Compositions and methods of constructing conjugates

Abstract

Methods of constructing conjugates are disclosed. The preparation method comprises the following steps: reacting an amino-containing component and a thiol-containing component with o-phthalaldehyde in one step under a considerable molar ratio to generate a conjugate with a substituted or unsubstituted 1-thio-isoindole structure in the presence of an additive. The method provides an efficient and practical platform for constructing the biological activity modular primitive.

Description

[0001] Related Applications Citations

[0002] This application claims the benefit of and priority to U.S. Serial No. 63 / 401,301, filed on August 26, 2022, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The disclosed invention generally relates to conjugates and methods of constructing the same, and more particularly to conjugates containing a 1-mercaptoisoindole structure formed by reacting an amine-containing component and a sulfur-containing component with o-phthalaldehyde, preferably in a one-step reaction. Background Art

[0004] Structures constructed from different modular groups are gaining increasing attention in the field of drug discovery, especially in the field of targeted cancer therapy. Conjugates endowed with cytotoxic and disease-targeting functions are designed to localize pharmacological activity to tumor cells and maximize the therapeutic index of cytotoxins (Srinivasarao & Low, Chem. Rev. 2017, 117, 12133–12164). For example, antibody-drug conjugates and peptide-drug conjugates have rapidly become promising treatments due to their enhanced local efficacy and reduced peripheral toxicity (Thomas, et al, Lancet Oncol. 2016, 17, 254-262; Cooper, et al., Chem. Soc. Rev. 2021, 50, 1480–1494). Although these structural conjugate groups are attractive, the chemistry of connecting different modular groups is not simple. First, the conjugation reaction between different modalities (such as drugs and targeting agents) must be chemically selective and effective. Furthermore, all other functional groups present in these modalities should not react, otherwise orthogonal protecting groups are required.

[0005] Different chemical linkers, such as amide, hydrazone, triazole, and disulfide bonds, have been reported for peptide / protein bioconjugation (Alas, et al., J. Med. Chem. 2020, 64, 216–232). Nevertheless, new linker strategies are still being explored to expand the current conjugation toolbox, including bioconjugation. The development of chemical selective reactions and modular strategies will be of great value for the efficient construction of conjugates.

[0006] Ortho-phthalaldehyde (OPA) has regained interest in protein bioconjugation and peptide cyclization. Roth first discovered that OPA could be used as a reagent for the fluorescence detection of α-amino acids in the presence of 2-mercaptoethanol (Roth, Anal. Chem. 1971, 43, 880–882). This highly fluorescent product generated by the OPA-amine-thiol reaction was later identified as 1-mercaptoisoindole. However, unlike the individual amino acids used by Roth, peptides showed greatly reduced fluorescence for unknown reasons at the time. Subsequently, Benson modified Roth's method in 1975 (Benson & Hare, Proc. Natl. Acad. Sci. USA 1975, 72, 619–622). The new protocol utilized excess 2-mercaptoethanol and React with OPA in borate buffer (pH 9.7) and then add amino acids. Since then, this optimized method has been widely adopted for amino acid detection due to its improved effectiveness and reproducibility. In addition, excess conditions using thiol groups are also often used (Zuman, Chem. Rev. 2004, 104, 3217–3238). At the same time, it has also been reported that peptides and proteins were successfully detected by adding 189-fold excess 2-mercaptoethanol compared to OPA (Weidekamm, et al., Anal. Biochem. 1973, 54, 102–114). However, the fundamental reason why large excess thiols are required in these reactions remains unclear.

[0007] In this study, the reaction of o-phthalaldehyde (OPA) with amine groups in peptides / proteins to form phthalimides (isoindol-1-ones) under aqueous buffer conditions was explored, which has been reported in the literature (Tung, et al., Org. Lett. 2016, 18, 2569–2578). Unlike the conditions in previous studies involving organic solvents (Thiele & Schneider, Liebigs Annalen 1909, 369, 287-299; Takahashi & Hatanaka, Heterocycles 1997, 45, 2475-2499; Perard-Viret, et al., Tetrahedron 2002, 58, 5103-5108; Kulla & Zuman, Org. Biomol. Chem. 2008, 6, 3771-3780), the reaction rate was significantly increased in aqueous buffer (pH 6-11) (Tung, et al., Org. Lett. 2016, 18, 2569-2578). In addition, this aqueous OPA-amine two-component reaction resulted in a rapid and quantitative conversion, making it promising in protein / peptide bioconjugation. Mechanistically, this finding helps explain the previous requirement for a large excess of thiol groups: in aqueous buffer, the OPA-amine two-component reaction competes with the OPA-amine-thiol three-component reaction, and an excess of thiol groups is required to ensure the formation of the desired three-component product. This finding is consistent with a recent study (Nie, et al., J. Org. Chem. 2022, 87, 2551–2558). The reaction between OPA, amine-containing DNA, and 4-tert-butylbenzenethiol was reported to produce a mixture of two-component and three-component products. Notably, the addition of 300-3000 equivalents of the thiol compound resulted in an 85% conversion to the three-component product. Further increasing the thiol compound to 3000 equivalents increased the conversion to 95%.

[0008] To complement the efficient OPA-amine two-component intermolecular reaction, the research group independently reported the intramolecular OPA-amine-thiol reaction for peptide cyclization (Zhang, et al., J. Am. Chem. Soc. 2019, 141, 12274–12279; Todorovic, et al., Angew. Chem., Int. Ed. 2019, 58, 14120–14124). In this intramolecular setting, an unprotected peptide, bearing both amine and thiol groups, reacts rapidly with OPA to undergo thiol-amine cyclization, which is much faster than the OPA-amine two-component reaction. As a result, only the desired three-component product is generated. In addition, the intramolecular OPA-amine-amine reaction for peptide immobilization has also been reported recently (Li, et al., Nat. Commun. 2022, 13, 311). However, a major problem with intramolecular reactions is that, in nature and in most chemical and / or biochemical synthetic settings, substrates containing thiol or amine groups are usually not present or purchased in an intramolecular form, i.e., the two groups reacting are not in the same molecule.

[0009] Although intramolecular OPA reactions with peptides are simple, rapid, and highly chemoselective, it remains to be determined whether this reaction can be re-transferred to intermolecular reactions, especially for bioactive modular construction. A major existing obstacle is that large numbers of thiol groups are traditionally required to achieve three-component products. For bioconjugation, the peptides, proteins, and drugs used are often limited in quantity and costly. Therefore, reacting these components in large excess is cost-prohibitive and unaffordable. Another issue is the low stability of the core of the conjugate, such as the 1-mercaptoisoindole product obtained from the OPA-amine-thiol reaction. Therefore, there remains a need to develop improved, practical, and efficient methods for the intermolecular reactions of modular groups to produce conjugates, such as bioconjugates.

[0010] Therefore, one object of the present invention is to provide improved methods for the intermolecular production of conjugates.Another object is to provide improved methods for the intermolecular production of bioconjugates, such as bioconjugates containing biomolecules, including peptides, carbohydrates, proteins, lipids and nucleic acids. Summary of the invention

[0011] Disclosed are conjugates, such as bioconjugates, and methods for making these conjugates by intermolecular reactions between modular groups in aqueous solution. The method involves reacting a first reactant having a structural formula I with (i) a second reactant containing a thiol nucleophile and (ii) a third reactant containing an amine nucleophile in the presence of an additive to form a conjugate,

[0012]

[0013] Preferably, the additive inhibits the formation of products formed solely by the reaction between the first reactant and the third reactant, wherein:

[0014] A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, preferably substituted aromatic or unsubstituted aromatic,

[0015] Preferably, m and n are 1,

[0016] Preferably, X and Y are absent,

[0017] Preferably, E1 and E2 are adjacent in the first reactant, ie, they are directly attached to adjacent atoms in the chemical ring of the first reactant,

[0018] Preferably, E1 and E2 are aldehydes.

[0019] Preferably, the first reactant is o-phthalaldehyde (OPA).

[0020] Traditionally, these intermolecular reactions involve the use of a large excess (e.g., at least 4.6 equivalents) of a thiol group-containing reactant to ensure that a desired product comprising both reactants is formed in a chemoselective manner. However, the use of large amounts of reactants results in inefficient reactions and is cost-prohibitive for bioconjugation. As described below, the presence of an additive reduces or eliminates the need to use large amounts of a thiol nucleophile-containing reactant. Preferably, the additive, such as guanidine in the form of its salt (e.g., a guanidine salt, e.g., guanidine hydrochloride), generates an OPA-guanidine adduct that inhibits the non-selective formation of imine-aldehyde intermediates common in two-component and three-component products. Thus, preferably, the molar ratio of the thiol group-containing reactant to the amine group-containing reactant is between 1:4 and 4:1.

[0021] Furthermore, the 1-mercaptoisoindole product obtained from the OPA-amine-thiol reaction has low stability. Preferably, the low stability problem is mitigated by using sterically hindered thiol groups that react with OPA and / or introducing substituents on the heteroaromatic groups of the 1-mercaptoisoindole core.

[0022] Preferably, forming the conjugate involves the following steps:

[0023] (ii) adding additives to form a solution, dispersion or suspension in a suitable buffer, such as PBS,

[0024] (i) adding a first reactant, such as OPA, and a second reactant, such as a reactant containing a thiol group, to the solution, dispersion or suspension in step (i) to form another solution, dispersion or suspension,

[0025] (iii) adding a third reactant containing an amine group to the solution of step (ii) to form another solution, dispersion or suspension.

[0026] Preferably, the conjugate has the following structure:

[0027]

[0028] in:

[0029] R1-R4 are hydrogen atoms, or at least one of R1-R4 independently comprises a targeting agent, a diagnostic agent, a preventive agent or a therapeutic agent, preferably, the targeting agent comprises a peptide,

[0030] R5 is a hydrogen atom, an unsubstituted C1-C10 alkyl group, a substituted C1-C10 alkyl group, an unsubstituted C1-C10 alkenyl group or a substituted C1-C10 alkenyl group,

[0031] R6 and R7 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl, provided that R6 and R7 are at least divalent, as permitted by the valence, optionally, R6 and R7 independently contain a stimulus responsive chemical group, wherein preferably R6 contains an optional stimulus responsive chemical group,

[0032] R8 and R9 are independently hydrogen atoms, unsubstituted C1-C10 alkyl groups or substituted C1-C10 alkyl groups, provided that at least one of R8 and R9 is not a hydrogen atom, such as unsubstituted C1-C10 alkyl groups or substituted C1-C10 alkyl groups, preferably, R8 and R9 are independently unsubstituted C1-C10 alkyl groups or substituted C1-C10 alkyl groups,

[0033] (i) PD1 and PD2 comprise a peptide, preferably an unprotected peptide, or

[0034] (ii) one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other PD1 and PD2 comprises a non-peptide therapeutic agent, a non-peptide diagnostic agent, a non-peptide preventive agent or a non-peptide targeting agent. Preferably, one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other PD1 and PD2 comprises a non-peptide therapeutic agent.

[0035] Additional advantages of the disclosed methods and compositions will be set forth in part in the description that follows, in part understandable from the description, or may be learned by practicing the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained by the elements and combinations particularly pointed out in the appended claims. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The drawings illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0037] Figure 1 is a schematic diagram of the synthesis of a conjugate consisting of a first reactant (eg, o-phthalaldehyde (OPA)), a second reactant (eg, a molecule containing a thiol group), and a third reactant (eg, a molecule containing an amine group).

[0038] Figures 2A to 2D is a schematic diagram of the OPA-based reaction. Figure 2A The three-component reaction of OPA-amine-thiol molecules reported by Roth (Roth, Anal. Chem. 1971, 43, 880-882), Benson (Benson & Hare, Proc. Natl. Acad. Sci. USA 1975, 72, 619-622) and Weidekamm (Weidekamm, Anal. Biochem. 1973, 54, 102-114) was demonstrated. Figure 2B A schematic diagram of the OPA-amine two-component reaction reported by Thiele (Thiele & Schneider, Liebigs Annalen 1909, 369, 287-299) and Li (Tung, et al., Org. Lett. 2016, 18, 2569-2578) is shown. Figure 2C A schematic diagram of the intramolecular three-component reaction of OPA-amine-thiol reported by Li (Zhang, et al., J. Am. Chem. Soc. 2019, 141, 12274–12279) and Perin (Todorovic, et al., Angew. Chem., Int. Ed. 2019, 58, 14120–14124) is shown. Figure 2D An intermolecular three-component reaction described in this work, promoted by a guanidine additive using stoichiometric amounts of the thiol component, is shown. "1°-thiol" and "3°-thiol" refer to primary and tertiary thiols, respectively.

[0039] Figures 3A to 3Q The OPA-amine-thiol intermolecular three-component reaction for the construction of peptide-peptide conjugates is demonstrated, as well as some peptide-peptide conjugates. Figure 3A Peptide sequences of thiol-containing peptides and amine-containing peptides are shown. Figure 3B A control reaction without additives, such as guanidine, condition [A] is shown. Figure 3C Shown is a liquid chromatogram of the crude reaction mixture of PPC-1 without additives. Figure 3D A test reaction in the presence of an additive, such as guanidine, is shown, condition [B]. Figure 3EShown is a liquid chromatogram of a crude PPC-1 reaction mixture with additives such as guanidine. Figures 3F to 3Q Representative sequences of PPC 1-12 and their respective isolated yields are shown. The concentration of applied additives, such as guanidine, ranged from 3 M to 8 M. The isolated yields were calculated based on HPLC purified products.

[0040] Figure 4A Schematic representation of a three-component conjugate containing a 1-mercaptoisoindole core. Figure 4B is a schematic diagram of a further functionalized conjugate containing a 1-mercaptoisoindole core. As shown, the linker between the drug and the 1-mercaptoisoindole core contains a stimuli-responsive bond (eg, a disulfide bond). Figures 4C to 4J is an example of a bioconjugate produced using the methods described herein. The reaction conditions were as follows: [C]: thiol (1.0 equiv), amine (1.5 equiv), OPA (1.2 equiv) in PBS buffer pH 7.4 containing 1 M guanidine, 0.5 mM, room temperature, 1 hour; [D]: [C], a little DMAC (2.0 equiv), 1 hour; [E]: 3°-thiol (1.0 equiv), OPA (1.2 equiv) in PBS buffer pH 7.4 containing 6 M guanidine, 5 mM, room temperature, 3 hours; [F]: 3°-thiol (3.0 equiv), amine (1.0 equiv), functionalized OPA-fluorescein (3.0 equiv) in PBS buffer pH 7.4 containing 6 M guanidine, 1 mM 37°C, 2 hours; [G] 3°-thiol (2.0 equiv), amine (1.0 equiv), functionalized OPA-peptide (1.2 equiv) in PBS buffer pH 7.4 contains 6 M guanidine, 1 mM room temperature, 2 hours. Figure 4K Conjugation of cytotoxins and targeting agents using OPA to form the conjugate PDC-1 is shown, as well as a liquid chromatogram of the crude PDC-1 reaction mixture. "3°-thiol" refers to a tertiary thiol.

[0041] Figures 5A to 5C is a line graph of stability testing and biological evaluation of PDC 1-6. Figure 5A Stability testing of PDC-1 and PDC-3 dissolved in 5% ACN water or 5% ACN PBS buffer pH 7.4 at a concentration of 0.5 mM, placed at room temperature or 37°C, as indicated, is shown. Compounds were checked by LCMS after 24 and 48 hours. Figure 5B The results of CCK-8 assays of U87-MG cells treated with PDCs1-6 are shown. Figure 5C Shown are the results of CCK-8 assays of HEK-293T cells treated with PDCs 1-6. DETAILED DESCRIPTION

[0042] I. Definitions

[0043] "Additive", as used herein, refers to a compound that inhibits undesirable side reaction pathways in a reaction while not preventing other desired reaction pathways from proceeding. For example, an additive binds to one reactant in a chemical reaction and inhibits the reaction between that reactant and another reactant, as compared to a different reactant involved in the chemical reaction. For example, an additive can inhibit a reaction pathway that results in a product being formed by the reaction between only an amine group and an electrophilic group in another reactant. An additive can be an inorganic salt, or an organic compound that is a conjugate acid of a base (e.g., a base in the form of a salt) or present in a chemical reaction as a free base.

[0044] "Inhibit", in relation to the additives described herein, means to reduce or decrease the rate of occurrence of a reaction pathway and / or product formation. This can be a complete or partial reduction. Inhibition can be compared to a control or standard level. A control can be reaction conditions involving the same reactants in the absence of the additive. Inhibition can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51, 52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99, or 100%.

[0045] "Steric hindrance", as it relates to nucleophiles in organic compounds, refers to the presence of one or more substituents (e.g., two or more substituents) at the α- or β-position of the nucleophile in the organic compound. More specifically, "steric hindrance" refers to the presence of one or more substituents (e.g., two or more substituents) at the α-position of the nucleophile in the organic compound. It is to be understood that the disclosed compounds, compositions and methods are not limited to specific synthetic methods, specific analytical techniques or specific reagents, unless otherwise specified, and therefore, these may vary. It is also to be understood that the terms used herein are used only to describe specific forms and embodiments and are not intended to be limiting.

[0046] "Substituted" as used herein refers to all permissible substituents of the compounds or functional groups described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogen, hydroxy, or any other organic combination containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally containing one or more heteroatoms such as oxygen, sulfur or nitrogen, in a linear, branched or cyclic structural format. Representative substituents include substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silicon, thiol, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxido, sulfoxy, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphoryl, amino acid. Such substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polyaryl, substituted or unsubstituted polyheteroaryl, substituted or unsubstituted aralkyl, halogen, hydroxy, alkoxy, phenoxy, aryloxy, silicon, thiol, thiol, alkylthio, substituted alkylthio, phenylthio, arylthio, cyano, isocyano, nitro, substituted or unsubstituted carbonyl, carboxyl, amino, amide, oxidizing group, sulfoxyl, sulfonyl, sulfonic acid, phosphonium, phosphanyl, phosphoryl, phosphoryl and amino acid may be further substituted.

[0047] Heteroatoms, such as nitrogen, may have hydrogen substituents and / or any permissible substituents of organic compounds that match the valence of the heteroatoms. It should be understood that "substitution" or "substituted" includes an implicit prerequisite that such substitution should be consistent with the permissible valence of the substituted atom and the substituent, and that the substitution result is a stable compound, i.e., a compound that does not spontaneously undergo changes such as rearrangement, cyclization, elimination, etc.

[0048] "Alkyl" refers to the free radical of a saturated aliphatic group in this article, including straight chain alkyl, branched chain alkyl and cycloalkyl (alicyclic). In some forms, straight chain or branched chain alkyl has 30 or less carbon atoms in its main chain (for example, straight chain is C1-C30, branched chain is C3-C30), 20 or less, 15 or less, or 10 or less. Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Similarly, cycloalkyl is a non-aromatic carbon-based ring composed of at least three carbon atoms, such as a non-aromatic monocyclic or non-aromatic polycyclic ring structure containing 3 to 30 carbon atoms, 3 to 20 carbon atoms or 3 to 10 carbon atoms, and there are 5, 6 or 7 carbon atoms in the ring. Cycloalkyl groups containing polycyclic ring systems may have two or more non-aromatic rings in which two or more carbon atoms are common to two adjacent rings (i.e., "fused cycloalkyl rings"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0049] "Substituted alkyl" refers to an alkyl moiety having one or more substituents replacing a hydrogen on one or more carbon atoms of the carbon-hydrogen backbone. These substituents can be any of the substituents described above, for example, halogen (e.g., fluorine, chlorine, bromine or iodine), hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformyl), aromatic, alkoxy, aralkyl, phosphonium, phosphanyl, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino, amide, guanidino, imino, cyano, nitro, azido, oxido, thiol, alkylthio, silicon, sulfonyl, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, aromatic or heteroaromatic moieties. NRR', where R and R' are independently hydrogen, alkyl or aromatic, wherein the nitrogen atom may be quaternized; SR, where R is phosphinyl, sulfonyl, silicon, hydrogen, alkyl or aromatic; CN; NO2; COOH; carboxylate; COR, COOR or CON(R)2, where R is hydrogen, alkyl or aromatic; imine, silicon, ether, halogenated alkyl (e.g., CF3, CH2CF3, CCl3); CN; NCOCOCH2CH2; NCOCOCHCH; and NCS; and combinations thereof.

[0050] It should be understood that the groups substituted on the hydrocarbon chain may themselves be substituted if appropriate. For example, the substituents of substituted alkyl groups may include halogen, hydroxyl, nitro, thiol, amino, aralkyl, azido, imino, amide, phosphonium, phosphanyl, phosphoryl (including phosphate and phosphate ester), oxidized, sulfonyl (including sulfate, sulfonamide, sulfonamide and sulfonate) and silicon groups, as well as ethers, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester), haloalkyl, CN, etc. Cycloalkyl groups may be substituted in the same manner.

[0051] Unless otherwise specifically indicated, the number of carbon atoms, "lower alkyl" herein refers to an alkyl as defined above, but having one to ten carbon atoms, more preferably one to six carbon atoms in its main chain structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

[0052] "Heteroalkyl" as used herein refers to a linear or branched, or cyclic carbon-containing alkyl radical, or a combination thereof, containing at least one heteroatom in the carbon skeleton. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms may be oxidized, and nitrogen heteroatoms may be quaternized. For example, a "heterocycloalkyl group" is a cycloalkyl group as defined above, wherein at least one carbon atom in the ring is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0053] The term "alkenyl" as used herein refers to a hydrocarbon group containing from 2 to 24 carbon atoms, the structural formula of which contains at least one carbon-carbon double bond. Alkenyl groups include straight chain alkenyls, branched chain alkenyls, and cycloalkenyls. Cycloalkenyls are non-aromatic carbon-based rings composed of at least three carbon atoms, containing at least one carbon-carbon double bond, such as non-aromatic monocyclic or non-aromatic polycyclic ring structures containing from 3 to 30 carbon atoms and at least one carbon-carbon double bond, 3 to 20 carbon atoms and at least one carbon-carbon double bond, or 3 to 10 carbon atoms and at least one carbon-carbon double bond, with 5, 6 or 7 carbon atoms in the ring structure and at least one carbon-carbon double bond. Cycloalkenyl groups containing polycyclic ring systems can have two or more non-aromatic rings, wherein two or more carbon atoms are common to two adjacent rings (i.e., "fused cycloalkenyl rings"), and contain at least one carbon-carbon double bond. Asymmetric structures, such as (AB)C=C(C'D), are intended to include E and Z isomers. This can be assumed when an asymmetric olefin is present in the formula, or explicitly indicated by the bond symbol C. As used herein, the term "alkenyl" is intended to include both "unsubstituted alkenyl" and "substituted alkenyl", the latter meaning that the alkenyl moiety has one or more substituents replacing a hydrogen on one or more carbons of the carbon-hydrogen backbone. The term "alkenyl" also includes "heteroalkenyl".

[0054] "Substituted alkenyl" refers to an alkenyl moiety having one or more substituents replacing one or more hydrogen atoms on one or more carbons in the carbon-hydrogen backbone. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformyl), alkoxy, phosphonium, phosphanyl, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (e.g., quaternized amino), amide, guanidino, imino, cyano, nitro, azido, oxide, thiol, alkylthio, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof.

[0055] The term "heteroalkenyl" as used herein refers to a linear or branched, or cyclic carbon-containing alkenyl radical, or a combination thereof, containing at least one heteroatom in the carbon skeleton. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms may be oxidized, and nitrogen heteroatoms may be quaternized. For example, a "heterocycloalkenyl group" is a cycloalkenyl group in which at least one carbon atom in the ring is replaced by a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0056] The term "alkynyl group" herein refers to a hydrocarbon group containing 2 to 24 carbon atoms and containing at least one carbon-carbon triple bond in the structural formula. Alkynyl groups include straight chain alkynyl groups, branched chain alkynyl groups and cycloalkynyl groups. Cycloalkynyl groups are non-aromatic carbon-based rings consisting of at least three carbon atoms and at least one carbon-carbon triple bond, such as non-aromatic monocyclic or non-aromatic polycyclic rings containing 3 to 30 carbon atoms and at least one carbon-carbon triple bond, containing 3 to 20 carbon atoms and at least one carbon-carbon triple bond, or containing 3 to 10 carbon atoms and at least one carbon-carbon triple bond, and there are 5, 6 or 7 carbon atoms in the ring structure and at least one carbon-carbon triple bond. Cycloalkynyl groups containing polycyclic ring systems can have two or more non-aromatic rings, wherein two or more carbon atoms are shared by two adjacent rings (i.e., "fused cycloalkynyl groups"), and contain at least one carbon-carbon triple bond. Asymmetric structures such as (AB)C=C(C"D) are intended to include both E and Z isomers. This may be assumed when an asymmetric alkyne is present in the formula, or may be explicitly indicated by the bond symbol C. As used herein, the term "alkynyl" is intended to include both "unsubstituted alkynyl" and "substituted alkynyl", the latter meaning that one or more substituents are substituted for hydrogen atoms on one or more carbon atoms in the hydrocarbon backbone in the alkynyl group. The term "alkynyl" also includes "heteroalkynyl".

[0057] "Substituted alkynyl" refers to an alkynyl group in which one or more substituents replace hydrogen atoms on one or more carbon atoms in the hydrocarbon backbone. These substituents can be any of the substituents described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphonate, phosphazene, phosphoryl, phosphoric acid, phosphate, phosphonate, amino (such as quaternary ammonium amino), carbamoyl, guanidino, imino, cyano, nitro, azido, thiol, alkylthio, sulfate, sulfonate, sulfonamide, sulfonic acid, sulfonyl, heterocyclic, alkylaryl, haloalkyl, -CN, aryl, heteroaryl, polyaromatic, polyheteroaromatic, and combinations thereof.

[0058] "Heteroalkynyl" herein refers to a linear or branched, or cyclic carbon-containing alkynyl radical, or a combination thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, and S, wherein nitrogen, phosphorus, and sulfur atoms may be oxidized, and nitrogen heteroatoms may be quaternized. For example, a "heterocycloalkynyl group" is a cycloalkynyl group in which at least one carbon atom of the ring is substituted with a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus.

[0059] "Aromatic" as used herein refers to aromatic or fused aromatic ring systems consisting of C5 to C26 members. Examples of aromatic groups include benzene, naphthalene, anthracene, phenanthrene, Pyrene, chrysene, chrysene, etc.

[0060] The term "substituted aromatic" refers to an aromatic group in which one or more hydrogen atoms on one or more aromatic rings are replaced by one or more substituents, including but not limited to halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (such as ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (or quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, imino, alkylthio, sulfate, sulfonic acid, sulfonamide, sulfinyl, sulfonyl, heterocyclic, alkyl aromatic, haloalkyl (such as CF3, CH2-CF3, -CCl3), -CN, aromatic, heteroaromatic, and combinations thereof.

[0061] "Heterocycle" and "heterocyclyl" are used interchangeably and refer to non-aromatic monocyclic or polycyclic ring radicals attached through ring carbon atoms or nitrogen atoms, containing 3 to 30 ring atoms, 3 to 20 ring atoms, 3 to 10 ring atoms or 5 to 6 ring atoms, each ring containing carbon and one to four heteroatoms, the heteroatoms selected from non-peroxide oxygen, sulfur and N(Y), wherein Y is hydrogen, oxygen, C1 to C10 alkyl, phenyl or benzyl, and optionally containing 1 to 3 double bonds, and optionally substituted with one or more alkyl and aromatic substituents as described above. Heterocyclyl is distinguished from heteroaromatic groups in definition. The heterocycle can be a heterocycloalkyl, a heterocycloalkenyl, a heterocycloalkynyl, etc., such as piperazinyl, piperidinyl, piperidinoyl, 4-piperidinoyl, dihydrofuran [2,3-b] tetrahydrofuran, morpholinyl, piperazinyl, piperidinyl, piperidinoyl, 4-piperidinoyl, piperidinoyl, pyranyl, 2H-pyrrolyl, 4H-quinolinyl, quinuclidine cyclohexyl, tetrahydrofuranyl, 6H-1,2,5-thiazinyl. The heterocyclic group can be optionally substituted by one or more substituents as described above.

[0062] The term "heteroaromatic" refers to an aromatic or fused aromatic ring system consisting of C5 to C26 members in which one or more of the carbon atoms in the aromatic ring structure is replaced by a heteroatom. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaromatic groups include, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridopyrimidine, and the like. Examples of heteroaromatic rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiophenyl, benzothiophenyl, phenoxyazolyl, phenoxyazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzothiazolyl, benzothiophenyl, carbazolyl, 4aHcarbazolyl, carbazolinyl, chromanyl, chromanyl, chromanone, chromanolinyl, decahydronaphthyridinyl, 2 H,6H-1,5,2-dithiazinyl, furanyl, furanoyl, imidazolyl, imidazolyl, imidazolyl, imidazolyl, 1H-indolyl, indolene, indolyl, indolyl, 3H-indolyl, isoindenyl, isobenzofuranyl, isochromanyl, isoindolyl, isoindolyl, isoquinolyl, isothiazolyl, isothiazolyl, methoxydioxyphenyl, naphthyridinyl, octahydroisoquinoline oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolinyl, oxazolyl, indolyl, benzopyridinyl, benzoquinolinyl, benzothienyl, phenoxythiazinyl, phenoxyoxazolyl, phthalazinyl, purinyl, pyrazinyl, pyrazolinyl, pyrazolone, pyrazolyl, pyridopyrimidinyl, pyridoimidazolyl , pyridothiazolyl, pyridinyl, pyridinyl, pyrimidinyl, piperidinyl, piperidonyl, pyrrolyl, quinolyl, quinazolyl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, 1,2,3-thiazinyl, 1,2,4-thiazinyl, 1,2,5-thiazinyl, 1,3,4-thiazinyl, thiazinyl, thiazolyl, thienyl, thienothiphenyl, thienoxazolyl, thienyl. One or more rings may be substituted with one or more substituents as defined below under "Substituted Heteroaromatic".

[0063] The term "substituted heteroaromatic" refers to a heteroaromatic group in which one or more hydrogen atoms on one or more heteroaromatic rings are replaced by one or more substituents, including but not limited to halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, carbonyl (such as ketone, aldehyde, carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogenphosphate, amino (or quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, imino, alkylthio, sulfate, sulfonic acid, sulfonamide, sulfinyl, sulfonyl, heterocyclyl, alkylaromatic, haloalkyl (such as CF3, CH2-CF3, -CCl3), -CN, aromatic, heteroaromatic, and combinations thereof.

[0064] The term "polyaromatic" refers to a chemical group that contains two or more fused aromatic groups. When two or more fused heteroaromatic groups are involved, the chemical group may be referred to as "polyheteroaromatic".

[0065] The term "substituted polyaromatic" refers to a polyaromatic group in which one or more aromatic groups are substituted with one or more substituents, including but not limited to halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (or quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, alkylthio, sulfate, sulfonic acid, sulfonamide, sulfinyl, sulfonyl, heterocyclic, alkyl aromatic, haloalkyl, -CN, aromatic, heteroaromatic, and combinations thereof. When referring to polyheteroaromatic, the chemical group may be referred to as "substituted polyheteroaromatic".

[0066] The term "cyclic ring" or "cyclic group" refers to a substituted or unsubstituted monocyclic ring or a substituted or unsubstituted polycyclic ring (such as formed by a single or fused ring system), such as a substituted or unsubstituted cycloalkyl, a substituted or unsubstituted cycloalkenyl, a substituted or unsubstituted cycloalkynyl, or a substituted or unsubstituted heterocyclyl, which has from three to thirty carbon atoms, subject to geometric constraints. Substituted cycloalkyl, cycloalkenyl, cycloalkynyl and heterocyclyl groups are substituted as described above for the definitions of alkyl, alkenyl, alkynyl and heterocyclyl.

[0067] The term "aralkyl" as used herein refers to an aromatic group or a heteroaromatic group having an alkyl, alkynyl or alkenyl group as defined above attached to an aromatic group, such as an aromatic, a heteroaromatic, a polyaromatic or a polyheteroaromatic. An example of an aralkyl group is a benzyl group.

[0068] The terms "alkoxy" or "alkyloxy", "aryloxy" or "aryloxy" generally describe compounds represented by the formula ORv, where Rv includes, but is not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted aralkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkyl aromatic, substituted or unsubstituted alkyl heteroaromatic, substituted or unsubstituted aralkyl, substituted or unsubstituted carbonyl, phosphonium, phosphoalkyl, phosphoryl, sulfinyl, silicon, thiol, amide and amino. Exemplary alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like. "Lower alkoxy" is an alkoxy group containing one to six carbon atoms. An "ether" is two functional groups covalently linked through an oxygen atom as defined below. Thus, the substituents that make an alkyl group an ether are or are similar to alkoxy groups, and can be represented by O-alkyl, O-alkenyl, O-alkynyl, O-aralkyl, O-aromatic, O-heteroaromatic, O-polyaromatic, O-polyheteroaromatic, O-heterocyclyl, etc.

[0069] "Substituted alkoxy" refers to an alkoxy group in which one or more substituents replace one or more hydrogen atoms on one or more carbon atoms on the alkoxy backbone. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphonium, phosphanyl, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, oxide, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, and combinations thereof.

[0070] The term "ether" is represented herein by the formula A2OA1, wherein A2 and A1 can independently be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted aralkyl, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, phosphonium, phosphane, phosphoryl, sulfinyl, silicon, thiol, substituted or unsubstituted carbonyl, alkoxy, amide or amino, as described above.

[0071] The term "polyether" is represented herein by the following formula:

[0072]

[0073] wherein A3 can independently be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted aralkyl, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, phosphonium, phosphanyl, substituted or unsubstituted carbonyl, alkoxy, amide or amino, as described above; and g can be a positive integer from 1 to 30.

[0074] "Phenoxy" is an industry-recognized term that refers to a compound with the formula OR v A compound wherein R v It is C6H5 (ie OC6H5). Those skilled in the art will recognize that phenoxy is a type of aryloxy.

[0075] "Substituted phenoxy" refers to a phenoxy group as defined above, wherein one or more hydrogen atoms on the phenyl ring are replaced by one or more substituents. Such substituents include, but are not limited to, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformyl), alkoxy, phosphonium, phosphanyl, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (e.g., quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, and combinations thereof.

[0076] The terms "aryloxy" and "aryloxy" are used interchangeably herein and are represented by Oaryl or Oheteroaryl, wherein aryl and heteroaryl are as defined above.

[0077] "Substituted aryloxy" and "substituted aryloxy", the two terms are used interchangeably herein, and refer to O aromatic or O heteroaromatic groups, wherein one or more hydrogen atoms on one or more ring atoms on the aromatic and heteroaromatic groups are replaced by one or more substituents, as defined herein. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphonium, phosphanyl, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof.

[0078] The term "amino" as used herein includes the following groups:

[0079]

[0080] wherein E may not exist, or E may be a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted aromatic, a substituted or unsubstituted heteroaromatic, a substituted or unsubstituted polyaromatic, a substituted or unsubstituted polyheteroaromatic, or a substituted or unsubstituted heterocyclic group, wherein, independently of E, Rx, Rxi, and Rxii each independently represent a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, hydroxy, alkoxy, phosphonium, phosphanyl, phosphoryl, sulfinyl, silicon, thiol, amide, amino, or (CH2)mR"; R" represents hydroxy, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, alkoxy, phosphonium, phosphanyl, amide or amino; m is zero or an integer from 1 to 8. "Quaternary ammonium amino" also includes nitrogen atoms, Rx, Rxi and Rxii together with the N+ to which they are attached to form a heterocyclic or heteroaromatic group, with 3 to 14 atoms in the ring structure. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0081] The terms "amide" or "amido" are used interchangeably herein to refer to both "unsubstituted amide" and "substituted amide" and are represented by the following general formula:

[0082]

[0083] wherein E may be absent, or E may be a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, or substituted or unsubstituted heterocyclic group, wherein, independently of E, R and R' each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aralkyl group (e.g., a substituted or unsubstituted alkylaryl group, a substituted or unsubstituted arylalkyl group), a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, a substituted or unsubstituted heterocyclic group, a hydroxyl group, an alkoxy group, a phosphonium group, a phosphane group, a phosphoryl group, a sulfinyl group, a silicon group, a thiol group, an amide group, an amino group, or -(CH2)m R"; R and R' together with the nitrogen atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R" represents hydroxy, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, alkoxy, phosphonium, phosphanyl, amide or amino; m is zero or an integer from 1 to 8. In certain forms, when E is oxygen, a carbonate is formed. Those skilled in the art understand that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0084] "Carbonyl", as used herein, refers to a recognized chemical structure including a group that can be represented by the following general formula:

[0085]

[0086] wherein X is a bond, or represents oxygen or sulfur, and R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, hydroxy, alkoxy, phosphonium, phosphanyl, amide, amino, or (CH2)m R", or a pharmaceutically acceptable salt thereof; E" is absent, or E" is substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclyl; R' represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkyl aromatic, substituted or unsubstituted aromatic alkyl), substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclyl, hydroxy, alkoxy, phosphonium, phosphanyl, amide, amino, or (CH2)m R"; R" represents a hydroxyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, an alkoxy group, a phosphonium group, a phosphine group, an amide group, an amino group; m is zero or an integer from 1 to 8. These substituents may be any of the substituents described above, for example, a halogen, an azido group, an alkyl group, an aralkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a hydroxyl group, a carbonyl group (such as a carboxyl group, an alkoxycarbonyl group, a formyl group or an acyl group), a silicon group, an ether, an ester, a thiocarbonyl group The term "carboxylic acid" refers to a group (e.g., a thioester, a thioacetate, or a thioformyl group), an alkoxy group, a phosphonium group, a phosphine group, a phosphoryl group, a phosphate group, a phosphate ester, a phosphate, a hydrogen phosphate, an amino group (e.g., a quaternized amino group), an amide group, a guanidine group, an imine group, a cyano group, a nitro group, an azido group, a thiol group, a sulfate group, a sulfonic acid group, a sulfonamide group, a sulfonyl group, a heterocyclic group, an alkyl aromatic group, a haloalkyl group, a CN group, an aromatic group, a heteroaromatic group, and combinations thereof. It is understood by those skilled in the art that the E" groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl). When X is oxygen and R is as defined above, the group is also referred to as a carboxyl group. When X is oxygen and R is hydrogen, the formula represents a "carboxylic acid". When X is oxygen and R' is hydrogen, the formula represents a "formic acid".When X is oxygen and R or R' is not hydrogen, the formula represents an "ester". Generally, when the oxygen atom in the above formula is replaced by a sulfur atom, the formula represents a "thiocarbonyl" group. When X is sulfur and R or R' is not hydrogen, the formula represents a "thioester". When X is sulfur and R is hydrogen, the formula represents a "thiocarboxylic acid". When X is sulfur and R' is hydrogen, the formula represents a "thioformic acid". When X is a bond and R is not hydrogen, the above formula represents a "ketone". When X is a bond and R is hydrogen, the above formula represents an "aldehyde".

[0087] The "phosphoalkyl" is represented by the following formula:

[0088]

[0089] wherein E may be absent, or E may be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, wherein Rvi and Rvii independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted heterocyclic, and independently of E, Rvi and Rvii each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl ... unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl group (e.g., substituted or unsubstituted alkylaryl group, substituted or unsubstituted arylalkyl group, etc.), substituted or unsubstituted aromatic group, substituted or unsubstituted heteroaromatic group, substituted or unsubstituted polyaromatic group, substituted or unsubstituted polyheteroaromatic group, substituted or unsubstituted heterocyclic group, hydroxyl group, alkoxy group, phosphonium group, phosphane group, phosphoryl group, sulfinyl group, silicon group, thiol group, amide group, amino group, or (CH2)m R"'; or Rvi and Rvii together with the phosphorus atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R" represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, an alkoxy group, a phosphonium group, a phosphoalkyl group, an amide group or an amino group; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0090] Phosphonium is represented by the following formula:

[0091]

[0092] wherein E may be absent, or E may be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, wherein, independently of E, Rvi, Rvii and Rviii each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl group (e.g., substituted or unsubstituted alkylaryl group, substituted or unsubstituted arylalkyl group, etc.), substituted or unsubstituted aromatic group, substituted or unsubstituted heteroaromatic group, substituted or unsubstituted polyaromatic group, substituted or unsubstituted polyheteroaromatic group, substituted or unsubstituted heterocyclic group, hydroxyl group, alkoxy group, phosphonium group, phosphane group, phosphoryl group, sulfinyl group, silicon group, thiol group, amide group, amino group, or (CH2)m R'', or Rvi, Rvii and Rviii together with the P+ atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R'' represents hydroxy, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, alkoxy, phosphonium, phosphanyl, amide or amino; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0093] The "phosphoryl group" is represented by the following formula:

[0094]

[0095] wherein E may be absent, or E may be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, oxygen, alkoxy, aryloxy or substituted alkoxy or substituted aryloxy, wherein, independently of E, Rvi and Rvii each independently represent hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aromatic group, substituted or unsubstituted heteroaromatic group, substituted or unsubstituted polyaromatic group, substituted or unsubstituted polyheteroaromatic group, substituted or unsubstituted heterocyclic group, hydroxyl group, alkoxy group, phosphonium group, phosphanyl group, phosphoryl group, sulfinyl group, silicon group, thiol group, amide group, amino group, or (CH2)m R'', or Rvi and Rvii together with the phosphorus atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R'' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, an alkoxy group, a phosphonium group, a phosphanyl group, an amide group or an amino group; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0096] "Phosphoryl" defines a phosphoryl group wherein E is absent, E is oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as described above, and Rvi and Rvii are independently hydroxy, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as described above, independent of E. When E is oxygen, the phosphoryl group cannot be attached to another chemical species, such as to form an oxygen-oxygen bond or other unstable bond, as will be appreciated by those skilled in the art. When E, Rvi and Rvii are substituted, the substituents include, but are not limited to, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (e.g., thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (e.g., quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (e.g., methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0097] A "sulfinyl group" is represented by the following formula:

[0098]

[0099] wherein E may be absent, or E may be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic, substituted or unsubstituted heterocyclic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, wherein independently of E, R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl group (e.g., substituted or unsubstituted alkylaryl group, substituted or unsubstituted arylalkyl group), substituted or unsubstituted aryl group, substituted or unsubstituted heteroaromatic group, substituted or unsubstituted polyaromatic group, substituted or unsubstituted polyheteroaromatic group, substituted or unsubstituted heterocyclic group, hydroxyl group, alkoxyl group, phosphonium group, phosphanyl group, phosphoryl group, silicon group, thiol group, amide group, amino group, or (CH2)m R'', or E and R together with the sulfur atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R'' represents a hydroxyl group, a substituted or unsubstituted carbonyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted cycloalkenyl group, a substituted or unsubstituted heterocyclyl group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, an alkoxy group, a phosphonium group, a phosphanyl group, an amide group or an amino group; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0100] A "sulfonyl group" is represented by the following formula:

[0101]

[0102] wherein E may be absent, or E may be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, etc.), substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic, substituted or unsubstituted heterocyclic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, wherein independently of E, R represents hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl), substituted or unsubstituted aromatic R'' represents hydroxyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted heterocyclic, hydroxyl, alkoxyl, phosphonium, phosphanyl, amide, amino, or (CH2)mR'', or E and R together with the sulfur atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R'' represents hydroxyl, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclic, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, alkoxyl, phosphonium, phosphanyl, amide or amino; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0103] The term "sulfonic acid group" refers to the sulfonyl group defined above, wherein R is hydroxy, E is absent, or E is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, or substituted or unsubstituted heteroaromatic. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0104] The term "sulfate" refers to the sulfonyl group as defined above, wherein E is absent, is oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as described above, and R is independently hydroxy, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as described above. When E is oxygen, the sulfate cannot be attached to another chemical species, such as to form an oxygen-oxygen bond or other unstable bond, as will be appreciated by those skilled in the art. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0105] The term "sulfonic acid group" refers to the sulfonyl group as defined above, wherein E is oxygen, alkoxy, aryloxy, substituted alkoxy or substituted aryloxy, as described above, R is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amino, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aralkyl, substituted or unsubstituted alkylaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, (CH2)mR"', R"' represents hydroxy, substituted or unsubstituted carbonyl, aromatic, cycloalkyl ring, cycloalkenyl ring, heterocyclyl, amide, amino or polycyclic group; m is zero or an integer from 1 to 8. When E is oxygen, the sulfonic acid group cannot be attached to another chemical species, such as to form an oxygen-oxygen bond or other unstable bond, as will be understood by those skilled in the art. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0106] The term "sulfonyl" refers to a sulfonamide or sulfonamide represented by the following formula:

[0107]

[0108] wherein E may be absent, or E may be a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, a substituted or unsubstituted aralkyl (e.g., a substituted or unsubstituted alkyl aromatic group, a substituted or unsubstituted cycloalkyl group, etc.), a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, a substituted or unsubstituted polyaromatic group, a substituted or unsubstituted polyheteroaromatic group, or a substituted or unsubstituted heterocyclic group, wherein R and R' independently of E each independently represent hydrogen, a substituted or unsubstituted alkyl, or a substituted or unsubstituted alkyl. substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbonyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aralkyl group (e.g., substituted or unsubstituted alkylaryl group, substituted or unsubstituted arylalkyl group, etc.), substituted or unsubstituted aryl group, substituted or unsubstituted heteroaromatic group, substituted or unsubstituted polyaromatic group, substituted or unsubstituted polyheteroaromatic group, substituted or unsubstituted heterocyclic group, hydroxyl group, alkoxy group, phosphonium group, phosphanyl group, amide group, amino group, or (CH2)m R'", or R and R' together with the nitrogen atom to which they are attached form a heterocyclic structure containing 3 to 14 atoms; R'" represents hydroxy, substituted or unsubstituted carbonyl, substituted or unsubstituted aromatic, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, alkoxy, phosphonium, phosphanyl, amide or amino; m is zero or an integer from 1 to 8. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaryl, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof. It is understood by those skilled in the art that the E groups listed above are divalent (for example, methylene, ethane-1,2-diyl, ethylene-1,2-diyl, 1,4-phenylvinyl, cyclohexane-1,2-diyl).

[0109] The term "silicon group" is represented herein by the formula SiRR'R", wherein R, R' and R" can independently be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkyl aromatic, substituted or unsubstituted aromatic alkyl, etc.), substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted carbonyl, phosphonium, phosphanyl, phosphoryl, sulfinyl, thiol, amide, amino, alkoxy or oxidized, as described above. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaromatic, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof.

[0110] "Thiol" may be used interchangeably and is represented by -SR, where R may be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic, substituted or unsubstituted aralkyl (e.g., substituted or unsubstituted alkyl aromatic, substituted or unsubstituted aromatic alkyl, etc.), substituted or unsubstituted polyaromatic, substituted or unsubstituted polyheteroaromatic, substituted or unsubstituted carbonyl, phosphonium, phosphanyl, amide, amino, alkoxy, oxidase, phosphoryl, sulfinyl or silicon, as described above. These substituents can be any of the substituents described above, for example, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), silicon, ether, ester, thiocarbonyl (such as thioester, thioacetate or thioformyl), alkoxy, phosphoryl, phosphate, phosphate ester, phosphate, hydrogen phosphate, amino (such as quaternized amino), amide, guanidine, imino, cyano, nitro, azido, thiol, sulfate, sulfonic acid, sulfonamide, sulfonyl, heterocyclic, alkylaromatic, haloalkyl, CN, aromatic, heteroaromatic, polyaromatic, polyheteroaromatic, and combinations thereof.

[0111] The disclosed compounds and substituent groups may independently possess two or more of the groups listed above. For example, if the compound or substituent group is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group may be substituted by a hydroxyl group, an alkoxy group, etc. Depending on the selected group, the first group may be embedded in the second group, or as a side chain (i.e., connected) to the second group. For example, in the phrase "an alkyl group containing an ester group", the ester group may be embedded in the main chain of the alkyl group. Alternatively, the ester group may be connected to the main chain of the alkyl group. The nature of the selected group will determine whether the first group is embedded in or connected to the second group.

[0112] The compounds and substituents may be independently substituted with substituents as described in the above definition of "substituted".

[0113] Numerical ranges disclose every possible number that the range may reasonably contain, as well as any subranges and combinations of subranges. For example, in a given carbon atom range of C3-C9, the range also discloses C3, C4, C5, C6, C7, C8 and C9, as well as any subranges between these numbers (e.g., C4-C6), and any possible range combinations between these values. Another example is that a given temperature range may be from about 25°C to 30°C, where the range also discloses temperatures that can be independently selected from about 25, 26, 27, 28, 29 and 30°C, as well as any ranges between these numbers (e.g., 26 to 28°C), and any possible range combinations between these values.

[0114] The term "about" is intended to describe a value above or below the stated value, which value is modified by the term "about" to be within a range of approximately plus or minus 10%. When the term "about" is used before a series of numbers (i.e., about 1-5) or a series of numbers (i.e., about 1, 2, 3, 4, etc.), it is intended to modify both ends of the numerical range and / or each number mentioned in the entire series, unless otherwise indicated.

[0115] "Oxo" means =O.

[0116] The compounds and substituents may be independently substituted with substituents as described in the above definition of "substituted".

[0117] The term "about" is intended to describe a value above or below the stated value, which value is modified by the term "about" to be within a range of approximately plus or minus 10%. When the term "about" is used before a series of numbers (i.e., about 1-5) or a series of numbers (i.e., about 1, 2, 3, 4, etc.), it is intended to modify both ends of the numerical range and / or each number mentioned in the entire series, unless otherwise indicated.

[0118] II. Conjugates, Preparation Methods and Reagents

[0119] Described herein are conjugates, such as bioconjugates, and methods for making these conjugates by intermolecular reactions between modular groups. Traditionally, these intermolecular reactions involve the use of a large amount of one modular group to ensure the formation of the desired product. However, the use of a large amount of reactants (e.g., at least 10 times the equivalent) limits the potential of this reaction and makes it impractical in bioconjugation. It is now found that the addition of additives to the intermolecular reaction can reduce or eliminate the need to use a large amount of one modular group. Therefore, a product formed by at least three components can be effectively generated and a stoichiometric reaction with a comparable molar ratio of reactants is achieved. Preferably, the reaction is carried out in an aqueous buffer.

[0120] A. Conjugate

[0121] In certain forms, the disclosed conjugates comprise the following structure:

[0122]

[0123] in:

[0124] R1 to R4 are independently hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted amide, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fused combination thereof;

[0125] R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl, substituted C1-C10 alkenyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted amide alkyl, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof;

[0126] R6 and R7 are independently unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or fused combinations thereof, provided that R6 and R7 are at least divalent, as permitted by valence;

[0127] R8 and R9 are independently hydrogen, unsubstituted C1-C10 alkyl, unsubstituted C1-C10 alkyl, provided that at least one of R8 and R9 is not hydrogen, such as unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl;

[0128] PD1 and PD2 independently comprise a peptide, a non-peptide therapeutic agent, a non-peptide diagnostic agent, a non-peptide preventive agent, or a non-peptide targeting agent.

[0129] In some forms, the conjugate is as described in Formula XA, except that R1-R4 are independently hydrogen, unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl. In some forms, the conjugate is as described in Formula XA, except that R1-R4 are hydrogen. In some forms, the conjugate is as described in Formula XA, except that at least one of R1-R4 independently comprises a targeting agent, a diagnostic agent, a prophylactic agent or a therapeutic agent. In some forms, the targeting agent comprises a peptide.

[0130] In certain forms, the conjugate is as described in formula XA, except that R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl, or substituted C1-C10 alkenyl.

[0131] In some forms, the conjugate is as described in Formula XA, except that R6 and R7 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl, provided that R6 and R7 are at least divalent, as allowed by the valence. In some forms, the conjugate is as described in Formula XA, except that R6 and R7 independently contain a stimulus-responsive chemical group. In some forms, the conjugate is as described in Formula XA, except that R6 contains a stimulus-responsive chemical group. A stimulus-responsive group refers to a chemical group or bond that responds to an external stimulus. A stimulus-responsive chemical group can be cleaved by stimuli such as pH (such as a change in pH), redox potential (such as a change in redox potential), reactive oxygen species (ROS), enzymes (such as overexpression of enzymes (e.g., proteases, esterases, etc.) due to a disease or disorder), ionic strength (such as a change in ionic strength), and hypoxia. Examples of stimulus-responsive chemical groups that are responsive to ROS are described in Saravanakumar et al., Adv. Sci. 2017, 4, 1600124, which is cited herein. In some forms, the stimulus-responsive chemical group independently comprises a disulfide bond, an orthogonal ester, a hydrazone, a hydrazone acid, a hydrazone amine, an imine, an acetal, a vinyl ether, a polyketone, a methyl maleic acid, an ester bond, an amide bond, a nitroaromatic group (e.g., nitrobenzyl), a nitroheteroaromatic group (e.g., nitroimidazole), a quinone group, an azoaromatic group (e.g., azophenyl), an azoheteroaromatic group (e.g., azopyridyl), a peroxyoxalate, an aminoacrylate, an alkyl thioether, or a selenide (e.g., a monoselenide bond, a diselenide bond, etc.), a thioacetal, a peroxyoxalate, or a dimethyl maleic acid. In some forms, the stimulus-responsive chemical group is independently a disulfide bond, an orthoester, a hydrazone, a hydrazone acid, a hydrazone amine, an imine, an acetal, methyl maleic acid, or dimethyl maleic acid. In some forms, the stimulus-responsive chemical group independently comprises a disulfide bond, an orthoester, or a combination thereof. In some forms, the stimulus-responsive chemical group is a disulfide bond.

[0132] In certain forms, the conjugate is as described in Formula XA, except that R8 and R9 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl.

[0133] In some forms, the conjugate is as described in Formula XA, except that PD1 and PD2 comprise a peptide. In some forms, the conjugate is as described in Formula XA, except that one of PD1 and PD2 comprises a peptide and the other comprises a non-peptide therapeutic agent, non-peptide diagnostic agent, non-peptide preventive agent or non-peptide targeting agent. In some forms, the conjugate is as described in Formula XA, except that one of PD1 and PD2 comprises a peptide and the other comprises a non-peptide therapeutic agent. In some forms, the peptide in PD1 or PD2 is a targeting peptide. In some forms, the peptide in PD1, PD2 or both is an unprotected peptide. An unprotected peptide refers to a peptide that does not contain an amino acid protecting group. Amino acid protecting groups are discussed in the review by Isidro-Llobet et al., Chem. Rev. 2009, 109 (6), 2455-2504, the contents of which are cited herein. "Non-peptide", with respect to therapeutic, prophylactic, diagnostic or targeting agents, refers to those agents that do not contain two or more amino acid monomers linked by a peptide bond (-C(O)NH-).

[0134] B. Preparation Methods and Reagents

[0135] In some forms, the method of making a conjugate involves reacting a first reactant with a second reactant and a third reactant in the presence of an additive to form a conjugate. Preferably, the first reactant comprises the following structure:

[0136]

[0137] The second reactant contains a nucleophile denoted as nu1,

[0138] The third reactant contains a nucleophile denoted as nu2,

[0139] nu1 and nu2 are preferably selected from thiol groups, amine groups and sulfide anions, preferably nu1 and nu2 are different,

[0140] m and n are independently integers from 1 to 5, such as 1, 2, 3, 4 or 5, preferably m and n are 1,

[0141] A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof, such as a fusion of substituted aromatic and substituted heteroaromatic, a fusion of substituted aromatic and unsubstituted heteroaromatic, a fusion of unsubstituted aromatic and substituted heteroaromatic, or a fusion of substituted aromatic and unsubstituted heteroaromatic,

[0142] X and Y are independently absent, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, unsubstituted ether, substituted ether, unsubstituted polyether, substituted polyether, substituted amino, unsubstituted amino, substituted amide, unsubstituted amide, substituted C3-C20 cycloalkyl, unsubstituted C3-C20 cycloalkyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, substituted C3-C20 cycloalkenyl, unsubstituted C3-C20 cycloalkenyl, substituted C3-C20 cycloalkynyl, unsubstituted C3-C20 cycloalkynyl, or a fused combination thereof, preferably X and Y are absent,

[0143] E1 and E2 are independently selected substituted carbonyl or unsubstituted carbonyl electrophiles, such as aldehydes, ketones, Michael acceptors; substituted carboxyls; or unsubstituted carboxyls. Preferably, the second reactant and the third reactant are not covalently linked prior to the reaction. Preferably, the first reactant, the second reactant, and the third reactant react with each other to form a conjugate containing a substituted 1-mercaptoisoindole moiety or an unsubstituted 1-mercaptoisoindole moiety. The phrase "intermolecular" means, with respect to the reactants, that at least the second reactant and the third reactant are not covalently bonded prior to the reaction.

[0144] In some forms, E1 and E2 are adjacent in the first reactant, i.e., they are directly or indirectly connected to adjacent atoms in the chemical ring or chain of the first reactant. Preferably, E1 and E2 are directly or indirectly connected to adjacent atoms in the chemical ring of the first reactant. More preferably, E1 and E2 are directly connected to adjacent atoms in the chemical ring or chain of the first reactant. Most preferably, E1 and E2 are directly connected to adjacent atoms in the chemical ring of the first reactant.

[0145] In some forms, the method of making a conjugate is as described above, except that the additive comprises a nucleophile. Preferably, the additive includes, but is not limited to, guanidine, urea, thiourea, sodium bisulfite, or a combination thereof. Preferably, the additive suppresses undesirable side reaction pathways in the reaction, compared to the desired reaction pathway. In some forms, the additive combines (e.g., reacts) with the first reactant to form an adduct, suppressing the reaction between the third reactant (e.g., containing an amine nucleophile) and the first reactant. In some forms, the additive suppresses the reaction pathway that leads to the formation of a two-component product involving only the first reactant and the third reactant. For example, adding an additive in the form of a guanidine salt (e.g., a guanidine salt, such as guanidine hydrochloric acid) produces an OPA-guanidine adduct that can suppress the formation of imine-aldehyde intermediates common in the non-selective formation of two-component and three-component products.

[0146] In certain forms, the method of making the conjugate is as described above, except that the concentration of the additive is between about 0.5 M and about 10 M, between about 1 M and about 8 M, or between about 3 M and about 8 M, such as 1 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M or 10 M, preferably 1 M, 3 M, 6 M and 8 M. As described above, such concentrations preferably refer to the concentration of the additive in the solvent system (e.g., buffer) prior to its addition to the solvent system in which the reaction will be carried out.

[0147] In some forms, the method of making the conjugate is as described above, except that the molar ratio of the first reactant to the second reactant is maintained between 1:8 and 8: 1. Preferably, the molar ratio of the first reactant to the second reactant is maintained between 1:5 and 5:1, more preferably between 1:4 and 4:1, or between 1:3 and 3:1, such as 1:1.2, 1:1 or 2:1.2.

[0148] In some forms, the method of making the conjugate is as described above, except that the molar ratio of the second reactant to the third reactant is maintained between 1:4 and 4: 1. Preferably, the molar ratio of the second reactant to the third reactant is between 1:4 and 4:1, or between 1:3 and 3:1, such as 1:1, 1:1.5, 3:1 or 2:1.

[0149] In some forms, the method of making a conjugate is as described above, except that the second reactant and the third reactant are independently a therapeutic agent, a prophylactic agent, a diagnostic agent, or a targeting agent. In some forms, the second reactant and the third reactant are independently a linear peptide, a cyclic peptide, a non-polymer small molecule drug (e.g., a drug with a molecular weight between 100 Da and 2,500 Da), a fluorophore, a chemiluminescent compound, or a combination thereof.

[0150] In some forms, the method of making the conjugate is as described above, except that the second reactant comprises a thiol group that reacts with the first reactant. In some forms, the second reactant comprises a sterically hindered thiol group that reacts with the first reactant.

[0151] In some forms, the methods of making the conjugate are as described above, except that the third reactant comprises an amine group that reacts with the first reactant.

[0152] In certain forms, the method of making the conjugate is as described above, except that the first reactant has the structure:

[0153]

[0154] in:

[0155] A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted C1-C20 heterocyclic, unsubstituted C1-C20 heterocyclic, or a fused combination thereof. Preferably, E1 and E2 are adjacent in the first reactant, i.e., they are attached to adjacent atoms in a chemical ring or chain. Preferably, E1 and E2 are attached to adjacent atoms in a chemical ring of the first reactant.

[0156] In certain forms, the methods of making the conjugate are as described above, except that for formula I (including the first reactant), A is substituted aromatic or unsubstituted aromatic.

[0157] In certain forms, the method of making the conjugate is as described above, except that structural formula I has the following structure:

[0158]

[0159] Preferably, in formula II, E1 and E2 are adjacent in the aromatic ring.

[0160] In certain forms, the methods of making the conjugate are as described above, except that for Formula I and Formula II, m and n are 1.

[0161] In certain forms, the method of making the conjugate is as described above, except that either Formula I or Formula II has the following structure:

[0162]

[0163] In certain forms, the method of making the conjugate is as described above, except that Formula I, Formula II, or Formula III has the following structure:

[0164]

[0165] In some forms, the method of making the conjugate is as described above, except that the method involves mixing the additive with an appropriate buffer (such as phosphate buffered saline, PBS) to form a solution, and then adding the first reactant, the second reactant, and the third reactant. Preferably, after the additive is added to the buffer, the pH of the resulting solution is adjusted to a desired value. In some forms, the pH of the resulting solution is adjusted to about 7.4. This pH adjustment can be achieved using a 1M sodium hydroxide solution.

[0166] In some forms, the method of making the conjugate is as described above, except that the method involves adding the first reactant and the second reactant to a solution containing an additive and an appropriate buffer prior to adding the third reactant. Preferably, the second reactant is added first to inhibit (preferably completely inhibit) the formation of an undesirable two-component product from the first reactant and the third reactant alone.

[0167] In some forms, the conjugate is prepared as described above, except that the pH of the buffer is between about 7.0 and about 7.5, such as about 7.4.

[0168] In some forms, the method of making the conjugate is as described above, except that the reaction is carried out at a temperature between about 23° C. and about 40° C. In some forms, the reaction is carried out at room temperature, such as between about 23° C. and about 28° C. In some forms, the reaction is carried out at about 30° C. and about 40° C., such as at about 37° C. It should be understood that the temperature selected should ensure that the relevant reactants (such as proteins, peptides, etc.) are not denatured during the reaction.

[0169] In some forms, the methods of making the conjugate are as described above, except that the reaction concentration is between about 0.1 mM and about 7.5 mM, about 0.1 mM and about 5 mM, about 0.1 mM and about 2.5 mM, about 0.1 mM and about 2 mM, about 0.5 mM and about 2.5 mM, or about 0.5 mM and about 2 mM.

[0170] In some forms, the method of making the conjugate is as described above, except that the reaction involves the following steps:

[0171] (i) adding additives to a suitable buffer (such as PBS) to form a solution, dispersion or suspension,

[0172] (ii) adding the first reactant and the second reactant to the solution, dispersion or suspension of step (i) to form another solution, dispersion or suspension, and

[0173] (iii) adding a third reactant to the solution of step (ii) to form another solution, dispersion or suspension. In some forms, the solution, dispersion or suspension of step (iii) is maintained at a temperature of about 23°C to about 40°C in an air atmosphere. In some forms, the solution, dispersion or suspension of step (iii) is maintained at room temperature, for example, between about 23°C to about 28°C. In some forms, the solution, dispersion or suspension of step (iii) is maintained at a temperature of about 30°C to about 40°C, for example, at about 37°C. It should be understood that the selected temperature should ensure that the relevant reactants (such as proteins, peptides, etc.) will not denature during the reaction.

[0174] The disclosed methods and conjugates can be further understood by referring to the following numbered paragraphs.

[0175] 1. A method for making a conjugate, the method comprising:

[0176] allowing the first reactant, the second reactant and the third reactant to undergo an intermolecular reaction in the presence of an additive to form a conjugate, preferably the additive inhibiting the formation of a product formed solely by the reaction between the first reactant and the third reactant,

[0177] in:

[0178] The first reactant contains the following structure:

[0179]

[0180] The second reactant contains a nucleophile denoted as nu1,

[0181] The third reactant contains a nucleophile denoted as nu2,

[0182] Preferably, the second reactant and the third reactant are not covalently linked prior to the reaction,

[0183] nu1 and nu2 are preferably selected from thiol groups, amine groups and sulfide anions, preferably nu1 and nu2 are different,

[0184] m and n are independently integers from 1 to 5, such as 1, 2, 3, 4 or 5, preferably, m and n are 1,

[0185] A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof, such as a fusion of substituted aromatic and substituted heteroaromatic, a fusion of substituted aromatic and unsubstituted heteroaromatic, a fusion of unsubstituted aromatic and substituted heteroaromatic, or a fusion of substituted aromatic and unsubstituted heteroaromatic,

[0186] X and Y are independently absent, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, unsubstituted ether, substituted ether, unsubstituted polyether, substituted polyether, substituted amino, unsubstituted amino, substituted amide, unsubstituted amino, substituted amide, unsubstituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, unsubstituted ether, substituted ether, unsubstituted polyether, substituted polyether, substituted amino, unsubstituted amino, substituted amide, unsubstituted alkylthio, ... acyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted C3-C20 cycloalkyl, unsubstituted C3-C20 cycloalkyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, substituted C3-C20 cycloalkenyl, unsubstituted C3-C20 cycloalkenyl, substituted C3-C20 cycloalkynyl, unsubstituted C3-C20 cycloalkynyl, or a fusion combination thereof,

[0187] E1 and E2 are independently selected electrophiles of substituted carbonyl or unsubstituted carbonyl, such as aldehydes, ketones, Michael acceptors; substituted carboxyl; or unsubstituted carboxyl.

[0188] 2. The method of paragraph 1, wherein the additive comprises a nucleophile, preferably selected from guanidine, urea, thiourea, sodium bisulfite and combinations thereof.

[0189] 3. The method of paragraph 1 or paragraph 2, wherein the concentration of the additive is between about 0.5M and about 10M, between about 1M and about 8M, or between about 3M and about 8M, for example 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M or 10M, preferably 1M, 3M, 6M and 8M.

[0190] 4. The method of any one of paragraphs 1 to 3, wherein the molar ratio of the first reactant to the second reactant is between 1:8 and 8:1, between 1:5 and 5:1, between 1:4 and 4:1, or between 1:3 and 3:1, for example, 1:1.2, 1:1 or 2:1.2.

[0191] 5. The method of any one of paragraphs 1 to 4, wherein the molar ratio of the second reactant to the third reactant is between 1:4 and 4:1 or between 1:3 and 3:1, for example 1:1, 1:1.5, 3:1 or 2:1.

[0192] 6. The method of any one of paragraphs 1 to 5, wherein the second reactant and the third reactant are independently a therapeutic agent, a prophylactic agent, a diagnostic agent or a targeting agent.

[0193] 7. The method described in any one of paragraphs 1 to 6, wherein the second reactant and the third reactant are independently linear peptides, cyclic peptides, non-polymer small molecule drugs (e.g., drugs with a molecular weight between 100 Da and 2,500 Da), fluorophores, chemiluminescent compounds, or a combination thereof.

[0194] 8. The method of any one of paragraphs 1 to 7, wherein the second reactant comprises a thiol group that reacts with the first reactant.

[0195] 9. The method of any one of paragraphs 1 to 8, wherein the second reactant comprises a sterically hindered thiol group that reacts with the first reactant.

[0196] 10. The method of any one of paragraphs 1 to 9, wherein the third reactant comprises an amine group that reacts with the first reactant.

[0197] 11. The method of any one of paragraphs 1 to 10, wherein the first reactant, the second reactant, and the third reactant react to form a conjugate containing a substituted 1-mercaptoisoindole moiety or an unsubstituted 1-mercaptoisoindole moiety.

[0198] 12. The method of any one of paragraphs 1 to 11, wherein the first reactant has the following structure:

[0199]

[0200] in:

[0201] A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted C1-C20 heterocyclic, unsubstituted C1-C20 heterocyclic, or a fusion combination thereof.

[0202] 13. The method of any one of paragraphs 1 to 12, wherein for formula I, A is substituted aromatic or unsubstituted aromatic.

[0203] 14. The method of any one of paragraphs 1 to 13, wherein the first reactant has the following structure:

[0204]

[0205] 15. The method of any one of paragraphs 1 to 14, wherein m and n are 1.

[0206] 16. The method of any one of paragraphs 1 to 15, wherein the first reactant has the following structure:

[0207]

[0208] 17. The method of any one of paragraphs 1 to 16, wherein the first reactant has the following structure:

[0209]

[0210] 18. The method of any one of paragraphs 1 to 17, wherein the reaction is carried out in an aqueous buffer.

[0211] 19. The method of any one of paragraphs 1 to 18, involving mixing the additive with a suitable buffer, such as phosphate buffered saline (PBS), to form a solution before adding the first reactant, the second reactant, and the third reactant.

[0212] 20. The method of any one of paragraphs 1 to 19, comprising adding the first reactant and the second reactant to a solution containing an additive and a suitable buffer before adding the third reactant.

[0213] 21. The method of any of paragraphs 19 or 20, wherein the pH of the buffer is between about 7.0 and about 7.5.

[0214] 22. The method of any one of paragraphs 1 to 21, wherein the reaction is carried out at room temperature, such as between about 23°C and about 37°C, or between about 23°C and about 28°C.

[0215] 23. The method of any of paragraphs 1 to 22, wherein the concentration of the reaction is between about 0.1 mM and about 7.5 mM, about 0.1 mM and about 5 mM, about 0.1 mM and about 2.5 mM, about 0.1 mM and about 2 mM, about 0.5 mM and about 2.5 mM, or about 0.5 mM and about 2 mM.

[0216] 24. A method as described in any one of paragraphs 1 to 23, which involves:

[0217] (i) adding additives to a suitable buffer such as PBS to form a solution, dispersion or suspension,

[0218] (ii) adding the first reactant and the second reactant to the solution, dispersion or suspension of step (i) to form another solution, dispersion or suspension, and

[0219] (iii) adding a third reactant to the solution of step (ii) to form another solution, dispersion or suspension.

[0220] 25. The method of paragraph 24, wherein the solution, dispersion or suspension of step (iii) is maintained at a temperature of about 23°C to about 40°C, between about 23°C and about 28°C, or between about 30°C and about 40°C in air to form the conjugate.

[0221] 26. A conjugate having the following structure:

[0222]

[0223] in:

[0224] R1 to R4 are independently hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted amide, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fused combination thereof,

[0225] R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl, substituted C1-C10 alkenyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted amide alkyl, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof,

[0226] R6 and R7 are independently unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fused combination thereof, provided that R6 and R7 are at least divalent, as permitted by the valence,

[0227] R8 and R9 are independently hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, provided that at least one of R8 and R9 is not hydrogen, such as unsubstituted C1-C10 alkyl, or substituted C1-C10 alkyl,

[0228] PD1 and PD2 independently comprise a peptide, a non-peptide therapeutic agent, a non-peptide diagnostic agent, a non-peptide preventive agent, or a non-peptide targeting agent.

[0229] 27. The conjugate of paragraph 26, wherein R1 to R4 are independently hydrogen, unsubstituted C1-C10 alkyl, or substituted C1-C10 alkyl.

[0230] 28. The conjugate of paragraph 26 or 27, wherein R1 to R4 are hydrogen.

[0231] 29. The conjugate of paragraph 26 or 27, wherein at least one of R1 to R4 independently comprises a targeting agent, a diagnostic agent, a prophylactic agent or a therapeutic agent, preferably the targeting agent comprises a peptide.

[0232] 30. The conjugate of any one of paragraphs 26 to 29, wherein R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl, or substituted C1-C10 alkenyl.

[0233] 31. The conjugate of any one of paragraphs 26 to 30, wherein R6 and R7 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl, provided that R6 and R7 are at least divalent, as permitted by valency.

[0234] 32. The conjugate of any one of paragraphs 26 to 31, wherein R6 and R7 independently comprise a stimulus responsive chemical group, preferably R6 comprises a stimulus responsive chemical group.

[0235] 33. The conjugate of any one of paragraphs 26 to 32, wherein R8 and R9 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl.

[0236] 34. The conjugate of any one of paragraphs 26 to 33, wherein PD1 and PD2 comprise a peptide, preferably an unprotected peptide.

[0237] 35. The conjugate of any one of paragraphs 26 to 34, wherein one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other comprises a non-peptide therapeutic agent, non-peptide diagnostic agent, non-peptide prophylactic agent or non-peptide targeting agent.

[0238] 36. The conjugate of any one of paragraphs 26 to 35, wherein one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other comprises a non-peptide therapeutic agent.

[0239] Example

[0240] Example 1: Efficient synthesis of conjugates containing 1-mercaptoisoindole core using a three-component reaction

[0241] Materials and methods

[0242] All commercially available amino acids and coupling reagents (purchased from Aldrich and CS Bio) were used without further purification. All reagent grade (RCI) or HPLC grade (DUKSAN) solvents were used without purification. Anhydrous dichloromethane (DCM) was freshly distilled from calcium hydride (CaH2) before use. Analytical HPLC was performed on a Waters system equipped with a photodiode array detector (Waters2996) using a Vydac 218TPTM C18 column (5 μm, 4.6×250 mm) at a flow rate of 0.6 mL / min. The Waters UPLC H-class system was equipped with an ACQUITY UPLC photodiode array detector and a Waters SQDetector 2 mass spectrometer, using a Waters ACQUITY BEH C18 column (1.7 μm, 2.1×50mm) at a flow rate of 0.4mL / min. Preparative HPLC was performed on a Waters system using a Vydac 218TPTM C18 column (10μm, 22×250mm) at a flow rate of 10mL / min or a Vydac 218TPTM C18 column (10μm, 30×250mm) at a flow rate of 20mL / min. The mobile phase used in HPLC was as follows: Solvent A: 0.1% TFA (v / v) in acetonitrile; Solvent B: 0.1% TFA (v / v) in water. Mass spectrometry was performed using a Waters 3100 mass spectrometer.

[0243] Solid Phase Peptide Synthesis

[0244] Synthesis was performed manually on rink amide resin (CS Bio) under standard Fmoc protocol. Fmoc protecting groups were removed using a 20 / 80 (v / v) piperidine / DMF mixture for 20 minutes. Coupling was performed using Fmoc-amino acids (4.0 equivalents), HATU (4.0 equivalents) and DIPEA (8.0 equivalents) in DMF at room temperature for 1 hour. Acetylation of the N-terminus of the peptide was performed using AcOH (8.0 equivalents) and DIPEA (16.0 equivalents) in DMF at room temperature for 1 hour. After the synthesis was complete, global deprotection was performed using 95:2.5:2.5 (%) TFA:TIPS:water (v:v:v). The peptide was then precipitated in cold diethyl ether and purified by preparative RP-HPLC.

[0245] General procedure for the three-component reaction of OPA

[0246] Preparation of stock solutions: Stock solutions of OPA (1 mg / 100 μL DMSO) were prepared fresh before use. For OPA derivatives, OPA-fluorescein and OPA-peptide, stock solutions were prepared at 1 mg / 10 μL DMSO. For peptide-drug conjugates, DM1 components were freshly dissolved in 3% ACN, MeOH, and DMSO (total reaction volume), respectively, before use.

[0247] Prepare PBS buffer containing guanidine: Add guanidine hydrochloride (1M to 8M) to PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4). Adjust the pH of the solution to 7.4 by adding 1 M NaOH solution.

[0248] Synthesis of peptide-peptide conjugates

[0249] Condition [A]: Thiol-containing peptide (1.0 eq.) and OPA (1.2 eq.) were first dissolved in PBS buffer at pH 7.4 at a concentration of 2 mM. Amine-containing peptide (1.0 eq.) was then added and reacted in air at room temperature (rt) for 1 hour. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0250] Condition [B]: The thiol-containing peptide (1.0 equiv) and OPA (1.2 equiv) were first dissolved in PBS buffer at pH 7.4 containing guanidine at a concentration of 2 mM. (The guanidine concentration used in the peptide-peptide conjugate model was determined on a case-by-case basis, ranging from 3 M to 8 M, as described in each item below.) The amine-containing peptide (1.0 equiv) was then added and reacted in air at room temperature (rt) for 1 hour. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0251] Synthesis of peptide-drug conjugates

[0252] Condition [C]: DM1 (1.0 eq.) and OPA (1.2 eq.) were first dissolved in PBS buffer pH 7.4 containing 1 M guanidine at a final concentration of 0.5 mM. Then c[RGDfK] or c[RGEfK] peptide (1.5 eq.) was added and reacted in air at room temperature for 1 hour. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0253] Condition [D]: DM1 (1.0 eq.) and OPA (1.2 eq.) were first dissolved in PBS buffer pH 7.4 containing 1 M guanidine at a final concentration of 0.5 mM. Then c[RGDfK] or c[RGEfK] peptide (1.5 eq.) was added and reacted in air at room temperature for 1 hour. Afterwards, guanidine was added to the crude reaction mixture to reach 8 M guanidine buffer, followed by DMAC (2.0 eq.) and reacted for another 1 hour. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0254] Condition [E]: c[RGDfPen] or c[RGEfPen] peptide (1.0 eq.) and OPA (1.2 eq.) were first dissolved in PBS buffer pH 7.4 containing 6 M guanidine at a concentration of 5 mM. H2N-CH2CH2-SS-DM1 (1.0 eq.) was then added and reacted in air at room temperature for 3 hours. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0255] Condition [F]: c[RGDf(K-Pen)] (3.0 equiv) and OPA-fluorescein (3.0 equiv) were first dissolved in PBS buffer pH 7.4 containing 6 M guanidine at a concentration of 1 mM. H2N-CH2CH2-SS-DM1 (1.0 equiv) was then added. The reaction was carried out in air at 37°C for 2 hours. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0256] Condition [G]: c[RGDf(K-Pen)] (2.0 eq.) and OPA-peptide (1.2 eq.) were first dissolved in PBS buffer pH 7.4 containing 6 M guanidine at a concentration of 1 mM. H2N-CH2CH2-SS-DM1 (1.0 eq.) was then added. The reaction was carried out at room temperature in air for 2 hours. The crude reaction mixture was then purified by preparative reverse phase HPLC.

[0257] Synthesis of PPC-2

[0258] The thiol-containing fragment (2.02 mg, 0.0016 mmol, 1.0 equiv) and OPA (0.26 mg, 26 μL stock solution in DMSO, 0.0019 mmol, 1.2 equiv) were first dissolved in PBS buffer pH 7.4 containing 3 M guanidine at a concentration of 2 mM (800 μL). The amine-containing fragment (0.97 mg, 0.0016 mmol, 1.0 equiv) was then added and reacted in air at room temperature for 1 hour. The crude reaction mixture was then purified by preparative reverse phase HPLC (with H2O and ACN containing 0.1% TFA as eluents) to give PPC-2 as a white powder (1.57 mg, 50% yield). Peptide-peptide conjugates were constructed following similar procedures as listed in condition [B], while peptide-drug conjugates were constructed following similar procedures as listed in conditions [C] to [G].

[0259] In vitro cytotoxicity assay

[0260] Cells were seeded in 96-well microplates at a density of 5 x 10^4 cells per well, cultured for 24 hours to adhere to the wall, and then 90 μl of DMEM medium containing 10% FBS and 100U / mL P / S was added to each well. PDCs 1-6 were first dissolved in DMSO at a concentration of 10mM, and then serially diluted in culture medium to reach the specified different concentrations. For U87-MG cells, the concentration range of the administered compounds was 1.37 to 27000nM. For HEK-293T cells, the concentration range of the administered compounds was 0.03 to 1000nM. The treated cells were cultured for another 48 hours. Then 10μl of CCK-8 solution was added to each well and cultured for 30 minutes. The absorbance was obtained at 450nm using a spectrophotometer (Synergy HTX, Biotek). The three experiments were performed in triplicate, and the data were processed using GraphPad Prism (GraphPad Software, La Jolla, CA).

[0261] Fluorescence confocal imaging analysis

[0262] U87-MG cells were seeded in 35 mm glass-bottomed culture dishes at a density of 2 x 10^5 cells per well to adhere to the wall. 1 mL of DMEM medium containing 10% FBS and 100 U / mL P / S was added to the culture dish. After 24 hours of culture, the culture medium was discarded. Freshly prepared DMEM medium (containing 10% FBS and 100 U / mL P / S) containing PDC-7 (final concentration: 10 μM / mL) and DAPI (final concentration: 1 μg / mL) was added to the cells and then the resulting solution was added to the cells and cultured for 15 minutes. Afterwards, the supernatant was discarded and the cells were washed three times with PBS. The cells were supplemented with PBS and fluorescent confocal imaging (Leica DMI3000b) was performed. Images were captured and processed by Leica Application Suit X under ×40 oil lens (Leica).

[0263] result

[0264] A series of compounds that react with aldehyde groups and inhibit side reaction pathways were screened, including guanidine, urea, thiourea, and sodium bisulfite (Table 1). The additives were dissolved in the buffer and adjusted to the desired pH before use. Then, the thiol-containing peptide Ac-QSQQTFSNLWRLLCQN-NH2 (SEQ ID NO: 1) (1.0 equiv) and OPA (1.2 equiv) were dissolved in the specified buffer, followed by the addition of the amine-containing peptide Ac-QSQQTFKNLWRLLPQN-NH2 (SEQ ID NO: 2) (1.0 equiv). The reaction was monitored by LCMS. Among the additives, guanidine showed the best results and exhibited excellent selectivity for the desired conjugate.

[0265] Table 1: Screening conditions for potential compounds

[0266]

[0267] aThe time required for the reaction to complete.

[0268] b Percent conversion calculated from LCMS analysis of the crude reaction mixture. Unless otherwise specified, most by-products were unreacted starting materials.

[0269] c Selectivity for three-component products.

[0270] Additional experiments involved performing the OPA-amine-thiol three-component reaction via synthetic peptide-peptide conjugates (PPCs). To test the chemoselectivity of the approach, a series of thiol-containing unprotected peptides (containing a Cys side chain) and amine-containing unprotected peptides (containing a Lys side chain or the N-terminus) consisting of all proteinogenic amino acids were prepared. The thiol fragment (1.0 equiv) and OPA (1.2 equiv) were first added to PBS buffer containing guanidine, followed by the amine fragment (1.0 equiv). Reactions were performed at a concentration of 2 mM and in air for 1 h at room temperature (RT). Control experiments were performed under the same conditions, but without the addition of guanidine.

[0271] Twelve representative model peptide-peptide conjugates PPC 1-12 were constructed (see Figure 3F-3Q ). Side chain functional groups present in unprotected peptides, including COOH (Glu, Asp), CONH2 (Gln, Asn), OH (Ser, Thr, Tyr), guanidine (Arg), imidazole (His), and S-StBu (Cys) are fully compatible. According to the results, when guanidine was not added, the average ratio of the two-component product to the isolated three-component product was 56:44. When guanidine was added, only the three-component product was isolated, and no two-component product was observed or its amount was too small to be purified by HPLC. Overall, this model study demonstrates the chemical selectivity and robustness of this chemical approach, as well as its ability to serve as a facile method for conjugating modular groups such as unprotected peptides.

[0272] Among the twelve representative entries, most required only 3 M of guanidine to show clear selectivity for the desired three-component product. However, in order to achieve higher reaction selectivity and efficiency, the added guanidine concentration may need to be adjusted due to the different electrophilicity of the imine and the nucleophilicity of the thiol in the intermediate.

[0273] With the successful peptide-peptide conjugate model, it is assumed that this reaction can be further extended to prepare conjugates with active biological properties, such as peptide-drug conjugates. For this purpose, maytansine (DM1) was selected as a cytotoxin. Although DM1 shows high antitumor activity, its clinical application is limited due to its significant systemic toxicity (Perrino et al., Cancer Res. 2014, 74, 2569-2578). To solve this problem, DM1 can be conjugated with a targeting agent. For example, trastuzumab-maytansine (T-DM1), a well-known antibody-maytansine derivative, was approved by the FDA in 2013 for the treatment of HER2-positive metastatic breast cancer (Amiri-Kordestani et al., Clin. Cancer Res. 2014, 20, 4436-4441). In this study, the free thiol group on DM1 makes it suitable for OPA-amine-thiol three-component reaction.

[0274] In addition to drug selection, the choice of therapeutic targets is also a factor to consider. Integrins are a class of cell surface receptors that are involved in a wide range of cell-extracellular matrix interactions, cell-cell interactions, and signal transduction (Hynes, Cell 1992, 69, 11-25; Cabodi, et al., Adv. Exp. Med. Biol. 2010, 674, 43-54). Among the twenty-four subtypes of integrins, αvβ3 has received the most attention for three reasons. First, it was found that integrin αvβ3 was highly overexpressed in clinical glioblastoma and melanoma, thereby distinguishing them from physiological normal cells (Desgrosellier & Cheresh, Nat. Rev. Cancer 2010, 10, 9–22). Second, αvβ3 plays an important role in cancer progression, tumor metastasis, and angiogenesis (Desgrosellier & Cheresh, Nat. Rev. Cancer 2010, 10, 9–22). Third, integrin αvβ3 is highly expressed on activated endothelial cells and new blood vessels, but is absent on most normal organ systems and quiescent endothelial cells (Liu, et al. Drug Dev. Res. 2008, 69, 329-339). These properties make αvβ3 an attractive integrin for tumor cell and tumor vascular therapy.

[0275] The arginine-glycine-aspartate (RGD) cell adhesion sequence was discovered in fibronectin more than two decades ago (Pierschbacher & Ruoslahti, Nature 1984, 309, 30–33; Ruoslahti & Pierschbacher, Science 1987, 238, 491–497). Subsequent studies have shown that it has strong binding affinity and selectivity for integrins, especially the αvβ3 subtype (Chen & Chen, Theranostics 2011, 1, 189–200). At the atomic level, this ligand binds to the primary interface between the αv and β3 subunits, and each residue is extensively involved in this interaction (Xiong, et al., Science 2002, 296, 151–155). Because this motif is highly conserved, it has been used as a carrier for drug delivery in active tumor targeted therapy (Kapp, et al., Sci. Rep. 2017, 7, 39805). A comprehensive study was conducted to optimize the structure of the RGD motif to further enhance its binding to αvβ3 integrin. Among the derivatives studied, the cyclic peptides c[RGDfK] and c[RGDfV] showed 39-fold and 58-fold increases in binding affinity over the linear RGD tripeptide, respectively (Kapp, et al., Sci. Rep. 2017, 7, 39805). The free amine present on the Lys side chain of c[RGDfK] makes it a suitable reaction counterpart for the method studied in this study. Overall, the OPA-c[RGDfK]-DM1 conjugate was used as an ideal test point for the current OPA-peptide-drug conjugation study.

[0276] The improved peptide-peptide conjugate conditions described above were first applied to the synthesis of OPA-c[RGDfK]-DM1, with the reaction conditions reduced to 0.5 mM to ensure complete drug solubility. Under these calibrated conditions, cytotoxin DM1 (1.0 equiv) and the cyclic peptide c[RGDfK] targeting integrin αvβ3 (1.5 equiv) were reacted with OPA (1.2 equiv) in PBS buffer containing 1 M guanidine at a concentration of 0.5 mM. The reaction was completed within 1 hour with excellent selectivity for the three-component conjugate product and generated PDC-1 in a yield of 51% ( Figure 4C ).

[0277] Although the reaction went smoothly, the 1-mercaptoisoindole conjugate obtained after HPLC purification was not stable. The obtained conjugate gradually degraded. The increase in the decomposition rate in acidic medium is consistent with the reported acid-catalyzed hydrolysis of ethylene sulfide (Simons & Johnson, Annals in Biochemistry 1977, 82, 250-254). In addition, the obtained conjugate and the two-component byproducts are susceptible to air oxidation over time (Zuman, Chem. Rev. 2004, 104, 3217-3238).

[0278] To enhance the stability of the conjugate, two methods were tested. In the first method, the isoindole moiety was modified with dimethylacetylene dicarboxylate (DMAC). It has been reported that DMAC-isoindole adducts are more stable than isoindole (Zhang, et al., J.Am.Chem.Soc.2019,141,12274–12279; Simons, et al., J.Org.Chem.1981,46,4739–4744). Therefore, OPA-c[RGDfK]-DM1-DMAC conjugate was prepared under condition [D] (condition [C], one point DMAC (2.0 equivalents), 1 hour), PDC-2 ( Figure 4D ). Compared with PDC-1, PDC-2 showed enhanced stability in aqueous solvents over a wide pH range.

[0279] The second approach was to replace the thiol group in the reaction with a more sterically hindered thiol substrate, under the assumption that the resulting bulkier conjugate might have enhanced stability. Given that the Lys residue in c[RGDfK] was blocked from conjugation, it was replaced with penicillamine with little effect on its integrin targeting ability. Thus, the cyclic peptide c[RGDfPen] was prepared.

[0280] A triggered drug release system was incorporated into the design, with the goal of maximizing the level of free DM1 in the cell and improving its therapeutic effect. This was achieved by using a reduction-sensitive linker based on the highly reducing environment in the cell, with glutathione (GSH) content in the cytoplasm 1000 times higher than in plasma (Meister & Anderson, Annu. Rev. Biochem. 1983, 52, 711–760). In addition, due to hypoxia, the concentration of GSH in cancer cells was found to be higher (Bansal & Simon, J. Cell Biol. 2018, 217, 2291–2298). Redox-sensitive links including disulfide bonds have been used in targeted drug delivery systems (Austin, et al., Proc. Natl. Acad. Sci. USA 2005, 102, 17987–17992; Wang, et al., Curr. Med. Chem. 2012, 19, 2976–2983). By utilizing the free thiol groups on DM1, disulfide bonds and amine groups were incorporated into it for conjugation, thus synthesizing a modified DM1, H2NCH2CH2-SS-DM1.

[0281] The two components of the OPA-amine-3°-thiol reaction were performed. Due to steric hindrance, the reaction efficiency was much lower than the OPA-amine-thiol reaction. Since DM1 is now attached to a free amine and has improved solubility, it is assumed that the reaction can be carried out at higher concentrations. Ultimately, it was observed that more than 90% conversion could be achieved within 3 h at a concentration of 5 mM. Condition [E] (3o-thiol (1.0 eq), OPA (1.2 eq) in PBS buffer pH 7.4 containing 6 M guanidine, 5 mM, room temperature, 3 h) provided the desired product PDC-3 ( Figure 4E ). Purified PDC-3 has improved stability compared to PDC-1 ( Figure 5A Another conjugate utilizing Pen for the reaction was also designed. The goal was to minimize the structural modifications of the c[RGDfK] targeting sequence to fully maintain its activity. The cyclic peptide c[RGDf(K-Pen)] with Pen installed on the Lys side chain was synthesized and PDC-4 ( Figure 4F ).

[0282] For biological evaluation, two conjugates were synthesized as negative controls. A single amino acid mutation Asp->Glu was introduced into the RGD integrin targeting motif, which minimally altered its structure and size. Although Asp and Glu confer similar charges, the RGD tripeptide is very specific in its binding to integrins and does not tolerate structural changes. Therefore, when the RGD motif is subjected to this modification, its targeting properties are completely abolished (Brake, et al., J. Cell Biol. 1990, 111, 1275–1281; Mondal, et al., Biomaterials 2013, 34, 6249–6260). PDC-5 ( ) containing the non-targeting control cyclic peptides c[RGEfK] and c[RGEfPen] were prepared according to conditions [C] (thiol (1.0 eq), amine (1.5 eq), OPA (1.2 eq) in PBS buffer pH 7.4 containing 1 M guanidine, 0.5 mM, room temperature, 1 h) and [E] (3o-thiol (1.0 eq), OPA (1.2 eq) in PBS buffer pH 7.4 containing 6 M guanidine, 5 mM, room temperature, 3 h). Figure 4G ) and PDC-6( Figure 4H Overall, the syntheses of PDCs 1-6 demonstrate the flexibility of the conjugation strategy used in the current study with respect to substrate scope ( Figures 4C-4H ). This reaction is compatible not only with linear peptides but also with cyclic peptides and drugs bearing amine, thiol, or larger 3°-thiol groups.

[0283] Next, the bioactivity of these conjugates was evaluated. U87-MG is reported to express high levels of αvβ3 integrin receptors and has been used as a positive control cell line for various RGD-containing targeted drugs, including Cilengitide (Maurer, et al., Neuro-Oncology 2009, 11, 747–756; Chen and Chen, Theranostics 2011, 1, 189-200). In contrast, the human embryonic kidney cell line HEK-293T, which expresses low levels of αvβ3 integrin, was used as a negative control (Hussain, et al., Nucleic Acid Ther. 2013, 23, 203–212).

[0284] Then, the CCK-8 assay ( Figure 5B) examined the in vitro antitumor effects of PDCs 1-6. In the same cell line, the IC50 of free DM1 and its conjugates were compared to determine the potency of the six compounds. In the tumor model U87-MG cell line, the cytotoxicity of PDC-3 and PDC-4 increased by 5.95-fold and 1.29-fold, respectively, compared with free DM1. These results clearly demonstrated the contribution of three factors to the improved activity: the αvβ3 targeting effect of the RGD peptide, the releasable cytotoxic DM1, and the improved stability of the conjugates. Functional conjugates can be easily constructed by our three-component reaction conjugation method, and the stability improvement promoted by the introduction of 3o-thiol showed superiority over the intolerant isoindole core modification technology. For PDC-1 with a 1-thiol isoindole structure and non-releasable DM1, the cytotoxicity was slightly reduced by 0.85-fold. The decreased activity may be due to the introduction of the RGD cyclic peptide on DM1, which hindered the drug from reaching the cells. For the two negative controls PDC-5 and PDC-6 with mutated RGE targeting motifs, their antitumor effects decreased by 0.49-fold and 0.48-fold, respectively. The least active compound was PDC-2, which showed only 0.09-fold activity.

[0285] The peptide-drug conjugates were tested in negative control cell lines. In HEK-293T cells ( Figure 5C ), the cytotoxicity of the conjugates was reduced, with an activity range of 0.01-fold to 0.41-fold that of DM1. In general, no significant differences were observed in the cytotoxicity of the RGD- or RGE-conjugates. Although PDC-3 and PDC-6, which carry releasable DM1, had relatively good activity among the six compounds, their cytotoxicity was still reduced compared to DM1. PDC-3 and PDC-4, which had the best activity in U87-MG cells, had reduced cytotoxicity to only 0.31-fold and 0.16-fold in αvβ3-negative cell lines, respectively. This result suggests that the targeting peptide is able to specifically deliver the drug to U87-MG cells, which express higher αvβ3 receptors. In addition, PDC-2 also had the lowest cytotoxicity in HEK-293T cells. The combined results suggest that the DMAC-isoindole adduct skeleton may not be the best core structure for biomedical applications.

[0286] In view of the encouraging cytotoxicity test results above, further experiments have adopted other types of modular groups and / or functions into the conjugate. Fluorescent targeting peptide conjugates have been reported as powerful tools for non-invasive imaging in preclinical models (Cheng, et al., Bioconjug. Chemistry 2005, 16, 1433-1441). When the targeting peptide binds to its specific receptor, tumor tissue can be visualized. This imaging method has an impact on studying diseases at the molecular level. In addition, targeted imaging agents have also been used in endocytosis studies (Garanger, et al., Mol. Ther. 2005, 12, 1168-1175).

[0287] These fluorescent and additional targeting compounds were used to further functionalize OPA( Fig. 4I and 4J ). The OPA derivative OPA-CH2-COOH was first coupled to fluorescein amine to generate an OPA-fluorophore linked via an amide bond. The reactions were performed under established conditions [C] (thiol (1.0 equiv), amine (1.5 equiv), OPA (1.2 equiv) in PBS buffer pH 7.4 containing 1 M guanidine, 0.5 mM, room temperature, 1 hour) and [E] (3o-thiol (1.0 equiv), OPA (1.2 equiv) in PBS buffer pH 7.4 containing 6 M guanidine, 5 mM, room temperature, 3 hours). No product was observed. The significantly reduced reaction efficiency may be due to steric hindrance of the OPA derivative and the 3o-thiol group. The hypothesized steric hindrance issue was addressed by adding a PEG3 linker between OPA and fluorescein. Extensive screening showed that further functionalized conjugates could be achieved under heating conditions, while the use of excess reactants could further accelerate the reaction. With the new conditions [F] (3o-thiol (3.0 equiv), amine (1.0 equiv), functionalized OPA-fluorescein (3.0 equiv) in PBS buffer containing 6 M guanidine pH 7.4, 1 mM 37 ° C, 2 h), PDC-7 ( Fig. 4I ). Next, fluorescence confocal imaging was performed on U87-MG cells treated with PDC-7, and the endocytosis of the conjugate was observed. This further functionalized conjugate with OPA-PEG3-fluorescein can be used to study the endocytosis process of the generated drug conjugate. In addition, OPA can be derivatized with near-infrared fluorophores to produce conjugates suitable for in vivo imaging (Cheng, et al., Bioconjug. Chem. 2005, 16, 1433-1441).

[0288] In addition to fluorophores, it was also evaluated whether OPA could be linked to other functional groups. Human epidermal growth factor receptor 2 (HER2) is overexpressed in various human cancers, such as breast cancer and ovarian cancer. HER2 plays an important role in proliferation and anti-apoptotic mechanisms, so it is an important diagnostic and therapeutic target (Tai, et al., J. Control. Release 2010, 146, 264-275). The peptide LTVSPWY identified by phage display technology was reported to be able to bind to the HER2 receptor (Pero, et al., Int. J. Cancer Res. 2004, 111, 951-960). The corresponding OPA-peptide derivatives were synthesized. Compared with the reaction of OPA-fluorescein, OPA-peptide showed better reactivity, probably due to its less crowded structure. The dual targeting peptide conjugate PDC-8 ( Figure 4J ), with a yield of 46%.

[0289] These further functionalized conjugates synthesized via OPA derivatives further expand the chemical scope of the compounds. These examples demonstrate that additional functional groups are highly versatile, with both fluorophores and peptides being compatible with established conjugation reactions. Although additional functional groups may introduce steric hindrance to the dialdehyde, this drawback was overcome by introducing the spacer groups described above. Furthermore, the reaction efficiency could be improved by increasing the number of reactants or heating the reaction to 37°C.

[0290] Summarize

[0291] In summary, the intermolecular OPA-amine-thiol reaction, which has been used for fluorescent detection of α-amino acids, was retested under new conditions. Compared with traditional conditions, which require large amounts of thiol (e.g., 10-3000 equivalents), additives such as guanidine or sodium bisulfite were shown to be effective additives to achieve stoichiometric reactions. This approach provides an efficient strategy for conjugation and to build complex molecular architectures in a modular manner. Moreover, the structural and functional complexity of the resulting conjugates can be further diversified by introducing additional functional groups on OPA.

[0292] A series of conjugates (such as peptide-peptide conjugates and peptide-drug conjugates) have been constructed, including RGD-DM1 conjugates. In addition, it is worth noting that the 1-mercaptoisoindole product generated by the OPA-amine-thiol reaction is not very stable. This problem was solved by using sterically hindered thiol substrates, such as replacing cysteine ​​with penicillamine, or generating 1-mercaptoisoindole cores containing further substituents on the pyrrole ring. From bioactivity studies in U87-MG cells expressing upregulated αvβ3 integrin, two RGD-DM1 conjugates containing 3o-thiol groups and releasable drug designs showed up to 5.95-fold improvement in cytotoxicity compared with free DM1.

[0293] The data show that these reactions are highly chemoselective. Furthermore, the isolated yields of peptide-peptide conjugates and peptide-drug conjugates range from 40-53%. This method is broadly applicable to a variety of substrates, including but not limited to linear or cyclic peptides, drugs, and fluorophores. Finally, by using pre-modified reactants (such as OPA derivatives), the structural and functional complexity of the resulting conjugates can be further diversified.

[0294] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, that there are many equivalents to the specific embodiments of the methods and combinations described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for preparing a conjugate, the method comprising: allowing the first reactant, the second reactant and the third reactant to undergo an intermolecular reaction in the presence of an additive to form a conjugate, preferably the additive inhibiting the formation of a product formed solely by the reaction between the first reactant and the third reactant, in: The first reactant contains the following structure: The second reactant contains a nucleophile denoted as nu1, The third reactant contains a nucleophile denoted as nu2, Preferably, the second reactant and the third reactant are not covalently linked prior to the reaction, nu1 and nu2 are preferably selected from thiol groups, amine groups and sulfide anions, preferably nu1 and nu2 are different, m and n are independently integers from 1 to 5, such as 1, 2, 3, 4 or 5, preferably, m and n are 1, A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof, such as a fusion of substituted aromatic and substituted heteroaromatic, a fusion of substituted aromatic and unsubstituted heteroaromatic, a fusion of unsubstituted aromatic and substituted heteroaromatic, or a fusion of substituted aromatic and unsubstituted heteroaromatic, X and Y are independently absent, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted alkynyl, unsubstituted alkynyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, unsubstituted ether, substituted ether, unsubstituted polyether, substituted polyether, substituted amino, unsubstituted amino, substituted amide, unsubstituted amino, substituted amide, unsubstituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, unsubstituted ether, substituted ether, unsubstituted polyether, substituted polyether, substituted amino, unsubstituted amino, substituted amide, unsubstituted alkylthio, ... acyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted C3-C20 cycloalkyl, unsubstituted C3-C20 cycloalkyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, substituted C3-C20 cycloalkenyl, unsubstituted C3-C20 cycloalkenyl, substituted C3-C20 cycloalkynyl, unsubstituted C3-C20 cycloalkynyl, or a fusion combination thereof, E1 and E2 are independently selected electrophiles of substituted carbonyl or unsubstituted carbonyl, such as aldehydes, ketones, Michael acceptors; substituted carboxyl; or unsubstituted carboxyl.

2. The method according to claim 1, wherein the additive comprises a nucleophile, preferably selected from guanidine, urea, thiourea, sodium bisulfite and combinations thereof.

3. The method according to claim 1 or 2, wherein the concentration of the additive is between about 0.5M and about 10M, between about 1M and about 8M, or between about 3M and about 8M, such as 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M or 10M, preferably 1M, 3M, 6M and 8M.

4. A method according to any one of claims 1 to 3, wherein the molar ratio of the first reactant to the second reactant is between 1:8 and 8:1, between 1:5 and 5:1, between 1:4 and 4:1, or between 1:3 and 3:1, such as 1:1.2, 1:1 or 2:1.

2.

5. The method according to any one of claims 1 to 4, wherein the molar ratio of the second reactant to the third reactant is between 1:4 and 4:1 or between 1:3 and 3:1, such as 1:1, 1:1.5, 3:1 or 2:

1.

6. The method according to any one of claims 1 to 5, wherein the second reactant and the third reactant are independently a therapeutic agent, a prophylactic agent, a diagnostic agent or a targeting agent.

7. The method according to any one of claims 1 to 6, wherein the second reactant and the third reactant are independently linear peptides, cyclic peptides, non-polymer small molecule drugs (e.g., drugs with a molecular weight between 100 Da and 2,500 Da), fluorophores, chemiluminescent compounds, or combinations thereof.

8. A method according to any one of claims 1 to 7, wherein the second reactant comprises a thiol group which reacts with the first reactant.

9. A process according to any one of claims 1 to 8, wherein the second reactant comprises a sterically hindered thiol group which reacts with the first reactant.

10. A method according to any one of claims 1 to 9, wherein the third reactant comprises an amine group which reacts with the first reactant.

11. The method according to any one of claims 1 to 10, wherein the reaction of the first reactant, the second reactant and the third reactant forms a conjugate comprising a substituted 1-thioisoindole structure or an unsubstituted 1-thioisoindole structure.

12. A method according to any one of claims 1 to 11, wherein the first reactant has the following structure: in: A is substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted C1-C20 heterocyclic, unsubstituted C1-C20 heterocyclic, or a fusion combination thereof.

13. The method according to any one of claims 1 to 12, wherein for formula I, A is substituted aromatic or unsubstituted aromatic.

14. A method according to any one of claims 1 to 13, wherein the first reactant has the following structure:

15. The method according to any one of claims 1 to 14, wherein m and n are 1.

16. A method according to any one of claims 1 to 15, wherein the first reactant has the following structure:

17. A method according to any one of claims 1 to 16, wherein the first reactant has the following structure:

18. The process according to any one of claims 1 to 17, wherein the reaction is carried out in an aqueous buffer.

19. A method according to any one of claims 1 to 18, comprising mixing the additive with a suitable buffer, such as phosphate buffered saline (PBS), to form a solution prior to adding the first reactant, the second reactant and the third reactant.

20. A method according to any one of claims 1 to 19 comprising adding the first and second reactants in a solution comprising an additive and a suitable buffer prior to adding the third reactant.

21. The method of claim 19 or 20, wherein the pH of the buffer is between about 7.0 and about 7.

5.

22. A method according to any one of claims 1 to 21, wherein the reaction is carried out at room temperature, such as between about 23°C and about 37°C, or between about 23°C and about 28°C.

23. The method according to any one of claims 1 to 22, wherein the concentration of the reaction is between about 0.1 mM and about 7.5 mM, between about 0.1 mM and about 5 mM, between about 0.1 mM and about 2.5 mM, between about 0.1 mM and about 2 mM, between about 0.5 mM and about 2.5 mM, or between about 0.5 mM and about 2 mM.

24. A method according to any one of claims 1 to 23, comprising: (i) adding additives to a suitable buffer such as PBS to form a solution, dispersion or suspension, (ii) adding the first reactant and the second reactant to the solution, dispersion or suspension of step (i) to form another solution, dispersion or suspension, and (iii) adding a third reactant to the solution of step (ii) to form another solution, dispersion or suspension.

25. The method according to claim 24, wherein the solution, dispersion or suspension of step (iii) is maintained at a temperature of about 23°C to about 40°C, or between about 23°C and about 28°C, or between about 30°C and about 40°C in an air atmosphere to form the conjugate.

26. A conjugate having the following structure: in: R1-R4 are independently hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted amide, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fused combination thereof, R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl, substituted C1-C10 alkenyl, unsubstituted alkoxy, substituted alkoxy, unsubstituted aryloxy, substituted aryloxy, unsubstituted alkylthio, substituted alkylthio, unsubstituted carbonyl, substituted carbonyl, unsubstituted carboxyl, substituted carboxyl, unsubstituted ester, substituted ester, substituted amino, unsubstituted amino, substituted acyl, Amine, unsubstituted amide, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fusion combination thereof, R6 and R7 are independently unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, substituted aromatic, unsubstituted aromatic, substituted heteroaromatic, unsubstituted heteroaromatic, substituted polyaromatic, unsubstituted polyaromatic, substituted polyheteroaromatic, unsubstituted polyheteroaromatic, substituted cycloalkyl, unsubstituted cycloalkyl, substituted cycloalkenyl, unsubstituted cycloalkenyl, substituted cycloalkynyl, unsubstituted cycloalkynyl, substituted C1-C20 heterocyclyl, unsubstituted C1-C20 heterocyclyl, or a fused combination thereof, provided that R6 and R7 are at least divalent, if the valence permits, R8 and R9 are independently hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, provided that at least one of R8 and R9 is not hydrogen, such as unsubstituted C1-C10 alkyl, or substituted C1-C10 alkyl, PD1 and PD2 independently comprise a peptide, a non-peptide therapeutic agent, a non-peptide diagnostic agent, a non-peptide preventive agent, or a non-peptide targeting agent.

27. The conjugate according to claim 26, wherein R1 to R4 are independently hydrogen, unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl.

28. The conjugate according to claim 26 or 27, wherein R1 to R4 are hydrogen.

29. The conjugate according to claim 26 or 27, wherein at least one of R1 to R4 independently comprises a targeting agent, a diagnostic agent, a prophylactic agent or a therapeutic agent, preferably the targeting agent comprises a peptide.

30. The conjugate according to any one of claims 26 to 29, wherein R5 is hydrogen, unsubstituted C1-C10 alkyl, substituted C1-C10 alkyl, unsubstituted C1-C10 alkenyl or substituted C1-C10 alkenyl.

31. The conjugate according to any one of claims 26 to 30, wherein R6 and R7 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl, provided that R6 and R7 are at least divalent, as valency permits.

32. The conjugate according to any one of claims 26 to 31, wherein R6 and R7 independently comprise a stimulus responsive chemical group, preferably wherein R6 comprises a stimulus responsive chemical group.

33. The conjugate according to any one of claims 26 to 32, wherein R8 and R9 are independently unsubstituted C1-C10 alkyl or substituted C1-C10 alkyl.

34. The conjugate according to any one of claims 26 to 33, wherein PD1 and PD2 comprise a peptide, preferably an unprotected peptide.

35. The conjugate according to any one of claims 26 to 34, wherein one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other comprises a non-peptide therapeutic agent, non-peptide diagnostic agent, non-peptide prophylactic agent or non-peptide targeting agent.

36. The conjugate according to any one of claims 26 to 35, wherein one of PD1 and PD2 comprises a peptide, preferably an unprotected peptide, and the other comprises a non-peptide therapeutic agent.

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