[0001] This application claims benefit of U.S. Provisional Application No. 63/046,438 filed June 30, 2020, the specification(s) of which is/are incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 1552568 awarded by NSF. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

[0003]The present invention relates to modifications of triazabutadiene molecules, more particularly to metal-based (e.g., palladium-based) coupling for the addition of carbon-carbon bonds to a triazabutadiene (TBD) scaffold. The present invention includes the synthesis, characterization, and use of said triazabutadiene molecules, in addition to the molecules themselves.

Background Art

[0004] Aryl diazonium ions are coveted electrophiles in organic and bioconjugation chemistry due to their highly reactive nature (FIG. 1, Pathway A). The inherent instability of diazonium ions make them incompatible with many common reaction conditions and creates limitations when planning efficient syntheses. At the same time, diazotization of a primary arylamine occurs under acidic conditions, a method which can cause problems when acid intolerant groups have already been installed onto an organic molecule. The triazabutadiene scaffold has been used to protect this electrophilic warhead, and it has been shown that aryl diazonium ion release can be achieved under mildly acidic aqueous or organic conditions. These conditions are generally compatible with a variety of functional groups when compared to the harsh acidic conditions necessary for its formation (FIG. 1, Pathway B). There are reports of triazabutadiene thermal decomposition occurring at high temperatures (<150 °C), with electron donating groups favoring this degradation pathway at room temperature. This scaffold is also a reported metal chelator, capable of binding in a bidentate fashion. With these considerations in mind, initial ventures into triazabutadiene functionalization excluded the use of well-defined catalytic reactions to maintain the protecting group’s integrity.

[0005] Inventors surprisingly discovered that triazabutadiene (TBD) scaffolds can be chemically modified using a Suzuki coupling reaction for the formation of a carbon-carbon bond, which allows for easy attachment of a variety of aryl groups.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention features compositions comprising a triazabutadiene scaffold featuring a carbon-carbon bond for attachment of an aryl group, as well as methods of preparation of said compositions. For example, the present invention provides methods comprising Suzuki coupling for installation of the carbon-carbon bond to the triazabutadiene scaffold (see FIG. 2). As previously discussed, the carbon-carbon bond allows for attachment of a variety of aryl groups. The aryl groups can remain protected by the triazabutadiene scaffold and reserved for future use. The present invention is not limited to palladium catalyzed Suzuki coupling, nor is the present invention limited to any particular aryl ring added to the triazabutadiene scaffold.

[0007]The present invention includes the synthesis, characterization, and use of said triazabutadiene compositions, in addition to the compositions themselves.

[0008]The methods and compositions herein, including synthetic intermediates as shown in FIG. 1, may be used for a variety of applications in a variety of different industries (e.g., biological research industry, textile industry, pharmaceutical industry, chemical research industry, chemical engineering industry, etc.), including but not limited to protein labeling, synthesis of pro-drugs, probe synthesis, drug discovery applications, mass spectrometry, fluorescence microscopy, synthesis of various conjugate molecules, etc. More specifically, the methods herein that create stable covalent bonds for irreversible binding to tyrosine may allow for strong and stable protein tagging to track proteins of interest. Or, the methods and compositions herein may be used to create covalent tags to pull down proteins of interest, for the covalent attachment of fluorophores to proteins, creating triazabutadiene-drug conjugates which could be used to pull down protein-protein interactions or selectively separate proteins from a mixture, or turn noncovalent drugs into covalent ones, etc. The present invention is not limited to the aforementioned applications. As depicted in FIG. 1, aryl diazoniums are coveted synthetic intermediates which can be transformed into a variety of functional groups allowing for simple and fast library diversification.

[00091 For example, the present invention features triazabutadiene compositions according to Compound A1 below. In certain embodiments, Ar is a 5, 6, or 7-member aryl ring. Compound A1 comprises R, and in certain embodiments at least one R. R is an aryl or a derivative thereof.

[0010] In certain embodiments, Ar is a 6-membered ring and R is at the ortho position. In certain embodiments, Ar is a 6-membered ring and, R is at the meta position. In certain embodiments, Ar is a 6-membered ring and, R is at the para position.

[0011]Triazabutadiene compositions of the present invention include those according to Compound A2.

[0012] Triazabutadiene compositions of the present invention include those according to Compound A3.

[0013]Triazabutadiene compositions of the present invention include those according to Compound A4.

[0014] In certain embodiments, Compound A1 comprises two R groups. In certain embodiments, both R groups are the same. In certain embodiments, the R groups are different.

[0015] In certain embodiments, D or E = H. In certain embodiments, D or E = alkyl (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, or (CH ₂ ) _n X. In certain embodiments, n = 1-8. In certain embodiments, E = C _1-16 alkyl. In certain embodiments, E = C _1-20 alkyl. In certain embodiments, D = C _1-16 alkyl. In certain embodiments, D = C _1-20 alkyl. In certain embodiments, X = SO ₃ -, CO ₂ - , NH ₂ , NMe ₂ , NEt ₂ , NPr ₂ , NBu ₂ , NHMe, NHEt, NHPr, NHBu, NMeEt, NMePr, NMeBu, NEtPr, NEtBu, NPrBu, PPh ₃ ⁺ , PO ₃ - , or CCH -terminal alkyne.

[0016] In certain embodiments, A = N. In certain embodiments, A = S. In certain embodiments, A = O. In certain embodiments, D = H or an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.; additional alkyls are disclosed herein). In certain embodiments, E = H or an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.; additional alkyls are disclosed herein). Additional non-limiting examples of alkyl groups are disclosed below.

[0017] In certain embodiments, X ¹ or Y ¹ is an aryl, an alkyl, a carboxylic acid, an alcohol, or an amine. In certain embodiments, X ¹ or Y ¹ is methyl. In certain embodiments, X ¹ or Y ¹ is t-butyl. In certain embodiments, X ¹ or Y ¹ is C _1-16 alkyl. In certain embodiments, X ¹ or Y ¹ is C _1-20 alkyl.

[0018] Triazabutadiene compositions of the present invention include those according to Compound B1.

[0019]Triazabutadiene compositions of the present invention include those according to Compound B2.

[0020] Referring to Compound B1 and B2, in certain embodiments, R ₁ = aryi or a derivative thereof. Referring to Compound B1 and B2, in certain embodiments, R ₂ is aryl or alkyl (e.g., C _1-16 alkyl, C _1-20 alkyl, etc.). Referring to Compound B1 and B2, in certain embodiments, R ₃ is aryl or alkyl (e.g., C _1-16 alkyl, C _1-20 alkyl, etc.). As an example, in certain embodiments, R ₂ is t-butyl and R ₃ is methyl.

[0021]The triazabutadiene compositions of the present invention may decompose into an aryl diazonium species. In certain embodiments, the triazabutadiene composition is a pro-drug and upon decomposition of the triazabutadiene composition, the aryl diazonium species is a drug. In certain embodiments, R is at least a portion of a drug. In certain embodiments, R is at least a portion of a fluorophore. In certain embodiments, the R is at least a portion of a label for a protein. In certain embodiments, the label is an affinity tag. In certain embodiments, the label is a tag for a pull-down assay. In certain embodiments, the label is a fluorescent moiety. In certain embodiments, the label is a colorimetric moiety.

[0022] The present invention also includes triazabutadiene compositions according to Compound C1, wherein R = aryll, and X = a halide (e.g., Br, I, CL F, etc.) or a pseudo-halide (e.g., tosylate, triflate, mesylate, etc.). The present invention also includes triazabutadiene compositions according to Compound C1, wherein R ~ vinyl, and X = a halide (e.g., Br, I, Cl, F, etc.) or a pseudo-halide (e.g., tosylate, triflate, mesylate, etc.}.

[0023] Triazabutadiene compositions of the present invention include those according to Compound C2.

[0024] Triazabutadiene compositions of the present invention include those according to Compound C3.

[0025] Triazabutadiene compositions of the present invention include those according to Compound C4.

[0026] Triazabutadiene compositions of the present invention include those according to Compound C5.

[0027]For any of the appropriate disclosed compounds, including appropriate compounds disclosed in the figures, the compounds with aryl rings featuring the -R functional group are not limited to -H at other carbon positions around the aryl ring. As an example, for Compounds A2, A3, A4, B1 , B2, etc., the 6-membered aryl ring comprises the -R functional group and is bonded to nitrogen (N). The additional 4 carbons of the aryl ring are not limited to C-H and include C-R ^* , wherein R ^* represents any other appropriate functional group.

[0028]For any of the appropriate disclosed compounds, including appropriate compounds disclosed in the figures, the compounds with aryl rings featuring the halide or pseudo-halide functional group are not limited to -H at other carbon positions around the aryl ring. As an example, for Compounds C2, C3, C4, and C5, the 6-membered aryl ring comprises the halide or pseudo-halide functional group and is bonded to nitrogen (N). The additional 4 carbons of the aryl ring are not limited to C-H and include C-R ^’ , wherein R’ represents any other appropriate functional group.

[0029]The present invention also features methods of synthesizing a triazabutadiene compositions. The method comprises subjecting a triazabutadiene scaffold according to Compound C1 (or C2, C3, C4, C5, or variations thereof, etc.) to a palladium catalyst and an organoboron molecule with an aryl group. In certain embodiments, the palladium catalyst comprises [1,1'-Bis(diphenylphosphino)ferrocene]palladium(ll) dichloride, palladium (II) acetate with triphenylphosphine (1:3 ratio), or bis(acetonitrile)palladium dichloride with triphenylphosphine (1:3 ratio).

[0030] In certain embodiments, the method is for synthesizing a pro-drug. In certain embodiments, the method is for synthesizing a label for a protein. In certain embodiments, the label is a tag for a pull-down assay. In certain embodiments, the label is an affinity tag. In certain embodiments, the label is a fluorescent moiety. In certain embodiments, the label is a colorimetric moiety. In certain embodiments, the method is for converting a non-covalent drug to a covalent drug.

[0031] The present invention also features the use of any of the triazabutadiene compositions herein.

[0032] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0033]The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0034] FIG. 1 shows two pathways by which aryl diazonium ions may be created (Pathway A, Pathway B).

[0035] FIG. 2 shows palladium catalyzed Suzuki coupling for installation of an aryl ring to a triazabutadiene scaffold (triazabutadiene scaffold, left molecule; new triazabutadiene composition, middle molecule), as well as the diazonium ion molecule (molecule on the right) released from degradation of the triazabutadiene molecule.

[0036] FIG. 3A shows examples of formulas for triazabutadiene scaffolds, which may then be modified using metal-based coupling (e.g., Suzuki coupling) for the installation of an aryl group via a carbon-carbon bond. Note Formulas I, II, III, and IV are examples of Formula A. The orientation of X ¹ and Y ¹ is case-by-case dependent. [0037] FIG. 3B shows examples of triazabutadiene scaffolds used in the methods herein. Examples of triazabutadiene scaffolds may feature a halide at the para-, ortho-, and meta- positions. Note the present invention is not limited to the use of the halides. Alternatives to halides include tosylate, triflate, mesylate, etc. Note R-X (X= Cl, Br, I, tosylate, triflate, mesylate, etc.) is an sp ² carbon part of an aryl or vinyl system.

[0038] FIG. 4 shows examples of triazabutadiene compositions of the present invention and non-limiting examples of R groups.

[0039] FIG. 5 shows non-limiting examples of triazabutadiene compositions of the present invention.

[0040] FIG. 6 shows non-limiting examples of triazabutadiene compositions of the present invention.

[0041] FIG. 7 shows an example of a triazabutadiene composition of the present invention Compound B1).

[0042] FIG. 8 shows an example of a triazabutadiene composition of the present invention Compound B2).

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention features triazabutadiene compositions comprising a triazabutadiene scaffold and the attachment of an aryl group via a carbon-carbon bond, as well as methods of preparation of said compositions.

[0044] Triazabutadienes (triazabutadiene scaffolds) have been previously described. Examples include but are not limited to those described in U.S. Pat. No. 9,458,143, U.S. Pat. No. 9,593, 080, U.S. Pat. No. 10, 125, 105, W.O. 2018/023130, U.S. Pat. Application No. 2017/0114033, U.S. Pat. Application No. 2018/023106, and W.O. 2018/165666, the disclosures of which are incorporated in their entirety by reference herein.

[0045] Non-limiting examples of triazabutadiene scaffolds are shown in the formulas in FIG. 3A. In certain embodiments, A = N. In certain embodiments, A = S. In certain embodiments, A = O. In certain embodiments, D = H or an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.), including alkyls with additional functional groups. In certain embodiments, E = H or an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.), including alkyls with additional functional groups. Additional alkyl groups are disclosed herein.

[0046] In certain embodiments, X ¹ is an aryl, an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.), including alkyls with additional functional groups, a carboxylic acid, an alcohol, or an amine. For example, in certain embodiments, X ¹ is methyl. In certain embodiments, X ¹ is t-butyl.

[0047] In certain embodiments, Y ¹ is an aryl, an alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, etc.), including alkyls with additional functional groups, a carboxylic acid, an alcohol, or an amine. In certain embodiments, Y ¹ is methyl. In certain embodiments, Y ¹ is t-butyl.

[0048] In certain embodiments, Z ¹ is an aryl, e.g., an aryl halide.

[0049] As used herein, the term “aryl” refers to a functional group derived from an aromatic ring. Aryls are not limited to 6-membered ring-based aryls. The present invention also includes other 3-, 4-, 5-membered ring-based aryls as well, including aryl rings of various sizes with additional functional groups.

[0050] As used herein, the term “alkyl” refers to an acyclic branched or unbranched hydrocarbon, such as ones according to the formula C _n H _2n , less one hydrogen. The term “alkyl” may also refer to branched or unbranched hydrocarbons comprising additional functional groups (or other hydrocarbons such as an alkyne, alkene, etc.). For example, alkyls may include but are not limited to Me (-CH ₃ ), Et (-CH ₂ -CH ₃ ), -CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH ₃ , -CH ₂ -CH, CH ₂ -CH ₂ -CH, -CH ₂ -CH ₂ -CH ₂ -CH,

-CH ₂ -CH ₂ -CH ₂ -CH ₂ -CH, -CH ₂ -C, -CH ₂ -CH ₂ -C, -CH ₂ -CH ₂ -CH ₂ -C, -CH ₂ -CH ₂ -CH ₂ -CH ₂ -C, Pr, e.g., (n,i)-Pr (e.g., isopropyl, -CH ₂ -CH ₂ -CH ₃ ), Bu, e.g., ( n , /, s, t)-Bu (e.g., t-butyl, s-butyl, isobutyl, -CH ₂ -CH ₂ -CH ₂ -CH ₃ ), or (CH ₂ ) _n X (e.g., n=1-8 or more) wherein X=S0 ₃ ^_ , C0 ₂ -, NH ₂ , NMe ₂ , NEt ₂ , NPr ₂ , NBu ₂ , NHMe, NHEt, NHPr, NHBu, NMeEt, NMePr, NMeBu, NEtPr, NEtBu, NPrBu, (and other variations of NH ₂ , NMe ₂ , NEt ₂ , NPr ₂ , NBu ₂ , etc., noting the examples of Pr and Bu may refer to (n,/)-Pr, (n, i, s, t)-Bu), PPh ₃ ⁺ , P0 ₃ -, CCH -terminal alkyne, etc., as well as any of the molecules and groups previously disclosed in U.S. Pat. No. 9,458,143, U.S. Pat. No. 9,593, 080, U.S. Pat. No. 10, 125, 105, W.O. 2018/023130, U.S. Pat. Application No. 2017/0114033, U.S. Pat. Application No. 2018/023106, and W.O. 2018/165666, the disclosures of which are incorporated in their entirety by reference herein.

[0051] The present invention is not limited to the aforementioned examples of A, D, E, X ¹ , Y ¹ , or Z ¹ .

[0052] The triazabutadiene scaffolds, e.g., as described above, may be chemically modified to install a carbon-carbon bond so as to incorporate an aryl group. The triazabutadiene compositions of the present invention may be used to protect or reserve the aryl group for future use. Thus, the triazabutadiene compositions of the present invention may be referred to as “protected” aryl diazonium molecules, “activatable” triazabutadienes, releasable triazabutadienes, pro-aryl diazonium molecules, or other similar terms. As used herein, the aforementioned terms may refer to an inactive form of an aryl diazonium that upon exposure to particular conditions or a trigger can yield an activated aryl diazonium ion. Triggers may include but are not limited to an enzymatic trigger, a pH trigger, a reducing environment, light exposure, etc.

[0053] Installation of the carbon-carbon bond on the triazabutadiene scaffold features methods comprising Suzuki coupling (see FIG. 2), or a variation thereof. As previously discussed, the carbon-carbon bond allows for attachment of a variety of aryl groups. The aryl groups can remain protected by the triazabutadiene scaffold and reserved for future use. Non-limiting examples of alternative compatible catalysts include: [1,T-Bis(diphenylphosphino)ferrocene]palladium(ll) dichloride, palladium (II) acetate with triphenylphosphine (1:3 ratio), and bis(acetonitrile)palladium dichloride with triphenylphosphine (1:3 ratio). The present invention is not limited to the aforementioned palladium catalysts.

[0054] In certain embodiments, triazabutadiene scaffold used for Suzuki coupling (or a variation of Suzuki coupling) comprises a halide as shown in FIG. 3B. The present invention is not limited to a halide; in certain embodiments, the halide is substituted with an alternative molecule such as but not limited to tosylate, inflate, mesylate, etc.

[0055] As used herein, the term “halide” may also refer to a pseudo-halide. Non-limiting example of pseudo-halides include a tosylate, triflate, or mesylate.

[0056] FIG. 4 shows examples of triazabutadiene compositions synthesized using methods of the present invention, including non-limiting examples of R groups.

[0057] FIG. 5, FIG, 6, FIG. 7, and FIG. 8 show additional non-limiting examples of triazabutadiene compositions synthesized using methods of the present invention.

[0058]Referring to FIG. 7 and FIG. 8, in certain embodiments, R ₂ and R ₃ may be aryl (e.g., mesityl, phenyl, etc.), or alkyl (e.g., as described herein, e.g., t-butyl, methyl, other long chain alkyl, alky! that promotes solubility, etc.). For example, in some embodiments, R, - t-butyl and R ₃ = methyl.

[0059] Note Mes as used herein refers to mesityl, as shown:

[0060] The present invention is not limited to triazabutadiene scaffolds with any particular halide, e.g., alternatives to Br may be considered, e.g., F, Cl, I, etc.

[0061] The present invention is not limited to the Suzuki reactions described herein. Other variations of Suzuki conditions and extensions (e.g., sources of Pd, ligands, solvents, temperatures, etc.) may be considered and are within the scope of the present invention.

[0062] The present invention includes the synthesis, characterization, and use of said triazabutadiene compositions, in addition to the compositions themselves.

[0063] Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0064] The disclosures of the following documents are incorporated in their entirety by reference herein: U.S. Pat. No. 8,617,827; U.S. Pat. Application No. 2009/0048222; U.S. Pat. No. 3,591,575. U.S. Pat. No. 3,607,542; U.S. Pat. No. 4107353; WO Pat. No. 2008090554; U.S. Pat. No. 4,218,279; U.S. Pat. App. No. 2009/0286308; U.S. Pat. No. 4,356,050; U.S. Pat. No. 8,603,451; U.S. Pat. No. 5,856,373; U.S. Pat. No. 4,602,073; U.S. Pat. No. 3,959,210, U.S. Pat. No. 9,458,143, U.S. Pat. No. 9,593, 080, U.S. Pat. No. 10,125,105, W.O. 2018/023130, U.S. Pat. Application No. 2017/0114033, U.S. Pat. Application No. 2018/023106, and W.O. 2018/165666. The disclosures of the following publications are incorporated in their entirety by reference herein: Kimani and Jewett, 2015, Angewandte Chemie international Edition (DOI: 10.1002/anie.201411277 - Online ahead of print). Zhong et al., 2014, Nature Nanotechnology 9, 858-866; Stewart et al., 2011, J Polym Sci B Polym Phys 49(11):757-771; Poulsen et al., 2014, Biofouling 30(4):513-23; Stewart, 2011, Appl Microbiol Biotechnol 89(1):27-33; Stewart et al., 2011 , Adv Colloid Interface Sci 167(1-2):85-93; Hennebert et al., 2015, Interface Focus 5(1):2014.

EXAMPLE

[0065] The following is a non-limiting example of a procedure for synthesis of compositions according to the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0066] Appropriate aryl borane (1.2 eq), K ₃ P0 ₄ (2.3 eq), and palladium catalyst (0.11 eq) were added to a flask. Triazabutadiene (0.05 mmol, 1 eq) was added followed by 1 mL of a 5:1 solution of dioxane:water. The set up was degassed with argon for 15 minutes. The resulting solution was heated to 95 °C for one hour. Upon completion, the reaction mixture was diluted with dichloromethane and passed through a celite pad, which was further rinsed with dichloromethane and ethyl acetate. After the solvent was removed under pressure reduced, the products were purified by column chromatography using basic alumina as a stationary phase using acetone/dichloromethane as the mobile phase.

[0067] The methods herein may work with aryl boronic acids and aryl pinacol boranes.

[0068]Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising" includes embodiments that could be described as “consisting essentially of or “consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of or “consisting of is met.

[0069] The reference numbers recited in the below claims are solely for ease of examination of this patent application, and are exemplary, and are not intended in any way to limit the scope of the claims to the particular features having the corresponding reference numbers in the drawings.