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New glycolipid inhibitors of Myt1 kinase Bing-Nan Zhou, a Shoubin Tang, a Randall K. Johnson, b Michael P. Mattern, b John S. Lazo, c Elizabeth R. Sharlow, c Kim Harich d and David G. I. Kingston a, * a Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA b GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA c Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA, USA d Department of Biochemistry, M/C 0459, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA Received 5 May 2004; revised 9 November 2004; accepted 9 November 2004Available online 10 December 2004 Abstract —A crude extract of a marine alga showed activity against the enzyme Myt1 kinase. Bioassay-directed fractionation led to theisolation of two bioactive glycoglycerolipids. Lipid 1 was identified as sn -1,2-dipalmityl-3-( N -palmityl-6-deoxy-6-amino- a - D -glucosyl)glycerol and lipid 2 as sn -1-palmityl-2-myristyl-3-( N -stearyl-6-deoxy-6-aminoglucosyl)glycerol. Compounds 1 and 2 had IC 50 values of0.12and 0.43 m g/mL, respectively, in the Myt1 kinase inhibitory bioassay, and were inactive against Akt and Chk1 kinases. q 2004 Elsevier Ltd. All rights reserved. 1. Introduction The enzyme Myt1 kinase, which is a Thr-14 and Tyr-15specific cdc2 kinase, has been shown to be an importantregulator of cdc2/cyclin B kinase activity. It has beenreported that the inhibitory phosphorylation of cdc2 isimportant for the timing of entry into mitosis, and studieshave shown that premature activation of cdc2 leads tomitotic catastrophe and cell death. 1–3 Inhibition of Myt1kinase is predicted to cause premature activation of cdc2,and inhibitors of this enzyme would thus be expected to killrapidly proliferating cells and abrogate normal cell cyclecheckpoints. Such inhibitors would thus be attractive for thetreatment of cancer, because they could be used inconjunction with conventional chemotherapies to overcomedrug resistance and enhance their cytotoxicity by abrogatingcell cycle checkpoints.As a part of our systematic search for potential anti-canceragents from plants and marine organisms, 4–6 the methanolextract of an alga designated UM 2972M was found to showactivity in a bioassay for inhibitors of Myt1 kinase, with anIC 50 of 4 m g/mL. It was thus selected for fractionation forisolation of its bioactive constituents.The algae comprise a large group of marine organisms, andhave been subjected to intensive chemical studies. Manybioactive compounds, including the neurotoxic amino acidskainic acid and domoic acid 7 and the hypocholesterolemicbetain lipids 8 have been isolated from them, as well as carotenoids, 9 steroids, 10 halogenated compounds, 11,12 fattyacids, 13–17 phenolic compounds, 18,19 and terpenoids. 20–22 2. Results and discussion The methanol extract (UM 2972 M) from an unknown algalspecies was subjected to partition between aqueous MeOHand organic solvents, and the aqueous MeOH fraction wasthen stripped of its MeOH and extracted with n -butanol togive a bioactive n -butanol fraction (IC 50 Z 1 m g/mL).Column chromatography of the n -butanol fraction onSephadex LH 20 with elution with CH 2 Cl 2 –MeOH (3:1),followed by repeated chromatography on RP-18 reversedphase silica gel with MeOH–H 2 O (9:1) gave the twobioactive compounds 1 and 2 . Compounds 1 (1.05 mg,0.025%) and 2 (1.46 mg, 0.033%) had activities of 0.12 and0.43 m g/mL, respectively, in the Myt1 kinase bioassay.Compound 1 had a molecular weight of 967, as indicated bya pseudomolecular ion (M C Na) C at m / z Z 990 in itsMALDI TOF mass spectrum, and a composition of C 57 H 109 NO 10 , as indicated by FAB-HRMS. Three spin–spin systems were identified from its 1 H NMR and COSYspectra (Table 1). The first spin system indicated thepresence of a 1,2 diacylated glyceryl moiety [ d H 4.51 ppm(1H, dd, J Z 11.9, 3.1 Hz, H sn -1a ), 4.18 ppm (1H, dd, J Z 12.1, 7.0 Hz, H sn 1a ), 5.30 ppm (1H, m, H sn -2 ), 4.11 ppm 0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.tet.2004.11.013Tetrahedron 61 (2005) 883–887 Keywords : Marine; Myt1 kinase; Glycolipid.* Corresponding author. Tel.: C 1 540 231 6570; fax: C 1 540 231 3255;e-mail:
[email protected] (1H, dd, J Z 11.8, 5.2 Hz, H sn -3a ), and 3.57 ppm (1H, dd, J Z 11.8, 6.4 Hz, H sn -3b )]. The cross-peaks in the HMBCspectrum [ d H / d C : 5.31 (H sn -2 )/174.4 (COO); 2.33 ( a -CH 2 )/ 174.4 (COO); 4.51 and 4.18 (H sn -1 )/175.2 (COO); 2.33( a -CH 2 )/175.2 (COO)] also indicated the presence of acylgroups on the 1 and 2 positions of a glycerol moiety. Thesecond group of spin systems contained signals for threelong chain fatty acids [ d H 0.89 ppm (9H, t, J Z 6.83 Hz, 3 ! CH 3 –), 1–1.5 ppm (multi –CH 2 –) 1.59 ppm (6H, m, 3 ! b -CH 2 –), 2.33 ppm (6H, m, 3 ! a -CH 2 –). The third spinsystem indicated the presence of a glycosyl moiety [ d H 4.76 ppm (1H, d, J Z 3.8 Hz, H G-1 ), 3.40 ppm (1H, dd, J Z 9.5, 3.8 Hz, H G-2 ), 3.63 ppm (1H, d, J Z 9.5, 9.1 Hz, H G-3 ),3.08 ppm (1H, dd, J Z 9.1, 9.7 Hz, H G-4 ), 4.07 ppm (1H, m,H G-5 ), and 2.91 (1H, dd, J Z 14.3, 9.3 Hz, H G-6a ), and3.34 ppm (1H, d, J Z 14.1, 2.1 Hz, H G-6b )]. The HMBCcross-peak d H 4.76 (H G-1 )/ d C 67.1 (C sn -3 ) showed thatglycosylation was on the 3 position of the glycerol moiety,and that 1 was a glycoglycerolipid. Its 13 C NMR spectrumsupported this conclusion. The chemical shifts and thecoupling constants of the G-1 to G-4 protons of the glycosylmoiety were very close to those of methyl a - D -glucoside,indicating that these hydroxyl groups were unacylated. Thechemical shifts of H G-6 in the glycosyl moiety suggestedthe presence of an aminoacyl group at this position. Thecoupling constant of the anomeric proton ( J Z 3.8 Hz)indicated the a configuration, and since glucose has the D -configuration in most cases in nature compound 1 couldbe assigned as an a - D -6-desoxy-6-aminoglucoside.When 1 was hydrolyzed under various conditions (1%NaOMe in MeOH, with lipase from Mucor javanicus , 23,24 or 0.5% HCl) the only ester that could be detected wasmethyl palmitate. The polar fraction remaining after acidichydrolysis was reduced by reaction with NaBH 4 and theresulting products acetylated and analyzed by GC–MS.Analysis of the retention times and fragmentation patternsof the two products indicated them to be glycerol triacetateand 6-acetylamino-1,2,3,4,5-penta-acetoxyhexane. Thisresult confirmed that 1 was a glycoglycero lipid. A negativeCotton effect between l max 200–250 nm indicated that 1 was an sn -1, sn -2 diacylglycosylglycero-lipid. 25 Thedifference between the 13 C NMR signals for the twocarbonyl carbons of the two glyceryl esters (0.6 ppm)indicated that 1 was an sn -1, sn -2 diacyl ester; had it been an sn -1, sn -3 diacyl ester these signals would have been closeror even overlapped. 26,27 Thus, compound 1 was assigned thestructure sn -1,2-di-palmityl-3-( N -palmityl-6 0 -desoxy-6 0 -amino- a - D -glucosyl)-glycerol.Compound 2 also had a molecular weight 967 and acomposition of C 57 H 109 NO 10 , as indicated by its MALDITOF mass spectrum and FAB-HRMS. Its 1 H NMR andCOSY spectra (Table 1) were very similar to those of 1 , andshowed the presence of a diacylglyceryl moiety [ d H 4.50 ppm (1H, dd, J Z 12.0, 3.0 Hz, H sn -1a ), 4.18 ppm (1H,dd, J Z 12.0, 6.9 Hz, H sn -1a ), 5.30 ppm (1H, m, H sn -2 ),4.10 ppm (1H, dd, J Z 10.7, 5.3 Hz, H sn -3a ), and 3.56 ppm(1H, dd, J Z 10.7, 6.3 Hz, H sn -3b )].The HMBC cross-peaks [ d H / d C : 5.30 (H sn -2 )/174.4 (–COO–);2.33( a -CH 2 –)/174.4 (–COOH); 4.50 and 4.18 (H sn -1 )/175.2(–COO–); 2.33 ( a -CH 2 –)/175.2 (–COO–)] supported thisresult. Overlapped signals for three long chain fatty acids[ d H 0.89 ppm (9H, t, J Z 6.83 Hz, 3 ! CH 3 –), 1–1.5 ppm(multi –CH 2 –), 1.59 ppm (6H, m, 3 ! b -CH 2 –), 2.33 ppm(6H, m, 3 ! a -CH 2 –) could also be found in its NMRspectrum. NMR signals for a similar glycosyl moiety to thatof 1 [ d H 4.75 ppm (1H, d, J Z 3.7 Hz, H G-1 ), 3.39 ppm (1H,dd, J Z 9.6, 3.7 Hz, H G-2 ), 3.62 ppm (1H, d, J Z 9.6, 9.7 Hz,H G-3 ), 3.07 ppm (1H, dd, J Z 9.3, 9.7 Hz, H G-4 ), 4.06 ppm Table 1 . Partial NMR data of lipids 1 and 2 a,b Position 1 2 d H and J d C d H and J d C sn -1 4.51 (dd, J Z 12.0, 3.0) 64.3 4.50 (dd, J Z 12.0, 3.0) 64.34.18 (dd, J Z 12.0, 6.9) 4.18 (dd, J Z 12.0, 6.9) sn -2 5.31 (m) 71.7 5.30 (m) 71.7 sn -3 4.10 (dd, J Z 10.8, 5.2) 67.1 4.10 (dd, J Z 10.7, 5.3) 67.13.57 (dd, J Z 10.8, 6.4) 3.56 (dd, J Z 10.7, 6.3)G-1 4.76 (d, J Z 3.6) 101.0 4.75 (d, J Z 3.7) 100.0G-2 3.40 (dd, J Z 3.6, 9.5) 73.5 3.39 (dd, J Z 3.6, 9.6) 73.5G-3 3.63 (dd, J Z 9.5, 9.6) 75.0 3.62 (dd, J Z 9.6, 9.7) 75.0G-4 3.08 (dd, J Z 9.6, 9.2) 74.9 3.07 (dd, J Z 9.7, 9.3) 74.9G-5 4.07 (ddd, J Z 9.2, 9.4, 2.0) 69.9 4.06 (ddd, J Z 9.3, 8.4, 2.0) 68.9G-6 3.34 (dd, J Z 14.4, 2.0) 54.2 3.34 (dd, J Z 14.3, 2.0) 54.22.91 (dd, J Z 14.4, 9.4) 2.91 (dd, J Z 14.3, 8.4)CH 3 – 0.89 (3 ! CH 3 –, t, J Z 6.8) 14.5 0.89 (3 ! CH 3 –, t, J Z 7.0) 14.5(–CH 2 –) n 1.0–1.5 1.0–1.5 a -CH 2 – 2.33 (3 ! –CH 2 –, m) 35.0, 35.2 2.33 (3 ! –CH 2 –, m) 35.0; 35.2 b -CH 2 – 1.59 (3 ! –CH 2 –, m) 26.0 1.60 (3 ! –CH 2 –, m) 26.0–COO– 174.4 174.4–COO– 175.2 175.2–COO– 175.2 175.1 a d values are in ppm and J values are in Hz. b Chemical shifts were assigned by DQ COSY, HMQC, and HMBC spectra. B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887 884 (1H, m, H G-5 ), and 2.91 (1H, dd, J Z 14.3, 8.4 Hz, H G-6a ),and 3.34 ppm (1H, d, J Z 14.3, 2.0 Hz, H G-6b ) were alsoobserved. All the spectroscopic data for 2 wereconsistent with its assignment as an a - D -6-desoxy-6-amino-glucoside.Hydrolysis of 2 with 0.5% HCl followed by 1% NaOMe inmethanol and then by treatment with diazomethane yieldedapproximately equimolar amounts of methyl myristate,methyl palmitate and methyl stearate as determined by GC–MS. The polar fraction remaining after hydrolysis wasreduced with NaBH 4 and acetylated, and the resultingproduct analyzed by GC–MS. Analysis of the retentiontimes and fragmentation patterns of the two productsindicated than to be glycerol triacetate and 6-acetylamino-1,2,3,4,5-penta-acetoxyhexane. These results indicatedthat 2 was a glycoglycero lipid with three different acylgroups, so the locations of these groups needed to bedetermined.When 2 was hydrolyzed with 1% NaOMe in absolutemethanol, only methyl palmitate and methyl myristate weredetected by GC–MS. Since amides are hydrolyzed moreslowly than esters under alkaline conditions, this resultindicated that the stearyl moiety acylated the 6-aminoposition of glucose, and that the myristyl and palmitylgroups both acylated the glyceryl moiety. When 2 washydrolyzed with lipase from M. javanicus 23 and theproducts treated with diazomethane, methyl stearate andmethyl palmitate were both isolated. This indicates thatthe myristyl group is located at the more hindered sn -2position. Compound 2 was thus assigned as sn -1-palmityl-2-myristyl-3-( N -stearyl-6 0 -desoxy-6 0 -amino- a - D -glucosyl)-glycerol.Three 6 0 -desoxy-6 0 -amino-glucosylglycerolipids havepreviously been reported in the literature, 28 but the acylgroups in these compounds were different from ours.Compounds 1 and 2 are thus new natural products.As noted earlier, compounds 1 and 2 had IC 50 values of 0.12 G 0.07 and 0.43 G 0.01 m g/mL, respectively, in theMyt1 kinase bioassay. The bioactivity of compounds of thistype as inhibitors of Myt1 kinase is new, althoughdiacylglycerol is well known as an activator of proteinkinase C. 29 The inhibition against Myt1 kinase appeared tobe selective as we observed no inhibition of two otherprotein kinases, Chk1 and Akt, with concentrations of compounds 1 and 2 as high as 100 m g/mL.Although simple acylglycerols are not normally thought of as ‘druggable’ entities, they can serve as prototypes forcompounds with inproved pharmacological properties.Thus, conformationally constrained diacylglycerol-bis-lactones have been investigated as diacylglycerol analogs, 30 and the conversion of an O -glycoside to a C -glycosideboosted the potency of a ceramide O -glycoside by two tothree orders of magnitude. 31 In addition, it is worthnoting that a sea alga has yielded a sulfoquinovosyl-diacylglycerol which acts as a potent inhibitor of eukaryotic DNA polymerases and HIV reverse transcriptasetype 1. 32 3. Experimental3.1. General experimental procedures CD and NMR spectra were obtained as previouslydescribed; 33 a 9 Hz optimization was employed for thelong-range coupling pathway in HMBC determinations.MALDI spectra were determined on a Kratos Kompact SEQ(Kratos Analytical, Manchester, UK) time of flight massspectrometer, and FAB mass spectra were obtained on aJEOL JMS-HX-110 instrument. The conditions for GC–MSconditions for fatty acid methyl ester: Hewlett PackardHP5710A Gas Chromatograph with HP-5 column (30 M ! 0.32 mM i.d.), oven temperature programmed from 80 to280 8 C at 8 8 C/min for 1 and at 4 8 C/min for 2 , with Hecarrier gas at 12 psi. A VG7070E-HF mass spectrometer,scanned from 50 to 500 amu at 1.5 s/scan, was used fordetection. Sephadex LH-20 (Sigma) was employed for gelpermeation chromatography. Column chromatography wascarried out on LRP-2 (RP-18) and reversed-phase TLC onMKC18F silica gel 60 A (RP-18) from Whatman. 3.2. Bioassay methods The crude extract, fractions, and pure compounds wereassayed for inhibitory activity against Myt1 kinase by amodification of a previously described microtiter-basedfluorescence polarization assay. 34 Briefly, 10 m g recombi-nant glutathione-S-transferase (GST) tagged-Myt1 proteinwas incubated in kinase buffer (50 mM Tris–HCl, pH 7.5,20 mM MgCl 2 and 1 mM DTT) containing 2.5 nMfluorescein-labeled Cdc2-derived peptide (containingMyt1 phosphorylation target sites Thr-14 and Tyr-15)(Molecular Devices, Sunnyvale, CA), 100 m M ATP, andcompounds solubilized in dimethyl sulfoxide. The kinasereaction was incubated for 1 h at room temperature afterwhich an anti-phospho-Cdc2 antibody (1 m g/mL) (CellSignaling Technology, Beverly, MA) was added andincubated for an additional hour. Fluorescence polarizationmeasurements were taken in black 96 well microtiter plates(Costar, Acton, MA) with a Wallac Victor 1420 multilabelmicrotiter plate reader (Perkin Elmer, Boston, MA).Duplicate samples were subjected to 25 flashes andexperiments were performed 2–3 times. Dimethyl sulfoxidewas used as the vehicle control; staurosporine (Sigma, St.Louis, MO) was used as the positive control and it had anIC 50 value of 1.9 m M in this assay.The effects of 1 and 2 on Akt and Chk1 activity were alsostudied with 384 well microtiter-based IMAP (MolecularDevices, Sunnyvale, CA) fluorescence polarization assays.Briefly, compounds 1 or 2 (0.1 to 100 m g/mL) wereincubated for 30–60 min at room temperature in the kinasebuffer (10 mM Tris–HCl, pH 7.2, 10 mM MgCl 2 , 0.1%bovine serum albumin, and 0.05% NaN 3 ) with 0.2 U/mLAkt or 0.4 U/mL Chk1, 100 nM fluorescently labeled Aktsubstrate or Chk1 crosstide substrate peptide, 5 m M ATP ina total volume of 20 m L. IMAP binding solution (15–60 m L)was added to each well and incubated at room temperaturefor 30 min. Data were collected on an Analyst GT(Molecular Devices) and analyzed using SoftMaxPro soft-ware. Triplicate experiments were performed 2–3 times.Staurosporine (Sigma-Aldrich, St. Louis,MO) was used as a B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887 885 positive control for Akt (IC 50 Zw 60 nM) and UCN-01(National Cancer Institute, Bethesda, MD) was the positivecontrol for Chk1 (IC 50 Zw 2 m M). 3.3. Isolation of bioactive compounds The crude algal extract UM 2972M 35 (4.04 g) waspartitioned between 50% aq MeOH and n -hexane, and theaq MeOH layer was then partitioned with CH 2 Cl 2 . The 50%aq MeOH fraction was then evaporated in vacuo to removeMeOH, and the residual H 2 O fraction was extracted withBuOH. The BuOH fraction (1.56 g) was active in the Myt1kinase bioassay (IC 50 Z 1.0 m g/mL) and was subjected tocolumn chromatography on Sephadex LH 20 (20 g, elutedwith CH 2 Cl 2 –MeOH (3:1). A total of 45 fractions (5 mL/ each fraction) was collected and fractions 8–12 (119.0 mg),which showed the characteristic 1 H NMR spectra of aglycoglycerolipid, were the most active (IC 50 Z 0.4–1.0 m g/ mL). The active fractions 9–10 (IC 50 Z 0.4 m g/mL) wererepeatedly purified by chromatography on an RP-18 columnwith MeOH–H 2 O (9:1) as eluant. Compound 1 (1.05 mg,0.026% based on crude extract) was isolated from fractions13–28, and compound 2 (1.35 mg, 0.033% based on crudeextract) from fraction 11. The inhibitory activities of theisolated compounds were IC 50 Z 0.12 m g/mL for 1 andIC 50 Z 0.43 m g/mL for 2 . 3.3.1. Compound 1. MALDI TOF mass spectrum: m / z 990(M C Na) C . HRFABMS m / z 968.8059 [M C H] C ; (calcd forC 57 H 110 NO 10 968.8130). [ a ] D23 Z C 41.8 ( c 0.12, MeOH). 1 H NMR and 13 C NMR spectra: see Table 1. 3.3.2. Alkaline hydrolysis of 1. Compound 1 (0.1 mg) inMeOH (0.2 mL) was treated with 1% NaOMe–MeOHsolution (0.8 mL) and the solution was stirred at roomtemperature for 5 h. After partition between n -hexane andMeOH, the n -hexane extract was subjected on GC–MS.Methyl palmitate was detected with retention time 15 0 14 00 and EIMS m / z 270 (M) C , 227, 143, 87, 74, 57, 55. Itsretention time and fragmentation peaks were same as thedata from a standard sample in the data base. 3.3.3. Enzymatic hydrolysis of 1. Compound 1 (0.1 mg)was mixed with lipase (1 mg) from M. javanicus (FlukaChemie AG, CH-9471, Switzerland, EEC No. 2326199) indioxane–H 2 O (1:1, 1 mL) and the reaction mixture wasincubated at 38 8 C for 4 h. After removal of the dioxane andH 2 O, the residue was dissolved in 2 mL MeOH andpartitioned with n -hexane. The n -hexane extract was treatedwith diazomethane and analyzed by GC–MS. Methylpalmitate was detected as described above. 3.3.4. Complete hydrolysis of 1. Compound 1 (0.12 mg)was treated with 0.5% HCl at 65 8 C overnight. The reactionmixture was evaporated to dryness in vacuo and the residuemixed with 1% NaOMe in MeOH and incubated at roomtemperature for 6 h. After partitioning with n -hexane, the n -hexane fraction was evaporated to dryness and treatedwith diazomethane and analyzed by GC–MS as describedabove. Methyl palmitate was the only ester detected. Theaqueous fraction was evaporated to dryness and treated withNaBH 4 in MeOH at room temperature for 4 h with stirring.After evaporation of the solvent, the reaction mixture wasacetylated with Ac 2 O (0.5 mL) and dry pyridine (20 m L)with stirring overnight. After removal of the reagents withN 2 , the reaction mixture was extracted with CH 2 Cl 2 . TheCH 2 Cl 2 extract was subjected to GC–MS and glyceroltriacetate (retention time: 7 0 20 00 , EIMS m / z 158 (M K 60) C ,145, 116, 115, 183, 74, 73, and 61) and 6-acetylamino-1,2,3,4,5-penta-acetoxyhexane (retention time: 17 0 47 00 , andEIMS m / z 433 (M) C , 374, 331, 304, 289, 259, 207, and 87)were detected. 3.3.5. Compound 2. MALDI TOF mass spectrum: m / z Z 990 (M C Na) C . HRFABMS m / z 968.8076 [M C H] C ;(calcd for C 57 H 110 NO 10 968.8130). [ a ] D23 Z C 31.1 ( c 0.10,MeOH). 1 H NMR and 13 C NMR spectrum data: see Table 1. 3.3.6. Alkaline hydrolysis of 2. Compound 2 (0.11 mg) inMeOH (0.2 mL) was treated with 1% NaOMe in MeOH(1.0 mL) and the solution was stirred at room temperaturefor 4 h. After extraction with n -hexane, the n -hexane extractwas analyzed by GC–MS. Methyl myristate was detectedwith retention time 17 0 46 00 and EIMS m / z 242 (M C ), 199,143, 87, 74, and 57 and methyl palmitate with retention time22 00 58 00 and EIMS m / z 270 (M C ), 227, 185, 143, 87, 74, and57. Their retention times and fragmentation patterns werethe same as those of standard samples in the database. 3.3.7. Enzymatic hydrolysis of 2. Compound 2 (0.12 mg)was hydrolyzed with lipase from M. javanicus and the n -hexane extract treated with diazomethane and analyzed byGC–MS as previously described. Two peaks were detected.One of them was identified as methyl palmitate by itsretention time (22 0 53 00 ) and EIMS ( m / z 270 (M C ), 239, 227,185, 171, 143, 129, 115, 99, 87, 74, 57). The second wasidentified as methyl stearate by its retention time (27 0 42 00 )and EIMS ( m / z 298 (M C ), 267, 255, 213, 199, 185, 157,143, 129, 97, 87, 74, and 57). The retention times andfragmentation patterns of these compounds were the sameas those of standard samples in the database.The MeOH fraction after partition was evaporated todryness in vacuo. The residue was treated with 1%NaOMe in MeOH at room temperature for 4 h. Afterextraction with n -hexane, the n -hexane fraction wasconcentrated, treated with diazomethane, and analyzed byGC–MS. The major peak was identified as methyl myristateby its retention time of 17 0 37 00 and EIMS m / z 242 (M C ),199, 143, 87, 74, 57, and 55. 3.3.8. Complete hydrolysis of 2. Compound 2 (0.10 mg)was treated with 0.5% HCl solution (1 mL) at 65 8 Covernight, the reaction mixture evaporated to dryness, andthe residue incubated with 1% NaOMe in MeOH (1 mL) atroom temperature for 6 h. After partition with n -hexane, the n -hexane fraction was evaporated to dryness and treatedwith diazomethane and analyzed by GC–MS. The first peak was identified as methyl myristate by its retention time of 17 0 20 00 and EIMS m / z 242 (M C ), 199, 143, 87, 74, 57, and55. The second peak was identified as methyl palmitate byits retention time of 22 00 55 00 and EIMS m / z 270 (M C ), 239,227, 185, 143, 87, 74, 57. The third peak was detected asmethyl stearate by its retention time of 27 0 46 00 and EIMS m / z 298 (M C ), 267, 255, 213, 199, 143, 87, 74, 57. The aqueousfraction was evaporated to a small volume and reduced with B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887 886 NaBH 4 at room temperature for 4 h with stirring. Afterevaporation of the solvent and drying under vacuumovernight, the reaction mixture was acetylated with Ac 2 O(0.5 mL) and dry pyridine (20 m L) with stirring overnight.After removal of the reagents with N 2 , the reaction mixturewas extracted with CH 2 Cl 2 . 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