Bioconjugate Chem. 1995, 6,473-482
473
Synthesis of Novel Phosphoramidite Reagents for the Attachment of Antisense Oligonucleotides to Various Regions of the Benzophenanthridine Ring System Jer-kang Chen,' H. Lee Weith,' Rupinder S. Grewal,? Guangyi Wang,' and Mark Cushman",' Departments of Medicinal Chemistry and Pharmacognosy and Biochemistry, Purdue University, West Lafayette, Indiana 47907. Received March 28, 1995@
Four benzophenanthridine phosphoramidite reagents have been prepared in which the linker chain between the benzophenanthridine and the phosphoramidite moiety is attached to C-2, C-6, C-9, and (2-12 of the benzophenanthridine ring system. These benzophenanthridine phosphoramidites should prove to be useful in the syntheses of antisense oligonucleotide-intercalator conjugates in which the linker chain is attached to various regions of the benzophenanthridine intercalator. One of the new benzophenanthridine phosphoramidite reagents was used to prepare an antisense oligonucleotideintercalator conjugate in which the oligonucleotide TCAGTGGTp was connected a t its 5'-hydroxyl group through a linker chain to the C-2 hydroxyl group of a benzophenanthridine.
INTRODUCTION
The control of gene expression by antisense oligonucleotides offers an exciting as well as rational strategy for the treatment of viral diseases, cancer, and genetic diseases. Antisense oligonucleotidesbind specifically to complementary sequences in DNA or RNA and interfere with either transcription or translation. The field has been surveyed repeatedly and several recent reviews are available (1-18). Despite many promising results in in vitro systems, however, the potential application of antisense oligonucleotides themselves as therapeutic agents is severely limited by their instability to nucleases and their poor membrane penetration. Additional problems relate to their binding site selectivity and affinity for the target sequence. In view of these considerations,various chemical modifications of oligonucleotides have been made in order to improve their properties as potential therapeutic agents. These have included the replacement of the phosphodiester linkages with methyl phosphonates, phosphorothioates, and phosphorodithioates. One of the possible structural modifications of antisense oligonucleotides which may improve their therapeutic potential is the attachment of intercalating agents to the 3' andlor 5' ends through linker chains. This may stabilize the ends of the oligonucleotidetoward hydrolysis by exonucleases (19-23). The increase in lipophilicity provided by the intercalator may also facilitate cell membrane penetration (19). The intercalation andlor stacking interactions of the intercalators with the base pairs of the miniduplex also increases its affinity for the target nucleic acid and (22-271, if chosen correctly, could also add to the binding site selectivity. Acridine (20,22, 23,26,28-37) intercalators have most often been linked to oligonucleotides,although oxazolopyridocarbazole(38, 391, anthraquinone (251, phenanthridine (401, phenazine (411, and ellipticine (27) conjugates have also been prepared. In certain cases, it has been demonstrated that oligonucleotide-acridine conjugates were effective in inhibiting gene expression, whereas the corresponding oligonucleotides themselves were inactive (19, 21, 42-
There are many variables which must be considered in the design of oligonucleotide-intercalator conjugates, including the sequence of the oligonucleotide, the point of attachment of the linker chain to the oligonucleotide, the length and type of linker chain, the point of attachment of the linker chain to the intercalating agent, and the choice of the intercalator. For potential use in the control of HIV gene expression, we felt that an ideal intercalator should (1)be a potent inhibitor of viral gene expression on its own, without any attached oligonucleotide, by binding to the template primers; (2) possess selectivity for binding to viral DNA polymerases as opposed to other DNA and RNA polymerases; (3) be nonmutagenic; and (4)have established binding site selectivity that could be taken advantage of in targeting the conjugate to specific sequences of viral RNA. Both nitidine chloride (1) and fagaronine chloride (2), as well as some structurally related benzophenanthridine alkaloids, inhibit RNA-directed DNA polymerase activity from avian myeloblastosis virus, Raucher murine leukemia virus, and simian sarcoma virus by binding to the template primers (45-50). They also inhibit viral DNA polymerase to a greater extent than mouse embryo RNA polymerase, DNA polymerase, and poly(A) polymerase (49). In a study of the activities of 15 benzophenanthridine alkaloids against avian myeloblastosis virus, fagaronine chloride (2) was found to be the most active, followed closely by O-methylfagaronine sulfate and nitidine chloride (1) (50). Viral DNA polymerase activity was greatly diminished when A T template primers were used; however, no inhibition occurred with G:C template primers (45,47-49). It has also been demonstrated that nitidine chloride is not mutagenic (511.
44). Department of Medicinal Chemistry and Pharmacognosy. t Department of Biochemistry. Abstract published in Advance ACS Abstracts, July 1, 1995. +
@
1043-1802/95/2906-0473$09.00/0
The goal of the present study was to synthesize a series reactive phosphoramidites attached through linker chains to multiple regions of the benzophenanthridine intercalator system. These molecules were designed to allow 0 1995 American Chemical Society
474 Bioconjugate Chem., Vol. 6,No. 4,1995
Chen et al.
1H), 7.61 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.30 (s, 1H), 7.11 (s, 1 H), 6.79 (s 1 H), 4.14 (s, 2 H), 4.13 (t, J = 6.8 Hz, 2 H), 4.03 (9, 3 H), 3.98 (s, 3 H), 3.93 (s, 3 H), 3.66 (t, J = 6.5 Hz, 2 H), 2.61 (s, 3 H) 1.94 (m, 2 H), 1.61 (m, 2 H), 1.56 (m, 2 H), 1.47 (m, 2 H); CIMS, mlz (relative intensity) 452 (MH+, 100); HREIMS calcd for C27H3305N (M+)451.2359, found 451.2359. 2-[(6-Hydroxy-n-hexyl)oxyl-5,6-dihydro-3,8,9-trimethoxy-N-methylbenzo[clphenanthridine6-042Cyanoethyl NJV-diisopropylphosphoramidite)(5). N,N-Diisopropylethylamine (0.11 mL) was added to the solution of compound 4 (28 mg, 0.06 mmol) in THF (2 EXPERIMENTAL PROCEDURES mL). The solution was stirred for 10 min. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite(69 pL, 0.31 mmol) Melting points were determined in capillary tubes and was added to the solution, and the reaction mixture was are uncorrected. lH NMR spectra were recorded a t 200 stirred a t room temperature for 1.5 h. The reaction or 500 MHz using CDC13 as the solvent, except where mixture was then subjected to preparative, centrifugally noted otherwise. Low-resolution chemical ionization accelerated, radial, thin-layer chromatography on silica mass spectra (CIMS) were determined using 2-methylgel, eluting with EtOAc-hexane-triethylamine (5:4:1), propane as the reagent gas. Various other types of mass to yield the phosphoramidite 5 (36.3 mg, 90%) as an oil: spectra are abbreviated as follows: FABMS (fast atom lH NMR 6 7.69 (d, J = 8.6 Hz, 1 HI, 7.61 (8, 1 HI, 7.49 bombardment mass spectrum), HRFABMS (high-resolu(d, J = 8.5 Hz, 1 H), 7.30 (s, 1 H), 7.11 (s, 1 H), 6.79 (s, tion fast atom bombardment mass spectrum), HRCIMS 1H), 4.14 (s, 2 H), 4.13 (t,J = 6.9 Hz, 2 H), 4.03 (s, 3 HI, (high-resolution chemical ionization mass spectrum), 3.98 (s, 3 HI, 3.93 (5, 3 HI, 3.83 (m, 1 H), 3.77 (m, 1 HI, HREIMS (high-resolution electron impact mass spec3.67 (m, 1H), 3.58 (m, 3 H), 2.61 (s, 3 H), 2.61 (t, J = 6.5 trum). Microanalyses were performed by the Purdue Hz, 2 H), 1.93 (m, 2 H), 1.65 (m, 2 H), 1.53 (m, 2 H), 1.47 Microanalytical Laboratory. Column chromatography (m, 2 H), 1.17 (d, J = 3.0 Hz, 6 H), 1.16 (d, J = 3.0 Hz, was carried out on silica gel (Merck, grade 60, 230-400 6 H); 31PNMR (202 MHz) 6 147.99. mesh). 2-[[5’-(Ethoxycarbonyl)-n-pentylloxyl-3,8,9-tri- 12-Acetoxy-5,6-dihydro-8,9-dimethoxy-N-methyl2,3-(methylenedioxy)-6-oxobenzo[clphenanthrimethoxy-N-methylbenzo[clphenanthridiniumChlodine (7). A solution of trans-N-methyl-8,9-dimethoxyride (3). Potassium tert-butoxide (1 M THF solution, 2,3-(methylenedioxy)-6,12-dioxo-4b,5,6,10b,ll, 12-hexa308.6 pL) was added dropwise to the solution of fagarhydrobenzo[c]phenanthridine (6)(3.81 g, 10 mmol) and onine chloride (2,80 mg, 0.208 mmol) in dry dimethyl p-toluenesulfonic acid monohydrate (0.95 g, 5 mmol) in sulfoxide (2.1 mL), and the mixture was stirred a t room isopropenyl acetate (150 mL) was heated a t reflux under temperature for 30 min. Ethyl 6-(tosyloxy)hexanoate air for 24 h and then heated a t 85 “C for another 24 h. (328.8 mg, 1.047 mmol) was then added, and the reaction The reaction mixture was diluted with ether, and the mixture was stirred a t room temperature for 24 h. The precipitate (2.52 g) was filtered and washed with ether. solvent was evaporated a t room temperature under The filtrate was concentrated and dissolved in chloroform reduced pressure. The residue was redissolved in MeOH (100 mL). The resulting solution was washed with 2 N and subjected to preparative, centrifugally accelerated, Na2C03 (3 x 50 mL), dried (Na2S04),and concentrated. radial, thin layer chromatography on silica gel, eluting The residue was chromatographed on silica gel using 25% with chloroform-MeOH (9:l) (52-55). Recrystallization ethyl acetate in methylene chloride to give an additional from chloroform-petroleum ether furnished the pure crop of 7 (1.40 g). The total yield was 3.92 g (93%). product 3 (100 mg, 91%): mp 221-223 “C dec; IR (KBr) Recrystallization from methylene chloridelethyl acetate 3060-3000, 2920, 1730, 1615, 1510, 1280, 1010 cm-l; gave a n analytically pure sample: mp 275-277 “C; IR NMR 6 10.57 (s, 1HI, 8.39 (d, J = 9.9 Hz, 1HI, 8.03 (br (KBr) 2915, 2820, 1757, 1641, 1608, 1502, 1469, 1387, d, 2 H), 7.92 (s, 1 H), 7.88 (s, 1 HI, 7.34 (s, 1 HI, 5.13 (s, 1362, 1305, 1253, 1205, 1033 cm-l; NMR 6 7.92 (s, 1H), 3 H), 4.26 (s, 3 H), 4.22 (t, J = 6.6 Hz, 2 H), 4.13 (9, J = 7.76 (s, 1 HI, 7.66 (s, 1 HI, 7.45 (s, 1 HI, 7.19 (s, 1 HI, 7.2 Hz, 2 H), 4.08 (s, 3 H), 4.07 (s, 3 HI, 2.36 (t, J = 7.5 6.13 (s, 2 H), 4.10 (s, 3 H), 4.06 (s, 3 H), 3.97 (9, 3 H), Hz, 2 H), 1.99 (m, 2 H), 1.75 (m, 2 HI, 1.59 (m, 2 HI, 1.25 2.52 (s, 3 HI; CIMS mlz (relative intensity) 422 (MH-, (t,J = 7.5 Hz, 3 H); FABMS, mlz (relative intensity) 492 100),381(111,380 (171,379 (9);HRCIMS calcd for C23HZO(MA- C1, 100); HRFABMS calcd for C29H34N06 (M’ NO, 422.1240, found 422.1230. C1) 492.2385, found 492.2372. 12-[[4’-(ethoxycarbo5,6-Dihydro-2-[(6-hydroxy-n-hexyl)oxyl-3,8,9-tri- 5,6-Dihydro-8,9-dimethoxynyl)-n-pentylloxyl-N-methyl-2,3-(methylenedioxy)methoxy-N-methylbenzo[clphenanthridine (4). benzo[c]phenanthridine (8). A mixture of 12-acetoxyLithium aluminum hydride (95%, 27.74 mg, 0.69 mmol) 5,6-dihydro-8,9-dimethoxy-N-methyl-2,3-(methylenedioxy)was added to a solution of compound 3 (61 mg, 0.12 6-oxobenzo[c]phenanthridine(7) (632 mg, 1.5 mmol) and mmol) in THF (6 mL). The reaction mixture was stirred sodium hydroxide (600 mg) in ethailol(30 mL) and water a t room temperature for 4 h. The reaction mixture was (15 mL) was stirred a t room temperature for 2 h. The cooled to 0 “C and decomposed by addition of water (0.1 solvent was evaporated in vacuo, and the residue was mL), 15%aqueous NaOH (0.1 mL), and finally water (0.3 thoroughly dried under vacuum. Dry DMSO (15 mL) was mL). The mixture was stirred for 15 min and filtered. added and the resulting mixture stirred for 20 min. The aluminate was washed with chloroform. The comEthyl 5-bromovalerate (2.0 g, 9.5 mmol) was added and bined organic layers were dried (NazSOJ and evaporated. the mixture stirred for 18 h. The resulting clear solution The residue was dissolved in chloroform and subjected was acidified with 2 N HCl under cooling with ice and to preparative, centrifugally accelerated, radial, thindiluted with chloroform (100 mL). The mixture was layer chromatography on silica gel, eluting with EtOAcwashed with water (3 x 60 mL), dried (Na2S04), and hexane (8:2) to afford pure product 4 (40 mg, 77%): mp concentrated. Most of the excess ethyl 5-bromovalerate 162-164 “C dec; IR (KBr)3540,3040,2920,2850,1600, was removed under vacuum. The residue was chromato1500, 1455, 1240, 1000 cm-l; NMR 6 7.69 (d, J = 8.7 Hz,
the eventual preparation of a series of benzophenanthridine-oligonucleotide conjugates in which the point of attachment of the linker chain to the benzophenanthridine intercalator is varied. In more specific terms, methodology was sought which would allow the attachment of the linker chain to the “top”, “bottom”, “righthand side”, and “left-hand side” of the intercalator. The effect of the point of attachment of the linker chain to the intercalator on affinity for the target sequence has not previously been investigated systematically with any oligonucleotide-intercalator conjugates.
Synthesis of Novel Phosphoramidite Reagents
Bioconjugate Chem., Vol. 6,No. 4, 1995 475
solution of 3-isopropoxy-4-methoxybenzaldehyde(12,6.28 graphed on silica gel using 25% ethyl acetate in methg, 0.0323 mol) and 3,4-dimethoxyacetophenone(13,5.83 ylene chloride to give a white solid (710 mg, 91%): IR g, 0.0323 mol) in ethanol (60 mL) was treated with 10% (KBr) 2924, 1734, 1629, 1506, 1474, 1424, 1390, 1315, aqueous sodium hydroxide (6.0 mL, 0.015 mol). The 1272, 1254, 1185, 1144, 1037 cm-l; CIMS mlz (relative was stirred a t room temperature for 20 h. Some intensity) 522 (MH+, 100); HRCIMS calcd for C Z Q H ~ ~ N Omixture ~ of the ethanol was removed on the rotary evaporator at 522.2128, found 522.2139. 5,6-Dihydro-12-[ (6'-hydroxy-n-hexyl)oxy] -8,9- room temperature. Water (100 mL) was added to the dimethoxy-5-N-methyl-2,3-(methylenedioxy)benzo- residue, and the mixture was extracted with chloroform (3 x 50 mL). The combined organic layer was washed [clphenanthridine (9). A suspension of the ester 8 with water (25 mL), dried (Na2S04),and concentrated to (0.71 g, 1.36 mmol) and lithium aluminum hydride (0.5 give 14 (11.40 g, 99%) as an oil: bp 245-250 "C (1.2 mm); g, 13.6 mmol) in dry THF (70 mL) was heated a t reflux IR (thin film) 2960,2930,2825,1650,1595,1580,1510, under nitrogen for 18 h. The reaction was quenched with 1440,1255,1130,1015cm-l; lH NMR 6 7.75 (d, J = 15.6 15%NaOH under cooling with ice. The organic layer was Hz, 1H), 7.70 (d, J = 9.0 Hz, 1HI, 7.62 ( 8 , 1HI, 7.41 (d, separated and the residue extracted with THF (3 x 10 J = 15.6 Hz, 1H), 7.28 (d, J = 9.0 Hz, 1H), 7.21 (s, 1H), mL). The combined extracts were dried (Na2S04) and 6.92 (d, J = 8.1 Hz, 1 H), 6.90 (d, J = 8.1 Hz, 1H), 4.60 the solvent evaporated. The residue was chromato(sept, J = 5.8 Hz, 1 H), 3.95 ( 8 , 6 H), 3.89 (6, 3 H), 1.40 graphed on silica gel using 20% ethyl acetate in meth(d, J = 5.8 Hz, 6 H); CIMS (isobutane) mlz (relative ylene chloride to give 9 (0.38 g, 60%) as a white solid. intensity) 357 (MH+, 1001, 356 (M+, 8). Recrystallization from ethyl acetate-hexane gave a n 2-(3-Isopropoxy-Q-methoxyphenyl)-4-(3,4~e~oxanalytically pure sample: mp 125 "C; IR (KBr) 3509, yphenyl)-4-oxobutyronitrile(15). Acetic acid (2.3 mL) 1939, 1869, 1607, 1526, 1501, 1460, 1399, 1345, 1316, was added to a solution of enone 14 (10.40 g, 0.0292 mol) 1279, 1236, 1201, 1118, 1037 cm-'; NMR 6 7.63 (s, 1HI, in 2-ethoxyethanol (53 mL) a t 100 "C. An aqueous 7.58 (s, 1 H), 7.24 (s, 1 H), 7.01 (s, 1 H), 6.81 (s, 1 H), solution of potassium cyanide (5.1 g, 0.078 mol) in water 6.06 (s, 2 H), 4.20 (t, J = 6.0 Hz, 2 HI, 4.11 (s, 2 H), 4.01 (9.4 mL) was then added to the solution a t 118 "C, and (s, 3 H), 3.96 (s, 3 H), 3.69 (t, J = 6.5 Hz, 2 H), 2.53 (s, 3 the mixture was stirred a t 118 "C for 7 min. The reaction H), 1.96 (m, 2 H), 1.69-1.47 (m, 6 H); CIMS mlz (relative mixture was cooled to room temperature and poured into intensity) 466 (MH+, loo), 465 (M+, 48); HRCIMS calcd ice water (150 mL). Recrystallization of the crude for C Z ~ H ~ 466.2230, ~ N O ~ found 466.2221. 12-[(6'-Hydroxy-n-hexyl)oxyl-5,6-dihydro-8,9- material from methanol gave nitrile 15 (10.47 g, 93%): dimethoxy.N-methyl-2,3-(methylenedioxy)benzo~cl- mp 104-105 "C; IR (KBr)2955,2920,2825,1668,1585, phenanthridine 6-0-(2~Cyanoethyl-N~-diisopro-1507, 1455, 1337, 1305, 1250, 1237, 1150, 1135, 1100, 1010 cm-'; 'H NMR 6 7.51 (d, J = 5.9 Hz, 1 HI, 7.50 (s, pylphosphoramidite)(10). A solution of the alcohol 9 1 HI, 6.95-6.98 (m, 4 HI, 4.46-4.58 (m, 2 HI, 3.94 (6, 3 (186 mg, 0.4 mmol) and Nfl-diisopropylethylamine (309 H), 3.92 (s, 3 H), 3.84 ( 8 , 3 H), 3.64 (dd, J = 17.7, 7.8 Hz, mg, 2.4 mmol) in dry methylene chloride (2 mL) was lH),3.44(dd,J=l7.7,6.5Hz,lH),1.38(d,J=6.2Hz, added dropwise to a solution of 2-cyanoethyl N f l 3 HI, 1.36 (d, J = 6.2 Hz, 3 HI; CIMS (isobutane) mlz diisopropylchlorophosphoramidite (188 mg, 0.8 mmol) in (relative intensity) 384 (MH+. 100): HRCIMS calcd for dry methylene chloride (1 mL) a t 0 "C under nitrogen. CzzHzsN05 384.1811, found 384.1815. Anal. Calcd for The solution was stirred a t room temperature for 50 min CzzH25N05: C, H. and diluted with methylene chloride a t 0 "C. The 2-(3-Isopropoxy-4-methoxyphenyl)-4-(3,4-dimethoxresulting solution was washed with cold 2N Na2C03(10 mL) and then water (3 x 10 mL), dried (Na2S04), and yphenyl)-4-oxobutyricAcid (16). A mixture containing nitrile 15 (4.50 g, 0.0117 mol), water (50 mL), ethanol concentrated. The residue was chromatographed on (21.6 mL), and sodium hydroxide (4.77 g, 0.119 mol) was silica gel (chromatotron) using 30% ethyl acetate in methylene chloride to afford 10 (163 mg, 61%) as a heated under nitrogen a t reflux for 8 h. The mixture was cooled, and water (25 mL) was added. The resulting semisolid material: IR (neat) 2964, 2936, 2869, 2251, 1608, 1527, 1501, 1460, 1238, 1221, 1202, 1121, 1036 mixture was washed with ether (35 mL). The aqueous layer was acidified with 10% HC1 and extracted with cm-l; NMR 6 7.63 (s, 1 H), 7.57 (s, 1 H), 7.25 (s, 1 H), chloroform (3 x 30 mL). The combined chloroform 7.01 (s, 1H), 6.81 (s, 1 H), 6.06 (s, 1 H), 4.20 (t, J = 6.0 extracts were dried (Na2S04)and concentrated to give Hz, 1H), 4.11 (5, 2 H), 4.02 (s, 3 H), 3.96 (9, 3 HI, 3.89acid 16 (3.75 g, 79%), which was recrystallized from 3.56 (m, 6 H), 2.62 (t, J = 6.5 Hz, 2 H), 2.53 (s, 3 H), benzene-hexanes: mp 162-163 "C; IR (KBr) 37001.99-1.92 (m, 2 H), 1.73-1.47 (m, 6 H), 1.18 (9, J = 6.5, 2400, 1705, 1674, 1590, 1510, 1448, 1420, 1325, 1255, 1.5 Hz, 12 H); FABMS mlz (relative intensity) 666 (MH+, 1135, 1015 cm-l; lH NMR 6 10.70 (br s, 1 H), 7.59 (d, J 441, 665 (M+, 68), 664 (761, 364 (100). = 8.5 Hz, 1 HI, 7.51 ( 8 , 1 H), 6.92-6.81 (m, 4 H), 4.53 3-Isopropoxy-4-metho~benzaldehyde (12). A mix(sept, J = 5.8 Hz, 1HI, 4.21 (dd, J =10.0, 3.9 Hz, 1 H), ture containing anhydrous potassium carbonate (15.0 g), 3.94 (s, 3 HI, 3.91 (s, 3 HI, 3.86 (m, 1 H), 3.83 (s, 3 H), isovanillin (11,5.00g, 0.0329 mol), 2-bromopropane (15.0 3.26 (dd, J = 17.8, 3.9 Hz, 1H), 1.36 (d, J = 5.8 Hz, 3 H), mL, 0.160 mol), and DMF (10 mL) was stirred a t room 1.35 (d, J = 5.8 Hz, 3 HI; CIMS (isobutane) mlz (relative temperature for 15 min and then heated under reflux for intensity) 403 (MH+, 100); HRCIMS calcd for C22H2707 2.5 h , cooled, and poured into water (20 mL). The 403.1756, found 403.1750. Anal. Calcd for C22H2607: C, mixture was extracted with chloroform (4 x 20 mL). The H. combined chloroform extracts were washed with water 2-(3-Isopropoxy-4-methaxyphenyl)-4-(3,40(5 x 10 mL), dried (Na2SO4),and concentrated to give 12 (6.29 g, 98%) as a liquid: bp 101-105 "C (0.6 mm) yphenyllbutyricAcid (17). A mixture of intermediate [lit. (56) bp 110-113 "C (1 mm)]; IR (thin film) 2970, 16 (3.50 g, 0.00870 mol) and 10% Pd/C (1.00 g) in acetic acid (112 mL) was hydrogenated a t atmospheric pressure 2920,1680,1575,1500,1425,1260,1120 cm-l; IH NMR at room temperature for 3 days. The catalyst was filtered S 9.84 (s, 1 H), 7.45 (d, J = 7.9 Hz, 1 H), 7.42 (s, 1 H), off. The solvent was removed under high vacuum a t 6.98 (d, J = 7.9 Hz, 1 H), 4.65 (sept, J = 6.2 Hz, 1 H), room temperature, and the residue was taken up in 3.95 (s, 3 H), 1.41 (d, J = 6.2 Hz, 6 HI; CIMS (isobutane) chloroform, dried (NaZSOd, and concentrated to give mlz (relative intensity) 195 (MH', 100). acid 17 (3.32 g, 98%), which was crystallized from ethyl 3-Isopropoxy-3,4,4'-trimethoxychalcone(14). A
476 Bioconjugate Chem., Vol. 6,No. 4, 1995
Chen et al.
cis-1,2,3,4-Tetrahydro-2(3-isopropoxy-4-methoxacetate-hexanes: mp 79-82 "C; IR (KBr) 3650-2400, yphenyl)-6,7-dimethoxy-l-(iV-methylformamido~1705, 1590, 1515, 1450, 1415, 1260, 1230, 1135, 1100, naphthalene (21). A mixture containing amine 20 1020 cm-l; lH NMR 6 11.63 (br s, 1H), 6.97-6.61 (m, 6 (570.2 mg, 1.479 mmol), chloral (0.40 mL, freshly preH), 4.52 (sept, J = 6.1 Hz, 1 H), 3.85 (s, 6 H), 3.84 (s, 3 pared by mixing chloral hydrate with an equal amount H), 3.48 (t, J = 7.6 Hz, 1 HI, 2.54 (t, J = 7.0 Hz, 2 HI, of concentrated sulfuric acid and then distilling it), and 1.97-1.88 (m, 2 H), 1.36 (d, J = 6.1 Hz, 6 H); CIMS dry chloroform (10 mL) was heated a t reflux for 3 h under (isobutane) mlz (relative intensity) 389 (MH+, 16), 347 nitrogen. Water (20 mL) was added, and the mixture (MH+ - C3&, 100);HREIMS calcd for CzzHzsO6 388.1886, was extracted with chloroform (3 x 30 mL). The chlofound 388.1878. 243-Isopropoxy-4-methoxyphenyl)-6,7-dimethoxy- roform extracts were dried (Na2SO4)and concentrated. The residue was chromatographed on two silica gel plates 1-tetralone(18). A mixture containing acid 17 (3.00 g, (2 mm thickness) using 60% ethyl acetate-hexanes to 0.00772 mol), potassium carbonate (14.0 g, 0.101 mol), give 21 (480.6 mg, 79%), which was recrystallized from and dry chloroform (18 mL) was stirred a t room temperbenzene-hexanes: mp 140-141 "C; IR (KBr)2930,2825, ature for 0.5 h under nitrogen. Phosphorus oxychloride 1670,1605,1515,1440,1425,1245,1110,1010 cm-l; 'H (6.6 mL, 0.0708 mol) was added, and the mixture was NMR 6 7.80 (s, V5 H), 7.63 ( ~ , ~H),6.97-6.56 /5 ( m , 4 H), heated a t 80 "C for 75 min. The reaction mixture was 6.52(s,lH),4.63(d,J=5.0Hz,lH),4.50(sept,J=6.1 poured into ice water (100 mL), made alkaline with 5% Hz, 1 H), 3.90 (s, 3 H), 3.84 (5, 3 H), 3.81 (s, 3 H), 3.27NaOH (50 mL), and extracted with chloroform (3 x 40 2.78 (m, 3 H), 2.56 (9, 3/5 H), 2.52 (s, 12/5H), 2.36-1.93 mL). The combined extracts were dried (Na2S04)and (m, 2 H), 1.36 (d, J = 6.1 Hz, 6 HI; CIMS (isobutane) mlz concentrated. The residue was chromatographed on a (relative intensity) 414 (MH+, 91, 355 (MH+ - CzHsNO, silica gel column using 25% ethyl acetate-hexanes to 100); HRCIMS calcd for C Z ~ H ~ Z N 414.2280, O~ found afford pure 18 (1.56 g, 55%): mp 115-116 "C; IR (KBr) 414.2238. Anal. Calcd for C24H31N05: C, H. 2970, 2930, 2830, 1672, 1603, 1518, 1450, 1370, 1338, 2-(3-Isopropoxy-4-methoxyphenyl)-6,7-dimethoxy1270, 1195, 1135, 1020 cm-l; lH NMR 6 7.58 (s, 1 H), 1-W-methylformamido)naphthalene(22). DDQ (767.7 6.95-6.66 (m, 4 H), 4.49 (sept, J = 6.0 Hz, 1 H), 3.95 (s, mg, 3.382 mmol) was added to a solution of formamide 3 H), 3.92 (s, 3 H), 3.84 (s, 3 H), 3.69 (t,J = 7.6 Hz, 1H), 21 (463.7 mg, 1.121 mmol) in dry benzene (25 mL) a t 3.19-2.83 (m, 2 H), 2.58-2.25 (m, 2 H), 1.34 (d, J = 6.0 room temperature under nitrogen. The mixture was Hz, 3 H), 1.33 (d, J = 6.0 Hz, 3 H); CIMS mlz (relative intensity) 371 (MH+, 100); HRCIMS calcd for C Z Z H Z ~ O ~heated a t reflux for 2 h. The resulting precipitate was filtered, and the filtrate was concentrated. The residue 371.1858, found 371.1862. Anal. Calcd for CzzH2605: C, was taken in chloroform (25 mL) and washed with 5% H. NaOH solution (20 mL). The aqueous layer was ex1,2,3,4-Tetrahydro-2-(3'.isopropoxy-4" tracted again with chloroform (4 x 25 mL). The comnyl)-6,7-dimethoxy-l-(methylimino)naphthalene (19). bined chloroform extracts were dried (Na2SO4) and Methylamine (5.0g) was bubbled through a chloroform concentrated to give 22 (419 mg, 91%), which was solution (10 mL) containing 18 (0.60 g, 0.00162 mol) at recrystallized from benzene-hexanes: mp 147- 148 "C; 0 "C for 10 min. The solution was added to a solution of IR (KBr)2970,2930,2830,1685,1510,1465,1420,1335, titanium(1V) chloride (0.40 mL, 0.00364 mol) in chloro1320,1260,1240,1220,1160,1135,1105,1020,995 cm-l; form (5 mL) a t 0 "C under nitrogen. The reaction mixture 'H NMR 6 8.42 (s, '/5 HI, 8.19 (s, 4/5 H), 7.76 (d, J = 8.3 was stirred a t room temperature for 18 h under nitrogen. H, 1 H), 7.38 (d, J = 8.3 Hz, 1 H), 7.21 (s, 1 H), 7.05The precipitate was filtered off, and the filtrate was 6.80 (m, 4 H), 4.53 (sept, J = 5.8 Hz, 1 HI, 4.04 (s, 3 HI, concentrated to give 19 (0.62 g, 100%) as a n oil. This 3.99 (s, 3 H), 3.89 (s, 3 H), 3.09 (s, "16 HI, 2.96 (s,3/5 H) material was used immediately in the next step without 1.38 (d, J = 5.8 Hz, 6 H); CIMS (isobutane) mlz (relative any further purification: lH NMR 6 7.90 (s, 1H), 6.79intensity) 410 (MH+, 651, 152 (100); HRCIMS calcd for 6.59 (m, 4 H), 4.41 (sept, J = 6.0 Hz, 1H), 4.28 (m, 1H), CZ4HzsNO5 410.1967; found 410.1925. Anal. Calcd for 4.01 (s, 3 H), 3.91 (s, 3 H), 3.82 (s, 3 H), 3.24 (s, 3 H), Cz4Hz7N05: C, H. 2.95-2.00 (m, 4 H), 1.32 (d, J = 6.0 Hz, 3 HI, 1.30 (d, J 9~Isopropoxy-2,3,8-trimethoxy-5-methylbenzo[cl= 6.0 Hz, 3 H); CIMS (isobutane) mlz (relative intensity) phenanthridinium Chloride (23). Phosphorus oxy384 (MH+, 100). cis- 1,2,3,4-Tetrahydro-2-(3-isopropoxy-4-methox-chloride (0.40 mL) was added to a solution of formamide 22 (200.2 mg, 0.489 mmol) in acetonitrile (10 mL) at room yphenyl)-6,7-dimethoxy1-(methylaminolnaphthatemperature under nitrogen. The mixture was heated lene (20). Sodium borohydride (0.35 g, 0.00925 mol) was a t reflux for 0.5 h. The mixture was cooled and poured added to a solution of imine 19 (0.62 g, 0.00162 mol) in into ice water (10 mL). The resulting precipitate was methanol (30 mL). The reaction mixture was stirred a t filtered and washed with cold water (3 mL) and benzene room temperature for 1.5 h. Methanol was removed on (10 mL). The precipitate was recrystallized from methathe rotary evaporator, and water (20 mL) was added to nol-chloroform to give 23 (187.1 mg, 89%): mp 276the residue. The mixture was extracted with chloroform 279 "C; IR (KBr) 3400, 2860, 1610, 1505, 1455, 1425, (3 x 25 mL). The combined chloroform extracts were 1380,1267,1210,1090,1000cm-l; lH NMR (CF3COOD) dried (Na2S04)and concentrated to give 20 (0.58 g, 93%) 6 9.39 (s, 1H), 8.58 (d, J = 8.8 Hz, 1H), 8.29 (d, J = 8.8 as an oil, which was used in the next step without any Hz, 1H), 8.26 (s, 1H), 8.18 (s, 1H), 7.81 (8,1H), 7.69 (s, further purification: IR (thin film) 3310, 2980, 2940, 1 H), 5.30 (sept, J = 5.8 Hz, 1 H), 5.10 (s, 3 H), 4.28 (s, 2840, 2780, 1610, 1520, 1470, 1460, 1445, 1335, 1265, 3 H), 4.25 (s, 3 H), 4.24 (s, 3 H), 1.68 (d, J = 5.8 Hz, 6 H); 1140, 1125, 1030 cm-'; 'H NMR 6 6.87-6.81 (m, 4 H), CIMS (isobutane) mlz (relative intensity) 378 (MH' 6.66 (s, 1 H), 4.48 (sept, J = 6.0 Hz, 1 HI, 3.88 (s, 3 HI, CH3C1, 100); HRCIMS calcd for C Z ~ H Z ~ 378.1705, NO~ 3.87 (s, 3 HI, 3.85 (s, 3 H), 3.62 (d, J = 3.6 Hz, 1H), 3.16 found 378.1654. (dt, J = 11.7, 3.6 Hz, 1 H), 3.00-2.64 (m, 2 H), 2.549-Hydroxy-2,3,8-trimethoxy-5-methylbenzo~cl2.25 (m, 1 H), 2.22 (s, 3 H), 2.10-1.86 (m, 1H), 1.36 (br phenanthridinium Chloride (24). A mixture contains, 1 H), 1.35 (d, J = 6.0 Hz, 6 HI; CIMS (isobutane) mlz ing 23 (60.4 mg, 0.141 mmol) and methanesulfonic acid (relative intensity) 386 (MH', 231,355 (MH+ - CH3NH2, (2.5 mL) was heated a t 57 "C for 2 h. The mixture was 100); HRCIMS calcd for C23H32N04 386.2331, found cooled and poured into diethyl ether (25 mL). The 386.2335.
Synthesis of Novel Phosphoramidite Reagents
Bioconjugate Chem., Vol. 6,No. 4, 1995 477
resulting solution was stirred for 40 min, diluted with resulting precipitate was filtered and stirred with 10% methylene chloride (20 mL), and washed with cold 2 N NaCl solution (8 mL) a t room temperature for 1h. The NaZC03 (4 x 10 mL). The organic layer was dried (Nazresulting solid was filtered, washed with cold water (1 SO4) and concentrated. The residue was chromatomL), and dried. This solid was recrystallized from graphed on silica gel (chromatotron) with 30% ethyl methanol to give 24 (35.1 mg, 64%): mp 250-252 "C (lit. acetate in methylene chloride to give 27 (106 mg, 79%) (57) mp 261-263 "C); IR (KBr) 3600-2200,1612, 1540, as a semisolid: IR (neat) 2935, 2251, 1606, 1503, 1477, 1450, 1410, 1320, 1302, 1275, 1210, 1172, 1125, 1010 1463, 1355, 1348, 1300, 1246, 1220, 1205, 1154, 1012 cm-l; 'H NMR 6 9.39 (s, 1H), 8.56 (d, J = 9.0 Hz, 1H), cm-l; lH NMR (CDC13) 6 7.70 (d, J = 8.5 Hz, 1H), 7.66 8.35 (s, 1H), 8.27 (d, J = 9.0 Hz, 1H), 8.17 (8,1H), 7.75 (s, 1H), 7.54 (d, J = 8.5 Hz, 1 H), 7.33 (5, 1 H), 7.14 (s, (s, 1H), 7.67 (s, 1 H), 5.09 (8,3 H), 4.27 (s, 3 H), 4.25 (9, 1 H), 6.81 (s, 1 H), 4.16 (s, 2 HI, 4.14 (t, J = 7.0 Hz, 2 6 H); CIMS (isobutane) mlz (relative intensity) 350 (M+ H), 4.08 (8, 3 H), 4.02 (5, 3 H), 3.94 (s,3 HI, 3.90-3.56 - C1-, 41), 336 (MH+ - CH3C1, 100). (m, 6 H), 2.63 (m, 5 H), 1.95-1.88 (m, 2 H), 1.71-1.44 9-[5~(Etho~carbonyl)-n-~n~~l-5,6-dihydr0-2,3,& (m, 6 H), 1.19-1.17 (m, 12 HI; FABMS mlz (relative trimethoxy-5-methylbenzo[clphenanthridine(25). intensity) 652 (MH+, 371, 651 (M+, 671, 650 (86), 350 NaH (60% in mineral oil, 200 mg, 5 mmol) was added in portions to a mixture of 9-hydroxy-5-methyl-2,3,8-tri- (100). 6-Hex-S'-eny1-5,6-dihydro-8,9-dimethoxy-5-methmethoxybenzo[c]phenanthridinium chloride (24,385 mg, (28). yl-2,3-(methylenedio~)benzo[clphenanthridine 1mmol) and dry DMSO (30 mL). The reaction mixture Magnesium (250 mg, 10.28 mmol) was added to a solution was stirred for 30 min and ethyl 6-bromohexanoate of 6-bromo-l-hexene (1.31g, 8.03 mmol) in dry THF (10 added. The reaction mixture was stirred overnight, and mL) and the mixture stirred a t room temperature for 30 a clear solution was obtained. Sodium borohydride (76 min. The reaction mixture was then heated a t reflux for mg, 2 mmol) was added and the resulting mixture stirred 1h. Nitidine chloride (1,300 mg, 0.78 mmol) was added. for 1h and diluted with methylene chloride (70 mL). The The reaction mixture was heated a t reflux for 1 h, the solution was washed with 2 N sodium carbonate (5 x 40 reaction was quenched with 1 N HC1 (30 mL), and the mL), dried (Na2SO4),and concentrated. The residue was resulting mixture was extracted with chloroform (3 x 40 chromatographed through a short column (silica gel) mL). The combined chloroform extracts were washed using methylene chloride and ethyl acetate to give 25 with water (25 mL), dried (Na2S04),and concentrated. (388 mg, 79%). Recrystallization from ethyl acetateThe residue was chromatographed on a silica gel plate hexane gave a n analytically pure sample: mp 133 "C; (1 mm thickness) using 25% ethyl acetate in hexane to IR (KBr)2942,2868,1733,1605,1504,1463,1364,1301, give 28 (248 mg, 74%) as a n oil: IR (neat) 2935, 2845, 1251, 1012 cm-l; NMR 6 7.93 (9, 1 H), 7.91 (d, J = 8.0 1635, 1603, 1522, 1500, 1470, 1397, 1350, 1250, 1175, Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.16 (s, 1H), 7.03 (s, 1145, 1035 cm-l; IH NMR 6 7.70 (s, 1 HI, 7.67 (d, J = lH),6.56(s,lH),4.13(~,2H),3.99(q,J=7.OHz,2H), 8.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.11 3.86 (t, J = 6.2 Hz, 2 H), 3.64 (8,3 H), 3.53 (s, 6 H), 2.66 (s, 1 H), 6.72 (s, 1 H), 6.04 (s, 2 HI, 5.74 (m, 1 HI, 4.90 (s, 3 H), 2.14 (t, J = 7.1 Hz, 2 H), 1.78-1.35 (m, 6 H), (m, 2 H), 3.98 (s, 3 H), 3.95 (s, 3 H), 3.84 (m, 1 H), 2.62 0.99 (t, J = 7.1 Hz, 3 HI; CIMS mlz (relative intensity) (s, 3 H), 1.97 (m, 2 H), 1.71-1.10 (m, 6 H); CIMS mlz 494 (MH-, 100); HRCIMS calcd for C29H36N06 494.2543, (relative intensity) 432 (MH+, 1001, 431 (M+, 25); HREfound 494.2523. Anal. Calcd for CzgH3E"s: C, H, N. IMS calcd for CzYHzgN04 431.2097, found 431.2101. 5,6-Dihydro-9[(6-hydroxy-n-hexyl)oxyl-2,3,8tri6-(6-Hydroxyhexyl)-8,9-dimethoxy-5-methyl=2,3methoxybenzo[clphenanthridine (26). Lithium alu(methylenedioxy)benzo[clphenanthridine (29). minum hydride (50 mg, 1.34 mmol) was added to a 9-BBN (6.4 mL, 0.5 M in THF, 3.2 "01) was added to solution of the ester 25 (329 mg, 0.66 mmol) in dry ether a solution of 28 (230 mg, 0.53 mmol) in dry THF (15 mL) (40 mL), and the resulting mixture was stirred a t room under nitrogen, and the mixture was stirred a t room temperature for 1h. The reaction mixture was quenched temperature for 3 days. NaOH (1.5 N, 4.0 mL) and 30% with 2 N sodium carbonate solution (3 mL) a t 0 "C. The hydrogen peroxide (2.0 mL) were added, and the mixture organic layer was separated, and the residue was diluted was heated a t 50 "C for 1h. Water (20 mL) was added, with 2 N sodium carbonate solution (30 mL) and exand the mixture was extracted with chloroform (3 x 25 tracted with methylene chloride (4 x 30 mL). The mL). The combined chloroform extracts were dried (Napcombined organic layer was dried (Na~S04)and concenSO4) and concentrated. The residue was chromatotrated to give 26 (242 mg, 80%). Chromatography on graphed on a silica gel plate (2 mm thickness) using 60% silica gel with 30% ethyl acetate in methylene chloride ethyl acetate in hexane to give 29 (185 mg, 77%) as an and recrystallization from ethyl acetate-hexane afforded oil: IR (neat) 3350, 2905, 2840, 1595, 1515, 1490, 1450, a n analytically pure sample: mp 150 "C; IR (KBr) 3529, 1390,1340,1235,1160,1135,1015cm-'; IH NMR 6 7.70 2935, 2860, 1605, 1561, 1503, 1463, 1350, 1300, 1247, (s, 1 H), 7.69 (d, J = 8.3 Hz, 1 H), 7.48 (d, J = 8.3 Hz, 1 1206, 1153, 1012 cm-l; 'H NMR 6 7.92 (s, 1HI, 7.90 (d, H), 7.32 (s, 1H), 7.11 (s, 1H), 6.73 (s, 1H), 6.05 (s, 2 H), J = 7.8 Hz, 1H), 7.53 (s, 1H), 7.02 (s, 1H), 6.55 (s, 1HI, 3.99 (s, 3 H), 3.95 (s, 3 H), 3.84 (t,J = 6.0 Hz, 1H), 3.56 6.04 (d, J = 8.4 Hz, 1 H), 4.12 (s, 2 H), 3.92 (m, 2 H), (t, J = 6.0 Hz, 2 HI, 2.62 (s, 3 HI, 2.00-1.00 (m, 11 HI; 3.63 (s, 3 H), 3.52 (5, 6 H), 3.43-3.22 (m, 2 HI, 2.65 (s, 3 CIMS m l z (relative intensity) 450 (MH+, 100); HRCIMS H), 1.86-1-63 (m, 2 H), 1.60-1.12 (m, 6 H); CIMS mlz calcd for C Z ~ H ~ Z450.2280, NO~ found 450.2234. (relative intensity) 452 (MH', 100); HRCIMS calcd for 6-[(6'-Hydroxy-n-hexyl)oxyl-5,6-dihydro-8,9C27H34N05452.2437, found 452.2418. Anal. Calcd for dimethoxy-5-methyl-2,3-(methylenedioxy)benzo~clC27H33N05: C, H, N. phenanthridine 6-042-CyanoethylNJV-diisopro9-[(6-Hydroxy-n-hexyl)oxyl-5,6-dihydro-2,3,8-tripylphosphoramidite)(30). A solution of 2-cyanoethyl methoxy-5-methylbenzo[clphenanthridine6'-0-(2N,N-diisopropylchlorophosphoramidite (123 mg, 0.52 CyanoethylNJV-diisopropylphosphoramidite) (27). mmol) in dry methylene chloride (2 mL) was added A solution of 2-cyanoethyl Nfl-diisopropylchlorophosdropwise to a stirred solution of the alcohol 29 (120 mg, phoramidite (107 mg, 0.45 mmol) was added dropwise 0.26 mmol) and N,N-diisopropylethylamine(134 mg, 1.04 to a solution of the alcohol 26 (102 mg, 0.22 mmol) and mmol) in dry methylene chloride (3 mL) a t 0 "C under N,N-diisopropylethylamine(174 mg, 1.35 mmol) a t 0 "C nitrogen. The reaction mixture was stirred a t room in dry methylene chloride (2 mL) under nitrogen. The
Chen et al.
478 Bioconjugate Chem., Vol. 6,No. 4, 1995
temperature for 50 min, diluted with methylene chloride, and washed with cold 10% NaHC03 (15 mL) and with cold water (3 x 15 mL). The organic layer was dried (Na2SO4)and concentrated in vacuo a t room temperature. The residue was chromatographed on silica gel (chromatotron) using 30% ethyl acetate in methylene chloride to give 30 (148 mg, 89%) as a semisolid: IR (neat) 2965, 2930, 2868, 1607, 1526, 1499, 1396, 1363, 1350, 1312, 1243, 1200, 1183, 1147, 1079, 1039 cm-l; NMR 6 7.70 (d, J = 8.5 Hz, 1H), 7.70 (s, 1H), 7.48 (d, J = 9.0 Hz, 1 H), 7.32 (s, 1 H), 7.12 (s, 1 H), 6.73 (s, 1 H), 6.08-6.05 (m, 2 H), 3.99 (s, 3 H), 3.96 (s, 3 €3.83-3.41 I), (m, 7 HI, 2.65-2.58 (m, 5 H), 1.60-1.23 (m, 1 H), 1.22-1.12 (m, 12 H); 31PNMR (202 MHz) 146.29; FABMS 650 (MH+, 5), 649 (M+, 15), 648 (M- - 1,21), 432 (12), 349 (49), 348 (100); HRFABMS calcd for C36H49N306P 650.3356, found 650.3231.
Benzophenanthridine-Oligonucleotide Conjugate 34. Automated solid phase oligonucleotide synthesis was performed on a modified Milligen 7500 DNA Synthesizer. A three-way valve was placed between the synthesis column and the synthesizer to permit manual introduction of small volumes of phosphoramidite solutions without removing the synthesis column from the machine. The synthesis reagents and deoxyribonucleoside phosphoramidites were obtained from Milligen. The solid support was (dimethoxytrity1)uridine CPG (30 pmoll g, Milligen). Oligonucleotide synthesis was conducted using a standard 1pmol scale synthesis protocol. After the final 5’-dimethoxytrityl group was removed, and the support washed extensively with acetonitrile, the synthesizer was halted to permit manual addition of the dihydrofagaronine phosphoramidite. Phosphoramidite 5 (20 pmol) was dissolved in 1H-tetrazole-acetonitrile solution (0.35 M, 540 pL) in a syringe and introduced to the system and allowed to react with the support-bound oligonucleotide for 5 min. The synthesizer was then allowed to continue a normal coupling cycle. The final phosphite triester linkage and the fagaronine moiety were oxidized by the synthesizer using standard aqueous 1 2 solution (0.05 M 1 2 in THF, water, pyridine (7:2:1), 1 min exposure). After the synthesis was completed by the machine, the support containing the fagaronine oligonucleotide conjugate was placed in a pressure vial and treated with 2 mL of concentrated ammonium hydroxide solution a t 55 “C for 12-14 h. The supernatant solution was then filtered to remove the controlled pore glass and concentrated to dryness in vacuo. The fagaronineoligonucleotide conjugate 33 was isolated by reverse phase HPLC. Conjugate 33 was loaded on the column (30 x 0.4 cm column MCG-lO/MicroPack,Varian) in HzO and eluted with a linear gradient from 100% solvent A (0.1 M triethylammonium acetate, pH 6.5) to 40% solvent B (95% aqueous acetonitrile) over 40 min a t a flow rate of 1mumin. The purified product was characterized by FAB mass spectrometry in the positive ion mode (dithiothreitol-dithioerythntol, 3:l v/v, calcd for C1&142N320& M’ mlz 3257, found mlz 3257). The 3’ ribonucleoside residue was then removed by periodate oxidation and /3-elimination (0.04 M NaI04,O “C, 1h in the dark; excess periodate removed by incubation with 0.05 M methionine, 0 “C, 30 min; p-elimination 0.04 M cyclohexylamine, 0.1 M HEPES, pH 8.0, 45 “C, 90 min) to give conjugate 34. The final product was then purified by anion exchange HPLC (58). RESULTS AND DISCUSSION
A. Attachment of the Linker Chain to the “RightHand” Side. Fagaronine (2) was reacted with the tosylate of ethyl 6-hydroxyhexanoate in the presence of
Scheme 1
3
n
”\
O’P, ? { ;
c~3.f10cH, CHsO
A
N. CH3 5
Reagents: (a) (1)KO-t-Bu, DMSO, room temperature (30 min), (2) TsO(CH&COOEt, room temperature (24 h); (b) LiAlH4, THF, room temperature (4 h); (c) (1) EtN(i-Pr)z, THF, room temperature (10 min), (2) 2-cyanoethyl Nfl-diisopropylchlorophosphoroamidite, room temperature (1.5 h). a
potassium tert-butoxide in dry DMSO a t room temperature to afford the alkylated product 3 (Scheme 1) (59). Initially, the 5,6-didehydro derivative of 4, having a charged N-methylisoquinolinium ring system, was prepared by a separate approach from that presented in Scheme 1 and was found to be practically insoluble in various organic solvents, causing the preparation of the corresponding phosphoramidite to proceed poorly. Furthermore, this phosphoramidite failed to react with the 5‘-hydroxyl group of synthetic oligonucleotides. In view of these difficulties, both the ester and iminium group of intermediate 3 were reduced with lithium aluminum hydride in tetrahydrofuran to give intermediate 4, which was readily converted to the phosphoramidite 5 in quantitative yield. It was anticipated that the dihydroisoquinoline ring system present in 5 would be oxidized to the desired aromatic, N-methylisoquinolinium moiety during the iodine oxidation of the phosphite linkage during the synthesis of the oligonucleotideintercalator conjugates (Scheme 5). This approach involving dihydroisoquinolines was also taken in the subsequent syntheses of additional phosphoramidites in which the linker chain was attached to various other regions of the benzophenanthridine alkaloid ring system. €5. Attachment of the Linker Chain to the “Top”. The starting material 6 was obtained as previously described in our total synthesis of nitidine chloride (60). Treatment of 6 with isopropenyl acetate in the presence ofp-toluenesulfonic acid and air afforded the aryl acetate 7 (Scheme 2) (61). Hydrolysis of the acetate 7 with sodium hydroxide in aqueous ethanol and alkylation of the resulting anion with ethyl 6-bromoacetate gave intermediate 8, which was reduced to 9 with lithium
Bioconjugate Chem., Vol. 6, No. 4, 1995 479
Synthesis of Novel Phosphoramidite Reagents
Scheme 3
11
12
13
18
CHI0
19
ma
h
w30
10 k
Reagents: (a) p-TsOH, isopropenyl acetate, air, reflux (24 h), 85 "C (24 h); (b) (1)NaOH, EtOH, HzO, room temperature (2 h), (2) DMSO, ethyl 6-bromohexanoate, room temperature (18 h), (c) LiAlH4,THF, reflux (18 h); (d) EtN(i-Pr)z, 2-cyanoethyl Nfl-diisopropylchlorophosphoramidite,CH2C12, 0 "C to room temperature (50 min).
aluminum hydride in refluxing THF. The primary alcohol 9 was converted to the corresponding phosphoramidite 10 with 2-cyanoethyl Nfl-diisopropylchlorophosphoramidite in the presence of Nfl-diisopropylethylamine in dry methylene chloride. C. Attachment of the Linker Chain to the "LeftHand" Side. A benzophenanthridine system having a phenolic hydroxyl group on the "left-hand" side was required in order to attach a linker chain using our approach. Since no suitable naturally occurring benzophenanthridines were available, the phenolic benzophenanthridine intermediate 24 was synthesized using a n approach established by Ishii et al. (Scheme 3) (56, 62, 63). This approach utilizes an isopropyl protecting group for the phenol, which can be removed under acidic conditions. Alkylation of the potassium phenoxide anion derived from the phenol 11 with 2-bromopropane in refluxing DMF afforded the isopropyl ether 12 (56).Condensation of the aldehyde 12 with 3,4-dimethoxyacetophenone(13) under basic conditions gave the chalcone 14,which was converted to the nitrile 15 in the presence of potassium cyanide. Hydrolysis of the nitrile 15 with sodium hydroxide in refluxing aqueous ethanol gave the corresponding acid 16. The ketone group of 16 was converted to a methylene by prolonged hydrogenolysis over palladium on charcoal to yield 17, which underwent a n intramolecular Friedel-Crafts reaction on treatment with phosphorus oxychloride to yield the substituted tetralone 18. The imine intermediate 19 was obtained from treatment of the tetralone 18 with methylamine in the presence of titanium tetrachloride. Reduction of the imine 19 with sodium borohydride in methanol yielded the amine intermediate 20, which was assigned the cis stereochemistry on the basis of the observed 3.5 Hz
'
22
20RrH 6 2 1 R=CHO
0
ma0
0N?
P
' maa
-
;H O & -
/
/N?
%o
ma 24
-
E = 0 t-
N.
WlO
W3
25
HO26
CN
27
Reagents: (a) CH3CHBrCH3, K&03, DMF, reflux (2.5 h); (b) NaOH, EtOH, room temperature (20 h); (c) KCN, CH~CHZOCHZCHZOH, AcOH, HzO, 118 "C (7 min); (d) NaOH, aqueous EtOH, reflux (8 h); (e) Hz, PUC, aqueous AcOH, room temperature (3 days); (0(1)KzCO3, CHC13 (30 min), (2) Poc13, 80 "C (75 min); (g) CH~NHZ, TiC14, CHCl3, room temperature (18 h); (h) NaBH4, MeOH, room temperature (1.5 h); (i) CC13CH0, CHC13, reflux (3 h); (j)DDQ, C6H6, reflux (2 h); (k) POC13, CH3CN, reflux (30 min); (1) CH3S03H, 57 "C (2 h); (m) (1)NaH, DMSO, room temperature (30 min), (2) ethyl 6-bromohexanoate, room temperature (12 h), (3) NaBH4, room temperature (1 h); (n) LiAlH4, EtzO, room temperature (1 h); (0)EtN(i-Pr)z, 2-cyanoethyl Nfl-diisopropylphosphoramidite, CHzC12, 0 "C to room temperature (40 min). a
coupling constant between the methine protons in the lH NMR spectrum, in contrast to the 10 Hz coupling
Chen et ai.
480 Bioconjugate Chem., Vol. 6,No. 4,1995
Scheme 5
Scheme 4
5
b
TCAGTGGTN
I
LCAACPG 31
PN
29
32
OH
LCAA-CPG
Reagents: (a) HC=CH(CHp)4MgBr, THF, reflux (1 h); (b) (1)9-BBn, THF, room temperature ( 3 days), (2) HpOp, NaOH, THF, 50 "C (1 h); (c) EtN(i-Pr)Z, 2-cyanoethyl Nfl-diisopropylphosphoramidite, CHZClp, 0 "C to room temperature (50 min).
constant expected for the corresponding trans isomer (56, 62, 63). Formylation of the amine 20 with chloral in refluxing chloroform provided the desired formamide 21, which, in the presence of phosphorus oxychloride, underwent Bischler-Napieralski cyclization to the benzophenanthridine system 23. The isopropyl protecting group was removed when 23 was heated a t 57 "C in methanesulfonic acid for 2 h (64). Alkylation of the sodium phenoxide anion derived from the phenol 24 with ethyl 6-bromohexanoate in DMSO, followed by reduction of the iminium functionality with sodium borohydride, afforded compound 25. The ester 25 was reduced to the primary alcohol 26 with lithium aluminum hydride. Treatment of the primary alcohol 26 with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite in the presence of N,N-diisopropylethylamine in dry methylene chloride gave the phosphoramidite 27. D. Attachment of the Linker Chain to the "Bottom". The naturally occurring benzophenanthridine alkaloid nitidine chloride (1)was used as the starting material for the attachment of the linker chain to the "bottom" region. Addition of 5-hexenylmagnesium bromide to the iminium ion 1 afforded the alkene 28,which underwent smooth hydroboration-oxidation to the primary alcohol 29 (Scheme 4). Treatment of the alcohol 29 with 2-cyanoethyl N,N-diisopropylphosphoramiditein the presence of N,N-diisopropylethylamine gave the phosphoramidite 30. E. Utilization of the Phosphoramidite 5 in the Synthesis of an Antisense Oligonucleotide-Intercalator Conjugate. In order to demonstrate the synthetic utility of these new benzophenanthridine phosphoramidite reagents, phosphoramidite 5 was used to prepare an oligonucleotide-fagaonine conjugate by semiautomated solid phase synthesis. The oligonucleotide containing a free 5'-hydroxyl group was constructed on an automated synthesizer using standard DNA synthesis procedures. After removal of the 5'-hydroxyl protecting group, the synthesizer was halted to permit manual addition of the fagaronine phosphoramidite. The phosphoramidite 5 was mixed with tetrazole in acetonitrile
33
-
,
W O
O 0
s
0N,*
a
3
-0Ac 34
Reagents: (a) HO-5'-TCAGTGGTrU-LCA-CPG, 1H-tetrazole in CHsCN, 20 "C (5 min); (b) Ip in THF, pyridine, HpO, 20 "C (1 min); (c) concentrated NH40H, 55 "C (12-14 h); (d) NaI04, 0 "C (dark, 1 h); cyclohexylamine, HEPES, pH 8 , 4 5 "C (90 min). a
solution and reacted with the oligonucleotide on the solid support for 5 min to yield intermediate 31 (Scheme 5). The final phosphite linkage as well as the dihydrofagaronine residue were then oxidized by treatment with standard aqueous IZ solution on the DNA synthesizer to provide 32. The oligonucleotide-fagaronine conjugate was then released from the glass support and deprotected with concentrated ammonium hydroxide to yield the product 33,which was isolated by reverse phase chromatography. FABMS in the positive ion mode displayed a peak a t mlz 3257, consistent with structure 33. Periodate oxidation and p-elimination afforded the final product 34,bearing a 3'-terminal phosphate, which was purified by anion exchange HPLC (Figure 1). As judged from the HPLC trace in Figure 1, the iodine-mediated dehydrogenation of the dihydropyridine ring of 31 during its conversion to 32 occurs in high yield. The major component isolated from HPLC showed a significant absorbance in the U V M S spectrum at 390 nm, which is a characteristic absorbance of fagaronine chloride. The oligonucleotide sequence TCAGTGGT was designed to be complementary to residues 490-497 of rabbit p-globin mRNA, and the 3' terminal phosphate residue was included to prevent priming cDNA synthesis when the conjugates are tested as inhibitors of HIV-1 reverse transcriptase (65).
Synthesis of Novel Phosphoramidite Reagents
Bioconjugate Chem., Vol. 6, No. 4, 1995 481 Anion axehonpa HPLC
250
w a
300
350
400
450
WAVELENGTH (nm)
4
*-
0.00
0
10
20
30
40
ELUTION TIME (min)
Figure 1. HPLC elution profile and WMS spectrum obtained during the purification of 34.
The binding of the conjugate 34 and the parent oligonucleotide TCAGTGGTp to a complementary RNA oligonucleotide were investigated by analysis of U V absorbance vs temperature melting curves. The T , of the oligonucleotide TCAGTGGTp was 37 f 1 "C, while the T,,, of the conjugate 34 was 50 f 1"C (66). The IC50 values vs HIV-1 reverse transcriptase (globin mRNA assay) of 23 and 7 pM for TCAGTGGTp and the conjugate 34,respectively, correlate with the T , values (65, 66). However, the IC50 value of fagaronine chloride (2) itself was 2.2 pM. The new benzophenanthridine phosphoramidite reagents described here should prove useful in the preparation of a n array of antisense oligonucleotide-benzophenanthridine conjugates in which the linker chain is connected to various regions of the intercalator. This may in turn help to define the optimal orientation of the intercalating moiety in the double helix for inhibition of gene expression by the antisense oligonucleotide-intercalator conjugates. ACKNOWLEDGMENT
This investigation was made possible by Grants AI24289, AI25712, and AI27713 awarded by the National Institute of Allergy and Infectious Diseases, DHHS. W e are grateful to Dr. Edward M. Acton, Developmental Therapeutics Program, Division of Cancer Treatement, National Cancer Institute, for a generous supply of fagaronine chloride. LITERATURE CITED (1) Whitton, J. L. (1994)Antisense Treatment ofviral Infection. Adu. Virus Res. 44, 267-303. (2) Milligan, J. F., Matteucci, M. D., and Martin, J. C. (1993) Current Concepts in Antisense Drug Design. J. Med. Chem. 36, 1923-1937. (3) Tonkinson, J . L., and Stein, C. A. (1993) Antisense Nucleic Acids - Prospects for Antiviral Intervention. Antiviral Chem. Chemother. 4 , 193-400. (4) Stein, C. A., and Cheng, Y.-C. (1993) Antisense Oligonucleotides as Therapeutic Agents - Is the Bullet Really Magic? Science 261, 1004-1012. ( 5 ) To, R. Y.-L., and Neiman, P. E. (1992) The Potential for Effective Antisense Inhibition of Retroviral Replication Mediated by Retroviral Vectors, pp 261-271, Raven Press, Ltd., New York. ( 6 ) Stein, C. A. (1992) Anti-sense Oligodeoxynucleotides Promises and Pitfalls. Leukemia 6, 967-974. (7) Degols, G., Leonetti, J.-P., Milhaud, P., Mechti, N., and Lebleu, B. (1992) Antisense Inhibitors of HTV: Problems and Perspectives. Antiviral Res. 17, 279-287. (8) Agrawal, S., and Sarin, P. S. (1991) Antisense Oligonucleotides: Gene Regulation and Chemotherapy of AIDS. Adv. Drug Del. Rev. 6, 251-270, (9) Mirabelli, C. K., Bennett, C. F., Anderson, K., and Crooke, S.T. (1991) I n vitro and I n vivo Pharmacologic Activities of Antisense Oligonucleotides. Anti-Cancer Drug Des. 6, 647661.
(10) Cook, P. D. (1991) Medicinal Chemistry of Antisense Oligonucleotides - Future Opportunities. Anti-Cancer Drug Des. 6 , 585-607. (11) Cohen, J. S.(1991) Antisense Oligodeoxynucleotides as Antiviral Agents. Antiviral Res. 16, 121-133. (12) Cohen, J. S. (1991) Oligonucleotides as Therapeutic Agents. Pharm. Ther. 52, 211-225. (13) Calabretta, B. (1991) Inhibition of Protooncogene Expression by Antisense Oligodeoxynucleotides: Biological and Therapeutic Implications. Cancer Res. 51, 4505-4510. (14) Helene, C. (1991) Rational Design of Sequence-specific Oncogene Inhibitors Based on Antisense and Antigene Oligonucleotides. Eur. J . Cancer 27, 1466-1471. (15) Uhlmann, E., and Peyman, A. (1990) Antisense Oligonucleotides: A New Therapeutic Principle. Chem. Rev. 90, 543-584. (16) Tidd, D. M. (1990) A Potential Role for Antisense Oligonucleotide Analogues in the Development of Oncogene Targeted Cancer Chemotherapy. Anticancer Res. 10,1169-1182. (17) ToulmB, J.-J., and Helene, C. (1988) Antimessenger Oligodeoxyribonucleotides: An Alternative to Antisense RNA for Artificial Regulation of Gene Expression. Gene 72, 51-58. (18) Zon, G. (1988) Oligonucleotide Analogues as Potential Chemotherapeutic Agents. Pharm. Res. 5, 539-549. (19) Verspieren, P. (1987) An Acridine-linked Oligodeoxynucleotide Targeted to the Common 5' End of Trypanosome mRNA Kills Cultured Parasites. Gene 61, 307-315. (20) Stein, C. A., Mori, K., Loke, S.L., Subasinghe, C., Shinozuka, K., Cohen, J . S.,and Neckers, L. M. (1988) Phosphorothioate and Normal Oligodeoxyribonucleotides with 5'Linked Acridine: Characterization and Preliminary Kinetics of Cellular Uptake. Gene 72, 333-341. (21) Cazenave, C., Chewier, M., Thuong, N. T., and Helene, C. (1987) Rate of Degradation of Alpha- and Beta-oligodeoxynucleotides in Xenopus Oocytes. Implications for Antimessenger Strategies. Nucleic Acids Res. 15, 10507-10521. (22) Thuong, N. T., Asseline, U., Roig, V., Takasugi, M., and Helene, C. (1987) Oligo(a-deoxynuc1eotide)sCovalently Linked to Intercalating Agents: Differential Binding to Ribo- and Deoxyribopolynucleotides and Stability Towards Nuclease Digestion. Proc. Natl. Acad. Sei. U.S.A. 84, 5129-5133. (23) Asseline, U., Thuong, N. T., and Helene, C. (1985) Oligonucleotides Covalently Linked to Intercalating Dyes. J . Biol. Chem. 260, 8936-8941. (24) Helene, C., Montenay-Garestier, T., Saison, T., Takasugi, M., Toulme, J. J.,Asseline, U., Lancelot, G., Maurizot, J. C., Toulmh, F., and Thuong, N. T. (1985) Oligodeoxynucleotides Covalently Linked to Intercalating Agents: A New Class of Gene Regulatory Substances. Biochemie 67, 777-783. (25) Mori, K., Subasinghe, C., and Cohen, J. (1989) Oligodeoxynucleotide Analogs with 5'-linked Anthraquinone. FEBS Lett. 249, 213-218. (26) Asseline, U., Delarue, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T., and Helene, C. (1984) Nucleic Acid-binidng Molecules with High Affinity and Base Sequence Specificity: Intercalating Agents Covalently Linked to Oligodeoxynucleotides. Proc. Natl. Acad. Sei. U.S.A. 81, 32973301. (27) Vasseur, J.-J.,Gauthier, C., Rayner, B., Paoletti, J., and Imbach, J.-L. (1988) Efficient and Easy Synthesis of Octathymidylate Covalently Linked to Intercalating 9-Aminoellipticine. Biochem. Biophys. Res. Commun. 152, 56-61. (28) Durand, M., Maurizot, J. C., Asseline, U., Barbier, C., Thuong, N. T., and Helene, C. (1989) Oligothymidylates Covalently Linked to an Acridine Derivative and with Modified Phosphodiester Backbone: Circular Dichroism Studies of Their Interactions with Complementary Sequences. Nucleic Acids Res. 17, 1823-1837. (29) Lancelot, G., Guesnet, J.-L., Asseline, U., and Thuong, N. T. (1988) NMR Studies of Complex Formation Between the Modified Oligonucleotide d(T*TCTGT) Covalently Linked to an Acridine Derivative and Its Complementary Sequence d(GCACAGAA). Biochemistry 27, 1265-1273. (30) Thuong, N. T., and Chassignol, M. (1987) Synthesis and Reactivity of Oligothymidylates Substituted with an Intercalating Agent and a Thiophosphate Group. TetrahedronLett. 28, 4157-4160.
Chen et al.
482 Bioconjugate Chem., Vol. 6,No. 4,1995 (31) Asseline, U.,and Thuong, N. T. (1988) Oligothymidylates Substituted with a n Acridine Derivative a t the 5’ Position, at Both the 5’ and 3’ Positions, or on a n Internucleotide Phosphate. Nucleosides Nucleotides 7, 431-455. (32) Asseline, U.,Thuong, N. T., and Helene, C. (1986) Oligonucleotides Substituted on the 5’ Position with an Acridine Derivative. Nucleosides Nucleotides 5, 45-63. (33) Asseline, U.,Toulme, F., Thuong, N. T., and HBlene, C. (1984) Oligodeoxynucleotides Covalently Linked to Intercalating Dyes as Base Sequence-specific Ligands. Influence of Dye Attachment Site. EMBO J . 3, 795-800. (34) Praseuth, D., Chassignol, M., Takasugi, M., Le Doan, T., Thuong, N. T., and Helene, C. (1987) Double Helices with Parallel Strands Are Formed by Nuclease-resistant oligo-[a]deoxynucleotides and Oligo-[a]deoxynucleotides Covalently Linked to a n Intercalating Agent with Complementary Oligo[PI-deoxynucleotides. J . Mol. Biol. 196, 939-942. (35) Sun, J., Asseline, U., Rouzaud, D., Montenay-Garestier, T., Thuong, N. T., and Helene, C. (1987) Oligo-[a]-deoxynucleotides Covalently Linked to an Intercalating Agent. Double Heleces with Parallel Strands Are Formed with Complementary oligo-ul-deoxynucleotides.Nucleic Acids Res. 15, 6149-6158. (36) Boidot-Forget, M., Chassignol, M., Takasugi, M., Thuong, N. T., and Helene, C. (1988) Site-specific Cleavage of Singlestranded and Double-stranded DNA Sequences by Oligodeoxyribonucleotides Covalently Linked to an Intercalating Agent and an EDTA-Fe Chelate. Gene 72, 361-371. (37) Asseline, U.,Thuong, N. T., and Helene, C. (1983) New Substances with High and Specific Affinity Toward Nucleic Acid Sequences. C. R. Acad. Sci. Paris 297, 369-372. (38) Gautier, C., Morvan, F., Rayner, B., Huynh-Ding, T., Igolen, J., Imbach, J.-L., Paoletti, C., and Paoletti, J. (1987) a-DNA IV: a-Anomeric and P-Anomeric Tetrathymidylates Covalently Linked to Intercalating Oxazolopyridocarbazole. Synthesis, Physicochemical Properties and Poly(rA) Binding. Nucleic Acids Res. 15, 6625-6641. (39) Bazile, D.,Gautier, C., Rayner, B., Imbach, J.-L., Paoletti, C., and Paoletti, J. (1989) a-DNA X: a and /3 Tetrathymidylates Covalently Linked to Oxazolopyridocarbazolium (OPC): Comparative Stabilization of Oligo /3-[dTl:Oligo and P-[dA] and Oligo a[dTl:Oligo /3-[dA] Duplexes by the Intercalating Agent. Nucleic Acids Res. 17, 7749-7759. (40) Letsinger, R. L., and Schott, M. E. (1981) Selectivity in Binding a Phenanthridinium-Dinucleotide Derivative to Homopolynucleotides. J . Am. Chem. SOC. 103, 7394-7396. (41) Kutyaven, I. V., Podyminogin, M. A., Bazhina, Y. N., Fedorova, 0. S., Knorre, D. G., Levina, A. S., Mamayev, S. V., and Zartova, V. F. (1988) N-(2-Hydroxyethyl)phenazinium Derivatives of Oligonucleotides as Effectors of the Sequencespecific Modification of Nucleic Acids with Reactive Oligonucleotide Derivatives. FEBS Lett. 238, 35-38. (42) Cazenave, C., Loreau, N., Thuong, N. T., Toulme, J.-J.,and Helene, C. (1987) Enzymatic Amplification of Translation Inhibition of Rabbit /?-Globin mRNA Mediated by Antimessenger Oligodeoxynucleotides Linked to Intercalating Agents. Nucleic Acids Res. 15, 4717-4736. (43) Zerial, A., Thuong, N. T., and Helene, C. (1987) Selective Inhibition of the Cytopathic Effect of Type A Influenze Viruses by Oligonucleotides Covalently Linked to a n Intercalating Agent. Nucleic Acids Res. 15, 9909-9919. (44) Toulme, J. J., Krisch, H. M., Loreau, N., Thuong, N. T., and HBlBne, C. (1986) Specific Inhibition of mRNA Translation by Complementary Oligonucleotides Covalently Linked to Intercalating Agents. Proc. Natl. Acad. Sci. U.S.A. 83, 1227-1231. (45) Sethi, V. S.,and Sethi, M. L. (1975) Inhibition of Reverse Transcriptase Activity of RNA-Tumor Viruses by Fagaronine. Biochem. Biophys. Res. Commun. 63, 1070-1076. (46) Sethi, M. L. (1985) Comparison of Inhibition of Reverse Transcriptase and Antileukemic Activities Exhibited by Protoberberine and Benzophenanthridine Alkaloids and Structureactivity Relationships. Phytochemitry 24, 447-454. (47) Sethi, M. L. (1985) Enzyme Inhibition VIII: Mode of Inhibition of Reverse Transcriptase Activity by Analogues, Isomers, and Related Alkaloids of Coralyne. J . Pharm. Sci. 74, 889-891.
(48) Sethi, M. (1979) Inhibition of Reverse Transcriptase Activity by Benzophenanthridine Alkaloids. J . Nut. Prod. 42,187196. (49) Sethi, V. S. (1976) Inhibition of Mammalian and Oncornavirus Nucleic Acid Polymerase Activities by Alkoxybenzophenanthridine Alkaloids. Cancer Res. 36, 2390- 1295. (50) Sethi, M. L. (1981) Screening of Benzophenanthridine Alkaloids for Their Inhibition of Reverse Transcriptase Activity and Preliminary Report on the Structure-activity Relationships. Can. J . Pharm. Sci. 16, 29-34. (51) Cheng, C. C., Engle, R. R., Hodgson, J. R., Ing, R. B., Wood, H. B., Jr., Yan, S.-J., and Zee-Cheng, R. K. Y. (1977) Absence of Mutagenicity of Coralyne and Related Antileukemic Agents: Structural Comparison with the Potent Carcinogen 7,12-Dimethylbenz[alanthracene.J . Pharm. Sci. 66, 17811783. (52) Harrison, I. T. (1990) Model 7924 Chromatotron Znstruction Manual, Harrison Research, 840 Moana Court, Palo Alto, CA. (53) Heimbel, A., and Bosold, F. (1990) The Chromatotron. Angew. Chem. 102, A298. (54) Hostettman, K., Hostettman, M., and Marston, A. (1988) Preparative Chromatography Techniques. Applications in Natural Products Isolation, Springer-Varlag, New York. (55) Pelletier, S. W. (1985) Advances in Medicinal Plant Reserch. In Advances in Medicinal Plant Research (A. J. Vlietinck and R. A. Domisse, Eds.) pp 153-195, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart. (56) Ishii, H., Chen, I.+., and Ishikawa, T. (1987) Basic Intramolecular Acylation. Synthesis of 2-Aryl-1-tetralones Bearing Isopropoxy or Benzyloxy Groups, Synthetic Intermediates for Phenolic Antileukemic Benzo[clphenanthridine Alkaloids, from 2,4-Diarylbutyric Acid Derivatives. J . Chem. SOC.,Perkin Trans. 1 2415-2420. (57) Stermitz, F. R., Gillespie, J. P., Amoros, L. G., Romero, R., Stermitz, T. A., Larson, K. A., Earl, S., and Ogg, J. E. (1975) Synthesis and Biological Activity of Some Antitumor Benzophenanthridinium Salts. J . Med. Chem. 18, 708-713. (58) Lawson, T. G., Regnier, F. E., and Weith, H. L. (1983) Separation of Synthetic Oligonucleotides on Columns of Microparticulate Silica Coated with Crosslinked Polyethylene Imine. Anal. Biochem. 133,85-93. (59) Fieser, M., and Fieser, L. (1967) Reagents for Organic Synthesis, Vol. 1, pp 914, John Wiley and Sons, Inc., New York. (60) Cushman, M., and Cheng, L. (1978) Total Synthesis of Nitidine Chloride. J . Org. Chem. 43, 286-288. (61) Wang, G., and Cushman, M. (1991) A Convenient Method fo the Conversion of a-Tetralones t o Aryl Acetates. Synth. Commun. 21, 989-996. (62) Ishii, H., Chen, I.-S., Ueki, S., Akaike, M., and Ishikawa, T. (1987) Studies on the Chemical Constituents of Rutaceous Plants. LXIV. Structural Establishment of Oxyterihanine, a Phenolic Benzo[clphenanthridine Alkaloid. Syntheses of Phenolic Benzo[clphenenthridine Alkaloids, Terihanine and Isoterihanine, and Related Compounds. Chem. Pharm. Bull. 35, 2717-2725. (63) Ishii, H., Chen, I.-S., and Ishikawa, T. (1987) Studies on the Chemical Constituents of Rutaceous Plants. Part 62. Efficient Synthesis of Fagaronine, and Phenolic Benzo[c]phenanthridine Alkaloid with Antileukemic Activity. J . Chem. SOC.,Parkin Trans. 1 671-676. (64) Cheng, R. K.Y., and Cheng, C. C. (1973) Synthesis of 5,6Dihydro-6-methoxynitidine and a Practical Preparation of Nitidine Chloride. J . Heterocycl. Chem. 10, 85-88. (65) Byrn, S. R., Carlson, D. V., Chen, J . K., Cushman, M. S., Goldman, M. E., Ma, W. P., Pidgeon, C. L., Ray, K. A., Stowell, J. G., and Weith, H. L. (1991) Drug-oligonucleotide Conjugates. Adv. Drug. Delivery Rev. 6, 287-308. (66) Chen, J.-k., Carlson, D. V., Weith, H. L., O’Brien, J. A., Goldman, M. E., and Cushman, M. (1992) Synthesis of an Oligonucleotide-IntercalatorConjugate in Which the Linker Chain is Attached Via the Phenolic Hydroxyl Group of Fagaronine. Tetrahedron Lett. 33, 2275-2278. BC9500397
