CROSS-LINKING COMPOUNDS AND METHODS OF USE THEREOF

FIELD

The present invention concerns cross-linking compounds and methods of use thereof.

BACKGROUND

The construction of nanostructures upon enzymatic action under physiological conditions constitutes a new research arena that might be termed “synthetic chemistry in vivo.” The formation of covalently cross-linked nanostructures in vivo is particularly attractive because the resulting scaffold can be exploited for further reactions such as bioconjugation.

However, new compounds and methods are needed.

SUMMARY

One aspect of the present invention is directed to compounds that can cross-link such as under physiological conditions and/or in vivo. A compound of the present invention may have a structure of Formula IA:

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wherein:

each PG is a protecting group and each protecting group is independently an enzyme labile group (e.g., a glycosyl group, glucoside, glucuronide, galactosyl, phosphate (e.g., a phosphoester group) group, sulfoester group, β-lactam, phosphoramidate, group that is labile to peroxidases, and/or a self-immolative linker);

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—; A is an aryl or heteroaryl that is multivalent (e.g., having a valence of 2, 3, 4, 5, 6, or more);

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each Z is independently an enzyme (e.g., single enzyme nanogel), polyiodide binding matrix (e.g., amylose), targeting agent (e.g., antibody, peptide, receptor, etc.), recognition motif, radionuclide (e.g., iodide), imaging agent (e.g., sonophore, chromophore, phosphor, etc.), water solubilizing group, therapeutic agent, or bioconjugatable group (e.g., azide, hydroxyl, amino, etc.);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG);

n is an integer of 1 to 6;

m is an integer of 1 to 4; and

p is an integer of 0 to 5;

or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed to compound of Formula IB:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

A is an aryl or heteroaryl that is multivalent (e.g., having a valence of 2, 3, 4, 5, 6, or more);

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG);

n is an integer of 1 to 6;

m is an integer of 1 to 4; and

p is an integer of 0 to 5;

or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed to a compound of Formula II:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —O— or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention is directed to a compound of Formula III:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —O— or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed to a compound of Formula IV:

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wherein:

D¹, D², D³, D⁴, D⁵, and D⁶each independently has a structure of Formula C or Formula D:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention is directed to a compound of Formula V:

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wherein:

M is a metal having a valency of greater than 2 (e.g., zinc, palladium, copper, etc.) or is two hydrogensor;

custom-character , in each instance, is a single bond or double bond;

each R²¹, R²², R²³, R²⁴, R²⁶, R²⁷, R²⁹, R³⁰, R³¹, R³², R³⁴, and R³⁵is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido, cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide, urea, alkoxylacylamino, aminoacyloxy, hydrophilic groups, linking groups, surface attachment groups, and targeting groups;

or one or more of R²¹and R²², R²³and R²⁴, R²⁹and R³⁰, and R³¹and R³², together are ═O or spiroalkyl;

or where one or more of R²⁶and R²⁷, R²⁷and R²⁸, R³⁴and R³⁵, and R³⁵and R²⁰together represent a fused aromatic or heteroaromatic ring system;

wherein when custom-character is a double bond R²²and R²³are absent;

wherein when custom-character is a double bond R³⁰and R³¹are absent;

each z is independently an integer of 1 or 2;

L²⁰, L²⁵, L²⁸, and L³³is each independently absent or a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each of R²⁰, R²⁵, R²⁸, and R³³independently has a structure of Formula C or Formula D:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is directed to a method of treating a subject (e.g., a subject having a solid tumor) and/or reducing the size of a solid tumor in a subject, the method comprising: administering a compound of the present invention (e.g., a compound of Formula IA, IB, II, III, IV, and/or V) to the subject, thereby treating the subject and/or reducing the size of the solid tumor in the subject.

A further aspect of the present invention is directed to a method of detecting a cell, tissue, and/or agent (e.g., an infecting agent, etc.) in a subject, the method comprising: administering to the subject a compound of the present invention, optionally wherein the compound associates with the cell, tissue, and/or agent; and detecting the compound or a portion thereof within the subject, thereby detecting the cell, tissue, and/or agent.

A further aspect of the present invention is directed to a method of forming a cross-linked compound, the method comprising: contacting a compound of the present invention and an enzyme, thereby forming the cross-linked compound.

It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination. Applicant reserves the right to change any originally filed claim and/or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and/or incorporate any feature of any other claim or claims although not originally claimed in that manner. These and other objects and/or aspects of the present invention are explained in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing enzyme-triggered reactions of molecule A protected with a protecting group (PG) (A-PG); (i) shows self-assembly of A; (ii) shows hetero-coupling of A with acceptor B; and (iii) shows homo-coupling of A in the presence of O₂according to aspects of the present invention.

FIG. 2 is an axial view of a polyiodide binding matrix comprising a helical structure with axis C.

FIG. 3 is a circumferential view of the polyiodide binding matrix of FIG. 2 along the axis C.

FIG. 4 is an illustration of a polyiodide binding compound according to embodiments of the present invention.

FIG. 5 is a schematic showing formation of indigo (4) from indoxyl β-glucoside (1) via indoxyl (2) and tautomer (3).

FIG. 6 is a schematic showing a general structure of β-indoxyl glucosides linked to a chemical/bioconjugatable handle.

FIG. 7 is an ORTEP drawing of the single-crystal X-ray structures of (panel A) compound 17 and (panel B) compound 18 as described in Example 1. All ellipsoids are contoured at the 50% level.

FIG. 8 shows BCN-dibromoindoxyl 34, which has limited solubility in aqueous buffer.

FIGS. 9A-9C show the results from a study of reaction conditions for the indigoid-forming reaction from indoxyl-glucoside 33 with β-glucosidase from Agrobacterium. FIG. 9A shows the effect of pH on the reaction progress [33 (100 μM), enzyme (200 nM), 0.05 M phosphate buffer, n=3]. FIG. 9B shows the effect of the enzyme concentration [33 (100 μM), 0.05 M phosphate buffer, 2 h, n=3]. FIG. 9C shows the effect of the concentration of 33 [enzyme (200 nM), 0.01 M phosphate buffer (pH 7, 0.05 M NaCl), 2 or 14 h, n=3). Yields were determined by absorption spectroscopy of solubilized indigoid dye.

FIG. 10A shows time course of oligomerization with 46 under reaction conditions listed in entry 5 of Table 2.

FIG. 10B is an optical microscopic image (×40) of the precipitate suspended in H₂O.

FIG. 10C shows the DLS analysis of the precipitate suspended in H₂O.

FIG. 10D shows the absorption spectra (normalized at 637 nm) of the precipitate in DMF/DMSO (9:1) (top line), the extracted supernatant in DMF (middle line), and 43 in DMF (bottom line).

FIG. 10E shows the analytical SEC traces for the supernatant and precipitate samples from 300 μM of 46.

FIGS. 11A-11B show a comparison of ¹H NMR spectra (in DMSO-d₆) with FIG. 11A being the spectra of 33, 46, and the precipitate, and FIG. 11B being the spectra of 46, 43, and the precipitate.

FIG. 12 is an ORTEP drawing of the single-crystal X-ray structure of F-5. All ellipsoids are contoured at the 50% level.

FIG. 13 shows images relating to the oligomerization of compound V upon enzymatic digestion with β-glucosidase. (panel A) Photographs of the reaction samples in a 300-min time course. (panel B) Optical microscopic image (×40) of the precipitate suspended in H₂O. (panel C) DLS analysis of the precipitate suspended in H₂O. (panel D) Absorption spectral MCA of the precipitate. (panel E) Absorption spectral MCA of the supernatant.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”

It will also be understood that, as used herein, the terms “example,” “exemplary,” and grammatical variations thereof are intended to refer to non-limiting examples and/or variant embodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.

The term “about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, “about X” where X is the measurable value, is meant to include X as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein.

Unless indicated otherwise, nomenclature used to describe chemical groups or moieties as used herein follow the convention where, reading the name from left to right, the point of attachment to the rest of the molecule is at the right hand side of the name. For example, the group “alkylamino” is attached to the rest of the molecule at the amino end, whereas the group “aminoalkyl” is attached to the rest of the molecule at the alkyl end.

Unless indicated otherwise, where a chemical group is described by its chemical formula, including a terminal bond moiety indicated by “—” or “— custom-character ”, it will be understood that the attachment is read from the side in which the bond appears. For example, —O-heteroaryl is attached to the rest of the molecule at the oxygen end.

“Alkyl” as used herein alone or as part of another group, refers to a fully saturated straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as a C1-C20 alkyl, and can be substituted or unsubstituted. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. “Loweralkyl” as used herein, is a subset of alkyl, and, in some embodiments, refers to a saturated straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms and that can be substituted or unsubstituted. Representative examples of loweralkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The term “alkyl” or “loweralkyl” is intended to include both substituted and unsubstituted alkyl or loweralkyl unless otherwise indicated and these groups may be substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, heteroaryl, hydroxyl, alkoxy, polyalkoxy such as polyethylene glycol, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(O)_a, haloalkyl-S(O)_a, alkenyl-S(O)_a, alkynyl-S(O)_a, cycloalkyl-S(O)_a, cycloalkylalkyl-S(O)_a, aryl-S(O)_a, arylalkyl-S(O)_a, heterocyclo-S(O)_a, heterocycloalkyl-S(O)_a, amido, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, aminoalkyl, alkylphosphonate, alkylnitrile, acyloxy, ester, amide, sulfonamide, urea, carbamate, carboxylate, alkoxyacylamino, aminoacyloxy, nitro or cyano where a is 0, 1, 2 or 3.

“Alkenyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkenyl 1 to 4 carbon atoms) that includes 1 to 8 double bonds in the normal chain, and can be referred to as a C1-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term “alkenyl” or “loweralkenyl” is intended to include both substituted and unsubstituted alkenyl or loweralkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and loweralkyl above.

“Alkynyl” as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triple bond in the normal chain, and can be referred to as a C1-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term “alkynyl” or “loweralkynyl” is intended to include both substituted and unsubstituted alkynyl or loweralkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.

“Hydrocarbon” as used herein refers to a moiety including carbon and hydrogen that may be substituted or unsubstituted. Exemplary hydrocarbons include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, and aryl groups as defined herein.

“Halo” as used herein refers to any suitable halogen, including —F, —Cl, —Br, and —I.

“Mercapto” as used herein refers to an —SH group.

“Azido” as used herein refers to an —N₃group.

“Cyano” as used herein refers to a —CN group.

“Hydroxyl” as used herein refers to an —OH group.

“Nitro” as used herein refers to an —NO₂group.

“Alkoxy” as used herein alone or as part of another group, refers to an alkyl or loweralkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, —O—. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

“Acyl” as used herein alone or as part of another group refers to a —C(O)R²⁰group, wherein R²⁰is an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl.

“Acyloxy” as used herein alone or as part of another group refers to a —OC(O)R²⁰group, wherein R²⁰is an alkyl, alkenyl, alkynyl, cycloalkyl, or aryl.

“Haloalkyl” as used herein alone or as part of another group, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.

“Alkylthio” as used herein alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a thio moiety, as defined herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

“Cycloalkyl” as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 1 to 20 carbon atoms (optionally with a carbon atom replaced in a heterocyclic group as discussed below). A cycloalkyl group may include 0, 1, 2, or more double or triple bonds. A cycloalkyl may be aromatic. Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may optionally be substituted with additional substituents as described herein such as halo or loweralkyl. The term “cycloalkyl” is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.

“Heterocyclic group” or “heterocyclo” as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic heterocyclo (e.g., heteroaryl) ring systems containing at least one heteroatom in a ring. A heterocyclic group may include 1, 2, 3, 4, 5, 6, or more ring systems and examples include monocyclic heterocycles, bicyclic heterocycles, tricyclic heterocycles, and a tetracyclic heterocycles. Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto, alkyl-S(O)_m, haloalkyl-S(O)_m, alkenyl-S(O)_m, alkynyl-S(O)_m, cycloalkyl-S(O)_m, cycloalkylalkyl-S(O)_m, aryl-S(O)_m, arylalkyl-S(O)_m, heterocyclo-S(O)_m, heterocycloalkyl-S(O)_m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m=0, 1, 2 or 3. Examples of tetracyclic heterocycles include, but are not limited to, tetrapyrroles.

“Aryl” as used herein alone or as part of another group, refers to a monocyclic, carbocyclic ring system or a bicyclic, carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. The term “aryl” is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and loweralkyl above.

“Arylalkyl” as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

“Amino” as used herein means the radical —NH₂.

“Alkylamino” as used herein alone or as part of another group means the radical —NHR⁵⁰wherein R⁵⁰is an alkyl group.

“Ester” as used herein alone or as part of another group refers to a —C(O)OR⁵¹radical, wherein R⁵¹is an alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

“Formyl” as used herein refers to a —C(O)H group.

“Carboxylic acid” as used herein refers to a —C(O)OH group.

“Carboxylic ester” as used herein refers to a —C(O)OR⁵²group, wherein R⁵²is an alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Boronate ester” as used herein refers to a —B(O)OR⁵³group, wherein R⁵³is an alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Phosphate ester” or “phosphoester” as used herein refers to a —P(O)(OR⁵³)₂group, wherein each R⁵³is independently an alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Sulfoester” as used herein refers to a —S(O)₂(OR⁵³) group, wherein R⁵³is an alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Heteroatom” as used herein refers to O, S or N.

“Pharmaceutically acceptable” as used herein means that the compound, anion, cation, or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

As used herein, the terms “increase,” “increases,” “increased,” “increasing,” “improve,” “enhance,” and similar terms indicate an elevation in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more.

As used herein, the terms “reduce,” “reduces,” “reduced,” “reduction,” “inhibit,” and similar terms refer to a decrease in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.

Provided according to embodiments of the present invention are cross-linking compounds and methods of use thereof. A compound of the present invention comprises a cross-linking moiety and a protecting group. The cross-linking moiety may comprise an indoxyl. The protecting group is an enzyme labile group such as, but not limited to, a glycosyl group, glucoside, glucuronide, galactosyl, phosphate (e.g., a phosphoester group) group, sulfoester group, β-lactam, phosphoramidate, group that is labile to peroxidases, and/or a self-immolative linker. In some embodiments, the protecting group may be a group that is cleaved by one or more endogenous enzymes in a subject and/or biological sample such as one or more endogenous enzymes in circulation, extracellular space (e.g., tumor extracellular space), and/or in a lysosome of a cell. Example protecting groups include, but are not limited to, amide groups, galactosyl, phosphate groups (e.g., phosphoester groups), sulfoester groups, glycosyl groups, glucosides, β-lactams, phosphoramidates, glucuronides, groups that are labile to peroxidases, and/or groups that are known as self-immolative linkers. In some embodiments, the protecting group comprises a sugar (e.g., a glucoside or glucuronide), a phosphate (e.g., a phosphoester group), or a sulfur (e.g., a sulfoester group). In some embodiments, the protecting group for a compound of the present invention comprises a glucoside or glucuronide. Such groups can conveniently be attached using standard techniques of bioconjugation such as, e.g., to a hydroxy group or an aldehyde moiety. Removal of a protecting group such as PG as defined in Formula IA (e.g., by native enzymatic action) can reveal one or more cross-linking moieties, which may undergo self-reaction to create a cross-linked compound and/or a deposit comprising the cross-linked compound. In some embodiments, a compound of the present invention can cross-link with itself and/or another compound under physiological conditions and/or in vivo. The cross-linking moiety and protecting group may be attached to each other via an oxygen atom, sulfur atom, or linker. In some embodiments, the linker attaching the cross-linking moiety and protecting group is a self-immolative linker.

In some embodiments, a cross-linking compound of the present invention may have a structure of Formula IA:

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wherein:

each PG is a protecting group and each protecting group is independently an enzyme labile group (e.g., a glycosyl group, glucoside, glucuronide, galactosyl, phosphate (e.g., a phosphoester group) group, sulfur (e.g., a sulfoester group) group, β-lactam, phosphoramidate, group that is labile to peroxidases, and/or a self-immolative linker);

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—; A is an aryl or heteroaryl that is multivalent (e.g., having a valence of 2, 3, 4, 5, 6, or more);

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG);

n is an integer of 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6);

m is an integer of 0 to 4 (i.e., 0, 1, 2, 3, or 4); and

p is an integer of 0 to 5 (i.e., 0, 1, 2, 3, 4, or 5);

or a pharmaceutically acceptable salt thereof.

In some embodiments, in a compound of Formula IA, Z is an enzyme that does not cleave PG and/or X¹.

In some embodiments, a cross-linking compound of the present invention may have a structure of Formula IB:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—; A is an aryl or heteroaryl that is multivalent (e.g., having a valence of 2, 3, 4, 5, 6, or more);

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG);

n is an integer of 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6);

m is an integer of 0 to 4 (i.e., 0, 1, 2, 3, or 4); and

p is an integer of 0 to 5 (i.e., 0, 1, 2, 3, 4, or 5);

or a pharmaceutically acceptable salt thereof.

A compound of the present invention may comprise one or more (e.g., 1, 2, 3, or more) cross-linking unit(s). In some embodiments, a compound of the present invention comprises at least two cross-linking units that are optionally attached via a linker and the compound may further comprise one or more of the following: an enzyme (e.g., single enzyme nanogel), polyiodide binding matrix (e.g., amylose), targeting agent (e.g., antibody, peptide, receptor, etc.), recognition motif, radionuclide (e.g., iodide), imaging agent (e.g., sonophore, chromophore, phosphor, etc.), water solubilizing group, therapeutic agent, bioconjugatable group, and any combination thereof. A crosslinking unit may comprise an indoxyl group. In some embodiments, a crosslinking unit may have a structure of:

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wherein R², R³, and R⁴, are each as defined herein.

In some embodiments, the compound has a structure of Formula IA or IB, A is a triazine, and n+p is an integer of 1, 2, or 3, optionally wherein n is 1, 2, or 3 and p is 2, 1, or 0, respectively. The triazine may be a 1,2,3-triazine, 1,2,4-triazine, or 1,3,5-triazine.

In some embodiments, the compound has a structure of Formula IA or IB, A is a substituted or unsubstituted porphyrin, and n+p is an integer of 1, 2, 3, 4, 5, 6, 7, or 8, optionally wherein n is 1, 2, 3, or 4 and p is 0, 1 or 2. The porphyrin may be a chlorin or bacteriochlorin.

In some embodiments, the compound has a structure of Formula IA or IB, A is a structure of Formula A:

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and n+p is an integer of 1, 2, 3, or 4, optionally wherein n is 1, 2, 3, or 4 and p is 3, 2, 1, or 0, respectively.

In some embodiments, the compound has a structure of Formula IA or IB and A is a structure of Formula B:

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and n+p is an integer of 1, 2, 3, 4, 5, or 6, optionally wherein n is 1, 2, 3, 4, 5, or 6 and p is 5, 4, 3, 2, 1, or 0, respectively.

In some embodiments, R¹in a compound of Formula IB is —CH₂OH. In some embodiments, R¹in a compound of Formula IB is —C(O)OH.

In some embodiments, R²and R³in a compound of Formula IA or IB are each a halogen and R⁴is a hydrogen. In some embodiments, R²and R³in a compound of Formula IA or IB are each bromine and R⁴is a hydrogen.

In some embodiments, X¹in the compound of Formula IA or IB is O. In some embodiments, X¹in the compound of Formula IA or IB is S. In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker.

Exemplarily self-immolative linkers include, but are not limited to, those described in “Self-Immolative Spacers: Kinetic Aspects, Structure-Property Relationships, and Applications,” Ahmed Alouane, Raphaël Labruère, Thomas Le Saux, Frédéric Schmidt, and Ludovic Jullien, Angew. Chem. Int. Ed. 2015, 54, 7492-7509 and “Self-immolative Chemistry in Nanomedicine,” M. Gisbert-Garzarán, M. Manzano, M. Vallet-Regi, Chem. Eng. J. 2018, 340, 24-31. In some embodiments, a self-immolative linker comprises a moiety having the structure

of any one of Formulas E-H:

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wherein:

each X⁵is independently —O— or —S—;

each L⁴is independently absent or a C1-C12 hydrocarbon (e.g., a C1-C12 alkyl);

R¹⁰is H, NH₂, NCH₃, or NO₂;

R¹¹is a C1-C12 hydrocarbon, —O—, or —N(CH₃)—.

In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker having a structure of any one of Formula E-H wherein each X⁵is independently —O— or —S—. In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker having a structure of any one of Formula E-H wherein each X⁵is —O—. In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker having a structure of any one of Formula E-H wherein R^mis hydrogen. In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker having a structure of any one of Formula E-H wherein R¹⁰is NO₂. In some embodiments, X¹in the compound of Formula IA or IB is a self-immolative linker having a structure of Formula E, wherein each X⁵is —O—, R¹⁰is NO₂, and at least one L⁴is a C1-C12 hydrocarbon. In some embodiments, X¹in the compound of Formula IA or IB comprises a self-immolative linker having a structure of:

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Exemplary linkers (such as “L¹”, “L²”, “L³”, “L²⁰”, “L²⁵”, “L²⁸”, and “L³³”) that may be used in a compound of the present invention include, but are not limited to, a hydrocarbon moiety, a peptoid moiety, an amino acid (e.g., lysine) moiety, an oligoethylene glycol group, triazine (e.g., 1,3,5-triazine), 1,3,5-trisubstituted benzene, self-immolative linkers, and/or a polyethylene glycol (PEG) group. A linker may be selected to provide an attachment to another portion of the compound via a carbon-carbon bond or a carbon-heteroatom (e.g., oxygen, sulfur, or nitrogen) bond. In some embodiments, the linker may be a linear or branched hydrocarbon moiety (e.g., an alkyl moiety) and/or a carrier protein. In some embodiments, a linker may be substituted with one or more substituents such as, but not limited to, an unsubstituted or substituted aryl, alkylamino, alkoxy, heterocycle. Further exemplary linkers are shown in Scheme I.

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In some embodiments, the compound of Formula IA or IB comprises a linker comprising a PEG. In some embodiments, the compound of Formula IA or IB comprises a linker comprising —(CH₂CH₂O)_x—, wherein x is an integer of 1, 5, 10, 25, or 50 to 55, 75, or 100. In some embodiments, the compound of Formula IA or IB comprises a linker that is a self-immolative linker, optionally wherein the linker has a structure of any one of Formula E-H. In some embodiments, the compound of Formula IA or IB comprises a linker that is an amino acid moiety such as, e.g., a tyrosine moiety or lysine moiety. A D-amino acid (rather than an L-amino acid) may be used to provide a linker in a compound of the present invention as a D-amino acid moiety may reduce or eliminate inadvertent protease activity.

In some embodiments, L³and/or B in the compound of Formula IA or IB may be absent. In some embodiments, both L³and B in the compound of Formula IA or IB are absent.

In some embodiments, B is present in the compound of Formula IA or IB and is a water solubilizing group. Exemplary water solubilizing groups include, but are not limited to, a phosphoester (phosphate), thiophosphoester (thiophosphate), dithiophosphoester (dithiophosphate), phosphoamidate, thiophosphoamidate, glycoside, glucuronide, peptide, and/or PEG. In some embodiments, the water-solubilizing group is a PEG, optionally having a molecular weight in a range of 100 daltons (Da) to about 300 kDa. In some embodiments, the PEG has a molecular weight of less than 100 Da. In some embodiments, the PEG has a molecular weight of about 44 Da to about 100, 200, or 300 Da. In some embodiments, the PEG has a molecular weight of about 1, 5, or 10 kDa to about 20, 40, 50, 100, 150, 200, 250, or 300 kDa. The PEG may be a PEG having a methyl group at the terminus (referred to herein as m-PEG) and thereby have a —CH₃or —OCH₃at the terminus. In some embodiments, the compound of Formula IA or IB may comprise a PEG (e.g., m-PEG), optionally having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa. In some embodiments, a compound of the present invention comprises a water solubilizing group and the water solubilizing group may increase water solubility and/or modify (e.g., decrease) the clearance rate of the compound in vivo.

In some embodiments, p is at least 1 and m is at least 1 in the compound of Formula IA or IB, thereby at least one Z is present. In the compound of Formula IA or IB, Z may be an enzyme (e.g., single enzyme nanogel), polyiodide binding matrix (e.g., amylose), targeting agent (e.g., antibody, peptide, receptor, etc.), recognition motif, radionuclide (e.g., iodide), imaging agent (e.g., sonophore, chromophore, phosphor, etc.), water solubilizing group, therapeutic agent, or bioconjugatable group (e.g., azide, hydroxyl, amino, etc.). In some embodiments, p is 1 and m is 1, 2, 3, or 4 in the compound of Formula IA or IB. In some embodiments, p is 2 and m is 2, 3, or 4 or in the compound of Formula IA or IB.

Exemplary targeting agents include, but are not limited to, antibodies, peptides, and/or receptors. In some embodiments, the targeting agent is an antibody or fragment thereof, optionally wherein the targeting agent is a monoclonal antibody (mAb) or fragment thereof. Examples of antibody fragments include, but are not limited to, camelid-derived heavy chain antibodies (HCAbs) and the variable domain of the heavy chain antibodies (VHH), also termed nanobodies. The latter are small (about 15 kDa) and may afford better tumor penetration than the larger full antibodies. In some embodiments, a targeting agent (e.g., antibody) may not recognize every tumor cell type, and instead may recognize only a subset (e.g., a subset that is present in every tumor and every metastasis). In some embodiments, the target for a targeting agent (e.g., an antibody) is a glucosidase (e.g., a β-glucosidase) and/or a glucuronidase (e.g., a β-glucuronidase). Binding of the targeting agent and target may allow for the target to maintain its activity. For example, when the target is a glucuronidase the targeting agent may bind the glucuronidase and the glucuronidase may be able to cleave one or more protecting groups from the compound and removal of the protecting groups may cause the compound to crosslink with one or more additional compound(s) of the present invention. In some embodiments, a targeting agent (e.g., antibody and/or nanobody) may be substituted with one or more substituent(s), linker(s), and/or water solubilizing group(s), optionally to modify the water solubility and/or clearance time of the compound. In some embodiments, a targeting agent (e.g., antibody and/or nanobody) may be substituted with one or more PEG group(s), optionally to modify the water solubility and/or clearance time of the compound.

“Dye” and “chromophore” are used interchangeably herein to refer to a luminophore (e.g., a fluorescent and/or phosphorescent molecular entity), sonophore, and/or a non-luminescent molecular entity (e.g., a non-fluorescent and/or non-phosphorescent molecular entity). Exemplary dyes include, but are not limited to, tetrapyrroles; rylenes such as perylene, terrylene, and quarterrylene; fluoresceins such as TET (Tetramethyl fluorescein), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX) and 5-carboxyfluorescein (5-FAM); phycoerythrins; resorufin dyes; coumarin dyes; rhodamine dyes such as 6-carboxy-X-rhodamine (ROX), Texas Red, and N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAN/IRA); cyanine dyes; phthalocyanines; boron-dipyrromethene (BODIPY) dyes; quinolines; pyrenes; acridine; stilbene; as well as derivatives thereof. In some embodiments, the dye is a tetrapyrrole, which includes porphyrins, chlorins, and bacteriochlorins, and derivatives thereof. Chlorins and bacteriochlorins may be regarded as derivatives of porphyrins. Exemplary tetrapyrroles include but are not limited to those described in U.S. Pat. Nos. 6,272,038; 6,451,942; 6,420,648; 6,559,374; 6,765,092; 6,407,330; 6,642,376; 6,946,552; 6,603,070; 6,849,730; 7,005,237; 6,916,982; 6,944,047; 7,884,280; 7,332,599; 7,148,361; 7,022,862; 6,924,375; 7,501,507; 7,323,561; 7,153,975; 7,317,108; 7,501,508; 7,378,520; 7,534,807; 7,919,770; 7,799,910; 7,582,751; 8,097,609; 8,187,824; 8,207,329; 7,633,007; 7,745,618; 7,994,312; 8,278,340; 9,303,165; and 9,365,722; and International Application Nos. PCT/US17/47266 and PCT/US17/63251. In some embodiments, Z and/or B in the compound of Formula IA or IB is a sonophore and/or a compound used in photoacoustic imaging. In some embodiments, Z and/or B in the compound of Formula IA or IB is a copper bacteriochlorin. In some embodiments, Z and/or B in the compound of Formula IA or IB is a luminophore (e.g., a fluorophore or phosphor).

Exemplary enzymes include, but are not limited to, proteins, ribozymes, abzymes, and/or abiological catalysts. In some embodiments, the enzyme may be an enzyme that has activity toward a substrate that is not native in a cell (e.g., a cancer cell). In some embodiments, the enzyme may lack activity toward native substrates in a cell (e.g., a cancer cell) and/or may be heterologous to a subject that the enzyme and/or compound is to be administered to. In some embodiments, the enzyme is a single enzyme nanogel. In some embodiments, the enzyme is an enzyme as described in International Application No. PCT/US19/19090, which is incorporated herein by reference in its entirety.

A “recognition motif” as used herein refers to a molecular entity that can bind to a binding entity such that the two entities have affinity for each other. In some embodiments, the binding of a recognition motif to a binding entity alters the absorption spectrum of a dye and/or turns on fluorescence for a dye. Recognition motifs and binding entities known to those of skill in the art may be used in a compound of the present invention. Exemplary recognition motifs include, but are not limited to, crown ethers, cryptands, pincers, and/or chelating motifs. An example binding entity is a metal ion (e.g., Hg, Cr, Li, etc.). Another exemplary recognition motif and binding entity is an antibody or fragment thereof (e.g., a scFv) and an antigen.

A “radionuclide” as used herein refers to a nuclide that is radioactive. Exemplary radionuclides include, but are not limited to a radioiodide isotope.

“Polyiodide binding matrix” as used herein refers to any compound or moiety that binds a polyiodide. “Polyiodide” as used herein includes iodine (I₂), I_n, wherein n is an integer of 3 to 12 and I_nmay or may not carry a charge such as a −1 or −2 negative charge, a radioiodide isotope, and/or a radical thereof (e.g., I₂^•, I_n^•, I_n^•−, etc.). In some embodiments, “polyiodide” refers to iodide atoms in a linear chain, for example, in the form of: I₃⁻, I₅⁻, I₇⁻, I₉⁻, and mixtures of these species. In some embodiments, polyiodide species may be formed in equilibrium upon reaction of F and molecular iodine, I₂, (e.g., I⁻+I₂→I₃⁻). As one of skill in the art will understand, iodide is simply the monoatomic anion, namely I⁻, but a mixture of iodine (i.e., I₂) and iodide forms multiple species collectively referred to herein as polyiodide, which can be a linear chain of triiodide pentaiodide (I₅⁻), and/or the like. In some embodiments, a polyiodide binding matrix binds and/or sequesters a radioiodide isotope such as ¹³¹I, ¹²³I, ¹²⁴I, and/or ¹²⁵I. In some embodiments, a method of the present invention localizes and/or deposits a compound of the present invention and/or derivative thereof (e.g., the polyiodide binding matrix) in and/or around a tumor, optionally in tumor extracellular space, and provides a bed or matrix for spontaneous sequestration of a radioiodide isotope (e.g., ¹³¹I).

In some embodiments, the polyiodide binding matrix comprises a polysaccharide. The polysaccharide may be a linear polysaccharide and/or a modified polysaccharide. A modified polysaccharide refers to a polysaccharide for which at least one hydrogen or functional group of the native polysaccharide has been substituted. For example, a modified polysaccharide comprises at least one unit (e.g., sugar moiety such as a glucose unit) that comprises a substituent not present in the native polysaccharide. In some embodiments, the polyiodide binding matrix comprises amylose or a derivative thereof, cyclitol, an L-sugar, and/or a non-natural L-sugar. The polyiodide binding matrix (e.g., amylose) may be water-soluble and/or suitable for intravenous injection. In some embodiments, an amylose derivative is a compound in which one or more functional groups have been substituted with a substituent such as an alkyl, alkoxy, acyloxy and/or water-solubilizing group. The polyiodide binding matrix may comprise amylose or a derivative thereof having a 6-turn helix (i.e., 6 glucose units per helical turn). The polyiodide binding matrix may comprise one or more anhydroglucose unit(s). In some embodiments, the polyiodide binding matrix comprises at least one anhydroglucose unit (AGU) comprising a protecting group and cross-linking moiety bound to the AGU via a linker. In some embodiments, an AGU comprises a glucose unit having a structure of:

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A polyiodide binding matrix may comprise one or more groups that aid in increasing the water solubility of the polyiodide binding matrix. For example, in some embodiments, the polyiodide binding matrix may comprise a 1, 2, 3, 4, or more water-solubilizing group(s). In some embodiment, the polyiodide binding matrix comprises a water-solubilizing group that comprises a sulfate, phosphate, PEG, and/or surfactant (e.g., a cationic and/or anionic surfactant) and/or the polyiodide binding matrix has undergone sulfation and/or phosphorylation. In some embodiments, a hydroxy group of the polyiodide binding matrix has been modified to comprise a water-solubilizing group. In some embodiments, a water-solubilizing group may increase water solubility of the compound and/or decrease enzyme (e.g., amylase such as exo-amylases and/or endo-amylases) digestion.

In some embodiments, the polyiodide binding matrix has an average molecular weight from about 5,000 or 10,000 Da to about 25,000, 50,000, 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, or 500,000 Da. In some embodiments, the polyiodide binding matrix has an average molecular weight of about 5,000, 10,000, 15,000, 25,000, 50,000, 75,000, 100,000, 150,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, or 500,000 Da. In some embodiments, the polyiodide binding matrix has an average molecular weight from about 5,000 or 10,000 Da to about 25,000 or 50,000 Da or about 200,000 or 300,000 Da to about 400,000 or 500,000 Da. In some embodiments, the polyiodide binding matrix is polydisperse.

The polyiodide binding matrix may comprise a helical structure as shown in FIG. 2. The helical structure may have a mass per helical turn from about 900 Da to about 1,200 Da. In some embodiments, the helical structure has a mass per helical turn of about 900, 950, 1,000, 1,050, 1,100, 1,150, or 1,200 Da.

The polyiodide binding matrix may have a structure in which it comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) helical turn(s). A “helical turn” as used herein refers to a structure that forms a circle as shown in FIG. 3 when viewed down the helix axis in the direction of C as shown in FIG. 2, even though the beginning and end portions of the structure forming the helix turn are not directly attached to each other. As can be seen in FIG. 2, the helix may comprise at least 4 helical turns. In some embodiments, the polyiodide binding matrix comprises at least 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 helical turns. In some embodiments, the polyiodide binding matrix comprises 1, 5, 10, 15, 20, or 25 to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 helical turns. In some embodiments, the polyiodide binding matrix comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 helical turns.

The polyiodide binding matrix may have a loading capacity of about 1 iodide atom per helical turn. As one of skill of art will understand, a polyiodide binding matrix comprising at least 7 helical turns may have a loading capacity sufficient for the polyiodide species I₇as each of the 7 iodide atoms in I₇may be encompassed by one of the seven helical turns. In some embodiments, the polyiodide binding matrix has a loading capacity of about 1, 5, 10, 15, 20, or 25 iodide atoms to about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 iodide atoms. In some embodiments, the polyiodide binding matrix has a loading capacity of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 iodide atoms.

Referring now to FIG. 4, in some embodiments, a polyiodide binding matrix comprises: one or more unmodified anhydroglucose units (AGUs), wherein the number of unmodified AGUs is ml and ml is an integer of 1 to 20,000; one or more AGUs comprising a —O-L1-X-L2-PG group, wherein L1 is absent or a linker as described herein, X is absent or a cross-linking moiety as described herein, L2 is absent or a linker as described herein, and PG is a protecting group as described herein, wherein the number of AGUs comprising a —O-L1-X-L2-PG group is n1 and n1 is an integer of 1 to 20,000; a modified terminal unit (e.g., a unit comprising an alkyl, alkoxy, acyloxy at the 2-position of the unit); and Q1 wherein Q1 is a cancer targeting agent and/or a circulation enhancing agent. L1 and L2 may be the same or different, and L1, X, and L2 are each independently present or absent. The unmodified AGUs and AGUs comprising a —O-L1-X-L2-PG group may be in any order and are shown for simplicity in a consecutive sequence in FIG. 4. Further, as one of skill in the art will readily recognize, the polyiodide binding matrix shown in FIG. 4 is depicted as a linear structure for simplicity, but the polyiodide binding matrix may be branched. For example, amylose may be mostly linear but slightly branched (e.g., about 1% or less branching points) and amylopectin may have a greater number of branching points than amylose.

“Bioconjugatable group” or “bioconjugate group” and grammatical variations thereof, refer to a moiety and/or functional group that may be used to bind or is bound to a biomolecule (e.g., a protein, peptide, DNA, RNA, polysaccharide, etc.). Thus, “bioconjugatable group” or “bioconjugate group” and grammatical variations thereof do not comprise a biomolecule. However, in some embodiments, a bioconjugatable group is used to bind to a biomolecule, or a bioconjugate group or derivative thereof is bound to a biomolecule (e.g., a protein, peptide, DNA, RNA, polysaccharide, etc.). Exemplary bioconjugatable groups include, but are not limited to, amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, etc.; acids or acid derivatives such as N-hydroxysuccinimide esters (more generally, active esters derived from carboxylic acids, e.g., p-nitrophenyl ester), acid hydrazides, etc.; and other linking groups such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyls, halo groups, biotin, 2-iminobiotin, etc.

Linking groups such as the foregoing are known and described in U.S. Pat. Nos. 6,728,129; 6,657,884; 6,212,093; and 6,208,553. For example, a compound of the present invention may comprise a bioconjugate group that comprises a carboxylic acid and the carboxylic acid may be used for bioconjugation to a biomolecule (e.g., via carbodiimide-activation and coupling with an amino-substituted biomolecule). In some embodiments, a bioconjugatable group comprises an alkyne (e.g., a strained alkyne and/or a functional group used in click chemistry). Exemplary bioconjugatable groups comprising an alkyne include, but are not limited to, alkyne compounds described in Gröst, C. and Berg T., Org. Biomol. Chem., 2015, 13, 3866-3870. In some embodiments, a bioconjugatable group has the structure:

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Provided according to some embodiments is a compound of Formula II:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —O— or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹in a compound of Formula II is —CH₂OH. In some embodiments, R¹in a compound of Formula II is —C(O)OH.

In some embodiments, R²and R³in a compound of Formula II are each a halogen and R⁴is a hydrogen. In some embodiments, R²and R³in a compound of Formula II are each bromine and R⁴is a hydrogen.

In some embodiments, X¹in the compound of Formula II is O. In some embodiments, X¹in the compound of Formula II is S. In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of any one of Formula E-H.

In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of any one of Formula E-H wherein each X⁵is independently —O— or —S—. In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of any one of Formula E-H wherein each X⁵is —O—. In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of any one of Formula E-H wherein R¹⁰is hydrogen. In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of any one of Formula E-H wherein R¹⁰is NO₂. In some embodiments, X¹in the compound of Formula II is a self-immolative linker having a structure of Formula E, wherein each X⁵is —O—, R¹⁰is NO₂, and at least one L⁴is a C1-C12 hydrocarbon. In some embodiments, X¹in the compound of Formula II comprises a self-immolative linker having a structure of:

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In some embodiments, X²in the compound of Formula II is —O— and L¹is a C1-C12 hydrocarbon (e.g., a C1-C12 alkyl).

In some embodiments, X²in the compound of Formula II is absent and L¹is a —CH₂CH₂O—, wherein the oxygen of the —CH₂CH₂O— is bound to the indoxyl ring.

In some embodiments, in the compound of Formula II, X³and/or X⁴is —NH—.

In some embodiments, in the compound of Formula II, L²is an amino acid moiety (e.g., tyrosine moiety, lysine moiety, etc.), optionally wherein the amino acid moiety is a D-amino acid moiety.

In some embodiments, in the compound of Formula II, X⁴is absent and L², Z, L³, and B together have a structure of:

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wherein Z, L³, B, and m are each as defined herein.

In some embodiments, in the compound of Formula II, X⁴is absent and L², Z, L³, and B together have a structure of:

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wherein Z, L³, B, and m are each as defined herein.

In some embodiments, in the compound of Formula II, L²is a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula II, L²is an arylalkyl. In some embodiments, in the compound of Formula II, L²is an -phenyl-C1-C4 alkyl-, optionally -phenyl-(CH₂)₂—.

In some embodiments, in the compound of Formula II, L³is a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula II, L³is an alkylamino substituted with a heteroaryl, optionally wherein the alkylamino is —(CH₂)₄—NH— and the heteroaryl is a triazole, further optionally wherein L³is -1,2,3-triazole-(CH₂)₄—NH—.

In some embodiments, L³and/or B in the compound of Formula II may be absent. In some embodiments, both L³and B in the compound of Formula II are absent.

In some embodiments, B is present in the compound of Formula II and is a water solubilizing group. In some embodiments, the compound of Formula II comprises a PEG (e.g., m-PEG), optionally having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa. In some embodiments, in the compound of Formula II, B is a m-PEG having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa.

In some embodiments, m is 1, 2, 3, or 4 in the compound of Formula II. In some embodiments, m is 1 or 2 in the compound of Formula II.

In some embodiments, the compound of Formula II has a structure of:

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Provided according to some embodiments of the present invention is a compound of Formula III:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —O— or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

In some embodiments, R¹in a compound of Formula III is —CH₂OH. In some embodiments, R¹in a compound of Formula III is —C(O)OH.

In some embodiments, R²and R³in a compound of Formula III are each a halogen and R⁴is a hydrogen. In some embodiments, R²and R³in a compound of Formula III are each bromine and R⁴is a hydrogen.

In some embodiments, in the compound of Formula III, X¹is —O—. In some embodiments, X¹in the compound of Formula III is S. In some embodiments, X¹in the compound of Formula III is a self-immolative linker. In some embodiments, X¹in the compound of Formula III is a self-immolative linker having a structure of any one of Formula E-H.

In some embodiments, in the compound of Formula III, X²is absent.

In some embodiments, in the compound of Formula III, L¹is —CH₂CH₂O—, wherein the oxygen of the —CH₂CH₂O— is bound to the indoxyl ring.

In some embodiments, in the compound of Formula III, X³is —O—.

In some embodiments, in the compound of Formula III, at least one X⁴is —O—.

In some embodiments, in the compound of Formula III, at least one X⁴is —NH—.

In some embodiments, in the compound of Formula III, at least one L²is a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula III, at least one L²is an aryl, optionally wherein L²is a phenyl.

In some embodiments, in the compound of Formula III, at least one L²is —(CH₂CH₂O)_q— that is substituted with an alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclo, or heteroaryl, and q is an integer of 1 to 20, wherein the oxygen of the —(CH₂CH₂O)_q— is bound to the cycloalkyl, heterocycloalkyl, aryl, heterocyclo, or heteroaryl, optionally wherein L²is —(CH₂CH₂O)₅—CH₂CH₂—.

In some embodiments, L³and/or B in the compound of Formula III may be absent. In some embodiments, both L³and B in the compound of Formula III are absent.

In some embodiments, B is present in the compound of Formula III and is a water solubilizing group. In some embodiments, the compound of Formula III may comprise a PEG (e.g., m-PEG), optionally having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa.

In some embodiments, each m is 1, 2, 3, or 4 in the compound of Formula III. In some embodiments, each m is 1 or 2 in the compound of Formula III.

In some embodiments, the compound of Formula III is:

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Provided according to some embodiments is a compound of Formula IV:

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wherein:

D¹, D², D³, D⁴, D⁵, and D⁶each independently has a structure of Formula C or Formula D:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

In some embodiments, in the compound of Formula IV, one, two, three, four, five or six of D¹, D², D³, D⁴, D⁵, and D⁶have a structure of Formula C. In some embodiments, in the compound of Formula IV, four of D¹, D², D³, D⁴, D⁵, and D⁶have a structure of Formula C. In some embodiments, in the compound of Formula IV, D¹, D², D⁵, and D⁶each have a structure of Formula C.

In some embodiments, in the compound of Formula IV, one or two of D¹, D², D³, D⁴, D⁵, and D⁶have a structure of Formula D. In some embodiments, in the compound of Formula IV, two of D¹, D², D³, D⁴, D⁵, and D⁶have a structure of Formula D. In some embodiments, in the compound of Formula IV, D³and D⁴each have a structure of Formula D.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, R¹is —CH₂OH. In some embodiments, in the compound Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, R¹is —C(O)OH.

In some embodiments, in the compound Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, R²and R³are each a halogen and R⁴is a hydrogen. In some embodiments, in the compound Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, R²and R³are each bromine and R⁴is a hydrogen.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, X¹is —O—. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, is —S—. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, X¹is a self-immolative linker. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, and D⁶has a structure of Formula C and, in Formula C, X¹is a self-immolative linker having a structure of any one of Formula E-H, optionally as described herein.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, X²is absent.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, L¹is a —(CH₂CH₂O)_q—, wherein the last oxygen of the —(CH₂CH₂O)_q— is bound to the indoxyl ring, and wherein q is an integer of 1 to 20, 24, 28, 30, or more, optionally wherein q is 3.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula C and, in Formula C, X³is —O—.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, X⁴is —O—. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, X⁴is —NH—.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, L³and/or B is absent. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, both L³and B are absent.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, B is a water solubilizing group. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, B comprises a PEG (e.g., m-PEG), optionally having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, L²is a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, L²is an aryl, optionally wherein L²is a phenyl.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, L²is —(CH₂CH₂O)_q— that is substituted with an alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclo, or heteroaryl, and q is an integer of 1 to 20, wherein the oxygen of the —(CH₂CH₂O)_q— is bound to the cycloalkyl, heterocycloalkyl, aryl, heterocyclo, or heteroaryl, optionally wherein L²is —(CH₂CH₂O)₅— CH₂CH₂—.

In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, m is 1, 2, 3, or 4. In some embodiments, in the compound of Formula IV, at least one of D¹, D², D³, D⁴, D⁵, and D⁶has a structure of Formula D and, in Formula D, m is 1 or 2.

In some embodiments, the compound of Formula IV has a structure of:

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Provided according to some embodiments of the present invention is a compound of Formula V:

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wherein:

M is a metal having a valency of greater than 2 (e.g., zinc, palladium, copper, etc.) or is two hydrogens;

custom-character , in each instance, is a single bond or double bond;

or one or more of R²¹and R²², R²³and R²⁴, R²⁹and R³⁰, and R³¹and R³², together are ═O or spiroalkyl;

or where one or more of R²⁶and R²⁷, R²⁷and R²⁸, R³⁴and R³⁵, and R³⁵and R²⁰together represent a fused aromatic or heteroaromatic ring system;

wherein when custom-character is a double bond R²²and R²³are absent;

wherein when custom-character is a double bond R³⁰and R³¹are absent;

each z is independently an integer of 1 or 2;

L²⁰, L²⁵, L²⁸, and L³³is each independently absent or a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each of R²⁰, R²⁵, R²⁸, and R³³independently has a structure of Formula C or Formula D:

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wherein:

each R¹is independently —CH₂OH or —C(O)OH;

each R²is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R³is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each R⁴is independently selected from a halogen, alkyl, alkenyl, alkynyl, —OH, alkoxy, acyloxy, carboxy, carboxylic ester, boronate ester, thioalkoxy, and amino;

each X¹is independently —O—, —S—, or a self-immolative linker;

each X²is independently absent or —NH—, —O—, or —S—;

each L¹is independently a linker (e.g., a hydrocarbon or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each X³is independently absent or —NH—, —O—, or —S—;

each X⁴is independently absent or —NH—, —O—, or —S—;

each L²is independently absent or a linker (e.g., an amino acid (e.g., a D-amino acid), hydrocarbon, or polymer such as polyethylene glycol (PEG) each of which may be unsubstituted or substituted);

each L³is independently absent or a linker (e.g., a hydrocarbon or polymer such as PEG each of which may be unsubstituted or substituted);

each B is independently absent or a water solubilizing group (e.g., a PEG); and

m is an integer of 1 to 4;

or a pharmaceutically acceptable salt thereof.

In some embodiments, in the compound of Formula V, M is two hydrogens with each hydrogen only attached to a nitrogen, and the compound is the free base. In some embodiments, in the compound of Formula V, M is a metal having a valency of greater than 2 (e.g., zinc, palladium, copper, etc.). As one of skill in the art will understand, an apical ligand can provide any the charge balance; for example, when the metal is X—In (III), where X is chloride or hydroxyl. In some embodiments, M is zinc and the compound can fluoresce. In some embodiments, M is palladium and the compound can phosphoresce. In some embodiments, M is copper and the compound can be used in imaging (e.g., photoacoustic imaging).

In some embodiments, in the compound of Formula V, one, two, three, or four of R²⁰, R²⁵, R²⁸, and R³³independently have a structure of Formula C. In some embodiments, in the compound of Formula V, two of R²⁰, R²⁵, R²⁸, and R³³independently have a structure of Formula C. In some embodiments, in the compound of Formula V, R²⁵and R³³have a structure of Formula C and optionally z is two and L²⁵and L³³are each independently a linker.

In some embodiments, in the compound of Formula V, one or two of R²⁰, R²⁵, R²⁸, and R³³have a structure of Formula D. In some embodiments, in the compound of Formula V, two of R²⁰, R²⁵, and R³³have a structure of Formula D, optionally wherein R²⁰and R²⁸have a structure of Formula D and z is one.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, R¹is —CH₂OH. In some embodiments, in the compound Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, R¹is —C(O)OH.

In some embodiments, in the compound Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, R²and R³are each a halogen and R⁴is a hydrogen. In some embodiments, in the compound Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, R²and R³are each bromine and R⁴is a hydrogen.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X¹is —O—. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X¹is —S—. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X¹is a self-immolative linker. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X¹is a self-immolative linker having a structure of any one of Formula E-H, optionally as described herein.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X²is —O—.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, L¹is a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, L¹is a —C(O)NH(CH₂CH₂O)_q—CH₂CH₂—, wherein q is an integer of 1 to 20, optionally wherein q is 2.

In some embodiments, in the compound of Formula V, one, two, three, or four of L²⁰, L²⁵, L²⁸, and L³³is absent. In some embodiments, in the compound of Formula V, two of L²⁰, L²⁵, L²⁸, and L³³are absent, optionally wherein L²⁰and/or L²⁸are absent.

In some embodiments, in the compound of Formula V, one, two, three, or four of L²⁰, L²⁵, L²⁸, and L³³is independently a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula V, two of L²⁰, L²⁵, L²⁸, and L³³are each independently a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula V, L²⁵and/or L³³is each independently a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate.

In some embodiments, in the compound of Formula V, L²⁵and L³³are each independently a cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, or heteroaryl, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula V, L²⁵and L³³have a structure of:

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wherein R is R²⁵for L²⁵and R³³for L³³, with R²⁵and R³³as defined above and z is two.

In some embodiments, in the compound of Formula V, L²⁰and L²⁸are each independently a C1-C12 alkyl, C1-C12 alkenyl, C1-C12 alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, alkylaryl, heterocyclo, heteroaryl, alkylamino, aminoalkyl, alkylphosphonate, alkylnitrile, optionally substituted with an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkylaryl, heterocyclo, heteroaryl, alkylamino, amido, alkoxy, halo, hydroxyl, carbamate, or carboxylate. In some embodiments, in the compound of Formula V, L²⁰and/or L²⁸comprises —C(O)HNCH₂CC—.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula C, X³is absent.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula C and, in Formula D, X⁴is absent.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, L³and/or B is absent. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, both L³and B are absent.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, B is a water solubilizing group. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, B comprises a PEG (e.g., m-PEG), optionally having a molecular weight in a range of about 100 daltons (Da) to about 300 kDa.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, L³and B are each absent and L²has a structure of:

embedded image

wherein Z and m are as defined herein.

In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, L³, B, and X⁴are each absent and L²has a structure of:

embedded image

wherein Z and m are as defined herein.

In some embodiments, in the compound of Formula V, L²⁰and/or L²⁸is absent and R²⁰and/or R²⁸is Formula D wherein L³, B, and X⁴are each absent and L²and Z have a structure of:

(Z)_m—C(O)HNCH₂CC—,

wherein Z and m are as defined herein.

In some embodiments, in the compound of Formula V, at least one of L²⁰, L²⁵, L²⁸, and L³³has a structure of:

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In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, m is 1, 2, 3, or 4. In some embodiments, in the compound of Formula V, at least one of R²⁰, R²⁵, R²⁸, and R³³has a structure of Formula D and, in Formula D, m is 1 or 2.

In some embodiments, a compound of Formula V has a structure of:

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In some embodiments, a compound of the present invention is a crosslinked compound that optionally has a structure of Formula VIa, VIb, VIa′ or VIb′:

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wherein R², R³, and R⁴are each as defined herein.

According to some embodiments provided is a method of using a compound of the present invention, optionally to form a cross-linked compound. In some embodiments, a method of using a compound of Formula IA, IB, II, III, IV, or V to form a cross-linked compound is provided. The cross-linked compound may be a cross-linked deposit. The cross-linked compound and/or cross-linked deposit may have a structure of Formula VIa, Formula VIb, Formula VIa′, or Formula VIb′. A cross-linked compound of Formula VIa, Formula VIb, Formula VIa′, or Formula VIb′ may be formed by two compounds of the present invention via reaction of the indoxyl unit of each compound. For example, upon removal of a protecting group of a compound of Formula IA, IB, II, III, IV, or V the indoxyl unit may be available to cross-link with the indoxyl unit of another compound of the present invention.

In some embodiments, the cross-linked compound comprises an enzyme, polyiodide binding matrix, targeting agent, recognition motif, radionuclide, imaging agent, water solubilizing group, therapeutic agent, and/or bioconjugatable group. In some embodiments, the cross-linked compound comprises a radionuclide and water solubilizing group. In some embodiments, the cross-linked compound comprises a targeting agent and therapeutic agent. In some embodiments, the cross-linked compound comprises an imaging agent. In some embodiments, the cross-linked compound comprises a radionuclide that may be used for therapy and/or imaging. In some embodiments, the cross-linked compound is formed and/or deposited at a site for imaging and/or for delivery of a therapeutic agent and/or radionuclide.

A compound of the present invention may be contacted with an enzyme that may cleave or remove a protecting group and/or linker (e.g., a self-immolative linker). In some embodiments, a compound of the present invention may be contacted with an enzyme that may cleave or remove a portion of a compound of Formula IA, IB, II, III, IV, or V. The portion of the compound that may be cleaved may be the protecting group (e.g., sugar portion such as a glucuronide or glucoside) and/or a linker (e.g., a self-immolative linker). Enzymes that may cleave and/or remove the sugar portion and/or linker from the compound include, but are not limited to, phosphatases, sulfatases, glucosidases, galactosidases, galacturonidases, and/or glucuronidases. In some embodiments, a glucuronidase (e.g., a β-glucuronidase) may enzymatically cleave a compound of Formula IA, IB, II, III, IV, or V comprising a glucuronide. In some embodiments, a glucosidase (e.g., a β-glucosidase) may enzymatically cleave a compound of Formula IA, IB, II, III, IV, or V comprising a glucoside. In some embodiments, a compound of Formula IA, IB, II, III, IV, or V comprises an enzymatically cleavable group that can be cleaved by an enzyme that is present at a concentration in tumor extracellular space that is greater than the concentration of the enzyme in extracellular space of non-cancerous cells. In some embodiments, a compound of Formula IA, IB, II, III, IV, or V may be enzymatically cleaved by a glucosidase (e.g., a β-glucosidase) and/or a glucuronidase (e.g., a β-glucuronidase).

As used herein “contact”, “contacting”, “contacted,” and grammatical variations thereof, refer to bringing two or more materials (e.g., composition(s), enzyme(s), and/or compound(s), etc.) together in sufficient proximity such that, under suitable conditions, a desired reaction can be carried out (e.g., cross-linking a compound of the present invention). Contacting the two or more materials may be carried out by adding, administering, combining, pouring, spraying, mixing, flowing, injecting, and/or the like the two materials or a portion thereof together. For example, contacting may comprise placing a compound of the present invention in contact with an enzyme, which may cause a compound of the present invention to cross-link and/or form a cross-linked compound. The compound may cross-link with itself (e.g., two or more cross-linking units of the compound may cross-link) and/or the compound may cross-link with another compound of the present invention (e.g., a cross-linking unit of a first compound may cross-link with a cross-linking unit of a second compound). In some embodiments, a compound of the present invention is administered to a subject and a native enzyme aids in cross-linking the compound.

A compound of the present invention may be water-soluble and/or may comprise one or more (e.g., 1, 2, or more) bioconjugatable groups. In some embodiments, a compound of the present invention comprises a 4,6-dibromo-substituted indoxyl unit and may provide an indigoid chromophore. One or more cross-linking units of the present invention may be linked and/or attached using a propargyloxy and/or PEG-O— on the 5-position of the indoxyl unit. A compound of the present invention may comprise a triazine. In some embodiments, a cross-linked compound of the present invention may be enzymatically triggered (e.g., using a glucosidase) and/or may cross-link under physiological conditions. The cross-linking may be bioorthogonal to the two bioconjugatable groups. A biomolecule may be attached before and/or after formation of a cross-linked compound and/or may be attached via standard bioconjugation (including click chemistry). In some embodiments, a compound of the present invention provides a means for creating a stabilized matrix of biomolecules including enzymes and/or recognition motifs including polyiodide binding matrixes.

According to some embodiments of the present invention provided is a method of treating a subject (e.g., a subject having a solid tumor) and/or reducing the size of a solid tumor in a subject, the method comprising: administering a compound of Formula IA, IB, II, III, IV, and/or V to the subject, thereby treating the subject and/or reducing the size of the solid tumor in the subject. In some embodiments, a compound of Formula IA, IB, II, III, IV, and/or V comprises a therapeutic agent and/or radionuclide and is administered to a subject, wherein the compound is delivered to a tumor and immobilizes the therapeutic agent and/or radionuclide in and/or around the tumor. In some embodiments, a subject may be treated with a single radiolabeled compound. Administration of the compound may be chronically or intermittently over 1, 2, 3, 4, 5, 6, 7, or more days to about 1, 2, 3, 4, or more weeks. In some embodiments, the compound may be administered in a manner to allow the compound and/or therapeutic agent and/or radionuclide to accumulate in and/or around a tumor mass. The compound may localize in an area where both an enzyme that can cleave a protecting group of the compound and the target of the compound are present.

In some embodiments, a method of detecting a cell, tissue, and/or agent (e.g., an infecting agent, etc.) is provided, the method comprising: contacting the cell, tissue, and/or agent with a compound of Formula IA, IB, II, III, IV, and/or V, optionally wherein the compound associates with the cell, tissue, and/or agent; and detecting the compound or a portion thereof, thereby detecting the cell, tissue, and/or agent. In some embodiments, a method of detecting a cell, tissue, and/or agent (e.g., an infecting agent, etc.) in a subject is provided, the method comprising: administering to the subject a compound of the present invention, optionally wherein the compound associates with the cell, tissue, and/or agent; and detecting the compound or a portion thereof within the subject, thereby detecting the cell, tissue, and/or agent. In some embodiments, a compound of Formula IA, IB, II, III, IV, and/or V is used for laser-guided surgery. In some embodiments, when a compound of Formula IA, IB, II, III, IV, and/or V comprises a recognition motif and dye, the recognition motif upon binding to a binding entity may cause a shift in the absorption spectrum for the dye. In some embodiments, a compound of the present invention is used as a histological stain.

In some embodiments, provided is a method of using a compound of the present invention in photoacoustic imaging. According to some embodiments, a method of the present invention comprises a method of performing photoacoustic imaging. Photoacoustic imaging (PAI) is attractive in not relying on optical emission for detection (Haisch, C., Quantitative analysis in medicine using photoacoustic tomography. Anal. Bioanal. Chem. 2009, 393, 473-479; Cox, B.; Laufer, J. G.; Arridge, S. R.; Beard, P. C. Quantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Optical emission can be affected by light-scattering. In PAI, laser irradiation (e.g., optionally carried out with non-ionizing laser pulses) is followed by thermoelastic expansion and an ultrasonic pressure wave. Detection of the ultrasonic pressure wave can be achieved via a conventional ultrasound detector. In essence, ultrasound imaging can be carried out with laser input. It is noteworthy that in contrast to X-ray imaging methods, PAI does not rely on ionizing radiation.

A method of the present invention may comprise administering a compound of the present invention to a subject, optionally wherein the compound associates with a tissue and/or cell in the subject; irradiating at least a portion or part of the subject using a laser, optionally wherein the portion or part of the subject contains the compound of the present invention; and imaging at least the portion or part of the subject, optionally wherein the imaging comprises ultrasound imaging.

In some embodiments, a radiotherapy method is provided herein. With radiotherapy only a tiny fraction of the cells needs to be reached given the bystander effect of radiotherapeutic cell killing. Hence, a targeting mechanism that is selective for cancer versus normal cells, but non-globally active against all cancer cell types (e.g., which may be achieved by a compound of the present invention), still affords a viable mechanism for treating cancer. For example, if 1 out of 100 cells is reached, and such “seeding” cells are uniformly distributed across the tumor, then each such seeding cell will have on average ˜4.6 cells (cube root of 99) along the axis in every direction in a spherical volume. The radiation required to eradicate all cells must be sufficient to reach a handful of cell diameters; for a typical cell diameter of 10 microns, this equates to up to ˜100 microns.

In some embodiments, a cell surface receptor, unique to a tiny fraction of cells in a tumor, would suffice for a viable radiotherapeutic approach. Hence, the targeting approach may avoid a generic entity such as transferrin even if taken up with 5-10 times the avidity by all cancerous cells versus all normal cells. Instead, the unique marker, even if rare, is useful. In contrast, in chemotherapy, one absolutely needs to hit every single cancerous cell, to the extent selectivity versus normal cells can be achieved. With chemotherapy, global targeting of cancer takes precedence over selectivity of cancer versus normal. A compound of the present invention, in some embodiments, may target cancer cells with absolute selectivity versus normal cells, even though this may represent only a fraction of the cancer cells.

In a molecular brachytherapy approach, selectivity takes precedence over broad targeting. Briefly, the self-amplifying molecular brachytherapy approach entails a sequential process. The deposition of the radiolabeled compound selectively in the extracellular space of the tumor localizes a radiation field that results in indiscriminate killing of cells in the vicinity. Such indiscriminate cell killing results in lysis of cells, releasing additional enzyme; also, neutrophils and other cells are recruited or attracted to the necrotic space, and such cells may release enzymes. The additional enzyme causes additional deposition of radiolabeled compound that in turn spurs the autocatalytic process of cell killing, enzyme release, radiodeposit accumulation, and so forth.

In some embodiments, a compound of the present invention is used as a contrast agent in PAI and/or comprises a dye that can be used as a contrast agent in PAI. Example dyes for use in PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconducting polymers, and naphthalocyanines (Chitgupi, U.; Lovell, J. F. Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging. Biomed. Eng. Lett. 2018, 8, 215-221; de la Zerda, A., et al., Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 2011, 6, 346-369). In some embodiments, a compound of the present invention comprises a tetrapyrrole macrocycle (e.g., a chlorin, bacteriochlorin, etc.) or a phthalocyanine. In some embodiments, a compound of the present invention comprises a porphyrin. In some embodiments, a compound of the present invention comprises a sonochrome (see, e.g., Duffy, M. J., et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49-61).

An established approach for radiodiagnosis relies on decay of ^99mTc. The ^99mTc isotope decays exclusively by gamma emission, which can be readily detected. Radioiodide decays by parallel pathways: gamma ray and beta particle; the former enables radiodetection but no therapy whereas the latter causes therapeutic benefit by cell killing. The ^99mTc decay by gamma ray emission (140 keV, 98.6%, 142.7, 1.4%; see Dilworth, J. R.; Pascu, S. I., The Radiopharmaceutical Chemistry of Technetium and Rhenium. In The Chemistry of Molecular Imaging, 1st ed.; Long, N.; Wong, W.-T., Eds. John Wiley & Sons: UK, 2015; pp 137-164) is only suitable for radioimaging, not therapy. The decay of ^99mTc (140 keV with t_1/2=6.0 h) can be compared with that of ¹³¹I (t_1/2=8.0 d), which has several gamma emission energies, chiefly of 364 keV. In some embodiments, a compound of the present invention may comprise ¹³¹I given the strong cell-killing effect and convenient half-life, although other isotopes of iodide can be employed (e.g., ¹²⁵I)¹³¹I is chiefly used for therapy whereas ¹²⁵I is chiefly used for diagnostics and imaging. Radioiodide decays by parallel pathways (gamma ray and beta particle; the former enables radiodetection but no therapy whereas the latter causes therapeutic benefit by cell killing). ^99mTc decays entirely by gamma emission. Gamma emission from ^99mTc and radioiodide are at distinct energies.

Several types of ligands have been developed for binding ^99mTc. A key requirement is that the ligand must be prepared and incorporated into the architecture in the absence of the radionuclide ^99mTc. The rationale is that the ^99mTc should be incorporated in the last step, and without purification other than simple extraction or chromatography, given the short half-life of the radionuclide. Additional requirements here are stability of the coordination complex under physiological conditions and inertness toward metabolism. In some embodiments, a ligand for ^99mTc or stable nuclide rhenium (Re) may have a structure of any one of:

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As shown above, ligand L-1 for ^99mTc or stable nuclide Re can be readily synthesized and contains a carboxylic acid as a bioconjugable handle (Barandov, A., et al., ChemBioChem 2014, 15, 986-994). Ligand L-2 for ^99mTc or stable nuclide Re contains a triazole unit, which is readily prepared by click chemistry from an R¹—N₃(azide) and an alkyne, and also contains an alkyl amine as a bioconjugable handle (Romhild, K.; Fischer, C. A.; Mindt, T. L., ChemMedChem 2017, 12, 66-74). Ligand L-3 for ^99mTc or stable nuclide Re contains two triazoles, which can be prepared sequentially via click chemistry hence R¹= or ≠R², which affords versatility in design, and also contains an alkyl amine (or carboxylic ester) as a bioconjugable handle (Mindt, T. L., et al., ChemMedChem 2009, 4, 529-539). The strategy of “click-to-assemble” the ligand to the targeted biomolecule or biomedical construct has been developed by the group of Mindt (Mindt, T. L., et al., ChemMedChem 2010, 5, 2026-2038). Ligand L-4 for ^99mTc or stable nuclide Re is representative of a relatively large family of so-called “scorpionate” ligands, which typically contain 3 pincer arms attached to a tetrahedral center, here shown as carbon but boronate has been explored extensively (Martini, P., et al., Molecules 2018, 23, 2039). The tetrahedral center in the scorpionate ligands provides a convenient site for elaboration of a bioconjugatable handle, here simply illustrated as R′. Other ligands for ^99mTc in various oxidation states that can be used in a compound of the present invention are available as described in a recent review (Dilworth and Pascu, 2015).

In some embodiments, a method of forming a cross-linked compound is provided, the method comprising: contacting a compound of the present invention and an enzyme, thereby forming the cross-linked compound. The enzyme may be a glucosidase (e.g., a β-glucosidase) and/or a glucuronidase (e.g., a β-glucuronidase). In some embodiments, the cross-linked compound is formed in a subject. The subject may be a mammal and, in some embodiments, is a human. In some embodiments, the cross-linked compound is formed in vitro.

The present invention is explained in greater detail in the following non-limiting examples.

EXAMPLES
Example 1

Here, an enzymatically triggered “click reaction” has been developed by exploiting the indigo-forming reaction from indoxyl β-glucoside. The covalent cross-linking proceeds in aqueous solution, requires the presence only of an oxidant (e.g., O₂), and is readily detectable owing to the blue color of the resulting indigoid dye. To achieve facile indigoid formation in the presence of a bioconjugatable tether, diverse indoxyl β-glucosides were synthesized and studied in enzyme assays. The latter include glucosidases from two sources; tritosomes; and rat liver homogenates. Altogether 35 new compounds including 17 new glycosyl-indoxyl compounds were prepared and fully characterized in the course of meeting four essential requirements: enzyme triggering, facile indigoid dye formation, bioconjugatability, and synthetic accessibility. The 4,6-dibromo motif in a 5-alkoxy-substituted indoxyl-glucoside was a key design feature for fast and high-yielding indigoid formation. Two attractive molecular designs include (1) an indoxyl-glucoside linked to a bicyclo[6.1.0]nonyl (BCN) group for Cu-free click chemistry, and (2) a bis(indoxyl-glucoside). In both cases the intervening linker between the reactive moieties is composed of a two short PEG groups and a central triazine derivatized with a sulfobetaine for water solubilization. Glucosidase treatment of the bis(indoxyl-glucoside) in aqueous solution gave oligomers that were characterized by absorption, optical, and ¹H NMR spectroscopy; mass spectrometry; dynamic light-scattering; and HPLC. Key attractions of indigoid dye formation, beyond enzymatic triggering under physiological conditions without exogenous catalysts or reagents, are the chromogenic readout and compatibility with attachment to diverse molecules.

Enzyme-triggered reactions, where a spontaneous chemical reaction follows an enzymatic process, are of great interest in the life sciences particularly for therapeutic and diagnostic applications.^1-4As a general strategy, a molecule to be released (A) upon action by the target enzyme is protected with a covalently attached enzyme-cleavable ligand PG (A-PG) (FIG. 1). This general description encompasses yet extends far beyond the design of prodrugs (drug-PG) in formats such as antibody-directed enzyme-prodrug therapy (ADEPT), where the drug is released upon contact with a site-localized enzyme. Recently, there is increasing interest in enzyme-triggered construction of nanostructures in living cells, which constitutes an example of synthetic chemistry in vivo. For example, in enzyme-instructed self-assembly (EISA), a peptide cleaved by an enzyme assembles to form a hydrogel due to hydrophobic interactions and hydrogen-bonding [FIG. 1, (i)].⁵The cyanobenzothiazole-cysteine (CBT-Cys) click reaction^6,7relies on enzyme-triggered covalent-bond formation, where covalent coupling occurs between an enzymatically deprotected cysteine (A) and CBT (B) as an acceptor for the cysteine [FIG. 1(ii)]. Nanostructures that have been prepared using compounds bearing cysteine and CBT moieties include nanorings formed by oligomerization followed by self-assembly⁸and nanocrystals immobilized by cross-linking.⁹Such enzyme-triggered covalent-bond forming reactions remain rare despite extensive development of click chemistry as a potent means for bioconjugation.¹⁰

Another example of enzyme-triggered covalent bond formation is the natural formation of indigo. Indoxyl-glucoside 1 (also known as indican) upon action of a glycosidase yields indoxyl (2); subsequent enol-keto tautomerism affords indoline (3), which in the presence of air undergoes homo-coupling to give indigo (4) (FIG. 5).¹¹Indigo is quite insoluble in water and typically precipitates upon formation. The homo-coupling of two molecules of A to afford indigo (A-A) occurs without an acceptor (B), and is irreversible owing to the oxidation process [FIG. 1, (iii)]. The conversion of indoxyl-glucoside to indigo is accompanied by profound changes in the absorption spectrum, which facilitates quantitative characterization of the products.

The attractive features of indigoid dye formation include enzymatic triggering, chromogenicity, insoluble deposition from aqueous solution at the site of reaction, and reaction under physiological conditions. Histological and bacteriological use has been extended to include indoxyls bearing enzymatically cleavable substituents other than glucosides, including glucuronides, carboxylic esters, phosphoesters, phosphodiesters, and sulfoesters.¹⁴However, indoxyls have been little explored as cross-linking agents for biomolecules in vitro or in vivo.

Here, we describe results from lengthy studies aimed at developing indoxyl-based chromogenic cross-linking agents of the general design illustrated in FIG. 6. We were surprised to find that the linkers we employed for attaching a bioconjugatable tether thwarted indigoid dye formation upon enzymatic cleavage of the indoxyl-glucoside. Hence, a first set of studies entailed syntheses of diverse indoxyl β-glucosides to identify the structural features compatible with facile indigoid dye formation while bearing a bioconjugatable tether. Next, each structure was examined for indigoid dye formation upon treatment to several enzymatic conditions including β-glucosidases, tritosomes, and rat liver homogenates; from these studies the 5-alkoxy-4,6-dibromoindoxyl nucleus was found to give superior results. Finally, oligomerization via the indigoid dye-forming reaction under physiological conditions was explored to understand the fundamental properties of this cross-linking motif.

1. Synthesis of Indoxyl Species.

The commercially available 5-benzyloxy-3-formylindole (8) provided the sole indole starting material for all 17 new synthetic indoxyl-glucosides described herein. Compound 8 was converted in 3 steps to the fully protected 5-hydroxyindoxyl β-glucoside 9 (Scheme 1) in accord with a patent.²¹Deprotection of the acetyl and benzyl groups of 9 provided 5-hydroxyindoxyl β-glucoside 10 in 89% yield, while debenzylation of 9 afforded acetyl-protected 5-hydroxyindoxyl β-glucoside 11 in 99% yield.

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1,3,5-Triazine²⁶and carbamate linkers were selected to derivatize the phenolic hydroxy group in 11 (Scheme 2). Thus, treatment of 11 with 2,4-dichloro-6-methoxy-1,3,5-triazine (12) replaced one of the two chlorines to form chlorotriazine 13 in 87% yield. The remaining chloride was substituted upon pilot reaction with morpholine and with the elaborate amine 14 bearing a bicyclo[6.1.0]nonyl (BCN) group²⁸for Cu-free click chemistry.¹⁰Subsequent deprotection of the sugar in the BCN-tethered indoxyl-glucoside gave 15 in 93% yield (Scheme 2). Treatment of 11 with p-nitrophenyl chloroformate afforded a carbonate intermediate, which upon reaction with benzylamine gave the carbamate. Reaction with NaOMe caused removal of the acetyl groups to give carbamate 16 in 53% yield.

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In initial studies with β-glucosidase from almonds, neither 15 nor 16 afforded the corresponding indigoid species in good yield. It appeared that the alkoxy group, necessary for later bioconjugation, inhibited the indigogenic process. Thus, bromine atoms were introduced onto the indole ring to overcome the inhibitory effect of the alkoxy group (Scheme 3). Treatment of 11 with N-bromosuccinimide (NBS, 1.05 equiv) afforded 4-bromoindoxyl 17 in 75% yield, whereas a larger quantity of NBS (2.3 equiv) gave 4,6-dibromoindoxyl 18 in 83% yield. Single-crystal X-ray structures of 17 and 18 confirmed the sugar stereochemistry and the positions of the bromine atoms (FIG. 7 panels A and B).

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The 4,6-unsubstituted indoxyls, the 4-bromoindoxyls, and the 4,6-dibromoindoxyls bearing a linker at the 5-position were prepared from 10, 11, 17, or 18 (Scheme 4). As indoxyls (and the corresponding indigoid dyes) bearing 5-oxy and bromine substituents have not been reported (although each is known separately), we compared the indigogenic reactions among these indoxyls to investigate the effects of the bromine substituents. The triazine linker was introduced into indoxyls 10, 17, and 18 via the successive substitution of the chloro groups in dichlorotriazine 12. 4,6-Unsubstituted indoxyl glucoside 10 was treated with 12 followed by morpholine to afford 19 in 59% yield. 4-Bromoindoxyl 20 bearing the triazine linker was prepared from acetyl-protected 4-bromoindoxyl 17. Treatment of 17 with 12 followed by morpholine and subsequent acetyl deprotection gave 20 in 77% yield. Similarly, 4,6-dibromoindoxyl 21 was prepared from acetyl-protected 18 in 67% yield. Indoxyls 22-26 possess a methoxycarbonyl group, which can function as an amine-reactive linker. This linker was introduced by alkylation of the 5-hydroxy group in 11, 17, and 18 with ethyl bromoacetate in the presence of NaH and subsequent treatment with NaOMe in MeOH. Indoxyl 10 was reacted with propargyl bromide in the presence of K₂CO₃to afford 4,6-unsubstituted indoxyl glucoside 25 in 30% yield, which bears a propargyl group for ensuing click chemistry. Propargylation of acetyl-protected indoxyl 17 and 18 followed by acetyl-deprotection with triethylamine in MeOH provided 4-bromoindoxyl 26 and 4,6-dibromoindoxyl 27 in 71 and 53% yield, respectively.

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4,6-Dibromoindoxyl 30, which possesses the BCN group instead of the propargyl group in 27, was prepared from 18 (Scheme 5). The Mitsunobu reaction between 18 and commercially available BCN-methanol 28 gave 29 in 63% yield. Deacetylation of 29 with K₂CO₃/MeOH afforded 30 in 99% yield. Additionally, 18 was treated with triethylene glycol mononosylate 31 to give 32 in 89% yield, which was deprotected to afford 4,6-dibromoindoxyl 33 bearing a triethylene glycol linker in 94% yield.

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Water solubility of the indoxyl glucoside is important for biological applications. Regardless of the presence of the polar glucosyl group, poor water-solubility of the indoxyl glucoside was observed in some cases. For example, we prepared 4,6-dibromoindoxyl glucoside 34 (FIG. 8), which contains the BCN group along with a fluorescent anilinotriazine moiety;^29,30however, 34 had limited solubility in aqueous buffers (<10 μM at room temperature). Therefore, this compound is not suitable for bioconjugation. The synthesis of 34 relies on successive substitution of the dichloroanilinotriazine unit (derived from N,N-bis(2-methoxyethyl)aniline³¹and cyanuric chloride) with indoxyl-glucoside-PEG-OH and BCN-PEG-NH₂building blocks.

To improve the water solubility of the indoxyl species, a sulfobetaine unit^32,33was incorporated as a water-solubilizing group. Sulfobetaines are stable zwitterions over a wide range of pH. Synthesis of an indoxyl bearing a sulfobetaine unit is illustrated in Scheme 6. Boc-piperazine (35) was treated with 1,3-propane sultone to afford 36 in 63% yield. Quaternization of the tertiary nitrogen atom in 36 with 3-bromopropanol gave Boc-protected sulfobetaine 37 in 69% yield. The Boc group was cleaved with trifluoroacetic acid (TFA) to afford piperazine-TFA salt 38 in 97% yield. The N-acetyl group of 32 was selectively deprotected with NaHCO₃/MeOH in 84% yield. The product 39, piperazine-TFA salt 38, and BCN-amine 14 were assembled at a triazine ring via one-flask, successive substitution of cyanuric chloride to afford 40 in 51% yield. Cleavage of the acetyl groups of sulfobetaine 40 provided 41 in 98% yield. Owing to the sulfobetaine unit, 41 showed superior solubility (>400 μM at room temperature) versus 34 in a 100 mM phosphate buffer (pH 7.4, containing 100 mM NaCl).

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2. Indigogenic Studies.

With diverse glucosyl-indoxyl compounds in hand, we carried out a set of studies to examine indigoid-dye formation upon enzymatic cleavage of the glucosyl unit under physiological conditions. Altogether, 14 new (15, 16, 19-27, 30, 33, 41) and 2 known (1, 42) synthetic indoxyl-glucosides (lacking acetyl protecting groups) were examined in an effort to identify suitable combinations of substituents to support both bioconjugation and indigoid dye formation. In initial studies, β-glucosidase from almonds was employed to trigger indigoid dye formation (Table 1). Thus, a mixture of this enzyme (1 unit/mL) and an indoxyl β-glucoside (0.1 μmol, 1 mM) in acetate buffer (pH 5, containing 5% DMF) was incubated at 37° C. for 16-19 h. All indigoid dye was dissolved in each case for quantitative evaluation. The parent indoxyl 1 afforded indigo only in 17% yield under the reaction conditions (entry 1). By contrast, 5-bromo-4-chloroindoxyl β-glucoside (42, also known as X-Glu used in a chromogenic assay for β-glucosides)¹⁴provided the corresponding indigoid dye in 74% yield (entry 2, yield calculated based on ε=2.00×10⁴M⁻¹cm⁻¹reported for 5,5′-dibromo-4,4′-dichloroindigo).³⁴These results are consistent with Holt's report that a bromo (and chloro) substituent(s) on the indoxyl facilitated indigoid dye formation.

No indigoid dye was detected with 15 (entry 3), whereas 16 formed the corresponding indigoid dye, albeit in low yield (24%, entry 4). We measured the molar absorption coefficient of the parent indigo 4 in DMF/water (2:1) and found the value at λ_maxnear 600 nm to be ε=1.27×10⁴M⁻¹cm⁻¹, to be compared with ε=1.66×10⁴M⁻¹cm⁻¹in a different solvent reported by Holt and Sadler.³⁴For consistency, we have used the value in DMF/water (2:1) for all studies here unless noted otherwise.

Indoxyls 19-21 bearing the triazine linker did not form the indigoid dye regardless of the presence or absence of a bromo atom (entries 5-7, respectively). In the case of 5-[(methoxycarbonyl)methoxy]indoxyls 22-24, the yield of indigoid dye was markedly improved as the number of bromine atoms increased (entry 8, 22, <1%; entry 9, 23, 68%; entry 10, 24, 122% yield). The same trend was observed for 5-(propargyloxy)indoxyls 25-27 (entry 11, 25, <5%; entry 12, 26, 56%; entry 13, 27, 105% yield). These results indicated a significant promoting effect of the bromine atoms on indigoid dye formation. No indigo product was detected with BCN-indoxyl 30 (entry 14) whereas PEG₃-indoxyl 33 afforded indigoid dye in 52% yield (entry 15). In summary, the structure of the 5-substituent controlled the indigo-forming reaction: indoxyls 20, 21, and 30 did not engender the formation of any indigoid product regardless of the presence of bromine atoms. This may be because these substrates have low affinity for the enzyme due to the presence of the bulky triazine or BCN moiety.

TABLE 1

Indigogenic reactions of indoxyl derivatives.

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Yield (%)^a

β-glucosidase
β-glucosidase
rat liver

from
from
homo-

Entry
Indoxyl
Structure
almonds^b

Agrobacterium
^c

genate^d

1
1

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17
97 ± 5
10

2
42

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74^e
116 ± 8^e
65

3
15

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<1

^f

^f

4
16

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24

^f

9

5
19

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<1
46 + 5
<5

6
20

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<1
150 ± 3
<5

7
21

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<1
63 ± 4 (31 ± 2)^g
<5

8
22

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<1
30 ± 6
<5

9
23

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68
84 ± 4
<5

10
24

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122 (59)^g
209 ± 6 (102 + 3)^g
<5

11
25

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<5
37 ± 2
11

12
26

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56
79 ± 1
17

13
27

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105 (51)^g
184 ± 3 (89 ± 2)^g
50

14
30

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<1
21 ± 1 (10 ± 0.4)^g

^f

15
33

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52^g
99 ± 5^g
<5^g 81 ± 5^g,h

16
41

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^f

106 ± 4^g

^f

^aThe yield was estimated by absorption spectroscopy with ε = 1.27 × 10⁻¹M⁻¹ cm⁻¹ (DMF/H₂O = 2:1) measured for 4 (see the ESI) unless otherwise noted. ^bA mixture of the indoxyl (1 mM) and β-glucosidase from almonds (1 unit/mL) in 0.01M acetate buffer (pH 5, containing 5% DMF) was incubated at 37° C. for 16-19 h. ^cA mixture of the indoxyl (100 μM) and β-glucosidase from Agrobacterium (200 nM) in 0.05M phosphate buffer (pH 7.0, containing 2% DMF) was incubated at 37° C. for 2 h. The reaction was repeated three times. ^dThe indoxyl (1 mM) in rat liver homogenate containing 5% DMF was incubated at 37° C. for 24 h. ^eThe yield was estimated from absorption spectroscopy with ε = 2.00 × 10⁴M⁻¹ cm⁻¹ reported for 5,5′-dibromo-4,4′-dichloroindigo.^{34 f}Not conducted. ^gThe yield was estimated from absorption spectroscopy with ε = 2.6 × 10⁻¹M⁻¹ cm⁻¹ (DMF/H₂O = 2:1) measured for 43. ^hThe reaction was carried out with 33 (100 μM) and β-glucosidase from Agrobacterium (200 nM) plus rat liver homogenate containing 2% DMF at 37° C. for 4 h.

The results from the glucosidase survey prompted several further experiments. First, a parallel set of studies was carried out with inclusion of several oxidants commonly employed in histochemical studies, given that the indigoid dye forming process requires the presence of an oxidant. No substantial increase in yield was observed for the substrates shown in entries 1-15 of Table 1. Also, the same set of substrates was examined with tritosomes (lysosomes isolated by loading with a non-ionic detergent) but the results were uniformly poor except for a low yield of indigoid dye from 16 and 19. The activity of the β-glucosidase was affected only slightly in the presence of a non-ionic detergent. To verify that the results observed in Table 1 were reliable, a 5-mg scale reaction of 33 was carried out to isolate indigo 43, which was obtained in 66% yield (Scheme 7). The 66% isolated yield corresponded well with the enzymatic yield of 52% (Table 1, entry 15).

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Next, the β-glucosidase from Agrobacterium sp. was investigated as the trigger enzyme for indigoid dye formation. In contrast to β-glucosidase from almonds, which works chiefly under acidic conditions³⁵(optimum pH 5.6),³⁶β-glucosidase from Agrobacterium has a neutral pH optimum and maintains partial activity under acidic (pH 4-5) and basic (pH 8-9) conditions as determined by measurement of the rate of hydrolysis of 4-nitrophenyl β-D-glucopyranoside.³⁷Given that the indigoid dye-forming reaction is reported to be faster at a basic rather than an acidic pH,^16,38the pH effect on the indigoid dye-forming reaction was studied with β-glucosidase from Agrobacterium. The reaction was carried out using the enzyme (200 nM) and indoxyl-glucoside 33 (100 μM) in phosphate buffer (pH 4-9, containing 2% DMF) at 37° C. The progress of indigoid dye formation as a function of pH is illustrated in FIG. 9A. High to quantitative yields were attained in 3 h at pH 6-9; within this range, pH 7 provided the best result. The reactions at pH 4 and 5 were also nearly complete in 3 h, although the yields were lower (6% and 38% at pH 4 and 5, respectively). The yields with different enzyme concentrations (200 versus 10 nM) at 2 h are shown in FIG. 9B. Good yields (50-71%) obtained at pH 6-8 with 10 nM enzyme suggested that indigoid dye formation from the indolinone intermediate was not very fast compared to the enzymatic cleavage of the sugar of 33. A relatively large decrease in the yield at pH 9 with 10 nM enzyme may be attributed to the importance of the enzymatic activity under the conditions. The effect of concentration of 33 on the indigo-forming reaction at pH 7.0 is illustrated in FIG. 9C. High yields were maintained when the concentration was >10 μM (89, 86, 85, and 90% at 56, 32, 18, and 10 μM, respectively), while lower yields were obtained at lower concentrations (62, 51, 42, and 41% at 5.6, 3.2, 1.8, and 1.0 μM, respectively). Lengthening the reaction time from 2 h to 14 h improved the yields (88, 79, and 57% at 3.2, 1.8, and 1.0 μM, respectively). Note that with 200 nM enzyme and 100 μM substrate, complete reaction requires 500 turnovers of each enzyme. Given that reaction was still observed at 10 nM enzyme, such a modest turnover appears reasonable. In other words, the cases where incomplete reaction was observed likely were not due to limiting enzyme concentration.

With the results in hand for indoxyl-glucoside 33, the 15 other indoxyl compounds shown in Table 1 (100 μM) were similarly treated with β-glucosidase from Agrobacterium (200 nM) in phosphate buffer (pH 7) at 37° C. for 2 h. Unsubstituted indoxyl 1 and the 4-chloro-5-bromo derivative 42 provided good yields (97 and 116%, entries 3 and 4, respectively). In contrast to β-glucosidase from almonds, the enzyme from Agrobacterium cleaved the glucoside in indoxyls containing the triazine linker to give indigoid dye (entry 5 or 6, 46 or 150% yield). In the reactions of 22-27, the order of the yield was unsubstituted indoxyl<4-bromoindoxyl<4,6-dibromoindoxyl as observed with β-glucosidase from almonds (entries 8-13). Indoxyl 30 again resulted in low yield (10%, entry 14), suggesting severe steric hindrance of the BCN group in the molecule. The indigoid dye was quantitatively formed from indoxyls 33 and 41 (entries 15 and 16, 99 and 106% yield, respectively).

Finally, indigoid-dye formation was carried out in rat liver homogenate (Table 1, rightmost column). Good yields were obtained in the case of 42 (65%, entry 4) and 27 (50%, entry 13). Indoxyl 33 did not form an indigo product in rat liver homogenate (<5%, entry 15). However, when β-glucosidase from Agrobacterium (200 nM) was present in rat liver homogenate, the indigoid dye was obtained in 81% yield (entry 15).

3. Oligomerization Study

We sought to carry out an enzyme-triggered oligomerization using a bis(glucosyl-indoxyl) species bearing a water-solubilization motif. The synthesis of the monomer for oligomerization is shown in Scheme 8. Treatment of acetyl-protected dibromoindoxyl-glucoside 32 (two molar equiv) with cyanuric chloride resulted in substitution of two of the three chloro groups in the latter to give chlorotriazine 44 in 53% yield. After removal of the N-acetyl groups of 44 by treatment with basic methanol, the reaction with 38 installed the water-solubilizing group to afford 45 in 55% yield. Deprotection of the glucosyl O-acetyl groups provided the target bis(glucosyl-indoxyl) species 46 in 77% yield.

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Oligomerization of 46 was carried out by treatment with β-glucosidase from Agrobacterium (200 nM) in 10 mM phosphate buffer (pH 7) at 37° C. for 2-4 h (Scheme 9). Precipitation occurred during the reaction. After centrifugation, the precipitate was separated from the supernatant, washed with H₂O, and dried to afford a blue solid.

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The efficacy of the indigogenic oligomerization was examined under a variety of conditions. As shown in entries 1-4 in Table 2, the yield of indigoid dye in the supernatant versus precipitate reversed as the concentration of 46 was decreased from 300 to 10 μM, although the total yields were in the range 17-29%. While the reactions in entries 1-4 were carried out in phosphate buffer containing NaCl (0.05 M), those of entries 5 and 6 (with 300 and 50 μM of 46, respectively) were conducted in NaCl-free phosphate buffer. The use of NaCl-free phosphate buffer facilitated extraction of the indigoid dye in the supernatant for analysis.

TABLE 2

Study of the oligomerization of bis(indoxyl-glucoside) species 46.

Yield of indigoid dye (%)^a

Entry
[46], μM
Time, h
supernatant
precipitate

1^b
300
2
6
11

2^b
100
3
10
14

3^b
50
2
13 ± 0.4^c
16 ± 0.6^c

4^b
10
4
22
6

5^d
300
3
13
13

6^d
50
3
19
8

^aThe yield was calculated from absorption spectroscopy with ε = 2.6 × 10⁴M⁻¹cm⁻¹.

^bThereaction was carried out in phosphate buffer containing NaCl (0.05 M).

^cThe reaction was repeated three times.

^dThe reaction was carried out in phosphate buffer containing DMF (0.1-0.6%).

The time course of the oligomerization was examined under the reaction conditions listed in entry 5 of Table 2, with 300 μM of 46. The visible course of the reaction is shown in FIG. 10A. Noticeable changes include observation of blue clouding and color deepening at 20 min. Centrifugation enabled isolation of the precipitate. Optical microscopic analysis of the precipitate suspended in H₂O showed small particles of up to several micron dimensions (FIG. 10B). Dynamic light scattering (DLS) analysis indicated that the particle size was ˜680 nm (number mean, FIG. 10C). The absorption spectrum of the precipitate was examined in DMF/DMSO (9:1) and compared with that of the extracted supernatant (in DMF) and 43 (in DMF) (FIG. 10D). All three samples showed a characteristic indigogenic peak in the range 550-700 nm. The greater absorbance at ca. 300 nm in the precipitate and supernatant extract versus that of 43 suggested contamination of impurities composed of indole derivatives. Size-exclusion chromatography (SEC) [DMF/DMSO (9:1) as eluent] was applied to the precipitate and to the supernatant extract prepared from 300 μM of 46 (FIG. 10E). The first peak appeared at 18-20 min, and then the second peak at 38.5 min. The ratio of peak areas in the supernatant extract was 11:89, whereas that in the precipitate was 93:7. On the basis of a calibration curve prepared with poly(2-vinylpyridine) standards, the molecular size for the first and second peaks was expected to be >265 and <1 kDa, respectively. The large molecular size indicated for the first peak likely implies aggregation or assembly of the oligomers. The chromatograms for the samples prepared with 50 μM of 46 gave similar results but with higher purity compared with those at higher concentration.

The ¹H NMR spectra of 33, 46, and the precipitate dissolved in DMSO-d₆are shown in FIG. 11A. The lack of signals from the glucosyl group (hydroxyl protons ˜5.0 ppm; the anomeric proton at 4.65 ppm) in the spectrum of the precipitate is consistent with smooth enzymatic cleavage of the sugar moiety. On the other hand, the signals in the aromatic region of the precipitate were complicated (FIG. 11B). One interpretation is that the precipitate includes the indigoid dye in distinct environments and/or unknown indole derivatives other than the indigoid dye and indoxyl β-glucoside.

Finally, the oligomerization of 46 was carried out on a larger scale (7.87 mg) under the same reaction conditions as those of entry 5 in Table 2. As a result, 3.01 mg of the precipitate was obtained, which corresponds to 49% yield based on the monomer formula weight.

We attempted to use mass spectrometry to gain information about the composition of the oligomeric indigoid products formed upon enzymatic treatment of 46. Analysis of the supernatant by ESI-MS revealed negative ion peaks at m/z 1214.0 and 2428.9, consistent with monomer cyclization (n=1) and cyclodimerization (n=2), respectively. Analysis of the supernatant by MALDI-MS revealed a progression of broad peaks extending to m/z>10,000 with m/z 1210-1250 increment. Although the progression implies a mixture of oligomers, the observed m/z values did not match the calculated values. Incomplete purification, decomposition by laser irradiation (especially the bromoheteroarene units), or complicated isotopic distribution caused by multiple bromine atoms may contribute to the broad peaks. Attempts to use MALDI-MS to analyze the precipitate, which was very insoluble, were unfruitful.

Experimental Section

General methods. ¹H NMR and ¹³C NMR spectra were collected at room temperature in CDCl₃unless noted otherwise. Chemical shifts for ¹H NMR spectra are reported in parts per million (6) relative to tetramethylsilane or solvent signal (CD₃OD, δ=3.31 ppm). Chemical shifts for ¹³C NMR spectra are reported in parts per million (6), and spectra were calibrated by using solvent signals [CDCl₃, δ=77.16 ppm; (CD₃)₂SO, δ=39.52 ppm; CD₃OD, δ=49.00 ppm]. Silica (40 μm), diol-functionalized silica (40-63 μm), and reverse phase silica (C18, 40-63 μm) were used for column chromatography. Preparative TLC separations were carried out on Merck analytical plates precoated with silica 60 F₂₅₄. All solvents were reagent grade and were used as received unless noted otherwise. Commercial compounds were used as received. The known compounds 9,²¹12²⁷and N,N-bis(2-methoxyethyl)aniline³¹were prepared generally following procedures described in the literature. Microscopic analysis was performed on a Zeiss Axio Imager M.2. DLS analysis was performed on a Zetasizer Nano ZS. Centrifugation was carried out at 20,000 G at 4° C.

5-Hydroxy-1H-indol-3-yl β-D-glucopyranoside (10). A suspension of 9 (917.4 mg, 1.50 mmol), having >99% stereochemical purity at the anomeric carbon, in MeOH (7.50 mL) at room temperature was treated with sodium methoxide (25 wt % solution in MeOH, 648 μL, 3.0 mmol). After 2 h, acetic acid (229 μL, 6.00 mmol) and palladium on carbon (10 wt %, 79.8 mg, 0.075 mmol) were added. The reaction mixture was stirred for 2 h under hydrogen atmosphere (balloon) at room temperature and then filtered through Celite. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (7:3)] to afford a pale yellow solid (417.6 mg, 89%): ¹H NMR [400 MHz, (CD₃)₂SO] δ 3.09-3.29 (m, 4H), 3.42-3.53 (m, 1H), 3.65-3.76 (m, 1H), 4.47-4.55 (m, 1H), 4.59 (br s, 1H), 5.07 (br s, 1H), 5.15 (br s, 1H), 5.38 (br s, 1H), 6.58 (dd, J=2.6, 8.6 Hz, 1H), 6.89 (d, J=2.0 Hz, 1H), 6.98 (d, J=2.6 Hz, 1H), 7.06 (d, J=8.6 Hz, 1H), 8.68 (br s, 1H), 10.21 (s, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 62.6, 71.5, 75.0, 78.02, 78.04, 102.5, 105.8, 112.9, 113.1, 113.5, 121.9, 130.4, 138.4, 160.0; ESI-MS obsd 334.0894, calcd 334.0897 [(M+H)⁺, M=C₁₄H₁₇NO₇].

1-Acetyl-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-13-D-glucopyranoside (11).²¹Following a reported debenzylation procedure,²¹a suspension of 9 (6.911 g, 11.3 mmol) and Pd/C (10 w/w %, 360.8 mg, 0.339 mmol) in ethyl acetate/EtOH (4:1, 113 mL) was stirred for 3 h at room temperature under H₂atmosphere (balloon). The reaction mixture was filtered through Celite. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/ethyl acetate (10:1)] to afford a pale yellow solid (5.85 g, 99%): mp 88-90° C.; ¹H NMR (400 MHz, CDCl₃) δ 2.05 (s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.12 (s, 3H), 2.56 (s, 3H), 3.77-3.88 (m, 1H), 4.23 (dd, J=5.0, 12.4 Hz, 1H), 3.34 (d, J=12.4 Hz), 4.93-5.03 (m, 1H), 5.11-5.23 (m, 1H), 5.23-5.34 (m, 2H), 5.82-6.02 (m, 1H), 6.85-6.96 (m, 2H), 7.10 (br s, 1H), 8.22 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 20.63, 20.66, 20.73, 20.8, 23.7, 62.1, 68.3, 71.1, 72.4, 72.6, 101.0, 103.2, 110.9, 115.1, 117.7, 125.4, 128.3, 141.3, 153.0, 168.2, 169.58, 169.63, 170.4, 171.0; ESI-MS obsd 544.1430, calcd 544.1426 [(M+Na)⁺, M=C₂₄H₂₇NO₁₂].

1-Acetyl-5-[(4-chloro-6-methoxy-1,3,5-triazin-2-yl)oxy]-1H-indole-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (13). A sample of i-Pr₂EtN (65.3 μL, 0.375 mmol) was added dropwise over 5 min to a suspension of 11 (130.4 mg, 0.250 mmol) and 12 (58.5 mg, 0.325 mmol) in CH₂Cl₂(1.25 mL) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was washed with aqueous citric acid (10%, 1 mL) followed by brine (1 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated and chromatographed [silica, hexanes/ethyl acetate (2:3)] to afford a white solid (188.3 mg, 87%): ¹H NMR (300 MHz, CDCl₃) δ 2.05 (s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.62 (s, 3H), 3.80-3.95 (m, 1H), 4.02 (s, 3H), 4.14-4.38 (m, 2H), 4.97-5.09 (m, 1H), 5.09-5.40 (m, 3H), 7.10-7.36 (m, 3H), 8.44 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 20.6, 20.68, 20.71, 23.8, 56.3, 61.9, 68.2, 70.9, 72.37, 72.43, 100.7, 110.2, 111.1, 117.7, 119.7, 124.8, 131.4, 141.0, 147.6, 168.1, 169.2, 169.4, 170.2, 170.5, 172.5, 172.8, 173.2; ESI-MS obsd 665.1499, calcd 665.1492 [(M+H)⁺, M=C₂₈H₂₉ClN₄O₁₃].

5-{[4-({1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa-4-azadodecan-12-yl}amino)-6-methoxy-1,3,5-triazin-2-yl]oxy}-1H-indole-3-yl β-D-glucopyranoside (15). A sample of 13 (20.3 mg, 0.0305 mmol) was added to a solution of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (10.4 mg, 0.0321 mmol) and i-Pr₂EtN (6.7 μL, 0.038 mmol) in CH₂Cl₂(150 μL) at room temperature. After 3 h, MeOH (750 μL) and K₂CO₃(13.3 mg, 0.096 mmol) were added. After 1 h, the reaction mixture was quenched by the addition of acetic acid (9.2 μL) and then filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (3:1)] to afford a pale yellow solid (21.0 mg, 93%): ¹H NMR (700 MHz, CD₃OD, ˜1:1 mixture of rotamers) δ 0.85-0.98 (m, 2H), 1.25-1.40 (m, 1H), 1.49-1.65 (m, 2H), 2.09-2.30 (m, 6H), 3.10-3.18 (m, 1H), 3.23-3.28 (m, 1H), 3.29-3.63 (m, 14H), 3.716 (dd, J=5.6, 11.9 Hz, 0.5H), 3.719 (dd, J=5.7, 11.9 Hz, 0.5H), 3.87-3.93 (m, 2.5H), 3.94 (s, 1.5H), 4.10 (d, J=8.1 Hz, 0.5H), 4.12 (d, J=8.2 Hz, 0.5H), 4.68 (d, J=8.1 Hz, 0.5H), 4.70 (d, J=8.1 Hz, 0.5H), 6.67-6.73 (m, 0.5H), 6.78-6.84 (m, 0.5H), 6.87 (dd, J=2.3, 8.7 Hz, 1H), 6.90 (dd, J=2.3, 8.7 Hz, 1H), 7.17 (s, 1H), 7.28 (d, J=8.7 Hz, 0.5H), 7.29 (d, J=8.7 Hz, 0.5H), 7.47 (d, J=2.3 Hz, 0.5H), 7.48 (d, J=2.3 Hz, 0.5H); ¹³C NMR (175 MHz, CD₃OD) δ 18.9, 21.4, 21.9, 30.1, 41.2, 41.5, 41.6, 41.7, 41.9, 55.2, 55.3, 62.6, 63.70, 63.74, 70.0, 70.3, 70.8, 70.88, 70.91, 71.16, 71.22, 71.5, 75.0, 78.0, 78.16, 78.19, 99.5, 105.9, 106.0, 110.8, 111.0, 112.7, 112.9, 114.0, 114.2, 117.3, 117.5, 121.4, 121.5, 132.9, 133.0, 139.18, 139.24, 146.4, 146.6, 159.2, 159.2, 169.3, 169.5, 173.4, 173.7, 173.9, 174.1; ESI-MS obsd 743.3249, calcd 743.3247 [(M+H)⁺, M=C₃₅H₄₆N₆O₁₂].

5-[(Benzylcarbamoyl)oxy)]-1H-indole-3-yl β-D-glucopyranoside (16). Samples of p-nitrophenyl chloroformate (6.0 mg, 0.029 mmol) and i-Pr₂EtN (6 μL, 0.03 mmol) were added to a solution of 11 (13.0 mg, 0.025 mmol) in CH₂Cl₂(1 mL) at room temperature. After 1 h, the reaction mixture was quenched by the addition of saturated aqueous NH₄Cl (2 mL) and stirred for 30 min at room temperature. After H₂O (2 mL) was added, the mixture was extracted with Et₂O (3×2 mL). The combined organic layer was washed with H₂O (2 mL), brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. The residue was dissolved in CH₂Cl₂(1 mL). Benzylamine (3 μL, 0.03 mmol) was added to the solution at room temperature. After 20 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in MeOH (1 mL). NaOMe (25% in MeOH, 5 μL, 0.02 mmol) was added to the solution at room temperature. After 45 min, the reaction mixture was quenched with ion exchange resin (DOWEX 50WX8-200), stirred for 20 min at room temperature, and filtered. The filtrate was concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (5:1)] afforded a colorless oil (5.9 mg, 53%): ¹H NMR (700 MHz, CD₃OD) δ 3.32-3.36 (m, 1H), 3.40 (t, J=9.0 Hz, 1H), 3.43 (t, J=9.0 Hz, 1H), 3.48 (dd, J=9.0, 8.0 Hz, 1H), 3.71 (dd, J=12.0, 6.0 Hz, 1H), 3.90 (dd, J=12.0, 2.0 Hz, 1H), 4.37 (s, 2H), 4.67 (d, J=8.0 Hz, 1H), 6.85 (dd, J=8.0, 2.0 Hz, 1H), 7.14 (s, 1H), 7.24-7.28 (m, 2H), 7.31-7.40 (m, 4H), 7.43 (d, J=2.0 Hz, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 45.7, 62.6, 71.5, 75.0, 78.0, 78.2, 106.0, 111.0, 112.7, 114.2, 117.5, 121.5, 128.2, 128.4, 129.6, 132.9, 139.0, 140.4, 145.4, 158.6; ESI-MS obsd 467.1419, calcd 467.1425 [(M+Na),⁺, M=C₂₂H₂₃N₂NaO₈].

1-Acetyl-4-bromo-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (17). A solution of N-bromosuccinimide in CH₂Cl₂(100 mM, 4.20 mL) was added dropwise over 5 min to a solution of 11 (208.6 mg, 0.400 mmol) and 2,6-di-tert-butylpyridine (88 μL, 0.40 mmol) in CH₂Cl₂(5.80 mL) at −78° C. After 1.5 h, the reaction mixture was allowed to warm to room temperature and stirred for 30 min. The reaction mixture was washed with saturated aqueous Na₂S₂O₃(3 mL) and brine (5 mL), dried (Na₂SO₄), and concentrated under reduced pressure. Column chromatography [silica, hexanes/CH₂Cl₂/MeCN (2:1:1)] afforded a white solid (180.6 mg, 75%): mp 130° C. (dec.); ¹H NMR (400 MHz, CDCl₃) δ 2.05 (s, 3H), 2.08 (s, 3H), 2.10 (s, 3H), 2.10 (s, 3H), 3.86-3.96 (m, 1H), 4.22 (dd, J=5.4, 12.3 Hz, 1H), 4.36 (dd, J=2.0, 12.3 Hz, 1H), 5.06 (d, J=7.8 Hz, 1H), 5.21 (dd, J=9.2, 9.2 Hz, 1H), 5.31 (dd, J=9.2, 9.2 Hz, 1H), 5.40 (dd, J=7.8, 9.2 Hz, 1H), 5.74 (s, 1H), 7.05 (d, J=8.8 Hz, 1H), 7.22 (br s, 1H), 8.29 (br s, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 20.8, 20.9, 21.0, 23.9, 62.1, 68.3, 71.0, 72.6, 72.8, 98.6, 100.3, 111.2, 114.7, 117.1, 122.6, 129.0, 140.5, 149.4, 167.9, 169.4, 169.5, 170.4, 170.6; ESI-MS obsd 600.0712, calcd 600.0711 [(M+H)⁺, M=C₂₄H₂₆BrNO₁₂]. Suitable crystals for X-ray analysis were obtained by recrystallization from cyclohexane/CHCl₃.

1-Acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (18). A solution of N-bromosuccinimide (3.987 g, 22.4 mmol) in CH₂Cl₂(240 mL) was added dropwise over 1 h to a solution of 11 (5.841 g, 11.2 mmol) and 2,6-di-tert-butylpyridine (2.47 mL, 11.2 mmol) in CH₂Cl₂(160 mL) at −78° C. The reaction mixture was allowed to warm to room temperature and stirred for 3.5 h. N-Bromosuccinimide (598 mg, 3.36 mmol) was added. After 1 h, the reaction mixture was washed with aqueous Na₂S₂O₃(10%, 50 mL) and brine (50 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. Column chromatography [silica, hexanes/ethyl acetate (1:1)] followed by recrystallization from CH₂Cl₂/MeOH afforded a white solid (6.32 g, 83%): mp 102-103° C.; ¹H NMR (700 MHz, CDCl₃) δ 2.05 (s, 3H), 2.07 (s, 3H), 2.10 (s, 3H), 2.10 (s, 3H), 2.59 (s, 3H), 3.91 (ddd, J=2.4, 5.2, 9.9 Hz, 1H), 4.22 (dd, J=5.2, 12.5 Hz, 1H), 4.37 (dd, J=2.4, 12.5 Hz, 1H), 5.05 (d, J=7.6 Hz, 1H), 5.21 (dd, J=9.5, 9.9 Hz, 1H), 5.31 (dd, J=9.3, 9.5 Hz, 1H), 5.38 (dd, J=7.6, 9.3 Hz, 1H), 6.02 (s, 1H), 7.22 (br s, 1H), 8.63 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 20.7, 20.9, 21.0, 23.7, 62.1, 68.4, 71.0, 72.7, 72.8, 98.4, 100.3, 108.6, 112.0, 120.0, 122.6, 128.7, 140.2, 146.3, 167.8, 169.4, 169.5, 170.3, 170.6; ESI-MS obsd 699.9645, calcd 699.9636 [(M+Na)⁺, M=C₂₄H₂₅Br₂NO₁₂]. Suitable crystals for X-ray analysis were obtained by recrystallization from cyclohexane/acetone.

5-[(4-Methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-indole-3-yl β-D-glucopyranoside (19). A sample of i-Pr₂EtN (13.1 μL, 0.075 mmol) was added to a solution of 10 (15.6 mg, 0.050 mmol) and 12 (9.9 mg, 0.055 mmol) in DMF (125 μL) at room temperature. After 3 h, morpholine (8.6 μL, 0.10 mmol) was added. After 2 h, the reaction mixture was passed through silica. The resulting solution was concentrated under reduced pressure. Column chromatography [silica, CHCl₃/MeOH (4:1)] afforded a white solid (15.0 mg, 59%): ¹H NMR (300 MHz, CD₃OD) δ 3.25-3.93 (m, 14H), 3.92 (s, 3H), 4.67 (d, J=7.2 Hz, 1H), 6.87 (dd, J=8.7, 1.8 Hz, 1H), 7.16 (s, 1H), 7.27 (d, J=8.7 Hz, 1H), 7.46 (d, J=1.8 Hz, 1H); ¹³C NMR (75 MHz, CD₃OD) δ 45.2, 55.3, 62.6, 67.5, 71.5, 75.0, 78.0, 78.2, 106.0, 110.7, 112.7, 114.1, 117.3, 121.4, 132.9, 139.2, 146.5, 167.9, 173.8, 174.0; ESI-MS obsd 506.1883, calcd 506.1880 [(M+H)⁺, M=C₂₂H₂₇N₅O₉].

1-Acetyl-4-bromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-indole-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (pre-20). A sample of i-Pr₂EtN (10.5 μL, 0.060 mmol) was added to a solution of 17 (24.0 mg, 0.040 mmol) and 12 (7.9 mg, 0.044 mmol) in CH₂Cl₂(200 μL) at room temperature. After 30 min, morpholine (6.9 μL, 0.080 mmol) was added. After 3 h, the reaction mixture was quenched with acetic acid (2.2 μL) and then passed through silica (ethyl acetate as an eluent). The eluent was concentrated under reduced pressure. Column chromatography [silica, hexanes/ethyl acetate (1:2)] afforded a white solid (27.6 mg, 87%): ¹H NMR (400 MHz, CDCl₃) δ 3.60-3.76 (m, 6H), 3.78-3.94 (m, 6H), 4.20 (dd, J=5.2, 12.5 Hz, 1H), 4.37 (dd, J=2.2, 12.5 Hz, 1H), 5.06 (d, J=7.6 Hz, 1H), 5.19 (dd, J=9.3, 9.3 Hz, 1H), 5.29 (dd, J=9.3, 9.3 Hz, 1H), 5.37 (dd, J=7.6, 9.3 Hz, 1H), 7.17 (d, J=8.8 Hz, 1H), 7.30 (br s, 1H), 8.30-8.50 (m, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 20.7, 20.9, 21.0, 24.0, 44.06, 44.11, 54.8, 62.0, 66.6, 66.7, 68.3, 70.8, 72.6, 72.7, 100.3, 106.4, 112.1, 116.3, 121.5, 123.5, 132.1, 140.8, 146.2, 166.9, 168.1, 169.3, 169.5, 170.3, 170.6, 171.9, 172.6; ESI-MS obsd 816.1321, calcd 816.1334 [(M+Na)⁺, M=C₃₂H₃₆BrN₅O₁₄.].

4-Bromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-indole-3-yl D-D-glucopyranoside (20). A suspension of pre-20 (15.9 mg, 0.020 mmol) and K₂CO₃(2.8 mg, 0.020 mmol) in MeOH was stirred at room temperature for 20 min. The reaction mixture was quenched with acetic acid (2.9 μL) and concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (1:2)] afforded a white solid (10.4 mg, 89%): ¹H NMR (300 MHz, CD₃OD) δ 3.34-4.00 (m, 17H), 4.76 (d, J=7.8 Hz, 1H), 7.21-7.34 (m, 2H); ¹³C NMR (175 MHz, CD₃OD) δ 45.1, 45.3, 55.29, 55.34, 62.65, 62.69, 67.5, 71.6, 75.3, 78.1, 78.3, 105.2, 106.1, 112.2, 114.7, 118.3, 119.8, 133.6, 139.2, 143.8, 167.9, 173.4, 173.8; ESI-MS obsd 584.0986, calcd 584.0987 [(M+H)⁺, M=C₂₂H₂₆BrN₅O₉].

4,6-Dibromo-5-[(4-methoxy-6-morpholino-1,3,5-triazin-2-yl)oxy]-1H-indole-3-yl β-D-glucopyranoside (21). A sample of i-Pr₂EtN (7.3 μL, 0.042 mmol) was added to a solution of 18 (19.0 mg, 0.028 mmol) and 2,4-dichloro-6-methoxy-1,3,5-triazine (5.54 mg, 0.031 mmol) in CH₂Cl₂(140 μL) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h. Morpholine (4.8 μL, 0.056 mmol) was added. After 3 h, MeOH (560 μL) and K₂CO₃(19.3 mg, 0.14 mmol) were added. The reaction mixture was heated at 35° C. for 1 h and cooled to room temperature. The reaction mixture was quenched by the addition of acetic acid (16 μL) and filtered. The filtrate was concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (6:1)] afforded a white solid (12.5 mg, 67%): ¹HNMR (300 MHz, CD₃OD) δ 3.34-4.02 (m, 17H), 4.77 (d, J=7.2 Hz, 1H), 7.30 (s, 1H), 7.56 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 45.1, 45.3, 55.4, 55.5, 62.6, 67.4, 71.5, 75.2, 78.1, 78.3, 105.00, 105.02, 107.6, 111.2, 115.2, 115.7, 119.5, 133.6, 139.2, 140.6, 167.9, 172.6, 173.9; ESI-MS obsd 662.0098, calcd 662.0092 [(M+H)⁺, M=C₂₂H₂₅Br₂N₅O₉].

5-(Methoxycarbonyl)methoxy-1H-indol-3-yl β-D-glucopyranoside (22). Ethyl bromoacetate (5.0 μL, 45 μmol) and NaH (2.0 mg, 83 μmol) were added to a solution of 11 (10.0 mg, 0.019 mmol) in DMF (1 mL) at room temperature. After 1 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (2 mL) and stirred for 10 min at room temperature. After H₂O (2 mL) was added, the mixture was extracted with Et₂O (3×2 mL). The combined organic layer was washed with H₂O (2 mL), brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (1 mL). NaOMe (25% in MeOH, 5 μL, 0.02 mmol) was added to the solution at room temperature. After 45 min, the reaction mixture was quenched with ion exchange resin (DOWEX 50WX8-200), stirred for 20 min at room temperature, and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (5:1)] to afford a colorless oil (3.9 mg, 53%): ¹H NMR (300 MHz, CD₃OD) δ 3.34-3.56 (m, 4H), 3.72 (dd, J=12.0, 5.0 Hz, 1H), 3.80 (s, 3H), 3.91 (dd, J=12.0, 2.0 Hz, 1H), 4.66 (d, J=7.5 Hz, 1H), 4.71 (s, 2H), 6.82 (dd, J=9.0, 2.5 Hz, 1H), 7.09 (s, 1H), 7.16-7.22 (m, 2H); ¹³C NMR (75 MHz, CD₃OD) δ 52.5, 62.7, 67.0, 71.5, 75.1, 78.0, 78.2, 101.5, 106.0, 113.3, 113.7, 113.9, 153.1, 172.1; ESI-MS obsd 406.1109, calcd 406.1109 [(M+Na)⁺, M=C₁₇H₂₁NO₉].

4-Bromo-5-(methoxycarbonyl)methoxy-1H-indol-3-yl β-D-glucopyranoside (23). Ethyl bromoacetate (4.0 μL, 36 μmol) and NaH (1.0 mg, 42 μmol) were added to a solution of 17 (15 mg, 0.025 mmol) in DMF (0.5 mL) at room temperature. After 1 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (2 mL) and stirred for 10 min at room temperature. After H₂O (2 mL) was added, the mixture was extracted with Et₂O (3×2 mL). The combined organic layer was washed with H₂O (2 mL), brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (0.5 mL). NaOMe (25% in MeOH, 5 μL, 0.02 mmol) was added to the solution at room temperature. After 45 min, the reaction mixture was quenched with ion exchange resin (DOWEX 50WX8-200), stirred for 20 min at room temperature, and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (5:1)] to afford a colorless oil (5.4 mg, 52%): ¹H NMR (300 MHz, CD₃OD) δ 3.34-3.56 (m, 4H), 3.72 (dd, J=12.0, 5.0 Hz, 1H), 3.80 (s, 3H), 3.92 (d, J=12.0 Hz, 1H), 4.66 (s, 2H), 4.75 (d, J=7.0 Hz, 1H), 6.91 (dd, J=9.0, 1.0 Hz, 1H), 7.20 (dd, J=9.0, 1.0 Hz, 1H), 7.24 (s, 2H); ¹³C NMR (175 MHz, CD₃OD) δ 52.5, 69.7, 69.8, 71.6, 75.4, 78.3, 103.3, 105.3, 112.2, 113.9, 115.0, 120.3, 132.5, 138.9, 149.7, 171.7; ESI-MS obsd 484.0205, calcd 484.0214 [(M+Na)⁺, M=C₁₇H₂₀BrNO₉].

4,6-Dibromo-5-(methoxycarbonyl)methoxy-1H-indol-3-yl β-D-glucopyranoside (24). Ethyl bromoacetate (3.0 μL, 27 μmol) and NaH (1.0 mg, 42 μmol) were added to a solution of 18 (12.7 mg, 0.019 mmol) in DMF (1 mL) at room temperature. After 1 h, the reaction mixture was quenched with saturated aqueous NH₄Cl (2 mL) and stirred for 20 min at room temperature. After H₂O (2 mL) was added, the mixture was extracted with Et₂O (3×2 mL). The combined organic layer was washed with H₂O (2 mL), brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (1 mL). NaOMe (25% in MeOH, 5 μL, 0.02 mmol) was added to the solution at room temperature. After 45 min, the reaction mixture was quenched with ion exchange resin (DOWEX 50WX8-200), stirred for 20 min at room temperature, and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (5:1)] to afford a colorless oil (8.3 mg, 82%): ¹H NMR (700 MHz, CD₃OD) δ 3.37-3.41 (m, 2H), 3.45 (t, J=9.0 Hz, 1H), 3.53 (dd, J=9.0, 8.0 Hz, 1H), 3.70 (dd, J=12.0, 5.0 Hz, 1H), 3.84 (s, 3H), 3.92 (d, J=12 Hz, 1H), 4.61 (s, 2H), 4.74 (d, J=8.0 Hz, 1H), 7.27 (s, 1H), 7.51 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 52.6, 62.7, 70.3, 71.6, 75.3, 78.2, 78.3, 105.1, 107.6, 111.6, 115.4, 116.1, 119.9, 133.2, 139.1, 145.6, 170.6; ESI-MS obsd 561.9310, calcd 561.9319 [(M+Na)⁺, M=C₁₇H₁₉Br₂NO₉].

5-Propargyloxy-1H-indol-3-yl β-D-glucopyranoside (25). A suspension of 10 (15.6 mg, 0.050 mmol), propargyl bromide (18.6 μL, 80% in toluene, 0.125 mmol), and K₂CO₃(17.2 mg, 0.124 mmol) in DMF (125 μL) was heated to 80° C. for 2.5 h. The reaction mixture was allowed to cool to room temperature and then passed through silica (CH₂Cl₂/MeOH=1:1 as an eluent). The eluent was concentrated under reduced pressure. Preparative thin layer chromatography [silica, 0.25 mm, 20×20 cm, CHCl₃/MeOH (4:1)] afforded a brown solid (5.3 mg, 30%): ¹H NMR (400 MHz, CD₃OD) δ 2.88 (t, J=2.4 Hz, 1H), 3.32-3.54 (m, 4H), 3.73 (dd, J=5.0, 11.8 Hz, 1H), 3.92 (dd, J=2.2, 11.8 Hz, 1H), 4.69 (d, J=7.6 Hz, 1H), 4.71 (d, J=2.4 Hz, 2H), 6.79 (dd, J=2.4, 8.8 Hz, 1H), 7.09 (s, 1H), 7.18 (d, J=8.8 Hz, 1H), 7.28 (d, J=2.4 1H); ¹³C NMR (100 MHz, CD₃OD) δ 57.6, 62.7, 71.5, 75.1, 76.1, 78.0, 78.2, 80.5, 102.2, 105.9, 113.1, 113.5, 114.1, 121.4, 131.0, 139.0, 152.8; ESI-MS obsd 372.1055, calcd 372.1054 [(M+Na)⁺, M=C₁₇H₁₉NO₇].

4-Bromo-5-propargyloxy-1H-indol-3-yl β-D-glucopyranoside (26). Propargyl bromide (8.9 μL, 80% in toluene, 0.060 mmol) was added to a suspension of 17 (30.0 mg, 0.050 mmol) and K₂CO₃(8.3 mg, 0.060 mmol) in DMF (200 μL) at room temperature. After 4.5 h, triethylamine (20.9 μL, 0.15 mmol) and MeOH (100 μL) were added. After 2 h, NaOMe (21.6 μL, 25% in MeOH, 0.10 mmol) was added. After 30 min, the reaction mixture was quenched by the addition of acetic acid (20 μL) and concentrated under reduced pressure. Column chromatography [silica (CH₂Cl₂/MeOH=8:1) followed by diol-functionalized silica (acetone)] afforded a white solid (15.3 mg, 71%): ¹H NMR (400 MHz, CD₃OD) δ 2.90 (t, J=2.4 Hz, 1H), 3.34-3.52 (m, 3H), 3.55 (dd, J=8.2, 8.2 Hz, 1H), 3.65-3.7 (m, 1H), 3.92 (dd, J=1.2, 11.8 Hz, 1H), 4.71 (d, J=2.4 Hz, 2H), 4.74 (d, J=8.0 Hz, 1H), 7.01 (d, J=8.8 Hz, 1H), 7.20 (d, J=8.8 Hz, 1H), 7.23 (s, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 60.2, 62.7, 71.5, 75.3, 76.7, 78.0, 78.2, 80.2, 103.6, 105.2, 112.0, 114.5, 114.9, 120.2, 132.5, 138.8, 149.2; ESI-MS obsd 450.0155, calcd 450.0159 [(M+Na)⁺, M=C₁₇H₁₉BrNO₇].

4,6-Dibromo-5-propargyloxy-1H-indol-3-yl β-D-glucopyranoside (27). Propargyl bromide (1.8 μL, 80% in toluene, 0.012 mmol) was added to a suspension of 18 (6.8 mg, 0.010 mmol) and K₂CO₃(1.7 mg, 0.012 mmol) in DMF (80 μL) at room temperature. After 2 h, triethylamine (4.2 μL, 0.30 mmol) and MeOH (40 μL) were added. After 4.5 h, NaOMe (4.3 μL, 25% in MeOH, 0.020 mmol) was added. After 30 min, the reaction mixture was quenched by the addition of acetic acid (4 μL) and concentrated under reduced pressure. Column chromatography [silica (CH₂Cl₂/MeOH=4:1) followed by diol-functionalized silica (acetone)] afforded a white solid (2.7 mg, 53%): ¹H NMR (700 MHz, CD₃OD) δ 2.94 (t, J=2.6 Hz, 1H), 3.37-3.42 (m, 2H), 3.42-3.49 (m, 1H), 3.54 (dd, J=7.9, 9.1 Hz, 1H), 3.68-3.74 (m, 1H), 3.92 (dd, J=1.5, 11.9 Hz, 1H), 4.68 (d, J=2.6 Hz, 2H), 4.75 (d, J=7.8 Hz, 1H), 7.25 (s, 1H), 7.50 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 61.6, 62.7, 71.6, 75.3, 76.8, 78.1, 78.3, 79.5, 105.1, 108.1, 112.3, 115.2, 115.9, 119.8, 133.1, 139.1, 145.8, ESI-MS obsd 527.9260, calcd 527.9264 [(M+Na)⁺, M=C₁₇H_uBr₂NO₇].

1-Acetyl-5-{[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (29). Diisopropyl azodicarboxylate (39.4 μL, 0.20 mmol) was added to a solution of 18 (67.9 mg, 0.10 mmol), 28 (16.5 mg, 0.11 mmol), and PPh₃(52.5 mg, 0.20 mmol) in CH₂Cl₂(0.50 mL) at room temperature. After 1.5 h, the reaction mixture was passed through silica (ethyl acetate as an eluent). The eluent was concentrated and again chromatographed [silica, hexanes/acetone (2:1) followed by hexanes/ethyl acetate (1:1)] to afford a white solid (51.1 mg, 63%): ¹H NMR (400 MHz, CDCl₃) δ 1.09-1.1 (m, 2H), 1.60-1.82 (m, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.11 (s, 3H), 2.18-2.40 (m, 6H), 2.60 (s, 3H), 3.89 (ddd, J=2.3, 5.1, 9.7 Hz, 1H), 4.10 (d, J=7.2 Hz, 2H), 4.20 (dd, J=5.1, 12.5 Hz, 1H), 4.38 (dd, J=2.3, 12.5 Hz, 1H), 5.06 (d, J=7.6 Hz, 1H), 5.21 (dd, J=9.2, 9.7 Hz, 1H), 5.31 (dd, J=9.2, 9.2 Hz, 1H), 5.39 (dd, J=7.6, 9.2 Hz, 1H), 7.25 (s, 1H), 8.70 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 19.2, 20.7, 20.8, 20.9, 21.1, 21.7, 23.9, 29.5, 62.0, 68.3, 70.9, 72.0, 72.6, 72.7, 99.1, 100.3, 107.9, 112.0, 116.6, 120.4, 123.2, 131.0, 140.5, 150.1, 168.0, 169.4, 169.6, 170.4, 170.6; ESI-MS obsd 810.0761, calcd 810.0755 [(M+H)⁺, M=C₃₄H₃₇Br₂NO₁₂].

5-{[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl β-D-glucopyranoside (30). K₂CO₃(2.8 mg, 0.020 mmol) was added to a solution of 29 (16.2 mg, 0.020 mmol) in MeOH/THF (4:1, 200 μL) at room temperature. After 1 h, the reaction mixture was diluted with CH₂Cl₂and passed through silica [CH₂Cl₂/MeOH (2:1) as an eluent] to afford a white solid (11.9 mg, 99%): ¹H NMR (700 MHz, CD₃OD) δ 0.97-1.06 (m, 2H), 1.65-1.77 (m, 3H), 2.14-2.21 (m, 2H), 2.21-2.34 (m, 4H), 3.37-3.44 (m, 2H), 3.44-3.52 (m, 1H), 3.55 (dd, J=8.1, 8.9 Hz, 1H), 3.68-3.75 (m, 1H), 3.92 (d, J=11.8 Hz, 1H), 4.08 (d, J=7.8 Hz, 2H), 4.74 (d, J=7.7 Hz, 1H), 7.24 (s, 1H), 7.49 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 20.1, 21.7, 22.0, 30.6, 62.7, 71.5, 72.8, 75.3, 78.1, 78.3, 99.6, 105.2, 107.9, 112.5, 115.2, 116.0, 119.9, 132.8, 139.0, 146.9; ESI-MS obsd 600.0238, calcd 600.0227 [(M+H)⁺, M=C₂₄H₂₈Br₂NO₇].

2-[2-(2-Hydroxyethoxy)ethoxy]ethyl 2-nitrobenzenesulfonate (31). Triethylamine (1.53 mL, 11.0 mL) was added to a suspension of 2-nitrobenzenesulfonic chloride (2.216 g, 10.0 mmol) in triethylene glycol (26.7 mL, 200 mmol) at 0° C. The reaction mixture was warmed to room temperature. After 30 min, the reaction mixture was diluted with CH₂Cl₂(50 mL), washed with aqueous citric acid (10%, 100 mL) and brine (50 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated and chromatographed [silica, hexanes/acetone (2:3)] to afford a clear pale yellow oil (2.929 g, 87%): ¹H NMR (700 MHz, CDCl₃) δ 2.41 (br s, 1H), 3.54-3.59 (m, 2H), 3.59-3.66 (m, 4H), 3.66-3.75 (m, 2H), 3.76-3.83 (m, 2H), 4.38-4.47 (m, 2H), 7.74-7.79 (m, 1H), 7.79-7.85 (m, 2H), 8.13-8.20 (m, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 61.9, 68.7, 70.4, 70.9, 71.2, 72.5, 125.0, 129.9, 131.5, 132.5, 134.9, 148.4; ESI-MS obsd 336.0735, calcd 336.0748 [(M+H)⁺, M=C₁₂H₁₇NO₈S].

1-Acetyl-4,6-dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (32). A sample of i-Pr₂EtN (66 μL, 0.38 mmol) was added to a suspension of 18 (172.0 mg, 0.253 mmol) and 31 (110.4 mg, 0.329 mmol) in CH₂Cl₂(253 μL) at room temperature. The reaction mixture was heated to 35° C. for 24 h and then allowed to cool to room temperature. The reaction mixture was diluted with ethyl acetate (2 mL), washed with aqueous HCl (1 M, 2 mL) and brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated and chromatographed (silica, ethyl acetate as an eluent) to afford a white solid (182.7 mg, 89%): ¹H NMR (700 MHz, CDCl₃) δ 2.05 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 2.44 (br s, 1H), 2.60 (s, 3H), 3.64 (t, 2H), 3.71-3.78 (m, 4H), 3.78-3.84 (m, 2H), 3.89 (ddd, J=2.5, 5.2, 9.9 Hz, 1H), 4.17-4.23 (m, 2H), 3.97 (t, 2H), 4.20 (dd, J=5.2, 12.4 Hz, 1H), 4.38 (dd, J=2.5, 12.4 Hz, 1H), 5.05 (d, J=7.6 Hz, 1H), 5.20 (dd, J=9.6, 9.9 Hz, 1H), 5.30 (dd, J=9.4, 9.6 Hz, 1H), 5.38 (dd, J=7.6, 9.4 Hz, 1H), 7.25 (s, 1H), 8.69 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 20.7, 20.9, 21.1, 23.9, 61.9, 62.0, 68.3, 70.3, 70.6, 70.9, 71.0, 72.6, 72.7, 100.3, 107.7, 112.1, 116.3, 120.4, 123.2, 131.1, 140.5, 149.8, 168.0, 169.4, 169.5, 170.3, 170.6; ESI-MS obsd 832.0399, calcd 832.0422 [(M+Na)⁺, M=C₃₀H₃₇Br₂NO₁₅].

4,6-Dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-3-yl β-D-glucopyranoside (33). A suspension of 32 (10.2 mg, 0.013 mmol) and K₂CO₃(0.4 mg, 0.003 mmol) in MeOH (250 μL) was stirred for 30 min at room temperature. The reaction mixture was quenched with AcOH (0.4 μL), diluted with CH₂Cl₂, and then passed through silica gel [CH₂Cl₂/MeOH (2:1) as an eluent]. The eluent was concentrated under reduced pressure. The residue was triturated with MeOH/ethyl acetate/hexanes to afford a white solid (7.1 mg, 94%): ¹H NMR (700 MHz, CD₃OD) δ7.49 (s, 1H), 7.25 (s, 1H), 4.74 (d, J=7.7 Hz, 1H), 4.14 (t, J=4.9 Hz, 2H), 3.95 (t, J=4.9 Hz, 2H), 3.92 (d, J=11.8 Hz, 1H), 3.82-3.77 (m, 2H), 3.74-3.65 (m, 5H), 3.59 (t, J=4.8 Hz, 2H), 3.54 (dd, J=7.8, 9.2 Hz, 1H), 3.50-3.43 (m, 1H), 3.43-3.37 (m, 2H); ¹³C NMR (175 MHz, CD₃OD) δ146.8, 139.0, 132.9, 119.9, 116.0, 115.2, 112.2, 107.7, 105.1, 78.3, 78.1, 75.3, 73.7, 73.6, 71.7, 71.54, 71.50, 71.3, 62.7, 62.3; HRMS (ESI-TOF) m/z: [M+Na]⁺ Calcd for C₂₀H₂₇Br₂NNaO₁₀621.9894; found 621.9891.

3-[4-(tert-Butoxycarbonyl)piperazin-1-yl]propane-1-sulfonic acid (36). A sample of 1-(tert-butoxycarbonyl)piperazine (35, 2.011 g, 10.8 mmol) was added to a solution of 1,3-propane sultone (1.319 g, 10.8 mmol) in 1,4-dioxane (5.40 mL) at room temperature. The reaction mixture was heated to 60° C. for 1 h, and then allowed to cool to room temperature. The precipitate was filtered and washed with ethyl acetate to afford a white solid (2.106 g, 63%): ¹H NMR (700 MHz, D₂O) δ 1.47 (s, 9H), 2.17-2.28 (m, 2H), 3.02 (t, J=7.3 Hz, 2H), 3.20-3.39 (m, 2H), 2.65-3.91 (m, 6H), 4.23 (br s, 2H); ¹³C NMR (175 MHz, D₂O) δ 20.3, 28.5, 41.5, 48.7, 52.5, 56.4, 83.8, 156.5; ESI-MS obsd 309.1474, calcd 309.1479 [(M+H)⁺, M=C₁₂H₂₄N₂O₅S].

3-(4-(tert-Butoxycarbonyl)-1-(3-hydroxypropyl)piperazin-1-ium-1-yl)propane-1-sulfonate (37). 3-Bromopropanol (2.17 mL, 24 mmol) was added to a mixture of 36 (1.234 g, 4.00 mmol), NaHCO₃(2.688 g, 32.0 mmol), KI (132.8 mg, 0.80 mmol) in H₂O (1.09 mL) at room temperature. The reaction mixture was heated to 80° C. for 15 h, allowed to cool to room temperature, and washed with Et₂O (20 mL). The residue was suspended in CH₂Cl₂/MeOH (4:1, 25 mL) and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (4:6)] to afford a white solid (1.011 g, 69%): ¹H NMR (700 MHz, CD₃OD) δ 1.48 (s, 9H), 1.95-2.03 (m, 2H), 2.14-2.24 (m, 2H), 2.90 (t, J=6.6 Hz, 2H), 3.46-3.60 (m, 6H), 3.64-3.72 (m, 4H), 3.81 (br s, 4H); ¹³C NMR (175 MHz, CD₃OD) δ 18.7, 25.5, 28.5, 37.8, 39.0, 48.4, 57.4, 57.7, 59.3, 82.4, 155.5, ESI-MS obsd 367.1896, calcd 367.1897 [(M+H)⁺, M=C₁₅H₃₀N₂O₆S].

3-(1-(3-Hydroxypropyl)piperazin-1-ium-1-yl)propane-1-sulfonate trifluoroacetic acid salt (38). A sample of 37 (980.2 mg, 2.67 mmol) was dissolved in trifluoroacetic acid (1.78 mL) at room temperature. After 2 h, the reaction mixture was concentrated under reduced pressure. The residue was triturated with EtOH/Et₂O to afford a pale yellow solid (985.4 mg, 97%): ¹H NMR (700 MHz, CD₃OD) δ 0.75-0.85 (m, 2H), 0.96-1.07 (m, 2H), 1.75 (t, J=6.5 Hz, 2H), 2.40-2.73 (m, 14H); ¹³C NMR (175 MHz, CD₃OD) δ 18.9, 25.6, 38.8, 48.2, 58.3 (br s), 58.8 (br s), 59.1, 163.1 (q, J=34.5 Hz), ESI-MS obsd 267.1371, calcd 267.1373 [(M−CF₃CO₂H+H)⁺, M=C₁₂H₂₃F₃N₂O₆S].

4,6-Dibromo-5-[1-hydroxy-3,6,9-trioxanon-9-yl]-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (39). A suspension of 32 (811.4 mg, 1.00 mmol) and NaHCO₃(8.4 mg, 0.0.10 mmol) in MeOH (5.00 mL) was stirred for 3.5 h at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was suspended in ethyl acetate. The suspension was passed through silica (ethyl acetate as an eluent). The eluent was concentrated under reduced pressure to afford a white solid (650.1 mg, 84%): ¹H NMR (700 MHz, CDCl₃) δ 2.04 (s, 3H), 2.05 (s, 3H), 2.096 (s, 3H), 2.103 (s, 3H), 2.50 (br s, 1H), 3.62-3.68 (m, 2H), 3.71-3.78 (m, 4H), 3.78-3.84 (m, 3H), 3.94-4.00 (m, 2H), 4.13-4.20 (m, 2H), 4.24 (dd, J=4.8, 12.3 Hz, 1H), 4.27 (dd, J=2.8, 12.3 Hz, 1H), 4.97 (d, J=7.8 Hz, 1H), 5.19 (dd, J=9.5, 9.6 Hz, 1H), 5.29 (dd, J=9.3, 9.5 Hz, 1H), 5.37 (dd, J=7.8, 9.6 Hz, 1H), 7.07 (d, J=2.7 Hz, 1H), 7.45 (s, 1H), 7.94 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 20.6, 20.7, 20.8, 21.1, 61.7, 61.9, 68.4, 70.2, 70.4, 70.7, 71.0, 71.9, 72.4, 72.5, 72.9, 101.0, 106.6, 112.0, 114.5, 115.2, 118.7, 131.3, 136.7, 145.9, 169.5, 169.6, 170.3, 170.7; ESI-MS obsd 768.0494, calcd 768.0497 [(M+H)⁺, M=C₂₈H₃₅Br₂NO₁₄].

4-({1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa-4-azadodecan-12-yl)amino}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)piperazin-1-yl])-2-{10-[1-acetyl-3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl}-1,3,5-triazine (40). Cyanuric chloride (20.3 mg, 0.11 mmol) was added to a mixture of 39 (76.9 mg, 0.10 mmol), 1,10-phenanthroline (36.0 mg, 0.20 mmol), and powdered molecular sieves 4 Å (50.0 mg) in CH₂Cl₂(0.50 mL) at room temperature. After 16 h, 38 (49.4 mg, 0.13 mmol) in DMF (0.50 mL) and ⁱPr₂EtN (70 μL, 0.40 mmol) were added. After 3 h, 14 (35.7 mg, 0.11 mmol) in CH₂Cl₂(300 μL) and ⁱPr₂EtN (35 μL, 0.20 mmol) were added. After 4 h, ⁱPr₂EtN (35 μL, 0.20 mmol) was added. After 15 h, the reaction mixture was diluted with CH₂Cl₂(3 mL) and filtered. The filtrate was washed with aqueous citric acid (10%, 3 mL) and brine (3 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. Column chromatography [diol-functionalized silica (ethyl acetate/MeOH=19:1 to CH₂Cl₂/MeOH=5:1) followed by silica (CH₂Cl₂/MeOH=5:1)] afforded a white solid (72.5 mg, 51%): ¹H NMR (700 MHz, CDCl₃, mixture of rotamers) δ 0.91 (m, 2H), 1.26-1.38 (m, 1H), 1.54 (br s, 2H), 1.80-2.00 (m, 2H), 2.01 (s, 3H), 2.04 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.11-2.33 (m, 9H), 2.90 (br s, 2H), 3.15-4.47 (m, 42H), 4.75 (br s, 1H), 4.92 (s, 1H), 5.16 (t, J=9.1 Hz, 1H), 5.21-5.36 (m, 2H), 5.43 (s, 0.5H), 5.53 (s, 0.5H), 5.81 (br s, 0.5H), 6.05 (br s, 0.5H), 7.13 (s, 1H), 7.62 (s, 1H), 10.3 (s, 1H); ¹³C NMR (175 MHz, CDCl₃, mixture of rotamers) δ 17.8, 17.9, 20.1, 20.7, 20.9, 21.1, 21.5, 24.6, 29.1, 36.8, 40.5, 40.7, 40.8, 41.5, 47.4, 53.5, 56.4, 57.0, 58.3, 61.9, 62.7, 66.0, 66.1, 68.4, 69.3, 69.4, 69.7, 69.8, 70.1, 70.20, 70.23, 70.7, 70.8, 71.1, 71.9, 72.5, 73.0, 98.9, 101.1, 106.2, 111.6, 114.7, 115.7, 118.3, 131.5, 136.5, 145.6, 156.9, 165.6, 165.8, 166.7, 167.2, 169.5, 169.6, 170.2, 170.4, 170.8; ESI-MS obsd 717.1884, calcd 717.1888 [(M+2H)²⁺, M=C₅₈H₈₂Br₂N₈O₂₂S].

4-({1-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]-3-oxo-2,7,10-trioxa-4-azadodecan-12-yl)amino}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)piperazin-1-yl])-2-{10-[β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl}-1,3,5-triazine (41). K₂CO₃(0.3 mg, 2 μmol) was added to a solution of 40 in MeOH/CH₂Cl₂(25:6, 310 μL) at room temperature. After 2 h, the reaction mixture was passed through diol-functionalized silica [CH₂Cl₂/MeOH (2:1) as an eluent]. The eluent was concentrated under reduced pressure to afford a white solid (12.5 mg, 98%): ¹H NMR (700 MHz, CD₃OD, mixture of rotamers) δ 0.85-0.97 (m, 2H), 1.28-1.41 (m, 1H), 1.50-1.63 (m, 2H), 1.88-1.99 (m, 2H), 2.07-2.29 (m, 8H), 2.79-2.91 (m, 2H), 3.24-3.33 (m, 2H), 3.37-4.25 (m, 44H), 4.42-4.61 (m, 2H), 4.78 (d, J=8.3 Hz, 1H), 7.27 (d, J=6.8 Hz, 1H), 7.53 (s, 1H); ¹³C NMR (175 MHz, CD₃OD, mixture of rotamers) δ 18.7, 19.0, 21.4, 21.9, 22.0, 25.5, 30.2, 37.9, 38.0, 41.5, 41.7, 48.3, 49.5, 54.8, 55.1, 57.6, 57.7, 59.3, 59.46, 59.52, 62.56, 62.62, 63.7, 67.2, 67.3, 70.6, 70.7, 71.0, 71.1, 71.3, 71.4, 71.50, 71.55, 71.85, 71.88, 73.8, 75.3, 78.2, 78.30, 78.32, 99.6, 105.1, 105.2, 107.7, 112.4, 116.2, 132.9, 139.0, 146.8, 159.2, 166.9, 167.2, 168.2, 168.6, 171.8, 172.2; ESI-MS obsd 633.1670, calcd 633.1677 [(M+2H)²⁺, M=C₅₀H₇₄Br₂N₈O₁₈S].

4,4′,6,6′-Tetrabromo-5,5′-bis[1-hydroxy-3,6,9-trioxanon-9-yl]indigo (43). Samples of 33 (4.98 mg, 8.28 μmol) in DMF (414 μL), β-glucosidase in water (10 units/mL, 828 μL), and acetate buffer (pH 5.0, 7038 μL) were mixed at room temperature. The reaction mixture was incubated at 37° C. under air for 22 h and then allowed to cool to room temperature. The reaction mixture was diluted with H₂O (20 mL) and extracted with CH₂Cl₂(20 mL). The organic layer was washed with brine (10 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. Preparative thin layer chromatography [silica, 0.25 mm, CHCl₃/MeOH (12:1)] afforded an indigo-blue solid (2.4 mg, 66%): ¹H NMR (700 MHz, CDCl₃/CD₃OD=9:1) δ 3.60-3.66 (m, 4H), 3.69-3.76 (m, 8H), 3.79-3.84 (m, 4H), 3.94-4.00 (m, 4H), 4.16-4.21 (m, 4H), 7.31 (s, 1H); ¹³C NMR (175 MHz, CDCl₃/CD₃OD=9:1) δ 61.6, 70.2, 70.4, 70.9, 72.78, 72.79, 115.3, 116.0, 118.1, 122.3, 127.4, 147.9, 149.6, 185.7; ESI-MS obsd 870.8692, calcd 870.8707 [(M+H)⁺, M=C₂₈H₃₀Br₄N₂O₁₀]. To measure the molar absorption coefficient, the title compound (1.6 mg) was dissolved in CHCl₃/MeOH (2:1, 2.93 mL). Then an aliquot (64.0 μL) was withdrawn from this solution and concentrated under reduced pressure. The residue was dissolved in DMF/H₂O (2:1, 1000 μL) to prepare a 40 μM solution. The absorption spectrum was recorded at room temperature: ε_{631 nm}=2.6×10⁴M ⁻¹cM⁻¹.

2,4-Bis{10-[1-acetyl-3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-chloro-1,3,5-triazine (44). Pempidine (38.1 μL, 0.21 mmol) was added to a mixture of cyanuric chloride (11.1 mg, 0.060 mmol), 32 (102.6 mg, 0.13 mmol), and powdered molecular sieves 4Å (12.0 mg) in 1,2-dichloroethane (120 μL) at room temperature. The reaction mixture was heated to 60° C. for 13 h, cooled to room temperature, and passed through a silica pad (ethyl acetate as an eluent). The eluent was concentrated under reduced pressure. Preparative thin layer chromatography [silica, 1.0 mm, 20×20 cm, hexanes/acetone (6:4)] afforded a white solid (55.7 mg, 53%): ¹H NMR (700 MHz, CDCl₃) δ 2.04 (s, 6H), 2.07 (s, 6H), 2.091 (s, 6H), 2.093 (s, 6H), 2.60 (s, 6H), 3.70-3.73 (m, 4H), 3.73-3.82 (m, 4H), 3.83-3.94 (m, 6H), 3.94-3.97 (m, 4H), 4.13-4.23 (m, 6H), 4.38 (dd, J=1.5, 12.3 Hz, 2H), 4.55-4.62 (m, 4H), 5.05, (d, J=7.6 Hz, 2H), 5.17-5.23 (m, 2H), 5.30 (dd, J=9.3, 9.3 Hz, 2H), 5.34-5.41 (m, 2H), 7.25 (s, 2H), 8.68 (br s, 2H); ¹³C NMR (175 MHz, CDCl₃) δ 20.7, 20.9, 21.1, 23.8, 62.0, 68.4, 68.5, 68.9, 70.3, 70.9, 71.0, 72.6, 72.7, 100.3, 107.7, 112.2, 116.3, 120.3, 123.1, 131.0, 140.5, 149.8, 167.9, 169.4, 169.5, 170.3, 170.6, 172.5, 172.7; ESI-MS obsd 1730.0752, calcd 1730.0757 [(M+H)⁺, M=C₆₃H₇₂Br₄C1N₅O₃₀].

2,4-Bis{10-[3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine (45). i-Pr₂EtN (19.2 μL, 0.11 mmol) was added to a solution of 44 (190.8 mg, 0.11 mmol) in CH₂Cl₂/MeOH (5:1, 0.84 mL) at room temperature. After 4 h, 38 (46.0 mg, 0.12 mmol) in MeOH (0.70 mL) and 2,6-lutidine (25.5 μL, 0.22 mmol) was added. After 4 h, the reaction mixture was quenched with acetic acid (12.6 μL, 0.22 mmol) and concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (7:1 to 5:1)] followed by trituration with H₂O afforded a pale yellow solid (114.7 mg, 55%): ¹H NMR (700 MHz, CDCl₃) δ 1.78 (br s, 2H), 1.96-2.16 (m, 26H), 2.88 (br s, 2H), 3.15 (br s, 2H), 3.23 (br s, 2H), 3.36 (br s, 2H), 3.54 (br s, 4H), 3.68-3.99 (m, 22H), 4.04 (br s, 4H), 4.16-4.52 (m, 9H), 4.89 (d, J=7.3 Hz, 2H), 5.16 (dd, J=9.4, 9.4 Hz, 2H), 5.22-5.33 (m, 4H), 7.13 (s, 2H), 7.59 (s, 1H), 7.59 (s, 1H), 9.96 (s, 1H), 9.99 (s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 17.9, 20.7, 20.9, 21.2, 24.6, 37.0, 47.4, 56.4, 56.8, 58.0, 58.2, 61.9, 67.0, 68.4, 69.2, 70.2, 70.6, 70.7, 71.1, 71.8, 72.4, 73.0, 101.2, 0106.2, 111.6, 114.9, 115.7, 118.3, 131.4, 136.5, 145.5, 166.4, 169.6, 169.6, 170.2, 170.8, 171.6; ESI-MS obsd 1876.2076, calcd 1876.2079 [(M+H)⁺, M=C₆₉H₈₉Br₄N₇O₃₂S].

2,4-Bis{10-[3-(β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl]-1,4,7,10-tetraoxadec-1-yl]}-6-[4-(3-hydroxypropyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine (46). K₂CO₃(0.6 mg, 4 μmol) was added to a solution of 45 (39.3 mg, 0.020 mmol) in MeOH/CH₂Cl₂(5:1, 600 μL) at room temperature. After 15 min, H₂O (50 μL) was added. After 2 h, H₂O (150 μL) and K₂CO₃(2.2 mg, 16 μmol) were added. After 1 h, reverse phase silica (320 mg) was added. The mixture was dried under reduced pressure. The residue was purified by column chromatography [reverse phase silica, H₂O to MeOH/H₂O (4:1)] afforded a pale yellow solid (23.9 mg, 77%): ¹H NMR [700 MHz, (CD₃)₂SO] δ 1.78-1.87 (m, 2H), 1.93-2.02 (m, 2H), 2.47-2.56 (m, 2H), 3.15 (t, J=9.1 Hz, 2H), 3.22-3.36 (m, 6H), 3.38-3.55 (m, 11H), 3.55-3.69 (m, 11H), 3.69-3.78 (m, 6H), 3.78-3.86 (m, 4H), 3.93-4.05 (m, 6H), 4.05-4.15 (m, 2H), 4.36-4.45 (m, 4H), 4.56-4.64 (m, 2H), 4.65 (d, J=7.6 Hz, 2H), 4.78 (br s, 1H), 4.95-5.14 (m, 6H), 7.23 (s, 2H), 7.55 (s, 2H), 10.92 (s, 2H); ¹³C NMR [175 MHz, (CD₃)₂SO] δ 17.7, 24.1, 36.8, 47.3, 56.9, 57.6, 60.9, 66.4, 68.4, 69.4, 69.8, 69.9, 70.0, 72.4, 73.5, 76.8, 77.2, 99.5, 103.3, 106.1, 110.4, 113.3, 114.9, 117.9, 131.0, 137.4, 144.8, 166.4, 171.5; ESI-MS obsd 770.5642, calcd 770.5653 [(M+2H)²⁺, M=C₅₃H₇₃Br₄N₇O₂₄S].

Procedure for ε determination for unsubstituted indigo. Indigo (13.1 mg, 50 μmol) was dissolved in DMF (200 mL) to prepare a 250 μM solution. An aliquot (320 μL) was withdrawn from the solution and diluted with DMF/H₂O (1680:1000, 2680 μL) to prepare a 40.0 μM solution. The absorption spectrum was recorded at room temperature. The averages of two runs were calculated.

Procedure for ε determination for 43. Indigo 43 (1.6 mg, 1.8 μmol) was dissolved in CHCl₃/MeOH (2:1, 2.93 mL). An aliquot (64.0 μL) was withdrawn from the solution and concentrated under reduced pressure. The residue was dissolved in DMF/H₂O (2:1, 1000 μL) to prepare a 40 μM solution. The absorption spectrum was recorded at room temperature. ε₆₃₁nm=2.6×10⁴M⁻¹cm⁻¹.

Procedures for Indigogenic Reactions in Table 1

General methods. β-Glucosidase from almonds (lyophilized powder, units/mg solid) and peroxidase from horseradish were purchased from Sigma-Aldrich. β-Glucosidase from Agrobacterium sp. (recombinant, suspension in 3.2 M (NH₄)₂SO₄) was purchased from Megazyme; the concentration in solution was determined by absorption spectroscopy with E^0.1%=2.20 cm⁻¹at 280 nm.³⁷Tritosomes were purchased from XenoTech. Rat liver homogenate was purchased from MP Biomedicals.

Reactions with β-glucosidase from almonds. An indoxyl compound in DMF (5 μL, 20 mM) and β-glucosidase from almonds in H₂O (10 μL, 10 units/mL) were mixed with acetate buffer (85 μL, 50 mM, pH 5.0). The reaction mixture was incubated at 37° C. for 16-19 h and then allowed to cool to room temperature. DMF (300 μL for the reactions of 15, 16, 1, 19, 22, and 25; or 900 μL for the reactions of 42, 20, 21, 23, 24, 26, 27, 30, and 31, respectively) was added to dissolve any indigoid precipitate. The resulting solution was analyzed by absorption spectroscopy.

Reactions with β-glucosidase from Agrobacterium. An indoxyl compound in DMF (2 μL, 5 mM) and β-glucosidase from Agrobacterium in 10 mM phosphate buffer [2 μL, 10 μM, pH 7.0, containing 50 mM NaCl and 0.6 M (NH₄)₂SO₄] were mixed with 50 mM phosphate buffer (96 μL, pH 7.0). The reaction mixture was incubated at 37° C. for 2 h and then centrifuged for 3 min. Any precipitate was separated from the supernatant and dissolved in DMF (200 μL). The resulting solution was analyzed by absorption spectroscopy. The experiment was repeated three times.

Reactions in rat liver homogenate. An indoxyl compound in DMF (5 μL, 20 mM) was mixed with rat liver homogenate (95 μL). The reaction mixture was incubated at 37° C. for 24 h. After allowing to cool to room temperature, the reaction mixture was diluted with DMF (900 μL). The mixture was heated at 70° C. for 2 min and then centrifuged for 2 min. The supernatant was separated from any precipitate. This extraction procedure was repeated two or three times with DMF (100-500 μL). The combined supernatant (1500-1800 μL) was analyzed by absorption spectroscopy.

Reaction of 33 with β-glucosidase from Agrobacterium in rat liver homogenate. A DMF solution of 33 (2 μL, 5 mM) and β-glucosidase from Agrobacterium in 10 mM phosphate buffer [2 μL, 10 μM, pH 7.0, containing 50 mM NaCl and 0.6 M (NH₄)₂SO₄] were mixed with rat liver homogenate (96 μL). The reaction mixture was incubated at 37° C. for 4 h and then centrifuged for 3 min. Any precipitate was separated from the supernatant and then suspended in DMF (200 μL). The suspension was centrifuged for 3 min. The supernatant was analyzed by absorption spectroscopy.

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(32) S.-L. Niu, G. Ulrich, P. Retailleau, J. Harrowfield and R. Ziessel, Tetrahedron Lett., 2009, 50, 3840-3844. “New Insights into the Solubilization of Bodipy Dyes.”

(33) A. Natrajan, D. Sharpe and D. Wen, Org. Biomol. Chem., 2012, 10, 1883-1895. “Zwitterionic Reagents for Labeling, Cross-Linking and Improving the Performance of Chemiluminescent Immunoassays.”

(34) S. J. Holt and P. W. Sadler, Proc. R. Soc. Lond. B, 1958, 148, 495-505. “Studies in Enzyme Cytochemistry. III. Relationships between Solubility, Molecular Association and Structure in Indigoid Dyes.”

(35) E. M. Bowers, L. O. Ragland and L. D. Byers, Biochim. Biophys. Acta, 2007, 1774, 1500-1507. “Salt Effects on β-Glucosidase: pH-Profile Narrowing.”

(36) M. P. Dale, H. E. Ensley, K. Kern, K. A. R. Sastry and L. D. Byers, Biochemistry, 1985, 24, 3530-3539. “Reversible Inhibitors of β-Glucosidase.”

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(38) D. J. Rabiger, M. Y. Chang, S. Matsukawa and K. C. Tsou, J. Heterocycl. Chem., 1970, 7, 307-311. “Synthesis of 5-Iodo- and 5-Nitro-3-Indolyl Phosphates as Cytochemical Substrates for Acid Phosphatase.”

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Example 2

Treatment of an indoxyl β-glucoside with β-glucosidase affords the free indoxyl in situ, which dimerizes to the corresponding indigo. An indoxyl has been examined that bears 4,6-dibromo groups for efficient indigo formation and a linker at the 5-position for further derivatization. Although only β-glucosidase was employed, other enzymes can trigger the cross-linking if the indoxyl bears an appropriate ligand instead of the β-glucoside. We herein describe indoxyl β-glucuronides as the chromogenic cross-linking agents triggered by the enzyme β-glucuronidase. β-Glucuronide was selected as the enzymatically cleavable moiety for the indoxyl for the following reasons: (1) β-glucuronide and β-glucoside are structurally (and thus synthetically) related, (2) 5-bromo-4-chloroindoxyl β-glucuronide has been successfully used for histochemistry,^Pear,Kie(3) the carboxy group in β-glucuronide is expected to afford higher solubility in aqueous solution compared to that of the β-glucoside, and (4) β-glucuronidase is an important target enzyme in cancer therapy.^Gra,Tra

1. Conversion of an Indoxyl β-Glucoside to the Corresponding Indoxyl β-Glucuronide.

Methyl α-glucoside or phenyl β-glucoside is known to be converted into the corresponding glucuronide by selective oxidation of the primary hydroxy group at the 6-position with 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and a co-oxidizing agent such as PhI (OAc)₂or t-BuOCl.^Lu,MelTherefore, direct conversion of indoxyl β-glucosides into the corresponding indoxyl β-glucuronides was investigated. Oxidation of indoxyl β-glucosides with TEMPO/PhI (OAc)₂in the presence of a free indole nitrogen or a free hydroxy group at the indole 5-position was unsuccessful (see the Appendix), which prompted conversion of 1 with use of protecting groups (Scheme 10). The acetyl group of the primary hydroxy group in 1 was selectively deprotected with [t-BuSnOH(Cl)]₂in MeOH to afford 2 in 61% yield.^OriThe phenolic hydroxy group in 2 was protected with t-butyldimethylsilyl (TBS) group to give 3 in 65% yield. Subsequently, the primary hydroxy group in 3 was oxidized with TEMPO/PhI (OAc)₂. After methylation of the resulting carboxylate and deprotection of the TBS group, protected indoxyl β-glucuronide 4 was obtained in 33% yield from 3. Introduction of (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl (BCN) group for copper-free click chemistry via the Mitsunobu reaction followed by removal of the acetyl groups afforded 5 in 44% yield. As the BCN group did not survive under the oxidation conditions with TEMPO/PhI (OAc)₂(see the Appendix), this group was introduced after the oxidation. Hydrolysis of methyl ester 5 gave BCN-indoxyl β-glucuronide sodium salt 6 in 86% yield. Although the conversion of 1 to 6 was achieved, the overall yield (˜5%) was unsatisfactory.

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2. Direct synthesis of indoxyl β-glucuronides. In an alternative route, a solution of 7 in toluene/nitromethane was treated with methyl acetobromo-α-D-glucuronate (8) in the presence of HgBr₂, HgO and molecular sieves 4Å at 40° C. to give 9 in 52% yield (Scheme 11). Debenzylation of 9 was then carried out in tetrahydrofuran/CH₂Cl₂/ethanol containing Pd/C under an atmosphere of H₂to give 10 following recrystallization in 75% yield. Even though the starting material 9 was the pure β-isomer, a small amount of α-10 (2-3% on the basis of ¹H NMR spectroscopy) was obtained after deprotection. This epimer byproduct was readily removed by recrystallization of the mixture in hexanes/CH₂Cl₂. The solution of 10 (in CH₂Cl₂) was then treated with NBS (in CH₂Cl₂) at −78° C. to give 10 in 64% yield. Following the similar procedure as the previous synthetic route (Scheme 10), the BCN group was introduced via a Mitsunobu reaction to afford 11 in 69% yield. Removal of the acetyl groups and hydrolysis of the methyl ester were conducted (as for 6 in Scheme 10) to give the same final product 6. The overall yield of this route is around 11%, which is slightly better than that in Scheme 10.

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3. Introduction of PEG tether between indoxyl and BCN group. A PEGylated chain was introduced with the aim to avoid steric hindrance for the enzymatic cleavage (Scheme 12). Compound 4 was treated with 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-nitrobenzenesulfonate and N,N-diisopropylethylamine (DIPEA) to give the PEGylated indoxyl 12 in 79% yield. Following the same manner for the synthesis of 6, treatment of 12 to a two-step deprotection process afforded PEG-Ind-Gln (14).

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Compound 12 also was treated with 4-nitrophenyl carbonochloridate in the presence of pyridine to give the activated carbonate 15 in 97% yield (Scheme 13). Subsequent reaction with the commercially available building block 16 in the presence of DIPEA gave compound 17. A similar procedure of deprotection was then conducted to give the desired product BCN-PEG-Ind-Gln (19).

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4. Installation of a self-immolative spacer between indoxyl and glucuronide. Based on a well-established strategy in the design of many prodrugs, several potential structures were proposed as shown in Chart 1. We here proposed general synthetic routes (shown in Scheme 14), where solid lines represent reported reactions and dashed lines are unknown steps. A more specific route toward to the targets with a shorter spacer is shown in Scheme 15. Due to the deprotection step of the benzyl group, the amino group is accessible via the reductive condition.

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Experimental Section

General methods. ¹H NMR (700 MHz) and ¹³C NMR (175 MHz) spectra were collected at room temperature in CDCl₃unless noted otherwise. Chemical shifts for ¹H NMR spectra are reported in parts per million (6) relative to tetramethylsilane (or by use of the solvent signal for CD₃OD, δ=3.31 ppm). Chemical shifts for ¹³C NMR spectra are reported in parts per million (6), and spectra were calibrated by using solvent signals [CDCl₃, δ=77.16 ppm; (CD₃)₂SO, δ=39.52 ppm; CD₃OD, δ=49.00 ppm]. Silica gel (40 μm) was used for column chromatography. Preparative TLC separations were carried out on Merck analytical plates precoated with silica gel 60 F₂₅₄. All solvents were reagent grade and were used as received unless noted otherwise. Commercial compounds were used as received.

1-Acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (2)

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Following a general procedure^Oriwith slight modification, dichlorotetrakis(1,1-dimethylethyl)di-μ-hydroxyditin^Dri(6.8 mg, 0.012 mmol) was added to a solution of 1-acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (81.5 mg, 0.12 mmol) in MeOH/CHCl₃(0.80 mL, 5:3) at room temperature. After 12 h, the reaction mixture was diluted with ethyl acetate and passed through silica gel (ethyl acetate as eluent). The eluent was concentrated under reduced pressure. Column chromatography [silica gel, hexanes/acetone (2:1)] followed by trituration with hexanes/acetone afforded a white solid (46.7 mg, 61%): ¹H NMR (700 MHz, CD₃OD) δ 2.00 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.55 (s, 3H), 3.65 (dd, J=6.9, 12.1 Hz, 1H), 3.75 (dd, J=1.5, 12.1 Hz, 1H), 3.90-3.97 (m, 1H), 5.08 (dd, J=9.5, 9.7 Hz, 1H), 5.22 (d, J=8.3 Hz, 1H), 5.32 (dd, J=8.3, 9.0 Hz, 1H), 5.38 (dd, J=9.0, 9.5 Hz, 1H), 7.50 (s, 1H), 8.54 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 20.57, 20.58, 21.0, 23.6, 61.9, 70.3, 72.5, 74.6, 76.3, 100.8, 101.4, 110.9, 112.9, 120.8, 123.9, 129.6, 141.8, 148.9, 170.4, 171.2, 171.3, 171.7; ESI-MS obsd 659.9532, calcd 659.9530 [(M+Na)⁺, M=C₂₂H₂₃Br₂NO₁₁].

1-Acetyl-4,6-dibromo-5-(tert-butyldimethylsilyl)oxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-13-D-glucopyranoside (3)

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Et₃N (14.9 μL, 0.107 mmol) was added to a suspension of 1-acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (34.1 mg, 0.0535 mmol) and tert-butylchlorodimethylsilane (16.1 mg, 0.107 mmol) in CH₂Cl₂(535 μL) at room temperature. After 18 h, 2,2,2-trifluoroacetic acid (16.4 μL, 0.214 mmol), pyridine (4.3 μL, 0.53 mmol), and MeOH (535 μL) were added. After 5 h, the reaction mixture was diluted with ethyl acetate and passed through silica gel (ethyl acetate as eluent). The eluent was concentrated under reduced pressure. Column chromatography [silica gel, hexanes/ethyl acetate (2:3)] afforded a white solid (26.3 mg, 65%): ¹H NMR (700 MHz, CDCl₃) δ 0.35 (s, 3H), 0.36 (s, 3H), 1.05 (s, 9H), 2.05 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.52 (s, 3H), 2.67 (br s, 1H), 3.71-3.85 (m, 3H), 4.99 (d, J=7.8 Hz, 1H), 5.15 (dd, J=9.3, 9.3 Hz, 1H), 5.32 (dd, J=8.6, 9.3 Hz, 1H), 5.37 (dd, J=8.6, 9.3 Hz, 1H), 7.22 (s, 1H), 8.57 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 19.2, 20.8, 21.0, 23.7, 26.4, 29.8, 61.5, 68.8, 70.9, 72.8, 75.1, 100.7, 104.6, 111.0, 114.5, 120.5, 123.0, 129.2, 141.0, 147.1, 168.0, 169.7, 170.1, 170.4; ESI-MS obsd 750.0581, calcd 750.0575 [(M+H)⁺, M=C₂₈H₃₇Br₂NO₁₁Si].

1-Acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid methyl ester (4, route A)

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Following a reported procedure^Luwith modification, (diacetoxyiodo)benzene (26.4 mg, 0.082 mmol) was added to a suspension of 1-acetyl-4,6-dibromo-5-(tert-butyldimethylsilyl)oxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranoside (28.0 mg, 0.037 mmol), 2,2,6,6-tetramethylpiperidine 1-oxyl (1.7 mg, 0.011 mmol), and NaHCO₃(3.1 mg, 0.037 mmol) in MeCN/H₂O (3:1, 273 μL) at room temperature. After 3 h, NaHCO₃(6.2 mg, 0.074 mmol) was added. After 2 h, 2,2,6,6-tetramethylpiperidine 1-oxyl (1.2 mg, 0.0077 mmol) was added. After 1.5 h, (diacetoxyiodo)benzene (12.0 mg, 0.037 mmol) was added. After 30 min, NaHCO₃(15.7 mg, 0.19 mmol) and dimethyl sulfate (28.3 μL, 0.30 mmol) were added. After 4 h, the reaction mixture was diluted with ethyl acetate and passed through silica gel (ethyl acetate as eluent). The eluent was concentrated under reduced pressure. The residue was dissolved in THF (317 μL). AcOH (4.3 μL, 0.075 mmol) and tetra-n-butylammonium fluoride (1.0 M in THF, 56 μL, 0.056 mmol) were added at room temperature. After 2 h, the reaction mixture was diluted with ethyl acetate and passed through silica gel (ethyl acetate as eluent). The eluent was concentrated under reduced pressure. Column chromatography [silica gel, hexanes/acetone (3:2)] afforded a pale brown solid (8.2 mg, 33%): mp 202-204° C.; ¹H NMR (400 MHz, CDCl₃) δ 2.07 (s, 3H), 2.07 (s, 3H), 2.10 (s, 3H), 2.57 (s, 3H), 3.78 (s, 3H), 4.26 (d, J=9.2 Hz, 1H), 5.13 (d, J=6.8 Hz, 1H), 5.28-5.48 (m, 3H), 6.00 (br s, 1H), 7.32 (s, 1H), 8.67 (br s, 1H); ¹³C NMR (175 MHz, CDCl₃) δ 20.7, 20.8, 21.0, 23.7, 53.3, 68.9, 70.7, 72.0, 72.6, 98.4, 100.3, 108.6, 112.3, 120.1, 122.6, 128.7, 139.9, 146.3, 167.0, 168.0, 169.3, 169.5, 170.2; ESI-MS obsd 685.9469, calcd 685.9479 [(M+Na)⁺, M=C₂₃H₂₃Br₂NO₁₂].

5-{[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl β-D-glucopyranosiduronic acid methyl ester (5, route A)

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Diisopropyl azodicarboxylate (3.5 μL, 0.018 mmol) was added to a solution of 1-acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid methyl ester (7.0 mg, 0.11 mmol), (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (1.9 mg, 0.013 mmol), and PPh₃(4.7 mg, 0.018 mmol) in CH₂Cl₂(105 μL) at room temperature. After 3 h, MeOH (420 μL) and K₂CO₃(1.5 mg) were added. After 1.5 h, the reaction mixture was passed through silica gel [CH₂Cl₂/MeOH (2:1) as eluent]. The eluent was concentrated under reduced pressure. Preparative thin layer chromatography [silica gel, 0.25 mm, CHCl₃/MeOH (10:1)] afforded a white solid (2.9 mg, 44%): ¹H NMR (700 MHz, CD₃OD) δ 0.98-1.09 (m, 2H), 1.64-1.80 (m, 3H), 2.13-2.23 (m, 2H), 2.23-2.38 (m, 4H), 3.49 (dd, J=8.8, 9.4 Hz, 1H), 3.59 (dd, J=8.0, 8.8 Hz, 1H), 3.66 (dd, J=9.4, 9.6 Hz, 1H), 3.78 (s, 3H), 3.96 (d, J=9.6 Hz, 1H), 4.09 (d, J=7.8 Hz, 2H), 4.83 (d, J=8.0 Hz, 1H), 7.12 (s, 1H), 7.50 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 20.2, 21.7, 22.0, 30.6, 52.9, 72.8, 73.0, 75.0, 76.8, 77.3, 99.6, 105.3, 107.9, 112.6, 115.4, 116.0, 120.1, 132.9, 138.5, 147.0, 171.1; ESI-MS obsd 650.0000, calcd 649.9996 [(M+Na)⁺, M=C₂₅H₂₇Br₂NO₈].

Sodium 5-{[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl β-D-glucopyranosiduronate (6, route A)

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Aqueous NaHCO₃(100 mM, 46 μL) was added to a solution of 5-(((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)-4,6-dibromo-1H-indole-3-yl β-D-glucopyranosiduronic acid methyl ester (2.9 mg, 0.0046 mmol) in MeOH (184 μL) at room temperature. The reaction mixture was heated to 40° C. for 13 h and then 60° C. for 23 h. The reaction mixture was allowed to cool to room temperature and then concentrated under reduced pressure to afford a white solid (2.5 mg, 86%): ¹H NMR (700 MHz, CD₃OD) δ 0.98-1.08 (m, 2H), 1.65-1.80 (m, 3H), 2.13-2.22 (m, 2H), 2.22-2.36 (m, 4H), 3.51 (dd, J=8.8, 9.2 Hz, 1H), 3.56 (dd, J=9.2, 9.5 Hz, 1H), 3.59 (dd, J=8.1, 8.8 Hz, 1H), 3.68 (d, J=9.5 Hz, 1H), 4.09 (d, J=7.8 Hz, 2H), 4.73 (d, J=8.1 Hz, 1H), 7.35 (s, 1H), 7.49 (s, 1H); ¹³C NMR (175 MHz, CD₃OD) δ 20.1, 21.7, 22.0, 30.6, 72.8, 73.7, 75.1, 76.6, 77.9, 99.6, 105.2, 107.8, 112.3, 115.9, 116.2, 120.0, 132.8, 138.7, 146.8, 176.6; ESI-MS obsd 635.9840, calcd 635.9839 [(M+Na)⁺, M=C₂₄H₂₅Br₂NO₈].

1-Acetyl-5-(benzyl)oxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid methyl ester (9)

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Activated molecular sieves 4Å (250 mg), 1-acetyl-5-(benzyloxy)indolin-3-one (7) (28 mg, 0.10 mmol), acetobromo-α-D-glucuronic acid methyl ester (8) (124 mg, 0.31 mmol) and HgO (34 mg, 0.16 mmol) were placed in a flask and treated with toluene/MeNO₂(4:1, 1.0 mL). The orange suspension was then treated with HgBr₂(7.0 mg, 19 μmol), heated to 40° C. and stirred for 5.5 h. The reaction was quenched by the addition of pyridine (200 μL) and filtered through a silica pad (2 cm×2 cm, acetone). The filtrate was concentrated to give a crude mixture. Column chromatography [silica gel, hexanes/acetone (7:3)] followed by recrystallization in hexanes/CH₂Cl₂afforded a white solid (31 mg, 52%): mp 189-191° C.; ¹H NMR (600 MHz, CDCl₃) δ 8.31 (b, 1H), 7.46 (d, J=7.0 Hz, 2H), 7.39 (t, J=7.7 Hz, 2H), 7.35-7.30 (m, 1H), 7.15 (b, 1H), 7.07-7.01 (m, 2H), 5.40-5.31 (m, 3H), 5.11 (s, 2H), 5.08 (d, J=7.0 Hz, 1H), 4.19 (d, J=8.9 Hz, 1H), 3.74 (s, 3H), 2.55 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 170.1, 169.4, 169.2, 168.0, 166.8, 155.6, 141.1, 137.0, 128.6, 128.0, 127.6, 124.7, 117.7, 115.7, 110.6, 101.5, 100.8, 72.7, 71.8, 71.0, 70.5, 69.0, 53.1, 23.7, 20.7, 20.6, 20.5.

1-Acetyl-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucuronic acid methyl ester (10)

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A suspension of 9 (170 mg, 0.28 mmol) and Pd/C (10 wt %, 30 mg, 28 μmol) in THF/CH₂Cl₂/EtOH (5:4:1, 11 mL) was stirred under an atmosphere of H₂(1 atm) for 1 h. The reaction mixture was filtered through a silica pad (2 cm×2 cm, acetone). The filtrate was concentrated and recrystallized (hexanes/CH₂Cl₂) to afford a white solid (106 mg, 75%): mp 200-202° C.; ¹H NMR (600 MHz, CDCl₃) δ 8.24 (b, 1H), 7.10 (b, 1H), 6.92 (d, J=2.5 Hz, 1H), 6.89 (dd, J=8.9, 2.5 Hz, 1H), 6.13 (s, 1H), 5.41-5.32 (m, 3H), 5.09-5.05 (m, 1H), 4.23-4.19 (m, 1H), 3.76 (s, 3H), 2.54 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 170.4, 169.7, 169.5 168.3, 167.1, 152.8, 141.1, 128.4, 125.1, 117.8, 115.2, 110.8, 103.0, 100.8, 72.7, 71.9, 71.0, 69.1, 53.3, 23.7, 20.79, 20.75, 20.6.

1-Acetyl-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid methyl ester (4, route B)

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A solution of NBS (156 mg, 0.88 mmol) in CH₂Cl₂(10.0 mL) was added dropwise over 30 min to a solution of 10 (215 mg, 0.42 mmol) and 2,6-di-tert-butylpyridine (92 μL, 0.42 mmol) in CH₂Cl₂(6.0 mL) at −78° C. The reaction mixture was allowed to warm to rom temperature, stirred for 2.5 h, and quenched by the addition of 10% aqueous Na₂S₂O₃. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated. Column chromatography (silica, CH₂Cl₂with 1% to 4% acetone) afforded a white solid (242 mg, 87%): the characterization data (¹H NMR) were consistent with the product from route A.

1-Acetyl-5-{[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-5-hydroxy-1H-indol-3-yl 2,3,4-tri-O-acetyl-β-D-glucopyranosiduronic acid methyl ester (11)

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Diisopropyl azodicarboxylate (13 μL, 63 μmol) was added to a solution containing 4 (34 mg, 51 μmol), (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (9.3 mg, 62 μmol), and PPh₃(17 mg, 65 μmol) in CH₂Cl₂(0.51 mL) at room temperature. The reaction mixture was stirred for 1 h and then quenched by the addition of H₂O. The organic layer was washed with brine, dried (Na₂SO₄), and concentrated. Column chromatography (silica, hexanes with 0% to 4% acetone) afforded a white solid (28 mg, 69%): ¹H NMR (500 MHz, CDCl₃) δ 8.71 (s, 1H), 7.35 (s, 1H), 5.47-5.31 (m, 3H), 5.14 (d, J=7.0 Hz, 1H), 4.27 (d, J=9.6 Hz, 1H), 4.10 (d, J=7.8 Hz, 2H), 3.78 (s, 3H), 2.58 (s, 3H), 2.37-2.28 (m, 4H), 2.28-2.20 (m, 2H), 2.11 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 1.79-1.68 (m, 2H), 1.06 (dd, J=11.4, 8.5 Hz, 2H).

5-{[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl β-D-glucopyranosiduronic acid methyl ester (5, route B)

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A suspension of 11 (28 mg, 35 μmol) and K₂CO₃(4.8 mg, 35 μmol) in CH₂Cl₂/MeOH (1:4, 1.8 mL) was stirred at room temperature for 1.5 h and then quenched by the addition of acetic acid (7.0 μL, 0.12 mmol). The crude mixture was filtered through a silica pad (2 cm×2 cm, methanol). The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (9:1)] to afford a pale-yellow solid (14 mg, 64%): the characterization data (′H NMR) were consistent with the product from route A.

Sodium 5-{[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl]methoxy}-4,6-dibromo-1H-indole-3-yl β-D-glucopyranosiduronate (6, route B).

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A solution of 5 (14 mg, 22 μmol) in MeOH (0.88 mL) was treated with aqueous NaHCO₃(100 mM, 0.22 mL) at room temperature and stirred at 60° C. for 32 h. The reaction mixture was allowed to cool to room temperature and then concentrated. The residue was washed with hexanes (3.0 mL×3) and CH₂Cl₂(3.0 mL×3) to give the title compound as a pale-brown solid (13 mg, 93%): the characterization data (′H NMR) were consistent with the product from route A.

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N,N-Diisopropylethylamine (DIPEA, 64 μL, 0.37 mmol) was added to a solution containing 4 (122 mg, 0.18 mmol) and 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-nitrobenzenesulfonate (100 mg, 0.30 mmol) in CH₂Cl₂(1.0 mL) at room temperature. The reaction mixture was stirred for 36 h and then concentrated. Column chromatography (silica, CH₂Cl₂with 0% to 70% ethyl acetate) afforded a white amorphous non-crystalline solid (116 mg, 79%): ¹H NMR (600 MHz, CDCl₃) δ 8.71 (s, 1H), 7.35 (s, 1H), 5.47-5.37 (m, 2H), 5.34 (t, J=8.9 Hz, 1H), 5.13 (d, J=7.0 Hz, 1H), 4.26 (d, J=9.7 Hz, 1H), 4.22-4.16 (m, 2H), 4.00-3.94 (m, 2H), 3.84-3.78 (m, 2H), 3.78 (s, 3H), 3.77-3.70 (m, 4H), 3.67-3.60 (m, 2H), 2.57 (s, 3H), 2.10 (s, 3H), 2.06 (s, 3H), 2.06 (s, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 170.1, 169.3, 169.2, 167.9, 166.8, 149.7 140.1, 131.0, 123.0, 120.3, 116.2, 112.4, 107.5, 100.2, 72.53, 72.50, 72.4, 71.9, 70.9, 70.61, 70.5, 70.2, 68.8, 61.8, 53.1, 23.7, 20.9, 20.64, 20.55].

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A suspension of 12 (15 mg, 19 μmol) and K₂CO₃(2.7 mg, 19 μmol) in CH₂Cl₂/MeOH (1:4, 0.95 mL) was stirred at room temperature for 40 min and then quenched by the addition of acetic acid (3.0 μL, 52 μmol). The crude mixture was filtered through a silica pad (2 cm×2 cm, methanol). The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/MeOH (9:1)] to afford a pale-yellow amorphous solid (10 mg, 84%): ¹H NMR (600 MHz, CD₃OD) δ 7.49 (s, 1H), 7.12 (s, 1H), 4.82 (d, J=7.6 Hz, 1H), 4.14 (t, J=4.9 Hz, 2H), 3.98-3.91 (m, 3H), 3.81-3.75 (m, 5H), 3.70-3.63 (m, 5H), 3.61-3.55 (m, 3H), 3.48 (t, J=9.1 Hz, 1H).

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A solution of 13 (10 mg, 16 μmol) in MeOH (0.64 mL) was treated with aqueous NaHCO₃(100 mM, 0.16 mL) at room temperature and then stirred at 60° C. for 18 h. The reaction mixture was allowed to cool to room temperature and then concentrated. Column chromatography [silica, CH₂Cl₂/MeOH (4:1 to 1:4)] afforded a white solid (8.8 mg, 86%): ¹H NMR (600 MHz, CD₃OD) δ 7.48 (s, 1H), 7.34 (s, 1H), 4.72 (d, J=7.7 Hz, 1H), 4.14 (t, J=4.8 Hz, 2H), 3.94 (t, J=4.8 Hz, 2H), 3.82-3.77 (m, 2H), 3.71-3.64 (m, 5H), 3.61-3.53 (m, 4H), 3.50 (t, J=9.0 Hz, 1H); ¹³C NMR (150 MHz, CD₃OD) δ 175.2, 145.3, 137.4, 131.6, 118.6, 114.8, 114.6, 110.6, 106.2, 103.7, 76.5, 75.2, 73.7, 72.32, 72.27, 72.2, 70.3, 70.1, 69.9, 60.8.

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A suspension of 12 (60 mg, 75 μmol), 4-nitrophenyl carbonochloridate (24 mg, 119 μmol), and activated molecular sieves 4Å (150 mg) in anhydrous CH₂Cl₂(3.0 mL) was treated with pyridine (12 μL, 150 μmol) and stirred at room temperature for 6 h. The crude mixture was filtered through a silica pad (2 cm×2 cm, ethyl acetate). The filtrate was concentrated and chromatographed (silica, CH₂Cl₂with 0% to 20% ethyl acetate) to afford a white amorphous non-crystalline solid (70 mg, 97%); ¹H NMR (600 MHz, CDCl₃) δ 8.69 (s, 1H), 8.26-8.20 (m, 2H), 7.39-7.36 (m, 2H), 7.35 (s, 1H), 5.47-5.31 (m, 3H), 5.13 (d, J=6.9 Hz, 1H), 4.48-4.43 (m, 2H), 4.27 (d, J=9.7 Hz, 1H), 4.23-4.14 (m, 2H), 4.00-3.94 (m, 2H), 3.88-3.83 (m, 3H), 3.85-3.80 (m, 3H), 3.80-3.74 (m, 6H), 2.57 (s, 3H), 2.10 (s, 3H), 2.06 (s, 6H).

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A solution of 13 (5.4 mg, 5.6 μmol) and 16 (3.8 mg, 12 μmol) in anhydrous CH₂Cl₂(0.50 mL) was treated with DIPEA (2.0 μL, 11 μmol) and stirred at room temperature for 24 h. The crude mixture was concentrated and chromatographed (silica, CH₂Cl₂to ethyl acetate) to afford a pale-yellow amorphous solid (5.6 mg, 87%): ¹H NMR (700 MHz, CDCl₃) δ 8.72 (s, 1H), 7.36 (s, 1H), 5.45-5.38 (m, 2H), 5.36-5.30 (m, 2H), 5.14 (d, J=7.1 Hz, 1H), 4.26 (d, J=9.7 Hz, 1H), 4.25-4.21 (m, 2H), 4.19-4.13 (m, 4H), 3.96 (t, J=5.0 Hz, 2H), 3.81-3.76 (m, 5H), 3.74-3.68 (m, 4H), 3.61-3.59 (m, 4H), 3.58-3.54 (m, 4H), 3.41-3.36 (m, 4H), 2.58 (s, 3H), 2.32-2.18 (m, 6H), 2.10 (s, 3H), 2.06 (s, 3H), 2.06 (s, 3H), 1.60-1.56 (m, 2H), 0.94 (t, J=9.8 Hz, 2H).

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A suspension of 17 (5.6 mg, 4.9 μmol) and K₂CO₃(1.3 mg, 9.4 μmol) in MeOH (1.0 mL) was stirred at room temperature for 1 h and then quenched by the addition of acetic acid (2.0 μL, 34 μmol). The crude mixture was concentrated and chromatographed [silica, CHCl₃/MeOH (9:1)] to afford a pale-yellow amorphous solid (3.4 mg, 71%): ¹H NMR (700 MHz, CD₃OD) δ 7.52 (s, 1H), 7.14 (s, 1H), 4.85 (d, J=7.7 Hz, 1H), 4.21-4.11 (m, 6H), 3.98 (d, J=9.8 Hz, 1H), 3.96 (t, J=4.9 Hz, 2H), 3.81-3.76 (m, 5H), 3.74-3.66 (m, 5H), 3.62-3.57 (m, 5H), 3.55-3.49 (m, 6H), 3.32-3.27 (m, 4H), 2.27-2.13 (m, 6H), 1.64-1.51 (m, 2H), 0.96-0.85 (m, 2H).

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A solution of 18 (3.4 mg, 3.5 μmol) in MeOH (0.25 mL) was treated with aqueous NaHCO₃(100 mM, 50 μL) at room temperature and then stirred at 60° C. for 18 h. The reaction mixture was allowed to cool to room temperature and then concentrated. Column chromatography [silica, CH₂Cl₂/MeOH (4:1 to 1:4)] afforded a white solid (2.8 mg, 81%).

APPENDIX

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REFERENCES

(Pea) Pearson, B.; Standen, A. C.; Esterly, J. R. Histochemical β-Glucuronidase Distribution in Mammalian Tissue as Detected by 5-Bromo-4-Chloroindol-3-yl-β-D-glucopyruroniside. Lab. Investig. 1967, 17, 217-224.

(Kie) Kiernan, J. A. Indigogenic Substrates for Detection and Localization of Enzymes. Biotech. Histochem. 2007, 82, 73-103.

(Gra) Graaf, M.; Boven, E.; Scheeren, H.; Haisma, H.; Pinedo, H. Beta-Glucuronidase-Mediated Drug Release. Curr. Pharm. Des. 2002, 8, 1391-1403.

(Tra) Tranoy-Opalinski, I.; Legigan, T.; Barat, R.; Clarhaut, J.; Thomas, M.; Renoux, B.; Papot, S. β-Glucuronidase-Responsive Prodrugs for Selective Cancer Chemotherapy: An Update. Eur. J. Med. Chem. 2014, 74, 302-313.

(Ori) Orita, A.; Hamada, Y.; Nakano, T.; Toyoshima, S.; Otera, J. Highly Efficient Deacetylation by Use of the Neutral Organotin Catalyst [tBu₂SnOH(Cl)]₂. Chem. Eur. J. 2001, 7, 3321-3327.

(Dri) Driguez, P.-A. Dichlorotetrakis (1,1-Dimethylethyl)Di-μ-Hydroxyditin. In Encyclopedia of Reagents for Organic Synthesis; John Wiley & Sons, Ltd: Chichester, U K, 2012; pp 1-3.

(Lu) Lu, H.; Drelich, A.; Omri, M.; Pezron, I.; Wadouachi, A.; Pourceau, G. Catalytic Synthesis of a New Series of Alkyl Uronates and Evaluation of Their Physicochemical Properties. Molecules 2016, 21, 1301.

(Mel) Melvin, F.; McNeill, A.; Henderson, P. J. F.; Herbert, R. B. The Improved Synthesis of β-D-Glucuronides Using TEMPO and t-Butyl Hypochlorite. Tetrahedron Lett. 1999, 40, 1201-1202.

Example 3

Enzymatic studies of β-glucuronidase enzymes from E. coli and bovine liver have been reported by Antunes, I. F., et al. “Synthesis and Evaluation of [18F]-FEAnGA as a PET Tracer for 13-Glucuronidase Activity,” Bioconjug. Chem. 2010, 21, 911-920. Compounds of the present invention can be tested with a β-glucuronidase enzyme from E. coli or bovine liver. Both enzymes are readily available for studies. Enzymatic cleavage of a glucuronide releases a nitrophenol, the absorption of which is readily detected by absorption spectroscopy.

Example 4

An A₂BC-functionalized molecule (V) (i.e., a molecule that includes two scaffold cross-linking units (A₂), a bioconjugatable handle (B), and a molecular entity (C) such as a dye, docking group, reactive handle, or biomolecule) has been prepared that contains a pair of alkoxyamino-substituted triazines for branching, two dibromoindoxyl β-glucosides (A₂), an azide (B), and an aminocoumarin dye (C). Compound V additionally bears a sulfobetaine moiety to impart greater water solubility. The rationale for the dibromoindoxyl moieties stems from systematic studies of various indoxyl-glucoside substrates with β-glucosidase enzymes to identify molecular designs that (1) are compatible with enzymatic cleavage, (2) undergo facile indigoid dye formation, (3) are synthetically accessible, and (4) support incorporation into larger architectures via bioconjugation chemistry.

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Refined synthesis of indoxyl species. A refined synthesis of the fully protected 5-hydroxyindoxyl-glucoside F-6 was developed (Scheme 16) to avoid a mixture of β/α epimers. Indole F-1 was acetylated using acetic anhydride and triethylamine in the presence of a catalytic amount of 4-dimethylaminopyridine (DMAP) to afford the N-acetylated indole F-2.^cmpdO-2The Baeyer-Villiger oxidation^Bourto convert F-2 to F-3 was carried out in toluene (rather than dichloromethane), affording a low yield (32%) but the starting material F-2 was recovered in 42% yield and recycled. The glucosidation of F-3 with acetobromo-α-D-glucose (F-4) was carried out in a mixture of toluene and nitromethane, affording stereoselective formation of β-glucoside F-5. ¹H NMR investigation of F-5 showed >99% stereochemical purity at the anomeric carbon. A single-crystal X-ray structure determination (recrystallization from hexanes/CH₂Cl₂) also showed the anomer (FIG. 12). Deprotection of the acetyl and benzyl groups of F-5 provided 5-hydroxyindoxyl-glucoside F-7 in 89% yield, while debenzylation of F-5 afforded acetyl-protected 5-hydroxyindoxyl-glucoside F-6 in 99% yield (see Example 1 where F-1 is compound 8, F-5 is compound 9, and F-7 is compound 10).

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Triazine-based A₂BC architectures. Cyanuric chloride (F-8) was treated with 2 equiv of F-7 followed by tyramine to afford F-9 in 56% yield (Scheme 17). The two indoxyl moieties in F-9 showed distinguishable signals in NMR spectroscopy owing to slow rotation of the C—N bond between the triazine ring and the tyramine unit. Treatment of N-(6-hydroxyhexyl)maleimide (F-10) (K. A. Keller, et al., Tetrahedron Lett., 2005, 46, 1181-1184. “A Thermally-Cleavable Linker for Solid-Phase Synthesis”) with cyanuric chloride (F-8) followed by F-9 in the presence of bases gave F-11 in 77% yield. Ultimately we found that F-9 did not form the corresponding indigoid dye upon treatment with β-glucosidase, while treatment with tritosomes gave the indigoid dye albeit in low yield (ca. 4%). While F-9 and F-11 were attractive from a synthetic standpoint given the ability to derivatize the 5-hydroxy group of the indoxyl unit without protection of the four hydroxy groups of the glucosyl unit, the reaction with a β-glucosidase was sine qua non in the molecular design. Accordingly, we moved to the design of a more suitable architecture.

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Compound V was designed for preparation through selective and successive substitution of cyanuric chloride (F-8) (C. Afonso, N. Lourenco and A. Rosatella, Molecules, 2006, 11, 81-102; M. B. Steffensen, et al., J. Polym. Sci. Part A Polym. Chem., 2006, 44, 3411-3433; A. E. Enciso, et al., Polym. Chem., 2014, 5, 4635-4640). The assembly relied on two protected indoxyl-glucoside units (F-12) and a sulfobetaine-amino alcohol (F-13). Indoxyl F-12 emerged from a lengthy study of the interplay of substituents that enable attachment of a bioconjugatable tether and facile formation of the corresponding indigoid dye (see Example 1). Sulfobetaine F-13 emerged from studies of bis(indoxyl-glucoside) molecules wherein the intervening linker imparts water solubility (see Example 1 where F-12 is compound 32 and F-13 is compound 38). The assembly of protected indoxyl F-12 (two units) and sulfobetaine-amino alcohol F-13 onto a triazine ring afforded F-14 in 29% yield in 2 steps from F-8, as described previously (see Example 1 where F-14 is compound 45). Selective substitution (M. Kunishima, et al., J. Fluorine Chem., 2016, 190, 68-74) of one of three chlorines in cyanuric chloride F-8 by alcohol F-14 was carried out with 1,10-phenanthroline as a base at room temperature (Scheme 18). Subsequently, azide-PEG₅-amine F-15 was added to the reaction mixture to replace the second chlorine. After the solvent was changed to DMF, the third substitution for chlorotriazine intermediate F-16 (used without isolation) with coumarin-amine F-17 (Y. Shiraishi, et al., Org. Biomol. Chem. 2010, 8, 1310-1314) afforded F-18 in 39% yield from F-14. Treatment of F-18 with basic methanol caused removal of the acetyl groups and gave V in quantitative yield.

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Oligomers of the triazine-based A₂BC construct.

The oligomerization of compound V was examined by treatment with the enzyme β-glucosidase. The reaction mixture contained 50 μM of compound V and 200 nM of β-glucosidase (250-fold ratio). The reaction mixture was only faintly blue but upon centrifugation after 5 h of incubation, a blue precipitate was obtained. According to the colors of samples (FIG. 13, panel A), the yield of precipitate is low by comparison with our previously reported enzyme-substrate pairs (see Example 1). Multiple additions of enzyme (4×0.20 μM of enzyme stock, every 2 h) and longer incubation times (10 h or 24 h) were also tested but no increase in solution intensity or precipitate was observed. For the samples after 300 min of incubation, the precipitates suspended in H₂O were screened by optical microscopy (FIG. 13, panel B) and DLS (FIG. 13, panel C). The size of aggregates is between 100 and 1000 nm as measured by DLS, which is consistent with the images obtained by optical microscopy (˜1 μm).

Quantitation of the indigogenic compounds in the precipitate and supernatant was carried out by absorption spectroscopy and multicomponent analysis (MCA) (M. Taniguchi, et al., Photochem. Photobiol., 2018, 94, 277-289) (FIG. 13, panels D and E). MCA analysis is essential due to the overlaid spectra of indigo and coumarin at 362 nm, the peak wavelength for the coumarin. Spectral deconvolution relied on knowledge of the molar absorption coefficient at 362 nm of coumarin (ϵ_cou362=2.0×10⁴M⁻¹cm⁻¹, measured in DMF) and indigo model compound F19 (see Example 1 where F-19 is compound 43) shown in Chart 2 (ϵ_ind362=0.81×10⁴M⁻¹cm⁻¹, measured in DMSO/H₂O, v/v=2/1); and the molar absorption coefficient of F19 at 630 nm (ε_ind630=2.6×10⁴M⁻¹cm⁻¹, measured in DMSO/H₂O, v/v=2/1). The residual upon MCA was smaller than any of the components, as required for accurate analysis (Y. Zhang, et al., Phytochem. Anal., 2018, 29, 205-216). In this manner, the precipitate is composed of 51.6 nmol of coumarin and 2.8 nmol of indigoid dye; the supernatant is composed of 11.1 nmol of coumarin and 0.3 nmol of indigoid dye.

The results obtained from the above analysis are as follows: (1) the total quantity of indigoid dye (3.1 nmol) corresponds to a yield of 3.1%; (2) the indigoid dye in the precipitate (2.8 nmol) is 10 times greater than that in the supernatant (0.3 nmol), hence aggregation ensued following the indigogenic reaction; (3) the total quantity of compound V added (100 nmol) exceeds the total amount of coumarin calculated by MCA (62.7 nmol) may stem from experimental error, loss on handling, and/or alteration of the molar absorption coefficients of the indigoid dye in DMF employed in the MCA method. Regardless, the extent of oligomerization was low, which may stem from toxicity of the substrate to the β-glucosidase or aggregation of compound V prior to or during the course of enzymatic action. Further studies are required to better understand the origin of this result.

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Experimental Section
Oligomerization of Compound V.

Materials. DMF (HPLC grade) was purchased from Alfa Aesar. H₂O (molecular biology grade) for buffer preparation was purchased from Millipore Sigma. Compound V (5.6 mg) was dissolved in DMF (26 μL) to prepare 100 mM stock solution. Pi buffer was prepared freshly at 10 mM, pH 7. The enzyme β-glucosidase from Agrobacterium sp. was purchased from MegaZyme and was dissolved in Pi buffer at 10 μM to prepare the stock solution.

The stock solution of compound V was 1000-fold and 2000-fold diluted with DMF for absorption screening, by which the molar absorption coefficients were estimated: ε_{310 nm}=1.37×10⁴M⁻¹cm⁻¹, ε_{362 nm}=2×10⁴M⁻¹cm⁻¹. The absorption coefficient of compound V in Pi buffers (containing 1-5% DMF with 1% DMSO) was not obtained due to aggregation.

To screen the oligomerization procedure of compound V, samples of Pi buffer (1859 μL), DMF (100 μL), □-glucosidase stock (40 μL) and compound V stock (1 μL) were mixed in a 2-mL Eppendorf tube. The resulting mixed sample (2.0 mL) contains 50 μM of compound V as the substrate and 0.20 μM of β-glucosidase as the enzyme. Three identical samples were prepared for different assays. For a control sample, 40 μL of Pi buffer was added as a replacement for the β-glucosidase stock solution. The tubes were incubated at 37° C. for 5 h, and pictures of the tubes were captured at 0, 15, 30, 60, 120, 180 and 300 min. After 300 min, the tubes were centrifuged at 20,000×g for 10 min to isolate any precipitate from the supernatant. No precipitate formed in the control sample.

Three samples were treated differently for three assays: (1) for sample 1, the precipitate was dissolved in 100 μL of DMF for absorption screening; (2) for sample 2, the precipitate was suspended in 100 μL of H₂O for microscopic imaging; and (3) for sample 3, the precipitate was suspended in 1000 μL of H₂O for dynamic light scattering (DLS) assay. The supernatant of sample 1 was freeze-dried under high vacuum and afterwards dissolved in 100 μL of DMF for absorption screening. The absorption spectra of the precipitates and supernatants were employed for quantitation of indigo and coumarin.

Microscopic imaging of the suspended aggregates was carried out using a Zeiss Axio Observer Z1 inverted microscope with 40× objective lens in the phase contrast mode. DLS assay was carried out with a Malvern Zetasizer Nano. Multicomponent analysis (MCA) was carried out using the software PhotoChemCAD 3 (M. Taniguchi, et al., Photochem. Photobiol., 2018, 94, 277-289) with the following parameters: Range: 290-700 nm, Selected points: 362, 576 and 631 nm.

General. ¹H NMR (400 MHz) and ¹³C NMR (175 MHz) spectra were collected at room temperature unless noted otherwise. Silica (40 μm) was used for column chromatography. All solvents were reagent grade and were used as received unless noted otherwise. Commercial compounds were used as received. Known compounds (F-12-F-14 (see Example 1), F-7 (see Example 1), F-10 (K. A. Keller, et al., Tetrahedron Lett., 2005, 46, 1181-1184), and F-17 (Y. Shiraishi, et al., Org. Biomol. Chem. 2010, 8, 1310-1314)) were prepared as described in the literature. Cyanuric chloride (F-8) was recrystallized from hexanes/CH₂Cl₂before use. Silica gel (40 μm) and Diol-functionalized silica gel (40-63 μm) were used for column chromatography. Preparative TLC separations were carried out on Merck analytical plates precoated with silica gel 60 F₂₅₄.

1-Acetyl-5-(benzyloxy)-1H-indole-3-carbaldehyde (F-2) (A. Andreani, et al., J. Med. Chem., 2001, 44, 4011-4014). 4-Dimethylaminopyridine (104.9 mg, 0.86 mmol) was added to a suspension of 5-(benzyloxy)-1H-indole-3-carbaldehyde (F-1, 21.57 g, 85.8 mmol), triethylamine (23.9 mL, 171 mmol), and Ac₂O (16.2 mL, 171 mmol) in CH₂Cl₂(215 mL) at room temperature. After 40 min, the reaction mixture was washed with aqueous HCl (2 M, 200 mL), saturated aqueous NaHCO₃(100 mL), and brine (100 mL). The organic layer was dried (Na₂SO₄) and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/ethyl acetate (40:1)] to afford a pale brown solid (20.44 g, 81%): mp 120-121° C.;¹H NMR (400 MHz, CDCl₃) δ 2.58 (s, 3H), 5.08 (s, 2H), 7.04 (d, J=8.0 Hz, 1H), 7.20-7.60 (m, 5H), 7.75 (s, 1H), 7.82 (s, 1H), 8.19 (d, J=8.0 Hz, 1H), 9.99 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 23.5, 70.4, 105.1, 116.3, 117.2, 122.2, 127.0, 127.7, 128.0, 128.6, 130.9, 135.6, 136.9, 156.8, 168.3, 185.6; ESI-MS obsd 294.1126, calcd 294.1125 [(M+H)⁺, M=C₁₈H₁₅NO₃].

1-Acetyl-5-(benzyloxy)indolin-3-one (F-3) (C. D. Nenitzescu and D. Râileanu, Chem. Ber., 1958, 91, 1141-1145). Peracetic acid (32 wt % solution in acetic acid, 17.4 mL, 73.3 mmol) was added to a suspension of F-2 (21.5 g, 73.3 mmol) and sodium acetate (12.0 g, 146 mmol) in toluene (293 mL) at 0° C. The reaction mixture was stirred at 0° C. for 30 min followed by room temperature for 18 h. The reaction mixture was quenched by the addition of aqueous Na₂S₂O₃(10%, 100 mL) and filtered through Celite. The filtrate was washed with saturated aqueous NaHCO₃(200 mL) and brine (100 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated and chromatographed [silica, CH₂Cl₂/ethyl acetate (25:1)] to afford recovered F-2 (8.94 g, 42%) and the title compound as a pale yellow solid (6.61 g, 32%): mp 163-164° C.; ¹H NMR (300 MHz, CDCl₃) δ 2.26 (s, 3H), 4.24 (s, 2H), 5.04 (s, 2H), 7.17 (d, J=2.1 Hz, 1H), 7.26-7.46 (m, 6H), 8.46 (d, J=9.3 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 24.0, 56.6, 70.5, 105.8, 119.8, 125.7, 126.8, 127.6, 128.3, 128.7, 136.2, 148.8, 155.6, 167.6, 194.5; ESI-MS obsd 282.1125, calcd 282.1125 [(M+H)⁺, M=C₁₇H₁₅NO₃].

1-Acetyl-5-benzyloxy-1H-indol-3-yl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (F-5). A sample of HgBr₂(0.937 g, 0.65 mmol) was added to a mixture of F-3 (3.657 g, 13.0 mmol), acetobromo-α-D-glucose (F-4, 10.69 g, 26.0 mmol), HgO (2.816 g, 13.0 mmol), powdered molecular sieves 4 Å (26.0 g), and toluene/MeNO₂(2:1, 130 mL) at room temperature. After 11 h, acetobromo-α-D-glucose (2.673 g, 6.5 mmol) was added. After 3 h, the reaction mixture was treated with pyridine (3.1 mL, 39 mmol) and filtered. The filtrate was concentrated and chromatographed [silica, hexanes/acetone (7:3)] to afford a white solid (6.98 g, 88%): mp 146-149° C.; ¹H NMR (700 MHz, CDCl₃) δ 2.05 (s, 3H), 2.06 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.58 (s, 3H), 3.83-3.89 (m, 1H), 4.22-4.30 (m, 2H), 5.01 (d, J=6.0 Hz, 1H), 5.07-5.15 (m, 2H), 5.15-5.22 (m, 1H), 5.28-5.35 (m, 2H), 7.01 (s, 1H), 7.06 (d, J=9.0 Hz, 1H), 7.11 (br s, 1H), 7.30-7.36 (m, 1H), 7.36-7.43 (m, 2H), 7.43-7.48 (m, 2H), 8.33 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 20.65, 20.68, 20.8, 23.8, 62.1, 68.3, 70.6, 71.2, 72.5, 72.6, 100.8, 101.7, 110.4, 115.7, 117.7, 125.0, 127.6, 128.1, 128.5, 128.7, 137.0, 141.4, 155.7, 167.9, 169.3, 169.5, 170.2, 170.6; ESI-MS obsd 612.2071, calcd 612.2076 [(M+H)⁺, M=C₃₁H₃₃NO₁₂]. Suitable crystals for X-ray analysis were obtained by recrystallization (hexanes/CH₂Cl₂).

2,4-Bis[3-(β-D-glucopyranosyloxy)-1H-indol-5-yloxy]-6-[2-(4-hydroxyphenyl)ethylamino]-1,3,5-triazine (F-9). Ethyldiisopropylamine (87.1 μL, 0.500 mmol) was added to a suspension of cyanuric chloride (F-8, 36.9 mg, 0.200 mmol) and F-7 (130.7 mg, 0.420 mmol) in MeCN (1.00 mL) at 0° C. After 10 min, DMF (0.40 mL) was added at 0° C. Then the reaction mixture was allowed to warm to rt and stirred for additional 2 h. Tyramine (30.2 mg, 0.220 mmol) and ethyldiisopropylamine (69.7 μL, 0.400 mmol) was added. After 19 h, the reaction mixture was treated with AcOH (23 μL, 0.40 mmol) and concentrated under reduced pressure. Column chromatography [diol-functionalized silica gel, CH₂Cl₂/MeOH (6:4)] followed by washing with H₂O afforded a pale yellow solid (94.1 mg, 56%): ¹H NMR [400 MHz, CD₃OD] δ 2.31-2.52 (m, 2H), 3.06 (t, J=7.8 Hz, 2H), 3.05-3.17 (m, 1H), 3.24-3.56 (m, 7H), 3.62-3.78 (m, 2H), 3.78-3.96 (m, 2H), 4.64 (d, J=8.0 Hz, 1H), 4.68 (d, J=7.6 Hz, 1H), 6.35-6.48 (m, 4H), 6.90 (dd, J=2.0, 8.8 Hz, 1H), 6.94 (dd, J=2.0, 8.8 Hz, 1H), 7.18 (s, 1H), 7.24 (s, 1H), 7.29 (d, J=8.8 Hz, 1H), 7.34 (d, J=8.8 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD) δ 35.6, 44.4, 62.5, 62.6, 70.0, 71.3, 71.5, 75.0, 77.9, 78.0, 78.2, 106.1, 111.0, 112.9, 114.4, 115.0, 116.1, 117.3, 117.5, 121.6, 121.7, 130.7, 131.0, 133.05, 133.15, 139.18, 139.21, 146.5, 146.8, 156.4, 169.3, 174.1, 174.6, ESI-MS obsd 835.2756, calcd 835.2781 [(M+H)⁺, M=C₃₉H₄₂N₆O₁₅]

2,4-Bis[3-(β-D-glucopyranosyloxy)-1H-indol-5-yloxy]-6-[2-(4-(2-(6-maleimidohexyloxy)-6-chloro-1,3,5-triazin-4-yloxy)phenyl)ethylamino]-1,3,5-triazine (F-11). Cyanuric chloride (F-8, 16.6 mg, 0.0900 mmol) was added to a suspension of N-(6-hydroxyhexyl)maleimide (F-10, 21.3 mg, 0.108 mmol), 1,10-phenanthroline (27.0 mg, 0.150 mmol), and molecular sieves 3A (45.0 mg) in MeCN (450 μL) at rt. After 12 h, F-9 (50.1 mg, 0.0600 mmol), DMF (180 μL), and ethyldiisopropylamine (31.4 μL, 0.180 mmol) were added. After additional 5 h, the reaction mixture was directly chromatographed [diol-functionalized silica gel, CH₂Cl₂/MeOH (17:3)] to afford a yellow solid (53.0 mg, 77%): ¹H NMR [400 MHz, CD₃OD] δ 1.18-1.40 (m, 4H), 1.44-1.59 (m, 2H), 1.60-1.74 (m, 2H), 2.47-2.66 (m, 2H), 3.08-3.53 (m, 12H), 3.66 (dd, J=5.2, 12.0 Hz, 1H), 3.72 (dd, J=5.2, 12.0 Hz, 1H), 3.81 (dd, J=2.4, 12.0 Hz, 1H), 3.90 (dd, J=2.4, 12.0 Hz, 1H), 4.30 (t, J=6.8 Hz, 2H), 4.57 (d, J=7.2 Hz, 1H), 4.68 (d, J=7.2 Hz, 1H), 6.66 (d, J=8.4 Hz, 2H), 6.76 (s, 2H), 6.82 (d, J=8.4 Hz, 2H), 6.91 (dd, J=2.0, 8.8 Hz, 1H), 6.97 (dd, J=2.0, 8.8 Hz, 1H), 7.18 (s, 1H), 7.21 (s, 1H), 7.30 (d, J=8.8 Hz, 1H), 7.34 (d, J=8.8 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.57 (d, J=2.0 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD/CD₃CN) δ 26.0, 27.1, 29.1, 29.2, 35.6, 38.4, 43.8, 62.5, 62.6, 70.6, 71.2, 71.3, 74.8, 74.9, 77.76, 77.80, 77.9, 78.0, 105.77, 105.80, 110.8, 110.9, 113.0, 114.1, 114.4, 117.4, 117.7, 118.3, 138.5, 139.0, 139.1, 146.4, 146.7, 151.2, 169.4, 172.6, 173.5, 173.6, 173.7, 174.0, 174.5; ESI-MS obsd 1143.3450, calcd 1143.3457 [(M+H)⁺, M=C₅₂H₅₅ClN₁₀O₁₈]

2,4-Bis[1-(3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl)-1,4,7,10-tetraoxadec-10-yl]-6-[4-(3-(2-(1-azido-3,6,9,12,15-pentaoxoheptadecylamino)-4-[2-((4-methyl-2H-chromen-2-one-7-yl)amino)ethylamino]-1,3,5-triazin-6-yloxy)propyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine (F-18). A sample of F-8 (4.4 mg, 0.024 mmol) was added to a suspension of F-14 (37.6 mg, 0.020 mmol), 1,10-phenanthroline (9.0 mg, 0.050 mmol), and molecular sieves 4Å (10 mg) in CH₂Cl₂(100 μL) at room temperature. After 21 h, F-15 (7.8 μL, 0.028 mmol) and i-Pr₂EtN (10.5 μL, 0.060 mmol) were added. After 22 h, the reaction mixture was diluted with CH₂Cl₂(2 mL), filtered, washed with aqueous citric acid (10%, 2 mL) and brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure to afford the crude F-16 (2,4-bis[1-(3-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl)-1,4,7,10-tetraoxadec-10-yl]-6-[4-(3-(2-(1-azido-3,6,9,12,15-pentaoxoheptadecylamino)-4-chloro-1,3,5-triazin-6-yloxy)propyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine), which was used as is in the next step. A sample of F-17 (12.0 mg, 0.040 mmol), DMF (100 μL), and Et₃N (22 μL, 0.16 mmol) were added to the residue at room temperature. After 18 h, the reaction mixture was diluted with CH₂Cl₂(2 mL) and filtered. The filtrate was washed with aqueous citric acid (10%, 2 mL) and brine (2 mL), dried (Na₂SO₄), and filtered. The filtrate was concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (15:1 to 9:1] afforded a pale brown solid (19.4 mg, 39%): ¹H NMR (CDCl₃, 400 MHz, mixture of rotamers) δ 1.70-1.90 (m, 2H), 1.95-2.17 (m, 26H), 2.29 (s, 3H), 2.93 (br s, 2H), 3.10-4.60 (m, 73H), 4.81-4.98 (m, 2H), 5.05-5.40 (m, 6H), 5.73-6.05 (m, 3H), 6.38-6.65 (m, 2H), 6.88 (br s, 1H), 7.14 (s, 2H), 7.50-7.68 (m, 2H), 10.13 (br s, 2H); ¹³C NMR (CDCl₃, 175 MHz, mixture of rotamers) δ 18.2, 18.7, 20.8, 21.2, 21.4, 36.9, 39.6, 39.8, 40.6, 40.7, 40.8, 43.0, 43.2, 43.3, 47.26, 47.32, 47.5, 50.8, 53.6, 56.4, 58.3, 62.0, 62.7, 67.1, 68.5, 69.2, 69.8, 70.1, 70.4, 70.6, 70.66, 70.71, 70.73, 70.9, 71.1, 72.0, 72.5, 73.1, 97.5, 101.3, 106.4, 108.48, 108.55, 109.9, 110.5, 111.8, 115.1, 115.7, 118.4, 125.6, 131.5, 136.5, 145.7, 152.1, 152.3, 153.7, 153.8, 156.0, 162.3, 166.4, 166.6, 166.8, 167.0, 167.2, 169.6, 169.7, 169.9, 170.3, 170.8, 171.7; ESI-MS obsd 1238.2465, calcd 1238.2484 [(M+2H)²⁺, M=C₉₆H₁₂₆Br₄N₁₆O₃₉S].

2,4-Bis[1-(3-(β-D-glucopyranosyloxy)-4,6-dibromo-1H-indol-5-yl)-1,4,7,10-tetraoxadec-10-yl]-6-[4-(3-(2-(1-azido-3,6,9,12,15-pentaoxoheptadecylamino)-4-[2-((4-methyl-2H-chromen-2-one-7-yl)amino)ethylamino]-1,3,5-triazin-6-yloxy)propyl)-4-(3-sulfopropyl)piperazin-1-yl]-1,3,5-triazine (V). K₂CO₃(0.9 mg, 7 μmol) was added to a solution of F-18 (15.9 mg, 6.4 μmol) in MeOH/CHCl₃(4:1, 916 μL) at room temperature. After 30 min, H₂O (366 μL) was added. After 1.5 h, the reaction mixture was diluted with CH₂Cl₂(2 mL), dried (Na₂SO₄), and passed through a silica pad [diol-functionalized silica, CH₂Cl₂/MeOH (2:1) as an eluent]. The eluent was concentrated under reduced pressure to afford a pale yellow solid (13.7 mg, 100%): ¹H NMR (CDCl₃, 400 MHz) δ 2.09 (br s, 2H), 2.15 (br s, 2H), 2.25-2.33 (m, 3H), 2.54-2.63 (m, 2H), 3.14 (t, J=9.1 Hz, 2H), 3.18-4.43 (m, 76H), 4.64 (d J=7.3 Hz, 2H), 5.85-5.95 (m, 1H), 6.40-6.66 (m, 2H), 7.21 (s, 2H), 7.36-7.47 (m, 1H), 7.55 (s, 2H); ¹³C NMR (DMSO-d₆, 175 MHz, mixture of rotamers) δ 17.9, 18.1, 20.8, 36.9, 47.3, 47.4, 48.6, 50.0, 52.9, 57.1, 57.2, 61.0, 66.5, 68.5, 68.8, 68.9, 69.3, 69.48, 69.54, 69.59, 69.64, 69.7, 69.8, 69.9, 70.0, 70.1, 72.4, 73.6, 76.9, 77.2, 103.4, 106.2, 107.5, 108.9, 110.5, 113.3, 114.9, 117.9, 126.0, 131.0, 137.4, 144.8, 152.4, 153.8, 153.86, 153.91, 155.7, 155.76, 155.80, 160.8, 160.9, 166.4, 166.7, 167.1, 169.8, 171.5; ESI-MS obsd 1070.2073, calcd 1070.2061 [(M+2H)²⁺, M=C₈₀H₁₁₀Br₄N₁₆O₃₁S].

Example 5

1. Molecular design of a glucuronide trigger. The newest design is based on a reported compound wherein a protected glucuronide (Gln) bears the precursor of a self-immolative linker (SIL), W5^Z1(Scheme A). Compound W5 is coupled with indoxyl derivatives W8a-c and W10a-c (Scheme B). Compounds W9a-c will be used in a strategy with [Gln-SIL-Ind]₂-Triazine-Tyr(PEG)-I*, where Tyr is tyrosine, PEG is a polyethylene glycol unit, and I* is a radioisotope of iodine. Compounds W11a-c will be used in studies of [Gln-SIL-Ind]₄-Porphyrin-(PEG)-I* and [Gln-SIL-Ind]₄-Porphyrin-Tyr(PEG)-I*.

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The synthesis shown in Scheme B is employed to construct the indoxyl unit bearing a tether, and for attachment of the glucuronide unit bearing the self-immolative linker (W5).

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2. Proposed synthetic route for triazine strategy—[Gln-SIL-Ind]₂-Triazine-Tyr(PEG)-I*. A key objective is to ensure a long circulation time (slow clearance) of the radiotherapeutic molecules. To facilitate long circulation, we have included provisions in the molecular design to accommodate PEG groups. The synthesis of one target architecture is shown in Schemes C and D. The target compound (S6) includes two glucuronide—self-immolative linker-indoxyl units (Gln-SIL-Ind) attached to a central triazine moiety. The triazine moiety also contains a tyrosine unit (D-configuration), which is amidated, via a triazole, to a PEG unit. The presence of two (Gln-SIL-Ind) units gives rise to a linear polymer upon glucuronidase action.

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The tyrosine unit provides the site for radioiodination; indeed, two iodine atoms are installed per phenol side chain. The PEG unit can be of lengths ranging from quite short (few hundred Da) to 50 kDa or more. The radioiodination strategy entails installation of the radioisotope of iodine at the penultimate step of the synthesis (Scheme E). In this step, the glucuronide moieties remain in the protected form, and the indoxyl units likewise are protected with the N-acetyl unit. Radioiodination is carried out followed immediately by protecting group removal.

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A similar synthetic approach can be applied to achieve compound, Tl, which is shown in Scheme F. Compound T1 contains glucosides instead of glucuronides. The key precursor F1, which is compound 32 in Example 1, was synthesized.

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3. Proposed synthetic route for triazine strategy—[Gln-SIL-Ind]₄-Porphyrin-(PEG)-I* and [Gln-SIL-Ind]₄-Porphyrin-Tyr(PEG)-I*. (A) Iodination of the porphyrin. This strategy (Schemes G and H) is based on the accessibility of iodination on porphyrin in the very last step of the synthesis.

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(B) Tyrosine iodination. This strategy entails a tyrosine motif (as shown in Scheme I), which is similar to the triazine design.

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4. Iodination tests. As the radioactive ¹³¹I has a lifetime of approximately 8 days, the most ideal installation stage of radioiodide would be in the very last step of the synthesis. To further ensure the accessibility, we carried out several model studies. As shown in Scheme L, compound W12 was synthesized following a reported method^HF2with some modification. Compound W12 contains a central triazine unit, two phenoxy units as surrogates for the glucuronide-self-immolative linker-indoxyl units, and a tyramine group. The subsequent iodination was conducted under a well-known condition (0.125 M of W12) to give the doubly iodinated tyramine moiety of compound W13 within 10 minutes. The facile iodination of the tyramine unit is promising for our synthetic strategy.

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Another test concerns the iodination of the indoxyl motif. A model study was designed as shown in Scheme M. Fully protected compound W14 (0.025 M solution) was treated to the standard conditions with chloramine-T and NaI for 1 hour. No iodination product—neither at the 4,6-positions (flanking the benzyloxy unit) or at the 2-position—was observed by TLC analysis and LC-ESI-mass spectrometry. Partially deprotected compound W16 was planned to avoid iodination of the 2-position, which if occurs would block subsequent indigo formation. The synthesis of W16 is still ongoing due to its low solubility in methanol, which causes a low deprotection rate as well as formation of over-deprotected product (on the basis of TLC analysis).

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Another proposed model for the iodination study is shown in Scheme N. Compound W18 was synthesized in our previous study. Compound W18 has superior solubility versus W14. The solubility imparted by the PEG group at position 5 would facilitate studies where the selective deprotection step must be carried out. The proposed iodination would examine compound W19, and assess whether the product W20 would form.

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For the direct iodination study of the porphyrin core, compound S12 (5.9 mM solution) was treated to the standard conditions with chloramine-T and NaI for 30 min. A single product was observed based on the TLC analysis. This single product was then confirmed by ¹H NMR spectroscopy and its absorption spectra to be W22 as shown in Scheme O. Further study will be investigated later.

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Example 6

For purposes of describing compounds of Example 6, the following abbreviations and definitions for portions of the compound are provided: (PG-X)_n-Nex-(R*)CTA where PG is a protecting group, X is a cross-linking agent, R* is a radiolabel, subscript n denotes the number of species, Nex is a nexus, and CTA is a cancer-targeting agent.

The following designs differ in the number of cross-linkable groups in each molecule, encompassing one (I-a), two (I-b), or three or more (I-c). Accordingly, design I-a would give “dimers” upon cross-linking, design I-b would give linear polymers, and design I-c would give 3-dimensional matrices.

Design I-a has one each of PG-X, CTA, and R* (Scheme P). In pre-I-a, Y is a bioconjugatable group for attachment of the CTA.

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An example of pre-I-a and the synthesis are shown in Scheme Q. A radioactive iodine atom (e.g., ¹³¹I) is introduced following a known method.^F1

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Design I-b has two PG-X units instead of the one in design I-a (Scheme R).

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An example of pre-I-b and the synthesis are shown in Scheme S.

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A preferred example of pre-I-c and the synthesis are shown in Scheme U.

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The β-glucuronide linker shown in Scheme V has been developed for antibody-drug conjugates to treat cancer (when the payload is an anti-cancer drug).^F6-F8There are patent documents of this linker for cancer therapy.^F9,F10A distinguishing feature from the standard design in Scheme V versus our work is that our payload is never released; instead, the payload is immobilized.

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The synthesis of a 1:1 conjugate of a monoclonal antibody (mAb) and human serum albumin (HSA) was carried out using the enzyme tyrosinase in the presence of a small-molecule cross-linking agent (caffeic acid).^F11Exploiting this method in our case, modification of the lysine residues of the conjugate mAB-HSA utilizing Cu-free click chemistry (or thiol/maleimide conjugation) with R*—Z-L gives design II with HSA as a carrier (Scheme W).

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REFERENCES

JL1 Liu, W.; Song, H.; Chen, Q.; Yu, J.; Xian, M.; Nian, R.; Feng, D. Recent Advances in the Selection and Identification of Antigen-Specific Nanobodies. Mol. Immunol. 2018, 96, 37-47.

JL2 Thomas, A.; Teicher, B. A.; Hassan, R. Antibody-Drug Conjugates for Cancer Therapy. Lancet Oncol. 2016, 17, e254-262.

JL3 Muyldermans, S. Nanobodies: Natural Single-Domain Antibodies. Annu. Rev. Biochem. 2013, 82, 775-797.

F1 Reilly, S. W.; Makvandi, M.; Xu, K.; Mach, R. H. Rapid Cu-Catalyzed [²¹¹At]Astatination and [¹²⁵I]Iodination of Boronic Esters at Room Temperature. Org. Lett. 2018, 20, 1752-1755.

F6 Jeffrey, S. C.; Andreyka, J. B.; Bernhardt, S. X.; Kissler, K. M.; Kline, T.; Lenox, J. S.; Moser, R. F.; Nguyen, M. T.; Okeley, N. M.; Stone, I. J.; et al. Development and Properties of β-Glucuronide Linkers for Monoclonal Antibody-Drug Conjugates. Bioconjug. Chem. 2006, 17, 831-840.

F7 Jeffrey, S. C.; De Brabander, J.; Miyamoto, J.; Senter, P. D. Expanded Utility of the β-Glucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents. ACS Med. Chem. Lett. 2010, 1, 277-280.

F8 Kolakowski, R. V.; Haelsig, K. T.; Emmerton, K. K.; Leiske, C. I.; Miyamoto, J. B.; Cochran, J. H.; Lyon, R. P.; Senter, P. D.; Jeffrey, S. C. The Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization for the Targeted Delivery of Alcohol-Containing Payloads with Antibody-Drug Conjugates. Angew. Chem. Int. Ed. 2016, 55, 7948-7951.

F9 Jeffrey, S. Beta-Glucuronide-Linker Drug Conjugates. WO2007011968 A2.

F10 Kolakowski, R.; Jeffrey, S.; Burke, P. Methylene Carbamate Linkers for Use with Targeted-Drug Conjugates. WO2015095755 A1.

F11 Rollett, A.; Thallinger, B.; Ohradanova-Repic, A.; Machacek, C.; Walenta, E.; Cavaco-Paulo, A.; Birner-Gruenberger, R.; Bogner-Strauss, J. G.; Stockinger, H.; Guebitz, G. M. Enzymatic Synthesis of Antibody-Human Serum Albumin Conjugate for Targeted Drug Delivery Using Tyrosinase from Agaricus Bisporus. RSC Adv. 2013, 3, 1460-1467.

HF1 “Enzymatically Triggered Chromogenic Cross-Linking Agents Under Physiological Conditions,” Fujita, H.; Dou, J.; Matsumoto, N.; Wu, Z.; Lindsey, J. S. New J. Chem. 2019, submitted Aug. 8, 2019.

HF2 “Chromogenic A₂BC-Building Blocks for Forming Scaffold Assemblies under Physiological Conditions,” Fujita, H. et al., New J. Chem. 2020, to be submitted.

Z1 Desbene, S.; Van, H. D.; Michel, S.; Tillequin, F.; Koch, M.; Schmidt, F.; Florent, J. C.; Monneret, C.; Straub, R.; Czech, J; Gerken, M. Application of the ADEPT strategy to the MDR resistance in cancer chemotherapy. Anti-cancer Drug Design 1999, 14, 93-106.

Z2 Leu, Y.-L.; Roffler, S. R.; Chern, J.-W. Design and synthesis of watersoluble glucuronide derivatives of camptothecin for cancer prodrug monotherapy and antibody-directed enzyme prodrug therapy (ADEPT). J. Med. Chem. 1999, 42, 3623-3628.

Z3 Duimstra, J. A.; Femia, F. J.; Meade, T. J. A gadolinium chelate for detection of β-glucuronidase: a self-immolative approach. J. Am. Chem. Soc. 2005. 127, 12847-12855.

Z4 Prijovich, Z. M.; Burnouf, P. A.; Chou, H. C.; Huang, P.-T.; Chen, K.-C.; Cheng, T.-L.; Leu, Y.-L; Roffler, S. R. Synthesis and antitumor properties of BQC-glucuronide, a camptothecin prodrug for selective tumor activation. Molecular pharmaceutics 2016, 13(1242-1250.

CROSS-LINKING COMPOUNDS AND METHODS OF USE THEREOF

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