Targeted Protein Degradation by Chimeric Compounds Using Hydrophobic E3 Ligands and Adamantane Moiety

pharmaceuticals

Article Targeted Protein Degradation by Chimeric Compounds using Hydrophobic E3 Ligands and Adamantane Moiety

1, , 2, 1 1,3 3 Takuji Shoda † *, Nobumichi Ohoka †, Genichiro Tsuji , Takuma Fujisato , Hideshi Inoue , 1 2 1, Yosuke Demizu , Mikihiko Naito and Masaaki Kurihara ‡ 1 Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa 210-9501, Japan; [email protected] (G.T.); [email protected] (T.F.); [email protected] (Y.D.); [email protected] (M.K.) 2 Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa 210-9501, Japan; [email protected] (N.O.); [email protected] (M.N.) 3 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-44-270-6579; Fax: +81-44-270-6579 These authors contributed equally to this study. † Current address: School of Pharmacy, Department of Pharmaceutical Sciences, International University of ‡ Health and Welfare, 2600-1 Kitakanemaru, Otawara, Tochigi, Japan.

Received: 20 January 2020; Accepted: 19 February 2020; Published: 25 February 2020

Abstract: Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and speciﬁc and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced signiﬁcant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR.

Keywords: protein degradation; chimeric compound; hydrophobic tagging; adamantane

1. Introduction A technology for selectively degrading a target protein is expected not only to elucidate the physiological function of proteins but also to develop a therapeutic drug for a disease caused by the aberrant protein. Previous studies reported a series of chimeric compounds that recruit E3 ubiquitin ligases to specifically degrade target proteins via the ubiquitin-proteasome system [1,2]. These chimeras, which are called specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) and proteolysis-targeting chimeras (PROTACs), contain two ligands connected with a linker: one ligand specific to an E3 ubiquitin ligase and another ligand specific to the target protein. These chimeric compounds are designed to cross-link the E3 ligase with the target protein within

Pharmaceuticals 2020, 13, 34; doi:10.3390/ph13030034 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 34 2 of 12

the target protein. These chimeric compounds are designed to cross-link the E3 ligase with the target protein within the cells, and thereby, induce ubiquitination and subsequent degradation by Pharmaceuticalsproteasomes.2020 , Several13, 34 chimeric degraders have successfully been employed to degrade several2 of 13 proteins such as nuclear receptors [3–7], oncogenic kinases [8], epigenetic regulators [9,10], transcription factors [11], and others [12,13]. Some compounds can also degrade target proteins in the cells, and thereby, induce ubiquitination and subsequent degradation by proteasomes. Several vivo, suggesting the possibility of clinical applications [5,9,10,14,15]. In contrast to traditional chimericinhibitor-based degraders pharmaceuticals, have successfully such been as employedreversible/irreversible to degrade inhibitor several proteins and antagonists, such as nuclear the receptorschimeras [3 –require7], oncogenic only the kinases transient [8 ],binding epigenetic to any regulators surface of [the9,10 target], transcription to catalytically factors induce [11], its and othersubiquitination. [12,13]. Some Thus, compounds this technology can also has degrade emer targetged as proteins a novelin therapeutic vivo, suggesting approach the possibilityknown as of clinical“undruggable” applications proteins. [5,9,10 ,14,15]. In contrast to traditional inhibitor-based pharmaceuticals, such as reversibleRecently,/irreversible we developed inhibitor a and new antagonists, class of small the molecu chimerasle chimeras require that only recruit the transient the aryl hydrocarbon binding to any surfacereceptor of the (AhR) target E3 toligase catalytically and induce induce the AhR-dependent its ubiquitination. degradation Thus, this of target technology proteins has [16]. emerged β-NF- as a novelATRA, therapeutic 1 (Figure approach1), a chimeric known compound as “undruggable” directed against proteins. cellular retinoic acid-binding proteins (CRABPs),Recently, induced we developed the AhR-depe a new classndent of degradation small molecule of CRABP-1 chimeras and that CRABP-2 recruit in the MCF-7 aryl hydrocarbon and IMR- receptor32 cells (AhR) expressing E3 ligaseAhR [16]. and Interestingly, induce the the AhR-dependent 1-induced significant degradation reduction of of target CRABP-2, proteins but not [16 ]. β-NF-ATRA,of CRABP-1,1 was(Figure also1 ),observed a chimeric in SH-SY5Y compound cells, directedwhich do against not express cellular AhR retinoic (Figure acid-bindingS1 and S2). proteinsSimilar (CRABPs), results were induced also obtained the AhR-dependent by the treatmen degradationt of the ITE-ATRA of CRABP-1 [16], and in CRABP-2which an inalternative MCF-7 and IMR-32AhR cellsligand expressing was used AhR(Figure [16 S3).]. Interestingly, The ligand-bound the 1-induced AhR forms signiﬁcant a CUL4B-based reduction E3 ligase of CRABP-2, complex but [17]. The NEDD8-activating E1 enzyme inhibitor MLN4924 [18,19] completely inhibited the 1- not of CRABP-1, was also observed in SH-SY5Y cells, which do not express AhR (Figures S1 and S2). induced reduction of CRABP-1 but partly that of CRABP-2 (Figure S4). These results suggested that Similar results were also obtained by the treatment of the ITE-ATRA [16], in which an alternative AhR these chimeric compounds degrade CRABP-1 and CRABP-2 via an AhR-dependent mechanism, ligand was used (Figure S3). The ligand-bound AhR forms a CUL4B-based E3 ligase complex [17]. whereas CRABP-2 is also degraded via an AhR-independent mechanism. The NEDD8-activatingIn this study, we E1 synthesized enzyme inhibitor several MLN4924compounds [ 18to, 19investigate] completely the inhibitedmechanisms the of1-induced AhR- reductionindependent of CRABP-1 degradation but partlyand evaluate that of their CRABP-2 activities. (Figure These S4).results These will provide results suggesteduseful information that these chimericfor the compounds development degrade of chimeric CRABP-1 compounds and CRABP-2 using a via highly an AhR-dependent hydrophobic E3 mechanism, ligand and whereastheir CRABP-2pharmaceutical is also degraded applications. via an AhR-independent mechanism.

FigureFigure 1. 1.Chemical Chemical structurestructure of ββ-NF-ATRA-NF-ATRA (1 (1).).

2. InResults this and study, Discussion we synthesized several compounds to investigate the mechanisms of AhR-independent degradation and evaluate their activities. These results will provide useful information2.1. Design forand theSynthesis development of Compounds of chimeric compounds using a highly hydrophobic E3 ligand and theirSince pharmaceutical CRABP-2 was applications. degraded by 1 and ITE-ATRA in SH-SY5Y cells that do not express AhR (Figure S1-S3), we assumed that the high hydrophobicity of the β-NF ligand module might play a 2. Resultsrole in the and AhR-independent Discussion degradation of CRABP-2. Appending a hydrophobic moiety to a protein surface induces protein degradation; the hydrophobic moiety mimics a state of partial unfolding, 2.1. Design and Synthesis of Compounds inducing the degradation of the target protein via an intracellular quality control system [20–22]. BocSince3-Arg- CRABP-2 and adamantane-based was degraded hydrophobic by 1 and ITE-ATRA tagging (H inyT) SH-SY5Y strategies cells have thatbeen do applied not express to a range AhR (Figuresof exogenous S1–S3), we[23,24] assumed and endogenous that the high proteins hydrophobicity [25,26]. To of investigate the β-NF ligandwhether module CRABP might proteins play are a role in thedegraded AhR-independent by the HyT system, degradation we designed of CRABP-2. and synthesized Appending 2 (Ad-ATRA-1) a hydrophobic and 3 moiety (Ad-ATRA-2) to a protein by surface induces protein degradation; the hydrophobic moiety mimics a state of partial unfolding, inducing the degradation of the target protein via an intracellular quality control system [20–22]. Boc3-Arg- and adamantane-based hydrophobic tagging (HyT) strategies have been applied to a range of exogenous [23,24] and endogenous proteins [25,26]. To investigate whether CRABP proteins are degraded by the HyT system, we designed and synthesized 2 (Ad-ATRA-1) and 3 (Ad-ATRA-2) by Pharmaceuticals 2020, 13, 34 3 of 13

Pharmaceuticals 2020, 13, 34 3 of 12 Pharmaceuticals 2020, 13, 34 3 of 12 conjugatingconjugating an an adamantane adamantane moiety moiety (Figure (Figure2 2).). Besides, Besides, we designed 4 4thatthat lacked lacked the the benzochromene benzochromene conjugating an adamantane moiety (Figure 2). Besides, we designed 4 that lacked the benzochromene moietymoiety of of1 because 1 because we we expected expected that that the the hydrophobicityhydrophobicity of of 44 wouldwould be be smaller smaller than than that that of of1. 1. moiety of 1 because we expected that the hydrophobicity of 4 would be smaller than that of 1.

FigureFigure 2. 2.Chemical Chemical structures of of designed designed compounds. compounds. Figure 2. Chemical structures of designed compounds.

SchemeScheme1 presents 1 presents the the synthetic synthetic route route of of 22.. 1-Adamantanecarboxylic1-Adamantanecarboxylic acid acid was was condensed condensed with with Scheme 1 presents the synthetic route of 2. 1-Adamantanecarboxylic acid was condensed with 12 12[27 [27]] to to obtain obtain5 .5. Adamantane-ATRA,Adamantane-ATRA, with with an an ester ester bond bond (Ad-ATRA-1, (Ad-ATRA-1, 2), was2), obtained was obtained after the after 12 [27] to obtain 5. Adamantane-ATRA, with an ester bond (Ad-ATRA-1, 2), was obtained after the reduction of the azide group in 5, the condensation with 13 [12], and the deprotection of 7. thereduction reduction of of the the azide azide group group in in 5,5 the, the condensation condensation with with 13 13[12],[12 and], and the the deprotection deprotection of 7 of. 7. Adamantane-ATRA, with an amide bond (Ad-ATRA-2, 3) was also prepared via the condensation of Adamantane-ATRA,Adamantane-ATRA, with with an an amide amide bond (Ad-ATRA-2, (Ad-ATRA-2, 3)3 was) was also also prepared prepared via viathe condensation the condensation of 8 with 13, following a similar procedure (Scheme 2). Bn-ATRA (4) was obtained by condensing the of 88 withwith 13,, following following a asimilar similar procedure procedure (Scheme (Scheme 2). 2Bn-ATRA). Bn-ATRA (4) was ( 4) wasobtained obtained by condensing by condensing the corresponding glycol amino linkers with 13, followed by deprotection (Scheme 3). thecorresponding corresponding glycol glycol amino amino linkers linkers with with 13, 13followed, followed by deprotection by deprotection (Scheme (Scheme 3). 3).

Scheme 1 (a) 12, EDC-HCl, DMAP, CH2Cl2; (b) H2 gas, 10% Pd/C, EtOAc; (c) 13, EDC-HCl, DMAP, SchemeScheme 1. (1a )(a) 12, 12, EDC-HCl, EDC-HCl, DMAP, DMAP, CH CH22ClCl2;;( (b)b)H H22 gas,gas, 10% 10% Pd/C, Pd/C, EtOAc; EtOAc; (c) ( c13,) 13, EDC-HCl, EDC-HCl, DMAP, DMAP, CH2Cl2; (d) TBAF, THF. CHCH2Cl22Cl;(2d; (d)) TBAF, TBAF, THF. THF. Pharmaceuticals 2020, 13, 34 4 of 13 PharmaceuticalsPharmaceuticals 2020 2020, ,13 13, ,34 34 44 ofof 1212

SchemeSchemeScheme 2. 22( a (a)(a)) 14, 14,14, EDC-HCl, EDC-HCl,EDC-HCl, HOBt, HOBt, CHCH CH22ClCl2Cl22;; 2(b)(b);( b 4M4M) 4M HCl/1,4-dioxane;HCl/1,4-dioxane; HCl/1,4-dioxane; (c)(c) 13,13, (c )HATU,HATU, 13, HATU, DIPEA,DIPEA, DIPEA, DMF;DMF; (d) DMF;(d) (d)TBAF, TBAF, THF.THF.

OO NCNC OO

OO NHNH22 aa OO OO NN 1010 OO HH OO NN OO OO 1111 OO

HOHO bb NN OO HH OO NN OO OO OO Bn-ATRABn-ATRA ((44)) SchemeSchemeScheme 3. 33 ( (a)(a)a) 13,13, HATU,HATU, DIPEA, DIPEA, DMF; DMF; DMF; (b)(b) (b TBAF,)TBAF, TBAF, THF.THF. THF.

2.2.2.2.2.2. Evaluation EvaluationEvaluation of of Proteinof ProteinProtein Degradation DegradationDegradation Activity ActivityActivity WeWeWe examined examined examined the the the protein protein protein reduction reduction activity activity activity of of of2 2,, 3 23,,, and and3, and 4 4 using using4 using various various various cell cell lines. lines. cell In In lines. MCF-7MCF-7 In cells; cells; MCF-7 cells;22 andand2 and 33 effectively3effectivelyeﬀectively reducedreduced reduced thethe the CRABP-2CRABP-2 CRABP-2 proteinprotein protein level,level, level, whereaswhereas whereas 44,, which4which, which containedcontained contained thethe the lessless less hydrophobichydrophobichydrophobic benzyl benzylbenzyl moiety, moiety,moiety, showed showedshowed attenuated attenuatedattenuated protein proteinprotein knockdown knockdownknockdown activity activityactivity (Figure (Figure(Figure3 A 3A3A and andand Figure FigureFigure S5). TheS5).S5). CRABP2 TheThe CRABP2CRABP2 reduction reductionreduction activity activityactivity of 3 was ofof similar33 waswas similarsimilar to that toto of that1thatin of MCF7of 11 inin cellsMCF7MCF7 (Figure cellscells (Figure(Figure S6). The S6).S6). CRABP2 TheThe knockdownCRABP2CRABP2 knockdownknockdown activities of activitiesactivities2, 3, and ofof 4 22,, in 3,3, andSH-SY5Yand 44 inin SH-SY5YSH-SY5Y cells were cellscells similar werewere similasimila to theirrr toto their activitiestheir activitiesactivities in MCF-7 inin MCF-MCF- cells (Figure77 cellscells3B). (Figure(Figure The protein 3B).3B). TheThe level proteinprotein of CRABP-1 levellevel ofof CRABP-1CRABP-1 was not signiﬁcantly waswas notnot significantlysignificantly reduced reduced byreduced2, 3, andbyby 22,4, 33in,, andand SH-SY5Y 44 inin SH-SH- cells (FigureSY5YSY5Y3 B). cellscells Besides, (Figure(Figure 3 3B).3B).tended Besides,Besides, to reduce 33 tendedtended CRABP-2 toto reducereduce in CRABP-2 IMR-32CRABP-2 cells inin IMR-32IMR-32 that expressed cellscells thatthat AhR expressedexpressed but not AhRAhR CRABP-1 butbut in contrastnotnot CRABP-1CRABP-1 to 1 (Figure inin contrastcontrast S1 and toto 11 Figure (Figure(Figure3C). S1S1 These andand 3C).3C). results TheseThese indicated resultsresults indicatedindicated that CRABP-2, thatthat CRABP-2,CRABP-2, but not CRABP-1,butbut notnot CRABP-1,CRABP-1, waswas susceptiblesusceptible toto HyT-mediatedHyT-mediated degradation.degradation. Thus,Thus, 11,, whichwhich containscontains thethe highlyhighly was susceptible to HyT-mediated degradation. Thus, 1, which contains the highly hydrophobic β-NF hydrophobichydrophobic ββ-NF-NF ligand,ligand, isis likelylikely toto degradedegrade CRABP-2CRABP-2 inin thethe absenceabsence ofof AhRAhR viavia thethe HyTHyT ligand, is likely to degrade CRABP-2 in the absence of AhR via the HyT mechanism. mechanism.mechanism. Pharmaceuticals 2020, 13, 34 5 of 13 Pharmaceuticals 2020, 13, 34 5 of 12

FigureFigure 3. HyT3. HyT compounds compounds induce induce the the degradationdegradation of CRABP-2 but but not not of of CRABP-1. CRABP-1. (A–C) (A– CProtein) Protein knockdownknockdown activities activities of of HyT HyT2 ,23, ,3 and, and4 4or orβ β-NF-ATRA-NF-ATRA 1 inin MCF-7 MCF-7 (A), (A), SH-SY5Y SH-SY5Y (B), (B or), orIMR-32 IMR-32 cells cells (C).(C). Cells Cells were were treated treated with with the the indicated indicated compounds compounds for 24 24 h. h. Whole-cell Whole-cell lysates lysates were were analyzed analyzed by by westernwestern blotting. blotting. The The numbers numbers below below the the CRABP-1 CRABP- and1 and CRABP-2 CRABP-2 panels panels represent represent CRABP-1 CRABP-1/actin/actin and CRABP-2and CRABP-2/actin/actin ratios, respectively,ratios, respectively, normalized normalized by designating by designating the expression the expression from from the vehicle the vehicle control control condition as 100%. condition as 100%.

WeWe also also examined examined the the involvementinvolvement of of the the pr proteasomeoteasome in HyT-mediated in HyT-mediated degradation. degradation. The 2 and The 2 and3-induced3-induced reductions reductions of ofCRABP-2 CRABP-2 were were completely completely inhibited inhibited in inthe the presence presence of ofthe the proteasome proteasome inhibitor MG132, suggesting that 2 and 3 induce the proteasomal degradation of CRABP-2 (Figure inhibitor MG132, suggesting that 2 and 3 induce the proteasomal degradation of CRABP-2 (Figure4). 4). This result clearly indicated the involvement of proteasome activity for the reduction of the target This result clearly indicated the involvement of proteasome activity for the reduction of the target protein. However, this result could not deny the other possibilities, such as lysosomal degradations, protein. However, this result could not deny the other possibilities, such as lysosomal degradations, or insolubilization by Lys63-linked ubiquitination. It is important to reveal the complexed pathway or insolubilizationleading to the proteasome. by Lys63-linked ubiquitination. It is important to reveal the complexed pathway leading to the proteasome. Pharmaceuticals 2020 13 Pharmaceuticals 2020, , ,13 34, 34 6 of 612 of 13 PharmaceuticalsPharmaceuticals 2020 2020, ,13 13, ,34 34 66 ofof 1212

Figure 4. Proteasomal inhibitor MG132 inhibits the reduction of CRABP-2 by HyT compounds. MCF- FigureFigure 4. Proteasomal4. Proteasomal inhibitor inhibitor MG132 MG132 inhibits inhibits thethe reductionreduction of CRABP-2 by by HyT HyT compounds. compounds. MCF- MCF-7 7Figure cells were 4. Proteasomal incubated inhibitor with the MG132 indicated inhibits concentr the reationsduction of ofHyT CRABP-2 compounds by HyT in compounds. the presence MCF- or cells7 werecells were incubated incubated with with the indicated the indicated concentrations concentrations of HyT of compoundsHyT compounds in the in presence the presence or absence or absence7 cells ofwere 10 µMincubated MG132 withfor 18 the h. Whole-cellindicated concentrlysates wereations analyzed of HyT by compounds western blotting. in the presence or of 10absenceabsenceµM MG132 of of 10 10 µM µM for MG132 MG132 18 h. Whole-cell for for 18 18 h. h. Whole-cell Whole-cell lysates were lysates lysates analyzed were were analyzed analyzed by western by by western western blotting. blotting. blotting. Next, we expanded the investigation to other target proteins. We previously developed the Next,Next, we we expanded expanded the investigationthe investigation to other to other target target proteins. proteins. Wepreviously We previously developed developed the chimeric the chimericNext, compound we expanded β-NF-JQ1 the thatinvestig is directedation to against other bromodomain-containingtarget proteins. We previously (BRD) proteinsdeveloped using the compoundchimeric βcompound-NF-JQ1 thatβ-NF-JQ1 is directed that is against directed bromodomain-containing against bromodomain-containing (BRD) proteins(BRD) proteins using usingβ-NF or βchimeric-NF or (+)-JQ1 compound as an β-NF-JQ1 AhR or that BRD is directedligand, againstrespectively. bromodomain-containing β-NF-JQ1 induced the(BRD) AhR-dependent proteins using (+)-JQ1β-NF asor an(+)-JQ1 AhR oras BRDan AhR ligand, or BRD respectively. ligand, respectively.β-NF-JQ1 induced β-NF-JQ1 the induced AhR-dependent the AhR-dependent degradation degradationβ-NF or (+)-JQ1 of BRD as proteinsan AhR inor MCF-7 BRD ligand, cells expressing respectively. AhR β [16].-NF-JQ1 However, induced the the β-NF-JQ1-induced AhR-dependent of BRDdegradation proteins of inBRD MCF-7 proteins cells in expressing MCF-7 cells AhR expressing [16]. However, AhR [16]. the However,β-NF-JQ1-induced the β-NF-JQ1-induced reduction of reductiondegradation of BRD3,of BRD but proteins not of inBRD2 MCF-7 or ceBRD4,lls expressing was also AhRobserved [16]. inHowever, SH-SY5Y the cells, β-NF-JQ1-induced which do not reduction of BRD3, but not of BRD2 or BRD4, was also observed in SH-SY5Y cells, which do not BRD3,expressreduction but AhR not of (Figure ofBRD3, BRD2 S7),but or suggestingnot BRD4, of BRD2 was that or also BRD3 BRD4, observed is alsowas degraded also in SH-SY5Y observed by the cells,in AhR-independent SH-SY5Y which cells, do not which mechanism. express do not AhR express AhR (Figure S7), suggesting that BRD3 is also degraded by the AhR-independent mechanism. (FigureTherefore,express S7), AhR suggesting we (Figure synthesized thatS7), suggesting BRD3 Ad-JQ1 is also ( 15that) degraded that BRD3 conjugated is also by the degraded AhR-independentan adamantane by the AhR-independent moiety mechanism. to JQ1 (Scheme mechanism. Therefore, 4). we Therefore, we synthesized Ad-JQ1 (15) that conjugated an adamantane moiety to JQ1 (Scheme 4). synthesizedFigureTherefore, 5 presents Ad-JQ1 we synthesized (the15) significant that conjugatedAd-JQ1 Ad-JQ1-induced (15) anthat adamantane conjugated reduction moietyan adamantane of toBRD3, JQ1 (Schemebut moiety not 4ofto). BRD2 FigureJQ1 (Scheme or5 presentsBRD4 4). Figure 5 presents the significant Ad-JQ1-induced reduction of BRD3, but not of BRD2 or BRD4 the(FigureFigure signiﬁcant 55). presents These Ad-JQ1-induced resultsthe significant suggested reduction Ad-JQ1-induced that of BRD3, BRD3, bu but reductiont not not ofothers, BRD2 of BRD3,is or susceptible BRD4 but not (Figure toof BRD2HyT-mediated5). These or BRD4 results (Figure 5). These results suggested that BRD3, but not others, is susceptible to HyT-mediated suggesteddegradation.(Figure that5). These BRD3, results but not suggested others, is susceptiblethat BRD3, tobu HyT-mediatedt not others, is degradation.susceptible to HyT-mediated degradation.degradation. N N N NN N NN N N N N N N S N N S N SS O N SS a O H HO N a O O NH N 8 + HO N a N O O NH N 8 + HO O N O N O N 8 + O HNN OO O O HH O

Ad-JQ1 (16) Cl Cl 15 Ad-JQ1Ad-JQ1 ( 16(16)) 15 ClCl ClCl 15 Scheme 4 (a) HBTU, DIPEA, and DMF. SchemeSchemeScheme 4.4 4 (a)(a) HBTU, HBTU,HBTU, DIPEA, DIPEA, and and and DMF. DMF. DMF.

FigureFigure 5. 5.Ad-JQ1 Ad-JQ1 (16 (16)) induces induces the the degradationdegradation of BRD3 BRD3 but but not not of of BRD2 BRD2 or or BRD4. BRD4. Protein Protein knockdown knockdown Figure 5. Ad-JQ1 (16) induces the degradation of BRD3 but not of BRD2 or BRD4. Protein knockdown activitiesactivitiesFigure of 5. of Ad-JQ1Ad-JQ1 Ad-JQ1 ( in16 in MCF-7) MCF-7induces cells. cells. the degradation CellsCells were treate treatedof BRD3d with with but the not the indicated of indicated BRD2 orconcentration concentrationBRD4. Protein of Ad-JQ1knockdown of Ad-JQ1 for for activities of Ad-JQ1 in MCF-7 cells. Cells were treated with the indicated concentration of Ad-JQ1 for 4848 h.activities h. Whole-cell Whole-cell of Ad-JQ1 lysates lysates in were MCF-7were analyzed analyzed cells. Cells byby westernwere treate blotting. blotting.d with the indicated concentration of Ad-JQ1 for 4848 h. h. Whole-cell Whole-cell lysates lysates were were analyzed analyzed by by western western blotting. blotting. Pharmaceuticals 2020, 13, 34 7 of 13

In the present study, we designed and synthesized compounds with adamantane moiety to investigate whether the AhR-independent degradation of CRABP-2 and BRD3 by the chimeric compounds using AhR ligands are induced by the HyT system. We observed that 2, 3, and 16 induced significant degradation of CRABP-2 and BRD3, but not of other target proteins, by the proteasome. Besides, Bn-ATRA 4, which is less hydrophobic than 1, 2, and 3, induced a weaker activity than 1, 2, and 3. In conclusion, these results indicated that CRABP-2 and BRD3 are susceptible to HyT-mediated degradation and that the AhR-independent reduction of these proteins was caused by the HyT system. These results also provided useful information for drug development using chimeric compounds with a highly hydrophobic E3 ligand, which may occasionally induce targeted protein degradation by the HyT system. In addition to our findings, it is important to clarify the types of ubiquitin chains on target proteins and the specific E3 ligases involved in the ubiquitination.

3. Materials and Methods All reagents and solvents were purchased from Sigma-Aldrich, Wako Pure Chemical, and Tokyo Chemical Industry and used without purification. Analytical TLC was conducted using Merck silica gel 60 F254 pre-coated plates, and visualized with a 254 nm UV lamp using phosphomolybdic acid, p-anisaldehyde, or ninhydrin stains. Column chromatography was performed using silica gel (spherical, neutral) purchased from Kanto Chemical. 1H and 13C NMR spectra were measured using a Varian AS 400 spectrometer or a JEOL ECZ 600R spectrometer, and measurements were carried out using deuterated solvents. Chemical shifts are expressed in ppm downfield from a solvent residual peak or the internal standard TMS. Mass spectra were measured using a Shimadzu IT-TOF MS equipped with an electrospray ionization source. (2E,4E,6E,8E)-9-((E)-3-(((14-((3r,5r,7r)-adamantan-1-yl)-2,13-dioxo-6,9,12-trioxa-3-azatetradecyl) oxy)imino)-2,6,6-trimethylcyclohex-1-en-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid (Ad-ATRA-1, 2) EDC-HCl (120 mg, 0.63 mmol), followed by DMAP (15 mg, 0.12 mmol), was added to a mixture of 1-adamantaneacetic acid (100 mg, 0.52 mmol) and 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (12)[27] (108 mg, 0.62 mmol) in CH2Cl2 (2 mL). After continuous stirring for two days at room temperature, the reaction mixture was diluted with EtOAc and washed with 2M HCl, saturated with aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/EtOAc = 7:3) to 1 obtain 5 as a colorless oil (47 mg, 26%). H NMR (400 MHz, CDCl3) δ 4.23–4.21 (m, 2H), 3.72–3.66 (m, 13 8H), 3.40–3.38 (m, 2H), 2.10 (s, 2H), 1.97 (br. s, 3H), 1.72–1.62 (m, 12H); C NMR (150 MHz, CDCl3) δ 171.8, 70.6, 70.5, 70.0, 69.2, 62.8, 50.5, 48.7, 42.2, 36.6, 32.7, 28.5. ESI-HRMS calcd for C18H30N3O4 [M+H]+: 352.2231, found: 352.2194. A mixture of 5 (12 mg, 0.03 mmol) in EtOAc (10 mL) with 10% Pd/C (7.3 mg) was stirred for 13 h under H2 gas at room temperature and then, filtered through Celite to remove the catalyst. The filtrate was concentrated under reduced pressure to obtain the crude product (10.5 mg), which was used in the following step without further purification. EDC-HCl (14.6 mg, 0.076 mmol), followed by DMAP (9.3 mg, 0.076 mmol), was added to the mixture of the crude product (10.5 mg as 0.03 mmol) and 13 [12] (14.4 mg, 0.033 mmol) in CH2Cl2 (2 mL) After continuous stirring for 23 h at room temperature, the reaction mixture was diluted with CH2Cl2 and washed with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexanes/EtOAc = 3:1) to obtain 7 as a yellow oil (7.9 mg, 31% for two steps). 1H NMR (400 MHz, CDCl3) δ 7.03 (dd, J = 15.0, 15.0 Hz, 1H), 6.67 (t, J = 5.4 Hz, 1H), 6.35 (d, J = 15.6 Hz, 1H), 6.33–6.30 (m, 1H), 6.22 (d, J = 15.0 Hz, 1H), 6.21 (d, J = 15.6 Hz, 1H), 5.82 (s, 1H), 4.59 (s, 2H), 4.33 (t, J = 6.0 Hz, 2H), 4.21 (t, J = 6.0 Hz, 2H), 3.67 (t, J = 4.8 Hz, 2H), 3.62–3.60 (m, 4H), 3.58 (t, J = 4.8 Hz, 2H), 3.54–3.51 (m, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.37 (s, 3H), 2.09 (s, 2H), 2.03 (s, 3H), 1.96 (s, 3H), 1.87 (s, 3H), 1.73–1.69 (m, 2H), 1.63–1.61 (m, 12H), 1.10 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 171.6, 170.1, 166.1, 158.6, 154.4, 150.0, 139.1, 139.1, 135.9, 131.3, 127.0, 125.0, 117.6, 117.0, 73.1, Pharmaceuticals 2020, 13, 34 8 of 13

70.4, 70.3, 70.0, 69.2, 62.8, 58.0, 48.8, 42.3, 38.6, 38.6, 36.7, 36.0, 34.8, 32.7, 28.6, 27.6, 20.2, 18.1, 14.8, 14.0, + 12.8. ESI-HRMS calcd for C43H62N3O8 [M+H] : 748.4531, found: 748.4529. TBAF (1 M in THF solution, 43 µL, 0.043 mmol) was added to a solution of 7 (8 mg, 0.010 mmol) in THF (0.19 mL). After continuous stirring for 1.5 h at room temperature, the reaction mixture was diluted with CHCl3, containing 0.1% AcOH and then, concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (CHCl3/MeOH = 99:1 to 98:2) to obtain 2 as 1 a yellow solid (4.0 mg, 54%, containing ca. 10% of the 13-cis isomer). H NMR (400 MHz, CDCl3) δ 7.03 (dd, J = 15.6, 15.6 Hz, 1H), 6.68 (t, J = 5.7 Hz, 1H), 6.36 (d, J = 15.0 Hz, 1H), 6.31 (d, J = 16.2 Hz, 1H), 6.23–6.21 (m, 2H), 5.83 (s, 1H), 4.59 (s, 2H), 4.21 (t, J = 5.1 Hz, 2H), 3.67 (t, J = 4.8 Hz, 2H), 3.61–3.52 (m, 8H), 2.67 (t, J = 6.6 Hz, 2H), 2.37 (s, 3H), 2.03 (s, 3H), 1.96 (br., 4H), 1.87 (s, 3H), 1.69 (d, J = 12.0 Hz, 3H), 13 1.63–1.61 (m, 12H), 1.10 (s, 6H); C NMR (150 MHz, CDCl3) δ 171.7, 170.6, 170.2, 158.7, 154.7, 150.1, 139.2, 139.0, 136.2, 131.4, 131.2, 127.0, 125.0, 118.1, 73.1, 70.4, 70.3, 69.9, 69.3, 62.8, 48.8, 42.3, 38.7, 36.7, + 36.0, 34.9, 32.8, 28.6, 27.6, 20.2, 14.9, 14.0, 12.9. ESI-HRMS calcd for C40H59N2O8 [M+H] : 695.4266, found: 695.4226. (2E,4E,6E,8E)-9-((E)-3-(((14-((3r,5r,7r)-adamantan-1-yl)-2,13-dioxo-6,9-dioxa-3,12- diazatetradecyl)oxy)imino)-2,6,6-trimethylcyclohex-1-en-1-yl)-3,7-dimethylnona-2,4,6,8-tetraenoic acid (Ad-ATRA-2, 3) EDC-HCl (144 mg, 0.75 mmol) was added to a mixture of 1-adamantaneacetic acid (97 mg, 0.50 mmol), N-(tert-butoxycarbonyl)-2,20-(ethylenedioxy)diethylamine 14 (149 mg, 0.60 mmol) and HOBt-H2O (0.75 mmol) in CH2Cl2 (1.0 mL). After continuous stirring for 1.5 h at room temperature, the reaction mixture was diluted with EtOAc and washed with 1M HCl, saturated aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure to give a colorless oil (238 mg). This crude product (200 mg, as 0.47 mmol) was treated with 4M HCl/1,4-dioxane (1 mL) for 8 h at room temperature. The volatiles were removed by evaporation under reduced pressure and subsequent co-evaporation with CH CN (1 mL 2) under reduced pressure. 3 × HCl salt was obtained as a colorless oil (8), which was used in the following reaction without further purification. HATU (76 mg, 0.20 mmol), followed by DIPEA (104 µL, 0.60 mmol), was added to a mixture of 13 (44 mg, 0.10 mmol) and 8 (54 mg, as 0.15 mmol) in DMF (0.3 mL). After continuous stirring for 12 h at room temperature, the reaction mixture was diluted with EtOAc and washed with 1 M HCl, saturated with aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography 1 (CHCl3/MeOH = 100:0 to 99:1) to obtain 9 as a yellowish amorphous solid (56 mg, 75%). H NMR (400 MHz, CDCl3) δ 7.04 (dd, J = 15.6, 15.0 Hz, 1H), 6.67 (t, J = 5.4 Hz, 1H), 6.35 (d, J = 15.0 Hz, 1H), 6.32 (t, J = 15.6 Hz, 1H), 6.24–6.21 (m, 2H), 5.91 (br. s, 1H), 5.83 (s, 1H), 4.59 (s, 2H), 4.33 (t, J = 6.0 Hz, 2H), 3.62–3.52 (m, 10H), 3.47–3.42 (m, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.67 (t, J = 6.6 Hz, 1H), 2.37 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H), 1.93 (s, 2H), 1.87 (s, 3H), 1.71–1.69 (m, 3H), 1.63–1.62 (m, 12H), 1.11 (s, 6H); 13C NMR (150 MHz, CDCl3) δ 170.9, 170.2, 166.1, 158.7, 154.3, 150.1, 139.1, 139.0, 135.9, 131.3, 131.2, 126.9, 124.8, 117.5, 116.9, 73.0, 70.2, 70.1, 70.0, 69.8, 58.0, 51.6, 42.5, 38.9, 38.6, 36.7, 35.9, 34.8, 32.6, 28.6, 27.5, 20.1, + 18.0, 14.8, 13.9, 12.8. ESI-HRMS calcd for C43H63N4O7 [M+H] : 747.4691, found: 747.4655. TBAF (1 M in THF solution, 117 µL, 117.0 µmol) was added to a solution of 9 (29 mg, 0.039 mmol) in THF (0.7 mL). After continuous stirring for 1.5 h at room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (CHCl3/MeOH = 100:0 to 98:2) to obtain 3 as a yellowish amorphous solid (11 mg, 1 42%, containing 10% of the 13-cis isomer). H NMR (400 MHz, CDCl3) δ 7.02 (dd, J = 15.0, 15.0 Hz, 1H), 6.69 (t, J = 6.0 Hz, 1H), 6.34 (d, J = 15.0 Hz, 1H), 6.32 (d, J = 16.8 Hz, 1H), 6.29–6.19 (m, 2H), 5.93 (br. t, 1H), 5.83 (s, 1H), 4.60 (s, 2H), 3.62–3.53 (m, 10H), 3.45 (t, J = 5.6 Hz, 2H), 2.67 (t, J = 6.6 Hz, 2H), 2.37 (s, 3H), 2.03 (s, 3H), 1.96 (br. s, 3H), 1.94 (s, 2H), 1.87 (s, 3H), 1.69 (d, J = 12.0 Hz, 3H), 1.63–1.61 (m, 13 11H), 1.10 (s, 6H); C NMR (150 MHz, CDCl3) δ 171.1, 170.8, 170.4, 158.8, 154.3, 150.2, 139.2, 138.9, 136.3, 131.5, 131.1, 126.8, 124.8, 118.5, 73.0, 70.2, 70.1 (1C overlapped), 69.9, 51.7, 42.6, 39.0, 38.7, 36.7, Pharmaceuticals 2020, 13, 34 9 of 13

+ 35.9, 34.8, 32.7, 28.6, 27.6, 20.2, 14.9, 14.0, 12.8. ESI-HRMS calcd for C40H60N3O7 [M+H] : 694.4426, found: 694.4391. (2E,4E,6E,8E)-3,7-dimethyl-9-((E)-2,6,6-trimethyl-3-(((12-oxo-1-phenyl-2,5,8-trioxa-11- azatridecan-13-yl)oxy)imino)cyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoic acid) (Bn-ATRA, 4) HATU (46 mg, 0.12 mmol), followed by DIPEA (42 µL, 0.24 mmol), were added to a mixture of 13 (44 mg, 0.10 mmol) and 10 [28,29] (48 mg, 0.20 mmol) in DMF (2 mL). After continuous stirring for 12 h at room temperature, the reaction mixture was diluted with EtOAc and washed with 1 M HCl, saturated with aqueous NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography 1 (CHCl3/MeOH = 100:0 to 99:1) to obtain 11 as a yellow oil (41 mg, 62%). H NMR (600 MHz, CDCl3) δ 7.34–7.33 (m, 4H), 7.29–7.27 (m, 1H), 7.03 (dd, J = 15.6, 15.0 Hz, 1H), 6.69 (br. t, J = 5.4 Hz, 1H), 6.33 (d, J = 15.0 Hz, 1H), 6.31 (d, J = 15.6 Hz, 1H), 6.22 (d, J = 15.0 Hz, 1H), 6.22 (d, J = 15.0 Hz, 1H), 5.82 (s, 1H), 4.58 (s, 2H), 4.56 (s, 2H), 4.32 (t, J = 6.0 Hz, 2H), 3.67–3.65 (m, 2H), 3.64–3.62 (m, 6H), 3.59–3.57 (m, 2H), 3.53–3.51 (m, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.66 (t, J = 6.6 Hz, 2H), 2.37 (s, 3H), 2.03 (s, 3H), 1.87 (s, 13 3H), 1.61 (t, J = 6.6 Hz, 2H), 1.09 (s, 6H); C NMR (150 MHz, CDCl3) δ 170.0, 166.1, 158.5, 154.3, 149.9, 139.0, 138.0, 135.8, 131.3, 131.2, 128.3, 127.6, 127.5, 127.0, 124.9, 117.5, 117.0, 73.2, 73.0, 70.6, 70.4, 70.2, 69.8, 69.3, 58.0, 38.6, 38.5, 35.9, 34.8, 27.5, 20.1, 18.0, 14.8, 13.9, 12.8. ESI-HRMS calcd for C38H52N3O7 [M+H]+: 662.3800, found: 662.3834. TBAF (1 M in THF solution, 99 µL, 0.099 mmol) was added to a solution of 11 (22 mg, 0.033 mmol) in THF (0.6 mL). After continuous stirring for 1.5 h at room temperature, the reaction mixture was diluted with CHCl3, containing 0.1% AcOH, and then, concentrated under reduced pressure. The residue was purified by flash silica gel column chromatography (CHCl3/MeOH = 100:0 to 98:2) to obtain 4 as 1 a yellow solid (8.6 mg, 44%, containing ca. 10% of the 13-cis isomer). H NMR (400 MHz, CDCl3) δ 7.34–7.33 (m, 5H), 7.06–6.99 (m, 1H), 6.66 (br. t, J = 5.6 Hz, 1H), 6.37–6.19 (m, 4H), 5.83 (s, 1H), 4.58 (s, 2H), 4.56 (s, 2H), 3.66–3.62 (m, 8H), 3.59–3.57 (m, 2H), 3.54–3.49 (m, 2H), 2.66 (t, J = 6.8 Hz, 2H), 2.37 13 (s, 3H), 2.02 (s, 3H), 1.87 (s, 3H), 1.61 (t, J = 6.8 Hz, 2H), 1.10 (s, 6H); C NMR (150 MHz, CDCl3) δ 171.3, 170.3, 158.7, 154.6, 150.0, 139.1, 139.0, 138.1, 136.2, 131.4, 131.2, 128.4, 127.7, 127.6, 127.0, 125.0, 118.4, 73.3, 73.1, 70.6, 70.5, 70.3, 69.9, 69.4, 38.7, 36.0, 34.8, 27.6, 20.2, 14.9, 14.0, 12.8. ESI-HRMS calcd for + C35H49N2O7 [M+H] : 609.3534, found: 609.3497. 2-((3r,5r,7r)-adamantan-1-yl)-N-(2-(2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)acetamide (Ad-JQ1, 16) HBTU (21 mg, 0.055 mmol), followed by DIPEA (19.6 mg, 0.15 mmol), was added to a mixture of 8 (10.8 mg, 0.03 mmol) and 15 [10] in DMF (2 mL). After continuous stirring for 0.5 h, the reaction mixture was diluted with EtOAc and washed with 1M HCl, saturated aqueous NaHCO3, and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (CHCl3/MeOH = 100:0 to 99:1) to obtain 16 as a yellow 1 oil (6.0 mg, 34%). H NMR (600 MHz, CDCl3) δ 7.40 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.9 Hz, 2H), 7.09 (dd, J = 5.4 Hz, 1H), 6.52–6.46 (m, 1H), 4.63 (dd, J = 8.1, 6.1 Hz, 1H), 3.66–3.53 (m, 10H), 3.52–3.41 (m, 3H), 3.32 (dd, J = 14.3, 6.1 Hz, 1H), 2.65 (s, 3H), 2.39 (s, 3H), 1.90 (s, 2H), 1.89–1.86 (m, 3H), 1.67 13 (d, J = 0.9 Hz, 3H), 1.65–1.53 (m, 12H). C NMR (150 MHz, CDCl3) δ 171.41, 170.57, 164.03, 155.74, 149.93, 136.94, 136.64, 132.15, 131.03, 131.00, 130.62, 129.93, 128.81, 77.32, 77.10, 76.89, 70.40, 70.37, 69.89, 54.61, 51.55, 42.71, 42.61, 39.47, 39.18, 36.87, 32.75, 28.73, 14.48, 13.19, 11.90, 0.08. ESI-HRMS calcd for + C37H48N6ClO4S [M+H] : 707.3141, found: 707.3210.

3.1. Cell Culture Human breast carcinoma MCF-7 cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 100 µg ml-1 kanamycin. Human neuroblastoma SH-SY5Y cells were maintained in Dulbecco’s modiﬁed Eagle’s medium, containing 10% FBS and 100 µg ml-1 kanamycin. Human neuroblastoma IMR-32 cells were maintained in Eagle’s minimum essential medium, containing 10% Pharmaceuticals 2020, 13, 34 10 of 13

FBS and 100 µg ml-1 kanamycin. Cells were treated with various concentrations of compounds for the indicated duration.

3.2. Western Blotting Cells were lysed with SDS lysis buffer (0.1 M Tris-HCl at pH 8.0, 10% glycerol, 1% SDS) and immediately boiled for 10 min to obtain clear lysates. Protein concentrations were measured using the BCA method (Pierce). Lysates containing equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA) for western blot analysis using the appropriate antibodies. Immunoreactive proteins were visualized using the Immobilon Western chemiluminescent HRP substrate (Millipore, MA, USA) or the Clarity Western ECL substrate (Bio-Rad, CA, USA). Light emission intensity was quantified using a LAS-3000 luminous-image analyzer equipped with Image Gauge 2.3 (Fuji, Japan). The antibodies used in this study were: anti-CRABP-2 rabbit polyclonal antibody (pAb) (Bethyl, A300-809A), anti-CRABP-1 rabbit monoclonal antibody (mAb) (Cell Signaling Technology, 13206), and anti-AhR rabbit pAb (Cell Signaling Technology, 13790). All Western blotting were performed several times to confirm reproducibility. The typical results were shown here.

3.3. Cell Viability Assay Cell viability was determined using water-soluble tetrazolium WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) in a spectrophotometric assay, according to the manufacturer’s instructions (Dojindo, Japan). Cells treated with compounds were incubated with WST-8 for 0.5 h at 37 ◦C in a humidiﬁed atmosphere of 5% CO2. The absorbance at 450 nm was measured using an EnVision Multilabel Plate Reader (PerkinElmer, USA).

Supplementary Materials: The following is available online at http://www.mdpi.com/1424-8247/13/3/34/s1, Figure S1: Levels of CRABP-1, CRABP-2, and AhR in the indicated cancer cells, Figure S2: Protein knockdown activity of β-NF-ATRA. SH-SY5Y cells were treated with β-NF-ATRA for 24 h. Whole-cell lysates were analyzed by western blotting, Figure S3: Chemical structure and protein knockdown activity of ITE-ATRA. SH-SY5Y cells were treated with ITE-ATRA for 8 h. Whole-cell lysates were analyzed by western blotting, Figure S4: Inhibitory effect of MLN4924 on IMR-32 cells. Cells were treated with β-NF-ATRA in the presence or absence of 10 µM MLN4924, Figure S5: Statistical analysis of protein knockdown activityof HyT 2, 3, and 4.in MCF-7 cells, Figure S6: Protein knockdown activity of the indicated compounds. MCF-7 cells were treated with the compounds for 24 h. Whole-cell lysates were analyzed by western blotting, Figure S7: Chemical structure and protein knockdown activity of β-NF-JQ1. SH-SY5Y cells were treated with β-NF-JQ1 for 24 h. Whole-cell lysates were analyzed by western blotting. Author Contributions: T.S. and N.O. conceived and designed the research; T.S., T.F., and M.K. designed the compounds; T.S., T.F., H.I., G.T., Y.D., and M.K. synthesized the compounds; T.S., G.T., and N.O. wrote the manuscript; and T.S., N.O., Y.D., M.N., and M.K. supervised all research. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported in part by grants from the Japan Society for the Promotion of Science (KAKENHI Grants JP19K07009 to T.S., JP18K06567 to N.O., JP17K08385 to Y.D., JP16H05090, JP16K15121 and JP18H05502 to M.N., and JP16K08340 to M.K.) and the Japan Agency for Medical Research and Development (AMED Grants JP19ak0101073 and JP19im0210616 to M.N. and JP19cm0106136 and JP19ak0101073 to N.O.). Conflicts of Interest: M.N. received a research fund from Daiichi Sankyo Pharmaceutical Co., Ltd. All other authors declare no conflict of interest. Pharmaceuticals 2020, 13, 34 11 of 13

Abbreviations

PROTAC proteolysis-targeting chimeras SNIPER specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers AhR aryl hydrocarbon receptor β-NF β-naphthoflavone ATRA all-trans retinoic acid CRABPs cellular retinoic acid-binding proteins ITE 2-(10H-indole-30-carbonyl)-thiazole-4-carboxylic acid methyl ester HyT hydrophobic tagging Ad adamantane 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-Oxide HATU Hexafluorophosphate DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide TBAF tetrabutylammonium Fluoride THF tetrahydrofuran HBTU 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-Oxide Hexafluorophosphate TLC thin-layer chromatography UV ultra-violet NMR nuclear magnetic resonance TMS tetramethylsilane IT-TOF MS ion trap-time-of-flight mass spectrometry calcd caluculated DMAP N,N-dimethyl-4-aminopyridine

References

1. Ohoka, N.; Shibata, N.; Hattori, T.; Naito, M. Protein Knockdown Technology: Application of Ubiquitin Ligase to Cancer Therapy. Curr. Cancer Drug Targets 2016, 16, 136–146. [CrossRef][PubMed] 2. Pettersson, M.; Crews, C.M. PROteolysis TArgeting Chimeras (PROTACs)-Past, present and future. Drug Discov. Today Technol. 2019, 31, 15–27. [CrossRef][PubMed] 3. Demizu, Y.; Okuhira, K.; Motoi, H.; Ohno, A.; Shoda, T.; Fukuhara, K.; Okuda, H.; Naito, M.; Kurihara, M. Design and synthesis of estrogen receptor degradation inducer based on a protein knockdown strategy. Bioorg. Med. Chem. Lett. 2012, 22, 1793–1796. [CrossRef][PubMed] 4. Bondeson, D.P.; Mares, A.; Smith, I.E.; Ko, E.; Campos, S.; Miah, A.H.; Mulholland, K.E.; Routly, N.; Buckley, D.L.; Gustafson, J.L.; et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 2015, 11, 611–617. [CrossRef] 5. Ohoka, N.; Okuhira, K.; Ito, M.; Nagai, K.; Shibata, N.; Hattori, T.; Ujikawa, O.; Shimokawa, K.; Sano, O.; Koyama, R.; et al. In Vivo Knockdown of Pathogenic Proteins via Speciﬁc and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs). J. Biol. Chem. 2017, 292, 4556–4570. [CrossRef] 6. Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.; Hattori, T.; Koyama, R.; Sano, O.; Imaeda, Y.; Nara, H.; et al. Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands. J. Med. Chem. 2018, 61, 543–575. [CrossRef] 7. Salami, J.; Alabi, S.; Willard, R.R.; Vitale, N.J.; Wang, J.; Dong, H.; Jin, M.; McDonnell, D.P.; Crew, A.P.; Neklesa, T.K.; et al. Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance. Commun. Biol. 2018, 1, 100. [CrossRef] 8. Shibata, N.; Shimokawa, K.; Nagai, K.; Ohoka, N.; Hattori, T.; Miyamoto, N.; Ujikawa, O.; Sameshima, T.; Nara, H.; Cho, N.; et al. Pharmacological diﬀerence between degrader and inhibitor against oncogenic BCR-ABL kinase. Sci. Rep. 2018, 8, 13549. [CrossRef] 9. Raina, K.; Lu, J.; Qian, Y.; Altieri, M.; Gordon, D.; Rossi, A.M.; Wang, J.; Chen, X.; Dong, H.; Siu, K.; et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl. Acad. Sci. USA 2016, 113, 7124–7129. [CrossRef] Pharmaceuticals 2020, 13, 34 12 of 13

10. Winter, G.E.; Buckley, D.L.; Paulk, J.; Roberts, J.M.; Souza, A.; Dhe-Paganon, S.; Bradner, J.E. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 2015, 348, 1376–1381. [CrossRef] 11. McCoull, W.; Cheung, T.; Anderson, E.; Barton, P.; Burgess, J.; Byth, K.; Cao, Q.; Castaldi, M.P.; Chen, H.; Chiarparin, E.; et al. Development of a Novel B-Cell Lymphoma 6 (BCL6) PROTAC To Provide Insight into Small Molecule Targeting of BCL6. ACS Chem. Biol. 2018, 13, 3131–3141. [CrossRef][PubMed] 12. Itoh, Y.; Ishikawa, M.; Naito, M.; Hashimoto, Y. Protein knockdown using methyl bestatin-ligand hybrid molecules: Design and synthesis of inducers of ubiquitination-mediated degradation of cellular retinoic acid-binding proteins. J. Am. Chem. Soc. 2010, 132, 5820–5826. [CrossRef][PubMed] 13. Okuhira, K.; Ohoka, N.; Sai, K.; Nishimaki-Mogami, T.; Itoh, Y.; Ishikawa, M.; Hashimoto, Y.; Naito, M. Speciﬁc degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein. FEBS Lett. 2011, 585, 1147–1152. [CrossRef][PubMed] 14. Han, Z.; Bondeson, J.C.; Lewis, J.H.; Mannarino, E.G.; Friesen, S.A.; Wagar, M.M.; Balboni, T.A.; Alexander, B.M.; Arvold, N.D.; Sher, D.J.; et al. Evaluation of initial setup accuracy and intrafraction motion for spine stereotactic body radiation therapy using stereotactic body frames. Pract. Radiat. Oncol. 2016, 6, e17–e24. [CrossRef] 15. Toure, M.; Crews, C.M. Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew. Chem. Int. Ed. Engl. 2016, 55, 1966–1973. [CrossRef] 16. Ohoka, N.; Tsuji, G.; Shoda, T.; Fujisato, T.; Kurihara, M.; Demizu, Y.; Naito, M. Development of Small Molecule Chimeras That Recruit AhR E3 Ligase to Target Proteins. ACS Chem. Biol. 2019, 14, 2822–2832. [CrossRef] 17. Ohtake, F.; Takeyama, K.; Matsumoto, T.; Kitagawa, H.; Yamamoto, Y.; Nohara, K.; Tohyama, C.; Krust, A.; Mimura, J.; Chambon, P.; et al. Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 2003, 423, 545–550. [CrossRef] 18. Soucy, T.A.; Smith, P.G.; Rolfe, M. Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer. Clin. Cancer Res. 2009, 15, 3912–3916. [CrossRef] 19. Soucy, T.A.; Smith, P.G.; Milhollen, M.A.; Berger, A.J.; Gavin, J.M.; Adhikari, S.; Brownell, J.E.; Burke, K.E.; Cardin, D.P.; Critchley, S.; et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 2009, 458, 732–736. [CrossRef] 20. Lai, A.C.; Crews, C.M. Induced protein degradation: An emerging drug discovery paradigm. Nat. Rev. Drug Discov. 2017, 16, 101–114. [CrossRef] 21. Burslem, G.M.; Crews, C.M. Small-Molecule Modulation of Protein Homeostasis. Chem. Rev. 2017, 117, 11269–11301. [CrossRef] 22. Cromm, P.M.; Crews, C.M. Targeted Protein Degradation: From Chemical Biology to Drug Discovery. Cell Chem. Biol. 2017, 24, 1181–1190. [CrossRef][PubMed] 23. Neklesa, T.K.; Tae, H.S.; Schneekloth, A.R.; Stulberg, M.J.; Corson, T.W.; Sundberg, T.B.; Raina, K.; Holley, S.A.; Crews, C.M. Small-molecule hydrophobic tagging-induced degradation of HaloTagfusion proteins. Nat. Chem. Biol. 2011, 7, 538–543. [CrossRef][PubMed] 24. Long, M.J.; Gollapalli, D.R.; Hedstrom, L. Inhibitor mediated protein degradation. Chem. Biol. 2012, 19, 629–637. [CrossRef] 25. Xie, T.; Lim, S.M.; Westover, K.D.; Dodge, M.E.; Ercan, D.; Ficarro, S.B.; Udayakumar, D.; Gurbani, D.; Tae, H.S.; Riddle, S.M.; et al. Pharmacological targeting of the pseudokinase Her3. Nat. Chem. Biol. 2014, 10, 1006–1012. [CrossRef] 26. Gustafson, J.L.; Neklesa, T.K.; Cox, C.S.; Roth, A.G.; Buckley, D.L.; Tae, H.S.; Sundberg, T.B.; Stagg, D.B.; Hines, J.; McDonnell, D.P.; et al. Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging. Angew. Chem. Int. Ed. Engl. 2015, 54, 9659–9662. [CrossRef] 27. Canaria, C.A.; Smith, J.O.; Yu, C.J.; Fraser, S.E.; Lansford, R. New syntheses for 11-(mercaptoundecyl)triethylene glycol and mercaptododecyltriethyleneoxy biotin amide. Tetrahedron Lett. 2005, 46, 4813–4816. [CrossRef] Pharmaceuticals 2020, 13, 34 13 of 13

28. Zhou, J.; Wu, J.; Liu, X.; Qu, F.; Xiao, M.; Zhang, Y.; Charles, L.; Zhang, C.C.; Peng, L. Cooperative binding and self-assembling behavior of cationic low molecular-weight dendrons with RNA molecules. Org. Biomol. Chem. 2006, 4, 581–585. [CrossRef] 29. Suthagar, K.; Fairbanks, A.J. A new way to do an old reaction: Highly eﬃcient reduction of organic azides by sodium iodide in the presence of acidic ion exchange resin. Chem. Commun. (Camb.) 2017, 53, 713–715. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).