Tagging the Untaggable: A Difluoroalkyl-Sulfinate Ketone-BasedReagent for Direct C−H Functionalization of Bioactive HeteroarenesSamer Gnaim,† Anna Scomparin,‡ Xiuling Li,§ Phil S. Baran,⊥ Christoph Rader,§,∥ Ronit Satchi-Fainaro,‡

and Doron Shabat*,†

†School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences and ‡Department of Physiology and Pharmacology,Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978 Israel§Department of Cancer Biology and ∥Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458,United States⊥Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States

*S Supporting Information

ABSTRACT: We have developed a new difluoroalkyl ketalsulfinate salt reagent suitable for direct derivatization ofheteroarene C−H bonds. The reagent is capable of introducinga ketone functional group on heteroarene bioactive compoundsvia a one-pot reaction. Remarkably, in three examples theketone analog and its parent drug had almost identicalcytotoxicity. In a representative example, the ketone analogwas bioconjugated with a delivery vehicle via an acid-labile semicarbazone linkage and with a photolabile protecting group toproduce the corresponding prodrug. Controlled release of the drug−ketone analog was demonstrated in vitro for both systems.This study provides a general approach to obtain taggable ketone analogs directly from bioactive heteroarene compounds withlimited options for conjugation. We anticipate that this sodium ketal-sulfinate reagent will be useful for derivatization of otherheteroarene-based drugs to obtain ketone-taggable analogs with retained efficacy.

Bioconjugation has become an essential tool in chemicalbiology for attaining controlled release of a small molecule

with medicinal activity.1,2 It is typically implemented throughchemoselective modification of native functional groups presenton the target molecule.3 For example, amines can bechemoselectively derivatized through amide linkages, azides,or acetylenes through the “click” reaction,4,5 carboxylic groupsthrough ester bonds,6 disulfide bridges through a thiolcoupling,7 and carbonyl groups through the oxime ligation.8

Although many medicinal agents contain traditional “taggable”functional groups, some compounds present the challenge ofnot having any apparent chemical handles. As a further layer ofcomplexity, some molecules require an orthogonal handle forbioconjugation. We have recently introduced a new method tomodify unactivated C−H bonds based on the controlleddecomposition of alkanesulfinate reagents to reactive radicals insitu via a simple, cheap oxidative process.9−13 This strategy hasopened a new gateway for derivatization of medicinal moleculesby a one-step synthetic route carried out in aqueous mixtures,open to the air with operationally simple thermal initiationconditions. The chemical functionalization of the inert C−Hbond of small molecules with a fluoroalkyl substituent bearing a“clickable” functional group was recently demonstrated usingan azido-alkyl handle.3 However, bioconjugation of suchmolecules with targeting carriers produces an irreversiblenonlabile linkage (triazole), which lacks the ability for site-selective release of the bioactive entity.

The introduction of a ketone onto a heteroarene would allowfor the conjugation of bioactive molecules via an acid-labilehydrazone linkage or through a photolabile ketal, forcontrolled-release applications. However, functionalization ofa bioactive molecule to produce an analogue with a taggablefunctional group will only be effective if the obtained analogueretains the efficacy of the native compound. The ketone is afunctional group bearing a lower potential for interacting withother functionalities, in comparison to the amine, hydroxyl, orthiol groups. Thus, we anticipate that in many instances theketone would be less susceptible to interfere with binding thenatural target of the native bioactive compound. Here we reportthe synthesis and evaluation of a new difluoroalkyl-sulfinateketone-protected reagent suitable for direct C−H bondfunctionalization of heteroarens that have either limited or notagging option.Sodium difluoroalkyl ketal sulfinate 1 is a ketone-protected

form of our designed reagent. Such a reagent is capable ofreacting with a hetereoarene C−H bond under oxidative acidicconditions to produce the corresponding ketone derivative I(Figure 1). Deprotection of the ketal should occur in situ underthe hydrolytic acidic conditions of the reaction to afford theketone functional group. The heteroarene−ketone analog can

Received: July 13, 2016Revised: August 4, 2016Published: August 5, 2016

Communication

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be readily masked through an acid-labile semicarbazonelinkage14−16 (II) or a photolabile ketal protecting group16,17

(III).The synthesis of ketal sulfinate 1 was achieved in an

analogous manner to that recently described for other sulfinatesalts (Figure 2).3,13 Thus, alkylation of pyridine derivative 1a byketal 1b generated sulfone 1c. The latter was reacted withsodium mercaptoethanol to afford sulfinate salt 1. Thissynthesis could be easily performed on a multigram scale toproduce sulfinate 1 with good yields. Heteroarene difluor-oalkylation by sulfinate salt 1 to produce the correspondingketone derivative was initially established on caffeine as a testsubstrate. Caffeine successfully reacted with sulfinate 1 throughdirect functionalization of its C−H bond to afford ketonederivative 1d in 87% yield.Next, we sought to evaluate the functionalization-capability

of sulfinate salt 1 on four selected known heteroareneantineoplastic drugs camptothecin (CPT), temozolomide(TMZ), bosutinib, and methotrexate (MTX). CPT (2) is atopoisomerase inhibitor with limited options for bioconjugationthrough its tertiary hydroxyl group. TMZ (3) is a cytotoxicalkylating agent, which is considered an untaggable compounddue to the absence of a suitable functional group. Likewise,bosutinib (4), an approved tyrosine kinase inhibitor based drug,has no functional group available for conjugation. MTX (5), an

antifolate-based chemotherapeutic drug, is a heteroarene withlimited bioconjugation options (Table 1).The four heteroarene drugs were successfully functionalized

by sulfinate salt 1 to afford the corresponding ketone analogs inmoderate to good yields. For CPT, bosutinib, and MTX,sulfinate salt 1 was able to selectively functionalize theheteroarenes at the most electron-deficient C−H bond.As mentioned above, this functionalization will be useful only

if the taggable ketone-derivative maintains the biological activityof the native drug. Therefore, in order to find out whether thenew derivatives retain their efficacy, the in vitro cytotoxicity ofthe ketone analogs was evaluated in comparison to that of theparent drugs. Figure 3 shows representative cell-growthinhibition plots for each drug and its ketone analog. Theapplied cell lines and the calculated IC50 values are presented inTable 2.Remarkably, for three of the four drugs, tumor cell-growth

inhibition assays showed almost identical cytotoxicity (IC50

values) for the ketone-derivatives and their native drugs.Functionalization of CPT, TMZ, and bosutinib using sulfinatesalt 1 resulted in ketone analogs that retained their cytotoxicactivity. However, the ketone analog of MTX lost itscytotoxicity by 3 orders of magnitude. The assays wererepeated with several human tumor cell lines and the obtainedresults were similar (see Supporting Information). These resultsindicate that it is possible for certain biologically relevant

Figure 1. Employing sodium ketal sulfinate 1 to tag of a heteroarene C−H inert bond with a ketone functional group and its bioconjugation throughan acid-labile semicarbazone linkage (II) or a photolabile ketal group (III) for controlled-release applications.

Figure 2. Chemical synthesis and test reaction of sodium ketal sulfinate 1. (a) Ketal sulfinate 1 synthesis with 63% overall yield. (b) Caffeinealkylation reaction with Sulfinate 1.

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heteroarenes to maintain their original activity, after C−Hfunctionalization by sulfinate salt 1, at an appropriate position.To determine the usefulness of these drug analogs harboring

a ketone for controlled release and bioconjugation, we selectedCPT-ketone 2a for further evaluation. As presented in Figure 1,a ketone functional group can be masked either through anacid-labile semicarbazone linkage or with a photolabile ketalprotecting group. CPT-ketone 2a was masked with aphotolabile protecting group17,18 to produce ketal derivative 6(Figure 4). This derivative can be considered the prodrug formof CPT-ketone 2a. Irradiation of prodrug 6 with UV (λ = 360nm) light over 2 h under physiological conditions resulted inthe release of CPT-ketone 2a (Figure 4A). No release wasobserved without irradiation. The cytotoxicity of prodrug 6 wasthen evaluated in a standard cell-growth inhibition assay beforeand after irradiation with UV light (Figure 4B). As expected,derivative 6 before irradiation exhibited typical prodrugbehavior, with an IC50 value of 350 nM. Prodrug 6 afterirradiation has shown significantly higher cytotoxicity with an

IC50 value of 10 nM, similarly to that obtained for CPT-ketone2a (IC50 = 5 nM). These results demonstrate how the newlyinstalled ketone of the heteroarene analogs can be used toobtain prodrugs with a photolabile controlled-release pathway.In this example, the prodrug was activated with UV light;however, there are analogous protecting groups, which can beremoved through a visible or near-infrared light.19 To the bestof our knowledge, this is the first demonstration of a prodrugphotoactivation, which is based on unmasking of a ketonegroup.18

Next, we sought to evaluate the acid-labile hydrazone linkage,for controlled-release and bioconjugation between CPT-ketone2a and a targeting vehicle. Hydrolysis via an acid-labile linkageis a useful controlled-release mechanism for cell-penetratingvehicles such as folic acid20 (FA) conjugate (Figure 5). Thus,CPT-ketone 2a was reacted with a semicarbazide derivative offolic acid (7a), via a semicarbazone linkage, to generate CPT−folic acid conjugate 7 with an acid-labile controlled-releasemechanism (see Supporting Information for synthesis). A

Table 1. Difluoroalkylation of Selected Chemotherapeutic Drugs by Sodium Ketal Sulfinate Salt 1 to Produce theCorresponding Ketone Derivatives

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polyethyleneglycol (PEG) linker derivative was used as a spacerto connect between the folic acid and CPT-ketone 2a moieties.To validate the release of CPT-ketone 2a from folic acid

conjugate 7, we monitored the stability of the semicarbazonelinkage under various pHs (physiological conditions−pH 7.4;early stage endosomes, pH 6.5 and 6.0; and late-stageendosomes, pH 4.8). The kinetic plots of CPT-ketone 2arelease are presented in Figure 6. The semicarbazone-basedconjugate exhibited excellent selective hydrolysis under acidicand physiological conditions. Fast release kinetics of CPT-ketone 2a were observed under acidic pHs (t1/2 < 1 h), whileno release at all was observed over 24 h at physiological pH.Importantly, at relatively mild acid conditions (early stageendosomes, pH 6.5), release of CPT-ketone 2a still effectivelytook place (t1/2 < 5 h).14

The ability of folate-conjugate 7 to bind to FR receptors wasevaluated in a [3H]-folic acid competition assay with high FR

KB cells (HiFR-KB).21 CPT-PEG derivative 7b, which lacks thefolic acid moiety, was used as a control (Figure 7A). Theobtained measurements showed 82% and 14% competition of[3H]-folic acid by conjugate 7 and control 7b, respectively,when compared to free folic acid (≡96.5%). Cytotoxicityevaluation of CPT-folic acid conjugate 7 on HiFR-KB cellsrevealed relatively similar IC50 values for CPT-ketone 2a and itsfolic acid conjugate 7 (Figure 7B). CPT-PEG derivative 7bshowed a 15-fold higher IC50 value compared to conjugate 7.These results support FR-mediated uptake of the folic acid-CPT conjugate followed by intracellular CPT-ketone con-trolled-release through the acid-labile semicarbazone linkage.The sodium difluoroalkyl ketal sulfinate 1 has recently

become commercially available from Sigma-Aldrich (productno. 792446). The reagent was demonstrated to be useful fordirect derivatization of bioactive heteroarenes. The obtainedanalogs are equipped with a new ketone functional group,suitable for bioconjugation. Such chemistry can open a newdoor for obtaining drug analogs that had either a limited or nopractical option for covalent controlled-release. The approachwas demonstrated to be effective for three chemotherapeuticdrugs, where the ketone analog fairly maintained the biologicalactivity of the native drug. In one option, the ketone functionalgroup of the analogs could be masked in form of aphotoremovable ketal to produce a corresponding prodrug.In another option, the ketone was used for bioconjugation viaan acid-labile hydrazone linkage with an appropriate targetingcarrier. If a more stable linkage is required, one can use theoxime ligation.8

Figure 3. Ketone-analogs of CPT, TMZ, and bosutinib maintained their tumor growth inhibitory effect of their original parent drugs, as opposed tothe ketone analog of MTX (cell viability is presented as % of control).

Table 2. IC50 Values Calculated from Cell-Growth InhibitionAssays for Chemotherapeutic Drugs and Their KetoneAnalogs

drug and ketone analog IC50 cell line

CPT (2) 7 nM U87CPT-Ketone (2a) 10 nM U87TMZ (3) 36 μM U251TMZ-Ketone (3a) 35 μM U251Bosutinib (4) 17 μM SF295Bosutinib -Ketone (4a) 17 μM SF295MTX (5) 28 nM SF295MTX-Ketone (5a) >1000 nM SF295

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In summary, we have developed a new difluoroalkyl ketalsulfinate salt reagent suitable for direct derivatization ofheteroarene C−H bonds. The reagent is capable of introducinga ketone functional group on heteroarene bioactive compoundsvia one pot reaction. In three out of four examples, the ketoneanalog and its parent drug had almost identical cytotoxicity.The ketone analog of CPT was conjugated with a deliveryvehicle via an acid-labile semicarbazone linkage and with a

photolabile protecting group to produce a correspondingprodrug. Controlled-release of the CPT-ketone analog wasdemonstrated in vitro for both systems. This study provides ageneral approach to obtain taggable ketone-analogs directlyfrom heteroarene bioactive compounds. We anticipate that oursodium ketal-sulfinate reagent will be useful for derivatization ofother heteroarene-based drugs, affording ketone-taggableanaloges.

Figure 4. (A) Release of CPT-ketone 2a with (blue) and without (orange) irradiation of prodrug 6 (150 μM in PBS 7.4:DMSO, 95:5 v/v). (B) Cellgrowth inhibition assay with human primary glioblastoma cell line U-87; prodrug 6 before (yellow) and after (gray) irradiation, CPT-ketone 2a(blue).

Figure 5. Release of CPT-ketone 2a from a folic acid conjugate via acid-labile controlled-release mechanism of a semicarbazone linkage.

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■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bioconj-chem.6b00382.

General information, synthesis procedures, and spectra(PDF)

■ AUTHOR INFORMATION

Corresponding Author

*E-mail: chdoron@post.tau.ac.il. Tel: +972 (0) 3 640 8340.Fax: +972 (0) 3 640 9293.

Notes

The authors declare no competing financial interest.

Figure 6. Hydrolysis rate of CPT-ketone-semicarbazone-PEG-FA 7 (150 μM) as a function of time after incubation in buffer at pH 4.8 (blue), pH6.0 (orange), pH 6.5 (yellow), and pH 7.4 (green) at 37 °C.

Figure 7. (A) [3H]FA competitive binding assays using HiFR-KB cells. (B) Cytotoxicity assays using HiFR-KB cells.

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■ ACKNOWLEDGMENTS

D.S. thanks the Israel Science Foundation (ISF), the BinationalScience Foundation (BSF), and the German Israeli Foundation(GIF) for financial support. This work is supported in part by agrant from the Israeli National Nanotechnology Initiative in theFocal Technology Area: Nanomedicines for PersonalizedTheranostics. R.S.-F. thanks the European Research Councilfor the ERC Consolidator Grant Agreement no. [617445]-PolyDorm.

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