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J . Am. Chem. SOC.1990, 112, 4524-4528
25 mL). NADt (4.0 mg, 6.0 pmol), [I-3H]cyclohex-2-en-I-ol (0.5 mCi), and HLADH (3.0 mg, 6.0 units) were added, and the mixture was incubated in the dark at room temperature for 6 h. The mixture was extracted with hexanes (2 X 30 mL) and the organic layers were dried (MgSO,), filtered, and concentrated by rotary evaporation. After addition of carrier farnesol (1.8 mg, 8.1 pmol), flash chromatography (gradient from hexanes to 10/90 ethyl acetate/hexanes) afforded 116 I-'Hlfarnesol. pCi (46%) of (IR)-[ (IR)-[l-~H,12,13-1~z]Farnesyl Pyrophosphate (2k). (lR)-[ I-'H]Farnesol ( 1 16 pCi) was converted to the pyrophosphate as for 2g. After addition of [12,13-14Cz]farnesylpyrophosphate (9.6 X IO5 dpm, 8.7 nmol), purification on a column of DEAE-Sephadex A-25 gave farnesyl pyrophosphate (2k): 3.5 X IO7 dpm of 'H (14%). 8.96 X IO'dpm of I4C. ( 1R)-(1-3H,12,13-'4C,]Famesyl Diphenylurethane (12k). Hydrolysis of 2k (2.4 X 104 dpm of I'C) with acid phosphatase gave [I-'H,12,1314C,]farnesol (1.6 X IO4 dpm of I4C, 68% radiochemical yield, 'H/I4C 4.72 f 0.1 3). which was converted to the corresponding diphenylurethane after dilution with unlabeled farnesol. 12k: 8680 dpm of I'C, 59% radiochemical yield; 'H/I4C 5.05 f 0.14. The product was further purified by preparative thin-layer chromatography (three plates, 1000 pm, three elutions of 4% EtOAc/hexanes) before recrystallization from MeOH to constant melting point, 'H and I4C specific activities, and 'H/"C ratio: specific activity 62 pCi/pmol 'H, 15.1 pCi/pmol 14C; 'H/I4C (average of last three crystallizations) 4.10 f 0.02. Cyclization of ( 1 R )-[ 1 -'H, 12,l3-14C2]FamesylPyrophosphate (2k)to (3R)-[3-3H,14,15-1'CC,]Pentalenene (3k). (lR)-[ 1-'H,12,13-14C2]Farnesyl pyrophosphate (2k,1.57 X IO5 dpm of 14C)was dissolved in 200 mM Tris buffer, pH 8.4, containing 20 mM MgCI, and 5 mM 8-mercaptoethanol (5 mL). Pentalenene synthase purified through the (3-100 size exclusion step (50 pL, total activity 26 nmol/h)8b was added, and the mixture was incubated at 30 OC for 2 h. Carrier pentalenene (2.0 mg) in hexanes (5 mL) was added and the mixture extracted. After further extraction with hexanes (2 X 5 mL), the hexanes layers were passed through a flash chromatography column (hexanes). Fractions were concentrated on the rotary evaporator at room temperature to afford 3k: 6.2 X IO4 dpm of I T , 39% radiochemical yield; 'H/14C 4.09 f 0.04. (3R)-[3-'H,14,15-14Cz]-(7S,8R)and -(7R,8S)-7,8-Dihydroxypentalenanes (13k and 14k). Labeled pentalenene (3k)(1.54 X 104 dpm of I4C) was diluted with carrier (f)-pentalenene (40 mg, 196 pmol) and converted to the mixture of cis-diols, which was purified by flash chromatography (30/70 EtOAc/hexanes) to afford 35.7 mg ( I 50 pmol, 55%) of product: 1.2 X 10' dpm of I4C, 78% radiochemical yield; 'H/I4C 4.25 f 0.05. The individual diols were separated by fractional crystallization and recrystallized to constant melting point, 'H and I4C specific activity, and ' H / W ratio: 13k.4.02 f 0.10,and 14k,4.10 f 0.14, respectively. Incorporation of (3R)~3-3H,14,15-~~2]Pentelenene (3k)into Oxidized Metabolites by Streptomyces UC5319. Feeding of labeled pentalenene (3k) ( I .O mg, 3.1 I X IO' dpm of I4C) to 12 flasks of Streptomyces UC53 I9 in the usual manner gave the following products. Pentalenolactone methyl ester (19k): 5.6 mg; 1660 dpm of 14C; 3.7 pCi/pmol I T ; specific incorporation 1.4%; ' H / W 0.065 f 0.002. Epipentalenolactone F methyl ester (18k): 0.4 mg; 153 dpm of I T ; 43 pCi/pmol "C; specific
incorporation 1.5%; 'H/I4C 3.73 f 0.05. Pentalenic acid methyl ester (15k): 90% pure by 'H NMR analysis. Chromatography (silica gel, 85:15 hexane/ethyl acetate) afforded 26.5 mg (42%) of 13b as a yellow oil: IH NMR (300 MHz, CDCI,) 6 1.35 (t, J = 7.0 Hz, 3 H), 1.70 (br m, 1 H), 1.83-2.05 (br m, 2 H), 2.32 (br m, 1 H), 2.44 (s, 3 H), 2.66 (br m, 2 H), 3.66 (m,1 H), 3.83 (m,1 H), 5.06 (dd, J = 6.1, 8.9 Hz, 1 H), 7.44 (m,2 H), 7.60 (d, J = 8.0 Hz, 1 H), 8.25 (d, J = 8.0 Hz, 1 H); I3C NMR (75 MHz, CDCI,) 6 15.5, 20.0, 20.6, 24.3, 27.7, 62.2, 77.2, 115.1, 120.1, 122.5, 123.9, 124.7, 126.8, 127.0, 127.1, 136.2, 150.9, 169.4; IR (neat) 3238, 2973, 2941, 1760, 1637, 1596, 1575, 1507, 1391, 1369, 1209, 1199, 1069,947, 763 cm-I; MS (FAB, positive ion, nitrobenzyl alcohol matrix) m/z 300 (M', 16), 255 (54). 212 (loo), 195 (S), 154 (6), 136 (6); HRMS calcd for CISH2004300.1362, found 300.1346. 1 -[ 10"- (Ace t y l o xy ) -9"-hydroxy - 1"- ,2",3",4"- tet ra hydroanthracenyl)-2',3'-isopropylideneadenosine (15). (a) Preparation in C D CI,. A solution of quinone methide 6c (from 51.8 mg, 0.202 mmol, of phenol 9) and CDCI, (1.1 mL) was added to 2',3'-isopropylideneadenosine (14) in a dry reaction vial. This solution was stirred at room temperature until all of the quinone methide was consumed (IO h, 'H NMR monitoring). The reaction mixture was concentrated and chromatographed (silica gel, 4: I hexane/2-propanol) to afford 47.1 mg (42%) of 15 as a white solid (mp 135-137 OC and 1 : l mixture of diastereomers by HPLC analysis). (b) Preparation in CH3CN/H20from Ethanol Adduct 13b. A solution of 2',3'-isopropylideneadenosine (24.8 mg, 0.081 mmol, 1.20 equiv) and
J . Am. Chem. SOC.1990, 112, 4528-4531
4528
H20/CH,CN ( l : l , v/v; 1.0 mL) as added toethanol adduct 13b (20.2 mg,0.067 mmol) in a 5-mL reaction flask. The resulting homogeneous solution was stirred at room temperature for 48 h. The mixture was then diluted with water (2 mL) and extracted with CHC13 (3 X 5 mL). The combined organic extracts were dried (Na,SO,), concentrated, and chromatographed (silica gel, 4: I hexane/2-propanol) to afford 10.6 mg (28%) of IS as a white solid ( I : 1 mixture of diastereomers by IH NMR analysis). (c) Preparation in CH3CN/H20from Quinone Methide 6c. A solution of 2',3'-isopropylideneadenosine (36.9 mg,0.121 mmol, 1.20 equiv) and H20/CH,CN (1 : 1, v/v; 1 .O mL) was added to quinone methide 6c (prepared from 25.8 mg, 0.101 mmol,of phenol 9 ) in a reaction flask. The resulting homogeneous solution was stirred at room temperature for 48 h. The mixture was then diluted with water (2 mL) and extracted with CHCI, (3 X 5 mL). The combined organic extracts were dried (Na2S04),concentrated, and chromatographed (silica gel, 4:l hexane/ 2-propanol) to afford 13.2 mg (24%) of 15 as a white solid (1:l mixture of diastereomers by 'H NMR analysis). I5 (mixture of diastereomers): 'H NMR (300 MHz, CDCI,) 6 1.35 (s, 3 H), 1.38 (s, 3 H), 1.60 (s, 3 H), 1.64 (s, 3 H), 2.09 (br s, 6 H), 2.26 (br m, 2 H), 2.46 (s, 6 H), 2.60-2.85 (br m, 2 H), 2.85-3.10 (br m, 2 H), 3.93 (d, J = 12.1 Hz, 1 H), 3.98 (d, J = 12.5 Hz, 1 H), 4.51 (s, 2 H), 5.10 (m, 3 H), 5.19 (t, J = 5.29 Hz, 1 H), 5.72 (br d, J = 7.21 Hz, 2H),5.76-5.82(m,2H),6.18(t,J= 11.5Hz,2H),6.50(d,J=8.2 Hz, 1 H), 6.63 (br s, 1 H), 7.34-7.48 (m,4 H), 7.58 (d, J = 8.1 Hz, 2 H), 7.72 (s, 1 H), 7.77 (s, 1 H), 8.31 (d, J = 8.0 Hz, 2 H), 8.45 (s, 1 H), 8.46 (s, 1 H), 11.48 (br s, 2 H); IR (CCI,) 3422, 3255, 2938, 1763, 1624, 1583, 1477, 1383, 1374, 1332, 1210, 1114, 1082, 1054, 852cm-I;
MS (FAB, positive ion, nitrobenzyl alcohol matrix) m / r 561 (M', 50), 518 (6), 398 (71, 346 (14), 308 (33), 212 (100); HRMS calcd for C,H31NS07561.2223, found 561.2238. Anal. Cald for C29H31N507: C, 62.02; H, 5.56; N, 12.47. Found: C, 61.34; H, 5.45; N, 12.50. The two diastereomers were subjected to HPLC (8-pm Rainin Dynamax silica gel column, 4.6 X 250 mm, 5:l hexane/2-propanol; flow rate, 0.8 mLmin-'; retention time, 15.62 and 16.76 min) to afford the high Rfdiastereomer analytically pure. High-R,diastereomer: 'H NMR (300 MHz, CDCI,) 6 1.38 (s, 3 H), 1.6 (s, 3 H), 2.10 (br s, 3 H), 2.2672.32 (m, 1 H), 2.46 (s, 3 H), 2.60-2.80 (br m, 1 H), 2.80-3.20 (br m, 1 H), 3.76 (t, J = 11.8 Hz, 1 H), 3.94 (d, J = 12.7 Hz, 1 H), 4.51 ( s , I H),5.10(d,J=5.8Hz,IH),5.19(t,J=5.3Hz,IH),5.72(br d , J = 7 . 1 Hz, 1 H),5.81 ( d , J = 4 . 8 H z , 1 H ) , 6 . 1 4 ( d , J = 11.2Hz, 1 H), 6.50 (d, J = 8.2 Hz, 1 H), 7.37-7.48 (m,2 H), 7.58 (d, J = 8.2 Hz, 1 H), 7.77 (s, 1 H), 8.31 (d, J = 8.0 Hz, 1 H), 8.46 (s, 1 H), 11.48 (br s, 1 H).
Acknowledgment. This work was supported by funds provided by the University of California Cancer Research Coordinating Committee and the Natioanl Institutes of Health (GM 39354). W e thank Mr. Ron New and Dr. Richard Kondrat for determination of mass spectra. Registry No. 6c, 126457-70-3; 7, 52103-68-1; 8, 126391-81-9; 9, 107866-26-2; 10, 126457-71-4; 11, 126457-72-5; 13a, 126457-73-6; 13b, 126457-74-7; 14, 362-75-4; 15 (isomer I ) , 126457-75-8; 15 (isomer 2), 126457-76-9; 9, IO-bis(acety1oxy)- 1,4-dihydroanthracene, 126457-77-0.
Efficient Synthetic Routes to Fluorinated Isosteres of Inositol and Their Effects on Cellular Growth Alan P. Kozikowski,*gt Abdul H. Fauq,+Garth Powis,$and Deborah C. Melded Contribution from the Departments of Chemistry and Behavioral Neuroscience, University of Pittsburgh, Chevron Science Center, Pittsburgh, Pennsylvania 15260, and the Department of Pharmacology, Mayo Clinic, 200 First Street, S . W., Rochester, Minnesota 55905. Received October 10. I989
Abstract: Efficient synthetic routes to several fluorinated isosteres of inositol have been developed that are based upon the unexpected selectivity observed in the (diethy1amido)sulfur trifluoride reaction of polyhydroxylated cyclohexane derivatives. The conversion of D-pinitol to 1D- 1,Sdideoxy- 1,5-difluoro-neo-inositoland to 10-1 -deoxy- 1 -fluoro-myo-inositol is reported along with a mechanistic rationale for their formation. Furthermore, the cell growth inhibitory properties of three fluorinated inositol analogues on NIH 3T3 (normal fibroblasts) and v-sis-transformed NIH 3T3 cells are described. These inositol isosteres hold promise as tools for furthering our understanding of the phosphatidylinositol cascade and may also offer a new strategy in the treatment of neoplastic diseases.
One of the major advances in cell biology in recent years has been the discovery of a series of intracellular signaling pathways that couple the messages derived from the binding of biologically active molecules with receptors in t h e cell surface t o effector mechanisms within the cell. These signaling pathways have been found in all cell types and modulate the actions of hormones, neurotransmitters, growth factors, and oncogenes.' O n e of the most extensively studied signaling pathways is the phospholipase C dependent hydrolysis of membrane phosphoinositide t o form inositol polyphosphates and diacylglycerol.2 As part of the larger effort to understand the mechanism of inositol-based intracellular signaling in relation to cell growth control, we have been interested in the synthesis of fluorinated isosteres of myeinositol which may act as antimetabolites for the inositol p a t h ~ a y . Such ~ isosteres could a c t either by blocking the formation of certain inositol 'University of Pittsburgh. Mayo Clinic.
*
0002-7863/90/l5l2-4528$02.50/0
phosphates or by forming fraudulent analogues. In this article we detail an expedient route t o I ~ - l - d e o x y - l fluoro-myo-inositol (1) as well as 1D-1,5-dideoxy-1,5-difluoroneo-inositol (2). T h e synthesis of the latter compound rests upon the surprising regioselectivity observed in the fluorination reaction of an inositol derivative with D A S T ((diethy1amido)sulfur trifluoride). While we have already reported a IO-step route to the ( 1 ) Abdel-Latif, A. A. Pharmacol. Res. 1986, 38, 228. Berridge, M. J. Biochim. Biophys. Acta 1987,907,33. Rasmussen, H. N.Engl. J. Med. 1986, 314, 1094 and 1164. Inositol Lipids in Cell Signalins Michell, R. H., Drummond, A. H., Downes, C. P.,Eds.; Academic Press: London, 1989. (2) Putney, J. W.; Takemura, H.; Hughes, A. R.; Horstman, D. A.; Thastrup, 0.FASEB J. 1989,3, 1899. Whitman, M.; Cantley, L. Biochim. Biophys. Acta 1988, 948, 327. (3) Kozikowski, A. P.;Xia, Y.;Rusnak, J. M. J. Chem. SOC.,Chem. Commun. 1988, 1301. Kozikowski, A. P.;Fauq, A. H.; Rusnak, J. M. Terrahedron Lett. 1989, 30, 3365 and references cited therein. For a recent review on the synthesis of myc-inositol phosphates, see: Billington, D. C. Chem. SOC. Rev. 1989, 18, 83.
0 I990 American Chemical Society
