Article pubs.acs.org/joc
De Novo Synthesis of Possible Candidates for the Inagami−Tamura Endogenous Digitalis-like Factor Atsuo Nakazaki,* Keiko Hashimoto, Ai Ikeda, Takahiro Shibata, and Toshio Nishikawa Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8601, Japan S Supporting Information *
ABSTRACT: De novo synthesis of possible candidates for the Inagami−Tamura endogenous digitalis-like factor (EDLF) was achieved to validate a previously proposed structure. Our synthetic approach involves a highly regio- and diastereoselective Mizoroki−Heck reaction and a Friedel−Crafts-type cyclodehydration to construct steroidal tetracycle 14 as a versatile common intermediate leading to seven 2,14βdihydroxyestradiol analogues 1a−c, 2a−c, and 3 as possible candidates. By comparing the potency of inhibitory activity against Na+/K+-ATPase between the synthesized candidates and the EDLF, it was found that the proposed structure is not likely to be a true structure of the Inagami−Tamura EDLF.
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INTRODUCTION Cardiotonic steroids are originally isolated from plants, toads, snakes, and insects,1 and they cause an increase in calcium concentration through the inhibition of Na+/K+-ATPase, leading to contraction of cells.2 Cardiotonic steroids possess common structural features: (1) a β-hydroxy group at C14 in a steroidal scaffold bearing cis-fused AB and CD rings, (2) a βconfigured 5- or 6-membered unsaturated lactone moiety at C17 (cardenolides or bufadienolides, respectively), and (3) the presence of a variable number of sugar residues at C3. Lactones are considered to be the most essential functional group for biological activity of these molecules.3 A variety of the related steroids has also been isolated from mammalian tissues and fluids. These still unidentified compounds are referred to as the endogenous digitalis-like factor (EDLF) (Figure 1).4 Ouabain, the most well-known EDLF candidate, has been isolated from human plasma5a and bovine hypothalamus5b and structurally characterized by FABMS and 1H NMR. Although EDLF plays a role in various disorders, most EDLF structures remain ambiguous because the obtained amounts are typically quite small. A candidate for EDLF was isolated from bovine adrenal by Inagami and Tamura in 1987.6 This compound, named Inagami−Tamura EDLF, exhibited all of the representative properties of an EDLF: (1) potent inhibitory activity against Na+/K+-ATPase (postulated IC50: 9.9−182 nM) comparable to ouabain (IC50: 23 nM), (2) competitive displacement activity against [3H]-ouabain bound to the enzyme, (3) inhibitory activity for 86Rb uptake into intact human erythrocytes, and (4) cross-reactivity with antidigoxin-specific antibody. Overall, this compound functioned in a manner similar to that of ouabain. FAB-LRMS spectra showed an m/z value of 336, and biological evaluation suggested that it possesses a steroidal scaffold rather © 2017 American Chemical Society
than a lipid or peptide composition. NMR spectra could not be acquired due to the insufficient amount isolated (multimicrograms), and therefore, the structure has not been elucidated. In 1996, Sakakibara and Uchida proposed a 2,14βdihydroxyestradiol analogue having two hydroxy groups, compound A, as a structural candidate for the Inagami− Tamura EDLF based on this limited information and their own deductions (Figure 2).7 Compound A possesses a unique structural motif not found in other reported EDLF candidates such as ouabain owing to its unsaturated A-ring moiety. Sakakibara and Uchida envisaged that the positions and stereochemistries of the residual hydroxy groups on the dihydroxyestradiol basic scaffold could be determined by comparison of the inhibitory activity of synthetic compounds. However, two possible candidates semisynthesized from estradiol, 1a and 1b, exhibited low contractile activity of isolated rat aorta and guinea pig left atrium, indicating insufficient potency for the Inagami−Tamura EDLF.7 The validity of the basic scaffold, as well as the positions and the stereochemistries of two hydroxy groups, have not been explored further. To validate the structure proposed by Sakakibara, we embarked on the synthesis of proposed 2,14βdihydroxyestradiol analogues and an evaluation of their biological activities. In this report, we describe the de novo synthesis8 of seven possible candidates and their inhibitory activity of Na+/K+-ATPase. Our synthetic approach involves a highly regio- and diastereoselective Mizoroki−Heck reaction and a Friedel−Crafts-type cyclodehydration to construct a steroidal tetracycle as a common intermediate. This steroidal intermediate allows facile construction of the proposed 2,14βReceived: July 1, 2017 Published: August 8, 2017 9097
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
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The Journal of Organic Chemistry
Figure 1. Structures of the reported EDLF candidates.
Figure 2. Proposed structure A of the Inagami−Tamura EDLF and its possible candidates 1a−c, 2a−c, and 3.
dihydroxyestradiol analogues such as 1a−c, 2a−c, and 3 (Figure 2) because this intermediate possesses an alkene on the B- and C-rings for the introduction of hydroxy groups. Furthermore, our de novo approach offers a new and facile entry to a variety of other estradiol analogues with different functionalities.9
intramolecular Diels−Alder reaction of enol ester 5 and subsequent optical resolution.
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RESULTS AND DISCUSSION Synthesis of Lactone 8. To obtain enantioenriched tricyclic lactone 8 at the outset, we synthesized enol ester 5, a substrate of intramolecular Diels−Alder reaction, from commercially available ethyl sorbate (Scheme 2). Thus, isomerization of ethyl sorbate was carried out under conventional conditions to obtain β,γ-unsaturated ester 4a, which was then hydrolyzed to afford corresponding carboxylic acid 4b. Transformation of 4b into an acid chloride and subsequent treatment with 2-methyl-1,3-cyclopentanedione in the presence of Et3N afforded enol ester 5 in good overall yield. Intramolecular Diels−Alder reaction of 5 was conducted at 200 °C with BHT in 1,2,4-trichlorobenzene to provide the desired endo-6 in 66% as a major diastereomer along with exo-6 in 19% yield.10 A decagram-scale synthesis of endo-6 could be performed from carboxylic acid 4b (34% in 3 steps). The ketone carbonyl group in endo-6 was then reduced with NaBH4
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SYNTHETIC PLAN Our synthetic plan is depicted in Scheme 1. In contrast to Sakakibara’s semisynthesis of 1a and 1b from estradiol,7 we decided to adopt the de novo synthesis of the possible candidates 1a−c, 2a−c, and 3 from a steroidal tetracycle like B as a common intermediate to introduce hydroxy groups into the B- and C-rings. Tetracycle B would be synthesized by the Friedel−Crafts-type cyclodehydration of lactol C. An aryl group in lactone D, a precursor of lactol C, would be stereoselectively introduced by means of the Mizoroki−Heck reaction of lactone 8 with bromoarene E from the convex face of the tricyclic lactone system. Tricyclic lactone 8 bearing a cis-hydroindane scaffold would in turn be furnished by a newly designed 9098
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
Article
The Journal of Organic Chemistry Scheme 1. Synthetic Plan for Candidates 1a−c, 2a−c, and 3
Scheme 2. Synthesis of Enantioenriched Acetate 8 and Alcohol 7
in MeOH to afford alcohol (±)-7 in 82% yield. The relative stereochemistry of endo-6 and (±)-7 was determined by NOESY analysis as shown in Figure 3, which revealed that the reduction took place preferentially from the convex face of the scaffold. With racemic alcohol (±)-7 in hand, optical resolution was conducted next. After several attempts at kinetic resolution with various enzymes, it was found that the resolution with lipase MY-30 (Meito Sangyo Co. Ltd., Japan) in the presence of vinyl acetate and subsequent recrystallization afforded enantiomerically pure acetate 8 and alcohol 7 in good yields on a multigram scale. The absolute configuration of 8 was
Figure 3. Diagnostic NOESY correlations of endo-6 and alcohol (±)-7.
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DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
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The Journal of Organic Chemistry
Scheme 3. Synthesis of Tetracycle 14 through the Mizoroki−Heck Reaction and Friedel−Crafts-type Cyclodehydration
indispensable intermediate, in good yield. A combination of Sc(OTf)3/MeOH was crucial for this transformation;15 other systems such as Sc(OTf)3/CH2Cl2, p-TsOH/(CH2Cl)2, TFA/ (CH2Cl)2, and BF3·OEt2/CH2Cl2 only furnished a complex mixture instead of desired 14. Synthesis of Possible Candidates 1a−c from Tetracycle 14. With key steroidal intermediate 14 in hand, we moved on to the synthesis of possible candidates 1a−c for the Inagami−Tamura EDLF, as depicted in Scheme 4. Hydrogenation of the CC bonds in tetracycle 14 with the Crabtree’s catalyst under a hydrogen atmosphere (1 atm) provided 15 in 65% yield. Oxidation of the secondary alcohol in 15 led to ketone 16, which was treated with TMSOTf and Et3N to provide the silyl enol ether with a TMS-protected tertiary hydroxy group. Subsequent the Ito−Saegusa reaction16 of the resulting silyl enol ether led to enone 17a in 88% yield (2 steps). After deprotection of the TMS group, treatment of the resulting enone 17b with NaBH4 and CeCl3·7H2O in MeOH at rt afforded 18 in 80% yield and epi-18 in 9% yield. Dihydroxylation of the resulting allylic alcohol 18 using OsO4/NMO provided known compounds 19a and 19b as a separable mixture in 17 and 42% yields, respectively. It is worth noting that diols 19a and 19b were directly separated using Chromatorex-DIOL (Fuji Silysia Chemical Ltd., Japan), whereas in the previous report,7 they were separated after derivatization into the corresponding acetates. Finally, deprotection of the benzyl groups in 19a and 19b was successfully carried out with Pd/BaSO4 under a hydrogen atmosphere (1 atm) to afford the corresponding 1a and 1b, respectively.7 The 1 H and 13C NMR spectra of synthesized compounds 19a, 19b, 1a, and 1b were in agreement with those reported; however, subtle differences in chemical shifts were observed. Therefore, we identified 19a and 19b after acetylation to triacetates 20a and 20b. The 1H and 13C NMR spectra of 20a and 20b were identical to those previously reported. To obtain 15,16-trans-diol 1c, epoxidation of allylic alcohol 18 under the Sharpless conditions [VO(acac)2, TBHP]17 was followed by ring-opening of the resulting epoxide 21 using
unambiguously established by single-crystal X-ray diffraction analysis by using the Bijvoet method.11 Synthesis of Tetracycle 14, a Common Intermediate. We next investigated a Mizoroki−Heck reaction using acetate 8 (Scheme 3). The Mizoroki−Heck reaction of acetate 8 and bromoarene 912 with palladacycle 10 as a catalyst, reported by Herrmann and co-workers,13 resulted in the formation of desired product 11a in 70% yield along with its regioisomer 12a in 20% yield. Both compounds were obtained as a single diastereomer. Alcohol (±)-7 also underwent the related transformation with 9 to afford 11b and 12b in 62 and 28% yields, respectively. The observed regioselectivity could be attributed to steric hindrance by the buttressing effect of the angular methyl group. The relative stereochemistries of alcohols 11b and 12b were confirmed by NOESY correlations, as shown in Figure 4. The relative stereochemistries of acetates 11a and
Figure 4. Diagnostic NOESY correlations of 11b and 12b.
12a were confirmed by derivatizations from 11b and 12b, respectively. The stereochemical outcomes indicate that the Mizoroki−Heck reactions took place from the convex face of the tricycic lactone system, as anticipated. Thus, the present Mizoroki−Heck reaction enables stereoselective installation of the A-ring fragment into 11a with a proper C9 stereochemistry for construction of a trans-BC ring junction in steroidal tetracyclic system 14.14 The second key reaction leading to the steroid scaffold was examined next. Aryl adduct 11a was converted into lactol 13 by DIBAL reduction, and subsequent Friedel−Crafts-type cyclodehydration of 13 provided tetracycle 14, a common and 9100
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
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The Journal of Organic Chemistry Scheme 4. Synthesis of 15,16-Diols 1a and 1b and 15,16-trans-Diol 1c from Tetracycle 14
PhCO2Na at 150 °C to provide trans-diol 22.18 Finally, deprotection of the benzyl groups by the same manner afforded corresponding 1c in good overall yields. The newly generated stereogenic centers of 22 were determined by NOESY correlation between the C9 and C15 protons, as shown in Figure 5.
remaining C11−C12 alkene in the resulting 24 and 25 were performed under the same conditions [Pd/BaSO4, H2 (1 atm), EtOAc] leading to the candidates 2a and 2b, respectively. On the other hand, Prevost reaction of tetracycle 14 provided the corresponding dibenzoate, which was then hydrolyzed to afford the other 6,7-trans-diol 26. Final deprotection and hydrogenation also succeeded to obtain 2c in the same manner. The relative stereochemistry of 24 and 26 were determined by NOESY analysis (Figure 6). Synthesis of Possible Candidate 3 from Tetracycle 14. Final candidate 3 was synthesized from tetracycle 14 (Scheme 6). Regioselective hydrogenation of the sterically less congested alkene in 14 was carried out with Wilkinson’s catalyst in 82% yield. Subsequent dihydroxylation with trimethylamine Noxide19 resulted in the exclusive formation of β-cis-diol 28 in moderate yield. The relative stereochemistry of 28 was determined by NOESY analysis, as depicted in Figure 7. Final deprotection of the benzyl groups in 28 furnished 11,12cis-diol 3 in 42% yield.
Figure 5. Diagnostic NOESY correlations of 15,16-diol 22.
Synthesis of Possible Candidates 2a−c from Tetracycle 14. Tetracycle 14 was then converted into 6,7-diols 2a−c (Scheme 5). The buttressing effect of the angular methyl group in 14 was expected to allow discrimination of the two alkenes at C6−C7 and C11−C12. Indeed, oxidation of 14 with OsO4 and NMO afforded desired 6,7-cis-diol 24 in 27% yield as a minor product and hydroxy ketone 23 in 50% yield. Hydroxy ketone 23 was not an expected product but a useful intermediate to obtain 6,7-trans-diol 25. Reduction of 23 with NaBH4 afforded a separable mixture of 6,7-trans-diol 25 and 6,7-cis-diol 24. Deprotection of the benzyl groups and hydrogenation of the
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EVALUATION OF INHIBITORY ACTIVITY We investigated the inhibitory activity of seven synthesized analogues, 1a−c, 2a−c, and 3, against Na+/K+-ATPase from porcine cerebral cortex by colorimetric phosphate quantitation using malachite green (Figure 8). Ouabain (OUA) was used as a positive control. The inhibitory activities of the seven compounds were lower than that of OUA at a final 9101
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
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The Journal of Organic Chemistry Scheme 5. Synthesis of 6,7-Diol 2a−c from Common Intermediate 14
Figure 6. Diagnostic NOESY correlations of 24 and 26.
3 μM (Figure 8b). In this study, the synthesis and evaluation of the inhibitory activities were not examined for all possible dihydroxylated regio- and stereoisomeric analogues. However, on the basis of the general trend observed here for the structure and activity, other 2,14β-dihydroxyestradiol analogues seem to exhibit less potent inhibitory activities than that of OUA as well, even by changing position and stereochemistry of the hydroxy groups. From the viewpoint of the potent inhibitory activity reported by Inagami and Tamura,6 the structure proposed by Sakakibara is not likely to be a true structure of the Inagami− Tamura EDLF, and the basic scaffold may be incorrect.
Scheme 6. Synthesis of 11,12-Diol 3 from Common Intermediate 14
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CONCLUSIONS We have developed the de novo synthesis of seven dihydroxylated 2,14β-dihydroxyestradiol analogues to validate Sakakibara’s proposed structure for the Inagami−Tamura EDLF. A newly designed intramolecular Diels−Alder reaction of enol ester 5 enables construction of functionalized tricyclic lactone endo-6 on a decagram scale. Optical resolution of alcohol (±)-7 with lipase MY-30 and subsequent recrystallization provided enantiomerically pure acetate 8 in good yield. Two key transformations, a highly regio- and stereoselective Mizoroki−Heck reaction using Herrmann palladacycle 10 and a Friedel−Crafts-type cyclodehydration with Sc(OTf)3/MeOH, allowed for synthesis of steroidal tetracycle 14 in good overall yield. Finally, tetracycle 14, a versatile intermediate, was successfully converted into seven 2,14β-dihydroxyestradiol analogues 1a−c, 2a−c, and 3. We are hopeful that this newly developed de novo approach will provide a facile entry to a wide variety of other estradiol analogues with functionalities on the B- and C-rings, which are otherwise difficult to obtain.9 As
Figure 7. Diagnostic NOESY correlations of 11,12-diol 28.
concentration of 3 μM (Figure 8a). At a final concentration of 300 μM, some of the synthesized compounds such as 1a−c and 3 showed inhibitory activities comparable to that of ouabain at 9102
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
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The Journal of Organic Chemistry
Figure 8. Inhibitory activity of synthesized compounds 1a−c, 2a−c, and 3 at final concentrations of (a) 3 and (b) 300 μM for synthesized compounds and 3 μM for OUA (ouabain). The results shown are the means of three independent experiments ± SD (error bars). Ltd. HU50710) was used for Chromatorex DIOL silica gel flash column chromatography. Preparative TLC separations were carried out on 0.5 mm silica gel plates 60F254 (Merck). DIOL Preparative TLC separations were carried out on 0.25 mm DIOL silica gel plates (Fuji Silysia Chemical Ltd.). Dry THF and CH2Cl2 were purchased from Kanto Chemical Co., Inc. Dry benzene, DMPU, DMF, toluene, Et3N, DME, DMSO, and diisopropylamine were distilled from CaH2. Celite (Hyflo Super-Cel Celite) was purchased from Nacalai Tesque, Inc. Florisil was purchased from Kanto Chemical Co., Inc. DISMIC13JP (disposable membrane filter unit, pore size 0.20 μm), purchased from Toyo Roshi Kaisha, Ltd., was used for 0.20 μm membrane filter. Lipase MY-30 was purchased from Meito Sangyo Co., Ltd. All other commercially available reagents were used as received. Ester 4a. A solution of LDA was prepared by slow addition of nBuLi (1.60 M solution in hexane, 255 mL, 408 mmol) to a solution of dry diisopropylamine (55.0 mL, 416 mmol) in dry THF (682 mL) at −78 °C. To this solution was added dropwise DMPU (54.0 mL, 447 mmol) at −78 °C. After being stirred at −78 °C for 2 h, ethyl sorbate (47.8 g, 341 mmol) was added to this mixture. After being stirred at −78 °C for 2 h, the reaction mixture was allowed to warm to room temperature. The reaction was quenched with 10% acetic acid (700 mL, v/v). The aqueous layer was extracted with pentane. The combined organic layer was washed with H2O (4×) and a saturated aqueous solution of NaHCO3 (1×), dried over Na2SO4, and concentrated carefully under reduced pressure. The residue was purified by distillation to afford ester 4a (31.4 g, bp 69−71 °C/15 mmHg, 66% yield) as a pale-yellow oil. 1H NMR, 13C NMR, and IR data were identical to those reported for this compound.20 IR (KBr) vmax 1736, 1653 cm−1. 1H NMR (300 MHz, CDCl3) δ 1.26 (3H, t, J = 7 Hz, CH3CH2OCO), 3.11 (2H, d, J = 7 Hz, CH2CO), 4.15 (2H, q, J = 7 Hz, CH3CH2OCO), 5.06 (1H, d, J = 10 Hz, CHAHB=CH), 5.16 (1H, d, J = 17 Hz, CHAHB=CH), 5.79 (1H, dt, J = 15.5, 7 Hz, CHCHCH 2 CO), 6.14 (1H, dd, J = 17, 15.5 Hz, CH=CHCH2CO), 6.34 (1H, ddd, J = 17, 17, 10 Hz, CHAHB=CH). 13 C NMR (75 MHz, CDCl3) δ 14.2, 38.0, 60.7, 116.9, 125.7, 134.2, 136.3, 171.4. Carboxylic Acid 4b. To a solution of 4a (31.4 g, 224 mmol) and EtOH (3 mL) in dry THF (450 mL) was added lithium hydroxide (1.0 M solution in H2O, 270 mL, 269 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. The reaction was quenched with 1.0 N HCl. The aqueous layer was extracted with EtOAc (2×). The combined organic layer was washed with brine (1×), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by neutral silica gel open column chromatography (hexane:EtOAc = 14:1 to 3:1) to afford carboxylic acid 4b (24.8 g, 99% yield) as a yellow oil. 1H NMR, 13C NMR, and IR data were identical to those reported for this compound.21 IR (KBr) vmax 3021, 1709 cm−1. 1H NMR (400 MHz, CDCl3) δ 3.17 (2H, d, J = 7 Hz, CH2CO), 5.09 (1H, d, J = 10.5 Hz, CHAHB=CH), 5.19 (1H, d, J =
all of these synthetic analogues of Sakakibara’s proposed structure exhibited low inhibitory activity against Na+/K+ATPase, the basic scaffold of the proposed structure may be incorrect. It is reasonable to expect that further confirmation of the structure for the Inagami−Tamura EDLF would eventually be achieved through reisolation and characterization based on NMR studies of the compound.
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EXPERIMENTAL SECTION
General Techniques. Infrared spectra (IR) were recorded on a JASCO FT/IR-4100 type A spectrophotometer and are reported in wavenumber (cm−1). Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Varian Gemini-2000 (300 MHz), a Bruker Avance-400 (400 MHz), or a Bruker ARX-400 (400 MHz) spectrometer. Chemical shifts of all compounds are reported in ppm relative to the residual undeuterated solvent (CDCl3 as δ = 7.26, CD3OD as δ = 3.31, DMSO-d6 as δ = 2.50, acetone-d6 as δ = 2.05). Data were reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broadened), coupling constant(s), and assignment. Carbon nuclear magnetic resonance (13C NMR) spectra were recorded on a Varian Gemini-2000 (75 MHz), a Bruker Avance-400 (100 MHz), a Bruker ARX-400 (100 MHz), or a Bruker Avance-600 (150 MHz) spectrometer. Chemical shifts of all compounds are reported in ppm relative to the solvent (CDCl3 as δ = 77.0, CD3OD as δ = 49.0, DMSO-d6 as δ = 39.5, acetone-d6 as δ = 29.8). All NMR were measured at 300 K. High-resolution mass spectra (HRMS) were recorded on an Applied Biosystems Mariner ESI-TOF spectrometer for ESI-MS and reported in m/z. Elemental analyses were performed by the Analytical Laboratory of Graduate School of Bioagricultural Sciences, Nagoya University. Melting points (mp) were recorded on a Yanaco MP-S3 melting point apparatus and are not corrected. Absorbance was measured on a SPECTRAmax 250 microplate spectrophotometer. All reactions were monitored by thin layer chromatography (TLC) on 0.25 mm silica gel-coated glass plates 60F 254 (Merck, #1.05715.0001). Visualization was achieved by using UV light (254 nm) and appropriate reagent (ethanolic phosphomolybdic acid or panisaldehyde solution in H2SO4/AcOH/EtOH) followed by heating. Silica gel 60 (particle size 0.063−0.200 mm, Merck, #1.07734.9025) was used for silica gel open column chromatography. Silica gel 60 (spherical, particle size 0.04−0.05 mm, Kanto, #37562-84) was used for silica gel flash column chromatography. Silica gel 60N (neutral, particle size 0.063−0.200 mm, Kanto, #37563-79) was used for neutral silica gel open column chromatography. Chromatorex-DIOL (particle size MB100-75/200, Fuji Silysia Chemical Ltd. HU50711) was used for Chromatorex DIOL silica gel open column chromatography. Chromatorex-DIOL (particle size MB100-40/75, Fuji Silysia Chemical 9103
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
Article
The Journal of Organic Chemistry 17 Hz, CHAHB=CH), 5.77 (1H, dt, J = 15.5, 7 Hz, CHCHCH2), 6.17 (1H, dd, J = 15.5, 10.5 Hz, CH=CHCH2), 6.34 (1H, ddd, J = 17, 10.5, 10.5 Hz, CHAHB=CH). 13C NMR (100 MHz, CDCl3) δ 37.6, 117.4, 124.5, 134.9, 136.1, 178.1. Enol Ester 5. Carboxylic acid 4b (1.00 g, 8.92 mmol) was dried azeotropically with toluene and dissolved in dry CH2Cl2 (50 mL). To this solution was added DMF (1 drop) followed by oxalyl chloride (1.30 mL, 15.2 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. The residue was diluted with dry CH2Cl2 (50 mL). This solution was added to a solution of 2-methyl-1,3-cyclopentanedione (770 mg, 6.86 mmol) and dry Et3N (1.10 mL, 7.55 mmol) in dry CH2Cl2 (30 mL) at room temperature immediately via cannula. After being stirred at room temperature for 1 h, the reaction mixture was washed with H2O (1×) and a saturated aqueous solution of NaHCO3 (1×) and brine (1×), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by neutral silica gel open column chromatography (hexane:EtOAc = 5:1) to afford enol ester 5 (1.18 g, 84% in 2 steps) as a yellow oil. IR (KBr) vmax 1768, 1707, 1668, 1319, 1184, 1105 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.62 (3H, t, J = 2 Hz, CH3CCO), 2.52 (2H, m, CH x2), 2.82 (2H, m, CH x2), 3.34 (2H, d, J = 7.5 Hz, CHCHCH2CO), 5.12 (1H, d, J = 10 Hz, CHAHB=CH), 5.22 (1H, d, J = 17 Hz, CHAHB=CH), 5.79 (1H, dt, J = 15, 7.5 Hz, CHCHCH2CO), 6.23 (1H, dd, J = 15, 10 Hz, CH=CHCH2CO), 6.35 (1H, ddd, J = 17, 10, 10 Hz, CHAHB=CH). 13C NMR (100 MHz, CDCl3) δ 6.6, 27.0, 34.3, 37.8, 117.9, 123.5, 126.3, 135.6, 135.9, 167.2, 175.6, 205.9. HRMS (ESI) [M + Na]+ calcd for C12H14O3Na, 229.0835; found, 229.0843. Intramolecular Diels−Alder Reaction of 5. A solution of 5 (140 mg, 0.676 mmol) and BHT (5.6 mg, 0.027 mmol) in 1,2,4trichlorobenzene (33 mL) was stirred at 200 °C for 4 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel open column chromatography (hexane to EtOAc). This mixture was purified by silica gel flash column chromatography (hexane:EtOAc = 4:1 to 3:1 to 2:1) to afford endo6 (92.0 mg, 66% yield) as a white solid and exo-6 (25.9 mg, 19% yield) as a white solid. Relative stereochemistry of compound endo-6 was determined by NOESY correlations (Figure 3). endo-6: IR (KBr) vmax 1782, 1742 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.14 (3H, s, CH3CCO), 1.89 (1H, ddd, J = 14, 7, 4 Hz, CHAHBCH2CO), 2.11−2.17 (2H, m, CHCHCH2), 2.22 (1H, dd, J = 14, 10.5 Hz, CHAHBCH2CO), 2.43−2.50 (2H, m, CHAHBCH2CO), 2.55 (1H, dd, J = 16.5, 14.5 Hz, CHCHAHBCO), 2.71 (1H, dd, J = 16.5, 7 Hz, CHCHAHBCO), 3.06 (1H, m, CHCHAHBCO), 5.73 (1H, ddd, J = 9.5, 7, 3.5 Hz, CHCHCH2), 5.94 (1H, ddd, J = 9.5, 4.5, 2 Hz, CH=CHCH2). 13C NMR (100 MHz, CDCl3) δ 15.2, 26.7, 32.6, 33.5, 35.8, 38.3, 50.2, 94.2, 124.9, 127.9, 175.1, 216.8. HRMS (ESI) [M + Na]+ calcd for C12H14O3Na, 229.0835; found, 229.0845. Mp 98−99 °C. exo-6: IR (KBr) vmax 1774, 1743, 1237 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.11 (3H, s, CH3CCO), 1.96 (1H, dd, J = 17.5, 4 Hz, CHCHCHAHB), 2.08 (1H, m, CHCHCHAHB), 2.17 (1H, m, CHAHBCH2CO), 2.31 (1H, ddd, J = 14, 9, 3 Hz, CHAHBCH2CO), 2.41 (1H, dd, J = 17.5, 10.5 Hz, CHCHAHBC O), 2.48−2.57 (2H, m, CHAHBCH2CO), 2.85 (1H, dd, J = 17.5, 10 Hz, CHCHAHBCO), 2.95 (1H, m, CHCHAHBCO), 5.75−5.78 (2H, m, CH=CHCHAHB). 13C NMR (100 MHz, CDCl3) δ 15.3, 31.66, 31.69, 33.9, 35.6, 38.1, 50.7, 91.6, 124.0, 125.6, 174.8, 216.3. HRMS (ESI) [M + Na]+ calcd for C12H14O3Na, 229.0835; found, 229.0836. Mp 87−89 °C. Decagram-Scale Synthesis of endo-6 from Carboxylic Acid 4b. To a solution of 4b (24.8 g, 221 mmol) in dry CH2Cl2 (1.5 L) was added DMF (0.05 mL) followed by oxalyl chloride (32.0 mL, 376 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. The residue was diluted with dry CH2Cl2 (100 mL). This solution was added to a solution of 2-methyl-1,3-cyclopentanedione (19.1 g, 170 mmol) and dry Et3N (26.1 mL, 187 mmol) in dry CH2Cl2 (850 mL) at room temperature immediately via cannula. After being stirred at room temperature for 1 h, the reaction mixture was washed with H2O (1×) and a saturated aqueous solution of NaHCO3 (1×) and brine
(1×), dried over Na2SO4, and concentrated under reduced pressure. The residue was passed through a column with neutral silica gel (hexane:EtOAc = 4:1) to afford enol ester 5 (27.6 g, contained approximately 8% of carboxylic acid 4b) as a yellow oil. A solution of 5 (27.6 g, 134 mmol) and BHT (1.20 g, 5.45 mmol) in 1,2,4trichlorobenzene (1340 mL) was stirred at 155 °C for 10 h. The reaction mixture was concentrated under reduced pressure at 83 °C (7 mmHg). The residue was purified by silica gel open column chromatography (hexane to EtOAc) to give an 83:17 mixture of endo-6 and exo-6. This mixture was purified by recrystallization from hexane/EtOAc and silica gel open column chromatography (hexane:EtOAc = 2:1) to afford endo-6 (16.4 g, 34% yield in 3 steps) as a white solid and exo-6 (3.68 g, 8% yield in 3 steps) as a white solid. Reduction of endo-6. To a solution of endo-6 (8.87 g, 43.0 mmol) in MeOH (717 mL) was added NaBH4 (3.38 g, 89.3 mmol) at 0 °C. After being stirred at 0 °C for 30 min, the reaction was quenched with acetic acid, and the reaction mixture was concentrated under reduced pressure. The residue was diluted with H2O, and the aqueous layer was extracted with EtOAc (2×). The combined organic layer was washed with brine (1×), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel flash column chromatography (hexane:EtOAc = 3:2) to afford major isomer (±)-7 (7.33 g, 82% yield) as a white solid and minor isomer (±)-epi-7 (788 mg, 9% yield) as a white solid. Relative stereochemistry of (±)-7 was determined by NOESY correlations (Figure 3). (±)-7: IR (KBr) vmax 3433, 1773, 1010 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.12 (3H, s, CH3CCOH), 1.38 (1H, m, CHAHBCHAHBCHOH), 1.56 (1H, m, CHAHBCHAHBCHOH), 1.73 (1H, dd, J = 18.5, 2.5 Hz, CHCHCHAHB), 2.02−2.14 (2H, m, CHAHBCHAHBCHOH), 2.43 (1H, dd, J = 16, 14 Hz, CHCHAHBCO), 2.54 (1H, ddd, J = 18.5, 4, 2 Hz, CHCHCHAHB), 2.61 (1H, dd, J = 16, 7 Hz, CHCHAHBC O), 2.95 (1H, ddd, J = 14, 7, 2 Hz, CHCHAHBCO), 4.13 (1H, dd, J = 9, 8 Hz, CHOH), 5.77−5.85 (2H, m, CH=CHCHAHB). 13C NMR (100 MHz, CDCl3) δ 19.0, 29.5, 29.9, 31.4, 32.7, 39.6, 46.9, 78.2, 96.1, 124.1, 130.0, 176.5. HRMS (ESI) [M + Na]+ calcd for C12H16O3Na, 231.0992; found, 231.1002. Mp 93−94 °C. (±)-epi-7: IR (KBr) vmax 3485, 1754 cm−1. 1H NMR (400 MHz, CDCl3) δ 1.16 (3H, s, CH3CCOH), 1.76−1.89 (3H, m, CH x3), 1.99−2.11 (3H, m, CH x3), 2.44 (1H, dd, J = 16, 14.5 Hz, CHCHAHBCO) 2.64 (1H, dd, J = 16, 7 Hz, CHCHAHBCO), 2.96 (1H, m, CHCHAHBCO), 3.78 (1H, d, J = 4 Hz, CHOH), 5.74 (1H, m, CHCHCH2). 5.84 (1H, ddd, J = 10, 4, 2.5 Hz, CH=CHCH2). 13C NMR (100 MHz, CDCl3) δ 16.2, 31.1, 31.7, 33.1, 38.4, 39.3, 48.3, 81.3, 97.2 124.3, 129.3, 175.9. HRMS (ESI) [M + Na]+ calcd for C12H16O3Na, 231.0992; found, 231.0983. Mp 112−113 °C. Optical Resolution of Alcohol (±)-7. To a solution of racemic alcohol (±)-7 (6.24 g, 30.0 mmol) in dry DME/toluene (278 mL, 1:4) were added vinyl acetate (52.3 g, 608 mmol) and lipase MY-30 (11.35 g) at room temperature. After being stirred at room temperature for 13 h, to the resulting mixture was added lipase MY-30 (11.33 g) at room temperature. After being stirred at room temperature for 10.5 h, to the resulting mixture was added lipase MY-30 (11.09 g) at room temperature. After being stirred at room temperature for 16 h, to the resulting mixture was added lipase MY-30 (11.02 g) at room temperature. After being stirred at room temperature for 7.5 h, the reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel open column chromatography (hexane:EtOAc = 2:1) to afford acetate 8 (3.50 g, 47% yield, 90% ee) as a white solid and alcohol 7 (3.10 g, 50% yield, 89% ee) as a white solid. Recrystallization from hexane/CH2Cl2 afforded 8 (1.92 g, 99% ee, 55% yield) as a white solid. Recrystallization from hexane/EtOAc afforded 7 (77%, >99% ee) as a white solid. Enantiomeric excess was determined by chiral HPLC analysis. Chiral HPLC analysis for 8 (CHIRALPAK IB column, hexane/2-propanol = 90:10, flow rate = 0.5 mL/min, detection 220 nm light, 30 °C) tR = 17.7 min (major isomer), 19.1 min (minor isomer). Chiral HPLC analysis for 7 (CHIRALPAK IB column, hexane/2-propanol = 90:10, flow rate = 0.5 mL/min, detection 220 nm light, 30 °C) tR = 20.9 min (minor isomer), 22.1 min (major 9104
DOI: 10.1021/acs.joc.7b01640 J. Org. Chem. 2017, 82, 9097−9111
Article
The Journal of Organic Chemistry
pressure. The residue was purified by silica gel flash column chromatography (hexane:EtOAc = 3:1 to 2:1 to 1:1) to afford major product 11b (462 mg, 62% yield, dr = >95:95:95:95:
