology, along with a highly irregular membrane (Figure 6). The addition of 41 resulted in bleb-like structures on the cell surface, and there was also an increase in the amount of extracellular debris, further demonstrating the antiviral activity of compound 41. Bleb-like disruption of the cell membrane is characteristic of antibiotics that target the cell membrane; therefore, we hypothesize that compound 41 and other vinyl isocyanides target the cell membrane in Gram-positive bacteria.

[0092] Interestingly, permeability of the outer membrane of E. coli strain DH5α with polymyxin B nanopeptide resulted in a decrease in the MIC of compound 41 from 280 μg / mL to 16 μg / mL. This result suggests that the inability of this class of compounds to inhibit Gram-negative bacterial species at low concentrations is likely due to their inability to permeate the additional outer membrane of these organisms. The assay also demonstrated that promising target sites for these compounds exist in Gram-negative species (i.e., the cell membrane).

[0093] To further confirm whether the membrane is actually targeted by compound 41, a LIVE / DEAD assay was performed by inoculating MRSA252 with two dyes: SYTO9 and propidium iodide (PI). Cells with compromised membranes were stained fluorescent red, while cells with intact membranes were stained fluorescent green. When treated with compound 41 at a concentration of 4×MIC, the few cells remaining on the surface appeared red, suggesting significant membrane damage, which supports the hypothesis that the cell membrane is involved in the mechanism of action of compound 41. Conversely, cells without antibiotics exhibited a stained fluorescent green (Figure 7).

[0094] Next, the inventors attempted to further confirm that the cell membrane is the target site for their compound by undertaking a membrane depolarization assay using the voltage-sensitive dye, 3,3'-dipropylthiacarbocyanine iodide (DiSC3(5)). Compounds targeting bacterial cell membranes are often investigated using LIVE / DEAD assay kits (as shown above), however, these DNA-binding dyes do not detect changes in membrane potential. The membrane potential assay revealed that after 180 minutes, 16 μg / mL of compound 41 resulted in a 70% reduction in the proton motility of MRSA bacteria, a result comparable to that of the nonspecific surfactant cetyltrimethylammonium bromide (CTAB) (Figure 8).

[0095] After demonstrating that compound 41 induces MRSA membrane depolarization, intracellular potassium levels were then measured to clarify whether or not the physical integrity of the cell membrane was affected. This particular assay revealed that 60 minutes of exposure of MRSA cells to compound 41 resulted in a 50% loss of cellular K+ ions, further demonstrating the rapid bactericidal properties of this novel class of compounds (Figure 9).

[0096] Next, serial passage studies were initiated to clarify the rate at which target bacteria could develop resistance or select for persister cells within a population after exposure to compound 36. However, after 18 serial passage cycles, no increase in the MIC of 36 against MRSA252 was observed, compared to ofloxacin, which showed a more than 10-fold increase in MIC over the same number of cycles (Figure 10) (LL Ling et al., A new antibiotic kills pathogens without detectable resistance (vol 517, pg 455, 2015). Nature 520, (2015)). The absence of an increase in MIC in serial passage experiments often suggests a nonspecific mechanism of action, but with corresponding eukaryotic toxicity. In this case, the absence of cytotoxicity against eukaryotic cells and the lack of hemolytic activity suggest that the novel vinyl isocyanide compound developed by the inventors specifically targets processes unique to prokaryotic cells.

[0097] 8. Antifungal activity While the demand for new antibiotics is well documented, there is also an urgent need for the development of new antifungal agents to treat invasive fungal infections that are becoming increasingly resistant to our current accumulation of antifungal substances. Fungal infections, specifically those caused by Candida, Cryptococcus, and Aspergillus, infect more than 1.5 million people annually, with a mortality rate exceeding 50% (GD Brown et al., Hidden Killers: Human Fungal Infections. Science Translational Medicine 4, 9 (2012)). However, since fungi are also eukaryotes, and therefore share many common biochemical and morphological characteristics with mammalian cells, discovering treatments for fungal infections is inherently more difficult than discovering treatments for bacterial infections (T. Roemer, DJ Krysan, Antifungal Drug Development: Challenges, Unmet Clinical Needs, and New Approaches. Cold Spring Harbor Perspectives in Medicine 4, 14 (2014)). The antifungal activity of many of our vinyl isocyanide compounds was performed by CO-ADD. Initial primary screening assays revealed extremely potent antifungal activity for many of our compounds. All seven compounds tested were shown to inhibit many fungal strains at concentrations with MIC values ​​similar to those of currently used antifungal drugs (Table 2).

[0098] [Table 2]

[0099] method chemistry Details of all preparations, including the general procedure and synthesis of vinyl isocyanides, as well as their complete characterization, are described in detail in the supplementary information.

[0100] biology The bacterial strains used in this study were recovered from the bacterial isolate collections of Professor Toby Jenkins and Dr. Maisem Laabei.

[0101] Bacterial growth Escherichia coli DH5α, Pseudomonas aeruginosa PA01, and Staphylococcus aureus strain 53 were recovered from frozen (-80°C) glycerol (15% v / v) stocks onto lysogeny agar (LA)-Gram-negative and trypticase soy agar (TSA), and plated at 37°C for 24 hours. Single colonies were placed in either 3 mL of lysogeny broth (LB)-Gram-negative or trypticase soy broth (TSB)-Gram-positive and incubated at 37°C at 250 rpm for 18 hours.

[0102] Disk spreading Antiviral activity was determined using the Kirby-Bauer method, according to the Clinical Standard Laboratory Institute (CSLI) Guidelines (2017). Briefly, 180 μL of a 200-fold dilution (in LB or TSB) of overnight cultures of selected Gram-negative and Gram-positive bacteria was inoculated onto agar plates containing solid growth medium and Mueller-Hinton agar (MHA). A sterile disk inoculated with 50 μL of antibiotic was first added to the agar plate, and the plate was then incubated at 37°C for 24 hours. After incubation, the inhibition area (if present) was recorded.

[0103] Minimum Inhibition Concentration (MIC) Antibiotic MICs were determined by the broth microdilution method according to the Clinical Standard Laboratory Institute (CSLI) Guidelines (2017). Briefly, 195 μL of 1:2 diluted antibiotic (in TSB) was placed in each 96-well microplate, and 5 × 10⁶ solutions were added. 5 5 μL of overnight cultured bacteria, diluted to obtain a starting bacterial concentration of CFU / mL, was inoculated. The optical density of each inoculated well was recorded every 12 minutes for 18 hours at 37°C. The data from here were plotted in OriginPro8 (OriginLab), and sigmoid curves were fitted using a dose-response function. The MIC was calculated using the values ​​fitted in each curve.

[0104] Systemic toxicity assay Waxworms of the honeycomb species, purchased from Livefood UK (www.livefoods.co.uk), were inoculated with 10 μL of serially diluted antibiotics synthesized in-house. The antibiotic concentrations selected in this study were: 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, and 31.25 μg / mL. Each dilution was injected into 10 individual waxworms via their last abdominal appendage. The injected waxworms were stored at 25°C for 5 days. Five days after inoculation, cytotoxicity was determined as the survival percentage of waxworms of the honeycomb species.

[0105] Tobacco hawk moths were initially allowed to grow to the fifth instar, and then 10 μL of a predetermined 1 mg / mL antibiotic was inoculated onto the underside of one abdominal proleg. Each antibiotic was injected into five individual hawk moths. The mass of each hawk moth larva was measured at predetermined 24-hour intervals before and 72 hours after injection. Systemic toxicity was determined as the percentage of survival and mass growth compared to a positive control at predetermined intervals.

[0106] Biofilm assay Prevention: 100 μL of a mixture of TSB supplemented with 0.5% glucose and antibiotic (1:1) was added to each well of a 96-well plate. Then, 2.5 liters of bacterial overnight culture were inoculated into the wells and incubated at 37°C for 24 hours. After a predetermined duration, the medium in each well was discarded, washed twice with PBS, then 150 μL of 1% crystal violet solution was added, and the plate was incubated at room temperature for a further 30 minutes. Then, each well was washed four more times with PBS, then 200 μL of 7% acetic acid was added. The absorbance of each well was then measured using OD. 595 It was measured using [this method].

[0107] Eradication: To induce biofilm formation, 2.5 μL of the bacterial overnight culture was added to each well of a 96-well plate containing 100 μL of TSB supplemented with 0.5% glucose, and incubated at 37°C for 24 hours. After this, the medium in each well was discarded, washed once with PBS, and then the antibiotic at the set concentration was added, and incubated for a further 18 hours at 37°C. Next, the medium in each well was discarded, washed four times with PBS, and then 150 μL of 1% crystal violet was added, and the plate was incubated at room temperature for a further 30 minutes. After incubation, the medium in each well was discarded, and then washed four times with PBS. Next, 200 μL of 7% acetic acid was added, and the wells were OD 595 It was measured using [this method].

[0108] Cytotoxic assay HEK293 cells were manually counted using a Neubauer hemocytometer and then plated into 384-well plates containing the compound to obtain a density of 6000 cells / well in a final volume of 50 μL. DMEM supplemented with 10% FBS was used as the growth medium, and the cells were incubated with the compound at 37°C in 5% CO2 for 20 hours.

[0109] After adding 5 μL of 25 μg / mL rezazulin (final concentration 2.3 μg / mL) and incubating for a further 3 hours at 37°C in 5% CO2, cytotoxicity (or cell viability) was measured by fluorescence, excitation: 560 / 10 nm, emission: 590 / 10 nm (F560 / 590). Using a Tecan M1000 Pro monochromator plate reader, fluorescence intensity was measured, and the inhibition value vs. log(concentration) was curve-fitted using a sigmoid dose-response function with variable fitting values ​​at the bottom, top, and gradient, using automatic dose-increase calculations. 50 The concentration at 50% cytotoxicity was calculated. Curve fitting was performed using the Pipeline Pilot dose-response component.

[0110] Time-Kill Assay By measuring the decrease in CFU over time, the mode of inhibitory action of our novel class of compounds was determined. A 10-minute overnight culture of MRSA252 bacteria was used. 7 The solution was diluted to the specified concentration, then centrifuged at 2000 rpm for 5 minutes, and washed with PBS. The pellet was resuspended in TSB supplemented with antibiotics at 2× and 4× MIC and incubated at 37°C. A bacterial suspension mixed with 1M physiological saline was used as a control. Viable bacteria were determined by plating serial dilutions onto TSA plates at 0, 1, 2, 4, 8, and 24 hours after incubation at 37°C.

[0111] Cell morphology image The cell morphology of MRSA252 cells present on Melinex® film with or without antibiotic treatment was determined by scanning electron microscopy (SEM). Single colonies of MRSA252 were added to individual wells containing Melinex® film and 3 mL of TSB, and incubated at 37°C for 18 hours with minimal agitation (70 rpm). The growth medium was then exposed to various concentrations of antibiotics and incubated for a further 8 hours. Wells treated with vancomycin were used as positive controls, while wells without antibiotics were used as negative controls. Prior to observation, samples were fixed for 90 minutes using 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (SCB) (pH 7.3). The samples were then rinsed in 0.1 M SCB, followed by the addition of 1% osmium tetroxide, and incubated at room temperature for 1 hour. Next, the samples were washed twice with water and exposed to an acetone dehydration series of 50, 70, 90, and 2 × 100% acetone (v / v) acetone, followed by a chemical dehydration series of hexamethyldisilazane (HMDS) with 100% acetone and 50 and 2 × 100% HMDS (v / v) HMDS, for 10 minutes at each concentration. After evaporating the HMDS for 2 hours, the samples were further dried overnight in a drying oven and then sputter-coated with a palladium-gold thin film. The samples were observed using a field emission scanning electron microscope (FESEM) (JEOL JSM6301F, operated at 5KV).

[0112] LIVE / DEAD Assay The LIVE / DEAD BacLight® bacterial survival kit was purchased from Thermo Fischer Scientific and the assay was performed according to the manufacturer's instructions. This kit provides two nucleic acid stains: SYTO-9 and propidium iodide (PI), which allow for the differentiation of live bacteria with intact membranes from those with defective membranes. Single colonies of MRSA252 were added to individual wells containing Melinex® film and 3 mL of TSB, and incubated at 37°C for 18 hours with minimal agitation (70 rpm). The growth medium was then exposed to varying concentrations of antibiotics and incubated for 10 hours. The medium was then discarded from each well, washed with PBS, and 200 μL of a solution containing both nucleic acid stains was added (50 μL of each component in 10 mL of PBS). The mixture was incubated in the dark at room temperature for 15 minutes. Afterward, the Melinex® film was removed, gently washed with PBS, and observed under a confocal microscope at 20x magnification.

[0113] DiSC3(5) assay The membrane potential of Staphylococcus aureus SH1000 cells was determined using the method detailed by Winkel et al. after exposing cells to 4×MIC compound 41 and a control for 1 hour at 37°C, followed by resuspending in HEPES and glucose buffer. SH1000 cultures were then subjected to a 0.2 OD test. 600 The cells were grown until they reached a certain size, and then further incubated with 0.1 M KCl and 2 μM DiSC3(5) at 37°C for 30 minutes. The cells were then exposed to control and compound 41(4×MIC) for 1 hour at 37°C. Subsequently, the cells were centrifuged, and 1 mL of the supernatant was mixed with 1 mL of DMSO; the centrifuged pellet was dissolved in DMSO for 10 minutes and added to equal volumes of HEPES and glucose buffer. Extracellular and intracellular fluorescence were measured using an LS45 luminescence spectrometer (PerkinElmer) at excitation and emission at 622 nm and 670 nm, respectively. The membrane potential was calculated using the Nernst equation and expressed as a percentage of the initial value.

[0114]

number

[0115] [In the formula, Δθ = membrane potential, R = gas constant, and F = Faraday constant]

[0116] Potassium leak detection Potassium leakage in Staphylococcus aureus SH1000 cells exposed to the compound was performed using a previously published method. Briefly, the compound was added to intermediate-stage Staphylococcus aureus SH1000 cells along with HEPES buffer (approximately 10%). 8 The cells were incubated in CFU / ml for 60 minutes. The cells were then removed by centrifugation, and the supernatant was analyzed using a Perkin-Elmer 1100B atomic absorption spectrometer in flame emission mode (wavelength, 766.5 nm; slit, 0.7 nm high; air-acetylene flame). + Efflux was assayed. Before measurement, the instrument was calibrated using an analytical-grade potassium standard.

[0117] Resistance testing To induce resistance through continuous passage, 5 μL of MRSA252 cells cultured overnight were added to individual wells in a 96-well plate containing 195 μL of TSB supplemented with a wide range of antibiotic concentrations, and incubated at 37°C for 18 hours. Ofloxacin was used as a control. Wells showing bacterial growth after 18 hours were plated onto a TSB plate and incubated at 37°C for a further 18 hours. Subsequently, single colonies obtained from overnight-cultured bacteria were inoculated into fresh TSB medium, incubated at 37°C for 18 hours, and then used for the next cycle of resistance testing. This process was repeated for 18 cycles.

[0118] antifungal activity The fungal strains were cultured for 3 days at 30°C on yeast extract-peptone dextrose (YPD) agar. 1 × 10 6 ~5×10 6Yeast suspension (OD) CFU / mL 530 (determined by) was prepared from 5 colonies. Subsequently, the suspension was diluted and added to each well of the compound-containing plate, resulting in 2.5 × 10⁶ 3 The final cell density and total volume of 50 μL of fungal suspension at CFU / mL were obtained. All plates were covered and incubated at 35°C for 36 hours without shaking.

[0119] After adding Lezazlin (final concentration 0.001%) and incubating at 35°C for 2 hours, the growth of Candida albicans (C. albicans) was inhibited at 630nm (OD 630 The absorbance at ) was measured to determine the inhibition of Cryptococcus neoformans growth between 600 and 570 nm (OD 600-570 The difference in absorbance was measured to determine the difference between the two wells. Absorbance was measured using a Biotek Multiflo Synergy HTX plate reader. In both cases, the percentage of growth inhibition was calculated for each well using a negative control (culture medium only) and a positive control (fungus without the inhibitor) on the same plate. MIC was determined as the minimum concentration at which growth was completely inhibited, defined as ≥80% inhibition for Candida albicans and ≥70% inhibition for Cryptococcus neoformans. Due to the high variability of growth and inhibition, a lower threshold was applied to the Cryptococcus neoformans data. In addition, the maximum percentage of growth inhibition is reported as DMax, indicating which compound has limiting activity. In either of the replicate tests (n=2 on different plates), hits were classified by MIC ≤ 16 μg / mL or MIC ≤ 10 μM.

[0120] Experiment details Detailed preparation procedures (including all general steps) for vinyl isocyanide compounds and, in the case of synthesis, their precursor aldehydes.

[0121] Biological data Results of the initial screening disk distribution (Table 3) Survival percentage of the wax moth (Table 4) Results of Staphylococcus aureus disk diffusion (Table 5) Cytotoxicity data in lead complexes (Table 6)

[0122] stability studies Compound 41 after 6 months of storage in an organic solvent 1 1H NMR spectrum (Figure 11) Compound 39 in the presence of glutathione at set intervals 1 1H NMR spectra (Figures 12-16) MIC curves plotted against complex 41 for MRSA252 before and after cysteine ​​addition (Figure 17) Complex 41 before and after exposure to 1M acetic acid 1 1H NMR spectrum (Figure 18)

[0123] General Procedure 1: Horner-Wadsworth-Emmons Protocol In a round-bottom flask, two equivalents of pre-prepared phosphonate isocyanide were dissolved in 5 mL of anhydrous THF, cooled to -78°C, and purged with N2. Then, 2.5 equivalents of LiHMDS were added dropwise to the reaction vessel, and the mixture was stirred for 20 minutes. After this, one equivalent of the aldehyde source was dissolved in the minimum amount of THF and added to the reaction mixture, and the mixture was stirred overnight. The reaction was monitored by TLC. After the reaction was complete (i.e., no starting material was present in the TLC), the reaction solution was opened to air, quenched with phosphate buffer, MgSO4 was added, filtered, and concentrated under reduced pressure. The crude mixture was then purified by silica gel chromatography to obtain the desired compound.

[0124] General Procedure 2: Heck Cross Coupling Triethylamine (1.5 equivalents) and styrene (1.5 equivalents) were added to a solution of aryl halide (1 equivalent), Pd(OAc)2 (0.1 equivalents), and tri(o-tolyl)phosphine (0.2 equivalents) in DMF. The reaction mixture was heated to 120°C and refluxed overnight. The reaction mixture was then cooled to 0°C, followed by the addition of a 1:1 mixture of ether and hexane, and the mixture was stirred for a further 30 minutes. The resulting precipitate was filtered using a Celite plug. The filtrate was collected, extracted with DCM, washed with H2O and brine, dried on MgSO4, and concentrated under vacuum. The desired product was purified using silica gel chromatography.

[0125] General Procedure 3: Amide Formation One equivalent of a suitable amine-containing compound in DCM was mixed with 1.2 equivalents of acetic anhydride. The resulting solution was stirred overnight at room temperature. The reaction mixture was then diluted with DCM and washed with saturated Na2CO3. The organic layer was then extracted, dried over MgSO4, and concentrated under vacuum to obtain the desired compound.

[0126] General Procedure 4: Nitro reduction / ketal removal A suitable nitro compound (1 equivalent) was initially suspended in a 5:1 mixture of ethanol and H2O, and then iron powder (4 equivalents) and 1 mL of saturated ammonium chloride were added. The mixture was heated at 80°C for 3 hours, then cooled, filtered through Celite, and concentrated under vacuum. The resulting residue was partitioned between DCM and H2O, the organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to obtain the title compound.

[0127] Diethyl(isocyanomethyl)phosphonate (1a) [ka] A solution of diethyl-N-(formyl)aminomethylphosphonate (8.18 g, 0.04 mol) in DCM was purged with N2, cooled to -78 °C, and then triethylamine (51.44 mL, 0.38 mol) and methanesulfonyl chloride (7.70 mL, 0.10 mol) were added dropwise. After 16 h, the resulting reaction mixture was quenched with aqueous NaHCO3, washed with DCM, dried over MgSO4, and concentrated under reduced pressure. The remaining malodorous brown oil was purified by silica gel chromatography (ethyl acetate:petroleum ether (50:50)) to give a pale yellow oil. 1 H NMR (300 MHz, CDCl3): δ H = 1.35 (t, J = 7.3 Hz, 6H), 3.75 (d, J = 1.0 Hz, 2H), 4.20 (q, J = 7.0 Hz, 4H). 13 C NMR (125 MHz, CDCl3): δ c = 16.3, 37.5 (d, J = 155.5 Hz), 63.9, 160.6. 31 P NMR (125 MHz, CDCl3): δ p = 14.2. IR (film, cm -1 ): ν = 2152.40 (N-C). R f Value: 0.34 (50% ethyl acetate:50% petroleum ether)

[0128] Diisopropyl (isocyanomethyl)phosphonate (1b)

Chemical Structure

[0129] 4-(2-Isocyanovinyl)phenol (2)

Chemical formula

[0130] (4-hydroxyphenyl)acrylonitrile (3) [ka] Following general procedure 1: Diethyl (cyanomethyl)phosphonate (435 mg, 2.45 mmol), 4-hydroxybenzaldehyde (100 mg, 0.82 mmol), and LiHMDS (3.28 mL, 3.28 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a deep yellow, irritating solid in a 6:1 ratio of E and Z isomers (31 mg, 21%). Main isomer (E) 1 1H NMR (500 MHz, CD3OD): δ H =5.95(d,J=16.6Hz,1H), 6.80(d,J=8.8Hz,2H), 7.40(d,J=16.6Hz,1H), 7.42(d,J=8.8Hz,2H). Minor isomer (Z) 1 1H NMR (500 MHz, CD3OD): δ H =5.38(d,J=12.2Hz,1H), 6.85(d,J=8.8Hz,2H), 7.14(d,J=12.2Hz,1H), 7.72(d,J=8.8Hz,2H), 7.89(br s,1H). 13 ¹³C NMR (125 MHz, CD3OD):δ c =93.0, 117.0, 120.3, 127.0, 130.7, 152.3, 162.0. IR(film,cm) -1 ):ν=3279.81(OH), 2220.42(CN). R f Value: 0.79 (20% ethyl acetate: 80% hexane). HRMS(ESI)C9H7NO[MH] + Calculated values: Theoretical value m / z = 144.0448, Measured value m / z = 144.0485

[0131] 4-(2-isocyanoethyl)phenol(4) [ka] First, N-(4-((tert-butyldimethylsilyl)oxy)phenethyl)formamide (0.98 g, 3.50 mmol) was dehydrated using MsCl (0.82 mL, 10.70 mmol) and Et3N (4.48 mL, 32.20 mmol) in DCM (15 mL) to obtain a crude silyl-protected phenol-isocyanide as a brown oil. This was then redissolved in ethanol (15 mL) and treated with excess KOH to prepare the title compound. After stirring at room temperature for 2 hours, the crude reaction evaporation residue was partitioned between ethyl acetate and H2O, the organic phase was dried over MgSO4, and then concentrated to obtain a light brown oil. This was purified by silica gel chromatography (ethyl acetate:pentane (50:50)) to obtain a pale yellow oil (0.42 g, 82%). 1 1H NMR (300 MHz, (CD3)2SO):δ H =2.74~2.81(m,2H), 3.62~3.70(m,2H), 6.72(d,J=8.7Hz,2H), 7.07(d,J=8.7Hz,2H), 9.33(br s,1H). 13 ¹³C NMR (75 MHz, (CD3)2SO):δ c =34.2, 43.3, 115.5, 127.8, 130.1, 155.9, 156.6. IR(film,cm) -1 ):ν=3030.65(OH), 2132.62(NC). R f Value: 0.55 (50% ethyl acetate: 50% petroleum ether)

[0132] 4-Isocyanophenol (5) [ka] First, N-(4-((tert-butyldimethylsilyl)oxy)phenyl)formamide (550 mg, 2.19 mmol) was dehydrated using MsCl (0.50 mL, 10.70 mmol) and Et3N (2.68 mL, 19.71 mmol) in DCM (20 mL) to obtain a crude O-silyl-protected phenol-isocyanide as a brown oil. This was then redissolved in ethanol (15 mL) and treated with excess KOH to prepare the title compound. After stirring at room temperature for 2 hours, the crude reaction evaporation residue was partitioned between ethyl acetate and H2O, the organic layer was dried over MgSO4, and then concentrated to obtain a light brown oil. This was purified by silica gel chromatography (pentane:ethyl acetate (80:20)) to obtain a pale yellow oil (180 mg, 69%). 1 1H NMR (300 MHz, CDCl3): δ H =6.85(d,J=8.6Hz,2H), 7.26(d,J=8.6Hz,2H). 13 ¹³C NMR (75 MHz, CDCl3):δ c =116.3, 128.0, 157.1, 160.4. IR(film,cm) -1 ):ν=3382.31(OH), 2908.25(CH), 2947.60(CH), 2124.90(NC). R f Value: 0.45

[0133] 2-Isocyanovinylbenzene (6) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (375 mg, 2.83 mmol), benzaldehyde (75 mg, 0.94 mmol), and LiHMDS (3.77 mL, 3.77 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (10% ethyl acetate / hexane) to obtain an irritating brown solid in a 2.5:1 ratio of E and Z isomers (46 mg, 38%). Main isomer (E) 11H NMR (500 MHz, CDCl3): δ H =6.31(d,J=14.2Hz,1H), 6.97(d,J=14.7Hz,1H), 7.34~7.45(m,5H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =5.86(d,J=9.3Hz,1H), 6.41(d,J=9.3Hz,1H), 7.34~7.45(m,4H), 7.71(d,J=8.8Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =126.9, 129.0, 129.3, 129.6, 130.1, 137.0. IR(film,cm) -1 ):ν=3063.88(CH), 3028.52(CH), 2925.75(CH), 2121.38(NC). R f Value: 0.89 (10% ethyl acetate: 90% hexane)

[0134] (E)-1-Bromo-4-(2-isocyanovinyl)benzene(7) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (276 mg, 1.35 mmol), 4-bromobenzaldehyde (100 mg, 0.54 mmol), and LiHMDS (1.62 mL, 1.62 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (5% ethyl acetate / hexane) to obtain a single E isomer (38 mg, 34%) as an irritating dark yellow solid. 1 1H NMR (500 MHz, CD3CN): δ H =6.55(d,J=14.7Hz,1H), 7.05(d,J=14.7Hz,1H), 7.37(d,J=8.3Hz,2H), 7.58(d,J=8.31Hz,2H). 13 ¹³C NMR (125 MHz, CD3CN):δ c=116.2, 118.9, 129.2, 132.2, 134.0, 151.6. IR(film,cm) -1 ):ν=2923.56(CH), 2853.13(CH), 2120.13(NC). R f Value: 0.57 (5% ethyl acetate:95% hexane)

[0135] (E)-4-methylphenylvinyl isocyanide (8) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (426 mg, 2.08 mmol), p-tolualdehyde (100 mg, 0.83 mmol), and LiHMDS (2.49 mL, 4.29 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (30% ethyl acetate / hexane) to obtain a dark yellow solid as a single E isomer (62 mg, 52%). 1 1H NMR (500 MHz, CD3CN): δ H =2.34(s,3H), 6.47(d,J=14.7Hz,1H), 7.05(d,J=14.7Hz,1H), 7.22(d,J=7.8Hz,2H), 7.35(d,J=7.8Hz,2H). 13 ¹³C NMR (125 MHz, CD3CN):δ c =21.4, 127.8, 130.7, 137.4, 137.5, 141.3. IR(film,cm) -1 ):ν=2924.42(CH), 2854.53(CH), 2164.4(NC). R f Value: 0.77 (30% ethyl acetate:70% hexane)

[0136] (2-isocyanovinyl)-4-methoxybenzene(9) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (520 mg, 2.94 mmol), 4-methoxybenzaldehyde (100 mg, 0.73 mmol), and LiHMDS (4.41 mL, 4.41 mmol) were stirred in anhydrous THF (5 mL) for 18 hours. The title compound was purified by silica column chromatography (10% ethyl acetate / hexane) to obtain an irritating dark red solid in a 2:1 ratio of E and Z isomers (41 mg, 35%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =3.83(s,3H), 6.10(d,J=14.5Hz,1H), 6.87(d,J=8.9Hz,2H), 6.95(d,J=13.6Hz,1H), 7.30(d,J=9.8Hz,2H). Minor isomer (Z) 1 1H NMR (300 MHz, CDCl3): δ H =3.85(s,3H), 5.65(d,J=9.2Hz,1H), 6.87(d,J=8.9Hz,2H), 7.30(d,J=9.8Hz,1H), 7.70(d,J=8.9Hz,2H). 13 ¹³C NMR (125 MHz, CDCl3):δ c = 55.6, 114.4, 114.7, 125.7, 128.4, 131.3, 131.7, 136.5, 160.8. IR (film, cm) -1 ):ν=2960.46(CH), 2118.44(NC). R f Value: 0.82 (10% ethyl acetate: 90% hexane)

[0137] 3-(2-isocyanovinyl)phenol(10) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (326 mg, 1.84 mmol), 3-hydroxybenzaldehyde (75 mg, 0.61 mmol), and LiHMDS (2.45 mL, 2.45 mmol) were stirred in anhydrous THF (7.5 mL) for 18 hours. The title compound was purified by silica column chromatography (30% ethyl acetate / hexane) to obtain an irritating brown solid in a 5:2 ratio of E and Z isomers (40 mg, 46%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =6.25(d,J=14.7Hz,1H), 6.80~6.95(m,3H), 6.90(d,J=14.2Hz,1H), 7.20~7.30(m,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =5.85(d,J=9.3Hz,1H), 6.80~6.95(m,3H), 7.22(d,J=8.8Hz,1H), 7.20~7.30(m,1H). 13 ¹³C NMR (125 MHz, CDCl3): 109.2, 115.6, 116.0, 128.5, 131.2, 131.5, 136.2, 157.0. IR (film, cm) -1 ):ν=3272.77(OH), 2924.35(CH), 2111.66(NC). R f Value: 0.63 (30% ethyl acetate: 70% hexane). HRMS(ESI)C9H7NO[MH] + Calculated values: Theoretical value m / z = 144.0448, Measured value m / z = 144.0471

[0138] 2-(2-isocyanovinyl)phenol(11) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (158 mg, 0.77 mmol), tert-butyl(2-(2-isocyanovinyl)phenoxy)dimethylsilane (100 mg, 0.38 mmol), and LiHMDS (0.85 mL, 0.85 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. After solvent extraction, ethanol and KOH were added to the compound and stirred for 3 hours to remove the tert-butyldimethylsilane protecting group. Subsequently, the title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain an irritating brown solid as a single isomer (24 mg, 25%). 1 1H NMR (300 MHz, (CD3)2CO):δ H =6.66(d,J=14.2Hz,1H), 6.77(d,J=8.5Hz,1H), 6.85(d,J=8.5Hz,1H), 6.99(d,J=14.2Hz,1H), 7.04~7.27(m,2H), 7.58(d,J=7.3Hz,1H). 13 ¹³C NMR (75 MHz, (CD3)2CO):δ c =79.6, 116.8, 117.2, 121.1, 130.5, 131.9, 134.4, 157.1. IR(film,cm) -1 ):ν=3361.04(OH), 2116.04(NC). R f Value: 0.20 (20% ethyl acetate: 80% hexane). HRMS(ESI)C9H8NO[MH] + Calculated values: Theoretical value m / z = 144.0448, Measured value m / z = 144.0471

[0139] (E)-2-(2-isocyanovinyl)-4-methoxyphenol(12) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (262 mg, 1.48 mmol), 2-hydroxy-5-methoxybenzaldehyde (75 mg, 0.49 mmol), and LiHMDS (1.97 mL, 1.97 mmol) were stirred in anhydrous THF (5 mL) for 18 hours. The title compound was purified by silica column chromatography (30% ethyl acetate / hexane) to obtain a brown solid in a 5:1 ratio of E and Z isomers (38 mg, 44%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =3.80(s,3H), 5.40(br s,1H), 6.60(d,J=14.5Hz,1H), 6.70~6.80(m,3H), 7.05(d,J=14.5Hz,1H). Minor isomer (Z) 1 1H NMR (300 MHz, CDCl3): δ H =3.80(s,3H), 5.40(br s,1H), 5.90(d,J=9.5Hz,1H), 6.70~6.80(m,3H), 7.60(d,J=9.5Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c = 55.8, 112.8, 113.8, 116.7, 117.2, 120.7, 133.2, 148.4, 153.9, 164.1. IR (film, cm) -1 ):ν=3289.73(OH), 2835.44(CH), 2119.56(NC). R f Values: E=0.71, Z=0.91 (30% ethyl acetate:70% hexane). HRMS(ESI)C 10 H9NO2[M+Na] + Calculated values: Theoretical value m / z = 198.0525, Measured value m / z = 198.0532

[0140] 5-Bromo-2-(2-isocyanovinyl)phenol(13) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (263 mg, 1.49 mmol), 5-bromosalicyaldehyde (100 mg, 0.49 mmol), and LiHMDS (1.99 mL, 1.99 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (15% ethyl acetate / hexane) to obtain a yellow solid in a 4:1 ratio of E and Z isomers (15 mg, 28%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =5.51(br s,1H), 6.57(d,J=14.3Hz,1H), 6.69(d,J=8.3Hz,1H), 7.00(d,J=14.3Hz,1H), 7.30(dd,J=2.5,8.3Hz,1H), 7.38(d,J=2.5Hz). Minor isomer (Z) 1 1H NMR (300 MHz, CDCl3): δ H =5.87(d,J=9.3Hz,1H), 6.72(d,J=9.3Hz,1H), 6.86~6.95(m,2H), 7.35(d,J=2.26Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =112.9, 117.5, 131.2, 131.3, 132.8, 152.6. IR(film,cm) -1 ):ν=3198.11(OH), 2924.14(CH), 2853.3(CH), 2147.9(NC). R f Value: 0.55 (15% ethyl acetate: 85% hexane). HRMS(ESI)C9H6BrNO[MH] + Calculated values: Theoretical value m / z = 221.9560, Measured value m / z = 221.9552

[0141] 3-Bromo-4-(2-isocyanovinyl)phenol(14) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (458 mg, 2.23 mmol), 3-bromo-4-hydroxybenzaldehyde (150 mg, 0.75 mmol), and LiHMDS (3.0 mL, 3.0 mmol) were stirred in anhydrous THF (7 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a deep yellow, irritating solid in a 7:1 ratio of E and Z isomers (52 mg, 31%). Main isomer (E) 1 1H NMR (500 MHz, (CD3)2CO):δ H =6.65(d,J=14.2Hz,1H), 7.05(d,J=14.7Hz,1H), 7.06(d,J=8.3Hz,1H), 7.40(dd,J=2.0,8.3Hz,1H), 7.72(d,J=2.0Hz,1H), 9.40(br s,1H). Minor isomer (Z) 1 1H NMR (500 MHz, (CD3)2CO):δ H =6.05(d,J=9.3Hz,1H), 7.05(d,J=9.3Hz,1H), 7.10(1H,d,J=8.3Hz,1H), 7.65(dd,J=2.0,8.8Hz,1H), 7.95(d,J=3.0Hz,1H). 13 ¹³C NMR (125 MHz, (CD3)2CO):δ c =110.0, 116.6, 126.3, 127.6, 129.9, 131.6, 134.0, 135.0, 155.4. IR(film,cm) -1 ):ν=3078.76(OH), 2923.8(CH), 2151.03(NC). R f Value: 0.31 (20% ethyl acetate: 80% hexane). HRMS(ESI)C9H6BrNO[MH] + Calculated values: Theoretical value m / z = 221.9560, Measured value m / z = 221.9556

[0142] 3-Bromo-4-(2-isocyanovinyl)phenol(15) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (202 mg, 0.99 mmol), 2-bromo-4-hydroxybenzaldehyde (100 mg, 0.49 mmol), and LiHMDS (1.08 mL, 1.08 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (50% ethyl acetate / hexane) to obtain a yellow solid in a 5:1 ratio of E and Z isomers (30 mg, 56%). Main isomer (E) 1 1H NMR (500 MHz, CD3CN): δ H =6.35(d,J=14.2Hz,1H), 6.83(dd,J=2.5,7.8Hz,1H), 7.10(d,J=2.5Hz,1H), 7.23(d,J=14.7Hz,1H), 7.41(d,J=8.8Hz,1H). Minor isomer (Z) 1 1H NMR (125 MHz, CD3CN):δ H =6.03(d,J=9.3Hz,1H), 6.81(d,J=9.3Hz,1H), 6.92(dd,J=2.5,8.8Hz,1H), 7.15(d,J=2.5Hz,1H), 7.81(d,J=8.8Hz,1H). 13 ¹³C NMR (125 MHz, CD3CN):δ c =116.5, 117.1, 121.1, 125.6, 125.7, 129.6, 132.5, 135.9, 160.5. IR(film,cm) -1 ):ν=3185.19(OH), 2923.40(CH), 2853.06(CH), 2152.93(NC). R f Values: E=0.65, Z=0.77 (50% ethyl acetate:50% hexane). HRMS(ESI)C9H6BrNO[MH] + Calculated values: Theoretical value m / z = 221.9560, Measured value m / z = 221.9558

[0143] 4-(2-isocyanovinyl)benzene-1,2-diol(16) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (111 mg, 0.54 mmol), 3,4-bis-((tert-butyldimethylsilyl)oxy)benzaldehyde (100 mg, 0.27 mmol), and LiHMDS (0.66 mL, 0.66 mmol) were stirred in anhydrous THF (10 mL) for 18 hours, followed by desilylation with ethoxide. The crude product was purified using silica gel chromatography (ethyl acetate 1:4 hexane) to obtain the title compound as a pale yellow oil (15 mg, 20%). 1 1H NMR (300 MHz, CD3CN): δ H =6.25(d,J=14.3Hz,2H), 6.60~6.70(m,2H), 6.73~6.81(m,2H). 13 ¹³C NMR (75 MHz, CD3CN):δ C =114.5, 116.9, 121.4, 124.0, 125.50, 138.8, 147.2, 149.1. IR(film,cm) -1 ):ν=3220.54(OH), 2982.63(CH), 2933.75(CH), 2119.57(NC). R f Value: 0.05

[0144] 2,6-Dibromo-4-(2-Isocyanovinyl)phenol (17) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (94 mg, 0.53 mmol), 3,5-dibromo-4-hydroxybenzaldehyde (75 mg, 0.26 mmol), and LiHMDS (0.54 mL, 0.54 mmol) were stirred in anhydrous THF (7 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a deep yellow, irritating solid in a 4:1 ratio of E and Z isomers (27 mg, 30%). Main isomer (E) 1 1H NMR (300 MHz, CD3OD): δ H=6.60(d,J=14.5Hz,1H), 6.95(d,J=14.5Hz,1H), 7.70(s,2H). Minor isomer (Z) 1 1H NMR (300 MHz, CD3OD): δ H =6.05(d,J=9.2Hz,1H), 6.95(d,J=9.5Hz,1H), 7.40(s,1H), 7.40(d,J=3.5Hz,1H). 13 ¹³C NMR (125 MHz, CD3OD):δ c =113.0, 117.2, 132.2, 133.7, 135.7, 154.1. IR(film,cm) -1 ):ν=3464.4(OH), 3071.2(CH), 2123.69(NC). R f Value: 0.70 (20% ethyl acetate: 80% hexane). HRMS(ESI)C9H5Br2NO[MH] + Calculated values: Theoretical value m / z = 299.8665, Measured value m / z = 299.8652

[0145] (E / Z)-2-(isocyanovinyl)-1,4-dimethoxybenzene(18) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (305 mg, 1.49 mmol), 2,5-dimethoxybenzaldehyde (100 mg, 0.59 mmol), and LiHMDS (1.79 mL, 1.79 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (10% ethyl acetate / hexane) to obtain a light brown solid in a 9:1 ratio of E and Z isomers (39 mg, 35%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =3.76(s,3H), 3.84(s,3H), 6.50(d,J=14.7Hz,1H), 6.80~6.90(m,3H), 7.05(d,J=14.2Hz,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H=3.76(s,3H), 3.84(s,3H), 5.85(d,J=9.3Hz,1H), 6.80~6.90(m,3H), 6.91(d,J=9.3Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c = 55.6, 55.8, 111.9, 113.8, 115.6, 121.2, 132.7, 151.8, 153.2. IR(film,cm) -1 ):ν=2944.15(CH), 2835.15(CH), 2118.40(NC). R f Value: 0.56 (10% ethyl acetate: 90% hexane)

[0146] (E)-1-Bromo-2-(2-isocyanovinyl)-4-methoxybenzene(19) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (237 mg, 1.16 mmol), 2-bromo,5-methoxybenzaldehyde (100 mg, 0.46 mmol), and LiHMDS (1.40 mL, 1.40 mmol) were stirred in anhydrous THF (7 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a single E isomer (51 mg, 47%) as an irritating yellow solid. 1 1H NMR (500 MHz, CD3CN): δ H =3.82(s,3H), 6.55(d,J=14.7Hz,1H), 6.90(dd,J=3.0,8.8Hz,1H), 7.12(d,J=3.0Hz,1H), 7.28(d,J=14.2Hz,1H), 7.55(d,J=8.8Hz,1H). 13 ¹³C NMR (125 MHz, CD3CN):δ c = 55.5, 113.6, 115.0, 118.7, 135.1, 136.1, 160.5. IR(film,cm) -1 ):ν=2924.48(CH), 2852.07(CH), 2121.60(NC). R fValue: 0.45 (20% ethyl acetate: 80% hexane)

[0147] (E)-2,4-Dichloro-1-(2-isocyanovinyl)benzene(20) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (293 mg, 1.43 mmol), 2,4-dichlorobenzaldehyde (100 mg, 0.57 mmol), and LiHMDS (1.72 mL, 1.72 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (10% ethyl acetate / hexane) to obtain a single E isomer (55 mg, 49%) as an irritating dark yellow solid. 1 1H NMR (500 MHz, CD3Cl): δ H =6.30(d,J=14.2Hz,1H), 7.25~7.27(m,1H), 7.28(d,J=14.7Hz,1H), 7.36(d,J=8.3Hz,1H), 7.46(d,J=2.0Hz,1H). 13 ¹³C NMR (125 MHz, CD3Cl):δ c =127.4, 127.7, 130.1, 132.2, 136.2. IR(film,cm) -1 ):ν=2923.98(CH), 2852.71(CH), 2124.87(NC). R f Value: 0.89 (10% ethyl acetate: 90% hexane)

[0148] (E)-4-(2-isocyanovinyl)-N,N-dimethylalanine(21) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (275 mg, 1.34 mmol), 4-dimethylaminobenzaldehyde (100 mg, 0.67 mmol), and LiHMDS (2.01 mL, 2.01 mmol) were stirred in anhydrous THF (7 mL) for 20 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a dark yellow solid as a 5:1 mixture of E and Z isomers (51 mg, 44%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =3.01(s,3H), 6.10(d,J=14.2Hz,1H), 6.65(d,J=8.8Hz,2H), 6.85(d,J=14.7Hz,1H), 7.20(d,J=8.8Hz,2H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =3.03(s,3H), 5.60(d,J=9.3Hz,1H), 6.70(d,J=9.3Hz,1H), 7.40(d,J=8.3Hz,2H), 7.65(d,J=8.3Hz,2H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =40.4, 111.9, 112.2, 128.2, 130.0, 131.1, 132.2, 137.1, 151.6, 163.6. IR(film,cm) -1 ):ν=2908.28(CH), 2818.73(CH), 2114.22(NC). R f Value: 0.85 (20% ethyl acetate: 80% hexane)

[0149] (2-Isocyanovinyl)-4-Acetamidobenzene(22) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (81 mg, 0.46 mmol), 4-acetamidobenzaldehyde (75 mg, 0.46 mmol), and LiHMDS (1.84 mL, 1.84 mmol) were stirred in anhydrous THF (5 mL) for 18 hours. The title compound was purified by silica column chromatography (15% ethyl acetate / hexane) to obtain a brown, irritating solid in an 8:1 ratio of E and Z isomers (27 mg, 32%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =2.20(s,3H), 6.24(d,J=14.3Hz,1H), 6.90(d,J=14.3Hz,1H), 7.46(br s,1H), 7.30(d,J=8.3Hz,2H), 7.54(d,J=8.7Hz,2H). Minor isomer (Z) 1 1H NMR (300 MHz, CDCl3): δ H =2.23(s,3H), 5.80(d,J=9.4Hz,1H), 7.30(d,J=9.4Hz,1H), 7.60(br s,1H), 7.67~7.72(m,2H), 7.84(d,J=8.3Hz,2H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =24.3, 109.6, 119.2, 119.6, 127.2, 129.6, 131.8, 135.6, 139.2, 164.6, 168.3. IR(film,cm) -1 ):v=3253.59(NH), 3071.34(CH), 2116.37(NC). R f Values: E=0.44, Z=0.48 (15% ethyl acetate:85% hexane)

[0150] tert-butyl(4-(isocyanovinyl)phenyl)carbamate(23) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (200 mg, 0.98 mmol), tert-butyl-4-formylphenylcarbamate (100 mg, 0.49 mmol), and LiHMDS (1.23 mL, 1.23 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (hexane:ethyl acetate (95:5)) to obtain the title compound as a brown oily substance, as a mixture of E and Z isomers (4:1) (38 mg, 31%). Main isomer (E) 1 1H NMR (300 MHz, (CD3)2CO):δ H =1.45(s,9H), 6.35(d,J=14.3Hz,1H), 6.87(d,J=14.3Hz,1H), 7.30(d,J=8.7Hz,2H), 7.50(d,J=8.7Hz,2H), 8.24(s,1H). Minor isomer (Z) 1 1H NMR (300 MHz, (CD3)2CO):δ H =1.45(s,9H), 5.82(d,J=9.4Hz,1H), 7.24(d,J=9.4Hz,1H), 7.43(d,J=10.7Hz,2H), 7.50(d,J=12.8Hz,2H). 13 ¹³C NMR (75 MHz, (CD3)2CO):δ C =28.3, 80.2, 118.7, 127.9, 136.6, 141.4, 153.1. IR(film,cm) -1 ):ν=2976.09(CH), 2857.55(CH), 2123.08(NC). R f Value: 0.30

[0151] N-(4-(1-isocyanopropane-1-en-2-yl)phenyl)methanesulfonamide (24) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (191 mg, 1.12 mmol), N-(4-acetylphenyl)methanesulfonamide (100 mg, 0.47 mmol), and LiHMDS (1.40 mL, 1.40 mmol) were stirred in anhydrous THF (5 mL) for 20 hours. The title compound was purified by silica column chromatography (ethyl acetate / hexane (1:3)) to obtain a red, irritating solid in a 6:1 ratio of E and Z isomers (40 mg, 37%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =2.24(d,J=1.5Hz,3H), 3.06(s,3H), 6.03(s,1H), 6.48(s,1H), 7.22(d,J=8.8Hz,2H), 7.33(d,J=8.8Hz,2H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =2.10(d,J=1.5Hz,3H), 3.08(s,3H), 5.84(s,1H), 7.06(s,1H), 7.24(d,J=8.8Hz,2H), 7.50(d,J=8.8Hz,2H). 13 ¹³C NMR (125 MHz, CDCl3):δ C =16.9, 39.8, 119.8, 120.2, 127.3, 127.6, 127.8, 128.0, 129.1, 137.6, 142.8. IR(film,cm) -1 ):ν=3249.99(NH)3024.61(NH), 2983.74(CH), 2930.51(CH), 2114.18(NC). R f Value s: 0.12 (25% ethyl acetate: 75% hexane). HRMS(ESI)C 11 H 12 N2O2S[M+Na] + Calculated values: Theoretical value m / z = 259.0512, Measured value m / z = 259.0513

[0152] 3-(2-isocyanovinyl)-1H-indole (25 and 26) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (487 mg, 2.75 mmol), indole-3-carboxyaldehyde (100 mg, 0.69 mmol), and LiHMDS (4.13 mL, 4.13 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica gel column chromatography (50% ethyl acetate / hexane) to obtain a mixture of E and Z isomers in a 3:1 ratio. The E isomer was isolated as a dark yellow solid (20 mg, 23%), and the Z isomer was isolated as a dark red solid (35 mg, 40%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =6.35(d,J=14.2Hz,1H), 7.14(d,J=14.2Hz,1H), 7.25~7.35(m,2H), 7.36(s,1H), 7.43(d,J=7.8Hz,1H), 7.70(d,J=7.3Hz,1H), 8.35(br s,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =5.75(d,J=8.8Hz,1H), 7.20~7.27(m,2H), 7.28(d,J=8.3Hz,1H), 7.45(d,J=8.3Hz,1H), 7.69(d,J=8.3Hz,1H), 8.16(d,J=2.5Hz,1H), 8.55(br s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =107.1, 111.1, 119.9, 121.4, 123.4, 126.3, 130.2, 136.9, 163.1. IR(film,cm) -1 ):ν=3288.17(NH), 2926.27(CH), 2116.18(NC). R f Value s: E=0.69, Z=0.78 (50% ethyl acetate:50% hexane)

[0153] N-methyl-indole-3-carboxyaldehyde [ka] Indole-3-carboxyaldehyde (1.00 g, 6.90 mmol) was treated with NaH (0.30 g, 8.30 mmol) in anhydrous THF (30 mL) at 0°C for 10 minutes. Then, iodomethane (0.5 mL, 8.20 mmol) was added to the resulting mixture and stirred for 5 hours. The reaction was then quenched with H2O and extracted with ethyl acetate. The organic layer was then washed with H2O and brine, dried over MgSO4, and concentrated under vacuum. The remaining residue was purified by silica column chromatography (50% ethyl acetate: 50% hexane) to obtain a pale creamy solid (0.70 g, 67%). 1 1H NMR (500 MHz, CDCl3): δ H =3.89(s,1H), 7.33~7.38(m,3H), 7.70(s,1H), 8.32(d,J=7.3Hz,1H), 10.01(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =33.7, 109.7, 122.0, 122.9, 124.0, 139.1, 184.4. IR(film,cm) -1 ):ν=1651.55(C=O). R f Value: 0.50 (50% ethyl acetate: 50% hexane). HRMS(ESI)C 10 H9NO[M+H] + Calculated values: Theoretical value m / z = 160.0762, Measured value m / z = 160.0760. Melting point: 72℃

[0154] (E)-3-(2-isocyanovinyl)-1-methyl-indole(27) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (338 mg, 1.91 mmol), N-methyl-indole-3-carboxyaldehyde (100 mg, 0.63 mmol), and LiHMDS (2.55 mL, 2.55 mmol) were stirred in anhydrous THF (6.0 mL) for 18 hours. The title compound was purified by silica column chromatography (50% ethyl acetate / hexane) to obtain a deep red solid as a single E isomer (32 mg, 28%). 1 1H NMR (300 MHz, CDCl3): δ H =3.81(s,3H), 6.31(d,J=14.3Hz,1H), 7.10(d,J=14.3Hz,1H), 7.20(s,1H), 7.22~7.38(m,4H), 7.67(d,J=7.5Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =33.0, 106.2, 109.4, 110.0, 119.9, 121.1, 122.9, 125.2, 130.0, 130.7, 137.1, 162.9. IR(film,cm) -1 ):ν=3051.80(CH), 2929.57(CH), 2116.00(NC). R f Value: 0.70 (50% ethyl acetate: 50% hexane)

[0155] N-ethyl-indole-3-carboxyaldehyde [ka] Indole-3-carboxyaldehyde (1.00 g, 6.9 mmol) was treated with NaH (0.33 g, 8.3 mmol) in anhydrous THF (30 ml) for 10 minutes at 0°C. Bromoethane (0.62 ml, 8.3 mmol) was then added to the resulting mixture and stirred for 5 hours. The reaction was then quenched with H2O and extracted with ethyl acetate. The organic layer was then washed with H2O and brine, dried over MgSO4, and concentrated under vacuum. The remaining residue was purified by silica column chromatography (50% ethyl acetate / hexane) to obtain a cream-colored solid (0.76 g, 64%). 1 1H NMR (300 MHz, CDCl3): δ H =1.57(t,J=7.3Hz,3H), 4.25(q,J=7.3Hz,2H)7.31~7.42(m,3H), 7.77(s,1H), 8.32(d,J=6.9Hz,1H), 10.02(s,1H). 13 ¹³C NMR (500 MHz, CDCl3): δ c =15.0, 42.0, 109.9, 118.2, 122.2, 122.9, 123.9, 137.0, 137.4, 184.4. IR(film,cm) -1 ):ν=1651.55(C=O). R f Value: 0.71 (50% ethyl acetate: 50% hexane). HRMS(ESI)C 11 H 11 NO[M+H] + Calculated values: Theoretical value m / z = 174.0918, Measured value m / z = 174.0918. Melting point = 105℃

[0156] (2-isocyanovinyl)-1-ethyl-indole(28) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (325 mg, 1.84 mmol), N-ethyl-indole-3-carboxyaldehyde (100 mg, 0.613 mmol), and LiHMDS (2.43 mL, 2.43 mmol) were stirred in anhydrous THF (7 mL) for 20 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain an irritating light brown solid in a 9:1 ratio of E and Z isomers (36 mg, 31%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =1.50(t,J=7.5Hz,3H), 4.18(q,J=7.5Hz,2H), 6.25(d,J=14.3Hz,1H), 7.05(d,J=14.3Hz,1H), 7. 22~7.25(m,1H), 7.30(td,J=1.0,6.9Hz,1H), 7.36(dt,J=1.0,8.3Hz,1H), 7.68(d,J=7.5Hz,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =1.53(t,J=7.3Hz,3H), 4.26(q,J=7.4Hz,2H), 5.70(d,J=9.3Hz,1H), 7.24(d,J=9.3H) z,1H), 7.27~7.32(m,2H), 7.40(d,J=7.3Hz,1H), 7.67(d,J=7.8Hz,1H), 8.06(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =15.2, 29.7, 110.1, 120.1, 122.9, 129.0, 130.4. IR(film,cm) -1 ):ν=2925.26(CH), 2115.43(NC). R f Value s: E=0.72, Z=0.90 (20% ethyl acetate:80% hexane)

[0157] N-isopropyl-indole-3-carboxyaldehyde [ka]

[0158] Indole-3-carboxyaldehyde (1.00 g, 6.8 mmol) was treated with NaH (0.6 g, 13.7 mmol) in anhydrous THF (30 mL) for 10 minutes at 0°C. Isopropyl iodide (1.4 mL, 13.7 mmol) was then added to the resulting mixture and stirred for 5 hours. The reaction was then quenched with H2O and extracted with ethyl acetate. The organic layer was then washed with H2O and brine, dried over MgSO4, and concentrated under vacuum. The remaining residue was purified by silica column chromatography (50% ethyl acetate:50% hexane) to obtain a pale yellow solid (0.68 g, 54%). 1 1H NMR (500 MHz, CDCl3): δ H =1.62(d,J=6.9Hz,6H), 4.75(septet,J=6.9Hz,1H), 7.31~7.37(m,2H), 7.43(d,J=7.3Hz,1H), 7.86(s,1H), 8.32(d,J=6.9Hz,1H), 10.03(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =22.5, 48.2, 110.2, 119.9, 122.1, 122.8, 123.7, 184.5. IR(film,cm) -1 ):ν=1642.76(C=O). R f Value: 0.56 (50% ethyl acetate: 50% hexane). HRMS(ESI)C 12 H 13 NO[M+H] + Calculated values: Theoretical value m / z = 188.1075, Measured value m / z = 188.1083. Melting point: 98℃

[0159] (E)-3-(2-isocyanovinyl)-1-isopropyl-indole(29)

[0160] [ka]

[0161] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (299 mg, 1.69 mmol), N-isopropyl-indole-3-carboxyaldehyde (100 mg, 0.56 mmol), and LiHMDS (2.25 mL, 2.25 mmol) were stirred in anhydrous THF (7 mL) for 20 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a dark yellow solid as a single E isomer (29 mg, 26%). 1 1H NMR (300 MHz, CDCl3): δ H =1.55(d,J=6.8Hz,6H), 4.68(septet,J=6.8Hz,1H), 6.33(d,J=14.3Hz,1H), 7.13(d,J=14. 3Hz,1H), 7.21~7.33(m,2H), 7.36(s,1H), 7.42(d,J=7.9Hz,1H), 7.67(d,J=7.91Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =22.6, 47.4, 106.0, 110.3, 120.1, 121.1, 122.6, 125.5, 125.9, 130.3. IR(film,cm) -1 ):ν=2925.21(CH), 2854.40(CH), 2116.07(NC). R f Value: 0.72 (20% ethyl acetate: 80% hexane)

[0162] 4-Bromo-3-(2-isocyanovinyl)-1H-indole(30 and 31) [ka] Following general procedure 1: Diethyl (isocyanomethyl)phosphonate (177 mg, 1.00 mmol), 4-bromoindole-3-carboxyaldehyde (75 mg, 0.33 mmol), and LiHMDS (1.33 mL, 1.33 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (50% ethyl acetate / hexane) to obtain an irritating light brown solid in a 3:1 ratio of E and Z isomers (28 mg, 35%). Main isomer (E) 1 1H NMR (300 MHz, CDCl3): δ H =6.07(d,J=14.3Hz,1H), 7.09(t,J=7.6Hz,1H), 7.34(d,J=3.0Hz,1H), 7.38(m,1H), 7.43(d,J=2.6Hz,1H), 7.97(d,J=14.3Hz,1H), 8.45(br s,1H). Minor isomer (Z) 1 1H NMR (300 MHz, CDCl3): δ H =5.75(d,J=9.2Hz,1H), 6.80(d,J=9.2Hz,1H), 7.22~7.25(m,1H), 7.27~7.31(m ,1H), 7.44~7.46(m,1H), 7.69(d,J=8.4Hz,1H), 8.15(d,J=2.9Hz,1H), 8.58(br s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =110.2, 114.5, 118.0, 121.0, 123.2, 124.1, 126.4, 126.8, 128.5, 132.0. IR(film,cm) -1 ):ν=3658.20(NH), 2979.39(CH), 2888.24(CH), 2139.04(NC). R f Value s: E=0.57, Z=0.80 (50% ethyl acetate:50% hexane)

[0163] (E / Z)-2-bromo-3-(2-isocyanovinyl)naphthalene(32)

[0164] [ka]

[0165] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (217 mg, 1.06 mmol), 1-bromo-2-naphthaldehyde (100 mg, 0.43 mmol), and LiHMDS (1.27 mL, 1.27 mmol) were stirred in anhydrous THF (7 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a light brown solid as a 2:1 mixture of E and Z isomers (49 mg, 45%). Main isomer (E) 1 1H NMR (500 MHz, CD3CN): δ H =6.65(d,J=14.7Hz,1H), 7.62~7.74(m,4H), 7.91~8.01(m,2H), 8.36(d,J=9.3Hz,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CD3CN): δ H =6.26(d,J=9.3Hz,1H), 7.62~7.74(m,4H), 7.91~8.01(m,2H), 8.41(d,J=9.8Hz,1H). 13 ¹³C NMR (125 MHz, CD3CN):δ c =123.4, 127.5, 127.8, 128.4, 128.5, 135.9. IR(film,cm) -1 ):ν=2924.88(CH), 2122.21(NC). R f Value: 0.68 (20% ethyl acetate: 80% hexane)

[0166] (E / Z)-2-bromo-3-(2-isocyanovinyl)pyridine(33) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (274 mg, 1.34 mmol), 2-bromo-3-pyridinecarboxaldehyde (100 mg, 0.53 mmol), and LiHMDS (1.62 mL, 1.62 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (15% ethyl acetate / hexane) to obtain a dark yellow solid as a 4:1 mixture of E and Z isomers (48 mg, 45%). Main isomer (E) 1 1H NMR (500 MHz, CD3CN): δ H =6.55(d,J=14.7Hz,1H), 7.15(d,J=14.2Hz,1H), 7.88(dd,J=2.0,7.8Hz,1H), 8.35(dd,J=2.0,4.9Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =123.3, 131.0, 132.8, 134.1, 135.0, 151.0. IR(film,cm) -1 ):ν=2923.79(CH), 2852.33(CH), 2111.64(NC). R f Value: 0.63 (20% ethyl acetate: 80% hexane)

[0167] (E)-(2-isocyanovinyl)cyclohexane(34) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (450 mg, 2.2 mmol), cyclohexylcarboxaldehyde (100 mg, 0.89 mmol), and LiHMDS (2.67 mL, 2.67 mmol) were stirred in anhydrous THF (7 mL) for 20 hours. The title compound was purified by silica column chromatography (50% ethyl acetate / hexane) to obtain a dark yellow solid as a single E isomer (48 mg, 40%). 1 1H NMR (500 MHz, CDCl3): δ H=1.05~1.30(m,5H), 1.65~1.80(m,4H), 1.99~2.09(m,1H), 5.60~5.65(m,1H), 6.06~6.12(m,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =25.5, 25.7, 31.6, 38.4, 86.6, 110.8, 142.4, 144.2, 161.9. IR(film,cm) -1 ):ν=2925.99(CH), 2853.28(CH), 2123.43(NC). R f Value: 0.83 (50% ethyl acetate: 50% hexane)

[0168] 4-Hydroxy-2-Styrylbenzaldehyde [ka] Following general procedure 2: Triethylamine (0.41 mL, 2.98 mmol), styrene (0.3 mL, 2.98 mmol), 2-bromo-4-hydroxybenzaldehyde (400 mg, 1.98 mmol), Pd(OAc)2 (45 mg, 0.19 mmol), and tri(o-tolyl)phosphine (120 mg, 0.39 mmol) were stirred overnight in DMF (10 mL). The title compound was obtained by purification by silica gel chromatography (15% ethyl acetate: 85% petroleum ether) to obtain an orange / yellow solid (195 mg, 44%). 1 1H NMR (500 MHz, CD3OD): δ H =6.85(dd,J=2.9,6.4Hz,1H),7.10(d,J=16.1Hz,1H),7.15(d,J=2.5Hz,1H),7.28(t,J=7.3Hz,1H),7 .35(t,J=7.8Hz,2H), 7.56~7.57(m,1H), 7.75(d,J=8.9Hz,1H), 8.12(d,J=16.1Hz,1H), 10.07(s,1H). 13 ¹³C NMR (125 MHz, CD3OD):δ c =112.8, 115.0, 124.7, 126.6, 128.3, 128.8, 133.1, 135.2, 191.2. IR(film,cm)-1 ):ν=3150.27(OH), 2926.00(CH), 1654.73(C=O). R f Value: 0.22 (15% ethyl acetate: 85% petroleum ether). HRMS(ESI)C 15 H 12 O[M+H] + Calculated values: Theoretical value m / z = 223.0757, Measured value m / z = 223.0759. Melting point: 197~199℃

[0169] 4-((E / Z)-2-isocyanovinyl)-3-((E)-styryl)phenol(36) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (317 mg, 1.55 mmol), 4-hydroxy-2-styrylbenzaldehyde (120 mg, 0.51 mmol), and LiHMDS (2.07 mL, 2.07 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a dark yellow solid as a 4:1 mixture of E / Z isomers (31 mg, 25%). Main isomer (E) 1 1H NMR (500 MHz, CD3OD): δ H =6.35(d,J=14.7Hz,1H), 6.72(d,J=8.8Hz,1H), 6.97(d,J=16.1Hz,1H), 7.02(s,1 H), 7.27(m,2H), 7.30~7.40(m,4H), 7.55(t,J=7.8Hz,1H), 7.60(d,J=7.8Hz,2H). Minor isomer (Z) 1 1H NMR (500 MHz, CD3OD): δ H =6.05(d,J=9.3Hz,1H), 6.77(d,J=8.3Hz,1H), 6.85(d,J=7.4Hz,2H), 7.00(s,1H), 7.10~7.20(m,3H), 7.30~7.40(m,3H), 7.65(d,J=8.8Hz,1H), 7.85(d,J=8.8Hz,1H). 13¹³C NMR (125 MHz, CD3OD):δ c =111.1, 113.5, 116.4, 125.8, 127.5, 128.7, 128.7, 129.4, 131.2, 133.2, 135.5, 138.1, 139.4, 160.1. IR(film,cm) -1 ):ν=3293.28(OH), 2924.82(CH), 2120.25(NC). R f Value: 0.27 (20% ethyl acetate: 80% hexane). HRMS(ESI)C 17 H 13 NO[M+H] + Calculated values: Theoretical value m / z = 246.0917, Measured value m / z = 246.0918

[0170] 5-Cinnamyl-2-hydroxybenzaldehyde [ka] Following general procedure 2, triethylamine (0.63 mL, 4.47 mmol), styrene (51 mL, 4.47 mmol), 2-bromo-5-hydroxybenzaldehyde (600 mg, 2.98 mmol), Pd(OAc)2 (65 mg, 0.29 mmol), and tri(o-tolyl)phosphine (180 mg, 0.59 mmol) were stirred in DMF (10 mL). The title compound was obtained as a yellow solid (340 mg, 48%) after silica gel chromatography (10% ethyl acetate: 90% petroleum ether). 1 1H NMR (500 MHz, CDCl3): δ H =7.01(d,J=8.3Hz,1H), 7.05(d,J=7.8Hz,1H), 7.28(t,J=7.4Hz,1H), 7.38(t,J=7.5Hz,3H), 7 .51(d,J=7.3Hz,2H), 7.66(d,J=2.5Hz,1H), 7.72(d,J=2.5Hz,1H), 9.95(s,1H), 11.02(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c=118.1, 120.6, 126.3, 126.6, 127.7, 128.1, 128.7, 129.7, 131.5, 134.6, 136.9, 161.1, 196.5. IR(film,cm) -1 ):ν=3025.10(OH), 2853.34(CH), 1663.39(C=O). R f Value: 0.42 (10% ethyl acetate: 90% petroleum ether). HRMS(ESI)C 15 H 12 O[M+H] + Calculated values: Theoretical value m / z = 223.0756, Measured value m / z = 223.0759. Melting point: 195℃

[0171] 4-Cinnamyl-2-((E / Z)-2-isocyanovinyl)phenol(37) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (221 mg, 1.08 mmol), 5-cinnamyl-2-hydroxybenzaldehyde (100 mg, 0.43 mmol), and LiHMDS (1.30 mL, 1.30 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica column chromatography (20% ethyl acetate / hexane) to obtain a dark yellow solid as a single E isomer (41 mg, 38%). 1 1H NMR (500 MHz, CD3CN): δ H =6.75(d,J=14.7Hz,1H), 6.93(d,J=8.3Hz,1H), 7.10(d,J=5.4Hz,2H), 7.30~7.4 0(m,2H), 7.36(t,J=7.3Hz,2H), 7.60(dd,J=2.5,8.8Hz,1H), 7.58~7.61(m,3H). 13 ¹³C NMR (125 MHz, CD3CN):δ c =117.8, 118.5, 118.9, 127.6, 126.9, 127.1, 128.3, 128.8, 128.9, 130.1, 130.2. IR(film,cm) -1):ν=3245.54(OH), 2925.84(CH), 2853.53(CH), 2117.67(NC). R f Value: 0.64 (20% ethyl acetate: petroleum ether)

[0172] (E)-2-Styrenebenzaldehyde [ka] Styrene (0.5 mL, 4.26 mmol) and 2-chlorobenzaldehyde (0.3 mL, 2.84 mmol) were added to a solution of Pd(OAc)2 (64 mg, 0.28 mmol), Dave-phosphonate (67 mg, 0.17 mmol), and TBAE (1.71 g, 5.68 mmol) in dioxane (10 mL). The reaction mixture was stirred at 80°C for 48 hours. After the reaction was complete (confirmed by TLC), the resulting mixture was diluted with ethyl acetate, filtered through Celite, and concentrated under vacuum. The crude substance was then purified by silica gel column chromatography (20% ethyl acetate: 80% petroleum ether) to obtain the title compound as a yellow oil (340 mg, 51%). 1 1H NMR (500 MHz, CDCl3): δ H =7.06(d,J=16.1Hz,1H), 7.34(t,J=7.3Hz,1H), 7.41(t,J=7.3Hz,2H), 7.45(t,J=7.3Hz,1H), 7.59 ~7.61(m,3H), 7.72(d,J=7.8Hz,1H), 7.85(d,J=7.8Hz,1H), 8.06(d,J=16.1Hz,1H), 10.34(s,1H). 13 ¹³C NMR (75 MHz, CDCl3):δ c =124.2, 126.5, 126.7, 127.1, 127.8, 128.3, 131.8, 132.3, 133.2, 133.4, 136.3, 139.3, 192.2. IR(film,cm) -1 ):ν=2923.50(CH), 2852.26(CH), 1692.38(C=O). R fValue: 0.56 (20% ethyl acetate: 80% petroleum ether). HRMS(ESI)C 15 H 12 O[M+H] + Calculated values: Theoretical value m / z = 209.0966, Measured value m / z = 209.0968

[0173] 1-((E)-2-isocyanovinyl)-2-((E)-styryl)benzene(38) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (200 mg, 0.96 mmol), (E)-2-styrenebenzaldehyde (100 mg, 0.48 mmol), and LiHMDS (1.2 mL, 1.2 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica column chromatography (60% ethyl acetate / hexane) to obtain a dark yellow solid as a single E isomer (21 mg, 39%). 1 1H NMR (500 MHz, CDCl3): δ H =6.20(d,J=14.2Hz,1H), 6.99(d,J=16.1Hz,1H), 7.28(d,J=16.1Hz,1H), 7.29~7.43(m,7H), 7.55(d,J=7.3Hz,2H), 7.61(d,J=7.8Hz,1H). 13 ¹³C NMR (75 MHz, CDCl3):δ c =124.9, 126.4, 126.5, 126.9, 127.6, 128.0, 128.6, 129.6, 132.7, 134.7, 136.5, 136.6. IR(film,cm) -1 ):ν=2923.48(CH), 2852.51(CH), 2122.62(NC). R f Value: 0.83 (60% ethyl acetate: 40% petroleum ether)

[0174] E)-2-(2-(pyridine-4-yl)vinyl)benzaldehyde [ka] Following step 2, 2-bromobenzaldehyde (600 mg, 3.24 mmol), Pd(OAc)2 (15 mg, 0.06 mmol), tri(o-tolyl)phosphine (40 mg, 0.13 mmol), 4-vinylpyridine (0.52 mL, 4.86 mmol), and triethylamine (1.30 mL, 9.72 mmol) were stirred in anhydrous DMF for 24 hours. The crude substance was purified using silica gel chromatography (50% ethyl acetate: 50% petroleum ether) to obtain the title compound as a yellow oil (400 mg, 59%). 1 1H NMR (500 MHz, CDCl3): δ H =6.95(d,J=16.1Hz,1H), 7.40(d,J=6.4Hz,1H), 7.40(d,J=2.9Hz,1H), 7.51(dt,J=1.5,7.8Hz,1H), 7.61(dt,J=1.0,7.8Hz,1H), 7.7 2(d,J=7.3Hz,1H), 7.83(dd,J=1.5,7.3Hz,1H), 8.29(d,J=16.1Hz,1H), 8.59(d,J=6.4Hz,1H), 8.59(d,J=2.9Hz,1H), 10.24(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =121.1, 127.2, 128.5, 129.8, 130.5, 133.1, 133.5, 133.7, 138.2, 144.1, 150.2, 192.7. IR(film,cm) -1 ):ν=3033.36(CH), 2833.44(CH), 1689.97(C=O). R f Value: 0.21 (50% ethyl acetate: 50% petroleum ether). HRMS(ESI)C 14 H 11 NO[M+H] + Calculated values: Theoretical value m / z = 210.0925, Measured value m / z = 210.098

[0175] 4-(E)-2-((E)-2-isocyanovinyl)styryl)pyridine(39) [ka] Following general procedure 1, diisopropyl(isocyanomethyl)phosphonate (196 mg, 0.96 mmol), (E)-2-(2-(pyridine-4-yl)vinyl)benzaldehyde (100 mg, 0.48 mmol), and LiHMDS (1.20 mL, 1.20 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica gel chromatography to obtain a dark red oily substance as a single E isomer (51 mg, 47%). 1 1H NMR (500 MHz, CDCl3): δ H =6.90(d,J=16.1Hz,1H), 7.31(d,J=14.2Hz,1H), 7.33(m,1H), 7.37~7.44(m,4H), 7.48 (d,J=16.1Hz,1H), 7.62(d,J=7.8Hz,1H), 8.63(d,J=6.4Hz,1H), 8.63(d,J=2.9Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =120.8, 126.5, 127.0, 128.6, 129.3, 129.7, 130.0, 131.1, 135.2, 143.7, 150.1. IR(film,cm) -1 ):ν=3025.88(CH), 2123.15(NC). R f Value: 0.41 (65% ethyl acetate: 35% hexane). HRMS(ESI)C 16 H 12 N2[M+H] + Calculated values: Theoretical value m / z = 233.1078, Measured value m / z = 233.1088

[0176] (E)-2-(2-(pyrazine-2-yl)vinyl)benzaldehyde [ka] Following general procedure 2, 2-bromobenzaldehyde (1.2 g, 6.48 mmol), Pd(OAc)2 (30 mg, 0.12 mmol), tri(o-tolyl)phosphine (80 mg, 0.26 mmol), 2-vinylpyrazine (1.0 mL, 9.72 mmol), and triethylamine (2.6 mL, 19.4 mmol) were stirred in anhydrous DMF for 24 hours. The crude substance was purified using silica gel chromatography (50% ethyl acetate: 50% petroleum ether) to obtain the title compound as a yellow oily substance (860 mg, 63%). 1 1H NMR (500 MHz, CDCl3): δ H =7.10(d,J=16.1Hz,1H), 7.51(t,J=7.8Hz,1H), 7.61(t,J=7.8Hz,1H), 7.76(d,J=7.3Hz,1H), 7.86(d,J=7.3Hz, 1H), 8.45(d,J=4.4Hz,1H), 8.57(d,J=1.5Hz,1H), 8.65(d,J=16.1Hz,1H), 8.72(d,J=3.4Hz,1H), 10.36(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =127.7, 129.0, 129.3, 131.7, 132.6, 133.7, 134.1, 138.6, 143.5, 144.1, 144.7, 151.0, 192.5. IR(film,cm) -1 ):ν=3063.44(CH), 2846.10(CH), 1686.49(C=O). R f Value: 0.44 (50% ethyl acetate: 50% petroleum ether)

[0177] 5-((E)-2-((E)-2-isocyanovinyl)styryl)pyrimidine (40) [ka] Following general procedure 1, diisopropyl(isocyanomethyl)phosphonate (175 mg, 0.86 mmol), (E)-2-(2-(pyrazine-2-yl)vinyl)benzaldehyde (100 mg, 0.43 mmol), and LiHMDS (1.07 mL, 1.07 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was purified by silica gel chromatography to obtain a dark red oily substance as a single E isomer (45 mg, 41%). 1 1H NMR (500 MHz, CDCl3): δ H =6.21(d,J=14.2Hz,1H), 7.06(d,J=15.7Hz,1H), 7.34~7.44(m,4H), 7.69(d,J=7.8Hz, 1H), 8.01(d,J=15.7Hz,1H), 8.46(d,J=2.5Hz,1H), 8.60(m,1H), 8.64(d,J=1.5Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =113.0, 126.6, 127.1, 127.5, 129.0, 129.9, 131.5, 131.7, 134.5, 135.4, 143.4, 144.0, 144.5, 150.5, 165.5. IR(film,cm) -1 ):ν=3064.81(CH), 2122.68(NC). R f Value: 0.58 (65% ethyl acetate: 35% hexane). HRMS(ESI)C 15 H 11 N3[M+H] + Calculated values: Theoretical value m / z = 234.1011, Measured value m / z = 234.1008

[0178] 2-Bromo-4-nitrophenyl)methanol [ka] To a solution of 2-bromo-4-nitrobenzoic acid (500 mg, 2.00 mmol) in THF, trimethylamine (0.3 mL, 2.00 mmol) and dimethyl borane sulfide (6.0 mL, 6.00 mmol) were added at 0°C. The reaction mixture was then heated under reflux for 3 hours. After this, the reaction mixture was cooled to room temperature, slowly quenched with H2O, acidified with concentrated HCl, and refluxed for a further 30 minutes. The reaction mixture was then extracted with DCM, dried over MgSO4, and concentrated under vacuum to obtain the desired product as a yellow solid (464 mg, 100%). 1 1H NMR (500 MHz, CD3OD): δ H =4.72(s,2H), 7.81(d,J=8.3Hz,1H), 8.25(dd,J=2.0,8.3Hz,1H), 8.41(d,J=2.4Hz,1H). 13 ¹³C NMR (75 MHz, CD3OD):δ c =64.5, 121.7, 122.7, 127.6, 128.5, 147.3. IR(film,cm) -1 ):ν=3271.69(OH), 2916.75(CH), 2851.62(CH). R f Value: 0.45 (30% ethyl acetate: 70% petroleum ether). Melting point: 29°C

[0179] 2-Bromo-4-nitrobenzaldehyde [ka] Oxalyl chloride (0.2 mL, 2.58 mmol) was dissolved in DCM and cooled to -78°C. Then, DMSO (0.4 mL, 5.16 mmol) was added dropwise, and the solution was stirred for 5 minutes. Next, (2-bromo-4-nitrophenyl)methanol (400 mg, 1.72 mmol) was added, and the mixture was stirred for a further 1.5 hours. After this, triethylamine (1.2 mL, 8.60 mmol) was added, and the mixture was stirred for a further 1.5 hours, during which time the reaction mixture was warmed to room temperature. The reaction solution was then quenched with NaHCO3, extracted with DCM, the organic phase was dried with MgSO4, and concentrated under vacuum to obtain the desired compound as fine brown needle-like material (350 mg, 87%). 1 1H NMR (500 MHz, (CD3)2CO):δ H =8.13(d,J=8.8Hz,1H), 8.39~8.40(m,1H), 8.57(d,J=2.0Hz,1H). 13 ¹³C NMR (75 MHz, (CD3)2CO):δ c =123.0, 125.7, 128.9, 130.9, 190.0. IR(film,cm) -1 ):ν=2955.86(CH), 2955.34(CH), 1518.35(C=O). R f Value: 0.57 (30% ethyl acetate: 70% hexane). Melting point: 94°C

[0180] 2-(2-bromo-4-nitrophenyl)-1,3-dioxane [ka] 1,3-propanediol (0.1 mL, 1.63 mmol) and p-TSA (20 mg, 0.11 mmol) were added to a solution of 2-bromo-4-nitrobenzaldehyde (250 mg, 1.09 mmol) in toluene. The reaction mixture was stirred overnight at 110°C. Once complete, the reaction mixture was first cooled to room temperature and then quenched with H2O. The reaction mixture was then washed with H2O and brine, extracted with toluene, dried over MgSO4, and concentrated under vacuum to obtain the compound as a brown solid in quantitative yield. 11H NMR (500 MHz, CDCl3): δ H =2.25(m,2H), 4.05(m,2H), 4.30(m,2H)5.77(s,1H), 7.89(d,J=8.8Hz,1H), 8.20(dd,J=2.0,8.3Hz,1H), 8.43(d,J=8.4Hz,1H). 13 ¹³C NMR (75 MHz, CD3OD):δ c =25.3, 68.6, 100.7, 123.3, 123.5, 128.4, 130.3, 145.2. IR(film,cm) -1 ):ν=2924.84(CH), 2880.65(CH). R f Value: 0.45 (25% ethyl acetate: 75% petroleum ether). Melting point: 120℃

[0181] (E)-2-(4-nitro-2-styrylphenyl)-1,3-dioxane [ka] Following general procedure 2, 2-(2-bromo-4-nitrophenyl)-1,3-dioxane (200 mg, 0.69 mmol), Pd(OAc)2 (16 mg, 0.07 mmol), tri(o-tolyl)phosphine (42 mg, 0.14 mmol), triethylamine (0.2 mL, 1.04 mmol), and styrene (0.1 mL, 1.04 mmol) were stirred in anhydrous DMF for 18 hours. The title compound was purified using silica column chromatography (10% ethyl acetate: 90% petroleum ether) to obtain a pale yellow solid (130 mg, 61%). 1 1H NMR (500 MHz, CDCl3): δ H=1.49~1.51(m,1H), 2.29~2.32(m,1H), 4.05~4.06(m,2H), 4.31~4.33(m,2H)5.75(s,1H), 7.15(d,J=16.1Hz,1H), 7.35(t,J=7.3Hz,1H), 7.4 2(t,J=7.3Hz,2H), 7.48(d,J=16.1Hz,1H), 7.56(d,J=7.8Hz,2H), 7.84(d,J=8.8Hz,1H), 8.12(dd,J=2.5,8.8Hz,1H), 8.47(d,J=2.5Hz,1H). 13 ¹³C NMR (75 MHz, CDCl3):δ c =25.1, 67.8, 99.3, 121.5, 122.0, 123.9, 127.2, 128.1, 128.8, 129.1. IR(film,cm) -1 ):ν=2985.85(CH), 2846.84(CH), 1522.11(C=O). R f Value: 0.40 (20% ethyl acetate: 80% petroleum ether). HRMS(ESI)C 18 H 17 NO4 [M+H] + Calculated values: Theoretical value m / z = 312.1235, Measured value m / z = 312.1203. Melting point: 160℃

[0182] (E)-4-amino-2-styrylbenzaldehyde [ka] Following general procedure 4, (E)-2-(4-nitro-2-styrylphenyl)-1,3-dioxane (600 mg, 1.95 mmol) and iron powder (460 mg, 8.00 mmol) were stirred in a 5:1 ethanol / H2O mixture, and then 1 mL of saturated ammonium chloride was added. After the reaction was complete, the title compound was obtained as a yellow oily substance, which was then continued without further purification (400 mg, 93%). 1 1H NMR (500 MHz, CDCl3): δ H=6.65(dd,J=2.3,8.3Hz,1H), 6.89(d,J=2.3Hz,1H), 7.00(d,J=16.2Hz,1H), 7.29~7.41(m,3H), 7.54~7.58(m,2H), 7.67(d,J=8.3Hz,1H), 8.06(d,J=16.2Hz,1H), 10.06(s,1H). IR(film,cm) -1 ):ν=3219.12(NH), 2998.84(CH), 2945.12(CH), 1677.95(C=O). R f Value: 0.46 (35% ethyl acetate: 65% petroleum ether). HRMS(ESI)C 15 H 13 NO[M+H] + Calculated values: Theoretical value m / z = 246.0894, Measured value m / z = 2.0873

[0183] (E)-N-(4-formyl-3-styrylphenyl)acetamide [ka] Following general procedure 3: To a solution of (E)-2-(4-amino-2-styrylphenyl)-1,3-dioxane (400 mg, 2.22 mmol) in DCM, acetic anhydride (0.3 mL, 2.60 mmol) was added and stirred overnight. The desired compound was isolated in quantitative yield as a yellow oily substance. 1 1H NMR (500 MHz, CDCl3): δ H =2.23(s,3H), 7.04(d,J=16.2Hz,1H), 7.28~7.32(m,1H), 7.37(apparently t,J=7.8Hz,2H), 7.53(d,J=7.8Hz ,2H), 7.58(d,J=8.3Hz,1H), 7.79(d,J=8.3Hz,1H), 7.95(s,1H), 8.03(d,J=16.1Hz,1H), 10.21(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c=24.8, 116.9, 118.0, 124.4, 126.1, 127.0, 128.4, 128.6, 128.8, 129.0, 133.0, 134.0, 134.2, 136.7, 141.5, 142.8, 191.3. IR (film, cm) -1 ):ν=2992.21(CH), 29115.14(CH), 1687.93(C=O). R f Value: 0.21 (35% ethyl acetate: 65% petroleum ether). HRMS(ESI)C 17 H 15 NO2 [M+H] + Calculated values: Theoretical value m / z = 266.1176, Measured value m / z = 266.1181

[0184] N-(4-((E)-2-isocyanovinyl)-3-((E)-styryl)phenyl)acetamide (41 and 42) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (150 mg, 0.69 mmol), (E)-2-(4-nitro-2-styrylphenyl)-1,3-dioxane (120 mg, 0.36 mmol), and LiHMDS (0.9 mL, 0.9 mmol) were stirred in anhydrous THF (6 mL) for 18 hours. The title compound was initially purified by silica gel chromatography, and then further purified using semi-preparative HPLC column chromatography (C18 reversed phase - 90% acetonitrile / 10% water) to obtain a dark yellow solid as a mixture of isomers in a 6:1 ratio (63 mg, 49%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =2.21(s,3H), 6.15(d,J=14.2Hz,1H), 6.96(d,J=15.7Hz,1H), 7.23(d,J=16.1Hz,2H), 7.28~7 .33(m,3H), 7.40(t,J=7.3Hz,2H), 7.43(d,J=8.3Hz,1H), 7.53(d,J=7.3Hz,1H), 7.79(s,1H). Minor isomer (Z) 11H NMR (500 MHz, CDCl3): δ H =2.22(s,3H), 5.93(d,J=9.3Hz,1H), 6.72(d,J=9.3Hz,1H), 7.01(d,J=16.1Hz,1H), 7.15(d, J=16.1Hz,1H), 7.30~7.40(m,5H), 7.50(t,J=7.3Hz,2H), 7.79(d,J=8.3Hz,1H), 7.97(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =24.8, 110.0, 117.5, 119.0, 124.7, 126.8, 127.4, 128.4, 128.8, 133.4, 134.2, 136.7, 137.7, 139.3. IR(film,cm) -1 ):ν=3298.05(NH), 2908.28(CH), 2818.73(CH), 2114.81(NC), 1670.67(C=O). R f Values: E=0.41, Z=0.50 (50% ethyl acetate:50% petroleum ether). HRMS(ESI)C 19 H 16 N2O[M+H] + Calculated values: Theoretical value m / z = 287.1184, Measured value m / z = 287.1182

[0185] (E)-N-(6-(2-isocyanovinyl)-(1,1-biphenyl)-3-yl)acetamide(43) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (171 mg, 0.83 mmol), N-(6-formyl-(1,1-biphenyl)-3-yl)acetamide (100 mg, 0.42 mmol), and LiHMDS (1.05 mL, 1.05 mmol) were stirred in anhydrous THF (6 mL) for 20 hours. The title compound was purified by silica gel chromatography to obtain a red / brown solid as a 1:1 mixture of isomers (51 mg, 47%). Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H=2.19(s,3H), 6.15(d,J=14.2Hz,1H), 6.98(d,J=16.1Hz,1H), 7.26(d,J=16.1Hz,1H), 7.27~7.28(m,1H), 7.45(m,4H), 7.50(br s,1H), 7.60(d,J=7.8Hz,1H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =2.20(s,3H), 5.75(d,J=9.3Hz,1H), 7.29(d,J=8.3Hz,1H), 7.41~7.46(m,4H), 7.49(d,J=8.3Hz,1H), 8.03(d,J=8.3Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =25.0, 118.5, 119.0, 121.1, 121.4, 126.8, 128.1, 128.5, 128.7, 129.7, 129.9, 131.3, 135.4, 139.2, 143.1, 168.5. IR(film,cm) -1 ):ν=3301.47(NH), 3083.77(CH), 2926.82(CH), 2110.25(NC). R f Values: E=0.67, Z=0.75 (70% ethyl acetate:30% petroleum ether). HRMS(ESI)C 17 H 14 N2O[MH] + Calculated values: Theoretical value m / z = 261.1027, Measured value m / z = 261.1019

[0186] (E)-4-(2-(1,3-dioxan-2-yl)-5-nitrostyryl)pyridine [ka] Following general procedure 2, 2-(2-bromo-4-nitrophenyl)-1,3-dioxane (400 mg, 1.42 mmol), Pd(OAc)2 (16 mg, 0.12 mmol), tri(o-tolyl)phosphine (51 mg, 0.17 mmol), 4-vinylpyridine (0.16 mL, 1.54 mmol), and sodium acetate (230 mg, 2.84 mmol) were stirred in anhydrous DMF at 120°C for 24 hours. The crude substance was purified using silica gel chromatography (80% ethyl acetate: 20% petroleum ether) to obtain the title compound as a yellow solid (420 mg, 95%). 1 1H NMR (500 MHz, CDCl3): δ H =1.59~1.61(m,1H), 2.30~2.32(m,1H), 4.11~4.13(m,2H), 4.29~4.31(m,2H)5.75(s,1H), 7.15(d,J=16.2Hz,1H), 7.40(d,J=6.0Hz,2H), 7.7 6(d,J=16.2Hz,1H), 7.42(d,J=8.7Hz,1H), 8.17(dd,J=2.3,8.7Hz,1H), 8.49(d,J=2.3Hz,1H), 8.65(d,J=6.0Hz,1H), 8.65(d,J=3.4Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =25.9, 67.9, 99.6, 121.4, 121.5, 123.0, 128.5, 128.6, 130.9, 150.6. IR(film,cm) -1 ):ν=2968.65(CH), 2855.14(CH), 1593.91(C=O). R f Value: 0.27 (80% ethyl acetate: 20% petroleum ether). HRMS(ESI)C 17 H 16 N2O4[MH] + Calculated values: Theoretical value m / z = 283.2614, Measured value m / z = 283.2676. Melting point: 159℃

[0187] (E)-4-amino-2-(2-(pyridine-4-yl)vinyl)benzaldehyde [ka] Following step 4, (E)-4-(2-(1,3-dioxan-2-yl)-5-nitrostyryl)pyridine (400 mg, 1.28 mmol) and iron powder (300 mg, 5.12 mmol) were stirred in an ethanol / H2O mixture for 3 hours to obtain the title compound as a yellow oily substance, which was then carried out to the next reaction without purification. 1 1H NMR (500 MHz, CDCl3): δ H =6.70(dd,J=2.3,8.3Hz,1H), 6.90(d,J=16.2Hz,1H), 6.90(d,J=2.3Hz,1H), 7.42(d,J=6.0Hz,1H), 7.42(d,J=3.0 Hz,1H), 7.65(d,J=8.7Hz,1H), 8.33(d,J=16.2Hz,1H), 8.60(d,J=6.0Hz,1H), 8.60(d,J=3.0Hz,1H), 9.99(s,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =112.0, 113.8, 121.0, 121.1, 121.2, 129.9, 130.7, 136.5, 140.7, 144.4, 150.1, 150.3, 151.4, 190.7. IR(film,cm) -1 ):ν=3353.83(NH), 3206.08(NH), 2924.62(CH), 2854.37(CH), 1593.69(C=O). R f Value: 0.30 (100% ethyl acetate). HRMS(ESI)C 14 H 12 N2O[M+H] + Calculated values: Theoretical value m / z = 225.1027, Measured value m / z = 225.1021

[0188] (E)-N-(4-formyl-3-(2-(pyridine-4-yl)phenyl)acetamide [ka] Following general procedure 3, (E)-4-amino-2-(2-(pyridine-4-yl)vinyl)benzaldehyde (100 mg, 0.49 mmol) and acetic anhydride (0.1 mL, 0.60 mmol) were stirred overnight in anhydrous DCM to obtain the title compound as a yellow oily substance (quantitatively). 1 1H NMR (500 MHz, CD3OD): δ H =2.20(s,3H), 7.12(d,J=16.2Hz,1H), 7.61(d,J=5.9Hz,2H), 7.72(dd,J=2.0,8.3Hz,1H), 7.87(d, J=8.3Hz,1H), 8.14(d,J=2.0Hz,1H), 8.45(d,J=16.1Hz,1H), 8.54(d,J=5.9Hz,2H), 10.16(s,1H). 13 ¹³C NMR (125 MHz, CD3OD):δ c =31.1, 114.5, 116.5, 126.5, 128.4, 129.6, 130.3, 134.7, 135.4, 139.0, 144.4, 164.6, 193.5. IR(film,cm) -1 ):ν=3068.97(NH), 2922.87(CH), 2852.51(CH), 1679.72(C=O). R f Value: 0.10 (100% ethyl acetate). HRMS(ESI)C 16 H 14 N2O2[M+H] + Calculated values: Theoretical value m / z = 267.1133, Measured value m / z = 267.1120

[0189] N-(4-((E)-2-isocyanovinyl)-3-((E)-2-(pyridine-4-yl)vinyl)phenyl)acetamide(44) [ka] Following general procedure 1: Diisopropyl(isocyanomethyl)phosphonate (100 mg, 0.48 mmol), (E)-N-(4-formyl-3-(2-(pyridine-4-yl)phenyl)acetamide (65 mg, 0.24 mmol) and LiHMDS (0.61 mL, 0.61 mmol) were stirred in anhydrous THF (6.5 mL) for 20 hours. The title compound was purified by silica gel chromatography (gradient from 100% ethyl acetate to 10% methanol:DCM) to obtain a red solid as a mixture of isomers (39 mg, 56%) with an E:Z ratio of 7:2. Main isomer (E) 1 1H NMR (500 MHz, CDCl3): δ H =2.23(s,3H), 6.17(d,J=14.2Hz,1H), 6.92(d,J=16.1Hz,1H), 7.31~7.40(m,5H), 7.45(d,J=16.3Hz,1H), 7.93(s,1H), 8.64(d,J=6.4Hz,2H). Minor isomer (Z) 1 1H NMR (500 MHz, CDCl3): δ H =2.18(s,3H), 5.97(d,J=9.3Hz,1H), 6.72(d,J=8.8Hz,1H), 6.95(d,J=16.1Hz,1H), 7. 25(s,1H)7.31~7.46(m,5H), 7.79(d,J=8.8Hz,1H), 8.11(s,1H), 8.61(d,J=5.9Hz,1H). 13 ¹³C NMR (125 MHz, CDCl3):δ c =24.7, 117.5, 119.5, 120.8, 127.2, 128.9, 130.5, 133.4, 133.6, 139.2, 143.6, 150.0, 150.1. IR(film,cm) -1 ):ν=2926.03(CH), 2855.11(CH), 2114.64(NC). R f Value: 0.2 (100% ethyl acetate). HRMS(ESI)C 18 H 15 N3O[M+H] + Calculated values: Theoretical value m / z = 290.1293, Measured value m / z = 290.1272

[0190] [Table 3] JPEG0007876210000079.jpg87158

[0191] Table 4 JPEG0007876210000081.jpg29152

[0192] Table 5 JPEG0007876210000083.jpg199136

[0193] Table 6

[0194] Development of novel (E)-selective HWE reagents for the synthesis of vinyl isocyanides and aglycone (E)-4. Initial synthetic efforts focused on preparing phenol vinyl isocyanide (E)-4 (TF Bumol, AM Watanabe, Genetic information, genomic technologies, and the future of drug discovery. Jama-Journal of the American Medical Association 285, 551-555 (2001)). Deprotonation of diethyl isocyanomethylphosphonate 11 (R. Falcon et al., High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes. International Journal of Antimicrobial Agents 47, 343-350 (2016)) was carried out at -78°C for 15 minutes using 2.2 equivalents of LHMDS in THF, allowing it to compete with the deprotection of the free phenol group of 12a. This allowed the desired phenol vinyl isocyanide to be (E)-4(J (2,3) =14.4Hz) and (Z)-4(J (2,3) (=8.8Hz) A 7:3 mixture of isomers was obtained in 56% yield (Scheme 4) (AL Harvey, R. Edrada-Ebel, RJ Quinn, The re-emergence of natural products for drug discovery in the genomics era. Nature Reviews Drug Discovery 14, 111-129 (2015)).

[0195] [ka] Scheme 4. The reaction of the anion of HWE reagent 11 with p-hydroxybenzaldehyde 12a yields an inseparable 7:3 mixture of phenol vinyl isocyanide (E)-4 / (Z)-4.

[0196] Unfortunately, this mixture of geometric isomers proved difficult to separate by chromatography (silica, alumina), and considerable mass loss occurred during attempts to purify them.

[0197] The inventors investigated whether the HWE reaction between the anion of diisopropyl isocyanomethylphosphonate reagent 13, which has higher steric requirements, and aldehyde 12a could lead to improved (E) selectivity (P. Monciardini, M. Iorio, S. Maffioli, M. Sosio, S. Donadio, Discovering new bioactive molecules from microbial sources. Microbial Biotechnology 7, 209-220 (2014)). This new diisopropyl reagent 13 was prepared using a modified version of the four-step literature protocol previously used to prepare Schollkopf's HWE reagent 11 (see SI for details) (R. Falcon et al., High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes. International Journal of Antimicrobial Agents 47, 343-350 (2016)).

[0198] In THF, the reaction of 1.1 equivalents of the lithium anion of novel HWE reagent 13 (generated using 2.2 equivalents of LHMDS) with 1 equivalent of p-hydroxybenzaldehyde 12a at -78°C yielded the selective formation of (E)-4 in a 9:1 ratio, surpassing its corresponding (Z)-4 isomer. Furthermore, the HWE reaction of 1.1 equivalents of the lithium anion of HWE reagent 13 with p-TBSO-benzaldehyde 12b yielded a further improved 95:5 mixture of the corresponding p-TBSO-phenol-vinyl isocyanide (E)-14 and p-TBSO-phenol-vinyl isocyanide (Z)-14. Subsequently, base-mediated O-silyl deprotection of this geometric isomer mixture by treatment with KOH in EtOH yielded the desired aglycone in two steps in 35% yield, in an unchanged 95:5 ratio of (E)-4 / (Z)-4 (Scheme 5). This aglycone was found to be highly reactive and readily polymerize at room temperature to yield a black polymer. Aglycone (E)-4 could be stored as a diluted solution in acid-free chloroform in the dark at -10°C for several weeks without decomposition. However, long-term storage (several months) of (E)-4 (95:5dr) resulted in slow geometric isomerization, yielding a gradual increase in its (Z)-4 isomer, ultimately resulting in a thermodynamic 7:3 mixture of (E)-4:(Z)-4 at equilibrium.

[0199] [ka] Scheme 5. Synthesis of HWE reagent 13 and its use for the (E)-selective synthesis of vinyl isocyanide (E)-4.

[0200] Diisopropyl isocyanide phosphonate 13 (R= iSince the anion of (Pr) showed improved diastereoselectivity in the HWE reaction with aldehydes 12a / 12b, it was decided to investigate its use for preparing a small series of (E)-vinyl isocyanides 15a-h useful in synthesis (Table 7) (HB Bode, in Insect Biotechnology, A. Vilcinskas, Ed. (Springer-Verlag Berlin, Berlin, 2011), vol. 2, pp. 77-93). The lithium enolate of HWE reagent 13 was reacted with a wide range of eight aldehydes in THF at -78°C to obtain their corresponding vinyl isocyanides 15a-h in yields of 50-92%, with (E)- / (Z)-diastereomer ratios greater than 90:10 in all cases (Table 7, column 3). Good (E)-selectivity was observed in the HWE reactions of electron-rich aldehydes (Table 7, entries 2-4), electron-deficient aromatic aldehydes (Table 7, entry 5), heteroaryl aldehydes (Table 7, entry 6), aliphatic aldehydes (Table 7, entry 7), and cyclic aldehydes (Table 7, entry 8). 13(R= i The (E)- / (Z)- ratios obtained in the HWE reaction of Pr) were all significantly higher than those obtained in the corresponding HWE reaction of the lithium anion of the corresponding diethyl isocyanide phosphonate 11(R=Et) (see the (E)- / (Z)- ratios reported in columns 3 and 4 of Table 7). For example, the reaction of the lithium anion of HWE reagent 11(R=Et) with p-nitrobenzaldehyde yielded vinyl isocyanide 15e with an insufficient 57:43 (E)-:(Z)- ratio, while 14(R= i The lithium anion of Pr) yielded 15e, with a significantly improved 95:5 ratio, resulting in a favorable (E)-isomer (Table 7, entry 5).

[0201] [Table 7] JPEG0007876210000088.jpg52134

[0202] method General Procedure for Conducting the HWE Reaction LHMDS (1.0 M in THF) (1.2 mL, 1.2 mmol) was added dropwise at -78°C to a solution of diisopropyl(isocyanomethyl)phosphonate 13 (0.23 mL, 1.1 mmol) in anhydrous THF (5 mL), and the resulting solution was stirred for 20 minutes. Then, aldehyde (1.0 mmol) was added dropwise at -78°C, and the stirred reaction mixture was slowly warmed to room temperature over 16 hours. The reaction mixture was then quenched with phosphate buffer pH 7.0 (approximately 0.2 mL), extracted with siRNA (10 mL), dried, and the solvent was removed under vacuum to obtain the crude product, which was purified by silica gel chromatography to obtain the desired (E)-vinyl isocyanide product.

[0203] (References) 1. J. O'Neil, "Review on Antimicrobial Resistance," (2016) 2. MJ Renwick, DM Brogan, E. Mossialos, A systematic review and critical assessment of incentive strategies for discovery and development of novel antibiotics. Journal of Antibiotics 69, 73-88 (2016) 3. L. Hall-Stoodley, JW Costerton, P. Stoodley, Bacterial biofilms: From the natural environment to infectious diseases. Nature Reviews Microbiology 2, 95-108 (2004) 4. D. Davies, Understanding biofilm resistance to antibacterial agents. Nature Reviews Drug Discovery 2, 114-122 (2003) 5. I. Olsen, Biofilm-specific antibiotic tolerance and resistance. European Journal of Clinical Microbiology & Infectious Diseases 34, 877-886 (2015) 6. A. S. Lynch, D. Abbanat, New antibiotic agents and approaches to treat biofilm-associated infections. Expert Opinion on Therapeutic Patents 20, 1373-1387 (2010) 7. M. Otto, Staphylococcal biofilms. Bacterial Biofilms 322, 207-228 (2008) 8. R. Falcon et al., High vancomycin MICs within the susceptible range in Staphylococcus aureus bacteraemia isolates are associated with increased cell wall thickness and reduced intracellular killing by human phagocytes. International Journal of Antimicrobial Agents 47, 343-350 (2016) 9. N. K. Qureshi, S. H. Yin, S. Boyle-Vavra, The Role of the Staphylococcal VraTSR Regulatory System on Vancomycin Resistance and vanA Operon Expression in Vancomycin-Resistant Staphylococcus aureus. Plos One 9, 7 (2014) 10. C. Veeresham, Natural products derived from plants as a source of drugs. J. Adv. Pharma. Technol. Res 3, 200-201 (2012) 11. T. F. Bumol, A. M. Watanabe, Genetic information, genomic technologies, and the future of drug discovery. Jama-Journal of the American Medical Association 285, 551-555 (2001) 12. A. L. Harvey, R. Edrada-Ebel, R. J. Quinn, The re-emergence of natural products for drug discovery in the genomics era. Nature Reviews Drug Discovery 14, 111-129 (2015) 13. G. M. Cragg, D. J. Newman, Natural products: A continuing source of novel drug leads. Biochimica Et Biophysica Acta-General Subjects 1830, 3670-3695 (2013) 14. D. J. Newman, G. M. Cragg, Natural Products as Sources of New Drugs from 1981 to 2014. Journal of Natural Products 79, 629-661 (2016) 15. P. Monciardini, M. Iorio, S. Maffioli, M. Sosio, S. Donadio, Discovering new bioactive molecules from microbial sources. Microbial Biotechnology 7, 209-220 (2014) 16. H. B. Bode, in Insect Biotechnology, A. Vilcinskas, Ed. (Springer-Verlag Berlin, Berlin, 2011), vol. 2, pp. 77-93 17. J. M. Crawford, C. Portmann, X. Zhang, M. B. J. Roeffaers, J. Clardy, Small molecule perimeter defense in entomopathogenic bacteria. Proceedings of the National Academy of Sciences of the United States of America 109, 10821-10826 (2012) 18. V. S. Somvanshi et al., A Single Promoter Inversion Switches Photorhabdus Between Pathogenic and Mutualistic States. Science 337, 88-93 (2012) 19. D. C. Davis et al., Discovery and characterization of aryl isonitriles as a new class of compounds versus methicillin- and vancomycin-resistant Staphylococcus aureus. European Journal of Medicinal Chemistry 101, 384-390 (2015) 20. D. A. Smith, Pharmacokinetics and Metabolism in Drug Design. (Wiley-VCH, ed. 3, 2012), vol. 51 21. N. Brown, Bioisosteres and Scaffold Hopping in Medicinal Chemistry. Molecular Informatics 33, 458-462 (2014) 22. I. Hoppe, U. Schollkopf, SYNTHESIS AND BIOLOGICAL-ACTIVITIES OF THE ANTIBIOTIC-B-371 AND ITS ANALOGS. Liebigs Annalen Der Chemie, 600-607 (1984) 23. L. L. Silver, Challenges of Antibacterial Discovery. Clinical Microbiology Reviews 24, 71-+ (2011) 24. I. Ugi, U. Fetzer, U. Eholzer, H. Knupfer, Offerman.K, ISONITRILE SYNTHESES. Angewandte Chemie-International Edition 4, 472-& (1965) 25. A. P. Desbois, P. J. Coote, in Advances in Applied Microbiology, Vol 78, A. I. Laskin, S. Sariaslani, G. M. Gadd, Eds. (Elsevier Academic Press Inc, San Diego, 2012), vol. 78, pp. 25-53 26. C. P. Silva et al., Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta. Cellular Microbiology 4, 329-339 (2002) 27. B. R. Boles, A. R. Horswill, agr-mediated dispersal of Staphylococcus aureus biofilms. Plos Pathogens 4, 13 (2008) 28. C. f. O. A. D. Discovery, "Hit Confirmation of Antibiotics," (2016) 29. H. Bilgin, A. Eren, S. Kara, Hemolytic Anemia and Heart Failure Caused by Anti-C and Anti-E Immunization. Jcpsp-Journal of the College of Physicians and Surgeons Pakistan 26, 539-540 (2016) 30. R. Ramozzi, N. Cheron, B. Braida, P. C. Hiberty, P. Fleurat-Lessard, A valence bond view of isocyanides' electronic structure. New Journal of Chemistry 36, 1137-1140 (2012) 31. L. L. Ling et al., A new antibiotic kills pathogens without detectable resistance (vol 517, pg 455, 2015). Nature 520, (2015) 32. G. D. Brown et al., Hidden Killers: Human Fungal Infections. 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[0204] All publications described herein are incorporated herein by reference. While exemplary embodiments of the present invention have been disclosed in detail herein, it will be understood by referring to the accompanying drawings that the invention is not limited to the exact embodiments and that various changes and modifications can be made thereto without departing from the scope of the invention as defined by the accompanying claims and their equivalents.

Claims

1. Compounds of formula (I) or formula (II) 【Chemistry 1】 or its salts, solvates, diastereomers, or tautomers [In the formula, Y １ , Y ２ and Y ３ , C-R １ or selected independently from N; Each R １ is independently selected from H, C １ -C ６ alkyl, OH, OR, NHCOR, NHSO ２ R, CONHR, CONHSO ２ R, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl or R ７ and each R is independently selected from H or C １ -C ６ alkyl; R ２ is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or R ７ Selected from; R ３ and R ４ is H, or C １ -C ６ Independently selected from alkyl groups; R ７ The basis for the following equation is: 【Chemistry 2】 And; R ａ and R ｂ They form a substituted or unsubstituted phenyl ring together with the atoms to which they are bonded; R ５ This is selected from substituted, unsubstituted, or heteroaryl compounds; R ６ is H, or C １ -C ６ [Selected from alkyl groups].

2. Compound of formula (III): 【Transformation 3】 [In the formula, R １ H, C １ -C ６ Alkyl, OH, OR, NHCOR, NHSO ２ R, CONHR, CONHSO ２ Selected from R, substituted or unsubstituted aryls, or substituted or unsubstituted heteroaryls; Y １ , Y ２ and Y ３ , C-R １ or selected independently from N; Each R is either H or C １ -C ６ [Selected independently from alkyl groups] The compound according to claim 1.

3. Compound of formula (IV): 【Chemistry 4】 [In the formula, R ６ is H or C １ -C ６ Selected independently from alkyl, and R １ H, C １ -C ６ [Independently selected from alkyl, OH, or OR] The compound according to claim 1.

4. Compound of formula (VI): 【Transformation 6】 The compound according to claim 1.

5. R ５ The compound according to claim 1, wherein the compound is a substituted or unsubstituted aryl, pyridyl, pyrazyl, pyridadyl, or pyrimidyl.

6. R ３ and R ４ The compound according to claim 1, wherein each of them is H.

7. Y １ , Y ２ and Y ３ Each of them is CR １ The compound according to claim 1.

8. at least one R １ However, OH, OR, NHCOR, NHSO ２ R, CONHR, CONHSO ２ Selected from R, each R is either H or C １ -C ６ A compound according to claim 1, independently selected from alkyl groups.

9. The compound shown below: 【Transformation 7】 【change】 A compound selected from among them.

10. A method for producing the vinyl isocyanide compound according to claim 1 or 9, a) Phosphonate of formula (X): 【Transformation 8】 [In the formula, R １１ and R １２ C ３ -C ５ The step of providing [independently selected from alkyl, and optionally independently selected from isopropyl, isobutyl, and t-butyl] b) The step of reacting the phosphonate with a carbonyl compound in the presence of a base. The method, including the method described above.

11. The carbonyl compound is a compound of formula (XI) or (XII): 【Chemistry 9】 [In the formula, Y １ , Y ２ and Y ３ , C-R １ or selected independently from N; Each R １ H, C １ -C ６ Alkyl, OH, OR, NHCOR, NHSO ２ R, CONHR, CONHSO ２ R, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or R ７ Independently selected from; each R is H, or C １ -C ６ Independently selected from alkyl groups; R ２ is a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or R ７ Selected from; R ３ is H, or C １ -C ６ Selected from alkyl groups; R ７ The basis for the following equation is: 【Chemistry 10】 And; Ra and Rb, together with the atoms to which they are bonded, form substituted or unsubstituted phenyl rings; R ５ This is selected from substituted or unsubstituted aryl or heteroaryl compounds; R ６ is H, or C １ -C ６ Selected from alkyl groups] The method according to claim 10.

12. The method according to claim 10, wherein the base comprises a non-nucleophilic base, optionally a Li base, and optionally a base selected from lithium bis(trimethylsilyl)amide (LHMDS), lithium tetramethylpiperidide (LiTMP), and lithium diisopropylamide (LDA).

13. R ３ The method according to claim 11, wherein H is

14. The method according to claim 10, wherein a phosphonate is reacted with a carbonyl in the presence of THF as a base and solvent.

15. Compound of formula (X): 【Chemistry 11】 [In the formula, R １１ and R １２ C ３ -C ５ [Independently selected from alkyl groups, and optionally independently selected from isopropyl, isobutyl, and t-butyl groups] A reagent for use in the method according to claim 10, comprising:

16. Compounds according to any one of claims 1 to 9, as well as salts and solvates thereof, for use as pharmaceuticals.

17. Compounds according to any one of claims 1 to 9, as well as salts and solvates thereof, for use in the treatment of infectious diseases.

18. The compound according to claim 17, as well as salts and solvates thereof, for use in the treatment of bacterial, fungal, or protozoan diseases.

19. The compound according to claim 18, as well as salts and solvates thereof, wherein the bacterial disease is caused by Gram-negative bacteria or Gram-positive bacteria.

20. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9, as well as salts and solvates thereof, and a pharmaceutically acceptable excipient, carrier, or diluent.

21. Use of the compound according to any one of claims 1 to 9, as well as salts and solvates thereof, in the manufacture of a pharmaceutical product for the treatment of an infectious disease.

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