Euodenine A: A Small-Molecule Agonist of Human TLR4Juliette E. Neve,† Hasanthi P. Wijesekera,† Sandra Duffy,† Ian D. Jenkins,† Justin A. Ripper,†

Simon J. Teague,‡ Marc Campitelli,† Agatha Garavelas,† George Nikolakopoulos,† Phuc V. Le,†

Priscila de A. Leone,† Ngoc B. Pham,† Philip Shelton,‡ Neil Fraser,‡ Anthony R. Carroll,† Vicky M. Avery,†

Christopher McCrae,§ Nicola Williams,‡ and Ronald J. Quinn*,†

†Eskitis Institute for Drug Discovery, Griffith University, Nathan, 4111 Queensland, Australia‡AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH Leicestershire, United Kingdom§AstraZeneca R&D Molndal, Translational Science, Respiratory & Inflammation iMed, Pepperedsleden 1, SE-431 83 Molndal, Sweden

*S Supporting Information

ABSTRACT: A small-molecule natural product, euodenine A (1), was identified as an agonist of the human TLR4 receptor.Euodenine A was isolated from the leaves of Euodia asteridula (Rutaceae) found in Papua New Guinea and has an unusualU-shaped structure. It was synthesized along with a series of analogues that exhibit potent and selective agonism of the TLR4receptor. SAR development around the cyclobutane ring resulted in a 10-fold increase in potency. The natural productdemonstrated an extracellular site of action, which requires the extracellular domain of TLR4 to stimulate a NF-κB reporterresponse. 1 is a human-selective agonist that is CD14-independent, and it requires both TLR4 and MD-2 for full efficacy. Testingfor immunomodulation in PBMC cells shows the induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40 as well assuppression of IL-5 from activated PBMCs, indicating that compounds like 1 could modulate the Th2 immune response withoutcausing lung damage.

■ INTRODUCTION

Steroids and antibodies dominate the current anti-inflammatorytreatment for allergy and asthma. Since the discovery of theToll-like receptors (TLRs) in 19881 and the identification oftheir role in adaptive immunity,2 the targeting of TLRs hasopened up new therapeutic possibilities for the prevention andtreatment of inflammatory diseases. TLRs are attractive targetsfor the prevention and treatment of cancer, infection, allergy,asthma, autoimmune disease, and chronic neuropathic pain.3−10

Activation of the innate immune system through mammalianTLRs has an instructive role for the responses of the acquiredimmune response and thus may influence allergic diseases, suchas asthma. The allergic response usually features a strong Th2response, which can be counterbalanced by TLR signalinginduction; this would induce a cytokine response, activatingantigen-presenting cells and a Th1 response.11 Agonists of Toll-like receptor 4 (TLR4) could be therapeutically useful fortreating the acute phase of asthma.4 There is evidence that theacquired immune response in asthma is polarized to a responsedominated by Th2 lymphocytes, which are involved in theorchestration of the allergic inflammatory response by secretinga variety of type-2 cytokines (interleukin (IL)-4, IL-5, and IL-13).

These cytokines enhance the migration and survival ofeosinophils and B cells and contribute to mucous cellhyperplasia and mast cell activation. Modulation of the type-2acquired immune response is an attractive approach incontrolling allergic disease because it offers the potential fordisease normalization and contrasts with existing steroidal andantibody treatments on the market and in late-stage develop-ment, which offer only symptomatic relief. There are very fewsmall-molecule inhibitors of the TLR4 receptor known,examples include TAK-242 and (+)-naloxone (Figure 1). TAK-242is a small-molecule inhibitor of protein−protein interactions of theToll/interleukin-1 receptor (TIR) domain of TLR4;12 however,this compound acts as a Michael acceptor, binding covalently toCys747 in the intracellular domain of TLR4.13 (+)-Naloxone is anopioid-inactive TLR4 signaling inhibitor that can reverse neuro-pathic pain in rats.7 Very recently, dimethyl 2-(2-nitrobenzylidene)-malonate has been found to inhibit LPS-mediated nuclear factor-kappaB (NF-κB) activation, the cytokine production of IL-1β,tumor necrosis factor (TNF-α), and nitric oxide (NO) in the

Received: August 27, 2013Published: January 28, 2014

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nanomolar to low micromolar range.14 However, as withTAK-242, dimethyl 2-(2-nitrobenzylidene)malonate is aMichael acceptor and almost certainly binds covalently toCys747 (the corresponding compound without the α,β-doublebond is inactive with an IC50 > 100 μM).14 TAK-242,(+)-naloxone, and dimethyl 2-(2-nitrobenzylidene)malonateare all TLR4 antagonists. There is clearly an urgent need forsmall-molecule, druglike agonists of the TLR4 receptor.

■ IDENTIFICATION OF A NOVEL CLASS OF TLR-4AGONISTS

Screening a small library of 750 pure natural products against apanel of TLRs led to the identification of a potent and selectivesmall-molecule agonist of the TLR4 receptor. Euodenine A (1)was originally isolated from the air-dried leaves of the treeEuodia asteridula (Rutaceae) collected in Papua New Guinea.The EL-Nino fires of 1997/8 destroyed the original tree fromwhich 1 was isolated, and subsequent recollection of the plantfrom the same and different areas failed to provide furtherquantities of 1, resulting in the need for total synthesis. Themolecule (C26H29NO5, relative molecular mass 435.51) consistsof two aromatic regions held together in a U-shape, as dictatedby the relative stereochemistry about the central cyclobutanering (Figure 2). The relative stereochemistry was determinedthrough ROESY correlations observed between the methyl at8′ and H-7′ and among H-7′, H-4, and H-3, placing the methyland trimethoxyphenyl groups trans and the pyranoquinolineand trimethoxyphenyl groups cis on the cyclobutane ring. Thecis relationship of the two aromatic groups was furthersupported by the significant upfield shift (by ∼0.5 ppm)

observed for trimethoxyaryl protons H-2′ and H-6′ because ofshielding by the adjacent pyranoquinolinone.Compound 1 is closely related to melicodenines C−F

(Figure 3) that have recently been isolated from the leaves ofMelicope denhamii, a rutaceous shrub indigenous to regionsfrom Borneo to the Solomon Islands.15

Compound 1 appears to have full activity on the desiredreadout in peripheral blood mononuclear cells (PBMCs) (IL-5suppression) while limiting the undesired proinflammatoryeffects of full/LPS-driven TLR4 agonism. Compound 1 wasalso selective for TLR4, exhibiting no activity against TLR2/6and TLRs 3, 5, 7, 8, and 9 at 10 μM (higher concentrationswould be required to determine if the level of selectivity issignificant, see the Supporting Information for details of TLR4selectivity). In addition, 1 showed minimal cytotoxicity againstPBMCs at concentrations greater than 30 μM (Table 1). It is

worth noting that closely related melicodenines C−F showedlimited cytotoxicity against DLD-1 human colon cancer cells.For example, melicodenine D, the compound that most closelyresembles 1, resulted in a 30% reduction in cell viability at50 μM but no reduction at 20 μM.15

In HEK cell line-secreted alkaline phosphatase (SEAP)reporter assays for human, mouse, and rat TLR4, 1 was foundto be human-selective in comparison to LPS, which shows

Figure 1. Small-molecule inhibitors of TLR4: Tak-242 (a),(+)-naloxone (b), and dimethyl 2-(2-nitrobenzylidene)malonate (c).

Figure 2. Structure and 3D shape of euodenine A (1) (relative stereochemistry and MacroModel/MMFF force field with H2O solvation of thelowest-energy conformer are shown).

Figure 3. Structures related to euodenine A, melicodenines C−F (relative stereochemistry shown).

Table 1. Activity Profiles and Toxicity of 1 and LPS

TLR receptor activity (EC50)

cell line 1 LPS

TLR4 4 μM 3 ng/mLTLR2/6 NA at 10 μM NA at 1.5 μg/mLTLR3 NA at 10 μM NA at 1.5 μg/mLTLR5 NA at 10 μM NA at 1.5 μg/mLTLR7 NA at 10 μM NA at 1.5 μg/mLTLR8 NA at 10 μM NA at 1.5 μg/mL

Toxicity (CC50)a

PBMC >33 μM >1000 ng/mLaToxicity data from IL-5-suppression experiments using PHA-stimulated PBMCs at a concentration of 20 μM showed no toxiceffect for 1. In PBMCs from 4 donors, 1 was tested up to 100 μM witha small effect at 33 μM and clear evidence of toxicity at 100 μM.

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species crossover to both rat and mouse. Subsequent reporterassays using chimeric human/mouse TLR4 receptors were usedto determine the receptor domains required for agonistfunction, with LPS used as a control. In the four possiblecombinations of chimeric human/mouse, intra/extracellularTLR reporter assays, 1 only stimulated the NF-κB reporterwhen the extracellular domain of human TLR4 was expressedas a fusion to either the mouse or human intracellular domain(Figure 4). When the murine extracellular domain was fused toeither the mouse or human intracellular domain, 1 was inactive.From these studies, and the lack of activity of 1 in the SEAPmouse assays, it can be concluded that 1 has an extracellular siteof action, as there is a requirement for the extracellular domainof human TLR4 to stimulate a NF-κB reporter response.TLR4 is a multicomponent receptor composed of TLR4,

myeloid differentiation factor 2 (MD-2), and cluster ofdifferentiation 14 (CD14), each of which has their owncostimulatory function (Figure 5).

Initial studies with 1 and LPS revealed that these agonistsrequire both MD-2 and TLR4 to function and do not workwith TLR4 alone, confirming the essential role of MD-2 inTLR4 signaling (Figure 6).Various rat, mouse, and human MD-2 and TLR4 combination

reporter assays were used to determine the dependency of 1 onMD-2. LPS was active at both human and mouse TLR4irrespective of the species of MD-2; however, reduced potencywas observed when mouse MD-2 was combined with humanTLR4 (Figure 7a). Compound 1 showed activity only whenhuman TLR4 was expressed with human MD-2 (Figure 7b).Mouse MD-2 could not substitute. Therefore, 1 is human-selectivefor both human proteins.Because 1 and LPS stimulate TLR4 by different mechanisms,

cytokine profiling was undertaken. Compound 1 was comparedto LPS, and as expected, LPS showed significant induction ofIL-8, IL-12p40, IL-10, and TNF-α, with little effect on IL-2,IL-4, and IL-5. In comparison to LPS, 1 showed reduced

Figure 4. Extracellular site of action of euodenine A (1). A HEK293/NFκB reporter cell line stably transfected with human MD-2 and CD14 wastransiently transfected with a chimeric construct consisting of either the human or mouse TLR4 extracellular domain fused to either the human ormouse transmembrane and intracellular domains. Cells were subsequently treated with LPS or 1, and reporter gene activity was measured 24 h later.Data are representative of at least two independent experiments and are expressed as the mean of triplicate determinations. (A) LPS is active in allhuman/mouse chimeric assays. (B) 1 is a human-selective agonist and only shows a response when the extracellular domain of human TLR4 ispresent.

Figure 5. LPS activation of TLR4 via the CD14, TLR4, and MD-2 complex proceeds via MyD88 and the IRAK/TRAF6 pathway, leading toactivation of proinflammatory cytokines.

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induction of IL-12p40, IL-10, and TNF-α as well as inductionof IL-8 from PBMCs and the suppression of IL-5 fromactivated PBMCs. In 8/8 donors tested, IL-8 induction wasequal to that observed for LPS, whereas IL-10, TNF-α, andIL-12p40 induction were less than that observed for LPS. In9/9 activated PBMCs tested, IL-5 was suppressed (Figure 8).These results suggest that 1 would have the potential to preventIL-5-mediated allergic inflammation while having a lower risk ofinducing IL-12- and TNF-α-mediated proinflammatory re-sponses compared to LPS.

■ SYNTHESIS OF 1 AND ANALOGUESCompared to other known TLR4 agonists such as LPS, 1 is muchsimpler in structure. Compound 1 was at first synthesized via aphotochemically induced [2 + 2] cycloaddition reaction from O-methyl flindersine (3) and (E)-1,2,3-trimethoxy-5-(prop-1-enyl)-benzene followed by cleavage of the methyl ether with potassiumiodide in acetic acid to afford the natural product (Scheme 1).Initially, the photochemistry was carried out in dichloro-

methane, and the product was isolated by laborious preparativethin-layer chromatography.16 Improved yields resulted whenthe photoaddition was performed without solvent. Evaporationof a chloroform solution of O-methyl flindersine17,18 and(E)-1,2,3-trimethoxy-5-(prop-1-enyl)benzene in a jacketed round-bottomed flask afforded a thin film of reagents on the walls ofthe flask. Irradiation of this thin film using a medium-pressureUV lamp (125 or 400 W) with water cooling for 16 h led to the

desired intermolecular cycloaddition product in modest yield(36%) with excellent chemo- and regioselectivity, possibly as aresult of a π-stacking interaction between the aryl group of thestyrene and the quinolinone ring. Demethylation withpotassium iodide in acetic acid afforded the desired naturalproduct in 85% yield. Subsequent photoaddition of flindersine(2a) and (E)-1,2,3-trimethoxy-5-(prop-1-enyl)benzene af-forded the natural product in one step. This method wasused for the generation of 53 cyclobutane-based analogues of 1.Closely related melicodenines C, D, and E (Figure 3) have alsobeen synthesized in our laboratory by this route.19

Most of the analogues were synthesized directly from thecorresponding flindersine analogue and functionalized styrene(Scheme 2). This general synthetic approach enabled variationof the pyranoquinoline, the alkyl carbon group R at C8′, andthe pendant aromatic attached at C7′. Because only a few of therequired styrenes were commercially available, most weresynthesized using one of four methods (Scheme 3) dependingon the availability of starting materials. The preferred methodof synthesis of the styrenes was from the aldehyde by reactionwith a Grignard reagent to afford the corresponding carbinol(method A). Subsequent dehydration using excess self-indicating silica gel in dioxane under microwave conditions20

predominately gave the trans styrene, which was purified byflash chromatography prior to the photochemical cycloaddition.Cyclopentyl and cyclohexyl styrenes were accessed via therequired benzylic alcohol, which had been treated with

Figure 6. LPS and 1 require MD-2 and TLR4 for agonism. A HEK293/NFκB reporter cell line stably transfected with human TLR4 was transientlytransfected with constructs containing either human MD-2, human CD14, a combination of human MD-2 and CD14, or empty vector. Cells weresubsequently treated with (A) LPS or 1, and reporter gene activity was measured 24 h later. TNF-α was used as a control to stimulate maximalreporter gene activity, and results were calculated as the percent of the maximal TNF-α response. Data are representative of at least two independentexperiments and are expressed as the mean of triplicate determinations. Signaling only occurs when both TLR4 and MD-2 are present.

Figure 7. 1 is human-selective. A HEK293 NFκB reporter cell line was transiently transfected with a construct containing either human or mouseTLR4 together with a construct containing human or mouse MD-2 and CD14. Cells were subsequently treated with LPS or 1, and reporter geneactivity was measured 24 h later. Data are representative of at least two independent experiments and are expressed as the mean of triplicatedeterminations. (A) LPS is nonselective against human and mouse TLR4. (B) 1 is human selective for both TLR4 and MD-2, and mouse MD-2cannot substitute.

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phosphorus tribromide followed by triethyl phosphite to affordthe phosphonate ester (Michaelis−Arbuzov reaction). Reactionwith cyclopentyl or cyclohexylcarbaldehyde gave the corre-sponding styrene (Horner−Wadsworth−Emmons procedure,method B). Nitrostyrenes produced via the Henry reaction

provided substituted styrenes upon treatment with thecorresponding alkyl iodide in the presence of triethylboronand oxygen (method C),21 and the Suzuki coupling ofcyclopropylvinylboronic acid pinacol ester with an aryl bromideafforded the cyclopropyl substituted styrenes (method D).

Figure 8. Cytokine induction time-course data: IL-8 (A), IL-12p40 (B), IL-10 (C), and TNF-α (D). (E) IL-5 suppression from activated PBMCs by1 and (F) IL-5 suppression from activated PBMCs by LPS. In PBMC, 1 suppresses PHA-induced IL-5 release and, compared to LPS, stimulateslower levels of three out of four cytokines tested. (A−F) Human PBMCs were treated with LPS or 1 for 20 h. Levels of IL-8 (A), IL-12p40 (B),IL-10 (C), and TNF-α (D) were measured in the cell supernatants by ELISA. (E, F) Human PBMC were stimulated for 44 h with 1 μg/mL ofphytohemeagglutinin (PHA) in the presence or absence of LPS or 1. Levels of IL-5 were measured in the cell supernatants by ELISA. Data are froma representative experiment in PBMC from 1 of 8 donors (cytokine stimulation) or 9 donors (IL-5 suppression) and are expressed as the mean ±standard deviation of triplicate determinations.

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Further functionalized styrenes were prepared to incorporatea solubilizing group onto the pendant aryl ring by the followingmethods (Scheme 4).

Modifications to the pyranoquinoline were investigated byutilizing flindersine and further functionalized analogues. Flindersine(2a), N-methylfindersine (2b), and 9-methylflindersine (2c) were

Scheme 1. Synthesis of 1a

aRelative stereochemistry shown. Reagents and conditions: (i) UV light (hv), 125 W, neat 16 h; (ii) KI, HOAc, 50 °C, 4 h.

Scheme 2. General Synthetic Approacha

aRelative stereochemistry shown. Reagents and conditions: (i) UV light (hv), 125 or 400 W, neat 16 h. Yields: 2−36% (generally, 5−10%).

Scheme 3. Methods for Synthesis of Styrenesa

aReagents and conditions: (i) RCH2MgX, MePh; (ii) 1,4-dioxan, silica, microwave, 160 °C, 30 min; (iii) PBr3, DCM; (iv) P(OEt)3, (Bu)4NI, 110 °C16 h; (v) NaH, RCHO, THF, 0 °C to rt, O/N; (vi) CH3NO2, NH4OAc, 48 h, reflux; (vii) RI, B(Et)3, rt, 24 h; (viii) Pd(OAc)2, 2 M K2CO3, DME,80 °C, 16 h. Overall yields for methods A−C were 40−60% and for D (Suzuki coupling), ∼80%.

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prepared by the method of Lee et al.22 utilizing a tandemKnoevenagel-electrocyclic reaction (Scheme 5).9-Methoxyflindersine (2d) was purchased from Chembridge

and afforded 57−58. 9-Methylflindersine (2c) afforded 47−49.Pruning of the pyranoquinoline core was also investigated.

Analogues 55 and 56, possessing a single methyl group at C-10,were prepared as a mixture from the corresponding flindersineanalogues (2e,f). These were obtained using the sameprocedure as that employed for flindersine but by replacing3-methyl-2-butenal with crotonaldeyde. Further modificationswere made to the pyranoquinolinone by pruning the phenylring to afford a pyranopyridinone (6) scaffold, which was usedto synthesize a few selected pyridinone analogues (59−61,Scheme 6). The pyranopyridinone scaffold (6) was synthesizedfrom 2,4-dihydroxypyridine. Unfortunately, these compounds(59−61) had no activity when tested in the TLR4 assay.Relative to the styrene starting material, all of the analogues

(9−61) of euodenine A exhibited a characteristic upfield shiftof ortho aryl protons H-2′ and/or H-6′ by ∼0.5 ppm (range0.2−0.75 ppm) in the 1H NMR spectrum. Meta aryl protonsH-3′ and/or H-5′ exhibited a smaller upfield shift of ∼0.3 ppm(range 0.1 − 0.6 ppm), but para aryl protons H-4′ displayed anegligible or very small (<0.2 ppm) upfield shift. This upfieldshift of the ortho and meta protons is due to shielding by theadjacent pyranoquinolinone (or pyranopyridinone) and servedas a diagnostic to confirm the cis relationship of the twoaromatic groups. The cis relationship between the two aromaticrings was confirmed by ROESY for the majority of analogues.

For example, using a combination of HSQC and ROESY,correlations were observed between the methyl group on thecyclobutane ring of 59 and H-3 and H-7′ and between H-7′and H-3 and H-4.In an attempt to avoid the low-yielding photochemical

addition step, the cyclobutane portion of 1 was replaced by anacetal. The acetals (62−64) were produced in good yield fromthe diol of flindersine (7) and the corresponding dimethylacetal (8a−c) of a functionalized benzaldehyde (Scheme 7).The relative cis stereochemistry of the two aromatic rings wasestablished by NMR (ROESY correlations were observedbetween the methine H at 11a and the methine H at 3a, andbetween H-11a and the H-7′ and H-8′ methylene hydrogens ofthe butyl group). Interestingly, the significant upfield shift (by∼0.5 ppm) observed for the ortho aryl protons in all of thecyclobutane analogues was not seen with the acetal analogues.For example, with compound 62, the upfield shift was verysmall (0.03 ppm), but there was a significant upfield shift(0.4 ppm) for the para hydrogen. It was originally thought thatthese acetals could mimic the shape of the cyclobutane portionof 1, thus providing a more synthetically tractable target andavoiding the photocycloaddition. However, the upfield shift forthe para hydrogen rather than the ortho hydrogen probablyreflects a slightly different shape or conformation for theseacetal analogues.

■ SCREENING RESULTS AND DISCUSSION

The cyclobutane series showed good SAR at the TLR4 receptor(Table 2). When the methyl group on the cyclobutane isremoved (9),16 activity is lost, whereas replacing the methylgroup by larger groups results in an increase in activity up toR = cyclopentyl. For example, in the dimethoxyphenylcyclo-butane series (1 and 9−43, R1 and R4 = H, R2 and R3 = OMe),there is a regular increase from R = Me (10, EC50 3.1 μM) to Et(19, EC50 1.2 μM) to Bu (37, EC50 1.2 μM) to i-Pr (26, EC501.0 μM) to cyclopropyl (31, EC50 0.63 μM) to cyclopentyl(34, EC50 0.39 μM). Cyclopentyl compound 34 was the mostactive analogue prepared. In the trimethoxphenylcyclobutaneseries (1 and 9−43, R1 = H, R2−R4 = OMe), there was a similarincrease in activity with increasing size of R, but it was not as

Scheme 4. Synthesis of Functionalized Styrenesa

aReagents and conditions: (i) DMAP, pyridine; (ii) K2CO3, acetone. Yields: 5a (78%), 5b (37%), 5c (20%), and 5d (96%).

Scheme 5. Synthesis of Flindersine Analoguesa

aReagents and conditions: (i) (a) Yb(OTf)3, MeCN, 80 °C, 12 h or(b) MgSO4, pyridine, reflux 16 h. Yields: 2a (42%), 2b (98%), 2c(95%), and 2e,f (18%).

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pronounced or as regular (1, Me, EC50 3.9 μM; 20, Et, EC50

0.79 μM; 36, Bu, EC50 2.0 μM; 22, i-Pr, EC50 0.79 μM; 32,cyclopropyl, EC50 0.63 μM; and 35, cyclopentyl, EC50 0.63 μM).When R is larger than cyclopentyl or with any group other thanalkyl, activity at TLR4 is reduced or lost altogether (e.g., 38, COMe,EC50 10 μM; 42, CH2OiPr, not active at 300 μM). This indicatesthat the methyl group occupies a hydrophobic pocket of limited size.Comparing the activities of the trimethoxyphenylcyclobutane

with the dimethoxyphenylcyclobutane series, the removal ofone of the meta methoxy groups had little effect on TLR4activity (e.g., 1, EC50 3.9 μM and 10, EC50 3.1 μM; 31, EC50

0.63 μM and 32, EC50 0.63 μM), whereas introduction ofgroups larger than OMe at the para position (R3) proveddetrimental to TLR4 activity (26, OMe, EC50 1.0 μM and 28,OEt, EC50 3.9 μM; 10, OMe, EC50 3.1 μM and 17, OBn, notactive at 300 μM). Replacing the para OMe group of 1 by thesmaller H did not affect the activity (11, EC50 3.1 μM), butreplacing it by the polar OH resulted in a significant loss ofactivity (18, EC50 >10 μM). Groups larger than OMe could,however, be introduced at the meta position (R2), allowing theintroduction of solubilizing groups and resulting in analogueswith improved solubility properties and sometimes improvedactivities (26, OMe, EC50 1.0 μM and 29, O(CH2)2OH, EC50

0.5 μM; 31, OMe, EC50 0.79 μM and 30, O(CH2)3OH, EC50

0.79 μM), but the larger solubilizing group O(CH2)3morpholine,resulted in reduced activity (31, OMe, EC50 0.63 μM; 33,

O(CH2)3morpholine, EC50 3.9 μM). Replacing a meta methoxygroup by an acyl group resulted in a significant reduction inactivity (26, OMe, EC50 1.0 μM and 25, OAc, EC50 >10 μM; 27,OCO(CH2)2CO2H, EC50 6.3 μM). Removal of both metamethoxy groups was detrimental to activity (10, R2 and R3 =OMe, EC50 3.1 μM and 15, R2 = H, R3 = OMe, EC50 >10 μM).Replacing a meta methoxy group in the dimethoxyphenyl-

cyclobutane series by a halogen resulted in a modest decrease inactivity, with the effect becoming more pronounced withincreasing electronegativity (10, OMe, EC50 3.1 μM and 12, Br,EC50 3.9 μM; 26, OMe, EC50 1.0 μM and 23, Cl, EC50 2.0 μM;19, OMe, EC50 1.2 μM and 21, F, EC50 3.1 μM).Alkylation of the pyridine nitrogen (R5, 44−54, Table 3) had

little effect on the intrinsic activity in most cases (10, R5 = H,EC50 3.1 μM and 44, R5 = Me, EC50 2.5 μM; 19, R5 = H, EC50

1.2 μM and 45, R5 = Me, EC50 2.0 μM; 21, R5 = H, EC50

3.1 μM and 46, R5 = Me, EC50 3.1 μM). Other modifications tothe pyranoquinolone portion of the molecule generally had adetrimental effect on the activity at the TLR4 receptor. Forexample, removal of either one of the gem-dimethyl groupsfrom parent compound 1 resulted in a significant loss in activity(1, EC50 3.9 μM and 55, 56, EC50 >10 μM). Assuming that theNH and NMe analogues (R5 = H or Me) have comparableactivity as noted above, introduction of a methyl group at C9(R6) results in a decrease in activity (20, R = Et, R6 = H, EC50

0.79 μM and 47, R = Et, R6 = Me, EC50 3.1 μM; 19, R = Et,

Scheme 6. Synthesis of Pyridinone Analoguesa

aReagents and conditions: (i) (a) Yb(OTf)3, MeCN, 80 °C, 12 h or (b) MgSO4, pyridine, reflux 16 h; (ii) UV light (hv), 125 or 400 W, neat 16 h.Yields: 6 (80%), 59 (6.6%), 60 (11%), and 61 (8.5%).

Scheme 7. Synthesis of Acetal Analoguesa

aReagents and conditions: (i) PropylMgBr, MePh, rt, 26 h; (ii) MnO2, DCM, rt, 24 h; (iii) H2SO4, MeOH, (MeO)3CH; (iv) DCM, PPTS, 18 h.Overall yields: 62 (50%), 63 (78%), and 64 (30%).

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R6 = H, EC50 1.2 μM and 48, R = Et, R6 = Me, EC50 3.1 μM;22, R = i-Pr, R6 = H, EC50 0.79 μM and 49, R = i-Pr, R6 = Me,EC50 7.9 μM). Introduction of a methoxy group at C9 (R6)resulted in a large decrease in activity (1, R = Me, R6 = H, EC50

3.9 μM and 58, R = Me, R6 = Me, EC50 >10 μM; 26, R = i-Pr,R6 = H, EC50 1.0 μM and 57, R = i-Pr, R6 = Me, EC50 > 10 μM).Pyridinone analogues (benzene ring removed from the quinoli-none) did not show any activity when tested in the TLR4 assay.In summary, modifications to the trimethoxyaryl ring, the

cyclobutane substituent R, the quinolinone, and pyran ringsdemonstrated the tight SAR requirements of the TLR4 receptor.A small number of analogues with the cyclobutane ring

replaced by an acetal were also examined. On testing, theseanalogues (62−64) did show some promising activity in the

human TLR4 SEAP assay (Table 4); however, they were lessactive than most of the cyclobutane compounds and were notfurther investigated.

■ CONCLUSIONS

Euodenine A, a natural product of unusual architecture, is ahuman TLR4-selective agonist that is CD14-independent, andit requires both TLR4 and MD-2 for full efficacy. Euodenine Aand a series of analogues were prepared in two to four stepsfrom readily available starting materials. The dimethoxyphe-nylcyclobutane series showed good SAR at the TLR4 receptor.Thus, when the methyl group on the cyclobutane is removed,activity is lost, whereas replacing the methyl group by largergroups results in an increase in activity up to R = cyclopentyl,

Table 2. Aryl and Alkyl Modifications

EC50 TLR4 (μM)a IAb R R1 R2 R3 R4

1 3.9 0.95 Me H OMe OMe OMe9c NA at 30 μM H H OMe OMe OMe10 3.1 0.69 Me H OMe OMe H11 3.1 0.46 Me H OMe H OMe12 3.9 0.28 Me H Br OMe H13 3.1 0.44 Me Cl OMe OMe H14 NA at 71.5 μM Me H H H H15 >10 Me H H OMe H16 >10 Me OMe OMe OMe H17 NA at 300 μM Me H OMe OBn H18 >10 Me H OMe OH H19 1.2 1.01 Et H OMe OMe H20 0.79 1.05 Et H OMe OMe OMe21 3.1 0.49 Et H F OMe H22 0.79 1.18 i-Pr H OMe OMe OMe23 2.0 0.47 i-Pr H Cl OMe H24 2.0 0.2 i-Pr H CH2Cl OMe H25 >10 i-Pr H OAc OMe H26 1.0 0.9 i-Pr H OMe OMe H27 6.3 0.29 i-Pr H OCO(CH2)2CO2H OMe H28 3.9 0.19 i-Pr H OMe OEt H29 0.5 0.46 i-Pr H O(CH2)2OH OMe H30 0.79 0.88 Cypr H O(CH2)3OH OMe H31 0.63 0.75 Cypr H OMe OMe H32 0.63 0.82 Cypr H OMe OMe OMe33 3.9 0.19 Cypr H O(CH2)3morpholine OMe H34 0.39 0.9 Cyp H OMe OMe H35 0.63 0.63 Cyp H OMe OMe OMe36 2.0 0.69 Bu H OMe OMe OMe37 1.2 1.01 Bu H OMe OMe H38 10 0.30 COMe H OMe OMe H39 7.9 0.62 COCypr H OMe OMe H40 NA at 25 μM CO2Me H OMe OMe OMe41 5.0 0.1 CH2OAc H OMe OMe OMe42 NA at 300 μM CH2OiPr H OMe OMe OMe43 NA at 333 μM CO2Et H H Br H

aEC50 values are mean values based on between two and eight determinations. The standard deviations for −log EC50 values were <0.1 in nearly allcases (range 0.00 to 0.23). bIntrinsic activity relative to LPS. cRef 16.

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which was the most active compound and 10-fold more activethan euodenine A. Replacing the methyl by groups larger thancyclopentyl resulted in reduced activity. Groups larger thanOMe could be introduced into the meta position, allowing theintroduction of solubilizing groups and resulting in analogues withimproved solubility properties and sometimes improved activities.Testing for immunomodulation in PBMC cells shows theinduction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40and suppression of IL-5 from activated PBMCs, indicating thatcompounds like 1 could modulate the Th2 immune responsewithout causing lung damage. The results suggest that 1 wouldhave the potential to prevent IL-5-mediated allergic inflammationwhile having a lower risk of inducing IL-12- and TNF-α-mediatedproinflammatory responses compared to LPS.

■ EXPERIMENTAL SECTIONChemistry, General Methods, and Materials. 1H NMR spectra

were recorded at either 500 or 600 MHz, and 13C NMR were recorded

at 125 MHz. Chemical shifts are reported in parts per million (ppm)relative to the solvent (DMSO-d6:

1H, 2.49; 13C, 39.51 ppm or CDCl3:1H, 7.26; 13C, 77.0 ppm), and coupling constants (J) are reported inhertz. HPLC−MS was run in ESI mode using a C18 50 mm × 4.6 mm,5 μm analytical column using a diode array detector over multiplewavelengths. Both HPLC−MS and HPLC were reverse-phase with aMeCN/H2O (0.01% TFA) gradient and a flow rate of 1.5 mL/min.The purity of all tested compounds was established as ≥95% by HPLCanalysis using two different gradients with UV detection on aphotodiode array detector (210, 254, and 280 nm). Flashchromatography was performed using silica gel (230−400 mesh) orneutral alumina.

Molecular Modeling. The structure of 1 was subjected togeometry optimization in MacroModel using the MMFF force fieldwith H2O solvation. Default parameters and convergence criteria wereused elsewhere. The reported structure is the lowest-energy conformerresulting from a mixed MCMM/Low-Mode search with 1000 stepsper rotatable bond (including tetrahydropyan ring bonds).

Isolation and Structure Determination of Euodenine A (1)from Euodia Asteridula. The leaves of Euodia asteridula (Rutaceae)were collected by Biodiversity Research Ltd. in PNG, and a vouchersample is lodged at the Lae Herbarium, Lae, PNG.

Extraction and Isolation. The air-dried material (100 g) wasground and extracted exhaustively with methanol (1 L) anddichloromethane saturated with ammonia (400 mL). The dichloro-methane extract was evaporated; the dry extract was then dissolved inthe methanol extract. Mass spectrometry was performed on thiscombined extract to confirm the target alkaloid compounds chosen bythe small-scale MS survey. Strong cation-exchange resin (SCX, 30 g)was added to the extract. The mixture was shaken by hand and left for15 min. Mass spectrometry was performed to confirm the targetalkaloids were completely bound to the SCX. The mixture was thenvacuum filtered with a sintered glass funnel. The load extract withoutalkaloid was thus separated from the alkaloid-bound SCX resin. Thebound alkaloids were eluted off the SCX resin by methanol-ammonia(10%) (300 mL) and dichloromethane saturated with ammonia(100 mL). This alkaloid-enriched fraction was then evaporated (6 g)and preabsorbed onto C18 (2 g) and packed dry into a small cartridge,

Table 3. Pyranoquinolinone Modifications

EC50 TLR4 (μM)a IAb R R1 R2 R3 R4 R5 R6 R7 R8

44 2.5 0.64 Me H OMe OMe H Me H Me Me45 2.0 0.61 Et H OMe OMe H Me H Me Me46 3.1 0.36 Et H F OMe H Me H Me Me47 3.1 0.3 Et H OMe OMe OMe Me Me Me Me48 3.1 0.29 Et H OMe OMe H Me Me Me Me49 7.9 0.41 i-Pr H OMe OMe OMe Me Me Me Me50 3.1 0.53 i-Pr H Cl OMe H Me H Me Me51 3.1 0.31 i-Pr H OAc OMe H Me H Me Me52 0.79 0.81 Cypr H OMe OMe OMe Me H Me Me53 1.0 0.67 Cypr H OMe OMe H Me H Me Me54 2.5 0.69 Cyp H OMe OMe OMe Me H Me Me55 >10 Me H OMe OMe OMe H H Me H56 >10 Me H OMe OMe OMe H H H Me57/ >10 i-Pr H OMe OMe H H OMe Me Me58 >10 Me H OMe OMe OMe H OMe Me Me

aEC50 values are mean values based on between two and eight determinations. The standard deviations for −log EC50 values were <0.1 in nearly allcases (range 0.00 to 0.23). bIntrinsic activity relative to LPS.

Table 4. Acetal Analogues

EC50 TLR4 (μM)a IA R R1

62 3.1 0.7 H OMe63 15.8 0.2 OMe H64 NA at 60 μM OMe OMe

aEC50 values are mean values based on between two and eightdeterminations. The standard deviations for −log EC50 valueswere <0.1 in nearly all cases (range 0.00 to 0.23).

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which was connected to a preparative C18 column (Betasil C18,150 × 21.2 mm, 5 μm, Thermo Electro Corporation). This fractionwas chromatographed using mass-directed purification on a HP1100LC−MS and eluted at 10 mL/min with a gradient solvent systemstarting with 100% water (1% TFA) gradient to 60% acetonitrile (1%TFA)/40% water (1% TFA) in 50 min, then gradient to 100%acetonitrile (1% TFA) in the next 10 min. Pure 1 (20 mg) was elutedat 53−55 min. Yellow oil, optically inactive, UV (MeOH) λmax (ε) 212(28 602), 217 (27 502), 286 (4602), 317 (4711), 329 nm (3971). IR(film) νmax 3424, 2949, 1647, 1605, 1507, 1457, 1427, 1396, 1238,1128, 756, 505 cm−1. For 13C and 1H NMR data, see Tables 5 and 6(numbering as in 1, Scheme 2). (+)-HRESIMS m/z 436.2126 (calcdfor C26H30NO5, 436.2119).Structure Determination. The 13C NMR spectrum (Table 5) of 1

displayed 23 carbon signals. Three of the carbons were double theintensity of the others, suggesting that there was some symmetry inthe molecule. The carbons could be assigned to six methyls(two coincident), four aliphatic methines, six aromatic methines(two coincident), nine downfield quaternary carbons (two coincident),and one oxygenated quaternary carbon from interpretation of g-HSQCand 1H NMR data. The most downfield carbon signal at δ 162.2 wasassigned to a conjugated amide because an IR absorption band wasobserved at 1647 cm−1. Out of the six methyls, three were aromaticmethoxy groups, two of which were coincident. Analysis of the 1HNMR spectrum of euodenine A indicated that the remaining methylgroups were two quaternary methyls at δ 1.14 and 1.48 and one was asecondary methyl signal at δ 1.13 (d, 7 Hz). Both quaternary methylprotons showed a HMBC correlation to the oxygenated quaternarycarbon C-2 (δ 76.5) and to an aliphatic methine C-3 at δ 45.7 ppm.This demonstrated that the molecule contained an isopropyloxy groupvicinal to an aliphatic methine, C-3. The secondary methyl proton onC-8′ also correlated to C-3 as well as to another two methine carbons,C-8′ (35.2 ppm) and C-7′ (49.4 ppm), in the HMBC spectrum.Interpretation of the COSY spectrum was complicated by theobservation that the signals for the protons attached to C-3(45.7 ppm) and C-8′ (35.2 ppm) were coincident (δ 2.33) in the

1H NMR spectrum. COSY correlation from the methyl protons onC-8′ to δ 2.33 indicated that the carbon at 35.2 ppm must be vicinal tothe methyl group and therefore the carbons at δ 45.7 and 49.4 ppmwere each vicinal to C-8′. If the secondary methyl group was vicinal toC-3, then it should have had a HMBC correlation to C-2. COSYcorrelations were also observed from the signal at δ 2.33 to methinesignals at δ 3.30 (H-7′) and 3.69 (H-4). This could only be possible ifthe proton attached to C-3 (δ 2.33) was vicinal to H-4 (δ 3.69). ACOSY correlation from H-4 to H-7′ indicated that they too werevicinal to each other and that AR-Q500206 contained a tetrasub-stituted cyclobutyl group. The molecule contained a symmetricaltetrasubsituted phenyl group because a two-proton aromatic singlet atδ 6.12 (H-2′ and H-6′) displayed both HMBC and HSQC correlationsto a carbon signal at 105.4 ppm (C-2′ and C-6′), indicating that thetwo protons were meta to each other. The chemical shift of thecarbons C-2′ and C-6′ suggested that they were both ortho to oxygensubstituents, and this was confirmed by the observation of HMBCcorrelations from these protons to oxygenated aromatic quaternarycarbons at δ 151.7 and 135.8 ppm. HMBC correlations were alsoobserved from the three methoxyl protons at δ 3.34 (2 methoxyls) and3.53 to the oxygenated aromatic quaternary carbons at δ 135.8 and151.7 ppm, respectively. The upfield chemical shift of one of theseoxygenated quaternary carbons C-4′ suggested that it was flanked oneither side by the other two methoxyl groups. It could therefore beconcluded that the molecule contained a 1,2,3-trimethoxylphenylgroup. This phenyl group was attached to C-7′ because HMBCcorrelations were observed from H-2′ and H-6′ to C-7′. HMBCcorrelations from δ 2.33 (H-3), 3.30 (H-7′), and 3.69 (H-4) to aquaternary carbon (C-4a) at 107.7 ppm indicated that this carbon wasvicinal to C-4. The proton H-4 also correlated to an additionaldownfield quaternary carbon at δ 155.5 (C-10b) and the amidecarbonyl carbon C-5 at 162.2 ppm. Both of these carbons couldlogically be attached only to the carbon (C-4a) at δ 107.7 ppm, andthe chemical shift of C-10b suggested that it was oxygenated. Adownfield exchangeable proton H-6 (δ 11.06) assigned to an amideNH also correlated to C-4a. The four remaining protons in the

Table 5. 1H (500 MHz) and 13C (125 MHz) NMR Data for Euodenine A in DMSO-d6

position 13C NMR 1H NMR g-COSY g-HMBC ROESY

2 76.53 45.7 2.33 (m) 4, 8′ 4a, 4, 2, 8′CH3, 8′, 7′ 7′, 4, 2′CH3, 2′CH3

4 31.7 3.69 (dd, 8.4, 8.0) 3, 7′ 5, 4a, 10b, 3, 8′, 7′ 7′, 3, 2′CH3

4a 107.75 162.26 (NH) 11.06 (s) 4a, 6a, 10a 96a 137.57 114.6 7.15 (d, 8.5) 8 10b, 9, 8, 10a 9, NH8 129.8 7.42 (dd, 8.5, 7.5) 9, 7 9, 6a 99 120.9 7.13 (dd, 8.0, 7.5) 10, 8 8, 10a 10, 810 121.7 7.82 (d, 8.0) 9 10b, 8, 7 910a 114.810b 155.51′ 135.92′ 105.4 6.12 (s) 7′, 1′, 3′, 4′, 6′ 8′,7′, OMe (C3′)3′ 151.74′ 135.85′ 151.76′ 105.4 6.12 (s) 7′, 1′, 2′, 4′, 5′ 8′, 7′, OMe (C5′)7′ 49.4 3.30 (dd, 8.4, 9.0) 4, 8′ 4a, 3, 8′CH3, 8′, 1′, 2′, 6′ 4,3, 8′CH3, 2′, 6′8′ 35.2 2.33 (m) 8′CH3, 7′, 3 4, 2, 8′CH3, 7′, 1′ 8′CH3, 2′, 6′2′CH3 23.2 1.14 (s) 3, 2, 2′CH3 13, 2′CH3

2′CH3 24.4 1.48 (s) 10b, 3, 2, 2′CH3 8′, 2′CH3

8′CH3 20.2 1.13 (d, 7.0) 8′ 3, 8′, 7′ 8′, 7′, 2′, 6′OMe (C3′) 55.0 3.34 (s) 3′ 2′OMe (C4′) 59.8 3.53 (s) 4′OMe (C5′) 55.0 3.34 (s) 5′ 6′

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1H NMR spectrum were part of a 1,2-disubstituted aromatic groupbecause correlations were observed in the COSY spectrum from H-10and H-8 to H-6 and H-7. One of the substituents of the disubstitutedaromatic group was the carbon at δ 155.5 ppm (C-10b) because a HMBCcorrelation was observed to this carbon from H-10. The other substituentwas deduced to be a nitrogen because H-10, H-8, and the amide proton,H-6, showed HMBC correlations to a heteroatom-substituted aromaticquaternary carbon at δ 137.5 ppm. The remaining quaternary aromaticcarbon C-10a correlated to H-9, H-6a, and the amide proton, H-6, andfrom this data, it was concluded that AR-Q500206 contained a3-cyclobutyl-substituted quinolinone. This data indicated that 12 of the13 degrees of unsaturation were accounted for, which dictated that themolecule must contain an extra ring. A weak HMBC correlation from thequaternary methyl proton on C-2 to the oxygenated quaternary carbonC-10b suggested that a carbon oxygen bond linked the isopropyloxygroup to the oxygenated quaternary carbon C-10b, thus forming adihydropyran ring. The gross structure of euodenine A was thereforeassigned.A change in deuterated solvents for NMR experiments from

DMSO-d6 to methanol-d4 helped to resolve the overlapping signals ofthe quaternary methyl (δ 1.14), the secondary methyl (δ 1.13), H-3 (δ2.33), and H8′ (δ 2.33) (Table 6). Correlations observed in a ROESYspectrum performed in methanol-d4 were used to deduce the relativestereochemistry of the substituents about the cyclobutyl group. Thesecondary methyl and the trimethoxyphenyl group were trans to eachother because ROESY correlations were observed between the methylat 8′ and H-7′. H-7′, H-4, and H-3 were all on the same face of thecyclobutyl group because ROESY correlations were observed amongthese three protons. The cis relationship of the quinolinone andtrimethoxy phenyl ring helps to explain the significant upfield shift ofthe aromatic protons H-2′ and H-6′ of the trimethoxyphenyl group. Ina cis configuration, the phenyl group lies in the shielding zone of thequinolinone. Thus, the structure of euodenine A was deduced as 1.Synthesis of 1 and Analogues. General Procedure for Synthesis

of Styrenes. Method A: (E)-1,2,3-Trimethoxy-5-(prop-1-enyl)-benzene [5273-85-8] (4a). 3,4,5-Trimethoxybenzaldehyde

(51 mmol) was dissolved in toluene (40 mL) and cooled to 0 °C.A solution ethylmagnesium chloride (3.0 M in Et2O, 91.8 mmol) wasadded dropwise, and the reaction mixture was stirred for a further16 h. To this was added 5% AcOH (200 mL), and the resultingmixture extracted with EtOAc (100 mL). The organic phase was thenextracted with water (100 mL), saturated NaHCO3 (100 mL), andbrine (100 mL). The organic phase was dried (MgSO4), filtered, andconcentrated to afford 1-(3,4,5-trimethoxyphenyl)propan-1-ol (10.86g, 94%). 1H NMR (CDCl3, 500 MHz) δ 0.97 (t, J = 7 Hz, 3H), 1.79(m, 2H), 3.87 (s, 3H), 3.90 (s, 6H), 4.56 (t, J = 6.5 Hz, 1H), 6.61(s, 2H). 13C NMR (CDCl3, 125 MHz) δ 10.44, 21.64, 32.16, 56.34,61.03, 76.44, 103.12, 137.50, 138.07, 140.67, 153.47. (+) LRMS (ESI)m/z 227 [M + H]+. (E)-(3,4,5-Trimethoxyphenyl)propan-1-ol (8 g)was dissolved in dioxan (40 mL), and orange silica gel (Riedel-de-Haen 13767, 40 g) was added. The resulting solution was heatedunder microwave conditions at 180 °C for 10 min. The reactionmixture was filtered, concentrated to dryness, and then purified byflash chromatography on silica gel using a gradient from 100% hexaneto 20% EtOAc over 20 min to afford (E)-1,2,3-trimethoxy-5-(prop-1-enyl)benzene (3.86 g, 53%) as a colorless oil. 1H NMR (CDCl3,500 MHz) δ 1.84 (d, J = 6.5 Hz, 3H), 3.82 (s, 3H), 3.90 (s, 6H), 6.19(dt, J = 6.5, 13.5 Hz, 1H), 6.37 (d, J = 16 Hz, 1H), 6.60 (s, 2H). (+)LRMS (ESI) m/z 209 [M + H]+.

(E)-1,3-Dimethoxy-5-(prop-1-enyl)benzene [146205-98-3] (4b).From 3,5-dimethoxybenzaldehyde and ethylmagnesium chloride. 1HNMR (CDCl3, 500 MHz) δ 1.74 (d, J = 6.5 Hz, 3H), 3.66 (s, 3H),3.68 (s, 3H), 6.08 (dt, J = 6.5, 13.5 Hz, 1H), 6.20 (d, J = 16 Hz, 1H),6.20 (s, 1H), 6.36 (s, 2H). (+) LRMS (ESI) m/z 179 [M + H]+.

(E)-2-Bromo-1-methoxy-4-(prop-1-enyl)benzene (4c). From 3-bromo-4-methoxybenzaldehyde and ethylmagnesium bromide. 1HNMR (DMSO-d6, 500 MHz) δ 1.83 (d, J = 7 Hz, 6H), 3.84 (s,3H), 6.21 (m, 1H), 6.34 (d, J = 15 Hz, 1H), 7.05 (d, J = 8 Hz, 1H),7.36 (dd, J = 1.5, 8 Hz, 1H), 7.60 (d, J = 1.5 Hz, 1H). LRMS (ESI)m/z 228 [M79Br + H]+, 230 [M81Br + H]+.

(E)-2-Chloro-3,4-dimethoxy-1-(prop-1-enyl)benzene (4d). From2-chloro-3,4-dimethoxy benzaldehyde and ethylmagnesium bromide.

Table 6. 1H (600 MHz) and 13C (125 MHz) NMR Data for Euodenine A in Methanol-d4

position 13C NMR 1H NMR g-COSY g-HMBC ROESY

2 78.73 47.7 2.39 (dd, 8.4, 9.0) 4, 8′ 4a, 4, 2, 2′CH3, 8′ CH3, 8′ 4, 2′CH3, 8′CH3, 7′4 33.5 3.81 (dd, 8.4, 9.0) 3, 7′ 5, 4a, 10b, 3, 8′, 7′ 7′, 3, 2′CH3

4a 108.95 165.46 (NH)6a 138.67 116.4 7.22 (d, 9.0) 8 10b, 9,10a 88 131.7 7.49 (ddd, 8.4, 9.0, 1.2) 9, 7 9, 7 9, 79 123.3 7.24 (dd, 8.4, 8.4) 10, 8 8, 7, 6a, 10a 10, 810 123.6 7.98 (dd, 1.2, 8.4) 9 10b, 8, 6a 910a 117.210b 159.01′ 137.62′ 107.0 6.14 (s) 4, 7′, 1′, 3′, 4′, 6′ 8′,7′, OMe (C3′)3′ 153.64′ 137.65′ 153.66′ 107.0 6.14 (s) 4, 7′, 1′, 2′, 4′, 5′ 8′, 7′, OMe (C5′)7′ 51.8 3.36 (dd, 10.2, 9.0) 4, 8′ 4a, 4, 8′CH3, 8′, 1′, 2′, 6′ 4,3, 8′CH3, 2′, 6′8′ 36.9 2.45 (ddq, 10.2, 9.0, 6.0) 8′CH3, 7′, 3 3, 7′, 2′ 8′CH3, 2′, 6′, 2′CH3

2′CH3 24.1 1.23 (s) 3, 2, 2′CH3 4, 3, 2′CH3

2′CH3 25.2 1.56 (s) 10b, 3, 2, 2′CH3 8′, 2′CH3

8′CH3 20.8 1.19, (d, 6.0) 8′ 3, 8′, 7′ 3, 8′, 7′OMe (C3′) 56.1 3.40 (s) 3′ 2′OMe (C4′) 61.1 3.64 (s) 4′OMe (C5′) 56.1 3.40 (s) 5′ 6′

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1H NMR (DMSO-d6, 500 MHz) δ 1.83 (d, J = 7 Hz, 6H), 3.74(s, 3H), 3.85 (s, 3H), 6.34 (m, 2H), 7.05 (m, 2H). LRMS (ESI) m/z213 [M35Cl + H]+, 215 [M37Cl + H]+.(E)-4-(But-1-enyl)-1,2-dimethoxybenzene [76252-28-3] (4e).

From 3,4-dimethoxybenzaldehyde and propylmagnesium chloride.1H NMR (d6-DMSO, 500 MHz) δ 1.05 (t, J = 6.5 Hz, 3H), 2.15(q, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.77 (s, 3H), 6.20 (dt, J = 6.5, 13.5Hz, 1H), 6.31 (d, J = 16 Hz, 1H), 6.87 (br s, 2H), 7.00 (br s, 1H). 13CNMR (d6-DMSO, 125 MHz) δ 13.6, 25.3, 55.4, 55.4, 108.9, 111.8,118.6, 128.4, 130.0, 130.4, 148.0, 148.8. (+) LRMS (ESI) m/z 193[M + H]+.(E)-5-(But-1-enyl)-1,2,3-trimethoxybenzene [357386-06-2] (4f).

From 3,4,5-trimethoxybenzaldehyde and propylmagnesium chloride.1H NMR (CDCl3, 500 MHz) δ 1.12 (t, J = 7.5 Hz, 3H), 2.25 (m, 2H),3.86 (s, 3H), 3.90 (s, 6H), 6.21 (dt, J = 6.5, 13.5 Hz, 1H), 6.33 (d, J =16 Hz, 1H), 6.60 (s, 2H). 13C NMR (CDCl3, 125 MHz) δ 14.2, 19.3,41.5, 56.3, 61.1, 74.8, 103.0,137.4, 141.0, 153.4. (+) LRMS (ESI) m/z223 [M + H]+.(E)-4-(But-1-enyl)-2-fluoro-1-methoxybenzene (4g). From 3-fluo-

ro-4-methoxybenzaldehyde and isobutylmagnesium chloride. 1H NMR(DMSO-d6, 500 MHz) δ 1.03 (t, J = 7.5 Hz, 3H), 2.16 (q, J = 7.5 Hz,2H), 3.82 (s, 3H), 6.18 (dt, J = 6, 14 Hz, 1H), 6.30 (d, J = 14 Hz, 1H),7.07 (dd, J = 8, 9 Hz, 1H), 7.11 (br d J 8.5 Hz, 1H), 7.25 (dd, J = 1.5,11 Hz, 1H). 13C NMR (DMSO-d6, 500 MHz) δ 14.2, 26.0, 56.6, 113.3(d, J = 18 Hz), 114.4 (d, J = 17 Hz), 123.0 (d, J = 12 Hz), 137.9, 131.6(d, J = 6 Hz), 132.2, 146.7 (d, J = 11 Hz), 152.4 (d, J = 242 Hz). (+)LRMS (ESI) m/z 181 [M + H]+.(E)-1,2,3-Trimethoxy-5-(3-methylbut-1-enyl)benzene (4h). From

3,4,5-trimethoxybenzaldehyde and isobutylmagnesium chloride. 1HNMR (DMSO-d6, 500 MHz) δ 1.06 (d, J = 6.5 Hz, 6H), 2.42 (m, 1H),3.63 (s, 3H), 3.77 (s, 6H), 6.24 (m, 2H), 6.67 (s, 2H). 13C NMR(DMSO-d6, 125 MHz) δ 13.7, 21.6, 30.9, 31.9, 55.8, 60.0, 103.2, 133.1,136.66, 152.89. (+) LRMS (ESI) m/z 236 [M + H]+.(E)-2-Chloro-1-methoxy-4-(3-methylbut-1-enyl)benzene (4i).

From 3-chloro-4-methoxybenzaldehyde and isobutylmagnesium chlo-ride. 1H NMR (DMSO-d6, 500 MHz) δ 1.03 (d, J = 7 Hz, 6H), 2.42(m, 1H), 3.83 (s, 3H), 6.18 (dd, J = 6.5, 13.5 Hz, 1H), 6.33 (d, J =13.5 Hz, 1H), 7.06 (d, J = 8 Hz, 1H), 7.30 (d J 8 Hz, 1H), 7.46 (s,1H). (+) LRMS (ESI) m/z 211 [M35Cl + H]+, 213 [M37Cl + H]+.(E)-2-(Chloromethyl)-1-methoxy-4-(3-methylbut-1-enyl)benzene

(4j). From 3-(chloromethyl)-4-methoxybenzaldehyde and isobutyl-magnesium chloride. 1H NMR (DMSO-d6, 600 MHz) δ 1.02 (d, J = 7Hz, 6H), 2.42 (m, 1H), 3.83 (s, 3H), 4.68 (s, 2H), 6.13 (dd, J = 6, 13Hz, 1H), 6.28 (d, J = 13 Hz, 1H), 6.97 (d, J = 8 Hz, 1H), 7.33 (d, J = 8Hz, 1H), 7.45 (s, 1H). LRMS (ESI) m/z 243 [M35Cl + H]+, 245[M37Cl + H]+

(E)-2-Methoxy-5-(3-methylbut-1-enyl)phenyl Acetate (4k). (E)-2-methoxy-5-(3-methylbut-1-enyl)phenol (1.21 g, 6.3 mmol) wasdissolved in pyridine (2 mL). Acetic anhydride (0.893 mL, 9.5 mmol)and a catalytic amount of DMAP were added, and the reaction mixturestirred at rt for 16 h. The reaction mixture was partitioned between 1N HCl (20 mL) and EtOAc (30 mL). The organic phase was washedwith brine (20 mL), dried (MgSO4), filtered, and concentrated toafford (E)-2-methoxy-5-(3-methylbut-1-enyl)phenyl acetate as a whitesolid (1.14 g, 77%). 1H NMR (CDCl3, 600 MHz) δ 1.08 (d, J = 6.6Hz, 6H), 2.31 (s, 3H), 2.43 (m, 1H), 3.83 (s, 3H), 6.04 (dd, J = 6, 13Hz, 1H), 6.24 (d, J = 13 Hz, 1H), 6.89 (d, J = 8 Hz, 1H), 7.07 (d, J =1.8 Hz, 1H), 7.15 (dd, J = 1.8, 8 Hz, 1H). LRMS (ESI) m/z 235[M + H]+.(E)-1,2-Dimethoxy-4-(3-methylbut-1-enyl)benzene [195192-74-6]

(4l). From 3,4,-dimethoxybenzaldehyde (30.6 mmol) and isobutyl-magnesium chloride. 1H NMR (DMSO-d6, 500 MHz) δ 1.06 (d, J =6.5 Hz, 6H), 2.42 (m, 1H), 3.63 (s, 3H), 3.78 (s, 3H), 6.15 (dd, J =6.5, 13.5 Hz, 1H), 6.28 (d, J = 16 Hz, 1H), 6.87 (s, 2H), 7.01 (s, 1H). 13CNMR (DMSO-d6, 125 MHz) δ 22.4, 30.8, 55.3, 55.4, 109.0, 111.7, 118.7,126.4, 130.4, 135.2, 148.0, 148.8. (+) LRMS (ESI) m/z 206 [M + H]+.(E)-2-Methoxy-5-(3-methylbut-1-enyl)phenol (4m). From 3-hydroxy-

4-methoxybenzaldehyde and isobutylmagnesium chloride. 1H NMR(DMSO-d6, 600 MHz) δ 1.01 (d, J = 6.6 Hz, 6H), 2.38 (m, 1H),3.73 (s, 3H), 5.98 (dd, J = 5.5, 13 Hz, 1H), 6.17 (d, J = 13 Hz, 1H),

6.71 (dd, J = 1.8, 8.4 Hz, 1H), 6.80 (s, 1H), 6.81 (d, J = 8 Hz, 1H),8.83(s, 1H). LRMS (ESI) m/z 193 [M + H]+.

(E)-5-(Hex-1-enyl)-1,2,3-trimethoxybenzene [208192-84-1] (4n).From 3,4,5-trimethoxybenzaldehyde and pentylmagnesium bromide.1H NMR (DMSO-d6, 500 MHz) δ 0.91 (t, J = 6.5 Hz, 3H), 1.34 (m,2H), 1.43 (m, 2H), 2.17 (q, J = 6.5 Hz, 2H), 3.64 (s, 3H), 3.78 (s,6H), 6.25 (dt, J = 6.5, 13.5 Hz, 1H), 6.32 (d, J = 16 Hz, 1H), 6.68 (brs, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 13.7, 21.6, 30.9, 31.9, 55.8,60.0, 103.2, 133.1, 136.7, 152.9. (+) LRMS (ESI) m/z 250 [M + H]+.

(E)-4-(Hex-1-enyl)-1,2-dimethoxybenzene [195192-75-7] (4o).From 3,4-dimethoxybenzaldehyde and pentylmagnesium bromide.1H NMR (DMSO-d6, 500 MHz) δ 0.91 (t, J = 6.5 Hz, 3H), 1.34 (m,2H), 1.41 (m, 2H), 2.17 (q, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.78 (s,3H), 6.15 (dt, J = 6.5, 13.5 Hz, 1H), 6.31 (d, J = 16 Hz, 1H), 6.86 (brs, 2H), 7.00 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 13.7, 21.7,31.1, 32.0, 55.3, 55.4, 109.0, 111.4, 111.7, 118.6, 128.3, 129.3, 130.4,148.0, 148.8. (+) LRMS (ESI) m/z 221 [M + H]+.

Method B: 4-[(E)-2-Cyclopentylvinyl]-1,2-dimethoxybenzene (4p).A solution of 3,4-dimethoxybenzyl alcohol (5.0 g, 4.26 mL, 29.7 mmol)in DCM (136 mL) was cooled to 0 °C under an atmosphere of N2,phosphorus tribromide (2.89 mL, 30.6 mmol) was added dropwise, andthe reaction mixture maintained at 0 °C for 90 min. The reactionmixture was warmed to rt, stirred for a further 30 min, and then pouredonto ice and extracted with DCM (2 × 100 mL). The organic phase wasdried (MgSO4), filtered, and concentrated to dryness to afford 3,4-dimethoxybenzyl bromide as a pale yellow solid (7 g), which was used inthe next step without further purification. 1H NMR (CD2Cl2, 500 MHz)δ 3.83 (s, 3H), 3.84 (s, 3H), 4.52 (s, 2H), 6.81 (d, J = 1.5 Hz, 1H), 6.92(d, J = 8 Hz, 1H), 6.94 (dd, J = 1.5, 8 Hz, 1H). A solution of 3,4-dimethoxybenzyl bromide (7.0 g, 29.7 mmol) and tetrabutyl ammoniumiodide in triethyl phosphite (7.75 mL, 45.6 mmol) was heated at 110 °Covernight. Ethyl bromide and excess triethyl phosphite was removed bydistillation at reduced pressure to afford an oil that was purified by flashchromatography to afford diethyl 3,4-dimethoxybenzylphosphonate(6.55 g). 1H NMR (DMSO-d6, 500 MHz) δ 1.18 (t, J = 7 Hz, 6 H),3.10 (s, 1 H), 3.15 (s, 1H), 3.72 (s, 6H), 3.92 (m, 4H), 6.79 (d, J = 1.5Hz, 1H), 6.88 (m, 2H). (+) LRMS (ESI) m/z 289 [M + H]+.

A suspension of sodium hydride (150 mg, 3.64 mmol) in THF(5 mL) was cooled to 0 °C. Cyclopentane carboxaldehyde (0.36 g,3.64 mmol) and 3,4-dimethoxybenzylphosphonate (1 g, 3.47 mmol)were added, and the reaction mixture was allowed to warm to rt andstirred O/N. Water (10 mL) was added, and the mixture was extractedwith EtOAc (2 × 50 mL). The organic phase was washed with brine,dried (MgSO4), filtered, and concentrated to dryness. The resulting oilwas purified by flash chromatography on silica gel (5% EtOAc in hexane)to afford 4-[(E)-2-cyclopentylvinyl]-1,2-dimethoxybenzene as a colorlessoil (0.39 g, 48%). 1H NMR (DMSO-d6, 500 MHz) δ 1.38 (m, 2H), 1.56(m, 2H), 1.67 (m, 2H), 1.81 (m, 2H), 2.58 (m, 1H), 3.73 (s, 3H), 3.77 (s,3H), 6.23 (dd, J = 6.5, 15 Hz, 1H), 6.32 (d, J = 15 Hz, 1H), 6.86 (s, 2H),7.00 (s, 1H). (+) LRMS (ESI) m/z 233 [M + H]+.

4-[(E)-2-Cyclopentylvinyl]-1,2,3-trimethoxybenzene (4q). From3,4,5-trimethoxy benzyl alcohol. 1H NMR (DMSO-d6, 600 MHz) δ1.38 (m, 2H), 1.56 (m, 2H), 1.67 (m, 2H), 1.81 (m, 2H), 2.58(m, 1H), 3.66 (s, 3H), 3.78 (s, 6H), 6.23 (dd, J = 6.5, 15 Hz, 1H), 6.32(d, J = 15 Hz, 1H), 6.67 (s, 2H). (+) LRMS (ESI) m/z 263 [M + H]+.

Method C: (E)-1-Ethoxy-2-methoxy-4-(3-methylbut-1-enyl)-benzene [195192-86-6] (4r). A suspension of 3-ethoxy-4-methox-ybenzaldehyde (1.50 g, 8.32 mmol) and NH4OAc (0.205g, 2.66mmol) in nitromethane (15 mL) was heated at reflux for 16 h. Thereaction mixture was cooled and filtered through a plug of SiO2 elutingwith hexane to afford (E)-1-ethoxy-2-methoxy-4- (2-nitrovinyl)benzene as a yellow solid (1.58 g, 85%). 1H NMR (CDCl3, 500 MHz)δ 1.51 (m, 3H), 3.92 (s, 3H), 4.17 (s, 2H), 6.90 (d, J = 7.0 Hz, 1H),7.00 (s, 1H), 7.17 (d, J = 7.0 Hz, 1H), 7.52 (d, J = 11 Hz, 1H), 7.96(d, J = 11 Hz, 1H). (+) LRMS (ESI) m/z 239 [M + H]+.

2-Iodopropane (4.32 mL, 43 mmol) was added to a solution of(E)-1-ethoxy-2-methoxy-4- (2-nitrovinyl) benzene (0.50 g, 2.24 mmol)in THF (20 mL). Air was bubbled through the solution for 5 min,triethylborane (6.48 mL, 1.0 M in hexanes, 6.48 mmol) was added, andthe resulting mixture stirred for 1 h, concentrated to dryness, and

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purified by flash chromatography on silica gel (hexane) to afford (E)-1-ethoxy-2-methoxy-4- (3-methylbut-1-enyl) benzene as a colorless oil(0.30 g, 61%). 1H NMR (CDCl3, 500 MHz) δ 1.08 (m, 3H), 1.09 (m,3H), 1.45 (t, J = 6.0 Hz, 3H), 2.44 (m, 1H), 3.89 (s, 3H), 4.08 (q, J =6.0 Hz, 2H), 6.90 (m, 1H), 6.27 (m, 1H), 6.80 (d, J = 7.0 Hz, 1H), 6.85(d, J = 7.0 Hz, 1H), 6.91 (s, 1H). (+) LRMS (ESI) m/z 221 [M + H]+.Method D: 5-[(E)-2-Cyclopropylvinyl]-1,2,3-trimethoxybenzene

(4s). Palladium acetate (0.023g, 0.1 mmol) was added to a solutionof 5-bromo-1,2,3-trimethoxybenzene (1.0 g, 4.05 mmol), cylopropyl-vinyl boronic acid pinacol ester (1 mL, 4.86 mmol), triphenylphos-phine (0.11 g, 4.05 mmol), and 2 M K2CO3 (5.6 mL) in 1,2-dimethoxyethane (4.6 mL). The reaction mixture was heated at refluxfor 18 h, cooled, poured into water (10 mL), and extracted withEtOAc (2 × 30 mL). The organic phases were combined, washed withbrine (30 mL), dried (MgSO4), filtered, and concentrated to dryness.The resulting oil was purified by flash chromatography on silica gelusing gradient elution (0−20% EtOAc in hexane over 20 min) toafford 5-[(E)-2-cyclopropylvinyl]-1,2,3-trimethoxybenzene (776 mg,81%) as a light yellow oil that solidified on standing. 1H NMR(DMSO-d6, 500 MHz) δ 0.51 (m, 2H), 0.81 (m, 2H), 1.55 (m, 1H),3.64 (s, 3H), 3.78 (s, 6H), 5.82 (dd, J = 9.5, 16 Hz, 1H), 6.38 (d, J =16 Hz, 1H), 6.65 (s, 2H). (+) LRMS (ESI) m/z 235 [M + H]+.(E)-4-(2-Cyclopropylvinyl)-1,2-dimethoxybenzene (4t). From 5-

bromoveratrole and cylopropylvinyl boronic acid pinacol ester. 1HNMR (DMSO-d6, 500 MHz) δ 0.47 (m, 2H), 0.77 (m, 2H), 1.53 (m,1H), 3.72 (s, 3H), 3.74 (s, 3H), 5.72 (dd, J = 9, 16 Hz, 1H), 6.37(d, J = 16 Hz, 1H), 6.80 (d, J = 8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 6.96(s, 1H). (+) LRMS (ESI) m/z 205 [M + H]+.(E)-5-(2-Cyclopropylvinyl)-2-methoxyphenol (4u). From 5-bromo-

2-methoxyphenyl acetate and cylopropylvinylboronic acid pinacolester. 1H NMR (CDCl3, 500 MHz) δ 0.50 (m, 2H), 0.81 (m, 2H),1.55 (m, 1H), 3.83 (s, 3H), 5.61 (dd, J = 9, 16 Hz, 1H), 6.37 (d, J = 16Hz, 1H), 6.78 (s, 2H), 6.94 (s, 1H), 6.96 (s, 1H). 13C NMR (CDCl3,125 MHz) δ −7.3, 14.5, 56.2, 110.9, 111.5, 118.1, 127.1, 132.0, 133.4,145.8, 145.9. (+) LRMS (ESI) m/z 191 [M + H]+.(E)-4-(2-Methoxy-5-(3-methylbut-1-enyl)phenoxy)-4-oxobuta-

noic acid (5a). (E)-2-Methoxy-5-(3-methylbut-1-enyl)phenol (0.54 g,2.8 mmol) was dissolved in pyridine (3 mL). Succinic anhydride (0.56 g,5.6 mmol) and a catalytic amount of DMAP were added, and thereaction mixture was allowed to stir at rtRT for 16 h. A further 2 equivof succinic anhydride was added, and the reaction mixture was stirred fora further 24h. The reaction mixture was partitioned between EtOAc(30 mL) and water (10 mL), and the organic phase washed with 1 NHCl (20 mL) and brine (20 mL). The organic phase was dried(MgSO4), filtered, and concentrated to dryness to afford (E)-3-(2-methoxy-5-(3-methylbut-1-enyl)phenoxy)propanoic acid as a white solid(640 mg, 78%). 1H NMR (DMSO-d6, 500 MHz) δ 1.02 (d, J = 7 Hz,6 H), 2.39 (m, 3H), 2.57 (dd, J = 5.5, 6.5 Hz, 2H), 2.77 (dd, J = 5.5,7 Hz, 2H), 3.73 (s, 3H), 5.98 dd (J 6.5, 16 Hz, 1H), 6.18 (d, J = 16 Hz,1H), 6.73 (dd, J = 1.5, 8 Hz, 1H), 6.80 (d, J = 1.5 HZ, 1H), 6.82 (d, J =8 Hz, 1H), 8.83 (s, 1H).(E)-4-(3-(5-(2-Cyclopropylvinyl)-2-methoxyphenoxy)propyl)-

morpholine (5b). (E)-5-(2-Cyclopropylvinyl)-2-methoxyphenol (1.0g,5.3 mmol) was dissolved in acetone (5 mL). Potassium carbonate(3.66 g, 26.5 mmol) and 4-(3-chloropropyl)morpholine (2.6 g, 15.9 mmol)were added, and the reaction mixture was heated at reflux for 16 h. Thereaction mixture was cooled, concentrated to dryness, and partitionedbetween EtOAc (30 mL) and water (20 mL). The organic phase was dried(MgSO4), filtered, concentrated, and purified by flash chromatography onsilica gel (EtOAc) to afford (E)-4-(3-(5-(2-cyclopropylvinyl)-2-methoxyphenoxy)propyl)morpholine as an orange oil (629 mg, 37%).1H NMR (DMSO-d6, 500 MHz) δ 1.03 (d, J = 7 Hz, 6 H), 2.37 (m, 5H),2.69 (m, 2H), 3.54 (m, 4H), 3.73 (s, 3H), 4.09 (t, J = 6 Hz, 2H), 6.12 dd(J 7.2, 16.2 Hz, 1H), 6.23 (d, J = 16.2 Hz, 1H), 6.85 (s, 2H), 7.03 (s, 1H).13C NMR (DMSO-d6, 125 MHz) δ 7.35, 14.6, 29.8, 53.9, 55.8, 56.3, 67.2,67.5, 110.9, 112.2, 119.0, 127.3, 131.4, 133.1, 148.7, 148.9. (+) LRMS(ESI) m/z 306 [M + H]+.(E)-2-(2-Methoxy-5-(3-methylbut-1-enyl)phenoxy)ethanol (5c).

(E)-5-(2-Isopropylopropylvinyl)-2-methoxyphenol (0.73g, 3.8 mmol)was dissolved in acetone (16 mL). Potassium carbonate (2.63 g, 19 mmol)

and bromoethanol (0.54 mL, 7.6 mmol) were added, and the reactionmixture was heated at reflux for 16 h. The reaction mixture was cooled,concentrated to dryness and partitioned between EtOAc (30 mL) andwater (20 mL). The organic phase was dried (MgSO4), filtered,concentrated and purified by flash chromatography on silica gel(EtOAc) to afford (5c) as a colorless oil (190 mg, 20%). 1H NMR(DMSO-d6, 500 MHz) δ 1.06 (d, J = 7 Hz, 6 H), 2.42 (m, 1H), 3.72(br s, 2H), 3.75 (s, 3H), 3.99 (t, J = 5 Hz, 1H), 4.81 (m, 1H), 6.14(dd, J = 7, 16 Hz, 1H), 6.27 (d, J = 16 Hz, 1H), 6.88 (m, 2H), 7.02(s, 1H). (+) LRMS (ESI) m/z 235 [M + H]+.

(E)-3-(5-(2-Cyclopropylvinyl)-2-methoxyphenoxy)propan-1-ol(5d). (E)-5-(2-Cyclopropylopropylvinyl)-2-methoxyphenol (1.0 g,5.26 mmol) was dissolved in acetone (15 mL). Potassium carbonate(3.6 g, 2.63 mmol) and 3-bromo-1-propanol (0.92 mL, 10.5 mmol)were added, and the reaction mixture was heated at reflux for 16 h.The reaction mixture was cooled, concentrated to dryness, andpartitioned between EtOAc (30 mL) and water (20 mL). The organicphase was dried (MgSO4), filtered, concentrated, and purified by flashchromatography on silica gel (EtOAc:Hexane 7:3) to afford (5d) as awhite solid (1.25 g, 96%). 1H NMR (DMSO-d6, 500 MHz) δ 0.48 (m,2 H), 0.76 (m, 1H), 1.52 (m, 1H), 1.85 (m, 2H), 3.56 (m, 2H), 3.72(s, 3H), 4.01 (t, J = 5 Hz, 2H), 4.50 (t, J = 5 Hz, 1H), 5.70 (dd, J = 7,16 Hz, 1H), 6.36 (d, J = 16 Hz, 1H), 6.80 (d, J = 8 Hz, 1H), 6.85(d, J = 8 Hz, 1H), 6.95 (s, 1H). (+) LRMS (ESI) m/z 249 [M + H]+.

Synthesis of Flindersine and Analogues. 2,4-Dichloroquino-line. Aniline (6.7 g, 72 mmol) and malonic acid (11.7 g, 112 mmol)were dissolved in POCl3 (60 mL), heated at reflux for 5 h, cooled, andpoured into ice (an exothermic reaction occurred, and some productwas lost). The crude product was filtered off and purified on a shortcolumn of silica gel using hexane (500 mL) and 90% hexane/EtOAc(500 mL) to afford 2,4-dichloroquinoline (5.56 g, 39%) as a whitesolid. 1H NMR (DMSO-d6, 500 MHz) δ 7.48 (s, 2H), 7.64 (t, J = 8Hz, 1H), 7.75 (t, J = 8 Hz, 1H), 8.03 (d, J = 8 Hz, 1H), 8.17 (d, J = 8Hz, 1H). (+) LRMS (ESI) m/z 198:200:202 (100:60:10) [M + H]+.

2,4-Dimethoxyquinoline. 2,4-Dichloroquinoline (5.56 g, 28 mmol)was heated under reflux in a methanolic sodium methoxide solution(6 g sodium in 150 mL of MeOH) for 20 h. The reaction mixture wascooled and poured into ice water, and the residue was filtered off andpurified on a short column of silica gel using 95% hexane/EtOAc toafford 2,4-dimethoxyquinoline (3.88 g, 73%) as a white solid and 4-chloro-2-methoxyquinoline as a white solid (1.06 g, 20%). 1H NMR(DMSO-d6, 500 MHz) δ 4.01 (s, 3H), 4.10 (s, 3H), 6.25 (s, 1H), 7.36(t, J = 8 Hz, 1H), 7.63 (t, J = 8 Hz, 1H), 7.82 (d, J = 8 Hz, 1H), 8.08(d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 190 [M + H]+. 4-Chloro-2-methoxyquinoline: 1H NMR (DMSO-d6, 500 MHz) δ 4.10 (s, 3H),7.06 (s, 1H), 7.49 (t, J = 8 Hz, 1H), 7.70 (t, J = 8 Hz, 1H), 7.89 (d, J =8 Hz, 1H), 8.13 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 194:196(3:1) [M + H]+.

2,4-Dimethoxy-3-(3-methylbut-2-enyl)quinoline. 2,4-Dimethoxy-quinoline (0.95 g, 5 mmol) was dissolved in dry THF (20 mL) andcooled to 0 °C under an atmosphere of argon. n-BuLi (2.5M, 2.8 mL,7 mmol) was added dropwise with stirring, and the reaction mixturewas stirred for a further 30 min at 0 °C. 1-Bromo-3-methylbut-2-ene(1.27 g, 1 mL, 8.55 mmol) was added dropwise, and the reaction wasstirred for a further 30 min at 0 °C. The reaction mixture was thenwarmed to rt and stirred for a further 1 h. The reaction mixture wasconcentrated to dryness and purified by flash chromatography on silicagel (9:1 hexane/EtOAc) to afford 2,4-dimethoxy-3-(3-methylbut-2-enyl)quinoline as a clear oil (0.870 g, 67%). 1H NMR (DMSO-d6,500 MHz) δ 1.72 (s, 3H), 1.84 (s, 3H), 3.49 (d, J = 7 Hz, 2H),3.99 (s, 3H), 4.12 (s, 3H), 5.25 (br t, J = 7 Hz, 1H), 7.39 (t, J = 7.5 Hz,1H), 7.59 (t, J = 7.5 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.94 (d, J = 7.5 Hz,1H). (+) LRMS (ESI) m/z 258 [M + H]+.

3-((3,3-Dimethyloxiran-2-yl)methyl)-2,4-dimethoxyquinoline.2,4-Dimethoxy-3-(3-methylbut-2-enyl)quinoline (0.870 g, 3.4 mmol)was dissolved in CHCl3 (10 mL), mCPBA (2.91 g, 16.9 mmol) wasadded, and the reaction mixture was stirred for 4 h. The reactionmixture was extracted with 2 N NaCO3 (4 × 50 mL), dried with MgSO4,filtered, and concentrated to afford the crude epoxide (0.956 g, 100%),which was used in the next reaction without further purification.

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1H NMR (CDCl3, 500 MHz) δ 1.33 (s, 3H), 1.48 (s, 3H), 2.94(dd, J = 13, 5 Hz, 1H), 3.08 (dd, J = 11.5, 6.5 Hz, 1H), 3.13 (t, J = 7.5Hz, 1H), 4.03 (s, 3H), 4.13 (s, 3H), 7.40 (t, J = 7.5 Hz, 1H), 7.62(t, J = 7.5 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H).13C NMR (CDCl3, 125 MHz) δ 19.23, 24.33, 25.03, 53.95, 59.40,62.97, 63.37, 113.73, 121.24, 122.29, 124.02, 127.59, 129.49, 146.72,162.55, 163.03. (+) LRMS (ESI) m/z 274 [M + H]+.5-Methoxy-2,2-dimethyl-2H-pyrano[3,2-c]quinoline (3). A solu-

tion of 3-((3,3-dimethyloxiran-2-yl)methyl)-2,4-dimethoxyquinoline(5.8 g, 21.2 mmol) in DMSO (150 mL) and 2 N KOH (30 mL)was heated at 90 °C for 20 h. The reaction mixture was poured intowater and extracted with CHCl3 (2 × 100 mL). The aqueous phasewas neutralized with HCl to pH 7. Extraction with CHCl3 (3 × 100 mL)gave a brown oil that was dried (MgSO4), filtered, and concentrated.Purification on silica gel using flash chromatography (15% EtOAc inhexane) gave 5-methoxy-2,2-dimethyl-2H-pyrano[3,2-c]quinoline (3)(2.3g, 45%) and the diol, 1-(2,4-dimethoxyquinolin-3-yl)-3-methyl-butane-2,3-diol (0.326 g). 3: 1H NMR (CDCl3, 500 MHz) δ 1.56 (s,6H), 4.10 (s, 3H), 5.60 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 7.34(t, J = 7.5 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H),8.03 (d, J = 7.5 Hz, 1H). (+) LRMS (ESI) m/z 242 [M + H]+. 1-(2,4-Dimethoxyquinolin-3-yl)-3-methylbutane-2,3-diol: 1H NMR (CDCl3,500 MHz) δ 1.50 (s, 6H), 3.94 (s, 3H), 4.14 (s, 3H), 6.86 (d, J = 15Hz, 1H), 6.93 (d, J = 15 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.60 (t, J =7.5 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.98 (d, J = 7.5 Hz, 1H). (+)LRMS (ESI) m/z 274 [M + H]+.2,2-Dimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one Flindersine

(2a). 2,4-Dihidroxyquinolinone (6.5 g, 40.3 mmol) and methylcroton-aldehyde (6.79 g, 7.7 mL, 80.6 mmol) were suspended in acetonitrile(400 mL). Ytterbium trifluoromethanesulfonate (2.5 g, 4.03 mmol)was added, and the reaction mixture was refluxed for 16 h. Thereaction mixture was cooled, filtered, and concentrated to dryness.Purification on silica using flash chromatography afforded 2a (3.8 g,42%) as a yellow solid. 1H NMR (DMSO-d6, 500 MHz) δ 1.50(s, 6H), 5.67 (d, J = 10 Hz, 1H), 6.53 (d, J = 10 Hz, 1H), 7.19 (t, J =8 Hz, 1H), 7.28 (d, J = 8 Hz, 1H), 7.51 (t, J = 8 Hz, 1H), 7.77 (d, J =8 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 27.67, 78.57, 105.45,114.05, 115.23, 116.58, 121.53, 121.87, 126.66, 130.78, 138.15, 155.36,160.18. (+) LRMS (ESI) m/z 228 [M + H]+.2,2,6-Trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2b). A mix-

ture of 3-methyl-2-butenal (1.64 mL, 17.1 mmol) and magnesiumsulfate (5.43 g) in pyridine (15 mL) was refluxed for a few minutes. Asolution of 4-hydroxy-1-methyl-2(1H)-quinolone (2.5 g, 14.3 mmol)in pyridine (9 mL) was then added slowly (over 20 min) to the abovemixture, and the mixture was refluxed overnight. The reaction mixturewas cooled to room temperature, and the pyridine was removed byevaporation. The resulting residue was diluted with water andextracted with DCM. The organic phase was washed with 1 N HCl,aqueous sodium bicarbonate, and brine solution and dried overanhydrous MgSO4. The organic phase was then concentrated todryness to afford 2,2,6-trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one(2b) as a brown oil (3.38 g, 98%). 1H NMR (500 MHz, DMSO-d6) δppm 1.48 (s, 6H), 3.59 (s, 3H), 5.68 (d, 1H, J = 10 Hz), 6.57 (d, 1H,J = 10 Hz), 7.27 (t, 1H, J = 7.5 Hz), 7.49 (d, 1H, J = 11 Hz), 7.62(t, 1H, J = 16 Hz), 7.86 (d, 1H, J = 8 Hz).2,2,9-Trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2c). A mix-

ture of 3-methyl-2-butenal (1.92 mL, 20 mmol) and magnesiumsulfate (6.34 g) in pyridine (17.5 mL) was refluxed for a few minutes.A solution of 4-hydroxy-6-methylquinolin-2(1H)-one (2.92 g, 16.7mmol) in pyridine (10.5 mL) was then added slowly (over 20 min) tothe above mixture, and the mixture was refluxed overnight. Thereaction mixture was cooled to room temperature, and the pyridinewas removed by evaporation. The resulting residue was diluted withwater and extracted with DCM. The organic phase was washed with1 N HCl, aqueous sodium bicarbonate, and brine solution and driedover anhydrous MgSO4. The organic phase was then concentrated todryness to afford 2,2,9-trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one(2c) as a brown oil (3.81 g, 95%). 1H NMR (500 MHz, DMSO-d) δ 1.48(s, 6H), 2.35 (s, 3H), 5.64 (d, 1H, J = 10 Hz), 6.51 (d, 1H, J = 9.5 Hz),

7.17 (d, 1H, J = 8.5 Hz), 7.32 (d, 1H, J = 7.5 Hz), 7.54 (s, 1H), 11.37(s, 1H).

2-Methyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2e,f). 2,4-Dihy-droxyquinolinone (1.27 g, 7.8 mmol) and crotonaldehyde (1.1 g, 1.4mL, 15.6 mmol) were suspended in acetonitrile (40 mL). Ytterbiumtrifluoromethanesulfonate (230 mg, 0.37 mmol) was added, and thereaction mixture was refluxed for 16 h. The reaction mixture wascooled, filtered, and concentrated to dryness. Purification on silicausing flash chromatography afforded 2e,f (341 mg, 18%) as a yellowsolid. 1H NMR (CDCl3, 500 MHz) δ 1.58 (s, 3H), 5.29 (m, 1H), 5.64(dd, J = 2, 9.5 Hz, 1H), 6.83 (d, J = 10 Hz, 1H), 7.23 (t, J = 8 Hz, 1H),7.41 (d, J = 8 Hz, 1H), 7.52 (t, J = 8 Hz, 1H), 7.89 (d, J = 8 Hz, 1H),11.82 (br s, 1H). 13C NMR (CDCl3, 125 MHz) δ 21.81, 73.83, 106.25,115.32 116.48, 118.53, 122.56, 122.77 122.78, 131.35, 137.98, 158.44,162.58. (+) LRMS (ESI) m/z 214 [M + H]+.

2,2-Dimethyl-2H-pyrano[3,2-c]pyridin-5(6H)-one (6). A mixtureof 3-methyl-2-butenal (2.59 mL, 27 mmol) and magnesium sulfate(8.55 g) in pyridine (23 mL) was refluxed for a few minutes. Asolution of 2,4-dihydroxypyridine (2.5 g, 22.5 mmol) in pyridine(14 mL) was then added slowly (over 30 min) to the above mixtureand refluxed overnight. The reaction mixture was cooled to roomtemperature, and the pyridine was removed. The resulting residue wasdiluted with water and extracted with DCM. The organic phase waswashed with 1 N HCl, aqueous sodium bicarbonate, and brine andthen dried over anhydrous MgSO4. The organic phase was thenconcentrated to dryness to afford 2,2-dimethyl-2H-pyrano[3,2-c]-pyridin-5(6H)-one (6) as a light yellow solid (3.18 g, 80%). 1H NMR(500 MHz, DMSO-d6) δ 1.37 (s, 6H), 5.46 (d, J = 10 Hz, 1H), 5.81(d, J = 7 Hz, 1H), 6.38 (d, 1H, J = 10 Hz, 1H), 7.18 (d, J = 7 Hz, 1H),11.18 (br s, 1H). (+) LRMS (ESI) m/z 178.1 [M + H]+.

Photochemical 2 + 2 Cycloaddition. General Procedure. Thecorresponding flindersine analogue (1 equiv) and styrene (2 equiv)were dissolved in CHCl3 (2 mL) and added to either a 250 mLjacketed round-bottomed flask or a pyrex test tube. The solvent wasevaporated to form a thin film, and the flask was then cooled andirradiated with UV light (125 or 400 W, 16 h). The resulting browngum was purified by reverse-phase HPLC to afford the title compound.

(1R,2R,2aR,10aS)-3-Methoxy-1,10,10-trimethyl-2-(3,4,5-trimethoxy-phenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano[3,2-c]-quinoline and (1S,2S,2aS,10aR)-3-Methoxy-1,10,10-trimethyl-2-(3,4,5-trimethoxyphenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano-[3,2-c]quinoline (Racemic Precursor of 1, Scheme 1). From 5-methoxy-2,2-dimethyl-4a,10b-dihydro-2H-pyrano[3,2-c]quinoline (3) (0.5 mmol)and 4a (1.0 mmol). Purified by isocratic elution MeOH/H2O (1% TFA,3:2) (102.3 mg, 36%). 1H NMR (DMSO-d6, 600 MHz) δ 1.12 (d, J = 6Hz, 3H), 1.16 (s, 3H), 1.50 (s, 3H), 2.36 (m, 2H), 3.23(s, 3H), 3.24 (s, 6H), 3.36 (t, J = 8 Hz, 1H), 3.51 (s, 3H), 3.71 (t, J =6.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.57 (d, J =7.5 Hz, 1H), 8.03 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz) δ20.97, 24.33, 25.64, 35.24, 46.00, 50.46, 53.08, 55.72, 60.47, 76.08, 102.64,105.88, 118.57, 122.90, 123.02, 126.18, 129.22, 135.18, 135.84, 145.38,156.97, 151.51, 161.79. (+) LRMS (ESI) m/z 450 [M + H]+. HRMS(ESI): calcd for C27H32NO5 [M + H]+, 450.2275; found, 450.2256.

(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (1). The previous compound (39 mg,0.07 mmol) was dissolved in acetic acid (2 mL). Potassium iodide(12 mg, 0.14 mmol) was added, and the reaction mixture was heated at50 °C for 4 h. The reaction mixture was extracted with a 10% NaHSO4solution and CHCl3 (3 × 30 mL), dried with MgSO4, filtered, andconcentrated to dryness to afford 1 (35.4 mg, 83%) as a white solid.1H NMR (DMSO-d6, 500 MHz) δ 1.12 (d, J = 6 Hz, 3H), 1.13 (s,3H), 1.47 (s, 3H), 2.33 (m, 2H), 3.16 (m, 1H), 3.29 (s, 3H), 3.33 (s,3H), 3.68 (t, J = 7.5 Hz, 1H), 4.06 (m, 1H), 6.11 (s, 2H), 7.13 (d, J =7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.81 (d,J = 8 Hz, 1H), 11.05 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 20.3,23.3, 24.4, 31.7, 35.2, 45.7 49.4, 55.0, 59.8, 76.5, 105.4, 107.8, 114.6,114.8, 120.9, 121.7, 129.8, 135.8, 135.9, 137.5, 151.7, 155.5, 162.2. (+)LRMS (ESI) m/z 436 [M + H]+. HRMS (ESI): calcd for C26H29NO5Na

Journal of Medicinal Chemistry Article

dx.doi.org/10.1021/jm401321v | J. Med. Chem. 2014, 57, 1252−12751266

[M + Na]+, 458.1938; found, 458.1928. (for other data, see theSupporting Information).Synthesis of 1 from flindersine (2a, 0.6 mmol) and 4a (1.2 mmol).

Purified by flash chromatography on neutral alumina (DCM, then 1:1DCM/CHCl3 then CHCl3). The fractions containing MH+ 436 werecombined and purified by isocratic elution with MeCN/H2O (55:45)(41 mg, 16%), which was identical to the sample above by 1H and 13CNMR and MS. There were small differences in the 1H NMR betweenthe synthetic 1 and the natural product (euodenine A), attributable tothe fact that the natural product contained some TFA (present in thesolvent used for HPLC purification). They were shown to be identicalby mixing the two NMR samples together and rerunning the 1H NMRspectrum. A single spectrum was obtained, identical to that ofeuodenine A.(1R,2R,2aR,10aS)-3-Methoxy-1,10,10-trimethyl-2-(3,4,-dimethoxy-

phenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano[3,2-c]-quinoline and (1S,2S,2aS,10aR)-3-Methoxy-1,10,10-trimethyl-2-(3,4,-dimethoxyphenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]-pyrano[3,2-c]quinoline (Racemic Precursor of 10). From 5-methoxy-2,2-dimethyl-4a,10b-dihydro-2H-pyrano[3,2-c]quinoline (3, 0.5mmol) and 1,2-dimethoxy-4-propenylbenzene (1.0 mmol). Purifiedby isocratic elution MeCN/H2O (1% TFA, 55:45) (36.4 mg, 17%). 1HNMR (DMSO-d6, 600 MHz) δ 1.12 (d, J = 5.5 Hz, 3H), 1.17 (s, 3H),1.52 (s, 3H), 2.38 (m, 2H), 3.06 (s, 3H), 3.26 (s, 3H), 3.23 (t, J = 9Hz, 1H), 3.63 (s, 3H), 3.73 (t, J = 9 Hz, 1H), 5.97 (d, J = 1.5 Hz, 1H),6.40 (dd, J = 2, 8 Hz, 1H), 6.65 (d, J = 8 Hz, 1H), 7.37 (t, J = 8 Hz,1H), 7.59 (m, 2H), 8.04 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 150MHz) δ 20.88, 24.52, 25.83, 33.12, 35.45, 46.53, 50.17, 53.38, 55.42,56.59, 77.07, 103.68, 111.82, 111.83, 119.56, 121.37, 122.05, 123.72,127.57, 129.83, 132.91, 145.69, 147.73, 148.26, 156.63, 162.28. (+)LRMS (ESI) m/z 420 [M + H]+. HRMS (ESI): calcd for C26H31NO4[M + H]+, 420.2169; found, 420.2159.(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4-dimethoxyphenyl)-

1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4-di-methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (10). The previous compound (16.6 mg,0.04 mmol) was dissolved in acetic acid (2 mL). Potassium iodide (9.4mg, 0.056 mmol) was added, and the reaction mixture was heated at50 °C for 20 h. The reaction mixture was partitioned between with a10% NaHSO4 solution and CHCl3 (3 × 30 mL), and the organic layerwas then extracted with aqueous ammonia solution (10%, 20 mL),dried with MgSO4, filtered, and concentrated to dryness to afford 10(9.3 mg, 57%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ1.16 (d, J = 6 Hz, 3H), 1.17 (s, 3H), 1.53 (s, 3H), 2.39 (m, 2H), 3.28(s, 3H), 3.38 (m, 1H), 3.67 (s, 3H), 3.74 (t, J = 7.5 Hz, 1H), 6.39 (brs, 1H), 6.52 (d, J = 8 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 7.19 (t, J = 8 Hz,1H), 7.19 (d, J = 7.5 Hz, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.87 (d, J = 8Hz, 1H), 11.07 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 21.66,22.95, 25.64, 32.97, 35.96, 46.72, 49.86, 55.54, 56.14, 77.19, 108.54,111.59, 112.59, 115.38, 115.59, 121.40, 121.74, 122.52, 130.77, 133.24,138.18, 147.87, 148.07, 156.17, 162.66. (+) LRMS (ESI) m/z 406[M + H]+. HRMS (ESI): calcd for C25H27NO4Na [M + Na]+, 428.1832;found, 428.1843.(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,5-dimethoxyphenyl)-

1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,5-di-methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (11). From flindersine (2a, 0.39 mmol)and (E)-1,3-dimethoxy-5-(prop-1-enyl)benzene (4b, 0.79 mmol). Theresidue was then partially purified by flash chromatography on neutralalumina using dichloromethane/chloroform (1:1) and then furtherpurified by isocratic elution MeCN/H2O (60:40) (10 mg, 6%). 1HNMR (CDCl3, 500 MHz) δ 1.23 (s, 6H), 1.57 (s, 3H), 2.32 (app t, J =8.5 Hz, 1H), 2.55 (m, 1H), 3.41 (app t, J = 9.5 Hz, 1H), 3.45 (s, 3H),3.91 (m, 1H), 6.14 (s, 2H), 6.17 (s, 1H), 7.14 (d, J = 8 Hz, 1H), 7.26(app t, J = 7.5 Hz, 1H), 7.49 (app t, J = 8 Hz, 1H), 8.00 (d, J = 8 Hz, 1H),10.25 (br s, 1H). 13C NMR (CDCl3, 125 MHz) δ 20.64, 24.24, 25.09,32.41, 35.12, 46.86, 50.63, 55.15, 77.95, 99.43, 106.55, 107.23, 115.95,116.40, 122.77, 122.84, 130.79, 136.62, 142.17, 158.60, 160.22, 163.77. (+)LRMS (ESI) m/z 406 [M + H]+. HPLC (Betasil C18 150 × 4.6 mm,

acetonitrile/water (70:30) at 1.5 mL/min) tR = 3.459 min. HRMS(ESI): calcd for C25H27NO4Na [M + Na]+, 428.1832; found, 428.1837.

(1R,2R,2aR,10aS)-2-(3-Bromo-4-methoxyphenyl)-10,10,dimethyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Bromo-4-methox-yphenyl)-10,10,dimethyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (12). From 2a (0.5 mmol)and 4c (1.0 mmol). Purified by flash chromatography on neutral alumina(DCM, then 1:1 DCM/CHCl3 then CHCl3). The fractions containingMH+ 454/456 were combined and purified by reverse-phase HPLC withisocratic elution MeCN/H2O (2:3) over 60 min to afford 30.1 mg (13%).1H NMR (DMSO-d6, 500 MHz) δ 1.10 (d, J = 6.5 Hz, 3H), 1.11 (s, 3H),1.47 (s, 3H), 2.32 (m, 2H), 3.32 (dd, J = 9, 10 Hz, 1H), 3.69 (s, 3H), 3.63(dd, J = 7.5, 8.5 Hz, 1H), 6.74 (d, J = 8 Hz, 1H), 6.80 (dd, J = 1.8, 8 Hz,1H), 7.05 (d, J = 1.8 Hz, 1H), 7.12 (dd, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5Hz, 1H), 7.41 (dd, J = 7.5 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 11.04(s, 1H). (+) LRMS (ESI) m/z 454 [M + H79Br]+, 456 [M + H81Br]+.HRMS (ESI): calcd for C24H25BrNO3Na [M + Na]+, 476.0832; found,476.0831.

(1R,2R,2aR,10aS)-2-(2-Chloro-3,4-dimethoxyphenyl)-10,10,di-methyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(2-Chloro-3,4-dimethoxyphenyl)-10,10,dimethyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (13).From flindersine (2a, 0.5 mmol) and 4d (1.0 mmol). Purified by flashchromatography on neutral palumina (DCM, then 1:1 DCM/CHCl3then CHCl3). The fractions containing MH+ 440 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O (2:3)over 60 min to give 20.4 mg (9%). 1H NMR (DMSO-d6, 500 MHz) δ1.14 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 2.36 (m, 2H), 3.36(dd, J = 9, 10 Hz, 1H), 3.63 (s, 3H), 3.69 (dd, J = 7.5, 8.5 Hz, 1H), 3.73(s, 3H), 6.34 (m, 1H), 7.16 (dd, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz,1H), 7.45 (dd, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.04 (s, 1H).(+) LRMS (ESI) m/z 440 [M + H35Cl]+, 442 [M + H37Cl]+ HRMS(ESI): calcd for C25H27ClNO4Na [M + Na]+, 462.1443; found, 462.1452.

(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and(1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(phenyl)-1,2,2a,4,10,10a-hex-ahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one: (14). From3 and (E)-prop-1-enylbenzene. Purified by isocratic elution MeOH/H2O (1% TFA, 7:3) to afford 20 mg (5%). 1H NMR (CDCl3, 500MHz) δ 1.24 (d, J = 6.5 Hz, 3H), 1.31 (s, 3H), 1.67 (s, 3H), 2.43(t, J = 9 Hz, 1H), 2.59 (m, 1H), 3.50 (t, J = 11.5 Hz, 1H), 3.63 (s,3H), 3.83 (t, J = 8.5 Hz, 1H), 6.77 (d, J = 7 Hz, 2H), 7.13 (t, J = 7 Hz,2H), 7.18 (t, J = 7 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.82 (t, J = 7.5Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.25 (d, J = 7.5 Hz, 1H). 13C NMR(CDCl3, 125 MHz) δ 19.8, 24.2 (2×), 24.2, 32.2, 34.0, 45.5, 58.8, 80.1,103.2, 118.0, 120.6, 122.4, 126.5, 127.3, 127.4 (2×), 128.3 (2×), 133.2,134.7, 137.9, 163.4. (+) LRMS (ESI) m/z 360 [M + H]+. HRMS(ESI): calcd for C24H26NO2 [M + H]+, 360.1958; found, 360.1969.This compound (10 mg, 0.028 mmol) was dissolved in acetic acid(2 mL). Potassium iodide (9.4 mg, 0.056 mmol) was added, and thereaction mixture was heated at 50 °C for 20 h. The reaction mixturewas extracted with a 10% NaHSO4 solution and CHCl3 (3 × 30 mL),dried with MgSO4, filtered, and concentrated to dryness to afford 14(2.6 mg, 27%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ1.13 (d, J = 6 Hz, 3H), 1.14 (s, 3H), 1.50 (s, 3H), 2.36 (m, 2H), 2.46(m, 1H), 3.39 (t, J = 8 Hz, 1H), 3.71 (t, J = 6.5 Hz, 1H), 6.89 (m, 2H),7.02 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.42 (t,J = 7.5 Hz, 1H), 7.82 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 346[M + H]+. HRMS (ESI): calcd for C23H24NO2 [M + H]+, 346.1801;found, 346.1791.

(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(4-methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(4-me-thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one (15). From 3 (0.5 mmol) and trans anethole(1.0 mmol). Purified by isocratic elution MeCN/H2O (1% TFA,55:45) (12.6 mg, 6%). 1H NMR (DMSO-d6, 500 MHz) δ 1.12 (d, J =5.5 Hz, 3H), 1.15 (s, 3H), 1.52 (s, 3H), 2.37 (m, 1H), 2.43 (m, 1H),3.28 (s, 3H), 3.40 (t, J = 9 Hz, 1H), 3.62 (s, 3H), 3.73 (t, J = 9 Hz,

Journal of Medicinal Chemistry Article

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1H), 6.58 (d, J = 8 Hz, 2H), 6.63 (d, J = 8 Hz, 2H), 7.36 (br t, J = 8Hz, 1H), 7.57 (m, 2H), 8.02 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z390 [M + H]+. HRMS (ESI): calcd for C25H28NO3 [M + H]+,390.2064; found, 390.2080. This compound (6.4 mg, 0.016 mmol)was dissolved in acetic acid (2 mL). Potassium iodide (9.4 mg, 0.056mmol) was added, and the reaction mixture was heated at 50 °C for 20h. The reaction mixture was partitioned between a 10% NaHSO4solution and CHCl3 (3 × 30 mL), and the organic layer was thenextracted with aqueous ammonia solution (10%, 20 mL), dried withMgSO4, filtered, and concentrated to dryness to afford 15 (4.6 mg,100%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ 1.16 (d, J =5.5 Hz, 3H), 1.17 (s, 3H), 1.53 (s, 3H), 2.37 (t, J = 9 Hz, 1H), 2.44(m, 1H), 3.37(t, J = 9 Hz, 1H), 3.67 (s, 3H), 3.72 (t, J = 9 Hz, 1H),6.63 (d, J = 8 Hz, 2H), 6.85 (d, J = 8 Hz, 2H), 7.18 (d, J = 8 Hz, 1H),7.20 (t, J = 8 Hz, 1H), 7.47 (t, J = 6.5 Hz, 1H), 7.86 (d, J = 8 Hz, 1H),11.02 (s, 1H). (+) LRMS (ESI) m/z 376 [M + H]+. HRMS (ESI):calcd for C24H25NO3Na [M + Na]+, 398.1727; found, 398.1727.(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(2,4,5-trimethoxyphenyl)-

1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(2,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (16). Two-hundred milligrams offlindersine (2a, 0.88 mmol) and α-asarone (1.66 mmol). Purified byisocratic elution MeCN/H2O (1% TFA, 55:45) (43.9 mg, 0.1 mmol,12%). 1H NMR (CDCl3, 500 MHz) δ 1.19 (d, J = 6.5 Hz, 3H), 1.25(s, 3H), 1.58 (s, 3H), 2.33 (t, J = 8.5 Hz, 1H), 2.50 (m, 1H), 3.04 (s,3H), 3.71 (s, 3H), 3.89 (s, 3H), 3.89 (m, 1H), 3.96 (dd, J = 8, Hz,1H), 6.02 (s, 1H), 6.40 (s, 1H), 7.19 (d, J = 8 Hz, 1H), 7.28 (t, J = 8Hz, 1H), 7.49 (t, J = 8 Hz, 1H), 8.03 (d, J = 8 Hz, 1H), 10.51 (br s,1H). 13C NMR (CDCl3, 125 MHz) δ 20.64, 24.12, 25.12, 32.10, 34.13,41.64, 46.84, 56.03, 56.42, 57.73, 78.17, 78.17, 98.59, 107.19, 112.09,116.27, 120.18, 122.70, 122.93, 130.77, 136.31, 142.53, 148.34, 153.09,158.70, 163.58. (+) LRMS (ESI) m/z 436 [M + H]+. HRMS (ESI):calcd for C26H29NO5 [M + H]+, 458.1938; found, 458.1928.(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3-methoxy-4-benzyloxy-

phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3-me-thoxy-4-benzyloxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (17). From flindersine(2a, 0.5 mmol) and isoeugenol benzylether (1.0 mmol). Purified byflash chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3) and then reverse-phase HPLC with isocratic elutionMeCN/H2O (1% TFA, 7:3) to afford 8.3 mg (3%). 1H NMR (DMSO-d6, 500 MHz) δ 1.10 (d, J = 6.5 Hz, 3H), 1.12 (s, 3H), 1.47 (s, 3H), 2.33(m, 2H), 3.09 (m, 1H), 3.23 (s, 3H), 3.68 (t, J = 9 Hz, 1H), 4.90 s, 2H),3.96 (dd, J = 8, Hz, 1H), 6.36 (s, 1H), 6.46 (d, J = 7.5 Hz, 1H), 6.70(d, J = 7.5 Hz, 1H), 7.13 (m, 2H), 7.27−7.43 (m, 6H), 7.80 (d, J = 7.5Hz, 1H), 11.02 (s, 1H). (+) LRMS (ESI) m/z 482 [M + H]+. HRMS(ESI): calcd for C31H31NO4Na [M + Na]+, 504.2145; found, 504.2129.(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3-methoxy-4-hydroxy-

phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3-me-thoxy-4-hydroxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (18). 17 (7 mg, 0.014 mmol) wasdissolved in MeOH, and Pd/C (10%, 2 mg) was added. Hydrogen gaswas bubbled through the reaction mixture for 20 min, and the resultingsolution was stirred under an atmosphere of hydrogen for 20 h.Filtration through Celite and concentration afforded 18 as a whitesolid (5.4 mg, 100%). 1H NMR (DMSO-d6, 500 MHz) δ 1.10 (d, J =6.5 Hz, 3H), 1.11 (s, 3H), 1.47 (s, 3H), 2.33 (m, 2H), 3.23 (m, 4H),3.68 (t, J = 9 Hz, 1H), 6.26 (s, 1H), 6.32 (d, J = 7.5 Hz, 1H), 6.41(d, J = 7.5 Hz, 1H), 7.13 (m, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.80 (d, J =7.5 Hz, 1H), 10.99 (s, 1H). (+) LRMS (ESI) m/z 392 [M + H]+.HRMS (ESI): calcd for C24H25NO4Na [M + Na]+, 414.1676; found,414.1665.(1R,2R,2aR,10aS)-1-Ethyl-10,10-trimethyl-2-(3,4,-dimethoxy-

phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and 1S,2S,2aS,10aR)-1-Ethyl-10,10-trimethyl-2-(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (19). From flindersine (2a, 0.5 mmol)and 4e (1.0 mmol). Purified by flash chromatography on neutral

alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractionscontaining MH+ 420 were combined and purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (3:2) to afford 12.2 mg(6%). 1H NMR (DMSO-d6, 500 MHz) δ 0.65 (t, J = 5.5 Hz, 3H), 1.12(s, 3H), 1.46 (s, 3H), 1.49 (m, 3H), 1.68 (m, 1H), 2.28 (m, 1H), 2.36(t, J = 9 Hz, 1H), 3.16 (s, 3H), 3.37 (t, J = 9 Hz, 1H), 3.60 (s, 3H),3.65 (t, J = 9 Hz, 1H), 6.28 (br s, 1H), 6.48 (d, J = 8.5 Hz, 1H), 6.58(d, J = 8.5 Hz, 1H), 7.12(m, 2H), 7.41 (t, J = 8 Hz, 1H), 7.80 (d, J = 8Hz, 1H), 10.97 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 12.59,24.55, 26.21, 29.41, 32.33, 41.79, 44.71, 48.05, 55.42, 56.81, 77.04,109.01, 111.33, 112.31, 115.48, 115.65, 121.49, 121.90, 122.60, 130.8,134.39, 137.97, 147.50, 148.59, 156.02, 162.57. (+) LRMS (ESI) m/z420 [M + H]+. HRMS (ESI): calcd for C26H29NO4Na [M + Na]+,442.1989; found, 442.1971.

(1R,2R,2aR,10aS)-1-Ethyl-10,10-dimethyl-2-(2,3,4-trimethoxy-phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-10,10-dimethyl-2-(2,3,4-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (20). From flindersine (2a, 0.5mmol) and 4f (1.0 mmol). Purified by isocratic elution MeCN/H2O(60:40) (29.3 mg, 13%). 1H NMR (DMSO-d6, 600 MHz) δ 0.65 (t,J = 7.2 Hz, 3H), 1.12 (s, 3H), 1.45 (s, 3H), 1.49 (m, 1H), 1.65 (m,1H), 2.27 (m, 1H), 2.36 (t, J = 9 Hz, 1H), 3.30 (s, 6H), 3.37 (t, J = 9Hz, 1H), 3.51 (s, 3H), 3.66 (dd, J = 8, 9 Hz, 1H), 6.09 (s, 2H),7.12(m, 2H), 7.41 (ddd, J = 1.2, 7.5, 8.4 Hz, 1H), 7.80 (dd, J = 1.2, 8.4Hz, 1H), 11.02 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 12.4, 24.3,25.8, 29.6, 32.4, 42.5, 44.5, 48.7, 56.1, 60.5, 77.6, 106.7, 107.5, 115.5,115.6, 121.8, 122.7, 130.7, 136.8, 138.6, 139.1, 153.3, 156.0, 163.5. (+)LRMS (ESI) m/z 450 [M + H]+. HRMS (ESI): calcd for C27H32NO5[M + H]+, 450.2275; found, 450.2257.

(1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methoxyphenyl)-10,10-di-methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methox-yphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (21). From flindersine (2a, 0.5mmol) and 4g (1.0 mmol). Purified by flash chromatography onneutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). Thefractions containing MH+ 408 were combined and purified by reverse-phase HPLC with isocratic elution MeCN/H2O (3:2) to MeCN over60 min to afford 24 mg (12%). 1H NMR (DMSO-d6, 600 MHz) δ0.65 (t, J = 8 Hz, 3H), 1.19 (s, 3H), 1.50 (s, 3H), 1.52 (m, 1H), 1.67(m, 1H), 2.32 (m, 1H), 2.35 (m, 1H), 3.42 (t, J = 9 Hz, 1H), 3.64(t, J = 9 Hz, 1H), 3.83 (s, 3H), 6.63 (d, J = 9.6 Hz, 1H), 6.67 (br d, J =8 Hz, 1H), 6.78 (dd, J = 6, 8 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.15 (t,J = 7.5 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 11.01 (s, 1H). (+) LRMS(ESI) m/z 408 [M + H]+. HRMS (ESI): calcd for C25H26FNO3Na[M + Na]+, 430.1789; found, 430.1776.

(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (22). From flindersine (2a, 0.88 mmol)and 4h (1.76 mmol). Purified by flash chromatography on neutralalumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractionscontaining MH+ 464 were combined and purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (7:3) to afford 27.6 mg(12%). 1H NMR (DMSO-d6, 500 MHz) δ 0.73 (d, J = 6.5 Hz, 3H),0.88 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 1.80 (m, 1H), 2.33(m, 2H), 3.31 (s, 6H), 3.52 (s, 3H), 3.52 (t, J = 9 Hz, 1H), 3.67 (t, J =9 Hz, 1H), 6.12 (s, 2H), 7.13 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz,1H), 7.43 (t, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.00 (s, 1H).(+) LRMS (ESI) m/z 464 [M + H]+. HRMS (ESI): calcd forC28H33NO5Na [M + Na]+, 486.2251; found, 486.2236.

(1R,2R,2aR,10aS)-2-(3-Chloro-4-methoxyphenyl)-10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Chloro-4-methoxyphenyl)-10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (23).From flindersine (2a, 0.88 mmol) and 4i (1.76 mmol). Purified byreverse-phase HPLC with gradient elution MeCN/H2O (3:7) toMeCN over 60 min. Then repurified by flash chromatography

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(2% MeOH/DCM) to give 45 mg (11.7%) as a white solid. 1H NMR(DMSO-d6, 600 MHz) δ 0.68 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.88 Hz,3H), 1.12 (s, 3H), 1.48 (s, 3H), 1.80 (dd, J = 12.8, 6.5 Hz, 1H), 2.30−2.36 (m, 1H), 2.50 (m, 1H), 3.53 (t, J = 9.4 Hz, 1H), 3.61 (t, J = 8.8Hz, 1H), 3.69 (s, 3H), 6.70−6.78 (m, 2H), 6.88 (d, J = 1.56 Hz, 1H),7.10−7.16 (m, 2H), 7.42 (t, J = 7.50 Hz, 1H), 7.81 (d, J = 7.82 Hz,1H), 11.00 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 17.16, 20.29,22.42, 24.42, 29.73, 30.16, 39.31, 41.19, 44.20, 54.56, 75.43, 106.79,110.07, 113.54, 119.78, 120.68, 126.23, 128.48, 128.65, 133.65, 136.22,151.25, 154.70, 160.80. (+) LRMS (ESI) m/z 437.8 [M + H]+. HRMS(ESI): calcd for C26H28ClNO3Na [M + Na]+, 460.1650; found,460.1657.(1R,2R,2aR,10aS)-2-(3-Chloromethyl-4-methoxyphenyl)-10,10-di-

methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Chlor-omethyl-4-methoxyphenyl)-10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (24). From flindersine (2a, 0.44 mmol) and 4j (0.88mmol). Purified by reverse-phase HPLC with gradient elution MeCN/H2O + 1% TFA (3:7) to MeCN over 60 min. Then flash chromatography(DCM to 2% MeOH/DCM) to give 6.7 mg (3.4%) as a white solid. 1HNMR (600 MHz, DMSO-d6) δ 0.68 (d, J = 6.9 Hz, 3H), 0.85 (d, J = 6.9Hz, 3H), 1.13 (s, 3H), 1.49 (s, 3H), 1.81 (td, J = 13, 6.3 Hz, 1H), 2.36(dd, J = 15.6, 9.4 Hz, 1H), 2.5 (m, 1H), 3.52 (t, J = 9.4 Hz, 1H), 3.58−3.64 (m, 1H), 3.69 (s, 3H), 4.30−4.38 (m, 2H), 6.6 (d, J = 0.5 Hz 1H),6.80−6.86 (m, 2H), 7.08−7.16 (m, 2H), 7.40 (t, J = 7.7 Hz, 1H), 7.81 (d,J = 7.8 Hz, 1H), 10.94 (s, 1H). (+) LRMS (ESI) m/z 452.3 [M + H]+.HRMS (ESI): calcd for C27H30ClNO3 [M + H]+, 452.1987; found,452.2002.5-[(1R,2R,2aR,10aS)-1-Isopropyl-10,10-dimethyl-3-oxo-

2,2a,3,4,10,10a-hexahydro-1H-cyclobuta[4,5]pyrano[3,2,-c]-quinolinyl]-2-methoxyphenylacetate and 5-[(1S,2S,2aS,10aR)-1-Iso-propyl-10,10-dimethyl-3-oxo-2,2a,3,4,10,10a-hexahydro-1H-cyclobuta[4,5]pyrano[3,2,-c]quinolinyl]-2-methoxyphenylacetate(25). From flindersine (2a, 0.88 mmol) and 4k (1.76 mmol). Purifiedby flash chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions containing MH+ 462 werecombined and purified by reverse-phase HPLC with gradient elutionMeCN/H2O (5:95) to MeCN/H2O (2:3) over 60 min (18.6 mg,4.6%). 1H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J = 6.5 Hz, 3H),0.86 (d, J = 6.5 Hz, 3H), 1.11 (s, 3H), 1.46 (s, 3H), 1.79 (m, 1H), 2.01(s, 3H), 2.22 (m, 1H), 2.31 (m, 1H), 3.50 (dd, J = 9.5, 10 Hz, 1H),3.59 (s, 3H), 3.61 (dd, J = 9, 10 Hz, 1H), 6.53 (br s, 1H), 6.68 (m,2H), 7.11 (m, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.78 (d, J = 8 Hz). (+)LRMS (ESI) m/z 462 [M + H]+. HRMS (ESI): calcd for C28H31NO5Na [M + Na]+, 484.2094; found, 484.2113.(1R,2R,2aR,10aS)-1-Isopropyl,10,10-dimethyl-2-(3,4,-dimethoxy-

phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyl,10,10-dimethyl-2-(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (26). From flindersine(2a, 0.88 mmol) and 4l (1.76 mmol). Purified by flash chromatographyon neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). Thefractions containing MH+ 434 were combined and purified by reverse-phase HPLC with isocratic elution MeCN/H2O (7:3) (14 mg, 6%). 1HNMR (DMSO-d6, 500 MHz) δ 0.68 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5Hz, 3H), 1.12 (s, 3H), 1.47 (s, 3H), 1.79 (m, 1H), 2.32 (m, 2H), 3.12 (m,3H), 3.49 (t, J = 9 Hz, 1H), 3.61 (s, 3H), 3.63 (t, J = 9 Hz, 1H), 6.26 (s,1H), 6.49 (d, J = 7.5 Hz, 1H), 6.57 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.80 (d, J =7.5 Hz, 1H), 10.95 (s, 1H). (+) LRMS (ESI) m/z 434 [M + H]+. HRMS(ESI): calcd for C27H31NO4Na [M + Na]+, 456.2145; found, 456.2140.(1R,2R,2aR,10aS)-2-[4-Methoxy-3-(oxobutanoicacid)phenyl]-

10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-[4-Methoxy-3-(oxobutanoicacid)phenyl]-10,10-dimethyl-1-(1-meth-ylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (27). From flindersine (2a, 0.55 mmol) and 5a (1.1mmol). Purified by flash chromatography (2% up to 5% MeOH/DCM). The resulting residue was repurified by reverse-phase HPLCwith isocratic elution MeCN/H2O + 1% TFA (1:1) to give 1.6 mg(0.56%) of a tan solid. 1H NMR (DMSO-d6, 500 MHz) δ 0.69

(d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H), 1.10−1.12 (s, 3H), 1.47(s, 3H), 1.77−1.82 (m, 1H), 2.30−2.36 (m, 1H), 2.41−2.46 (m, 2H),2.49−2.51 (m, 1H), 2.58 (t, J = 6.9 Hz, 2H), 3.51 (t, J = 9.5 Hz, 1H),3.58−3.64 (m, 4H), 6.57 (s, 1H), 6.64−6.70 (m, 2H), 7.09−7.14 (m,2H), 7.40 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 10.96 (s, 1H).(+) LRMS (ESI) m/z 520.3 [M + H]+.

(1R,2R,2aR,10aS)-1-Isopropyl-2-(4-ethoxy-3-methoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyl-2-(4-ethoxy-3-methoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexa-hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (28). Fromflindersine (2a, 0.5 mmol) and 4r (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 448 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O(2:3) over 60 min to afford 11 mg (5%). 1H NMR (DMSO-d6,500 MHz) δ 0.71 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.5 Hz, 1H), 1.15 (s,3H), 1.25 (t, J = 6.5 Hz, 3H), 1.50 (s, 3H), 1.81 (m, 1H), 2.34 (m,1H), 2.45 (m, 1H), 3.45 (m, 1H), 3.52 (t, J = 9 Hz, 1H), 3.63 (m,1H), 3.84 (q, J = 6.5 Hz, 2H), 6.23 (d, J = 1.5 Hz, 1H), 6.43 (dd, J =1.5, 7.5 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H),7.16 (d, J = 7.5 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1H), 7.83 (d, J = 7.5 Hz,1H), 10.96 (s, 1H). (+) LRMS (ESI) m/z 448 [M + H]+.

(1S,2S,2aS,10aR)-2-[4-Methoxy-3-(2-hydroxyethyl)phenyl]-10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1R,2R,2aR,10aS)-2-[4-Methoxy-3-(2-hydroxyethyl)phenyl]-10,10-dimethyl-1-(1-methyl-ethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (29). From flindersine (2a, 0.4 mmol) and 5d (0.8mmol). Purified by reverse-phase HPLC with gradient elution (3:7)MeCN/H2O + 1% TFA to (3:2) MeCN/H2O + 1%TFA over 60 minto give 32.4 mg (17%) of white solid. 1H NMR (500 MHz, DMSO-d6)δ 0.70 (d, J = 7 Hz, 3H), 0.85 (d, J = 7 Hz, 3H), 1.13 (s, 3H), 1.48 (s,3H), 1.77−1.81 (m, 1H), 2.29−2.36 (m, 1H), 2.49−2.54 (m, 1H),3.1−3.16 (m, 2H), 3.41−3.50 (m, 3H), 3.61− 3.64 (m, 5H), 6.31 (s,1H), 6.47 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 7.10−7.15 (m,2H), 7.41 (t, J = 7.4 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 10.94 (s, 1H).(+) LRMS (ESI) m/z 464.0 [M + H]+

(1R,2R,2aR,10aS)-2-[4-Methoxy-3-(3-hydroxypropyl)phenyl]-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-[4-Methoxy-3-(3-hydroxypropyl)phenyl]-10,10-dimethyl-1-(1-cyclo-propyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (30). From flindersine (2a, 0.88 mmol) and 5c (1.76mmol). Purified by reverse-phase HPLC with gradient elution (7:13)MeCN/H2O + 1% TFA to (13:7) MeCN/H2O + 1%TFA over 60min. Resulting residue was repurified by flash chromatography (1:99MeOH/DCM to 4:96 MeOH/DCM) to give 10.4 mg (2.5%) of alight yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 0.01−0.05 (m,2H), 0.26−0.36 (m, 2H), 0.90−0.94 (m, 1H), 1.14 (s, 3H), 1.47−1.57(m, 5H), 2.06 (d, J = 8.5 Hz, 1H), 2.46 (t, J = 9 Hz, 1H), 3.10−3.12(m, 1H), 3.25−3.28 (m, 1H), 3.36−3.41 (m, 1H), 3.49−3.52 (m, 1H),3.60−3.62 (m, 4H), 4.36 (t, J = 5 Hz, 1H), 6.23 (s, 1H), 6.46 (d, J = 8Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 7.12−7.16 (m, 2H), 7.42 (t, J = 7.69Hz, 1H), 7.81 (d, J = 7.96 Hz, 1H), 10.97 (s, 1H). (+) LRMS (ESI)m/z 476.0 [M + H]+. HRMS (ESI): calcd for C29H34NO5 [M + H]+,476.2432; found, 476.2424.

(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphen-yl)-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (31). From flinder-sine (2a, 0.88 mmol) and 4t (1.76 mmol). Purified by reverse-phaseHPLC with isocratic elution MeCN/H2O + 1% TFA (53:47) and thenflash chromatography (1% up to 3% MeOH/DCM) to give 11.9 mg(3.1%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ 0.01−0.08(m, 2H), 0.25−0.30 (m, 1H), 0.33−0.39 (m, 1H), 0.89−0.96 (m, 1H),1.14 (s, 3H), 1.53 (s, 3H), 2.06−2.12 (m, 1H), 2.47 (t, J = 8.8 Hz,1H), 3.19 (s, 3H), 3.35−3.41 (m, 1H), 3.60−3.65 (m, 4H), 6.27(d, J = 1.7 Hz, 1H), 6.45 (dd, J = 8.2, 1.7 Hz, 1H), 6.59 (d, J = 8.2 Hz,1H), 7.11−7.15 (m, 2H), 7.39−7.44 (m, 1H), 7.81 (d, J = 8.2 Hz,1H), 10.99 (s, 1H). (+) LRMS (ESI) m/z 431.8 [M + H]+. HRMS

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dx.doi.org/10.1021/jm401321v | J. Med. Chem. 2014, 57, 1252−12751269

(ESI): calcd for C27H29NO4Na [M + Na]+, 454.1989; found,454.1986.(1R,2R,2aR,10aS)-2-(3,4,5-Trimethoxyphenyl)-10,10-dimethyl-1-

(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4,5-Trime-thoxyphenyl)-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (32).From flindersine (2a, 0.88 mmol) and 4s (1.76 mmol). Purified byreverse-phase HPLC isocratic elution MeCN/ H2O + 1% TFA (1:1)over 60 min. Then flash chromatography (0% up to 2% MeOH/DCM) to give 8.8 mg (2.2%) as a white solid. 1H NMR (DMSO-d6,500 MHz) δ 0.03−0.12 (m, 2H), 0.28−0.40 (m, 2H), 0.90−0.97 (m,1H), 1.15 (s, 3H), 1.53 (s, 3H), 2.09 (q, J = 8.9 Hz, 1 H) 2.45−2.49(m, 1H), 3.35−3.41 (m, 1H), 3.53 (s, 3H), 3.64 (t, J = 8.5 Hz, 1 H)6.08 (s, 2H), 7.11−7.16 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.81 (d, J =8 Hz, 1H), 11.03 (s, 1H). (+) LRMS (ESI) m/z 461.6 [M + H]+.HRMS (ESI): calcd for C28H31NO5Na [Na]+, 484.2094; found,484.2085.(1R,2R,2aR,10aS)-2-(4-Methoxy-3-(2-morpholine-4-ylpropoxy)-

phenyl)-10,10,6-trimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexa-hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and(1S,2S,2aS,10aR)-2-(4-Methoxy-3-(2-morpholine-4-ylpropoxy)-phenyl)-10,10,6-trimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexa-hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (33). Fromflindersine (2a, 0.5 mmol) and 5b (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 545 were combined andpurified by reverse-phase HPLC with XBridge gradient elutionMeCN/H2O (10 mM NH4OAc) 95:5 to MeCN over 60 min toafford 5.8 mg (2%). 1H NMR (DMSO-d6, 500 MHz) δ 0.02 (m, 2H),0.27 (m, 1H), 0.35 (m, 1H), 0.92 (m, 1H), 1.13 (s, 3H), 1.53 (s, 3H),2.07 (m, 3H), 2.47 (m, 1H), 2.54 (m, 6H), 3.60 (m, 5H), 3.62 (s, 3H),3.98 (m, 2H), 6.46 (d, J = 1.5 Hz), 6.47 (dd, J = 1.5, 8 Hz, 1H), 6.60(d, J = 8.5 Hz, 1H), 7.12 (d, J = 8 Hz, 1H), 7.13 (dd, J = 7.5, 8 Hz,1H), 7.42 (dd, J = 7.5, 8.5 Hz, 1H), 7.57 (dd, J = 7.5, 8.5 Hz, 1H), 7.93(d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 545 [M + H]+. HRMS (ESI):calcd for C33H41N2O5 [M + H]+, 545.3010; found, 545.3010.(1R,2R,2aR,10aS)-1-Cyclopentyl-2-(3,4-dimethoxyphenyl)-10,10-

dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopentyl-2-(3,4-dime-thoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (34). From flindersine(2a, 0.50 mmol) and 4p (1.0 mmol). Purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (3:2) (19.4 mg, 8.5%). 1HNMR (DMSO-d6, 600 MHz) δ 0.89 (m, 1H), 1.11 (m, 1H), 1.55 (m,2H), 1.44 (m, 4H), 1.12 (s, 3H), 1.49 (s, 3H), 1.97 (m, 1H), 2.41 (m,1H), 2.42 (m, 1H), 3.12 (s, 3H), 3.48 (m, 1H), 3.60 (s, 3H), 3.64(t, J = 9 Hz, 1H), 6.25 (br s, 1H), 6.48 (dd, J = 1.5, 8.4 Hz, 1H), 6.57(d, J = 8.4 Hz, 1H), 7.13 (t, J = 8 Hz, 1H), 7.14 (d, J = 8 Hz, 1H), 7.42(t, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 10.95 (s, 1H). 13C NMR(CDCl3, 150 MHz) δ 24.2, 25.2, 26.2, 29.1, 31.5, 32.1, 40.5, 44.0, 45.2,45.6, 55.0, 55.8, 78.1, 109.5, 111.1, 112.0, 115.3, 115.8, 121.5, 121.7,122.5, 130.5, 135.9, 138.4, 147.6, 148.3, 155.8, 162.7. (+) LRMS (ESI)m/z 460 [M + H]+. HRMS (ESI): calcd for C29H33NO4Na [M + Na]+,482.2302; found, 482.2322.(1R,2R,2aR,10aS)-2-(3,4,5-Trimethoxyphenyl)-10,10-dimethyl-1-

(1-cyclopentyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4,5-Trime-thoxyphenyl)-10,10-dimethyl-1-(1-cyclopentyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (35).From flindersine (2a, 0.88 mmol) and 4q (1.76 mmol). Purified byreverse-phase HPLC with isocratic elution MeCN/H2O (1:1) over60 min. Then flash chromatography (DCM to 2% MeOH/DCM) togive 1 mg (0.23%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ1.12−1.19 (m, 5H), 1.39−1.48 (m, 4H), 1.51 (s, 3H), 1.56−1.63 (m,2H), 1.95−2.01 (m, 1H), 2.37−2.51 (m, 2H), 3.29−3.41 (m, 6H),3.49−3.53 (m, 4H), 3.68 (t, J = 8.7 Hz, 1H), 6.10 (s, 2H), 7.11−7.15(m, 2H), 7.41−7.43 (m, 1H), 7.81 (d, J = 8 Hz, 1 H) 11.00 (s, 1H).13C NMR (DMSO-d6, 150 MHz) δ 23.18, 24.18, 24.34, 25.31, 30.91,39.51, 43.64, 44.71, 44.85, 54.76, 59.56, 76.08, 105.36, 108.24, 114.26,114.64, 120.55, 121.38, 129.52, 135.49, 137.30, 137.63, 151.30, 155.14,

161.83. (+) LRMS (ESI) m/z 490.3 [M + H]+. HRMS (ESI): calcd forC30H35NO5Na [M + Na]+, 512.2407; found, 512.2426.

(1R,2R,2aR,10aS)-1-Butyl,10,10-trimethyl-2-(3,4,5-trimethoxy-phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Butyl,10,10-trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (36). From flindersine (2a, 0.5mmol) and 4n (1.0 mmol). Purified by isocratic elution MeCN/H2O(60:40) (34 mg, 14%). 1H NMR (DMSO-d6, 600 MHz) δ 0.72 (t, J =7 Hz, 3H), 0.99 (m, 1H), 1.06 (m, 1H), 1.13 (s, 3H), 1.14 (m, 2H),1.48 (s, 3H), 1.52 (m, 1H), 1.64 (m, 1H), 2.32 (m, 1H), 2.38 (t, J = 9Hz, 1H), 3.31 (s, 6H), 3.39 (t, J = 9 Hz, 1H), 3.53 (s, 3H), 3.69 (t, J =9 Hz, 1H), 6.11 (d, J = 8 Hz, 2H), 7.12 (d, J = 7.5 Hz, 1H), 7.13 (t, J =7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.83 (d, J = 8 Hz, 1H), 11.03 (s,1H). 13C NMR (DMSO-d6, 150 MHz) δ 14.60, 22.69, 24.38, 25.50,29.23, 32.20, 36.62, 40.45, 44.75, 49.08, 55.94, 60.40, 77.19, 106.32,108.78, 115.46, 115.52, 121.79, 122.61, 130.48, 136.36, 137.60, 137.69,152.20, 156.16, 162.76. (+) LRMS (ESI) m/z 478 [M + H]+. HRMS(ESI): calcd for C29H36NO5 [M + H]+, 478.2588; found, 478.2596.

(1R,2R,2aR,10aS)-1-Butyl,10,10-trimethyl-2-(3,4,-dimethoxyphen-yl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1-Butyl,10,10-trimethyl-2-(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (37). From flindersine (2a, 0.5mmol) and 4o (1.0 mmol). Purified by flash chromatography onneutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). Thefractions containing MH+ 448 were combined and purified by reverse-phase HPLC with isocratic elution MeCN/H2O (7:3) (13.2 mg, 7%).1H NMR (DMSO-d6, 500 MHz) δ 0.69 (t, J = 7 Hz, 3H), 0.94 (m,1H), 0.99 (m, 1H), 1.11 (s, 3H), 1.11 (m, 2H), 1.48 (s, 3H), 1.49 (m,1H), 1.63 (m, 1H), 2.34 (m, 2H), 3.14 (s, 3H), 3.36 (t, J = 9 Hz, 1H),3.60 (s, 3H), 3.65 (t, J = 9 Hz, 1H), 6.26 (s, 1H), 6.48 (d, J = 7.5 Hz,1H), 6.58 (d, J = 7.5 Hz, 1H), 7.12(d, J = 7.5 Hz, 1H), 7.13 (t, J = 7.5Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 10.97 (s,1H). 13C NMR (DMSO-d6, 150 MHz) δ 13.54, 21.56, 23.23, 24.60,28.31, 35.36, 31.17, 39.23, 43.81, 47.65, 53.92, 54.92, 76.36, 107.98,110.06, 111.08, 114.47, 114.49, 120.42, 120.72, 121.54, 129.54, 133.42,137.11, 146.63, 146.98, 155.30, 161.43. (+) LRMS (ESI) m/z 448 [M+ H]+. HRMS (ESI): calcd for C28H33NO4Na [M + Na]+, 470.2302;found, 458.2289.

(1R,2R,2aR,10aS)-1-Ethanone-2-(3,4-dimethoxyphenyl)-10,10-di-methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethanone-2-(3,4-dimethox-yphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (38). From flindersine (2a, 0.5 mmol)and (E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (1.0 mmol). Purified byflash chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 434 were combined andpurified by reverse-phase HPLC with gradient elution MeCN/H2O (3:7) toMeCN over 60 min to give 12.3 mg (6%). 1H NMR (DMSO-d6, 500MHz) δ 0.73 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H),1.49 (s, 3H), 1.86 (m, 3H), 2.98 (dd, J = 9 Hz, 1H), 3.23 (s, 3H), 3.31 (m,1H), 3.41 (dd, J = 10.5 Hz, 1H), 3.66 (s, 3H), 3.74 (dd, J = 10 Hz, 1H),6.44 (d, J = 1.5 Hz, 1H), 6.62 (dd, J = 1.5, 8.5 Hz, 1H), 6.67 (d, J = 8.5 Hz,1H), 7.16 (d, J = 7.5 Hz, 1H), 7.17 (dd, J = 7.5 Hz, 1H), 7.46 (dd, J = 7.5Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 11.07 (s, 1H). (+) LRMS (ESI)m/z 434[M + H]+. HRMS (ESI): calcd for C26H27NO5 [M + H]+, 434.1962; found,434.1942.

(1R,2R,2aR,10aS)-1-Cyclopropylmethanone-2-(3,4-dimethoxy-phenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopro-pylmethanone-2-(3,4-dimethoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (39). From flindersine (2a, 0.5 mmol) and (E)-1-cyclopropyl-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (1.76 mmol).Purified by flash chromatography on neutral alumina (DCM, then1:1 DCM/CHCl3, then CHCl3). The fractions containing MH+ 460were combined and purified by reverse-phase HPLC with gradientelution MeCN/H2O (3:7) to MeCN over 60 min to give 24.6 mg(11%). 1H NMR (DMSO-d6, 600 MHz) δ 0.75 (m, 4H), 1.16 (s, 3H),1.36 (s, 3H), 1.67 (m, 1H), 2.98 (dd, J = 8.4, 9.5 Hz, 1H), 3.20

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dx.doi.org/10.1021/jm401321v | J. Med. Chem. 2014, 57, 1252−12751270

(s, 3H), 3.52 (dd, J = 8.4, 9.6 Hz, 1H), 3.61 (s, 3H), 3.64 (dd, J = 8.4,9.5 Hz, 1H), 3.77 (dd, J = 8.4, 9 Hz, 1H), 6.44 (br s, 1H), 6.59 (br d,J = 8.4 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H),7.14 (dd, J = 7.8 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5Hz, 1H), 11.06 (s, 1H). 13C NMR (CDCl3, 150 MHz) δ 11.01, 19.73,23.54, 25.35, 31.94, 40.41, 46.01, 50.79, 55.19, 55.91, 76.74, 108.25,111.31, 111.95, 115.42, 115.66, 121.32, 121.68, 122.72, 130.72, 132.36,138.25, 148.08, 148.28, 156.74, 162.87, 210.44. (+) LRMS (ESI) m/z460 [M + H]+. HRMS (ESI): calcd for C28H29NO5Na [M + Na]+,482.1938; found, 482.1947.(1R,2R,2aR,10aS)-1-Carbomethoxy-10,10-dimethyl-2-(3,4,5-tri-

methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Carbome-thoxy-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (40).From flindersine (2a, 0.5 mmol) and (E)-methyl-3-(3,4,5-trimethoxyphenyl)-acrylate (1.0 mmol). Purified by gradient elution MeCN/H2O (1%TFA, 3:7 for 45 min then gradient elution to 75:25 over 25 min) toafford a white solid (5.4 mg, 2.2%). 1H NMR (DMSO-d6, 500 MHz) δ1.18 (s, 3H), 1.46 (s, 3H), 3.02 (t, J = 8.5 Hz, 1H), 3.20 (t, J = 8.5 Hz,1H), 3.37 (s, 6H), 3.54 (s, 3H), 3.62 (s, 3H), 3.76 (t, J = 8 Hz, 1H),3.95 (t, J = 9 Hz, 1H), 6.19 (s, 2H), 7.13 (m, 2H), 7.46 (t, J = 7.5 Hz,1H), 7.84 (d, J = 8 Hz, 1H), 11.14 (s, 1H). (+) LRMS (ESI) m/z 480[M + H]+. HRMS (ESI): calcd for C27H30NO7 [M + H]+, 480.2017;found, 480.1997.(1R,2R,2aR,10aS)-1-Acetoxymethyl-10,10-dimethyl-2-(3,4,5-tri-

methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Acetoxymeth-yl-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexa-hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (41). Fromflindersine (2a, 0.5 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)-allylacetate (1.0 mmol). Purified by gradient elution MeCN/H2O(1% TFA, 3:7 for 45 min then gradient elution to 75:25 over 25 min)to afford a white solid (44.3 mg, 9%). 1H NMR (DMSO-d6, 500 MHz)δ 1.17 (s, 3H), 1.47 (s, 3H), 1.89 (s, 3H), 2.60 (t, J = 9 Hz, 1H), 2.66(t, J = 9 Hz, 1H), 3.34 (s, 6H), 3.51 (s, 3H), 3.58 (t, J = 9 Hz, 1H),3.74 (t, J = 8 Hz, 1H), 6.13 (s, 2H), 7.13 (m, 2H), 7.42 (t, J = 7.5 Hz,1H), 7.81 (d, J = 8 Hz, 1H), 11.08 (s, 1H). (+) LRMS (ESI) m/z 494[M + H]+.(1R,2R,2aR,10aS)-1-Isopropyloxymethyl-10,10-dimethyl-2-(3,4,5-

trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyloxy-methyl-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (42).From flindersine (2a, 0.5 mmol) and (E)-5-(3-isopropoxyprop-1-enyl)-1,2,3-trimethoxybenzene (1.0 mmol). Purified by gradientelution MeCN/H2O (1% TFA, 35:65 for 30 min then gradientelution to 75:25 over 30 min) to afford a white solid (34.2 mg, 13%).1H NMR (CDCl3, 500 MHz) δ 1.13−1.22 (m, 9H), 1.45 (s, 3H), 2.96(t, J = 9 Hz, 1H), 3.10 (t, J = 9 Hz, 1H), 3.37 (s, 6H), 3.52 (s, 3H),3.61 (m, 1H), 3.75 (m, 1H), 3.95 (t, J = 9 Hz, 1H), 6.16 (s, 2H), 7.13(m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 11.11 (s,1H). (+) LRMS (ESI) m/z 504 [M + H]+. HRMS (ESI): calcd forC29H33NO7 [M + Na]+, 530.2149; found, 530.2125.(1R,2R,2aR,10aS)-1-Carboethoxy-10,10-dimethyl-2-(3,4,5-trime-

thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Carboethoxy-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (43). From 3 and(E)-ethyl-3-(4-bromophenyl)acrylate. Purified by isocratic elutionMeOH/H2O (1% TFA, 6:4) (59.7 mg, 12%). 1H NMR (CDCl3,500 MHz) δ 1.11 (t, J = 7 Hz, 3H), 1.16 (s, 3H), 1.49 (s, 3H), 3.04 (t,J = 9 Hz, 1H), 3.23 (t, J = 10 Hz, 1H), 3.28 (s, 3H), 3.79 (t, J = 8 Hz,1H), 3.99 (m, 1H), 4.08 (m, 2H), 6.72 (d, J = 7 Hz, 2H), 7.21 (d, J = 7Hz, 2H), 7.37 (t, J = 7 Hz, 1H), 7.58 (m, Hz, 2H), 8.02 (d, J = 8 Hz,1H). 13C NMR (CDCl3, 125 MHz) δ 13.95, 22.90, 24.46, 31.71, 41.13,41.37, 43.78, 52.19, 60.23, 75.74, 101.53, 118.51, 119.54, 121.46,123.43, 126.43, 129.43, 129.77, 130.1, 137.47, 145.00, 156.34, 160.99,172.53. (+) LRMS (ESI) m/z 496:498 (1:1) [M + H]+. HRMS (ESI):calcd for C26H27BrNO4 [M + H]+, 496.1118; found, 496.1121. Thiscompound was (26.3 mg, 0.050 mmol) was dissolved in acetic acid

(2 mL). Potassium iodide (0.066 mmol) was added, and the reactionmixture was heated at 50 °C for 20 h. The reaction mixture wasextracted with a 10% NaHSO4 solution and CHCl3 (3 × 30 mL), driedwith MgSO4, filtered, and concentrated to dryness to afford 43 (20 mg,83%) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 1.18 (t, J = 7Hz, 3H), 1.14 (s, 3H), 1.46 (s, 3H), 2.99 (t, J = 9 Hz, 1H), 3.19 (t, J =10 Hz, 1H), 3.72 (t, J = 8 Hz, 1H), 3.99 (m, 2H), 4.08 (m, 1H), 6.89(d, J = 7 Hz, 2H), 7.14 (t, J = 7 Hz, 2H), 7.22 (d, J = 7.5 Hz, 2H), 7.43(t, J = 7 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.10 (s, 1H). (+) LRMS(ESI) m/z 482:484 (1:1) [M + H]+. HRMS (ESI): calcd for C25H24BrNO4 [M + Na]+, 504.0781; found, 504.0777.

(1R,2R,2aR,10aS)-1,4,10,10-Tetramethyl-2-(3,4-dimethoxyphen-yl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,4,10,10-Tetramethyl-2-(3,4-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one (44). From N-Me-flindersine (2b, 0.93mmol) and 4a (1.87 mmol). Purified by reverse-phase HPLC withisocratic elution MeCN/H2O (55:45) over 60 min to give 15.4 mg(3.7%) as a light brown solid. 1H NMR (DMSO-d6, 600 MHz) δ1.10−1.17 (m, 6H), 1.49 (s, 3H), 2.30−2.37 (m, 2H), 3.23 (s, 3H),3.30−3.32 (m, 7H), 3.51 (s, 3H), 3.71 (t, J = 7.8 Hz, 1H), 6.06 (s,2H), 7.26 (t, J = 7.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.8Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz) δ20.04, 22.91, 24.09, 32.04, 34.58, 45.39, 49.17, 54.68, 59.53, 76.29,104.91, 107.10, 114.83, 115.49, 120.98, 121.85, 130.11, 135.63, 135.79,138.32, 151.29, 154.14, 161.15. (+) LRMS (ESI) m/z 449.8 [M + H]+.HRMS (ESI): calcd for C26H31NO5Na [M + Na]+, 472.2094; found,472.2104.

(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-1-ethyl-4,10,10-tri-methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphenyl)-1-ethyl-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (45). From N-Me-flindersine (2b,0.50 mmol) and 4e (1.0 mmol). Purified by flash chromatography onneutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). Thefractions containing MH+ 435 were combined and purified by reverse-phase HPLC with isocratic elution MeCN/H2O (3:2) over 60 min toafford 21.1 mg (10%). 1H NMR (DMSO-d6, 500 MHz) δ 0.64 (t, J =6.5 Hz, 3H), 1.51 (m, 1H), 1.67 (m, 1H), 1.13 (s, 3H), 1.47 (s, 3H),2.29 (m, 1H), 2.37 (t, J = 9 Hz, 1H), 3.13 (s, 3H), 3.20 (s, 3H), 3.60(s, 3H), 3.69 (t, J = 9 Hz, 1H), 6.25 (br s, 1H), 6.44 (dd, J = 1.5, 7.5Hz, 1H), 6.58 (d, J = 7.5 Hz), 7.25 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H). 13C NMR(CDCl3, 150 MHz) δ 12.50, 23.82, 25.32, 29.32, 30.68, 33.28, 41.82,48.51, 55.84, 56.90, 77.68, 109.51, 112.00, 113.11, 115.02, 115.97,122.31, 123.08, 123.82, 132.39, 135.88, 140.10, 148.68, 149.26, 156.38,163.88. (+) LRMS (ESI) m/z 435 [M + H]+. HRMS (ESI): calcd forC27H31NO4Na [M + Na]+, 456.2145; found, 456.2138.

(1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-2-(3-fluoro-4-me-thoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (46). From N-Me-flin-dersine (2b, 0.50 mmol) and 4g (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 422 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O(3:2) to MeCN over 60 min to afford 9.8 mg (5%). 1H NMR (DMSO-d6, 600 MHz) δ 0.63 (t, J = 8 Hz, 3H), 1.11 (s, 3H), 1.46 (s, 3H), 1.52(m, 1H), 1.66 (m, 1H), 2.27 (m, 1H), 2.36 (t, J = 10 Hz, 1H), 3.20 (s,3H), 3.43 (t, J = 9 Hz, 1H), 3.64 (t, J = 9 Hz, 1H), 3.75 (s, 3H), 6.54 (brd, J = 9.6 Hz, 1H), 6.59 (br d, J = 8 Hz, 1H), 6.73 (dd, J = 6, 8 Hz, 1H),7.25 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 8 Hz, 1H),7.93 (d, J = 8 Hz). (+) LRMS (ESI) m/z 422 [M + H]+. HRMS (ESI):calcd for C26H28FNO3Na [M + Na]+, 444.1945; found, 444.1951.

(1R,2R,2aR,10aS)-1-Ethyl-7,10,10-trimethyl-2-(3,4,5-trimethoxy-phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-7,10,10-trimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (47). From 2c (0.5 mmol) and 4a(1.0 mmol). Purified by flash chromatography on neutral alumina

Journal of Medicinal Chemistry Article

dx.doi.org/10.1021/jm401321v | J. Med. Chem. 2014, 57, 1252−12751271

(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractionscontaining MH+ 464 were combined and purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (55:45) to MeCN over60 min (12.6 mg, 6%). 1H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J =8.5 Hz, 3H), 1.14 (s, 3H), 1.49 (s, 3H), 1.52 (m, 1H), 1.67 (m, 1H),2.28 (m, 2H), 2.36 (s, 3H), 3.31 (s, 6H), 3.38 (t, J = 9 Hz, 1H), 3.52(s, 3H), 3.66 (t, J = 9 Hz, 1H), 6.11 (s, 2H), 7.05 (d, J = 7.5 Hz, 1H),7.26 (d, J = 7.5 Hz, 1H), 7.63 (br s, 1H), 10.95 (s, 1H). (+) LRMS(ESI) m/z 464 [M + H]+. HRMS (ESI): calcd for C28H33NO5Na[M + Na]+, 486.2251; found, 486.2273.(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-1-ethyl-7,10,10-tri-

methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphenyl)-1-ethyl-7,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one (48). From 2c (0.83 mmol) and 4e(1.67 mmol). Purified by flash chromatography on neutral alumina(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractionscontaining MH+ 434 were combined and purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (3:2) to MeCN over 60 min(9.7 mg, 3%). 1H NMR (DMSO-d6, 600 MHz) δ 0.65 (t, J = 8 Hz,3H), 1.13 (s, 3H), 1.48 (s, 3H), 1.53 (m, 1H), 1.67 (m, 1H), 2.31 (m,1H), 2.34 (s, 3H), 2.49 9m, 1H), 3.20 (s, 3H), 3.38 (t, J = 9 Hz, 1H),3.62 (s, 3H), 3.64 (t, J = 9 Hz, 1H), 6.30 (s, 1H), 6.48 (d, J = 8 Hz,1H), 6.59 (d, J = 8 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 7.25 (d, J =7.5 Hz, 1H), 7.62 (br s, 1H), 10.91 (s, 1H). (+) LRMS (ESI) m/z 434[M + H]+. HRMS (ESI): calcd for C27H31NO4 [M + H]+, 434.2326;found, 434.2329.(1R,2R,2aR,10aS)-7,10,10-Trimethyl-1-(1-methylethyl)-2-(3,4,5-

trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7,10,10-Trimeth-yl-1-(1-methylethyl)-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (49).From 2c (0.83 mmol) and 4h (1.67 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 480 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O(58:42) to MeCN over 60 min to afford 17.3 mg (4%). 1H NMR(DMSO-d6, 600 MHz) δ 0.72 (d, J = 8 Hz, 3H), 0.88 (d, J = 8 Hz, 3H),1.15 (s, 3H), 1.49 (s, 3H), 1.80 (m, 1H), 1.67 (m, 1H), 2.29 (m, 1H), 2.36(s, 3H), 2.49 (m, 1H), 3.32 (s, 6H), 3.38 (t, J = 9 Hz, 1H), 3.52 (s, 3H),3.66 (t, J = 9 Hz, 1H), 6.11 (s, 2H), 7.04 (d, J = 7.5 Hz, 1H), 7.25 (d, J =7.5 Hz, 1H), 7.62 (br s, 1H), 10.92 (s, 1H). 13C NMR (CDCl3, 150 MHz)δ 18.26, 20.39, 21.12, 23.22, 25.17, 30.52, 31.17, 40.94, 43.66, 45.45, 77.20,105.34, 109.12, 114.2, 115.51, 120.93, 130.52, 130.72, 136.11, 136.23,136.40, 155.96, 162.63. (+) LRMS (ESI) m/z 480 [M + H]+. HRMS(ESI): calcd for C29H35NO5Na [M + Na]+, 500.2407; found, 500.2421.(1R,2R,2aR,10aS)-2-(3-Chloro-4-methoxyphenyl)-4,10,10-tri-

methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Chloro-4-methoxyphenyl)-4,10,10-trimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (50). From N-Me-flindersine (2b, 0.5 mmol) and 4i(1.0 mmol). Purified by flash chromatography on neutral alumina(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractionscontaining MH+ 452 were combined and purified by reverse-phaseHPLC with isocratic elution MeCN/H2O (3:2) over 60 min to give12.4 mg (5.5%). 1H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J = 8.4Hz, 3H), 0.86 (d, J = 8.4 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 1.52 (m,1H), 1.81 (m, 1H), 2.30 (m, 1H), 2.51 (m, 1H), 3.20 (s, 3H), 3.55 (t,J = 9 Hz, 1H), 3.65 (t, J = 9 Hz, 1H), 3.70 (s, 3H), 6.73 (m, 3H), 7.26(t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.58 (t, J = 8 Hz, 1H),7.94 (d, J = 8 Hz). (+) LRMS (ESI) m/z 452 [M + H35Cl]+, 454 [M +H37Cl]+. HRMS (ESI): calcd for C27H30ClNO3Na [M + Na]+,474.1806; found, 458.1795.(1R,2R,2aR,10aS)-2-(3-Acetoxy-4-methoxyphenyl)-4,10,10-tri-

methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Ace-toxy-4-methoxyphenyl)-4,10,10-trimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (51). From N-Me-flindersine (2b, 0.83 mmol) and 4k(1.76 mmol). Purified by flash chromatography on neutral alumina(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions containing

MH+ 476 were combined and purified by reverse-phase HPLC withgradient elution MeCN/H2O (5:95) to MeCN/H2O (2:3) over 60 minto afford 17.6 mg (4.5%). 1H NMR (600 MHz, DMSO-d6) δ 0.69 (d, J= 6.6 Hz, 3H), 0.85 (d, J = 6.57 Hz, 3H), 1.13 (s, 3H), 1.48 (s, 3H),1.79 (m, Hz, 1 H), 1.95 (s, 3H), 2.29 (m, 1H), 2.26 (m, 1H), 3.21 (s,3H), 3.52 (m, 1H), 3.61 (s, 3H), 3.67 (m, 1H), 6.35 (s, 1 H), 6.67−6.76(m, 2 H), 7.24 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8 Hz, 1H), 7.56 (dd, J =7.5, 8 Hz, 1H), 7.92 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 476 [M +H]+. HRMS (ESI): calcd for C29H33NO5 [M + H]+, 476.2432; found,476.2425.

(1R,2R,2aR,10aS)-1-Cyclopropyl-2-(3,4,5-trimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopropyl-2-(3,4,5-trimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahy-dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (52). From N-Me-flindersine (2b, 0.5 mmol) and 4s (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 476 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O(42.5:57.5) over 60 min to give 8.6 mg (4%). 1H NMR (DMSO-d6,500 MHz) δ 0.07 (m, 2H), 0.30 (m, 1H), 0.36 (m, 1H), 0.93 (m, 1H),1.15 (s, 3H), 1.53 (s, 3H), 2.06 (m, 1H), 2.47 (m, 1H), 3.20 (s, 3H),3.28 (s, 6H), 3.39 (dd, J = 10 Hz, 1H), 3.50 (s, 3H), 3.65 (dd, J = 8.5,10 Hz, 1H), 6.01 (s, 2H), 7.24 (dd, J = 7.5, 8 Hz, 1H), 7.38 (d, J = 8.5Hz, 1H), 7.57 (ddd, J = 1.5, 7.5, 8.5 Hz, 1H), 7.93 (d, J = 8 Hz, 1H).(+) LRMS (ESI) m/z 476 [M + H]+. HRMS (ESI): calcd forC29H33NO5 [M + H]+, 476.2432; found, 476.2452.

(1R,2R,2aR,10aS)-1-Cyclopropyl-2-(3,4-dimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopropyl-2-(3,4-dimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahy-dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (53). From N-Me-flindersine (2b, 0.5 mmol) and 4t (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 446 were combined andpurified by reverse-phase HPLC with isocratic elution MeCN/H2O(42.5:57.5) over 60 min to afford 9.4 mg (4%). 1H NMR (DMSO-d6,500 MHz) δ 0.03 (m, 2H), 0.27 (m, 1H), 0.35 (m, 1H), 0.91 (m, 1H),1.14 (s, 3H), 1.53 (s, 3H), 2.06 (m, 1H), 2.47 (m, 1H), 3.15 (s, 3H),3.20 (s, 3H), 3.38 (dd, J = 9.5, 10 Hz, 1H), 3.60 (s, 3H), 3.65 (dd, J =8.5, 10 Hz, 1H), 6.22 (br s, 1H), 6.39 (br d, J = 8.5 Hz, 1H), 6.57(d, J = 8.5 Hz, 1H), 7.24 (dd, J = 7.5, 8 Hz, 1H), 7.37 (d, J = 8.5 Hz,1H), 7.56 (dd, J = 7.5, 8.5 Hz, 1H), 7.92 (d, J = 8 Hz, 1H). (+) LRMS(ESI) m/z 446 [M + H]+. HRMS (ESI): calcd for C28H31NO5 Na[M + Na]+, 484.2094; found, 484.2113.

(1R,2R,2aR,10aS)-1-Cyclopentyl-2-(3,4,5-trimethoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopentyl-2-(3,4,5-trimethoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahy-dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (54). From N-Me-flindersine (2b, 0.5 mmol) and 4q (1.0 mmol). Purified by flashchromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,then CHCl3). The fractions containing MH+ 504 were combined andpurified by reverse-phase HPLC with gradient elution MeCN/H2O(5:95) to MeCN/H2O (2:3) over 60 min to afford 18.6 mg (4.6%).1H NMR (DMSO-d6, 600 MHz) δ 95 (m, 1H), 1.16 (m, 1H), 1.16 (s,3H), 1.45 (m, 4H), 1.52 (s, 3H), 1.62 (m, 2H), 2.01 (m, 1H), 2.41(ddd, J = 7.5, 9.5 Hz, 1H), 2.48 (dd, J = 8.5 Hz, 1H), 3.21 (s, 3H),3.28 (s), 3.51 (s, 6H), 3.54 (dd, J = 8.5, 9 Hz, 1H), 3.71 (t, J = 8.5, 9Hz, 1H), 6.06 (s, 3H), 7.27 (dd, J = 7.5, 8.5 Hz, 1H), 7.40 (d, J = 8.5Hz, 1H), 7.59 (dd, J = 7.5, 8.5 Hz, 1H), 7.96 (d, J = 8.5 Hz). (+)LRMS (ESI) m/z 504 [M + H]+. HRMS (ESI): calcd for C31H37NO5[M + H]+, 504.2745; found, 504.2736.

(1R,2R,2aR,10aS)-1,10-Dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one and (1S,2S,2aS,10aR)-1,10-Dimethyl-2-(3,4,5-trime-thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one (55 and 56). From 2e−f (0.5 mmol) and 4a(1.0 mmol). Purified by gradient elution MeCN/H2O (55:45 for 45min then gradient elution to 100% MeCN over 5 min) to afford awhite solid (6.3 mg, 4%). 56: 1H NMR (d6 DMSO, 500 MHz) δ 1.08

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(d, J = 6 Hz, 3H), 1.16 (d, J = 6 Hz, 3H), 2.37 (m, 2H), 3.35 (s, 6H),3.52 (s, 3H), 3.66 (m, 1H), 3.78 (m, 1H), 4.62 (m, 1H), 6.17 (s, 2H),7.16 (m, 2H), 7.41 (m, 1H), 7.82 (m, 1H), 11.05 (s, 1H). (+) LRMS(ESI) m/z 422 [M + H]+. HRMS (ESI): calcd for C25H28NO5 [M +H]+, 422.1962; found, 422.1976. The next fraction afforded thealternate isomer (55) (4.5 mg, 3%). 55: 1H NMR (d6 DMSO, 500MHz); 1.22 (d, J = 6 Hz, 3H), 1.43 (d, J = 6 Hz, 3H), 2.42 (m, 2H),3.33 (s, 6H), 3.58 (s, 3H), 3.64 (m, 1H), 3.75 (m, 1H), 4.03 (m, 1H),6.16 (s, 2H), 7.18 (m, 2H), 7.43 (m, 1H), 7.83 (m, 1H), 11.03 (s,1H). (+) LRMS (ESI) m/z 422 [M + H]+. HRMS (ESI): calcd forC27H30NO7Na [M + Na]+, 444.1781; found, 444.1780.(1R,2R,2aR,10aS)-7-Methoxy-10,10-dimethyl-1-(1-methylethyl)-

2-(3,4-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7-Methoxy-10,10-dimethyl-1-(1-methylethyl)-2-(3,4-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-quinolin-3-one (57). 2d (0.15 mmol) and 4l (0.30 mmol). Purified byisocratic elution MeCN/H2O (1% TFA, 55:45 for 60 min) to afford awhite solid (10.9 mg, 16%). 1H NMR (CDCl3, 500 MHz) δ 1.12 (s,H), 1.12 (d, J = 7 Hz, 3H), 1.47 (s, 3H), 2.32 (m, 2H), 3.28 (m, 1H),3.35 (s, 6H), 3.52 (s, 3H), 3.76 (m, 1H), 3.78 (s, 3H), 6.11 (s, 2H),7.09 (m, 2H), 7.25 (m, 1H), 10.95 (s, 1H). (+) LRMS (ESI) m/z 466[M + H]+. HRMS (ESI): calcd for C28H34NO5 [M + H]+, 466.2224;found, 466.2224.(1R,2R,2aR,10aS)-7-Methoxy-1,10,10-trimethyl-2-(3,4,5-trime-

thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7-Methoxy-1,10,10-tri-methyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (58). From 2d (0.19mmol) and 4a (0.38 mmol). Purified by isocratic elution MeCN/H2O (1% TFA, 2:3 for 60 min) to afford a white solid (12.7 mg, 14%).1H NMR (CDCl3, 500 MHz) δ 0.69 (d, J = 6.5 Hz, 3H), 0.85 (d, J =6.5 Hz, 3H), 1.13 (s, 3H), 1.48 (s, 3H), 1.80 (m, 1H), 2.30 (m, 2H), 3.15(s, 3H), 3.49 (m, 1H), 3.59 (s, 3H), 3.63 (m, 1H), 3.79 (s, 3H), 6.27 (s,1H), 6.48 (d, J 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 7.07 (m, 2H), 7.24 (m,1H), 10.84 (s, 1H). (+) LRMS (ESI) m/z 464 [M + H]+. HRMS (ESI):calcd for C27H32NO6 [M + H]+, 464.2432; found, 464.2432.(1R,2R,2aR,8aS)-2-(3,4,5-Trimethoxyphenyl)-1,8,8-trimethyl-

1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]pyridine-3-one and (1S,2S,2aS,8aR)-2-(3,4,5-Trimethoxyphenyl)-1,8,8-trimeth-yl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-pyridine-3-one (59). From 6 (0.088 g, 0.5 mmol) and 4a (0.21 g, 1mmol). Purified by reverse-phase HPLC with isocratic elution MeCN/H2O (1:1) to give 12.8 mg (6.6%) of an off-white solid. 1H NMR (500MHz, DMSO-d6) δ 1.06 (s, 3H), 1.09 (s, 3H), 1.33 (s, 3H), 2.18 (m,1H), 2.21 (m, 1H), 3.17 (t, J = 8.5 Hz, 1H), 3.75−3.50 (m, 10H), 5.83(d, J = 7 Hz, 1H), 6.16 (apparent s, 2H), 7.07 (d, 1H, J = 7 Hz), 10.75(bs, 1H). Using a combination of HSQC and ROESY, correlationswere observed between the methyl group on the cyclobutane ring (δ1.07 ppm) and H-3 (δ 2.18 ppm) and H-7′ (δ 3.17 ppm) and betweenH-7′ and H-3 and H-4 (δ 3.50 ppm). LRMS (ESI) m/z 386.4 [M + H]+.(1R,2R,2aR,8aS)-1-Ethyl-2-(3,4,5-trimethoxyphenyl)-8,8-dimeth-

yl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-pyridine-3-one and (1S,2S,2aS,8aR)-1-Ethyl-2-(3,4,5-trimethoxy-phenyl)-8,8-dimethyl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]-pyrano[3,2-c]pyridine-3-one (60). From 6 (0.088 g, 0.5 mmol) and 4f(0.21 g, 1 mmol). Purified by reverse-phase HPLC with isocraticelution MeCN/H2O (1:1) to give (22 mg, 11%). 1H NMR (500 MHz,DMSO-d6) δ 0.66 (t, J = 7.5 Hz, 3H), 1.07 (s, 3H), 1.32 (s, 3H), 1.45(m, 1H), 1.60 (m, 1H), 2.20 (m, 1H), 2.26 (m, 1H), 2.19 (m, 2H),3.30 (t, J = 9 Hz, 1H), 3.77−3.59 (m, 10H), 5.83 (t, J = 5.5 Hz, 1H),6.16 (apparent s, 2H), 7.06 (d, J = 6.5 Hz, 1H), 10.75 (bs, 1H). Usinga combination of HSQC and ROESY, correlations were observedbetween one of the methylene hydrogens (δ 1.45 ppm) and H-3 (δ2.26 ppm) and between H-3 and H-4 (δ 3.50 ppm). LRMS (ESI) m/z400.4 [M + H]+.(1R,2R,2aR,8aS)-1-Ethyl-2-(3,4-dimethoxyphenyl)-8,8-dimethyl-

1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]pyridine-3-one and (1S,2S,2aS,8aR)-1-Ethyl-2-(3,4-dimethoxyphenyl)-8,8-di-methyl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-pyridine-3-one (61). From 6 (0.088 g, 0.5 mmol) and 4e (0.21 g,1 mmol). Purified by reverse-phase HPLC with isocratic elution

MeCN/H2O (1:1) to give an off-white solid (15.6 mg, 8.5%). 1HNMR (500 MHz, DMSO-d6) δ 0.63 (t, J = 7.5 Hz, 3H), 1.06 (s, 3H),1.32 (s, 3H), 1.46 (m, 1H), 1.60 (m, 1H), 2.25 (m, 2H), 3.29 (t, J =9.5 Hz, 1H), 3.51 (s, 3H), 3.65−3.62 (m, 4H), 5.83 (d, J = 7.5 Hz,1H), 6.35 (s, 1H), 6.35 (d, J = 8.5 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H),7.05 (d, J = 7.5 Hz, 1H), 10.75 (br s, 1H). Using a combination ofHSQC and ROESY, correlations were observed between H-7′ (δ 3.29ppm) and the methyl of the ethyl group (δ 0.63 ppm), H-3 (δ 2.25ppm), H-4 (δ 3.50 ppm), and one of the methylene hydrogens (δ 1.46ppm). LRMS (ESI) m/z 370.4 [M + H]+.

General Procedure: Preparation of Acetals 62−64. To asolution of flindersine (2a, 1.00 g, 4.4 mmol) and 4-methylmorpholineN-oxide (1.90 g, 16 mmol) in 2-methyl-2-propanol (100 mL),tetrahydrofuran (30 mL), and water (10 mL) was added a solution of2.5% osmium tetroxide in 2-methyl-2propanol (2.45 mL). Theresulting solution was stirred for 2 days. Saturated sodium hydrogensulfite (50 mL) was added, and the mixture was extracted withdichloromethane (3 × 200 mL). The combined organic phase wasdried (MgSO4) and filtered, and the solvent was removed in vacuo.The residue was purified by flash chromatography using methanol/chloroform (2:98) to give the flindersine diol 7 as a white solid (1.10g, 96%). 1H NMR (CDCl3, 500 MHz) δ 1.37 (s, 3H), 1.46 (s, 3H),3.67 (dd, J = 4.5, 4.5 Hz, 1H), 4.78 (d, J = 4.5 Hz, 1H), 4.78 (dd J =1.5, 4.5 Hz, 1H), 5.63 (d, J = 1.5 Hz, 1H), 7.18 (app t, J = 8 Hz, 1H),7.31 (d, J = 8.5 Hz, 1H), 7.52 (app t, J = 8 Hz, 1H), 7.76 (d, J = 7.5Hz, 1H), 11.56 (s, 1H). 13C NMR (CDCl3, 125 MHz) δ 23.25, 25.96,62.71, 70.67, 80.97, 106.78, 115.05, 115.93, 122.29, 123.28, 131.48,138.54, 156.14, 164.36. (+) LRMS (ESI) m/z 262 [M + H]+. HPLC(Betasil C18 150 × 4.6 mm, 0−95% acetonitrile (0.1% TFA) in water(0.1% TFA)/9 min at 1.5 mL/min). tR = 7.040 min.

1-(3,5-Dimethoxyphenyl)butan-1-ol. 3,5-Dimethoxybenzalde-hyde (2 g, 12 mmol) was dissolved in toluene (30 mL) and cooledto 0 °C. Propylmagnesium chloride (2.0 M in Et2O, 30 mL, 60 mmol)was added dropwise, and the reaction mixture was allowed to warm tort and stirred for a further 16 h. To this was added 5% AcOH (30 mL),and the resulting mixture was extracted with EtOAc (80 mL). Theorganic phase was then extracted with water (50 mL), saturatedNaHCO3 (50 mL), and brine (50 mL). The organic phase was dried(MgSO4), filtered, and concentrated to afford 1-(3,5-dimethoxyphenyl)-butan-1-ol (2.22 g, 88%) as a colorless oil. 1H NMR (DMSO-d6,500 MHz) δ 0.85 (t, J = 7 Hz, 3H), 1.23 (m, 1H), 1.34 (m, 1H), 1.57(m, 1H), 1.68 (m, 1H), 3.70 (s, 6H), 4.50 (m, 1H), 6.28 (dd, J = 1.5, 2Hz, 1H), 6.41 (d, J = 2.5 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz) δ14.19, 19.27, 41.37, 74.68, 99.58, 104.08, 147.90, 161.10. (+) LRMS(ESI) m/z 211 [M + H]+.

1-(3,5-Dimethoxyphenyl)butan-1-one. A solution of 1-(3,5-dimethoxyphenyl)butan-1-ol (2.22 g, 10.6 mmol) and manganese(IV)oxide (14.59 g, 176 mmol) in dichloromethane (20 mL) was stirredfor 18 h. The reaction mixture was then filtered through a plug ofsilica, the residue was washed with chloroform, and the solvent wasremoved from the combined filtrate in vacuo. The residue was purifiedby flash chromatography using ethyl acetate/hexane (1:9) to give 1-(3,5-dimethoxyphenyl)butan-1-one (1.74 g, 79%). 1H NMR (DMSO-d6, 500 MHz) δ 0.94 (t, J = 7 Hz, 3H), 1.63 (app hx, J = 7 Hz, 2H),2.98 (t, J = 7 Hz, 2H), 3.82 (s, 6H), 6.76 (app t, J = 2 Hz, 1H), 7.08(d, J = 2 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 14.29, 17.98,40.56, 56.20, 105.55, 106.35, 139.63, 161.32, 200.38. (+) LRMS (ESI)m/z 209 [M + H]+.

2-Butyl-2-(3,5-dimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-tet-rahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (62). To asolution of 1-(3,5-dimethoxyphenyl)butan-1-one (790 mg, 3.79mmol) in methanol (4 mL) were added trimethyl orthoformate(2.00 mL, 18.28 mmol) and concentrated sulfuric acid (1 drop), andthe resulting mixture was stirred under nitrogen for 18 h. Saturatedsodium hydrogen carbonate (20 mL) and dichloromethane (50 mL)were then added, and the resulting mixture was filtered through a 1PSfilter paper. The solvent was then removed from the organic phase invacuo to give acetal 8a. Without purification, 8a was dissolved indichloromethane (2 mL), and 7 (100 mg, 0.38 mmol) and pyridiniump-toulenesulfonate (220 mg) were added. The resulting solution was

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allowed to stir for 18 h, after which saturated sodium hydrogencarbonate (20 mL) and dichloromethane (50 mL) were added. Themixture was filtered through a 1PS filter paper, and the solvent wasremoved from the organic phase in vacuo. The residue was purified byflash chromatography using chloroform to give 62 (86 mg, 50%). 1HNMR (DMSO-d6, 500 MHz) δ 0.86 (t, J = 7 Hz, 3H), 1.21 (s, 3H),1.32 (m, 2H), 1.66 (s, 3H), 1.75 (m, 2H), 3.51 (s, 6H), 4.40 (d, J = 5.5Hz, 1H), 5.36 (d, J = 5.5 Hz, 1H), 5.82 (s, 1H), 6.38 (s, 2H), 7.01(app t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.39 (app t, J = 7.5Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 11.18 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 14.64, 17.19, 21.94, 25.85, 44.05, 55.43, 68.57, 78.47,79.33, 99.16, 103.29, 105.55, 110.92, 114.37, 115.26, 121.40, 122.91,131.21, 138.95, 147.15, 156.49, 159.88, 162.48. ROESY correlationswere observed between the 11a methine H (δ 4.40 ppm) and the 3amethine H (δ 5.36 ppm) and between the 11a methine H (δ 4.40ppm) and the methylene hydrogens of the butyl group (δ 1.75 and1.32 ppm), confirming the cis relationship of the dimethoxyphenyl andthe pyranoquinolinone ring systems. (+) LRMS (ESI) m/z 474 [M +Na]+. HRMS (ESI): calcd for C26H30NO6 [M + H]+, 452.2068; found,452.2069.2-Butyl-2-(3,4-dimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-tet-

rahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (63). From 1-(3,4-dimethoxyphenyl)butan-1-one (790 mg, 3.79 mmol) and 7 (100mg, 0.38 mmol) was obtained 63 (171 mg, 99%). 1H NMR (DMSO-d6, 500 MHz) δ 0.85 (t, J = 7 Hz, 3H), 1.20 (s, 3H), 1.32 (m, 2H),1.65 (s, 3H), 1.78 (m, 2H), 3.37 (s, 3H), 3.57 (s, 3H), 4.40 (d, J = 6.5Hz, 1H), 5.35 (d, J = 6.5 Hz, 1H), 6.46 (d, J = 8.5 Hz, 1H), 6.75(dd, J = 8.5, 1.5 Hz, 1H), 6.80 (d, J = 1.5 Hz, 1H), 7.01 (app t, J = 7.5Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.37 (app t, J = 8 Hz, 1H), 7.50 (d, J= 8 Hz, 1H), 11.18 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 14.66,17.35, 22.04, 25.78, 44.17, 55.71, 56.18, 68.50, 78.61, 79.25, 105.82,109.40, 111.04, 111.82, 114.47, 115.32, 117.46, 121.51, 122.90, 131.18,137.50, 138.86, 148.12, 148.19, 156.34, 162.47. ROESY correlationswere observed between the 11a methine H (δ 4.40 ppm) and the 3amethine H (δ 5.35 ppm) and between the 11a methine H (δ 4.40ppm) and the methylene hydrogens of the butyl group (δ 1.78 and1.32 ppm), confirming the cis relationship of the dimethoxyaryl andthe pyranoquinolinone ring systems. (+) LRMS (ESI) m/z 474[M + Na]+. HRMS (ESI): calcd for C26H30NO6 [M + H]+, 452.2068;found, 452.2072.2-Butyl-2-(3,4,5-trimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-

tetrahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (64). From1-(3,4,5-trimethoxyphenyl)butan-1-one (238 mg, 3.29 mmol) and 7(100 mg, 0.38 mmol) was obtained 64 (56 mg, 30%). 1H NMR(DMSO-d6, 500 MHz) δ 0.87 (t, J = 7.5 Hz, 3H), 1.19 (s, 3H), 1.37(m, 2H), 1.70 (s, 3H), 1.74 (m, 2H), 2.96 (s, 3H), 3.63 (s, 6H), 4.39(d, J = 6.5 Hz, 1H), 5.36 (d, J = 6.5 Hz, 1H), 6.51 (s, 2H), 6.99 (app t,J = 7.5 Hz, 1H), 7.05 (d, J = 8 Hz, 1H), 7.33 (app t, J = 7.5 Hz, 1H),7.47 (d, J = 8 Hz, 1H), 11.16 (s, 1H). 13C NMR (DMSO-d6, 125MHz) δ 19.39, 21.83, 26.67, 30.68, 48.99, 60.86, 64.71, 73.29, 83.36,84.34, 107.40, 110.49, 115.62, 119.12, 120.04, 126.25, 127.44, 135.84,141.39, 143.64, 145.63, 156.75, 161.22, 167.13. ROESY correlationswere observed between the 11a methine H (δ 4.39 ppm) and the 3amethine H (δ 5.36 ppm) and between the 11a methine H (δ 4.39 ppm)and H-8′ (methylene hydrogens of the butyl group, δ 1.37 ppm),confirming the cis relationship of the trimethoxyaryl and thepyranoquinolinone ring systems. (+) LRMS (ESI) m/z 482 [M + H]+,504 [M + Na]+. HRMS (ESI): calcd for C27H32NO7 [M + H]+,482.2173; found, 482.2168.Biological Assays. Human TLR4 Reporter Assays. Stable TLR4/

CD14/MD-2 Transfectants. HEK293-human TLR4/CD14/MD-2cells, stably transfected with human TLR4 (pUNO expression vector),CD14, MD-2 (pDUO expression vector), and pNiFty2-SEAP reporterplasmid were maintained in Dulbecco’s modified Eagle’s mediumsupplemented with 10% FCS (v/v), 2 mM L-glutamine, nonessentialamino acids, 10 μg/mL blasticidin S, 50 μg/mL hygromycin, and100 μg/mL zeocin. Cells were seeded in tissue culture-treated clearflat-bottom polystyrene 96- or 384-well plates (Costar, 3598) at 1 ×104 cells/well. Dose−response curves were generated by addition oftest compounds and incubation for 20 h at 37 °C in an atmosphere of

5% CO2. LPS (Escherichia coli 0111:B4 (Ultrapure, Invivogen) wasused as a positive control. The SEAP released was quantified using p-nitrophenyl phosphate as a substrate, and the absorbance at 405 nmwas measured at 30 s intervals over several minutes using a microplatereader. Data is expressed as Vmax calculated over the linear portion ofthe kinetic absorbance measurements.

Transient Transfections with Human CD14 and MD-2. TLR4-expressing HEK293 cells (stably transfected with human TLR4(pUNO expression plasmid) plus pNiFty2-SEAP reporter plasmidwere transiently transfected using either pUNO-human MD-2, pUNO-human CD14, pDUO-CD14/MD-2, or pUNO-(empty) and thenseeded in tissue culture-treated clear flat-bottom polystyrene 96-wellplates (Costar, 3598) at 10 000 cells per well in 100 μL of media(DMEM, 10% FCS, and 2 mM L-glutamine). Cells were incubatedovernight at 37 °C/5% CO2 in a humidified incubator. Compoundtreatments and SEAP assay were performed as above.

Transient Transfections with Different Combinations of Humanand Mouse TLR4, CD14, and MD-2. HEK293 cells stably transfectedwith pNiFty2-SEAP reporter plasmid were transiently transfected witheither human or mouse TLR4 (pUNO expression vector) incombination with either human or mouse CD14/MD-2 (pDUOexpression vector) and then seeded in tissue culture-treated clear flat-bottom polystyrene 96-well plates (Costar, 3598) at 30 000 cells perwell in 100 μL of media (DMEM, 10% FCS, and 2 mM L-glutamine).Cells were incubated overnight at 37 °C/5% CO2 in a humidifiedincubator. Compound treatments and SEAP assay were performed asabove. Results are expressed as the percent of the maximal response toTNF-α in the same experiment.

Testing Human/Mouse Chimeric TLR4 Receptors. Chimeric TLR4constructs were made that expressed the human TLR4 extracellulardomain fused to the mouse transmembrane and cytoplasmic domainsor the mouse TLR4 extracellular domain fused to the humantransmembrane and cytoplasmic domains. Site-directed mutagenesis ofhuman and mouse TLR4 was performed to introduce a PciI restrictionsite at a conserved position in the coding region for the extracellulardomain, close to the predicted transmembrane domain, without affectingthe amino acid sequence. Constructs encoding the chimeric receptorswere generated in the pUNO expression vector using this restriction siteto join the coding sequences for the human and mouse domains.

HEK293 cells stably transfected with human CD14/MD-2 (pDUOexpression vector) and the pNiFty2-SEAP reporter plasmid weretransiently transfected with either human, mouse, human/mouse, ormouse/human TLR4 construct and then seeded in tissue culture-treated clear flat-bottom polystyrene 96-well plates (Costar, 3598) at30 000 cells per well in 100 μL of media (as described above). Cellswere incubated overnight at 37 °C/5% CO2 in a humidified incubator.Compound treatments and SEAP assay were performed as above.

TLR Selectivity Testing. HEK293 cells stably expressing pNiFty2-SEAP reporter plasmid and a pUNO or pDUO expression plasmidcontaining either human TLR2, 2/6, 3, 5, 7 or 8 were maintained asabove for the human TLR4/CD14/MD-2 cells. Cells were seeded intissue culture-treated clear polystyrene 384-well plates at 7500 cells perwell. Compound treatment was performed as above. The SEAPreleased was quantified using DDAO phosphate as a substrate, andfluorescence was measured at an excitation of 620 nm and emission of665 nm in a PerkinElmer Envision spectrophotometer. Positive controlcompounds for each cell line are given in Table 7.

Human PBMC Preparation. Blood from healthy, consentingvolunteers was collected into heparin, layered onto Lymphoprep(Axis-Shield Diagnostics), and centrifuged at 700g for 25 min. The

Table 7

TLR2, 2/6 Pam3cysk4 (Invivogen, France)TLR3 Poly(I:C) (Invivogen, France)TLR5 Flagellin (Invivogen, France)TLR4 Ultrapure LPS (Invivogen, France)TLR7 and 8 R848 (AstraZeneca, UK)

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PBMC layer was removed, diluted to 50 mL with PBS, and centrifugedat 400g for 10 min. The supernatant was removed, and the pellet wasresuspended in 50 mL of PBS and centrifuged at 300g for 5 min. Thecells were then washed in 50 mL of PBS and recovered by centrifugingat 200g for 5 min. PBMCs were resuspended in assay medium (RPMI1640 with 25 mM HEPES, 10% FCS (v/v), 2 mM L-glutamine,10 U/mL penicillin, and 10 μg/mL streptomycin).PBMC Incubations. Twenty microliters of test compound or assay

medium with 1% DMSO (v/v), vehicle control, were added to each wellof a 96-well polystyrene tissue culture-treated clear flat-bottom plate(Costar 3598) followed by 180 μL of PBMC cell suspension (prepared asabove) in assay medium (200 000 cells). PBMCs and compounds wereincubated at 37 °C in an atmosphere of air/CO2 (95:5 v/v) for 20 h forcytokine stimulation and 44 h for IL-5 suppression studies (see below).Cytokine Determinations. Cytokines were determined from PBMC

supernatants using the following commercial kits (OptEIA kits, BD,Oxford, UK) following the manufacturer’s instructions: human IL-5(555202), human TNF-α (555212), human IL-12p40 (555171),human IL-8 (555244), and human IL-10 (555157).Human PBMC IL-5 Suppression Assay. Human PBMCs were

prepared and plated out with compounds as described above.Phytohemeagglutinin (PHA) (Sigma, Dorset, UK) was added at afinal concentration of 1 μg/mL, and the PBMCs were then incubatedfor 44 h before the supernatant was removed for determination of theamount of IL-5 produced.Cytotoxicity Measurements. Cytotoxicity was determined in

PBMC assays by measuring cellular metabolic activity using theWST-1 cell proliferation reagent (Roche Diagnostics, UK).

■ ASSOCIATED CONTENT*S Supporting InformationRepresentative NMR data for the 10 most active compounds fromthe cyclobutane series (compounds 1, 20, 22, 26, 31, 32, 34, 35,52, and 53) and two of the acetal analogues (compounds 63 and64). Selectivity data for euodenine A, LPS, and the positive controlsacross the TLR lines. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +61 7 37356009. Fax: +61 7 37356001. E-mail:R.Quinn@griffith.edu.au.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Hoan T. Vu for HRMS measurements and MarkFurber for assistance in the preparation of this manuscript.

■ ABBREVIATIONS USEDCD14, cluster of differentiation 14; MD-2, MD-2 is a 160-aminoacid protein that is associated with TLR4 on the cell surface andenables TLR4 to respond to LPS; PBMC, peripheral bloodmononuclear cells; SEAP, secreted embryonic alkaline phosphatase

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■ NOTE ADDED AFTER ASAP PUBLICATIONDr. George Nikolakopoulos' name was misspelled in theversion published ASAP on February 7, 2014. The correctedversion was re-posted on February 11, 2014.

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