J. Med. Chem. 1992,35,48664857
4854
N-Methylcarbamate Derivatives of Ellipticine and Olivacine with Cytotoxic Activity against Four Human Lung Cancer Cell Lines John C. RuckdeschelJp* Sandeep P. Modi,# Wageeh El-Hamouly,$Enrico Portuese,? and Sydney Archer**# Cogswell Laboratory, Chemistry Department, Rensseber Polytechnic Institute, Troy, New York 12180-3590, and Division of Medical Oncology, Albany Medical College, Albany, New York 12208 Received August 10,1992
A series of analogues of the antitumor alkaloids ellipticine and olivacine were tested for cytotoricity against four human lung cancer cell lines: H69, N417, H460, and H358. Adriamycin (doxorubicin), ellipticine, olivacine, and celiptinium were used as standards. Adriamycin was cytotoxic at 2 p M and celiptinium was inactive at the highest concentrations tested (ICs0 > 48 pM). N-methylcarbamates of 9-methoxy-6H-pyrido[4,3-blcarbazole 1-,5-, and 11-methanols gave ICs0 values ranging from 0.02 to 0.11 FM against N417, H460, and H358 and were only slightly less effective against H69. Introduction Following the report of Dalton et al.l on the clinical activity of ellipticine (1) and related compounds, there has been a continued interest in the development of therapeutially useful compounds related to ellipticineand olivacine (3). Congeners of these alkaloids are cytotoxic in several in vivo and in vitro systems.2 Earlier efforta were focused on analogues on 9-hydroxyellipticine (2h2t3 This emphasis was based on the hypothesis that 2, which is a metabolite of 1, is then oxidized further to a highly reactive quinoneimine which r e a h covalentlywith DNA.3 These studies led to the development of celiptinium (4): dateliptinium (6): and the 9-methoxy compound 6.s Archer and colleagues7proposed an alternate mechanism wherein it was suggested that ellipticine or ita metabolic product 2 was enzymically hydroxylated at the 5-methyl group to give 7, followed by enzymic esterification to furnish either the phosphate or sulfate ester 8 which then alkylated DNA. Although hydroxylation at other methyl groups such as C-11 in ellipticine or C-1 in olivacine was not discussed, there is no apparent reason why metabolic hydroxylationat these positions cannot occur. The major difference between the Auclair-Paoletti mechanism and that of Archer et al. is that the latter group considered + Division of Medical Oncology, Albany Medical College, Albany, NY 12208. *Present addrese: H. Lee Moffitt Cancer Center and Research Institute, P.O. Box 280179, Tampa,FL 33682-0519. 1 CogswellLaboratory, Chemietry Department, Rensselaer Polytechnic Institute, Troy, NY 12180-3590. (1)D$ton, L. K.; Demerac, S.; Elmes, B. C.; Swan, J. M.; Teitel, T. Synthesis of the Tumor-inhibitory Alkaloids, Ellipticine, BMethoxyellipticine and Related Pyrido[4,3-blcarbazolea. A u t . J. Chem. 1967, 20,2715-2727. (2) LePecq, J.-B.; Gosse, C.; Dat-Xuong, N.; Paoletti, C. Two New AntitumorDerivatives. l-Hydroxy-9-methyl-2-eUipticiniumAcetateand 9-Dimethyl-2,6-ellipticinium(chloride). Action on Lizio Leukemia of Mice. C. R. Acad. Sci. Paris 1975,281, 1365-1367. (3)Auclair, C.; Paoletti, C. Bioactivation of the Antitumor Drug 9-Hydroryellipticineand Derivativesby a PeroxikHydrogen Peroxide System. J. Med. Chem. 1981,24, 28S295. (4) Juret, P.; Tanguy, A.; Girard, A.; Letalaeu, J. Y.; Arbatucci, J. S.; DatrXuong,N.; LePecq, J.-B.; Paoletti, C. Preliminary Trial of 9-Hydroxy2-methylellipticinium (NSC 264137) in Advanced Human Cancers. Eur. J. Cancer 1978,14,205-206. (5) Kuayat, D.; Borel, C. H.; Merle, S.; Vu, C.; Myle, J. H.; Creton, R.; Oudart, S.;Bouloux, C.; Weil, M.; Piedbois, P.; Auclerc, G.; Thill,L.; Soubane, CI.; Jacquillat, C1. Phase I Studyof a New Ellipticine Derivative SR 961568 (Dateliptinium) Using a 24 Hour Continuous Infusion. Proc. AACR 1989,30,996. (6)Atassi, G.; Dumont, P.; Pepin, 0.; Gros, 0.;Groe, P. SR95325B, A New Ellipticine Derivative Highly Active Against Established Murine Solid Tumors. Proc. AACR 1989,30, 2458.
OO22-2623/92/ 1835-4854$03.00/0
P=Meth&ydlipti&
'
3. R=R?=CH,.R,=R,=H. Olivacine
that the key oxidation occurred at the methyl groups, whereas Auclair and Paoletti3considered oxidation of the 9-hydroxyl group to be of prime importance. Because of the possible chemical instability of esters such as 8, Archer et aL7prepared the N-methylcarbamate 9 as a surrogate for 8 for testing as an antitumor agent, a tactic that was used successfully in studies on the schistmomicide, hycanthone.8 The in vivo antitumor activity vs murine P-388 lymphocytic leukemia of 9 was superior to that of ellipticine 1 and the alcohol 7.' 9-Methoxyellipticine 2a along with ellipticine was isolated from the stems of Ochrosia acuminatas and in vitro callus cultures derived from the stems of Ochroda elliptica.lOJ1 In in vitro assays, 2a appears to be equally (7) Archer, S.; Ran, B. S.; Pica-Mattoccia, L.; Cioli, D. Synthesis and B i o l ~ P ~ ~ ~ e ~ o f S o m e 6 H - P y r i & [ J.Med. 4 , ~ bCkm. l~~l~ 1987,30, 1204-1210. Evidence, which tended to favor our mechanism over the Auclair-Paoletti mechanism, was the ineffectivenese of 9-hydroxy-6If-pyrido[4,3-blcarble 88 an antitumor agent in the P388 system. Chemically, this compound behaves like 9-hydroxyellipticine. Since it i devoid of both the 5- and 11-methyl groups, our m e c l " would predict such aa result while the Auclnir-Paolitti mechanirm would tend to suggest that it should be active. That biooxidationof the 5-methyl group in ellipticine in metabolically poesible wae shown by the transformation of ellipticine to 5-formylellipticine by Chohya ternata. Kouadio, K.; Rideau, M., Ganser, C.; Chenieux, J. C.; Viel, C. Biotranaformation of Ellipticine to 5-Formylellipticineby Choieya ternata. Plant Cell Rep. 1984,3,203-205. (8) Archer, S.; Pica-Mattoccia,L.; Cioli,D.; Seyed-Mdari,A.;Zayed, A.-H. Preparation and Antiechiatmod and Antitumor Activity of Hycanthone and Some of ita Congeners. Evidence for the Mode of Action of Hycanthone. J. Med. Chem. 1988,31,264-260. (9) Lin, Y. M.; Juichi, M.; Wu, R. Y.; Lee,K. H. Antitumor Agenta. LXM. Alkaloids of Ochrosia Acuminata. Planta Med. 1985,646-6. (10) Weber, J. F.; Gamier, S.; TAM, T. H.; Viel, C. Formation of Antitumoral Pyridocarbazole Alkaloids by In Vitro Cultures of Ochroein Elliptica Labill. Plant Med. Phytother. 1987,21,99-105. (11)Kouadio, K.; Creche, J.; Chenieux, J. C.; Rideau, M.; Viel, C. Alkaloid Production of Ochraia Elliptica Cell Suspension Cultures. J. Plant Phyniol. 1985,118,277-284.
@ 1992 American Chemical Society
Journal of Medicinal Chemistry, 1992, Vol. 35, No.26 4866
Cytotoxic Ellipticine and Olivacine Derivatives
Scheme I
I I
' 16
I
0
C6HSS02
C6H5S02
15
I N
17
C6H5S02
o
18
21
cytotoxic as ellipticine itself.12 Thus it seemed reasonable to prepare and test Q-methoxy-6H-pyrido[4,3bl carbazole derivatives such as 10, 14,and 26. Prediction of in vivo drug sensitivity has been the goal of several investigators. A bioassay developed by Salmon et al.13 has been a standard method for in vitro drug selection since 1978. More recently, Weisenthal et al.14 and Ruckdeschel and co-workers16 developed in vitro bioassayswhich appear to overcome some of the problems associatad with the Salmon assay. In this paper we report the synthesis of 9-methoxy-5methyl-6H-pyrido[4,3-b]carbazole-1l-methanol N-methylcarbamate (14)and in vitro bioassays of cytotoxicity of in four human lung cancer ten 6H-pyrido[4,3-blcarbazoles cell lines.I5 With the exception of 14, the syntheses of the other 6H-pyrido[4,3-blcarbazoleswere reported else~here.~J"'~ Chemistry A general synthesis of 5-methyl-GH-pyrido[4,3-b]carbazole-ll-methanol8 was reported earlier,17J8but the
preparation of 14 was not described previously. Ita synthesis is outlined in Scheme I. N-(Phenylsulfonyl)-5methoxyindole(15)was converted to ita lithio derivative with the aid of BuLi and then condensed with pyridine-3,4-dicarboxylicanhydride (16) to give a mixture of isomers 17 and 18 in which the former largely predominated. Treatment of 17, which waa obtained pure by fractional crystallization, with methylmagnesium bromide, afforded a mixture of the lactone 19 and the acid 20. Hydrolysis of 19 with NaOH followed by acidificationwith HC1 gave the lactone 21. Better yields of 21 were obtained by using the crude magnesium salt of 20. The lithio derivative of formaldehyde diethyl mercaptal was condensed with 21 and the unisolated intermediate was reduced with NaBH4 to give 11. Oxidation with bis(triflu0roacetoxy)iodoeobenzeneas described previ~usly~~J~furnished the aldehyde 12which, on reduction with NaBH4, afforded 13. Reaction of the latter with methyl isocyanate gave the target compound 14. Biological Results
(12) Meunier, G.;de Montauzon, J.; Bernardou, J.; Grassy, G.; Bonnafow, M.;Cros, 5.;Meunier, B. T h e Biooxidation of Cytotoxic The cytotoxicities of adriamycin (Doxorubicin), ellipEllipticineDerivatives: A Key toStructure-Activity RelationehipStudies? ticine (I),olivacine (31,celiptinium (41,and the newer Mol. Phortnacol. 1988,33,93-102. (13) Salmon, S. E.; Hamburger, L. W.; Soehnlein, B.; Quantitation of 6H-pyrid0[4,3-bI~arbezoles against four human l u g ~811Differential Sensitivityof Human-TumorStem Cella to AnticancerDrugs. cer cell lines are reported in Table I. Celiptiniumis active N. Engl. J. Med. 1978,298,1321-1327. (14) Weisenthal, L. M.; Lippmau, M.E. Clonogenicand Nonclonogenic in vivo against L1210 and P388 in mice2and waa reported In Vitro chemosensitivityh y . Cancer Treat.Rep. 1985,69,615632. (15) Ruckdemhel,J.C.;Camey,D.N.;Ole,H.K.;Ruseell,E.K.;Gazdar, to be clinically active in a limited number of patients in A. F. In Vitro Chemosensitivity of Human Lung- Cancer Cell Lines.Cancer a Phase I trial.lg In the bioassays reported here, the drug Treat. Rep. 1987, 71,697-704. was inactive against all four cell lines at the higheet (16) Modi, S. P.; McComb, T.; Zayed, A.-H.; Ogleeby, R. C.; Archer, S. Synthesisof GH-PyridoN,3-blcarkles. Tetrahedron ISSO,. 46,5555. concentration tested (ICs0 > 48 NM). A phase 11study in 5562. 43 patients with non-small cell lung cancer waa disap(17) Modi,S. P.; Carey, J. J.; Archer, S. Synthesis of &Methyl-6HPyrido[4,3-blcarb~le-ll-methanol. TetrahedronLett. 1990,41,58455848.
(18) Modi, S. P.; Michael, M.A.; Archer, 5.;Carey, J. J. An Efficient
Synthesisof C-11Subetituted6H-Pyrido[4,3-blcarboles. Tetrahedron
1990,47,6539-6548.
(19) E*, A. I.; Grella, R. J.; LeylandJonea, B. R; Kehn, D. P.; Cibes, I.; Lewis, E.; Greenberg, E. Phase I Study of Elliptinium (2-NMethyl-9-hydroxyelliptic~~). Cancer Znuest. 19M,S, 236-241.
Ruckdeschel et al.
4866 Journal of Medicinal Chemistry, 1992, Vol. 36, No.26
Table I. ICm Values for 6H-Pyrido[4,3-b]carbazolesin Four Human Lung Cancer Celle Linea
compound adriamycin 1, ellipticine 2,9-OH-ellipticine 4,celiptinium
R H OH
H H CHsO 14 CH3O 3, olivacine H 22b CHsO 23b CH30 24b CHaO 2Sb CHsO 26b CHsO a Reference 7. Reference 16. 7'
Y 1Ob
Ri
R2
R3
CH3 CH3
CHs CHs
H H
CH3 CH3 CH3 CHzOCONHCH, H H H H H H
CHzOH CHzOCONHCHs CHzOCONHCHs CH3 CHI CHs CHs CH3 CH3 CH3
H H H H CH3 CH3 CHO CHzOH CHzOAc CHzOCONHCHs
H69 0.87 1.80 1.70 >48.0 1.60 0.86
0.22 0.17 1.70 1.20 1.80 1.30 1.40 0.14
ICw OtM) H460
N417 0.23 0.87 3.60 >48.0 1.80 0.33 0.09 0.04 3.40 1.80 0.90 1.30 2.80 0.10
0.23 0.51 2.60 >4&0 0.95 0.17 0.06 0.02 1.80 2.10 1.60 1.90 1.70 0.07
H368 0.62 0.20
5.60 >48.0 1.60 0.19 0.10 0.09 2.30 2.10 1.60 1.30 2.00 0.11
Unless specified otherwise, for lithiation reactions, a three neck round-bottom flask equipped with a magnetic stirring bar, an inlet adapter for nitrogen or argon,and a rubber septum were used. All reactions were carried out in an atmosphere of nitrogen or argoninflamadriedgkware. THF wasdistibdoveraodium benzophenone ketyl before use. Methyl isocyanate and diieopropylaminewere dietilled before me. Low temperatumreactione were carried out in liquid nitrogen or dry ice-methanol cooling bathe. 3-Carbo.y-4-pyridyl l'-(Phenylaulfonyl)-V-"tho.y-2'indolyl Ketone and 4-Carbosy-3-pyridyl1'-(Phenylaulfonyl)-S'-metho.y-2'-indolyl Ketone (I7 and 18). A 2-L flask equipped with a mechanical stirrer was cooled to -70 O C and charged with a solution of 15.5 mL (11.2 g) of diisopropylamine in 100 mL of THF. Then 44.5 mL of a 2.5 M solution of BuLi in hexane was placed in the dropping funnel, and the solution was added dropwise over a period of 15 min. After stirring for 30mina solutionof 31.7g ofN-(phenyleulfonyl)-S-metho~dole in 80 mL of THF was added dropwise over a period of 20 min. The resulting clear yellow solution was stirred for 30 min during which time a yellow precipitate appeared. The reaction mixture was allowed to warm to -10 "C and stirred at that temperature for 30min. The eontenta of the flask were cooled to-100 O C , and a solution of 16.5 g of pyridina3,4-dicarboxylicanhydride in 100 Experimental Section mL of THFwad added dropwise. After the addition was complete, Adriamycin (doxorubicin) and ellipticine were obtained from the cooling bath was removed and the reaction mixture was commercial sources. Celiptinium was a gift from Dr. Bernard allowed to stir overnight. Meunier. Compoundsto be tested for cytotoxicitywere dissolved The resulting red-brownsolution was concentratedto dryness, in DMSO and diluted with 10 volumes of bovine serum albumin and the residue was treated with 1 L of cold HnO. The aqueous to give stuck solutions which were then diluted to proper solution was acidified to pH 2.0 with 10% HC1, stirred for 15 concentrations with a phosphate buffer. min, and fiitered. The filter cake was washed thoroughly with Meltingpoints were determined on a Mel-Tempopen capillary H10 and dried to give 41 g of a mixture of the isomericketo acide melting point apparatus and are uncorrected. 'H NMFt spectra which were readily separated by fractional crystallization from were obtained in the solvents indicated on a Varian &a00 (200 ethyl acetate. The less soluble isomer was the major product II: MHz) spectrometer. Chemical shifts are expressed in parts per mp 232-234 "C; IR (KBr) 3460, 1674, 1536, 1449,1356, 1220, milliondownfieldfromtetramethy~ilaneastheinte~stau~. 1172,1089,1032,726,684cm-l; N M R (DMSO-&) 6 9.10 (1 H, Infrared spectrawere recorded on a Perkin-Elmer Model 298 or a), 8.91 (1 H, d, J = 5.0),8.15-8.08 (3H, m), 7.74-7.67 (4H, m), adperkin-Elmer 1800 Fourier transform infrared speetropho7.55 (1 H, d, J = 5.0), 7.24-7.15 (3H, m), 3.77 (3 H, 8, O C b ) . tometer. Maas spectra were obtained on a Hewlett-Packard H P Anal. (CaHisNzOeS) C, H, N. 5987 GCIMS spectrometer usingisobutaue or methane as the CI The more soluble isomermelted at 246-247 O C , NMR (DMSOgas. Elemental analyses were performed by Atlantic Microhb, &) 6 8.92 (1 H, d, J = 5.0), 8.80 (1 H, a), 8.09-8.02 (3 H,m), Inc. Despite careful drying, some of the compounds retained 7.7*7.60(4H,m),7.19-7.18(3H,m),3.73(3H,s,OCHs).Anal. water tenaciously. Proton signals attributed to water were (C22HisNzOeS)C, H,N. observed in the NMR spectra. 1-(3-Corbosy-4-pyridyl)-1-[ 1'-(phenylaulfonyl)-S'-metho~y-2~-indolyl]ethano1 (20) and Ita Lactone 19. A stirred (20) Anderson, G.; Clavel, M.;Smyth, J.; Giacgone, G.;Gracia, M.; solution of 2.06 g of the keto acid 17 in 80 mL of THF wan cooled Planting, A. 5.;Dalesio, 0.; Kirkpatrick, A.; McVie, G. Phase I1 Study to 0 OC and treated with 8.1 mL of a 3.0 M solution of CH&igBr of %Hydroxy-2-methylEllipticiniumAcetate (Ellipticinium)in Patients in ether. After being heated under reflux for 24 h, the mixture with Advanced Carcinomaof the Lung,EORTCLung CancerCooperative was cooled in ice and acidified with diluted HBO4 to pH 1.0. The Group. Eur. J. Cancer Clin. Oncol. 1989,25,909-910. pointing. Celiptiniumshowed only one partial response,2° a result that could have been expected on the basis of the present resulta. 9-Hydroxyellipticine (2), which LePecq et al.2reported to be more effective than ellipticine in L1210 murine lymphocytic leukemia, was less active than 1 in three of the lung cancer cell lines and equiactive in a fourth. It was reported' that 7 was slightly more active than 1 in P388-infected mice and that the carbamate 9 was more active than either. Roughly the same order activity was observed in the four lung cancer cell lixies (Table 1). Since the 9-methoxy analogue 10 of the carbamate 9 was more cytotoxic than 9, only the 9-methoxyderivatives of N-methylcarbamate esters of the GH-pyrido[4,3-b1carbazoleC-1and C-11methanola26 and 14 were prepared. The trio, 10,14, and 26, were the most cytotoxic members of the series. Against N417,H460,and H358,the ICW values were in the 0.02-0.11 rM range and these compounds were only slightly less effective against H69.
Journal of Medicinal Chemistry, 1992, Vol. 36,No.26 4867
Cytotoxic Ellipticine and Olivacine Derivatives
solution was stirred at room temperature for 2 h and then S-Methoqrd-metthy1-6a-pyrido[4%b]carbarole- 1I-mathconcentrated in vacuo. The residue wan extracted with CHCb, anol(13). To an orange solution of 41 mg of the aldehyde 12 and the extract was washed with water, dried, and evaporated was added 110 mg of NaBH4. T h e suspension turned to a yellow to dryness. The residual oil was chromatographedon silica gel solution with a few minutes. After stirring at r c ” temperature using a gradient of ethyl acetate-hexane (20-60 % ). The lactone for 6 h, 40 mL of CHCla was added and the solution waa washed 19 was obtained as a foam which cryatallized from MeOH wt with HnO and brine and dried. On concentration, a yellow 750mg (37%);mp 203-206 OC; IR (KBr) 1776,1608,1476,1364, cryetalline solid separated, wt 26.6 mg (62%),mp 262-266 OC. 1206,1183,1089,1038,1016,927,809 cm-l; NMR (CDCla) 6 9.23 Evaporation of the fitrate gave an additional 16 mg (36%) (1 H, s), 8.86 (1 H, d, J = 6.0), 8.16 (1 H, d, J 9.2), 7.83 (1 H, suitable for use in the next step: IR (KBr) 3428, 1660, 1484, d, J = 7.41, 7.48-7.26 (6 H, m), 7.01 (1 H, dd, J 2.6, J = 9.21, 1401,1296,1220,1147,1036,806~m-’; NMR (DMSO-&) 6 11.26 6.89 (1 H, d, J 2.2). 6.79 (1 H, E), 3.81 (3 H, S, OCHs), 2.79 (3 (1 H, 8, NH),9.77 (1 H, 8, Hi), 8.40 (1 H, d, J 6.8 &),7.94 (1 H, 8, CHs); MS m/e = 436 (M + 1). Anal. (CzsHd~0Ss) C, H, N. H, d, J 6.0 It),7.89 (1 H, d, J 2.0 Hi& 7.49 (1 H, d, J = 8.6 Further elution of the column with ethyl acetatemethanol HT),7.19 (1 H, dd, J I2.0, J 8.7 &),6.56 (3 H, S, CHnOH, OH (10%)gave the hydroxy acid 20 mp 175-178 OC; IR (KBr) 3088 exchangeable with DzO), 3.89 (3 H, 8, OCHs),3.34 (e, HnO), 2.80 (br), 1702,1699,1478,1448,1369,1210,1179,1094,109,766,727, (3 H, S, CHs). Anal. (CieHiaN2029.26Hfi) C, H, N. 687 cm-l; NMR (DMSO-&) 6 8.76 (1 H, s), 8.32 (1 H, d, J 6.2), S - ~ h o ~ b m e t t h y l - 6 ~ - p y r i d ob]carbamlal [4~1-math7.93-7.89 (3 H, m), 7.59-7.43 (3 H, m), 7.12 (1 H, d, J = 2.4), 6.934.84 (3 H, m), 3.76 (3 H, 8, OCHa); MS m/e 436 (base peak, anol N-me thy la arb am ate^ (14). A mixture of 26 mg of 18,O.S M + l),452 (parent peak). mL of methyl isocyanate, 0.2 mL of pyridine, and 20 mL of dry 1-(3-Carboxy-4-pyridyl)-l-(S’-metho.y-2’-indolyl)et~CHCla (hydrocarbon stabilized)was stirred at room temperature no1 Lactone (21). A suspension of the crude magnesium salt of for 12 h. The clear solution was taken to dryness and the residue the hydroxy acid 20 and 0.7 g of NaOH pelleta in 76% ethanol chromatographed on a silica gel column using ethyl acetate aa was heated for 10 h. The resulting solution was concentrated, the developing solvent. The pure carbamate melted at 242-243 acidified to pH 2.0 with 20% HC1, and extraded with C H C L O C : wt 25 mg (84%1; IR (KBr) 3300,1686,1480,1260,1140,1130 CHsOH (10%). T h e dried extract was evaporated and the cm-I; NMR (DMSO-&) 6 11.72 (1 H, 8 , NH), 9.30 (1 H, 8, HI), residual foam was chromatographed on a silica gel column using 8.40 (1 H, d, J 6.0 Hs), 7.90 (1 H, d, J = 6.8, Hd,7.86 (1 H, an elution gradientof hexane-ethylacetate 10-60 % T h e lactone d, J m 2.0 Hie), 7.20 (1 H, d, J = 8.8 HT),7.10 (1 H, dd, J = 2.0, 21 crystallized after trituration with ether: wt 210 mg (36 % ); mp 193-196 OC; IR (KBr) 3827,1766,1606,1493,1467,1422,1366, J 8.7 He), 6.20 (2 H, 8, CHa), 3.83 (3 H, 8 , OCHs), 3.26 1276,1227,1206,1139,1026,914,861,793,726 cm-’; MS m/e (8, H a ) , 2.80 (3 H, S, CHa), 2.60 (3 H, 8, NCHs). Anal. 296 (M + 1); NMR (CDCb) 6 11.11 (1 H, a), 9.17 (1 H, e), 8.31 (C&14N303*0.6HzO) C, H, N. (1 H, d, J = 6.2), 7.74 (1 H, d, J = 6.0), 7.17 (1 H, d, J = 8.0), Cell Liner. All cell lines for cytotoxicity studies were 7.00 (1 H, d, J = 1.8), 6.74 (1 H, dd, J = 2.2, J = 9.0), 6.42 (1 H, graciously provided by Dr. A. G d a r of the NC1-Navy Medical 1.6), 3.71 (3 H, 8, OCHs), 3.33 (8, HzO), 2.10 (3 H, 8, CHa). d, J Oncology Branch and had been studied in earlier 8 8 8 a y ~of drug Anal. (Ci~HirNzOs.0.2H20)C, H, N. 9-Methoxy-S-methyl-6a-Pyrdo[4,3-b]carbarole-11-carsensitivity.i6 H69 is a classic small cell lung cancer (SCLC) cell boxaldehyde Diethyl Mercaptal (11). A solution of 14 mL of line and N417 is a ‘variant” SCLC line. H460 and H368 are 2.6 M n-BuLi in hexane was added to a hexane solution of 460 non-SCLCcelllineswithvaryingdegreesofseneitivitytostanderd mg of formaldehyde diethyl mercaptal at -56 OC. The solution chemotherapeuticagenta.16 Cella were maintainedin a 6 % C o p was stirred for 1 h while the temperature was allowed to rise to 96% air atmosphere at 37 O C and 85-100% humidity in RPMI -40 OC. The lactone 21 (130 mg) was added in one portion. T h e 1640 medium (Gibco Lab., Grand Island, NY). Mycoplasma colorless solution turned orangered and after 3 h became yellow. contamination was periodicallyevaluatedby the New York State Three millilitersof water was added,and the solvent were removed Department of Health Laboratories (Dr. Jerry Koratowki). in vacuo. The residue was dieeolved in 50 mL of EtOH, and 106 mg of NaBK was added. The mixture was heated under reflux Cytotoxicity Assay. Adherent cell linea (H460, H368) were for 6 h, cooled, and concentrated to small volume. Water was trypsinized and resuspended in RF’MI-1640/2% FBS prior to added, and the yellow insoluble solid was collected on a filter, counting. Floating cultures (H69, N417) were dieaggregated by washed with 2 X 2 mL of cold water, and dried, wt 70 mg (40%) gentle pipetting and collectedby sedimentation. Cella were plated of almost pure 11. Chromatographyof 30 mg of this material on onto half-well microliter plates (Corning) in concentratione a silica gel column using EtOAc-hexane as the eluant gave 28 mg previously determined to be optimal for aeeeeeing growth of pure 11: mp 222-224 OC; IR (KBr) 2926,1602, 1482, 1370, inhibition; 2600 cella/well for non-SCLC and 8OOO celle/well for 1217,1146,1043,803 cm-l; NMR (CDCb) 6 11.40 (1 H, 8, NH), 10.23 (1 H, S, Hi), 8.40 (1 H, d, J 6.4 Hs), 7.94 (1 H, d, J 8.8 SCLC cell lines. Twenty microliters of the appropriate drug H,), 7.79 (1 H, d, J = 2.4 Hlo), 7.56 (1 H, d, J 6.8 It),7.27 (1 solution or control were added to each well and the platee were H, dd, J = 2.0, J = 8.9 He), 6.57 (1 H, 8, (EtS)aCH) , 3.90 (3 H, incubated for 1 h, centrifuged, washed with RPMI 1640 twice, 8, OCHs), 3.34 (e, HzO), 2.79 (3 H, 8, CHd, 2.76 (4 H, m, (S(CH2and kept at 37 OC for 4 days. On the fourth day 50 aL of an CHs)2), 1.65 (6 H, t, (S(CH2CHa)d;MS m/e 397 (M + 1). Anal. aqueous solution of 5-(dimethylthiazol-2-yl)-2,3-diphenyltetra(CnH&.yOSfi.SHnO) C, H, N. 9-Methoxy-S-methyl-6H-pyrido[4,3-b]carba~ole-1l-car- zolium bromide (2 mg/mL) (Sigma Chemical Co., St. Louis,MO) was added and the plates were incubated for an additional 4 h. boxaldehyde (12). To a solution of 185 mg of the mercaptal 11 The plate were centrifugedand after removal of the supernatant, in 80 mL of CHsCN-H20 (91) was added 60 mg of bisthe purple dye remainingwas suspended in DMSO and the platee (trifluoroacetoxy)iodwbemnein one portion. After 1 hat room were shaken for 6 min. Absorbance at 540 nm waa determined temperature 50 mL of a saturated NaHCOa solution was added followed by CHaCN-MeOH (91). The layers were separated using a Biotek EL310 Microplate Autoreader. Appropriate and the organic layer was concentrated to dryness. Chromaconcentratione of each drug was testad to permit the calculation tography of the residue on a silica gel column using EtOAc of ICs0 values, i.e., the concentrationat which 60% inhibition of CHsOH (955) as the eluant furnished 57 mg (42%) of pure cellgrowth occurred. ICw valuea were calculated usingthe Crickorangered material, mp 290-291 OC,and 20 mg (18%)of darker Graph program. material suitable for use in the next step: IR (KBr) 3432,1673, 1599,1485,1384,1224,1036cm-l; NMR (DMSO-&) 6 1160 (1 H, 8, NH), 11.48 (1 H, S, Hi), 10.35 (1 H, 8, CHO), 8.62 (1 H, d, Acknowledgment. This investigation wm supported J 6.0 Ha), 8.10 (1 H, d, J = 2.4 Hio), 8.03 (1 H, d, J = 6.0 Hd, in part by grantafrom the National Cancer Inatitute (S.A.), 7.53(1H,d,J=9.0H7),7.29(1H,dd,J=2.4,J=8.8,He),3.89 t h e National Institutes of Health (J.C.R.), and the Potta (3 H, S, OCHs), 3.34 (8, HzO), 2.86 (3 H, S, CHa). Anal. Foundation (J.C.R.). (CieHirNzOTO.SHz0)C, H, N.
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