Synthesis of Homoverrucosanoid-Derived Esters and Evaluation as

Article pubs.acs.org/joc

Synthesis of Homoverrucosanoid-Derived Esters and Evaluation as MDR Modulators Andreas Schaf̈ er,*,† Sebastian C. Köhler,‡ Markus Lohe,† Michael Wiese,*,‡ and Martin Hiersemann† †

Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, 44227 Dortmund, Germany Pharmazeutisches Institut, Pharmazeutische Chemie II, Universität Bonn, 53121 Bonn, Germany

‡

S Supporting Information *

ABSTRACT: The synthesis of the A−B-cis,B−C-trans-annulated cyclohepta[e]hydrindane core of a gagunin E analogue is reported in detail. The tricarbocyclic scaffold was assembled starting from an easily accessible A ring building block by a (4 + 2)cycloaddition for annulation of the B ring. A ring-closing metathesis served for construction of the seven-membered C ring. The angular methyl groups were attached by electrophilic cyclopropanation−ring opening. A library based on the most active lead compound was made accessible by esterification of the terpenols with commercially available acids. A transannular etherification reaction gave access to tetracyclic derivatives of the synthetic inhibitors. The members of the compound library of non-natural homoverrucosanoid-derived esters were examined as modulators of the membrane transporter proteins ABCB1 (P-gp), ABCG2 (BCRP), and ABCC1 (MRP1), which are involved in the formation of multidrug resistance (MDR) in cancer chemotherapy.

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INTRODUCTION In 2002, Shin and co-workers reported the isolation and structural elucidation of the marine homoverrucosanoid gagunin E (1) in minute amounts (3.1 mg) from a sponge (1.8 kg dry weight) of the genus Phorbas (Figure 1).1 The relative configuration of 1 was proposed only in analogy to gagunin A for which extensive ROESY and 1D NOESY experiments formed the foundation for structural assignment. The absolute configuration of the members of the family of gagunins remains in question. It was also noted that 1 exerts a very notable LC50 (0.03 μg/mL) against the human leukemia cell line K562. Establishing an independent access to the natural product by total synthesis would certainly support further and broader biological profiling and finalization of the structural assignment. The intricate molecular architecture of the richly ornamented tricyclic ring system of gagunine E poses significant challenges to scaffold synthesis and asymmetric synthesis. Accordingly, studies aiming at the total synthesis of 1 are worthwhile endeavors to pursue. Here, we outline in detail the results of our own efforts that culminated in the synthesis of the simplified analogue 2 of gagunin E (1) featuring the A−Bcis,B−C-trans-annulated cyclohepta[e]hydrindane core with a fully functionalized B−C ring system.2 We delineate principles of specific reactivity of the scaffold and suggest validated synthetic tactics. Furthermore, we merged our interests in total © 2017 American Chemical Society

synthesis and in the discovery of terpenoid-derived entities with multidrug resistance (MDR) modulating properties and reveal the results of this merger by reporting the inhibitory activity of cyclohepta[e]hydrindanoids toward the ABCB1 (P-gp), ABCG2 (BCRP), and ABCC1 (MRP1) proteins.

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RESULTS AND DISCUSSION

Our initial synthetic campaign relied on a scaffold-first strategy toward the A−B-cis,B−C-trans-annulated cyclohepta[e]hydrindane core using an adaptable A ring building block (Figure 1). According to this strategy, we envisioned assembling of the cis-hydrindanone segment by (4 + 2)cycloaddition and subsequent annulation of the sevenmembered C ring by ring-closing metathesis (RCM). After assembly, functionalization of the tricyclic scaffold would be required. Our scaffold-first synthetic strategy was specifically designed to effectively control the relative configuration of the all-carbon-substituted stereogenic carbon atoms at the ring junctions. (4 + 2)-Cycloaddition. In accordance with our synthetic planning, the (4 + 2)-cycloaddition using an electron-rich silyl Received: August 10, 2017 Published: September 26, 2017 10504

DOI: 10.1021/acs.joc.7b02012 J. Org. Chem. 2017, 82, 10504−10522

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The Journal of Organic Chemistry

procedure by usage of EtAlCl2 resulted in the decomposition of the alkynoates. To circumvent the intrinsic site-selectivity issues and to further increase the electron-deficiency of the dienophile component, we turned to dimethyl acetylenedicarboxylate (10) (Table 1). Fortunately, our first exploratory experiment Table 1. Optimization of the (4 + 2)-Cycloaddition Using the Parent Diene 5aa

Figure 1. Retrosynthetic analysis of gagunin E (1) and our simplified analogue (2) leading to second-generation diene 5b and parent diene 5a, respectively. entry

10 (equiv)

1 2 3 4 5 6 7 8 9 10c 11 12 13

1 1 1 1 1 1 1 2 2 2 2 2 2

enol ether derived diene and an electron-poor dienophile was studied first. For initial exploration, we turned to the easily obtainable parent diene 5a. The diene 5a was made available in four steps and on gram scale starting from commercially available 1,2-cyclohexanediol.3 Initially, we selected the known alkyne dienophiles 6,4,5 7,6 8,7 and 98,9 featuring constitutionally differentiated end groups for exploratory studies of the chemo- and site-selectivity of the envisioned (4 + 2)cycloaddition (Scheme 1). Scheme 1. Attempted (4 + 2)-Cycloadditions Using the Parent Diene 5aa

additive (0.05 equiv)

TBHQ TBHQ TBHQ TBHQ TBHQ BHT BHTd

solvent

T (°C)

time (h)

yieldb (%)

C6H5CH3 CF3CH2OH CH2Cl2 CH2Cl2 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3 C6H5CH3

rt rt rt 60 rt 140 140 140 140 140 reflux 140 140

70 1 45 3.5 70 1 1 1 1 1 1 1 1

46 14 32 38 44 46 49 63 68 83 49 41 51

a

Reactions were run on a 1.1 mmol scale (0.25 g 5a) at a concentration of 0.75 M (1.5 mL of argon-saturated solvent) in sealable glass pressure tubes (Ace pressure tube). bIsolated yields. c Executed using 8.0 g of diene 5a. d1 equiv of BHT. TBHQ: tertbutylhydroquinone. BHT: 2,6-di-tert-butyl-4-methylphenol.

a

using equimolar amounts of diene 5a and dienophile 10 in toluene proceeded even at room temperature and delivered the desired (4 + 2)-cycloadduct 11 in already moderate yields (entry 1). However, the protocol was hampered by extensive reaction times. Double-bond isomerization as well as oxidative aromatization were feared as main cycloadduct-consuming pathways at prolonged reaction times. Thus, we set out to carefully optimize the reaction conditions (Table 1). During optimization, we immediately noticed that the rigorous exclusion of oxygen was mandatory to suppress oxidative aromatization of 11 to the 2,3-dihydro-1H-indene 12. Therefore, all experiments were performed using argonsaturated solvents in sealable glass pressure tubes. Solvent effects were briefly studied.10 2,2,2-Trifluoroethanol11 triggered solvolysis of silyl dienol ether 5a to the corresponding enone, and the desired product 11 was isolated in low yield (entry 2). Running the reaction in CH2Cl2 at ambient (entry 3) or increased (entry 4) temperature led to slightly improved yields. To further suppress oxidative aromatization, antioxidants such as tert-butylhydroquinone (TBHQ) and 2,6-di-tert-butyl-4-

EWG = electron-withdrawing group.

For initial screening, mixtures of the diene 5a and the dienophiles 6, 7, and 9 were stirred in toluene at temperatures ranging from ambient to 140 °C using sealable glass pressure tubes. At elevated temperatures, consumption of the starting materials was accompanied by the formation complex product mixtures. However, heating a mixture of the dienophile 8 and the diene 5a in toluene to 140 °C afforded a product mixture from which the corresponding cycloadduct was isolated in low yields (20−28%) and as a mixture of constitutional isomers (4:1). In our hands, attempts to establish a low temperature 10505


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as a single diastereomer. Subjecting 17 to Furukawa conditions of the Simmons−Smith reaction triggered regioselective cyclopropanation of the silyl enol ether double bond.15 The resulting siloxy cyclopropane was obtained as a single diastereomer but contaminated with the corresponding 2,3dihydro-1H-indene by undesired oxidative aromatization of 17. Fortunately, careful optimization of the reaction conditions including exclusion of light and variation of reagent stoichiometry (6 equiv of Et2Zn and 12 equiv of CH2I2) largely suppressed oxidative aromatization. The thus accessible mixture (10:1) of siloxy cyclopropane and 2,3-dihydro-1Hindene was treated with p-TsOH to provide cis-hydrindanone diol 18 by siloxy cyclopropane ring opening16 and isopropylidene acetal cleavage, but all attempts to isolate 18 from a complex product mixture were yet unsuccessful. Elaboration of the B Ring. With the elaboration of the A ring of 17 still under investigation, we headed to the structural development of the B ring in the parent compound 11 (Scheme 3). Hence, the bicyclic (4 + 2)-cycloadduct 11 was

methylphenol (BHT) were tested. However, no beneficial effect of TBHQ was noticed in toluene at room temperature (entry 5). Running the cycloaddition in toluene at elevated temperature allowed for significant shorter reaction times, but the outcome was still hampered by a mediocre yield (entry 6), even in the presence of TBHQ (entry 7). Notably, performing the reaction at elevated temperature for prolonged reaction times promoted an undesired double-bond isomerization, and the conjugated diene 13 was exclusively obtained after 24 h at 140 °C. Using a 2-fold excess of the dienophile 5a finally led to useful yields (entry 8), which improved further in the presence of TBHQ (entry 9) and, luckily, on larger scale (entry 10). An attempt to execute the reaction in a standard reflux apparatus led to markedly diminished yields (entry 11). Finally, BHT turned out to be ineffective in the prevention of the oxidative aromatization (entry 12), even when used in stoichiometric amounts (entry 13). Having established conditions for the (4 + 2)-cycloaddition of the parent diene 5a and dimethyl acetylenedicarboxylate (10), we turned our attention to the second-generation diene 5b to allow for asymmetric induction12 and for downstream elaboration of the A ring (Scheme 2). The synthesis of 5b

Scheme 3. Introduction of the 5a-Methyl Group

Scheme 2. Asymmetric (4 + 2)-Cycloaddition Using the Second-Generation Diene 5b

commenced with enantioenriched (1R,2S)-cyclohex-3-ene-1,2diol (14) (∼33% ee),13 which was converted into the corresponding isopropylidene acetal by transacetalization with 2,2-dimethoxypropane (2,2-DMP). Ozonolysis with reductive workup then delivered dialdehyde 15 and set the stage for the construction of the A ring by intramolecular aldol condensation using Corey’s conditions.14 Thus, 15 was treated with dibenzylammonium trifluoroacetate to afford the corresponding cyclopentene carbaldehyde, which was transformed to enone 16 by addition of methylmagnesium bromide and subsequent manganese dioxide oxidation. Synthesis of the secondgeneration diene 5b from enone 16 was best accomplished using TBSOTf and Et3N. With bicyclic diene 5b in hand, we targeted the asymmetric (4 + 2)-cycloaddition between 5b and dimethyl acetylenedicarboxylate (10). Fortunately, the (4 + 2)-cycloaddition under the previously established conditions furnished the tricyclic silyl enol ether 17

subjected to regioselective cyclopropanation to deliver the corresponding siloxy cyclopropane as a single diastereomer. Subsequent ring opening was best accomplished with p-TsOH to afford the initially unexpected enone 19 as a mixture of C10a epimers.17 With the enone 19 in hand, we envisioned to introduce the 5a-methyl group by conjugate addition to afford ketone 20. To this end, enone 19 was subjected to organolithium cuprate Me2CuLi·LiI (Gilman reagent)18 under various conditions and in the presence of the additives TMSCl,19 TMSOTf,20 or F3B·OEt2.21 Furthermore, we examined the copper-catalyzed conjugate addition of Me3Al.22 Unfortunately, in our hands, all attempts for nucleophilic introduction of the 5a-methyl group failed, and the much10506


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CH2Cl2 (5:1) at −100 °C (thawed to −50 °C) allowed for diastereomeric ratios up to 9:1. Interestingly, performing the reduction in a noncoordinating solvent such as CH2Cl2 led to inferior diastereoselectivities (dr = 6:4). Subjecting 23 to lithium aluminum hydride, borane complexes, or borohydrides under various conditions delivered the corresponding C4 alcohol in diastereoselectivities ranging from 1:1 to 3:1. The C4 alcohol from the DIBAL-H reduction of 23 was subsequently converted to the C4 p-bromobenzoate using Steglich’s conditions and employing an excess of DMAP.26 Differentiation of the primary hydroxyl groups at C6 and C10 by site-selective silyl ether cleavage was required next. Unfortunately, however, screening of fluoride-based reagents (TBAF, TBAF−AcOH, NH4F, HF−MeCN, and HF·pyridine) or Brønsted acids (p-TsOH, CSA, PPTS, ClCH2CO2H, TFA, and HCO2H) inevitably led to mixtures of the monosilylated constitutional isomers and the corresponding diol. Consequently, we turned to site-selective silyl ether formation. Hence, the primary TBS ethers were cleaved with TBAF in THF and the resulting diol was treated with tert-butyldiphenylsilyl chloride (TPSCl) and Et3N to afford the C10 TPS ether as a single diastereomer (after SiO2 chromatography) and, fortunately, contaminated only with trace amounts of the C6 TPS ether. We found no evidence for silyl group migration under the reaction conditions as subjecting the product mixture to Et3N did not alter the original product ratio. Notably, the use of TBSCl, TIPSCl, TIPSOTf, and TrCl led to inferior site selectivity. Having accomplished the differentiation of the C6 and C10 hydroxyl groups, we were positioned to screen for conditions for the introduction of an isopentenyl group at C6 in the presence of the C4 benzoate. Dess−Martin oxidation of the C6 hydroxyl group provided aldehyde 24. Subjecting 24 to isopentenyllithium, prepared by lithium−halogen exchange of tert-butyllithium and isopentenyl bromide,27 led to the cleavage of the benzoate. Usage of a putative isopentenylcerium reagent, prepared by transmetalation of isopentenyllithium and cerium(III) chloride,28 suppressed cleavage of the benzoate but, in the event, led to no conversion at all. Treatment of 24 with an excess of the Grignard reagent 25 finally afforded the desired product as a 89:11 mixture of diastereomers. Pursuing an orthogonal protecting group strategy, the C6 alcohol 26 was subsequently converted into the corresponding benzyloxymethyl (BOM) acetal and ensuing desilylation as well as oxidation afforded the aldehyde 27 as a single diastereomer after SiO2 chromatography. Synthesis of the 10aS-configured diene 28 was accomplished by Wittig methylenation of 27 using freshly prepared LiHMDS under high dilution conditions (c = 5 mM) to avoid cleavage of the benzoate. Configurational diversification at C10a was made possible by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-mediated epimerization of 27 in toluene− THF (10:1) to deliver the corresponding 10aR-configured aldehyde as a single diastereomer. Subsequent Wittig methylenation furnished the 10aR-configured diene 29. With the diastereomeric dienes 28 and 29 in hand, we were well positioned to study the annulation of the seven-membered C ring by RCM. A literature survey revealed that ruthenium carbene complexes are capable catalyzing the RCM for construction of cycloheptenoids containing a tri- or even tetrasubstituted double bond.29,30 Therefore, the commercially available ruthenium complexes Grubbs catalyst first-generation 30, Grubbs catalyst second-generation 31, Hoveyda−Grubbs catalyst second-generation 32, and catMETium catalyst RF1 33 were deployed in our studies (Table 2). All experiments were

desired ketone 20 was never observed. This experimental outcome was a frustrating setback to our synthetic design and enforced a laborious rerouting of the synthetic pathway to ketone 23. Forced by the failure of nucleophilic methylation of 19, we turned to an electrophilic introduction of the 5a-methyl group by diastereoface-differentiating cyclopropanation and subsequent cyclopropane ring-opening. Accordingly, 19 was subjected to Luche conditions, and the resulting allylic alcohol (mixture of four diastereomers) was protected as the TMS ether.23 Subsequent treatment with DIBAL-H delivered the corresponding diol, which was converted into the bis-TBS ether 21. Electrophilic cyclopropanation of 21 then provided the desired 5,6,3-tricarbocyclic scaffold. In preparation of the cyclopropane ring opening, the TMS ether was cleaved selectively with acetic acid in THF−H2O, and the resulting alcohol (single diastereomer after SiO2 chromatography) was oxidized to provide cyclopropyl ketone 22. Subsequent cyclopropane ring opening by hydrogenolysis of 22 in glacial acetic acid furnished cis-hydrindanone 23 on gram scale (88% isolated yield, 2.78 g isolated mass).24 Notably, the cyclopropane ring opening could also be triggered photochemically by irradiation with UV−C light (λ = 254 nm) in the presence of Et3N and LiClO4 to deliver 23 on a milligram scale (62% isolated yield, 28 mg isolated mass).25 When the reaction was performed in the absence of LiClO4, ketone 23 (48%, 22 mg) was partially reduced to the corresponding alcohol (20%, 9 mg, dr = 75:25). Annulation of the C Ring by RCM. Having established a reliable route to cis-hydrindanone 23, we targeted annulation of the seven-membered C ring by RCM. Accordingly, the synthesis of a suitable diene was required (Scheme 4). Diastereoselective reduction of C4 ketone 23 from the convex diastereoface using an excess of DIBAL-H (10 equiv) in THF− Scheme 4. Synthesis of Dienes 28 and 29

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The Journal of Organic Chemistry performed using sealable glass pressure tubes in argon-saturated solvents at 25 mM to prevent dimerization of the dienes (Tables 2 and 3).29b

Table 3. Ring-Closing Metathesis of 10aR-Configured Diene 29a

Table 2. Ring-Closing Metathesis of 10aS-Configured Diene 28a

entry 1 2 3 4 5 6 7 8 9

catalyst (equiv)b 31 31 33 32 31 32 32 32 32

(3 × 0.1) (3 × 0.1) (3 × 0.1) (0.1) (0.1) (0.1) (0.1) (0.1) (2 × 0.05)

solvent

time (days)

CH2Cl2 (CH2Cl)2 (CH2Cl)2 C6 F 6 C6 F 6 C6 F 6 C6H6 C6H5CH3 C6 F 6

6.0 6.0 6.0 2.5 3.0 2.5 4.0 4.0 5.5

yield (%) (mg) 78 92 89 97 27 96 78 46 96

(11) (11) (11) (42) (15)c (172) (195)c (170)c (437)

a

Reactions were run in argon-saturated solvents at 25 mM in sealable glass pressure tubes at oil bath temperatures of 60 °C for CH2Cl2 or 100 °C for (CH2Cl)2, C6F6, C6H6, and C6H5CH3. bCatalyst was added in portions as indicated. cIncomplete conversion: Percentage yields were determined from the 1H NMR spectrum of an inseparable mixture of 35 and 29.

entry 1 2 3 4

catalystb (equiv) 30 31 32 32

(2 × 0.2 + 0.4) (3 × 0.1) (0.1 + 0.2) (0.1)

solvent

time (days)

yield (%) (mg)

CH2Cl2 CH2Cl2 CH2Cl2 C6F6

3.5 6.0 5.5 4.5

no convc 92 (29) 84 (8) 90 (20)

catalyst pollution, we again switched to C6F6. Subjecting 29 to complex 32 in C6F6 led to a remarkable acceleration of the reaction rate at a decreased catalyst loading, and 35 was obtained in a nearly quantitative yield (entry 4). Performing the reaction with complex 31 under otherwise identical conditions led to incomplete conversion (entry 5). Having identified appropriate conditions for trans annulation on an exploratory scale, RCM on a larger scale was studied next (Table 3). In the event, subjecting 29 to a solution of 32 (0.1 equiv) in C6F6 at 100 °C for 2.5 days delivered 35 in 96% yield (entry 6). Cost-effectiveness called for nonfluorinated aromatic solvents. However, running the RCM in C6H6 or C6H5CH3 led to incomplete conversions at comparable reaction times (entries 7 and 8). The RCM was further scaled to 0.5 g of 29 (entry 9). In the event, the Hovyeda−Grubbs complex 32 was added in two portions, and the reaction time was extended to ensure complete conversion. The RCM product 35 was thus isolated in 96% in analytically pure form after purification by chromatography with visible impurities by ruthenium species from the metathesis catalyst (pale amber oil). Transannular Etherification. Having completed the assembly of the A−B-cis,B−C-trans-annulated cyclohepta[e]hydrindane core, we turned to the gagunin E-like elaboration of the C ring. Exploiting the masked 6-OH group as a handle for C ring functionalization required chemoselective removal of the BOM protecting group in the presence of the C4 bromobenzoate and the electrophilic trisubstituted C9/C10 double bond (Table 4). Subjecting 35 to boron trifluoride diethyl etherate and thiophenol in CH2Cl2 afforded the desired alcohol 36 in 49% yield accompanied by ether 37 in 36% yield (entry 1).33 Speculating about a Brønsted acid triggered transannular etherification, we turned to methylsulfide for capturing electrophilic degradation products. In the event, formation of 37 was suppressed and the desired alcohol 36 was isolated in 60% yield (entry 2). Increased Lewis acidity was

a

Reactions were run in argon-saturated solvents at 25 mM in sealable glass pressure tubes at oil bath temperatures of 60 °C for CH2Cl2 or 100 °C for C6F6. bCatalyst was added in portions as indicated. c86% (3 mg) of 28 was recovered.

The cis annulation of the seven-membered C ring was studied first. Hence, 10aS-configured diene 28 was subjected to the ruthenium carbene complexes 30−32 (Table 2). Complex 30 in CH2Cl2 was ineffective, and 28 was recovered (entry 1). Complex 31 catalyzed substrate conversion and formation of 34 in excellent yield (entry 2). Complex 32 in CH2Cl2 also triggered formation of 34 albeit in slightly lower yield (entry 3). Aiming to decrease catalyst pollution, we turned to C6F6 as solvent.31 In the event, a substantial decrease of complex 32 and reaction time was possible (entry 4). Opportunities for trans annulation of the 10aR-configured diene 29 by RCM were evaluated next (Table 3). Subjecting 29 to a solution of complex 31 in CH2Cl2 at elevated temperature (60 °C, sealed tube) delivered 35 in useful yield (entry 1). Performing the reaction in (CH2Cl)2 at further increased temperature (100 °C, sealed tube) improved the yield, but a substoichiometric catalyst loading was still required for complete consumption (entry 2). The structurally related but lower cost32 complex 33 also catalyzed formation of 35 (entry 3). Comparing the results from Tables 1 and 2 illustrated to our surprise that catalyst loadings and reaction times for cis or trans annulation in halocarbon solvents are comparable. To attenuate 10508


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infra). The esters 39−41 represent structural analogues of 36 regarding C4 ester substitution and were synthesized in three steps starting from RCM product 35 (Scheme 6).

Table 4. Chemoselective BOM Acetal Cleavage

Scheme 6. Synthesis of Esters 39−41

yield (%) (mg) entry

conditions

(±)-36

(±)-37

1

F3B·OEt2 (2 equiv), PhSH (10 equiv), CH2Cl2, −30 °C to rt, 1 h F3B·OEt2 (2 equiv), SMe2 (10 equiv), CH2Cl2, −30 °C to rt, 0.5 h Cl3B·SMe2 (2 equiv), CH2Cl2, 0 °C, 0.5 h Cl3B·SMe2 (20 equiv), Et2O, rt, 19 h

49 (52)

36 (38)

2 3 4

60 (4) 75 (5) 92 (319)

expected from BCl3.34 Exposure of 35 to boron trichloride methyl sulfide complex in CH2Cl2 delivered 36 in 75% yield on an exploratory scale (entry 3); the yield was improved to 92% at a serviceable scale by using a large excess of Cl3B·SMe2, running the reaction at room temperature and by switching the solvent to diethyl ether (entry 4).35 Cleavage of the BOM acetal by hydrogenolysis was considered as a viable alternative to the deployment of a large excess of Cl3B·SMe2. In the event, exposure of 35 to Pd(OH)2 under an atmosphere of hydrogen (balloon) in EtOH36 or EtOAc37 afforded complex mixtures containing products from the hydrogenation of the C9/C10 double bond. We briefly studied the propensity of the A−B-cis,B−C-cisannulated 34 for transannular etherification (Scheme 5).

Subjecting 35 to methanolic NaOH solution (1% w/w NaOH) at 60 °C afforded the corresponding C4 alcohol by transesterification of the bromobenzoate.40 Subsequent esterification with 2-naphthoic acid, quinoline-6-carboxylic acid, and 4-methoxybenzoic acid was performed using N-(3(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC) and an excess of 4-(dimethylamino)pyridine (DMAP) at elevated temperatures (40 °C) and proceeded uneventful.41 Cleavage of the BOM acetal was accomplished under the previously established conditions employing Cl3B·SMe2 in diethyl ether to deliver 39−41 in valuable yields of 70−78% from 35. The lead compound structure 36 was transformed into the bis-quinolinecarboxylate 42 by transesterification of the bromobenzoate and esterification of the corresponding diol using an excess of quinoline-6-carboxylic acid (Scheme 7). Site-

Scheme 5. Transannular Etherification of A−B-Cis,B−C-CisAnnulated 34

Scheme 7. Synthesis of Bis-quinolinecarboxylate 42

Notably, exposure of 34 to aqueous HCl−THF triggered cleavage of the BOM acetal and transannular etherification to deliver 38 as the sole product.38 Turning to boron trifluoride diethyl etherate and thiophenol in CH2Cl2 increased the effectiveness of the transannular etherification and 38 was isolated in 95% yield. The initially unconsidered transannular etherification poses a threat for synthetic passages to the gagunins on the one hand but on the other hand offers unique opportunities for structural diversification in the context of biological profiling (vide infra). Derivatization by Esterification. Considering our interest in terpenoid-derived entities with multidrug resistance (MDR) modulating properties, we synthesized an ensemble of esters based on the A−B-cis,B−C-trans-annulated cyclohepta[e]hydrindane scaffold. According to the principles of ligandbased drug design,39 structural diversification was guided by the lead compound structure 36, which exhibited a respectable IC50 value (2.9 μM) in the inhibition of ABCG2 (BCRP) (vide

selectivity of the monoesterification was briefly investigated by exposure of the diol to 1 equiv of quinoline-6-carboxylic acid. In the event, formation of a product mixture indicated a comparable reactivity of the 4-OH and 6-OH groups. Derivatization of bromobenzoate 37 from transannular etherification was achieved by cleavage of the bromobenzoate and subsequent esterification to afford quinolinecarboxylate 43 in 82% yield from 37 (Scheme 8). For a control experiment (vide infra), simple but unreported cyclohexyl quinoline-6-carboxylate (45) was prepared in excellent yield (Scheme 9). 10509


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unsuccessful, and ketone 46 was obtained as the sole product.43 Therefore, we considered access to dienone 47 from 46 by Saegusa oxidation.44 Accordingly, alcohol 36 was oxidized to ketone 46, and ensuing conversion to the corresponding silyl enol ether was achieved with TBSOTf and Et3N. Saegusa oxidation of the silyl enol ether was only effective when a large excess of Pd(OAc)2 (5 equiv) in CH3CN at 40 °C was used to afford 47 in 61% from 46;45 performing the reaction at 80 °C triggered nonspecific decomposition, and switching the solvent to THF led to diminished yields (44%). Pd(tfa)2(DMSO)2 in THF at 40 °C initially led to improved yields (67−78%),46 but using Pd(tfa)2 from a newly acquired packaging unit resulted in competing silyl enol ether hydrolysis. Efforts to oxidize the intermediate silyl enol ether according to Rubottom delivered the corresponding acyloin as a 1:1 mixture of C7 diastereomers in 81% yield.47 Subjecting the silyl enol ether to Corey’s conditions for allylic oxidation was unsuccessful.48 Attempts to access the diastereomerically pure acyloin by asymmetric hydroxylation of the sodium enolate of 46 with (−)-(8,8dichlorocamphorylsulfonyl)oxaziridine failed.49 DIBAL-H reduction of 47 in THF at −78 °C (thawed to −20 °C) delivered 6R-configured dienol 48. The resulting alcohol was transformed into the TBS ether, and the C4 bromobenzoate was reductively removed. The resulting alcohol was converted to the C4 butyrate. Site-selective and diastereoface-differentiating dihydroxylation of the less substituted C7/ C8 double bond was required next.50 Notably, nondirected OsO4-catalyzed dihydroxylation proceeded site- and diastereoselectively to deliver the 7S,8S-configured diol 49 as a single diastereomer.51,52 Site-selective acetylation of the allylic C8 hydroxyl group was achieved by subjecting 49 to an excess of acetic anhydride and catalytic amounts of DMAP at ambient temperature. Formation of the C7 butyrate required a large excess of butyric anhydride and DMAP at increased temperature (CH2Cl2, 60 °C, sealed tube). Exposure of the silyl protected tris-ester to TBAF in THF delivered a complex mixture of products, whereas various acidic conditions led to no conversion (HF·pyridine, TBAF−AcOH, TASF, NH4F in HFIP, NH4F−HF in DMF−NMP, and CeCl3·7H2O in CH3CN), or nonspecific decomposition (HCl in MeOH). Some acidic conditions triggered slow conversion, but product formation was accompanied by rapid transesterification of the C7 butyrate to deliver 50 as the major product (TBAF−NH4F, TBAF−HF, and THF−HCO2H−H2O (2:1:1)). Exposure of 49 to CH3CN−HF−H2O (2:1:1), slowly thawed from 0 to 10 °C, best affected silyl ether cleavage to provide a chromatographically separable mixture of the tris-esters 2 (11 mg) and 50 (25 mg); it merits mention that the ratio of the tris-esters 2 and 50 mirrors the ratio of gagunin E (1) and gagunin F from Phorbas sp. which are also only distinguished by the position of the butyrate residue. MDR Modulation. A major challenge in cancer chemotherapy is the emergence of multidrug resistance (MDR), a multifactorial phenomenon characterized by a resistance of cancer cells to chemically and mechanistically diverse chemotherapeutics.53 As a result, anticancer drugs become ineffective and the success of chemotherapeutic cure is compromised. One of the most important MDR mechanisms rests on the increased drug efflux from cancer cells mediated by members of the superfamily of ATP binding cassette (ABC) transporters.54 These membrane proteins actively transport numerous structurally unrelated cytostatic agents across the cell membrane utilizing energy derived from ATP hydrolysis.

Scheme 8. Synthesis of Quinolinecarboxylate 43

Scheme 9. Synthesis of Parent Quinolinecarboxylate 45

Structural Elaboration of the B−C Ring Segment. With the tricyclic A−B-cis,B−C-trans-annulated alcohol 36 available, we continued our efforts toward the gagunin E-like elaboration of the C ring. Tedious efforts to functionalize 34, 35, or 36 by site-selective allylic oxidation failed,42 and we decided to adopt a route in which dienone 47 would serve as a key intermediate for further oxidative functionalization of the C ring (Scheme 10). Initial attempts to convert bis-homoallylic alcohol 36 into dienone 47 using an excess of 2-iodoxybenzoic acid (IBX) (10 equiv) in toluene−DMSO (2:1) at 85 °C for 4 days were Scheme 10. Structural Elaboration of 36

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concentrations up to 30 μM. Concentration−response curves were generated by plotting the fluorescence values against logarithmic concentrations of the modulators to calculate the IC50 values for each compound (Figure 2).

Three prominent representatives of ATP-dependent transporters closely associated with MDR are the P-glycoprotein (Pgp, ABCB1), the breast cancer resistance protein (BCRP, ABCG2), and the multidrug resistance protein 1 (MRP1, ABCC1).55 They are naturally expressed in tissues with protective functions like the kidney, liver, lung, intestine, placenta, or blood−brain barrier, where they mediate the efflux of potentially cell-damaging xenobiotics. Affected cancer cells overexpress the efflux proteins, thereby decreasing the effective intracellular drug concentration in tumor tissues below therapeutic levels. A strategy to overcome MDR in cancer chemotherapy rests on the coadministration of ABC transporter modulators that inhibit the activity of the proteins to restore the cellular drug uptake.56 Several ABC transporter inhibitors were administered in combination with chemotherapeutics in clinical studies and third-generation modulators like tariquidar or elacridar show promising results. However, their clinical success in phase III trials is hampered by increased toxicity and interaction with the anticancer drugs; thus, the quest for more potent and selective inhibitors is ongoing.57 A promising approach for identification of novel ABC transporter modulators aims at phytochemicals with anticarcinogenic properties as they may provide less toxicity than medical drugs.58 In recent years, jatrophane and lathyrane diterpenoid esters isolated from Euphorbia species were evaluated for their in vitro activity as ABCB1 inhibitors.59 These macrocyclic diterpenoids turned out to be valuable lead compounds for natural product-based drug development; however, deeper structure−activity relationship (SAR) studies and better understanding of their interaction with ABC transporters are still needed. Studies from Ferreira et al.,60,61 Shao et al.,62 and Hiersemann et al.63 describe the synthesis of non-natural lathyranoid (Ferreira and Shao) and jatrophanoid (Hiersemann) derived esters and their evaluation as MDR modulators. These studies demonstrate that derivatization of natural products does indeed improve the inhibitory potency and, furthermore, allows for SAR studies to expand the knowledge of the underlying ligand−protein interactions. Evaluation as MDR Modulators. The members of our library of non-natural homoverrucosanoid-derived esters were examined as modulators of the MDR related transporter proteins ABCB1, ABCG2, and ABCC1. To this end, cyclohepta[e]hydrindanoids 35−43, 46−50, 2, the control compound 45, as well as the reference inhibitor cyclosporin A (Cs A) and the fumitremorgin analog Ko 143 were subjected to biological studies. To investigate the inhibitory activity against ABCG2, the Hoechst 33342 accumulation assay using the ABCG2 overexpressing MDCK II BCRP cell line was performed. The fluorescent dye Hoechst 33342 is a substrate of ABCG2, and it exerts much higher fluorescence intensity when bound to DNA.64 To explore the inhibitory activity against ABCB1 and ABCC1, the calcein acetoxymethyl ester (AM) accumulation assay using ABCB1 overexpressing A2780ADR cells and ABCC1 overexpressing H69AR cells, respectively, was performed. The nonfluorescent dye calcein AM is a substrate of ABCB1 and ABCC1. After diffusion through the plasma membrane the acetoxymethyl esters are hydrolyzed by unspecific esterases and the fluorescent calcein anion remains inside the cell.65 The observed fluorescence in the accumulation assays correlates with the increased intracellular concentration of the dye and, therefore, with the inhibitory activity of the modulator. The relative fluorescence intensity was measured in the presence of the modulator at

Figure 2. Concentration−response curves exemplified for 40 toward ABCG2 (A: MDCK II BCRP cells, Hoechst 33342 Assay) and ABCB1 (B: A2780ADR cells, calcein AM assay).

Our studies with respect to ABCC1 activity demonstrated that only quinolinecarboxylate 43 inhibited the ABCC1mediated efflux of calcein AM in concentrations up to 30 μM. Compound 43 showed a 4.5 fold weaker potency (IC50 ± SD = 13.2 ± 4.5 μM) than reference compound cyclosporin A (IC50 ± SD = 2.97 ± 0.38 μM). The IC50 values for the inhibition of ABCG2 and ABCB1 are summarized in Table 5. Notably, the homoverrucosanoidderived quinoline-6-carboxylic acid ester 40 (entry 6) exhibited an inhibitory activity against ABCB1 (IC50 = 1.39 μM) comparable to that of the first-generation modulator cyclosporin A (IC50 = 1.21 μM). Furthermore, 40 efficiently inhibited ABCG2-mediated efflux of Hoechst 33342 (IC50 = 1.56 μM). The analogous 4-bromobenzoic acid ester 36 (entry 2) inhibited ABCG2 activity (IC50 = 2.91 μM) without affecting ABCB1-mediated efflux up to 30 μM. Thus, quinolinecarboxylate 40 represents the most potent dual ABCG2/ABCB1 inhibitor and bromobenzoate 36 the most potent selective ABCG2 inhibitor of our compound library. In general, the inhibitory activity of the synthetic cyclohepta[e]hydrindanoids against ABCG2 seems to increase in the presence of an aromatic ester at C4 and an S-configured unmasked alcohol at C6. Initial studies showed that C4 bromobenzoate 36 (C6-OH) (entry 2) efficiently inhibited 10511


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selectivity was achieved with C4/C6-bis-quinolinecarboxylate 42 (entry 8), which exclusively inhibited ABCB1 activity (IC50 = 6.0 μM). The comparison between quinolinecarboxylate 43 (entry 9) and its almost inactive analogous bromobenzoate 37 (entry 3) highlights the promoting effect of the quinoline moiety on ligand−protein interaction: 43 efficiently inhibited the efflux mediated by ABCG2 (IC50 = 3.34 μM) and ABCB1 (IC50 = 2.02 μM). Additionally, 43 was capable of modulating ABCC1 activity (IC50 = 13.2 μM). The Brønsted basic nitrogen atom of the C4 quinolinecarboxylate possibly supports specific binding to the ABC transporters. As previously found, the quinoline moiety is an important structural element for molecular recognition and frequently present in ABCB1 and/ or ABCG2 modulatory compounds like tariquidar and its analogues.66 MDR profiling of the C10a diastereomers 37 and 38 (entries 3 and 4) revealed a dependence of the transporter selectivity on the mode of annulation: B−C-trans-configured 37 affected ABCG2 (IC50 = 25.8 μM), whereas B−C-cis-configured 38 interfered with ABCB1 activity (IC50 = 17.9 μM). The control compound 45 (entry 10) exhibited IC50 values of 16.1 and 27.8 μM for the inhibition of ABCG2 and ABCB1, respectively. The decreased inhibitory activity of 45 in comparison to quinolinecarboxylate 40 underlines the importance of the cyclohepta[e]hydrindane scaffold for molecular recognition. We calculated the octanol−water partition coefficients (C log P) of the tested compounds. A simple correlation between the overall lipophilicity and the extent of ABCG2/ABCB1 modulation was not observed, but strikingly, the homoverrucosanoid-derived esters exhibited C log P values in the range of 5.66−9.04, whereas the C log P value for cyclohexyl quinolone-6-carboxylate (45) is 4.14. The weaker lipophilicity of 45 may explain its decreased inhibitory activity against the ABC transporters. Lipophilic substrates and modulators enter the drug binding pocket directly from the plasma membrane.67 As a result, lipophilic drugs accumulating in the lipid bilayer possess an increased affinity toward the membrane proteins. The simplified gagunin E analogue (2) (C6-OH) (entry 16) exhibited a 2-fold higher inhibitory activity against ABCG2 (IC50 = 8.1 μM) than its constitutional isomer 50 (C6butyrate) (IC50 = 16.4 μM) (entry 15), highlighting the significance of an unmasked 6-OH group for efficient ABCG2 inhibition. Additionally, both compounds affected ABCB1mediated efflux at comparable rates (2: IC50 = 7.1 μM; 50: IC50 = 8.5 μM). In comparison to the most active inhibitor 40, the natural product analogues 2 and 50 feature a more highly oxygen atom functionalized C ring; however, they are less efficient in modulating the activity of ABCG2/ABCB1. The MTT cytotoxicity assay was used to determine the intrinsic cytotoxicity of the modulators. To this end, the most active compounds 36 and 40 as well as the most elaborated

Table 5. Inhibitory Activities of HomoverrucosanoidDerived Esters and Reference Compounds toward ABCG2 (MDCK II BCRP Cell Line, Hoechst 33342 Assay) and ABCB1 (A2780ADR Cell Line, Calcein AM Assay) entry

compd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a

(±)-35 (±)-36 (±)-37 (±)-38 (±)-39 (±)-40 (±)-41 (±)-42 (±)-43 (±)-45 (±)-46 (±)-47 (±)-48 (±)-49 (±)-50 (±)-2 Ko143 Cs Ac

ABCG2 IC50 ± SDa (μM) b

n.e. 2.91 ± 0.24 25.8 ± 3.9 n.e. 5.07 ± 1.13 1.56 ± 0.16 2.47 ± 0.22 n.e. 3.34 ± 0.33 16.1 ± 1.7 7.11 ± 1.24 10.1 ± 3.8 23.1 ± 6.8 27.2 ± 0.8 16.4 ± 1.3 8.10 ± 2.12 0.221 ± 0.024

ABCB1 IC50 ± SDa (μM) n.e. n.e. n.e. 17.9 n.e. 1.39 6.23 6.00 2.02 27.8 n.e. n.e. n.e. n.e. 8.52 7.10

± 4.6 ± ± ± ± ±

0.16 1.91 1.77 0.47 6.6

± 0.71 ± 1.73

1.21 ± 0.16

n = 3. bn.e. = no inhibitory effect up to 30 μM. cCs A: cyclosporin A.

ABCG2 mediated efflux (IC50 = 2.91 μM). In comparison, precursor 35 (C6-OBOM) (entry 1) had no inhibitory effect up to 30 μM and ether 37 (C6−O−C9) (entry 3) was almost inactive (IC50 = 25.8 μM). The C6 ketones 46 and 47 (entries 11 and 12) possessed a decreased inhibitory activity against ABCG2 with IC50 values of 7.11 μM and 10.1 μM, respectively. Interestingly, the 6R-configured dienol 48 (C6-OH) (entry 13) had almost no inhibitory potency (IC50 = 23.1 μM), showing the importance of a 6S-configuration for effective ABCG2 inhibition. Masking the 6-OH group by esterification (42, entry 8) resulted in complete loss of activity toward ABCG2; however, 42 affected ABCB1-mediated efflux. Considering 36 as a lead compound, the esters 39−41 (C6OH) were investigated next (entries 5−7). Introduction of a naphthoyl moiety (39, entry 5) decreased the inhibitory effect on ABCG2 (IC50 = 5.07 μM). Quinolinecarboxylate 40 (entry 6) showed the highest inhibitory activity against ABCG2 in this study (IC50 = 1.56 μM), but a loss of selectivity was observed: 40 inhibited ABCB1-mediated efflux (IC50 = 1.39 μM) at a rate comparable to that of cyclosporin A. Methoxybenzoate 41 (entry 7) showed a similar behavior and inhibited the activity of ABCG2 (IC50 = 2.47 μM) and ABCB1 (IC50 = 6.23 μM) at slightly higher concentrations. The introduction of a C4 quinolinecarboxylate significantly improves the inhibitory effect on ABCG2, but cross reactivity with ABCB1 was observed (40). Complete reversal of

Table 6. Intrinsic Toxicity of Compounds 36, 40, and 2 on MDCK II Cells (BCRP and Wild-type) and A2780 Cells (ADR and Wild-type) Determined by the MTT Cytotoxicity Assay MDCK II cell lines GI50 ± SDa (μM)

a

A2780 cell lines GI50 ± SDa (μM)

entry

compd

BCRP

wild-type

ADR

wild-type

1 2 3

(±)-36 (±)-40 (±)-2

25.8 ± 2.3 13.9 ± 1.2 13.9 ± 3.6

30.6 ± 4.5 18.0 ± 2.9 17.1 ± 1.6

11.2 ± 2.6 11.6 ± 3.7 11.4 ± 2.7

14.3 ± 5.3 10.0 ± 4.2 9.28 ± 2.96

n = 3. 10512


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cells; GI50,ADR = 11.6 and GI50,wild‑type = 10.0 μM for A2780 cells) which is around 8 to 10-fold higher than its IC50 values toward ABCB1 (1.39 μM) and ABCG2 (1.56 μM), respectively. The effect of 36 and 40 on cell viability is around 10-fold weaker than their inhibitory potency; however, larger GI50/IC50 ratios are required for the development of less toxic ABC transporter inhibitors.

gagunin E analogue (2) were selected. The cell lines MDCK II BCRP and A2780ADR as well as their corresponding wildtypes were exposed to different concentrations of modulator for a period of 72 h (Table 6). For all (used) cell lines, representative concentration−cell viability curves for compound 40 are shown in Figure 3. They were generated by

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CONCLUSIONS We have reported dead ends and detours on the way to the synthesis of simplified analogues of gagunin E. Lessons learned from the B-ring construction by (4 + 2)-cycloaddition, the introduction of the 5a-methyl group, as well as the oxidative functionalization of the C ring were outlined. Specific reactivities of the functionalized tricyclic scaffold, for instance a propensity for transannular etherification, were revealed. The synthesis of an ensemble of non-natural homoverrucosanoidderived esters provided opportunity for their evaluation as MDR modulators with respect to the ABCB1, ABCG2, and ABCC1 transporter proteins. SAR studies revealed that specific molecular constitution and configuration as well as general lipophilicity of the homoverrucosane scaffold are important factors for effective molecular recognition. The presence of an S-configured alcohol at C6 and a quinoline-6-carboxylic acid ester at C4 are most beneficial for binding to ABCG2 and ABCB1. The quinolinecarboxylate 40 represents the most potent dual ABCG2/ABCB1 modulator of our compound collection; 40 showed an inhibitory activity against ABCB1 comparable to that of standard inhibitor cyclosporin A. The analogous bromobenzoate 36 exhibited the strongest inhibitory effect on ABCG2 without affecting ABCB1-mediated efflux. Our synthetic modulators showed up to 10-fold lower cytotoxicity than their inhibitory activity and may serve as lead structures for future development of potent inhibitors based on the polycyclic homoverrucosane scaffold.

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Figure 3. Representative concentration−cell viability curves of the most active ABCG2/ABCB1 modulator 40 obtained in the MTT cytotoxicity assay with (A) MDCK II cells (transfected and wild-type) and (B) A2780 cells (resistant and wild-type).

EXPERIMENTAL SECTION

Chemistry. General Information. Unless otherwise stated, commercially available reagents, catalysts, and solvents were used as purchased. Toluene, tetrahydrofuran, diethyl ether, dichloromethane, 1,2-dichloroethane, and acetonitrile were dried deploying a commercially available solvent purification system. Triethylamine and pyridine were distilled from CaH2 and stored under an atmosphere of argon over activated 4 Å molecular sieves. Methanol was distilled from magnesium and stored under an atmosphere of argon over activated 4 Å molecular sieves. CDCl3 was passed through a short column of basic aluminum oxide and stored over activated 4 Å molecular sieves. DMP was prepared according to the literature and stored in the refrigerator.68 TBSOTf was prepared according to the literature and stored under an atmosphere of argon in the freezer.69 All moisturesensitive reactions were performed in flame-dried septum-sealed glassware under an atmosphere of argon. Reagents were transferred by means of syringe. Solids were introduced under a counter-flow of argon. Preparative irradiations were carried out in a Luzchem photoreactor (Model LZC-4V) equipped with 14 fluorescent lamps (Luzchem, Model LZC-UVC, 8 W, centered at 254 nm) using sealable quartz tubes (10 mL volume). Analytical TLC was performed using precoated silica gel foils (4 cm). Visualization was achieved using 254 nm ultraviolet irradiation followed by staining with the Kägi−Miescher reagent (p-anisaldehyde 2.53% v/v, acetic acid 0.96% v/v, ethanol 93.06% v/v, concd H2SO4 3.45% v/v).70 Chromatographic purification71 was performed on silica gel (particle size 0.040−0.063 mm). Mixtures of cyclohexane and ethyl acetate were used as eluents. 1H NMR spectra were recorded at 400, 500, or 600 MHz. Chemical shifts (δ) are reported in ppm relative to chloroform (7.26 ppm).72 Signal

plotting the cell viability as percentage against logarithmic concentrations of the modulator to calculate the GI50 values. Generally, both canine kidney cell lines (MDCK II) tolerated slightly higher concentrations of the investigated compounds than both human ovarian cancer cell lines (A2780). Simplified gagunin E analogue (2) (entry 3) exerted an intrinsic cytotoxicity with GI50 values of GI50,BCRP = 13.9 μM and GI50,wild‑type = 17.1 μM on the MDCK II cell line, which is around 2-fold higher than its IC50,ABCG2 value (8.1 μM). On the contrary, analogue 2 showed GI50 values against A2780 cells in a similar range of its IC50,ABCB1 (7.1 μM). The most potent selective ABCG2 inhibitor 36 (entry 1) showed the lowest cytotoxicity toward the MDCK II cell lines (GI50,BCRP = 25.8 μM; GI50,wild‑type = 30.6 μM), which is around 10-fold higher than its IC50,ABCG2 value of 2.91 μM. In comparison, the cytotoxic effect of 36 against the A2780 cells were 2-fold higher as we observed GI50 values of GI50,ADR = 11.2 μM for resistant and GI50,wild‑type = 14.3 μM for wild-type cells. The most potent dual ABCG2/ABCB1 inhibitor 40 (entry 2) had similar effects on cell viability as compound 2 (GI50,BCRP = 13.9 μM and GI50,wild‑type = 18.0 μM for MDCK II 10513


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and diethyl ether]) that was used immediately without further purification. Rf 0.22 (ethyl acetate). To a solution of the above crude dialdehyde 15 (186.21 g/mol, 22.749 g [contaminated with triphenylphosphine oxide and diethyl ether], assumed to be 20.797 mmol, 1 equiv) in CH2Cl2 (250 mL) was added dibenzylammonium trifluoroacetate (C16H16F3NO2, 311.30 g/mol, 6.474 g, 20.797 mmol, 1 equiv) at room temperature. The resulting colorless reaction mixture was stirred for 22 h at room temperature and the volatiles were removed under reduced pressure. Purification by flash chromatography (n-pentane−diethyl ether 20:1−2:1) and removal of the solvents to a volume of about 50 mL provided a clear colorless ethereal solution of (3aR,6aS)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxole5-carbaldehyde (15A) (C9H12O3, 168.19 g/mol, about 50 mL [contaminated with diethyl ether]). Rf 0.47 (cyclohexane−ethyl acetate 2:1). The above solution of 15A (168.19 g/mol, about 50 mL [contaminated with diethyl ether], assumed to be 20.797 mmol, 1 equiv) was diluted with THF (50 mL) and activated 4 Å molecular sieves (10 g, beads) were added. The solution was cooled to 0 °C and methylmagnesium bromide (CH3MgBr, 1 M in THF, 24.0 mL, 24.0 mmol, 1.15 equiv) was added dropwise at 0 °C over a period of 25 min. The resulting suspension was carefully diluted by the addition of saturated aqueous NH4Cl solution at 0 °C. The phases were separated and the aqueous layer was extracted with diethyl ether (3×). The combined organic phases were dried (MgSO4) and the volatiles were removed under reduced pressure. Purification by flash chromatography (n-pentane−diethyl ether 5:1−0:1) afforded a mixture of C1 epimers of allylic alcohol 15B (C10H16O3, 184.24 g/mol, 2.123 g, 11.523 mmol, dr = 55:45, 55% from 14A) as a colorless oil. The ratio of C1 epimers was determined by integration of the 1H NMR signals at 1.31 ppm (major epimer) and 1.29 ppm (minor epimer): Rf 0.29 (cyclohexane− ethyl acetate 2:1); 1H NMR (CDCl3, 600 MHz) δ 1.29 (d, J = 6.6 Hz, 3Hminor), 1.31 (d, J = 6.6 Hz, 3Hmajor), 1.33 (s, 3Hmajor+3Hminor), 1.39 (s, 3Hminor), 1.40 (s, 3Hmajor), 1.78−1.94 (m, OHmajor+OHminor), 2.45− 2.52 (m, 1Hmajor+1Hminor), 2.53−2.62 (m, 1Hmajor+1Hminor), 4.38 (q, J = 6.6 Hz, 1Hmajor), 4.39 (q, J = 6.6 Hz, 1Hminor), 4.76−4.79 (m, 1Hmajor+1Hminor), 5.06−5.11 (m, 1Hmajor+1Hminor), 5.61−5.63 (m, 1Hmajor), 5.63−5.65 (m, 1Hminor); 13C NMR (CDCl3, 151 MHz) δ 21.9 (CH3major), 22.0 (CH3minor), 25.70 (CH3minor), 25.75 (CH3major), 27.6 (CH3minor), 27.7 (CH3major), 37.80 (CH2major), 37.85 (CH2minor), 66.7 (CHmajor), 67.0 (CHminor), 78.15 (CHmajor), 78.20 (CHminor), 85.15 (CHminor), 85.20 (CHmajor), 109.8 (Cminor), 109.9 (Cmajor), 123.4 (CHminor), 123.6 (CHmajor), 150.3 (Cmajor), 150.5 (Cminor); IR ν 3420, 2980, 2930, 1370, 1205, 1155, 1035, 865 cm−1; HRMS (ESI) m/z could not be detected. A copy of the HRMS spectrum is provided as part of the Supporting Information. Acetyl Cyclopentene 16. A sealable glass pressure tube was charged with a solution of allylic alcohol 15B (C10H16O3, 184.24 g/mol, 1.075 g, 5.835 mmol, 1 equiv) in CH2Cl2 (28 mL). Pretreated (2 days at 150 °C and ambient pressure) manganese dioxide (90% w/w MnO2, 86.94 g/mol, 7.70 g, 6.93 g of MnO2, 79.710 mmol, 13.66 equiv) was added at room temperature, and the tube was sealed with a Teflon screw cap. The black suspension was placed in an oil bath and vigorously stirred at 60 °C for 2 h. The reaction mixture was cooled to room temperature and filtered through a plug of Celite using CH2Cl2 for rinsing. The volatiles were removed under reduced pressure, and the residue was purified by flash chromatography (n-pentane−diethyl ether 20:1−5:1) to provide acetyl cyclopentene 16 (C10H14O3, 182.22 g/mol, 873 mg, 4.791 mmol, 82%) as a colorless oil: Rf 0.44 (cyclohexane−ethyl acetate 2:1); 1H NMR (CDCl3, 600 MHz) δ 1.35 (s, 3H), 1.40 (s, 3H), 2.35 (s, 3H), 2.69 (dddd, J1 = 18.0 Hz, J2 = 5.7 Hz, J3 = 2.3 Hz, J4 = 0.8 Hz, 1H), 2.77 (dddd, J1 = 18.0 Hz, J2 = 1.6 Hz, J3 = 1.4 Hz, J4 = 0.8 Hz, 1H), 4.80 (dddd, J1 = J2 = 5.7 Hz, J3 = 0.8 Hz, J4 = 0.6 Hz, 1H), 5.22 (dddd, J1 = 5.7 Hz, J2 = 1.9 Hz, J3 = 1.6 Hz, J4 = 0.8 Hz, 1H), 6.52 (dddd, J1 = 2.3 Hz, J2 = 1.9 Hz, J3 = 1.4 Hz, J4 = 0.6 Hz, 1H); 13C NMR (CDCl3, 151 MHz) δ 25.5 (CH3), 27.0 (CH3), 27.4 (CH3), 36.6 (CH2), 77.7 (CH), 85.3 (CH), 110.4 (C), 139.6 (CH), 144.6 (C), 197.3 (C); IR ν 2995, 2955, 1675, 1375, 1295, 1210, 1080, 1060, 945 cm−1; HRMS (ESI) calcd for C10H15O3 ([M + H]+) 183.10157, found 183.10145; HRMS (ESI) calcd for C10H14O3Na ([M + Na]+) 205.0835, found 205.0835.

splitting patterns are labeled by the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or overlap of nonequivalent resonances. Coupling constants (Hz) are given as reported by the NMR processing and analysis software. 13C NMR spectra were recorded at 101, 126, or 151 MHz. Unless otherwise reported, all 13C NMR spectra were obtained with broadband proton decoupling. Chemical shifts are reported in ppm relative to CDCl3 (77.16 ppm); the total number of reported 13C atom signals may fall short of the expected number because of coincidental chemical shifts, even for constitutopic or diastereotopic carbon atoms. The NMR peak assignment as well as the assignment of the relative configuration rests on the interpretation of 13C DEPT, 1H13C HSQC, 1H1H COSY, and 1 1 H H NOESY experiments. Unless stated otherwise, IR spectra were recorded as a thin film on a KBr disk. Infrared absorptions are reported in reciprocal wavelength ν (cm−1) and are adjusted down- or upward to 0 or 5 cm−1. Molecular formula assignment was confirmed by combustion elemental analysis using the elemental analyzers Leco CHNS-932 or Elementar Vario Micro Cube. High-resolution mass spectra were recorded on a LTQ Orbitrap mass spectrometer using electrospray ionization (ESI). Melting points (mp) are uncorrected and were recorded on a Büchi B-540 melting point apparatus. Isopropylidene Acetal 14A: (3aR,7aS)-2,2-Dimethyl-3a,4,5,7atetrahydrobenzo[d][1,3]dioxole. To a solution of (1R,2S)-cyclohex3-ene-1,2-diol (14) (C6H10O2, 114.14 g/mol, 3.502 g, 30.682 mmol, ∼33% ee, 1 equiv) in CH2Cl2 (116 mL) were successively added pyridinium p-toluenesulfonate (PPTS, C12H13NO3S, 251.30 g/mol, 1.156 g, 4.60 mmol, 0.15 equiv) and 2,2-dimethoxypropane (2,2-DMP, C5H12O2, 104.15 g/mol, 0.85 g/mL, 6.66 mL, 5.661 g, 54.354 mmol, 1.77 equiv) at room temperature. The colorless reaction mixture was stirred at room temperature for 75 min and diluted by the addition of saturated aqueous NaHCO3 solution. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and the volatiles were removed under reduced pressure (600 mbar, 40 °C). Purification by flash chromatography (n-pentane−diethyl ether 100:1−20:1) and removal of the solvents (800 mbar, 40 °C) provided a colorless ethereal solution of isopropylidene acetal 14A (C9H14O2, 154.21 g/mol, 8.055 g [contaminated with diethyl ether: Mtotal 262.56 g/mol calculated from the 1H NMR spectrum], 30.679 mmol, 99%). An analytically pure sample of 14A was obtained by complete removal of diethyl ether under reduced pressure: Rf 0.63 (cyclohexane−ethyl acetate 5:1); 1H NMR (CDCl3, 500 MHz) δ 1.37 (s, 3H), 1.42 (s, 3H), 1.75−1.95 (m, 3H), 2.13−2.22 (m, 1H), 4.27 (ddd, J1 = 6.2 Hz, J2 = 5.4 Hz, J3 = 3.9 Hz, 1H), 4.44 (dd, J1 = 3.9 Hz, J2 = 3.2 Hz, 1H), 5.71 (dddd, J1 = 9.9 Hz, J2 = 3.4 Hz, J3 = J4 = 1.8 Hz, 1H), 5.92 (ddd, J1 = 9.9 Hz, J2 = 4.4 Hz, J3 = 3.2 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ 20.9 (CH2), 25.7 (CH2), 26.6 (CH3), 28.2 (CH3), 71.5 (CH), 73.2 (CH), 108.5 (C), 125.5 (CH), 131.4 (CH); IR ν 2985, 2935, 1375, 1215, 1155, 1050, 1025, 860, 680 cm−1; HRMS (ESI) calcd for C9H15O2 ([M + H]+) 155.1067, found 155.1064. Anal. Calcd for C9H14O2: C, 70.1; H, 9.2. Found: C, 69.3; H, 9.0 (unstable weight). Allylic Alcohol 15B: 1-((3aR,6aS)-2,2-Dimethyl-3a,6a-dihydro-4Hcyclopenta[d][1,3]dioxol-5-yl)ethan-1-ol. In a two-necked roundbottomed flask was placed a solution of isopropylidene acetal 14A (C9H14O2, 154.21 g/mol, 8.557 g [contaminated with diethyl ether: Mtotal 411.45 g/mol calculated from the 1H NMR spectrum], 20.797 mmol, 1 equiv) in MeOH (240 mL) and CH2Cl2 (80 mL). A spatula tip of Sudan Red B was added at room temperature, and the resulting pale red colored solution was cooled to −78 °C. Ozone was bubbled through the stirred reaction mixture at −78 °C until decolorization (about 15 min). Argon was bubbled through the reaction mixture for 2 min, and triphenylphosphine (C18H15P, 262.29 g/mol, 6.546 g, 24.957 mmol, 1.2 equiv) was subsequently added at −78 °C in small portions. The cooling bath was removed, and the resulting suspension was stirred at room temperature for 1 h. The reaction mixture was transferred to a single-necked round-bottomed flask using CH2Cl2 for rinsing, and the volatiles were removed under reduced pressure. Purification by a rapidly performed flash chromatography (n-pentane− diethyl ether 5:1−0:1) delivered crude dialdehyde 15 (C9H14O4, 186.21 g/mol, 22.749 g [contaminated with triphenylphosphine oxide 10514


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The Journal of Organic Chemistry (4 + 2)-Cycloadduct 17. To a solution of acetyl cyclopentene 16 (C10H14O3, 182.22 g/mol, 465 mg, 2.552 mmol, 1 equiv) and triethylamine (C6H15N, 101.19 g/mol, 0.726 g/mL, 2.12 mL, 1.539 g, 15.210 mmol, 6 equiv) in CH2Cl2 (25 mL) was carefully added tertbu ty ld im ethy lsilyl tr ifluoro me thanesulfonate (TBSOTf, C7H15F3O3SSi, 264.33 g/mol, 1.151 g/mL, 1.76 mL, 2.026 g, 7.665 mmol, 3 equiv) at 0 °C. The clear colorless solution was stirred at 0 °C for 1 h and was then diluted by the addition of saturated aqueous NaHCO3 solution at 0 °C. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were extracted with saturated aqueous NaCl solution (2×) and dried (MgSO4). The volatiles were removed under reduced pressure to deliver crude diene 5b (C16H28O3Si, 296.48 g/mol, 860 mg [contaminated with solid impurities]) as a pale yellow oil that was used without further purification. Rf 0.68 (cyclohexane−ethyl acetate 5:1). A sealable glass pressure tube was charged with a solution of the above crude diene 5b (296.48 g/mol, 860 mg [contaminated with solid impurities], assumed to be 2.552 mmol, 1 equiv) in toluene (5 mL). Dimethyl acetylenedicarboxylate (10) (DMAD, C6H6O4, 142.11 g/mol, 1.156 g/mL, 0.63 mL, 728 mg, 5.123 mmol, 2 equiv) was added at 0 °C and the resulting light yellow solution was stirred at 0 °C for 1 h while argon was bubbled through the reaction mixture. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (140 °C). After being stirred for 1.5 h at 140 °C, the reaction mixture was cooled to ambient temperature and transferred to a round-bottomed flask using ethyl acetate for rinsing. The volatiles were removed under reduced pressure, and the residue was purified by flash chromatography (cyclohexane−ethyl acetate 50:1−10:1) to provide 17 (C22H34O7Si, 438.59 g/mol, 970 mg, 2.212 mmol, dr >95:5, 87%) as a white solid. Assignment of the relative configuration rests on the interpretation of a 1H1H NOESY experiment; NOE correlations visible between 1-H and acetal-CH3Si, 2-H and acetalCH3Si/1-H/3-HSi as well as 10b-H and acetal-CH3Re/5-HRe. Rf 0.65 (cyclohexane−ethyl acetate 2:1): mp 102−104 °C; 1H NMR (CDCl3, 500 MHz) δ 0.12 (s, 3H, TBS-CH3), 0.14 (s, 3H, TBS-CH3), 0.93 (s, 9H, TBS-CH3), 1.29 (s, 3H, acetal-CH3Si), 1.48 (s, 3H, acetal-CH3Re), 2.25 (dddd, J1 = 16.2 Hz, J2 = 6.5 Hz, J3 = 2.5 Hz, J4 = 1.0 Hz, 1H, 3CH2Re), 2.87−3.01 (m, 3H, 3-CH2Si+5-CH2), 3.44 (dddd, J1 = 9.5 Hz, J2 = 8.5 Hz, J3 = 7.4 Hz, J4 = 2.5 Hz, 1H, 10b-CH), 3.75 (s, 3H, esterCH3), 3.84 (s, 3H, ester-CH3), 4.29 (ddd, J1 = 7.4 Hz, J2 = 6.5 Hz, J3 = 1.0 Hz, 1H, 1-CH), 4.53 (ddd, J1 = J2 = 6.5 Hz, J3 = 2.7 Hz, 1H, 2CH); 13C NMR (CDCl3, 126 MHz) δ −4.0 (TBS-CH3), −3.9 (TBSCH3), 18.2 (TBS-C), 25.7 (TBS-CH 3), 25.9 (acetal-CH3Si), 28.1 (acetal-CH3Re), 30.8 (3-CH2), 32.0 (5-CH2), 49.9 (10b-CH), 52.4 (ester-CH3), 52.5 (ester-CH3), 78.4 (2-CH), 83.4 (1-CH), 112.5 (C), 112.9 (C), 126.3 (C), 139.7 (C), 140.5 (C), 165.9 (ester carbonyl-C), 169.2 (ester carbonyl-C); IR ν 2930, 2855, 1715, 1275, 1215, 1145, 1070, 845, 780 cm−1; HRMS (ESI) calcd for C22H35O7Si ([M + H]+) 439.2147, found 439.2141; HRMS (ESI) calcd for C22H34O7SiNa ([M + Na]+) 461.1966, found 461.1960. Anal. Calcd for C22H34O7Si: C, 60.3; H, 7.8. Found: C, 60.1; H, 7.8 cis-Hydrindanone (±)-23. Cyclopropane Ring Opening by Hydrogenolysis. To a solution of (±)-22 (452.81 g/mol, 3.155 g, 6.97 mmol, 1 equiv) in glacial acetic acid (≥99.7% w/w C2H4O2, 209 mL) was cautiously added palladium on activated carbon (10% w/w Pd on carbon, Type 487, dry, 106.42 g/mol, 1.854 g, 1.74 mmol, 0.25 equiv) at room temperature in several portions. Gaseous hydrogen from a balloon (football bladder) was vigorously bubbled through the stirred suspension via cannula for 10 min at room temperature (one balloon filling). The flask was subsequently placed in a preheated oil bath (80 °C), and stirring was continued at 80 °C for 1 h while a vigorous flow of hydrogen was bubbled through the reaction mixture (six balloon fillings). After removal of residual hydrogen with an argon flush, the black suspension was cooled to ambient temperature and filtrated through a plug of Celite. The filter cake was washed with ethyl acetate (300 mL), and the organic phases were combined. The volatiles were removed under reduced pressure, and the residue was purified by flash chromatography (cyclohexane−ethyl acetate 1:0− 75:1) to provide (±)-23 (C25H50O3Si2, 454.83 g/mol, 2.78 g, 6.11 mmol, 88%) as a colorless oil. Cyclopropane Ring Opening by

Photochemical Electron Transfer. A sealable quartz tube was charged with a solution of (±)-22 (452.81 g/mol, 45.3 mg, 100.0 μmol, 1 equiv) and triethylamine (C6H15N, 101.19 g/mol, 0.7255 g/mL, 0.21 mL, 152 mg, 1.506 mmol, 15 equiv) in argon-saturated acetonitrile (0.05 M, 2.0 mL, argon was bubbled through the solvent for 30 min prior to use). Lithium perchlorate (LiClO4, 106.39 g/mol, 10.6 mg, 99.6 μmol, 1 equiv) was added at room temperature in one portion. The tube was sealed with a Teflon screw cap and placed in a Luzchem photoreactor. Irradiation of the colorless suspension was performed at 254 nm under stirring for 1 day at 28 °C. The resulting light yellow suspension was transferred to a separatory funnel using ethyl acetate for rinsing. The organic layer was sequentially extracted with aqueous NaHSO4 solution (0.5 M, 1×), aqueous phosphate buffer (pH 7, 1×), and saturated aqueous NaCl solution (1×). The organic phase was dried (MgSO4), and the volatiles were removed under reduced pressure. Purification of the residue by flash chromatography (cyclohexane−ethyl acetate 1:0−75:1) delivered (±)-23 (C25H50O3Si2, 454.83 g/mol, 28.0 mg, 61.6 μmol, 62%) as a colorless oil: Rf 0.55 (cyclohexane−ethyl acetate 10:1); 1H NMR (CDCl3, 500 MHz) δ −0.01 (s, 3H), 0.01 (s, 3H), 0.04 (s, 3H), 0.05 (s, 3H), 0.88 (s, 9H), 0.90 (s, 9H), 1.07 (s, 3H), 1.24 (s, 3H), 1.31 (ddd, J1 = 12.8 Hz, J2 = J3 = 7.8 Hz, 1H), 1.47−1.64 (m, 3H), 1.74−1.83 (m, 1H), 2.10 (ddd, J1 = 9.5 Hz, J2 = 5.4 Hz, J2 = 4.4 Hz, 1H), 2.21 (d, J = 13.9 Hz, 1H), 2.30 (d, J = 13.9 Hz, 1H), 2.32−2.40 (m, 2H), 3.26 (d, J = 10.3 Hz, 1H), 3.39 (d, J = 10.3 Hz, 1H), 3.75 (dd, J1 = 10.1 Hz, J2 = 9.5 Hz, 1H), 3.91 (dd, J1 = 10.1 Hz, J2 = 4.4 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ −5.6 (CH3), −5.5 (CH 3), −5.20 (CH3), −5.18 (CH3), 18.3 (C), 18.4 (C), 23.7 (CH2), 25.8 (CH3), 26.0 (CH3), 26.1 (CH3), 26.8 (CH3), 28.0 (CH2), 36.8 (CH2), 43.5 (C), 46.8 (CH), 48.7 (CH2), 50.1 (CH), 55.0 (C), 62.5 (CH2), 67.9 (CH2), 216.8 (C); IR ν 2955, 2925, 2885, 2855, 1710, 1470, 1255, 1090, 835, 775 cm−1. Anal. Calcd for C25H50O3Si2: C, 66.0; H, 11.1. Found: C, 66.1; H, 10.7. Bromobenzoates (±)-36 and (±)-37. To a solution of BOM ether (±)-35 (C32H39BrO4, 567.55 g/mol, 134.0 mg, 236.1 μmol, 1 equiv) and thiophenol (C6H6S, 110.18 g/mol, 1.08 g/mL, 0.24 mL, 259 mg, 2.353 mmol, 10 equiv) in CH2Cl2 (28 mL) was added boron trifluoride diethyl etherate (F3B·OEt2, C4H10BF3O, 141.93 g/mol, 1.12 g/mL, 60 μL, 67.2 mg, 47% w/w BF3, 67.81 g/mol, 32.1 mg of BF3, 473.5 μmol, 2 equiv) at −30 °C. The cooling bath was removed, and the clear solution was stirred at room temperature for 1 h. The reaction mixture was diluted by the addition of saturated aqueous NaHCO3 solution and H2O at 0 °C. Vigorous stirring of the biphasic mixture was continued at room temperature for 30 min. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification of the residue by flash chromatography (cyclohexane−ethyl acetate 100:1− 35:1) delivered (±)-36 (C24H31BrO3, 447.41 g/mol, 52.0 mg, 116.2 μmol, 49%) and (±)-37 (C24H31BrO3, 447.41 g/mol, 38.0 mg, 84.9 μmol, 36%) as colorless oils. (±)-36: Rf 0.26 (cyclohexane−ethyl acetate 10:1); 1H NMR (CDCl3, 400 MHz) δ 0.94 (s, 3H), 0.99 (s, 3H), 1.37 (dd, J1 = 12.7 Hz, J2 = 4.9 Hz, 1H), 1.43 (ddd, J1 = 12.4 Hz, J2 = 9.4 Hz, J3 = 3.2 Hz, 1H), 1.49 (ddd, J1 = 13.1 Hz, J2 = 9.5 Hz, J3 = 3.6 Hz, 1H), 1.54−1.68 (m, 4H), 1.68−1.89 (m, 3H), 1.74 (s, 3H), 1.96 (dddd, J1 = 13.1 Hz, J2 = 11.1 Hz, J3 = 7.2 Hz, J4 = 6.8 Hz, 1H), 2.08 (ddd, J1 = 12.4 Hz, J2 = 11.0 Hz, J3 = 8.8 Hz, 1H), 2.25 (dd, J1 = 12.7 Hz, J2 = 12.2 Hz, 1H), 2.28 (dd, J1 = 11.2 Hz, J2 = 4.0 Hz, 1H), 2.61 (dd, J1 = 14.4 Hz, J2 = 12.4 Hz, 1H), 3.32 (d, J = 4.0 Hz, 1H), 4.80 (br s, 1H), 5.30 (dd, J1 = 12.2 Hz, J2 = 4.9 Hz, 1H), 7.53−7.58 (m, 2H), 7.84−7.89 (m, 2H); 13C NMR (CDCl3, 101 MHz) δ 18.2 (CH3), 20.0 (CH2), 25.7 (CH2), 25.9 (CH3), 26.1 (CH3), 28.6 (CH2), 29.0 (CH2), 29.8 (CH2), 35.2 (CH2), 37.7 (CH), 41.1 (C), 45.9 (C), 51.4 (CH), 78.1 (CH), 78.3 (CH), 127.9 (C), 129.9 (C), 130.1 (CH), 131.2 (CH), 131.8 (CH), 139.5 (C), 165.9 (C); IR ν 3520, 2960, 2930, 1715, 1590, 1285, 1170, 1115, 1010, 760, 735 cm−1; HRMS (ESI) calcd for C24H31BrO3Na ([M + Na]+) 469.1349, found 469.1392; HRMS (ESI) calcd for C24H3181BrO3Na ([M + Na]+) 471.1332, found 471.1363. (±)-37: Rf 0.38 (cyclohexane−ethyl acetate 10:1); 1H NMR (CDCl3, 400 MHz) δ 0.88 (s, 3H), 0.97 (dd, J1 = J2 = 12.8 Hz, 1H), 0.99 (s, 3H), 1.37 (s, 3H), 1.43 (ddd, J1 = J2 = 12.5 Hz, 10515


Article

The Journal of Organic Chemistry J3 = 4.8 Hz, 1H), 1.41−1.89 (m, 12H), 1.95 (ddd, J1 = 12.2 Hz, J2 = 9.1 Hz, J3 = 2.7 Hz, 1H), 2.14 (ddd, J1 = 12.8 Hz, J2 = 10.5 Hz, J3 = 8.4 Hz, 1H), 3.83 (d, J = 6.6 Hz, 1H), 5.31 (dd, J1 = 11.2 Hz, J2 = 6.1 Hz, 1H), 7.54−7.60 (m, 2H), 7.83−7.89 (m, 2H); 13C NMR (CDCl3, 101 MHz) δ 15.3 (CH3), 19.8 (CH2), 26.45 (CH3), 26.50 (CH2), 26.7 (CH2), 28.4 (CH3), 30.5 (CH2), 35.6 (CH), 37.9 (C), 39.7 (CH2), 39.8 (CH2), 41.6 (CH2), 47.0 (C), 47.1 (CH), 78.8 (C), 79.0 (CH), 83.5 (CH), 128.0 (C), 129.9 (C), 131.2 (CH), 131.8 (CH), 165.8 (C); IR ν 2960, 2925, 1715, 1590, 1270, 1100, 1010, 755, 730 cm−1; HRMS (ESI) calcd for C24H31BrO3Na ([M + Na]+) 469.1349, found 469.1351; HRMS (ESI) calcd for C24H3181BrO3Na ([M + Na]+) 471.1332, found 471.1315. Ether (±)-38. Transannular Etherif ication Mediated by Aqueous HCl−THF. To a solution of (±)-34 (567.55 g/mol, 6.5 mg, 11.45 μmol, 1 equiv) in THF (2 mL) and H2O (1 mL) was added aqueous HCl (5 M in H2O, 1 mL) at 0 °C. The cooling bath was allowed to warm to room temperature and the clear colorless solution was stirred for 1 day at room temperature. The monophasic reaction mixture was diluted by the successive addition of saturated aqueous NaHCO3 solution and diethyl ether at 0 °C. The biphasic mixture was vigorously stirred for 15 min at room temperature, and the phases were separated. The organic phase was extracted with saturated aqueous NaHCO3 solution (2×) and dried (MgSO4). The volatiles were removed under reduced pressure and the residue was purified by flash chromatography (cyclohexane−ethyl acetate 100:1−20:1) to provide (±)-38 (C24H31BrO3, 447.41 g/mol, 4.0 mg, 8.94 μmol, 78%) as a colorless oil. Transannular Etherif ication Mediated by Boron Trifluoride Diethyl Etherate and Thiophenol. To a solution of (±)-34 (567.55 g/mol, 32 mg, 56.4 μmol, 1 equiv) and thiophenol (C6H6S, 110.18 g/mol, 1.08 g/mL, 58 μL, 63 mg, 568 μmol, 10 equiv) in CH2Cl2 (5 mL) was added boron trifluoride diethyl etherate (F3B·OEt2, C4H10BF3O, 47% w/w BF3, 7.5 mg of BF3, 141.93 g/mol, 1.12 g/mL, 14 μL, 15.7 mg, 110.5 μmol, 2 equiv) at −30 °C. The clear solution was allowed to warm to 0 °C over a period of 1.5 h under stirring. The reaction mixture was diluted by the successive addition of saturated aqueous NaHCO3 solution, H2O, and CH2Cl2 at 0 °C. Vigorous stirring of the biphasic mixture was continued at room temperature for 15 min. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification of the residue by flash chromatography (cyclohexane−ethyl acetate 100:1− 20:1) delivered (±)-38 (C24H31BrO3, 447.41 g/mol, 24 mg, 53.6 μmol, 95%) as a colorless oil: Rf 0.41 (cyclohexane−ethyl acetate 10:1); 1H NMR (CDCl3, 500 MHz) δ 0.96 (s, 3H), 1.00 (s, 3H), 1.33 (s, 3H), 1.38 (dd, J1 = 12.2 Hz, J2 = 4.4 Hz, 1H), 1.41 (ddd, J1 = 13.0 Hz, J2 = 5.4 Hz, J3 = 1.3 Hz, 1H), 1.47−1.68 (m, 7H), 1.71−1.81 (m, 1H), 1.82−1.93 (m, 2H), 1.94−2.06 (m, 2H), 2.12 (dd, J1 = J2 = 7.3 Hz, 1H), 2.29 (dd, J1 = 13.0 Hz, J2 = 12.4 Hz, 1H), 3.65 (dd, J1 = 6.4 Hz, J2 = 1.5 Hz, 1H), 5.37 (dd, J1 = 12.4 Hz, J2 = 5.4 Hz, 1H), 7.55− 7.59 (m, 2H), 7.85−7.89 (m, 2H); 13C NMR (CDCl3, 126 MHz) δ 20.8 (CH3), 21.4 (CH2), 26.2 (CH2), 26.8 (CH2), 27.0 (CH3), 28.3 (CH3), 31.0 (CH2), 33.1 (CH2), 34.2 (CH2), 36.9 (CH2), 37.1 (CH), 39.0 (C), 46.1 (CH), 46.6 (C), 79.2 (CH), 80.6 (C), 84.4 (CH), 127.9 (C), 130.0 (C), 131.2 (CH), 131.8 (CH), 165.9 (C); IR ν 2965, 2925, 1715, 1590, 1285, 1115, 1010, 755, 735 cm−1; HRMS (ESI) calcd for C24H32BrO3 ([M + H]+) 447.1529, found 447.1516; HRMS (ESI) calcd for C24H3281BrO3 ([M + H]+) 449.1509, found 449.1495; HRMS (ESI) calcd for C24H31BrO3Na ([M + Na]+) 469.1349, found 469.1341; HRMS (ESI) calcd for C24H3181BrO3Na ([M + Na]+) 471.1332, found 471.1320. Naphthoate (±)-39. The following procedure was performed once and is unoptimized. A sealable glass pressure tube was charged with freshly pestled sodium hydroxide (NaOH, 40.00 g/mol, 190 mg, 4.75 mmol, 50 equiv) in dry MeOH (12 mL). The suspension was stirred at room temperature until a clear, colorless solution resulted (about 10 min). A solution of (±)-35 (C32H39BrO4, 567.55 g/mol, 54.0 mg, 95.1 μmol, 1 equiv) in dry MeOH (12 mL) was added at room temperature. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (60 °C). After being stirred for 17 h at 60 °C, the pale yellow solution was cooled to ambient temperature and transferred to a

separatory funnel using CH2Cl2 for rinsing. The biphasic mixture was extracted once with aqueous NH4Cl solution (3.5 M). The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Subsequent flash chromatography (cyclohexane−ethyl acetate 20:1−5:1) provided the alcohol (±)-35A (C25H36O3, 384.56 g/mol, 35.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (about 10%). Rf 0.53 (cyclohexane−ethyl acetate 2:1). A sealable glass pressure tube was charged with a suspension of 2-naphthoic acid (C11H8O2, 172.18 g/mol, 94 mg, 545.9 μmol, 6 equiv) in CH2Cl2 (2 mL). 4-Dimethylaminopyridine (DMAP, C7H10N2, 122.17 g/mol, 133 mg, 1.089 mmol, 12 equiv) was added at room temperature, and stirring was continued at room temperature for 5 min until a clear colorless solution appeared. N-(3(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 105 mg, 547.7 μmol, 6 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of (±)-35A (384.56 g/mol, 35.0 mg, assumed to be pure, 91.0 μmol, 1 equiv) in CH2Cl2 (3 mL) was added at 0 °C. The cooling bath was removed, and the colorless solution was stirred at room temperature for 1.5 h. The tube was sealed with a Teflon screw cap, and the reaction mixture was stirred at 40 °C for 16 h. The mixture was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 100:1−50:1) delivered the ester (±)-35B (C36H42O4, 538.73 g/mol, 48.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (ca. 10%). Rf 0.38 (cyclohexane−ethyl acetate 10:1). To a solution of (±)-35B (538.73 g/mol, 48.0 mg, assumed to be pure, 89.1 μmol, 1 equiv) in Et2O (9 mL) was added boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.27 mL, 540 μmol, 6.1 equiv) at room temperature. The white suspension was stirred at room temperature for 1.5 h. Additional boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.27 mL, 540 μmol, 6.1 equiv) was added at room temperature, and stirring was continued at room temperature for 2 h. Further boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.58 mL, 1.160 mmol, 13 equiv) was added at room temperature, and stirring was continued at room temperature for 18 h. The resultant mixture consisted of a solid white precipitate adhered to the walls of the flask and a supernatant clear solution. The reaction mixture was diluted by the addition of saturated aqueous NaHCO3 solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 50:1−20:1) delivered (±)-39 (C28H34O3, 418.58 g/mol, 31.0 mg, 74.1 μmol, 78% from (±)-35) as a colorless oil: Rf 0.24 (cyclohexane−ethyl acetate 10:1); 1H NMR (CDCl3, 500 MHz) δ 0.97 (s, 3H), 1.04 (s, 3H), 1.44 (dd, J1 = 12.8 Hz, J2 = 4.9 Hz, 1H), 1.52 (ddd, J1 = 13.2 Hz, J2 = 9.3 Hz, J3 = 3.1 Hz, 1H), 1.52−1.71 (m, 3H+OH), 1.68 (dd, J1 = 11.5 Hz, J2 = 6.8 Hz, 1H), 1.71−1.90 (m, 3H), 1.76 (s, 3H), 1.99 (dddd, J1 = 13.2 Hz, J2 = 11.1 Hz, J3 = 7.4 Hz, J4 = 6.8 Hz, 1H), 2.17 (ddd, J1 = 12.4 Hz, J2 = 11.1 Hz, J3 = 8.8 Hz, 1H), 2.31 (dd, J1 = 12.8 Hz, J2 = 12.1 Hz, 1H), 2.32 (dd, J1 = 11.5 Hz, J2 = 4.0 Hz, 1H), 2.63 (dd, J1 = 14.5 Hz, J2 = 12.5 Hz, 1H), 3.46 (br s, 1H), 4.82 (br s, 1H), 5.39 (dd, J1 = 12.1 Hz, J2 = 4.9 Hz, 1H), 7.52− 7.60 (m, 2H), 7.87 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 8.04 (dd, J1 = 8.6 Hz, J2 = 1.6 Hz, 1H), 8.57 (s, 1H); 13C NMR (CDCl3, 126 MHz) δ 18.2 (CH3), 20.0 (CH2), 25.7 (CH2), 26.0 (CH3), 26.1 (CH3), 28.6 (CH2), 29.0 (CH2), 29.9 (CH2), 35.3 (CH2), 37.7 (CH), 41.1 (C), 46.0 (C), 51.4 (CH), 77.7 (CH), 78.4 (CH), 125.4 (CH), 126.7 (CH), 127.9 (CH), 128.20 (CH), 128.25 (CH), 128.30 (C), 129.5 (CH), 130.2 (CH), 131.0 (CH), 10516


Article

The Journal of Organic Chemistry 132.6 (C), 135.6 (C), 139.5 (C), 166.8 (C); IR ν 3505, 2960, 2925, 1695, 1285, 1230, 1195, 1130, 1095, 965, 910, 780, 730 cm−1; HRMS (ESI) calcd for C28H35O3 ([M + H]+) 419.2581, found 419.2580. Quinolinecarboxylate (±)-40. The following procedure was performed once and is unoptimized. A sealable glass pressure tube was charged with freshly pestled sodium hydroxide (NaOH, 40.00 g/mol, 200 mg, 5.00 mmol, 50 equiv) in dry MeOH (12.5 mL). The suspension was stirred at room temperature until a clear colorless solution resulted (about 10 min). A solution of (±)-35 (C32H39BrO4, 567.55 g/mol, 57.0 mg, 100.4 μmol, 1 equiv) in dry MeOH (13 mL) was added at room temperature. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (60 °C). After being stirred for 15 h at 60 °C, the pale yellow solution was cooled to ambient temperature and transferred to a separatory funnel using CH2Cl2 for rinsing. The biphasic mixture was extracted once with aqueous NH4Cl solution (3.5 M). The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3 × ). The combined organic phases were dried (MgSO4), and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Subsequent flash chromatography (cyclohexane−ethyl acetate 20:1− 5:1) provided the alcohol (±)-35A (C25H36O3, 384.56 g/mol, 38.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (ca. 10%). Rf 0.53 (cyclohexane−ethyl acetate 2:1). A sealable glass pressure tube was charged with a suspension of quinoline-6-carboxylic acid (C10H7NO2, 173.17 g/mol, 103 mg, 594.8 μmol, 6 equiv) in CH2Cl2 (2 mL). 4-(Dimethylamino)pyridine (DMAP, C7H10N2, 122.17 g/mol, 145 mg, 1.187 mmol, 12 equiv) was added at room temperature, and stirring was continued at room temperature until a clear beige solution appeared (about 5 min). N-(3(Dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 114 mg, 594.7 μmol, 6 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of (±)-35A (384.56 g/mol, 38.0 mg, assumed to be pure, 98.8 μmol, 1 equiv) in CH2Cl2 (3 mL) was added at 0 °C. The cooling bath was removed and the light brown solution was stirred at room temperature for 1 h. The tube was sealed with a Teflon screw cap and the reaction mixture was stirred at 40 °C for 20 h. The mixture was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4) and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Purification by flash chromatography (cyclohexane−ethyl acetate 20:1−5:1) delivered the ester (±)-35C (C35H41NO4, 539.72 g/mol, 46.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (ca. 10%). Rf 0.47 (cyclohexane−ethyl acetate 2:1). To a solution of (±)-35C (539.72 g/mol, 46.0 mg, assumed to be pure, 85.2 μmol, 1 equiv) in Et2O (8.5 mL) was added boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.26 mL, 520 μmol, 6.1 equiv) at room temperature. The white suspension was stirred at room temperature for 2.5 h. The resultant reaction mixture consisted of a solid white precipitate adhered to the walls of the flask and a supernatant clear solution. The mixture was diluted by the addition of saturated aqueous NaHCO3 solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4) and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 10:1−2:1) provided (±)-40 (C27H33NO3, 419.57 g/mol, 32.0 mg, 76.3 μmol, 76% from (±)-35) as a colorless oil: Rf 0.40 (cyclohexane−ethyl acetate 1:1); 1H NMR (CDCl3, 500 MHz) δ 0.97 (s, 3H), 1.04 (s, 3H), 1.44 (dd, J1 = 12.8 Hz, J2 = 4.9 Hz, 1H), 1.52 (ddd, J1 = 13.1 Hz, J2 = 9.2 Hz, J3 = 3.0 Hz, 2H), 1.56−1.72 (m, 3H+OH), 1.72−1.90 (m, 3H), 1.76 (s, 3H), 1.99 (dddd, J1 = 13.1 Hz, J2 = 11.1 Hz, J3 = 7.2 Hz, J4 = 6.8 Hz, 1H), 2.16 (ddd, J1 = 12.4 Hz, J2 = 11.0 Hz, J3 = 8.8 Hz, 1H), 2.33 (dd, J1 = 11.2

Hz, J2 = 4.0 Hz, 1H), 2.33 (dd, J1 = 12.8 Hz, J2 = 12.2 Hz, 1H), 2.64 (dd, J1 = 14.4 Hz, J2 = 12.4 Hz, 1H), 3.46 (dd, J1 = J2 = 4.0 Hz, 1H), 4.82 (br s, 1H), 5.40 (dd, J1 = 12.2 Hz, J2 = 4.9 Hz, 1H), 7.47 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 8.27 (dd, J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 8.28 (dd, J1 = 8.8 Hz, J2 = 1.8 Hz, 1H), 8.54 (d, J = 1.8 Hz, 1H), 9.00 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ 18.2 (CH3), 19.9 (CH2), 25.7 (CH2), 25.9 (CH3), 26.2 (CH3), 28.6 (CH2), 29.0 (CH2), 29.9 (CH2), 35.3 (CH2), 37.7 (CH), 41.1 (C), 46.0 (C), 51.4 (CH), 78.2 (CH), 78.3 (CH), 121.9 (CH), 127.5 (C), 129.0 (C), 129.2 (CH), 129.8 (CH), 130.1 (CH), 130.9 (CH), 137.6 (CH), 139.5 (C), 150.1 (C), 152.5 (CH), 166.1 (C); IR ν 3370, 2960, 2925, 1710, 1275, 1245, 1190, 1095, 965, 910, 785, 730 cm−1; HRMS (ESI) calcd for C27H34NO3 ([M + H]+) 420.2533, found 420.2535. Methoxybenzoate (±)-41. The following procedure was performed once and is unoptimized. A sealable glass pressure tube was charged with freshly pestled sodium hydroxide (NaOH, 40.00 g/mol, 229 mg, 5.725 mmol, 50 equiv) in dry MeOH (14.5 mL). The suspension was stirred at room temperature until a clear colorless solution resulted (about 10 min). A solution of (±)-35 (C32H39BrO4, 567.55 g/mol, 65.0 mg, 114.5 μmol, 1 equiv) in dry MeOH (14.5 mL) was added at room temperature. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (60 °C). After being stirred for 15 h at 60 °C, the pale yellow solution was cooled to ambient temperature and transferred to a separatory funnel using CH2Cl2 for rinsing. The biphasic mixture was extracted once with aqueous NH4Cl solution (3.5 M). The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3 × ). The combined organic phases were dried (MgSO4), and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Purification by flash chromatography (cyclohexane−ethyl acetate 20:1−5:1) provided the alcohol (±)-35A (C25H36O3, 384.56 g/mol, 44.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (ca. 10%). Rf 0.53 (cyclohexane−ethyl acetate 2:1). To a suspension of 4-methoxybenzoic acid (C8H8O3, 152.15 g/mol, 52 mg, 341.8 μmol, 3 equiv) in CH2Cl2 (2 mL) was added 4-(dimethylamino)pyridine (DMAP, C7H10N2, 122.17 g/mol, 84 mg, 687.6 μmol, 6 equiv) at room temperature. The reaction mixture was stirred for 5 min at room temperature until a clear colorless solution appeared. N-(3-(Dimethylamino)propyl)-N′ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 66 mg, 344.3 μmol, 3 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of (±)-35A (384.56 g/mol, 44.0 mg, assumed to be pure, 114.4 μmol, 1 equiv) in CH2Cl2 (3 mL) was added at 0 °C. The cooling bath was removed, and the colorless solution was stirred at room temperature for 4.5 h. The flask was sealed with a polyethylene plug, and the reaction mixture was stirred at 40 °C for 19 h. After TLC analysis showed incomplete conversion, the reaction mixture was transferred to a sealable glass pressure tube containing an excess of reagents [4-methoxybenzoic acid (C8H8O3, 152.15 g/mol, 104 mg, 683.5 μmol, 6 equiv); DMAP (C7H10N2, 122.17 g/mol, 168 mg, 1.375 mmol, 12 equiv); EDC (C8H18ClN3, 191.70 g/mol, 132 mg, 688.6 μmol, 6 equiv); prepared in CH2Cl2 (2 mL) as described above] at 0 °C using CH2Cl2 (1 mL) for rinsing. The tube was sealed with a Teflon screw cap, and the reaction mixture was stirred at 40 °C for 5 h. The mixture was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Subsequent flash chromatography (cyclohexane−ethyl acetate 50:1− 20:1) delivered the ester (±)-35D (C33H42O5, 518.69 g/mol, 57.0 mg) as a colorless oil which contained NMR-visible but inseparable impurities (ca. 10%). Rf 0.44 (cyclohexane−ethyl acetate 5:1). To a solution of (±)-35D (518.69 g/mol, 57.0 mg, assumed to be pure, 109.9 μmol, 1 equiv) in Et2O (11 mL) was added boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.33 mL, 660 μmol, 6 10517


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and the reaction mixture was stirred at room temperature for 17 h. The light brown solution was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 30 min. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and silica gel (1 g) was added to afford a slurry. Removal of the volatiles under reduced pressure afforded a powdery solid that was loaded onto a silica gel column. Subsequent flash chromatography (cyclohexane−ethyl acetate 2:1−0:1) provided (±)-42 (C37H38N2O4, 574.72 g/mol, 29.0 mg, 50.5 μmol, 65% from (±)-36) as a colorless oil: Rf 0.32 (ethyl acetate); 1H NMR (CDCl3, 500 MHz) δ 1.06 (s, 3H), 1.15 (s, 3H), 1.54 (ddd, J1 = 12.5 Hz, J2 = 9.3 Hz, J3 = 2.3 Hz, 1H), 1.57 (dd, J1 = 12.9 Hz, J2 = 4.8 Hz, 1H), 1.62−1.73 (m, 2H), 1.77 (dd, J1 = 11.2 Hz, J2 = 6.3 Hz, 1H), 1.81 (s, 3H), 1.84−2.17 (m, 6H), 2.04 (dd, J1 = 12.9 Hz, J2 = 12.1 Hz, 1H), 2.65 (dd, J1 = 11.2 Hz, J2 = 3.8 Hz, 1H), 2.70 (ddd, J1 = 14.5 Hz, J2 = 12.5 Hz, J3 = 1.8 Hz, 1H), 4.96 (br s, 1H), 5.02 (dd, J1 = 4.8 Hz, J2 = 1.8 Hz, 1H), 5.42 (dd, J1 = 12.1 Hz, J2 = 4.8 Hz, 1H), 7.41 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 7.50 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.17−8.22 (m, 3H), 8.26 (dd, J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 8.37 (dd, J1 = 8.8 Hz, J2 = 1.8 Hz, 1H), 8.46 (d, J = 1.8 Hz, 1H), 8.64 (d, J = 1.8 Hz, 1H), 8.95 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H), 9.02 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ 18.0 (CH3), 20.1 (CH2), 25.5 (CH2), 25.9 (CH3), 26.2 (CH3), 26.8 (CH2), 29.1 (CH2), 29.8 (CH2), 35.1 (CH2), 38.8 (CH), 40.7 (C), 46.1 (C), 51.4 (CH), 77.2 (CH), 81.7 (CH), 121.9 (CH), 122.1 (CH), 127.4 (C), 127.7 (C), 128.7 (C), 128.8 (C), 128.9 (CH), 129.1 (CH), 129.8 (CH), 130.0 (CH), 130.2 (CH), 131.0 (CH), 131.2 (CH), 137.50 (CH), 137.55 (CH), 139.9 (C), 150.1 (C), 150.2 (C), 152.5 (CH), 152.8 (CH), 165.0 (C), 166.0 (C); IR ν 2960, 2930, 1710, 1270, 1245, 1180, 1095, 970, 905, 785, 730 cm−1; HRMS (ESI) calcd for C37H39N2O 4 ([M + H]+) 575.2904, found 575.2915. Quinolinecarboxylate (±)-43. The following procedure was performed once and is unoptimized. A sealable glass pressure tube was charged with freshly pestled sodium hydroxide (NaOH, 40.00 g/mol, 156 mg, 3.90 mmol, 50 equiv) in dry MeOH (9.9 mL). The suspension was stirred at room temperature until a clear colorless solution resulted (about 10 min). A solution of bromobenzoate (±)-37 (C24H31BrO3, 447.41 g/mol, 35.0 mg, 78.2 μmol, 1 equiv) in dry MeOH (9.9 mL) was added at room temperature. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (60 °C). After being stirred for 19 h at 60 °C, the pale yellow solution was cooled to ambient temperature and transferred to a separatory funnel using CH2Cl2 for rinsing. The biphasic mixture was extracted once with aqueous NH4Cl solution (3.5 M). The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 10:1−2:1) delivered the alcohol (±)-37A (C17H28O2, 264.41 g/mol, 18.5 mg, 70.0 μmol, 89%) as a white solid. Rf 0.41 (cyclohexane−ethyl acetate 1:1); mp not determined. To a suspension of quinoline-6-carboxylic acid (C10H7NO2, 173.17 g/mol, 36.3 mg, 209.6 μmol, 3 equiv) in CH2Cl2 (1.5 mL) was added 4-(dimethylamino)pyridine (DMAP, C7H10N2, 122.17 g/mol, 51.3 mg, 419.9 μmol, 6 equiv) at room temperature. The reaction mixture was stirred for 5 min at room temperature until a clear beige solution appeared. N-(3(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 40.2 mg, 209.7 μmol, 3 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of alcohol (±)-37A (264.41 g/mol, 18.5 mg, 70.0 μmol, 1 equiv) in CH2Cl2 (2.5 mL) was added at 0 °C. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 19 h. The light brown solution was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 15 min. The phases were separated and the aqueous layer was extracted with CH2Cl2 (3 × ). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced

equiv) at room temperature. The white suspension was stirred at room temperature for 5 h. Additional boron trichloride dimethyl sulfide complex (2 M in CH2Cl2, 0.37 mL, 740 μmol, 6.7 equiv) was added at room temperature, and stirring was continued at room temperature for 22 h. The resultant mixture consisted of a solid white precipitate adhered to the walls of the flask and a supernatant clear solution. The reaction mixture was diluted by the addition of saturated aqueous NaHCO3 solution, H2O and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 20:1−10:1) delivered (±)-41 (C25H34O4, 398.54 g/mol, 32.0 mg, 80.3 μmol, 70% from (±)-35) as a colorless oil: Rf 0.31 (cyclohexane−ethyl acetate 5:1); 1H NMR (CDCl3, 500 MHz) δ 0.94 (s, 3H), 0.99 (s, 3H), 1.38 (dd, J1 = 12.7 Hz, J2 = 4.8 Hz, 1H), 1.44 (ddd, J1 = 12.4 Hz, J2 = 9.0 Hz, J3 = 2.4 Hz, 1H), 1.48 (ddd, J1 = 12.9 Hz, J2 = 9.4 Hz, J3 = 3.2 Hz, 1H), 1.53− 1.68 (m, 3H+OH), 1.68−1.88 (m, 3H), 1.74 (s, 3H), 1.96 (dddd, J1 = 12.9 Hz, J2 = 11.3 Hz, J3 = 7.3 Hz, J4 = 6.8 Hz, 1H), 2.08 (ddd, J1 = 12.4 Hz, J2 = 11.1 Hz, J3 = 8.8 Hz, 1H), 2.22 (dd, J1 = 12.7 Hz, J2 = 12.1 Hz, 1H), 2.28 (dd, J1 = 11.2 Hz, J2 = 3.8 Hz, 1H), 2.61 (dd, J1 = 14.5 Hz, J2 = 12.5 Hz, 1H), 3.43 (dd, J1 = J2 = 4.0 Hz, 1H), 3.85 (s, 3H), 4.80 (br s, 1H), 5.28 (dd, J1 = 12.1 Hz, J2 = 4.8 Hz, 1H), 6.88− 6.93 (m, 2H), 7.94−7.99 (m, 2H); 13C NMR (CDCl3, 126 MHz) δ 18.2 (CH3), 19.9 (CH2), 25.6 (CH2), 25.9 (CH3), 26.1 (CH3), 28.5 (CH2), 29.0 (CH2), 29.8 (CH2), 35.3 (CH2), 37.7 (CH), 41.0 (C), 45.9 (C), 51.3 (CH), 55.5 (CH3), 77.1 (CH), 78.3 (CH), 113.6 (CH), 123.4 (C), 130.2 (CH), 131.6 (CH), 139.4 (C), 163.3 (C), 166.4 (C); IR ν 3305, 2960, 2925, 1690, 1605, 1255, 1165, 1105, 1030, 965, 845, 770, 730 cm−1; HRMS (ESI) calcd for C25H35O4 ([M + H]+) 399.2530, found 399.2537. Bis-quinolinecarboxylate (±)-42. The following procedure was performed once and is unoptimized. A sealable glass pressure tube was charged with freshly pestled sodium hydroxide (NaOH, 40.00 g/mol, 156 mg, 3.90 mmol, 49.9 equiv) in dry MeOH (9.9 mL). The suspension was stirred at room temperature until a clear colorless solution resulted (about 10 min). A solution of bromobenzoate (±)-36 (C24H31BrO3, 447.41 g/mol, 35.0 mg, 78.2 μmol, 1 equiv) in dry MeOH (9.9 mL) was added at room temperature. The tube was sealed with a Teflon screw cap and placed in a preheated oil bath (60 °C). After being stirred for 18 h at 60 °C, the pale yellow solution was cooled to ambient temperature and transferred to a separatory funnel using CH2Cl2 for rinsing. The biphasic mixture was extracted once with aqueous NH4Cl solution (3.5 M). The phases were separated, and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic phases were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 10:1−1:1) afforded the diol (±)-36A (C17H28O2, 264.41 g/mol, 14.0 mg, 53.0 μmol, 68%) as a white solid. Rf 0.31 (cyclohexane−ethyl acetate 1:1); mp not determined. To a suspension of quinoline-6-carboxylic acid (C10H7NO2, 173.17 g/mol, 9.2 mg, 53.1 μmol, 1 equiv) in CH2Cl2 (1 mL) was added 4(dimethylamino)pyridine (DMAP, C7H10N2, 122.17 g/mol, 12.9 mg, 105.6 μmol, 2 equiv) at room temperature. The reaction mixture was stirred for 5 min at room temperature until a clear beige solution appeared. N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 10.2 mg, 53.2 μmol, 1 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of diol (±)-36A (264.41 g/mol, 14.0 mg, 53.0 μmol, 1 equiv) in CH2Cl2 (2 mL) was added at 0 °C. The clear light brown solution was stirred at 0 °C for 1 h and at room temperature for 6 h. After TLC analysis indicated formation of two monoesters and the bis-ester (±)-42, the reaction mixture was transferred to a second flask containing an excess of reagents [quinoline-6-carboxylic acid (C10H7NO2, 173.17 g/mol, 45.8 mg, 264.5 μmol, 5 equiv); DMAP (C7H10N2, 122.17 g/mol, 64.7 mg, 529.6 μmol, 10 equiv); EDC (C8H18ClN3, 191.70 g/mol, 50.7 mg, 264.5 μmol, 5 equiv); prepared in CH2Cl2 (1 mL) as described above] at 0 °C using CH2Cl2 (1 mL) for rinsing. The cooling bath was removed, 10518


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The Journal of Organic Chemistry pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 10:1−2:1) provided (±)-43 (C27H33NO3, 419.57 g/mol, 27.0 mg, 64.4 μmol, 82% from (±)-37) as a colorless oil: Rf 0.50 (cyclohexane−ethyl acetate 1:1); 1H NMR (CDCl3, 500 MHz) δ 0.91 (s, 3H), 0.98 (dd, J1 = J2 = 12.8 Hz, 1H), 1.04 (s, 3H), 1.38 (s, 3H), 1.47 (ddd, J1 = J2 = 12.5 Hz, J3 = 4.8 Hz, 1H), 1.48−1.91 (m, 12H), 1.96 (ddd, J1 = 12.2 Hz, J2 = 9.0 Hz, J3 = 2.9 Hz, 1H), 2.23 (ddd, J1 = 12.8 Hz, J2 = 10.5 Hz, J3 = 8.4 Hz, 1H), 3.85 (d, J = 6.8 Hz, 1H), 5.40 (dd, J1 = 11.2 Hz, J2 = 6.0 Hz, 1H), 7.47 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 8.28 (dd, J1 = 8.8 Hz, J2 = 1.8 Hz, 1H), 8.28 (dd, J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 8.54 (d, J = 1.8 Hz, 1H), 9.00 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ 15.3 (CH3), 19.8 (CH2), 26.45 (CH3), 26.50 (CH2), 26.7 (CH2), 28.4 (CH3), 30.5 (CH2), 35.5 (CH), 37.9 (C), 39.6 (CH2), 39.8 (CH2), 41.6 (CH2), 47.1 (CH), 47.1 (C), 78.8 (C), 79.2 (CH), 83.5 (CH), 121.9 (CH), 127.5 (C), 129.0 (C), 129.3 (CH), 129.7 (CH), 130.9 (CH), 137.8 (CH), 149.9 (C), 152.4 (CH), 166.0 (C); IR ν 2960, 2925, 1710, 1275, 1245, 1190, 1090, 975, 910, 785, 730 cm−1; HRMS (ESI) calcd for C27H34NO3 ([M + H]+) 420.2533, found 420.2537. (±)-Cyclohexyl Quinoline-6-carboxylate ((±)-45). The following procedure was performed once and is unoptimized. To a suspension of quinoline-6-carboxylic acid (C10H7NO2, 173.17 g/mol, 167 mg, 0.964 mmol, 2 equiv) in CH2Cl2 (23 mL) was added 4-(dimethylamino)pyridine (DMAP, C7H10N2, 122.17 g/mol, 235 mg, 1.924 mmol, 4 equiv) in one portion at room temperature. The reaction mixture was stirred for 5 min at room temperature until a clear beige solution appeared. N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC, C8H18ClN3, 191.70 g/mol, 185 mg, 0.965 mmol, 2 equiv) was added at 0 °C, and the resultant suspension was stirred for 10 min at 0 °C. A solution of (±)-cyclohexanol ((±)-44) (C6H12O, 100.16 g/mol, 0.951 g/mL, 0.051 mL, 48.5 mg, 0.484 mmol, 1 equiv) in CH2Cl2 (3 mL) was added at 0 °C. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 17 h. The light brown solution was diluted by the addition of saturated aqueous NH4Cl solution, H2O, and CH2Cl2 at room temperature. Vigorous stirring of the biphasic mixture was continued at room temperature for 1 h. The phases were separated and the aqueous layer was extracted with CH2Cl2 (3×). The combined organic layers were dried (MgSO4), and the volatiles were removed under reduced pressure. Purification by flash chromatography (cyclohexane−ethyl acetate 20:1−5:1) provided (±)-45 (C16H17NO2, 255.32 g/mol, 119 mg, 0.466 mmol, 96%) as a white solid: Rf 0.35 (cyclohexane−ethyl acetate 2:1); mp 96−98 °C; 1H NMR (CDCl3, 500 MHz) δ 1.31− 1.41 (m, 1H), 1.46 (ddddd, J1 = 13.4 Hz, J2 = J3 = 10.0 Hz, J4 = J5 = 3.5 Hz, 2H), 1.55−1.68 (m, 3H), 1.77−1.86 (m, 2H), 1.95−2.03 (m, 2H), 5.08 (dddd, J1 = J2 = 9.0 Hz, J3 = J4 = 3.8 Hz, 1H), 7.45 (dd, J1 = 8.3 Hz, J2 = 4.2 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.26 (dd, J1 = 8.3 Hz, J2 = 1.8 Hz, 1H), 8.30 (dd, J1 = 8.8 Hz, J2 = 1.8 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.99 (dd, J1 = 4.2 Hz, J2 = 1.8 Hz, 1H); 13C NMR (CDCl3, 126 MHz) δ 23.8 (CH2), 25.6 (CH2), 31.8 (CH2), 73.7 (CH), 121.9 (CH), 127.5 (C), 129.1 (C), 129.2 (CH), 129.8 (CH), 130.9 (CH), 137.4 (CH), 150.1 (C), 152.5 (CH), 165.6 (C); IR ν 2935, 2860, 1710, 1275, 1240, 1180, 1095, 1015, 785 cm−1; HRMS (ESI) calcd for C16H18NO2 ([M + H]+) 256.1332, found 256.1332. Biological Investigation. Chemicals. The reference compound Ko143 ((3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2methylpropyl)-1,4-dioxopyrazino[1′,2′:1,6]pyrido[3,4-b]indole-3propanoic acid 1,1-dimethylethyl ester) was purchased from Tocris Bioscience (Bristol, UK). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). Stock solutions of the compounds (10 mM) in DMSO were used to prepare the test samples for all cell-based assays. Aqueous Krebs-HEPES buffer (KHB) was prepared from 118.6 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 1.2 mM MgSO4, 11.7 mM Dglucose monohydrate, and 10.0 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The buffer solution was adjusted to pH 7.4 at 37 °C using sodium hydroxide solution and then sterilized by filtration through a membrane filter with 0.2 μm pore size (Whatman, Maidstone, UK).

Cell Culture. MDCK II BCRP cells were generated by transfection of the canine kidney epithelial cell line MDCK II with the human wildtype cDNA C-terminally linked to the cDNA of the green fluorescent protein (GFP). Cells were cultured in DMEM (Dulbecco’s modified Eagle medium) with 10% FCS (fetal calf serum), 50 μg/mL streptomycin, 50 U/mL penicillin G, and 2 mM L-glutamine. Human ovarian carcinoma cell line A2780ADR, which is doxorubicin-resistant and overexpresses ABCB1, was purchased from the European collection of animal cell culture (ECACC, No 93112520). The cell line was grown in RPMI-1640 medium supplemented with 10% FCS, 50 μg/mL streptomycin, 50 U/mL penicillin G, and 2 mM L-glutamine. The small cell lung cancer cell line H69AR, which is doxorubicin-resistant and overexpresses ABCC1, was purchased from American Type Culture Collection (ATCC, No CRL-11351). This cell line was grown in RPMI-1640 medium supplemented with 20% FCS, 50 μg/mL streptomycin, 50 U/mL penicillin G, and 2 mM Lglutamine. All cell lines were incubated under a 5% CO2-humidified atmosphere at 37 °C. After reaching confluence of at least 90%, cells were harvested for subculturing and using in cell-based assays. The cells were treated gently with a 0.05% trypsin/0.02% EDTA solution to detach them from the inner surface of the culture flask and transferred to a 50 mL tube followed by a centrifugation (266 g, 4 °C, 4 min). After aspiration of the supernatant, the cell pellet was suspended in fresh culture medium, and the cell density was determined using a CASY1 model TT cell counter with 150 μm capillary (Schärfe System, Reutlingen, Germany). The volume of suspension containing the required quantity of cells for an assay (see below) was transferred to a vial, further centrifuged, and the cells were washed three times with KHB. Hoechst 33342 Accumulation Assay. To investigate the inhibitory effect on ABCG2, the Hoechst 33342 accumulation assay was performed as described earlier.73 A methanolic dilution of compound stock solution (1 mM) was used to prepare different dilutions in KHB. The highest concentration was 31.62 μM used in this assay. After preparation of different concentrations of test compounds, 20 μL of each concentration was placed into black 96-well plates (Greiner, Frickenhausen, Germany). Black plates were chosen as they yielded much smaller background fluorescence than colorless plates when irradiated in the UV. The prepared cells were added into each well of the plate at a density of approximately 30,000 cells per well to a total volume of 180 μL. The 96-well plate was stored under 5% CO2 at 37 °C for 30 min. After this preincubation period, 20 μL of a 10 μM Hoechst 33342 solution (protected from light) was added to each well. Fluorescence was measured immediately in constant intervals (60 s) for a period of 120 min with an excitation of 355 nm and an emission wavelength of 460 nm at 37 °C using microplate readers (POLARstar and FLUOstar optima by BMG Labtech, Offenburg, Germany). For analysis of the data obtained from the assay, fluorescence of KHB was subtracted first from the total fluorescence detected for MDCK II cells. Average of fluorescence values in the steady state (from 100 up to 109 min) was calculated for each concentration. These values were plotted against logarithmic concentrations of tested compounds. Concentration−response curves were generated by nonlinear regression analysis using the four-parameter logistic equation with variable or fixed (= 1) Hill slope. The statistically preferred model was chosen for calculating IC50 values (GraphPad Prism, version 7.0, San Diego, CA). CalceinAM Assay. Compounds were further tested for their ABCB1 and ABCC1 inhibition using the calceinAM assay as described earlier.73 The dilution series of compounds were prepared as mentioned above. Twenty μL of the prepared solutions containing a test compound (see above) were pipetted into colorless 96-well plates (Greiner, Frickenhausen, Germany). Then, A2780ADR cells for ABCB1 and H69AR cells for ABCC1, respectively, were seeded into a plate at a density of approximately 30000 cells in a volume of 160 μL per well. After a preincubation time of 30 min, 20 μL of a 3.125 μM calceinAM solution (protected from light) was added to each well of the plate. The fluorescence was measured immediately in constant time intervals (60 s) for a period of 60 min at 37 °C with an excitation of 485 nm and an emission of 520 nm using microplate readers (see above). The first linear part of the fluorescence-time curves was used 10519


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(6) Tsui, G. C.; Villeneuve, K.; Carlson, E.; Tam, W. Organometallics 2014, 33, 3847−3856. (7) Yerino, L. V.; Osborn, M. E.; Mariano, P. S. Tetrahedron 1982, 38, 1579−1591. (8) Dötz, K. H.; Kuhn, W. J. Organomet. Chem. 1985, 286, C23. (9) Wulff, W. D.; Yang, D. C. J. Am. Chem. Soc. 1984, 106, 7565− 7567. (10) Cativiela, C.; García, J. I.; Mayoral, J. A.; Salvatella, L. Chem. Soc. Rev. 1996, 25, 209−218. (11) (a) Kim, W. H.; Lee, J. H.; Danishefsky, S. J. J. Am. Chem. Soc. 2009, 131, 12576−12578. (b) Cativiela, C.; García, J. I.; Gil, J.; Martínez, R. M.; Mayoral, J. A.; Salvatella, L.; Urieta, J. S.; Mainar, A. M.; Abraham, M. H. J. Chem. Soc., Perkin Trans. 2 1997, 2, 653−660. (c) Breslow, R.; Guo, T. J. Am. Chem. Soc. 1988, 110, 5613−5617. (12) Fisher, M. W.; Hehre, W. J.; Kahn, S. D.; Overman, L. E. J. Am. Chem. Soc. 1988, 110, 4625−4633. (13) (a) Takano, S.; Yoshimitsu, T.; Ogasawara, K. J. Org. Chem. 1994, 59, 54−57. (b) Takano, S.; Yoshimitsu, T.; Ogasawara, K. J. Org. Chem. 1995, 60, 1478. (14) (a) Corey, E. J.; Danheiser, R. L.; Chandrasekaran, S.; Siret, P.; Keck, G. E.; Gras, J.-L. J. Am. Chem. Soc. 1978, 100, 8031−8034. (b) Birman, V. B.; Danishefsky, S. J. J. Am. Chem. Soc. 2002, 124, 2080−2081. (c) Sun, C.; Lee, H.; Lee, D. Org. Lett. 2015, 17, 5348− 5351. (15) (a) Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron Lett. 1966, 7, 3353−3354. (b) Furukawa, J.; Kawabata, N.; Nishimura, J. Tetrahedron 1968, 24, 53−58. (16) (a) Arnó, M.; González, M. A.; Zaragozá, R. J. Tetrahedron 1999, 55, 12419−12428. (b) Abad, A.; Agulló, C.; Cuñat, A. C.; Navarro, I. Tetrahedron Lett. 2001, 42, 8965−8968. (c) Abad, A.; Agulló, C.; Cuñat, A. C.; De Alfonso Marzal, I.; Gris, A.; Navarro, I.; Ramírez de Arellano, C. Tetrahedron 2007, 63, 1664−1679. (17) However, it turned out that the double-bond isomerization of dimethyl acetylenedicarboxylate-derived (4 + 2)-cycloadducts is well precedented: (a) Kazan, J.; Greene, F. D. J. Org. Chem. 1963, 28, 2965−2970. (b) Howell, S. C.; Ley, S. V.; Mahon, M. J. Chem. Soc., Chem. Commun. 1981, 507−508. (c) Mori, K.; Watanabe, H. Tetrahedron 1986, 42, 273−281. (d) Caballero, E.; Longieras, N.; Zausa, E.; del Rey, B.; Medarde, M.; Tomé, F. Tetrahedron Lett. 2001, 42, 7233−7236. (18) Gilman, H.; Jones, R. G.; Woods, L. A. J. Org. Chem. 1952, 17, 1630−1634. (19) (a) Corey, E. J.; Boaz, N. W. Tetrahedron Lett. 1985, 26, 6015− 6018. (b) Corey, E. J.; Boaz, N. W. Tetrahedron Lett. 1985, 26, 6019− 6022. (c) Bertz, S. H.; Miao, G.; Rossiter, B. E.; Snyder, J. P. J. Am. Chem. Soc. 1995, 117, 11023−11024. (d) Bertz, S. H.; Chopra, A.; Eriksson, M.; Ogle, C. A.; Seagle, P. Chem. - Eur. J. 1999, 5, 2680− 2691. (20) (a) Corey, E. J.; Hannon, F. J.; Boaz, N. W. Tetrahedron 1989, 45, 545−555. (b) Bertz, S. H.; Smith, R. A. J. Tetrahedron 1990, 46, 4091−4100. (21) (a) Maruyama, K.; Yamamoto, Y. J. Am. Chem. Soc. 1977, 99, 8068−8070. (b) Yamamoto, Y.; Maruyama, K. J. Am. Chem. Soc. 1978, 100, 3240−3241. (c) Nakamura, E.; Yamanaka, M.; Mori, S. J. Am. Chem. Soc. 2000, 122, 1826−1827. (22) (a) Westermann, J.; Nickisch, K. Angew. Chem. 1993, 105, 1429−1431. (b) Westermann, J.; Nickisch, K. Angew. Chem., Int. Ed. Engl. 1993, 32, 1368−1370. (c) Kabbara, J.; Flemming, S.; Nickisch, K.; Neh, H.; Westermann, J. Chem. Ber. 1994, 127, 1489−1493. (23) (a) Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226−2227. (b) Gemal, A. L.; Luche, J. L. J. Am. Chem. Soc. 1981, 103, 5454−5459. (24) (a) Altman, J.; Becker, D.; Ginsburg, D.; Leewenthal, H. J. E. Tetrahedron Lett. 1967, 8, 757−758. (b) Chakrabortty, P. N.; Dasgupta, R.; Dasgupta, S. K.; Ghosh, S. R.; Ghatak, U. R. Tetrahedron 1972, 28, 4653−4665. (c) Ghatak, U. R.; Chakraborti, P. C. J. Org. Chem. 1979, 44, 4562−4566. (d) Attah-Poku, S. K.; Alward, S. J.; Fallis, A. G. Tetrahedron Lett. 1983, 24, 681−684. (e) Attah-Poku, S. K.; Antczak, K.; Alward, S. J.; Fallis, A. G. Can. J. Chem. 1984, 62, 1717−1721.

for calculating slopes. These slopes were plotted against logarithmic concentrations of tested compounds. Data analysis was performed as described above. MTT Assay for Determining Cytotoxicity. Intrinsic cytotoxicity of selected compounds was determined using the MTT cytotoxicity assay as described previously with minor modifications.73 Cells were seeded into 96-well tissue-culture treated plates (Starlab GmbH, Hamburg, Germany) in a volume of 180 μL per well (MDCK II BCRP and wildtype: 3000 per well; A2780ADR and wild-type: 8000 per well) and kept under 5% CO2 atmosphere at 37 °C for 3−6 h. Attachment of cells was controlled under a microscope. Different concentrations of test compounds were made in culture medium. The highest concentration of DMSO in dilutions used for the assays was not more than 1.0%. Then, 20 μL of test compound was added to achieve the required final concentration in a volume of 200 μL. Additionally, wells were prepared containing only medium (negative control) and 10% (v/v) of DMSO (positive control). After an incubation period of 72 h, a solution of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) in phosphate buffered saline (5 mg/mL) was added to each well (20 μL). Plates were further incubated for 1 h, after which MTT is reduced to a water-insoluble formazan. The supernatants were removed, and the cells were lysed with 100 μL DMSO per well. The color intensity of the formed formazan was determined spectrophotometrically by measuring absorbance at 570 nm and background correction at 690 nm using a Multiscan Ex microplate photometer (Thermo Fisher Scientific, Waltham, MA). The cell viability was calculated as percentage of the highest mean in the data set. GI50 values were calculated by nonlinear regression analysis, assuming a sigmoidal concentration−response curve with variable Hill slope (GraphPad Prism).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.joc.7b02012. NMR, MS, and IR spectra for all new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Michael Wiese: 0000-0002-5851-5336 Martin Hiersemann: 0000-0003-4743-5733 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the TU Dortmund and the Fonds der Chemischen Industrie (Ph.D. fellowship to A.S.) is gratefully acknowledged. MDCK II BCRP and wild-type cell lines were a generous gift from Alfred Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands).

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REFERENCES

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