PL,\NT ,1T\'TICXXCER XGEN'X X. ISOLA4TIOSO F CX ;\I P T 0THE CI S XN D 9-11E TH OXI'CX 11P T 0 THE C I S FRO11 E R T'A T A 1111-4HE YA17EdAITA S.IR.ITHP. (;CS;\SEKER.I,
?\IOH.UIED 11.BADAKI,GEOFFREY -1.CORDELL and 1ORMAS I?,F.II:SSII-ORTH
Departnren: of Plioriiiacogiiosg iiiid Pliariiiacologg, College 0.f Phoriiincy, L'iikersiiy of I1li)iois rrt ilia M e d i c i i l Ceiiter, Chicago, Illiiiois 80612, I;.S..-l.
and (in p a r t j
~IASIK CHIISIS Iiidioii Caiicer Reseiirch Ceiiter, Tiitir X e m o r i a l H o s p i t a l , Boiiibay, Iiidio .IBSTR.iCT.-The anticancer activity of the roots of Er;otaniia h e y i e o ) i o i . 1 ~ 0 c p a c e a e ) was found t o be due principally t o the knov-n alkaloid, camptothecin ( 1 ) . T h e closely related, hiit less active, 9-methoxj-camptothecin 12) x a s also obtained. This is t h e first reported isolation of these biogenetically interesting alkaloids from t h e indole alkaloid rich - 1 p o c p a c e a e .
-\lcjueou$ alcoholic extracts of tlie \\-ood and item bark of Eri,ata?i?ia heyiieaiiu (Kall.) T. Cooke (-1pocpnaceae) exhibited activity' in the P-:3S5 l>-mpliocytic leukemia s>-atem iu 'i i ; o and Eagles carcinoma of the riasopharynx (KB),s\->teniin cell culture. Fractionation with conconlitant bioassay afforded several ac.ive alkaloids ( 3 ) . but tlie principal source of activity was traced t o a neutral conipoilent identified as carnptothecin (1). Chromatography of the mother liquor, after removal of 1. afforded 9-nietlios!-caniptotliecin (2). Tlie compounds were identified by comparison \\-itli autheiitic samples.
EXPERII\IESTA\L3 PL.ISTir.iTERI.iL.-The
plant material4 was collected in India in December 19iG.
EXTR-ICTIOS ISD ISITIAL FR.ICTIOX.ITIOS.-.~ sample (35 k g j of the x-ood and s t e m bark of E . heyneana was extracted successively with light petroleum and methanol; after evaporation i n i~aczio,t h e residues weighed 180 g and 960 g, respectively. T h e methanol extract was parti, and tioned between water (3 liters) and ethyl acetate (3 liters): after drying ( S a 2 S 0 4 )filtering, evaporating i n mciio t h e eth>-l acetate residue weighed 115 g. Separation of t h e alkaloid fraction from this material was accomplished by extraction with 55: tartaric acid (3XZOO m l ) ; and t h e neutral fraction, after evaporation, n-eighed 107.2 g. 'For t h e previous paper in this series see Ref. 1. ?Extracts, fractions and compounds n-ere tested under the auspices of the D r u g Research and Development Program of the S a t i o n a l Cancer Institute ( 2 ) . -In isolate is considered active if it s h o x s a T C value of greater than 1305; in the P-38s lymphocytic leukemia system in mice or an ED:,( of 4 / i g nil or less in the H B or P-388 test s!-stenis in cell culture. W e l t i n g points IT-ere determined using a Kofler hot plate and are uncorrected. T h e uv spectra were olitaiiied w i t h a Beckman model IIB-C; grating spectrophotometer. T h e ir .spectra -were ohiained with a Beelinmi model IS-.% spectrophotometer \Tit11 polystyrene P m r spectra were recorded in CDC1- o r DlIEO-ds solutions Lvith a ca~ihrariulla t 1691 em-'. \-arian mc'dpl T-62.1 instrriment, operating a t 60 3IHz \\-it11 a Sicolet: model TT-7, Fourier an internal standard and chemiral Transform a t t a c h m e n t , Tetraniethylsilane was lis .pectra were obtained ~ i t ah Hirachi shifts are reported i n 6 !ppm,i i l i i i l ? . Lo\\- reso~ilTion t e r operating a t TO ey. single-fociising spec Perkin Elmer. model Rl 'The plant material ppli.?d Throllgh the allspices Of The Ilriig Research and Development Program. Satioiinl Cancer Insritutej hl- the Economic Botany Laboratory. Plant Genetics :;nd Germplasm Institiire: Igriciiltiiral Research Service, C.S.D.-1.; Beltsville, 11D. -4 herliariiim specimen dociimenting this collection is deposited in t h e Herbarium of the S a t i o n a l .irhoretuni, .1gricultiiral Eesearch Service, C.S. Department of Igricultiire, K a s h ington, D.C. ~
475
JOURSAL OF SA4TUR-4LPRODUCTS
476
[VOL.
42, so. 5
~EP.~R.~TIO OFS THE S E E T R I L F R . ~ C T I O X.-The neutral fraction 1107 g) R-as chromatographed on a column of silica gel PF 254j (2 kg) packed in chloroform. Elution x-ith chloroformmethanol (982, and concentration yielded a green residue (1.1 g) which, n-hen rechromatographed on F l o r i d 6 (20 g) packed in chloroform, yielded crude camptothecin. Crystallization from chloroform afforded pale yellow crystals (15 mg, 0.00013%) of 1 having t h e folloning physical properties: mp 270-271' d , [c1]*~D+42.8"(c. 0.28, CHC13-hIeOH: 4 : l ) [Lit. (4) 268~42~ 4:1)]; ir, vmax (KBr) 3420, 3220, 2940, 2915, 1745, 1655, 1605, 270" d, [ c 1 ] ~ ~ D(CHC13-MeOH: 1585 and 1157 cmlj uv, Xmax (AIeOH) 368 (log e 4.341, 358 (4.34), 289 (3.81), 253 (4.51), and 218 nm (4.63): pmr, (ds-D3ISO) 6 0.89 (3H, t , J = 6 . 9 Hz,. C-18 H ) , 1.88 (2H, q, J=6.9 Hz, C-19 H): 5.28 (2H, s, C-5 H ) , 5.42 (2H, s, C-17 H ) , 6.49 ( l H , s,C-20 O H ) , 7.36 ( I H , s, '2-14 Hj, 7.68-8.31 (4H. m , C-9-12 H) and 8.66 (lH, s, C-7 H ) : ms, 7 y e hl-318 ilOO%,),319 (24), 304 (201, 291 ,(13), 275 (14), 261 (31, 248 (?l),219 (19), 205 (8), 191 (9), 178 ( 5 ) and 140 ( 4 ) . T h e compound was identical with an authentic natural sample kindlj- supplied by Drs. [Tall and Wani.
R, 1
N
2
3
d
is
R, H
R* OH
OCH, OH H OCOCH,
I"k5fo 2
0
.%CETIL.\TIOS OF CAMPTOTHECIS (1).-Camptothecin (1, 10 mg) was treated with pyridine ( 2 m l ) and acetic anhydride (0.1 ml) a t room temperature for 3 days. Work-up in t h e usual way follon-ed b y crystallization from hexane yielded acetyl camptothecin (3) a s pale yellow needles, nip 289-290" d , [ L i t . (4) 288-290" d]; ir, vmax (KBr) 3480, 1755, 1675, 1665, 1625 and 1235 cm-1: uv, Xmas (NeOH) 368 (log c 4.07), 356 (4.07), 288 (3.52), 252 (4.26) and 218 nm (4.12); pmr, (CDCI3)6 0.97 (3H, t , J = 7 . 3 Hz, C-18 H ) , 2.23 (2H, m , C-19 Hj, 2.31 (3H, s, -OCOCH,), 5.28 ( 2 H , s, C-5 H ) , 5.35 (lH, d , J=16.6 Hz, C-17 H ) , 5.73 (lH, d , J = l G . B Hz, C-liH), 7.25 (lH, s, C-14 H i l 7.67-8.38 (5H, m, C-7, 9-12 H ) : ms i i 7 ; e 31' 390 (65%), 330 (SG), 315 (501, 302 (loo), 287 (48), 275 ( 2 0 ) , 246 (E),219 (15))205 ( l a ) , and 191 (10).
ISOL.ITIOS A S D CH;\R.ICTERIZ.ITIOS OF ~-\IETHOXTC.IXIPTOTHECIS (2) .-The mother liquor, after crl-stallization of camptothecin, was chromatographed on a plate of silica gel. -1uv florescent band n-as separated and, on crystallization from chloroform-hexane (1:2), afforded campthothecin (2) (14 mg, 0.00004~c)having t h e following physical yellow cr?-stals of 9-meth properties: m p 258-259" jD-76.1' (c. 0.5, pyridine) [Lit. (7) mp 258-259", [ ~ ~ ] ? ~ D - i i . 5 ~ j ; ir, vmax ( K B r ) 3420, 2940, 1761, 1668, 1622, 1603, 1450, 1372, 1270, 1237, 1195, 1160, 1112, 1090, 1050 and 813 ern-'; uv, Xmax (3IeOH) 371 sh (log e 4.28), 366 (4.30), 320 14.05), 305 (3.85), 262 (4.38) and 218 nm (4.52):pmr, (CDC13) 6 1.03 13H, t , J = 7 . 4 H z , C-18 H), 1.91 (2H, q , J = ? A Hz, C-19 H ) , 4.05 (3H, s, C-9 OCH8), 5.25 (2H, s, C-5 H ) , 5.25 (lH, d, J = 1 6 . 1 Hz, C-li H ) : 5.76 (lH, d , J=lG.4 Hz, C-17 H ) , 6.93 ( l H , d d , J = 6 . 2 , 2.5 Hz, C-10 H ) , 7.61-7.77 (2H, m , C-11 and C-12 H ) , 7.74 ( l H , bd, C-20 O H ) and 8.78 (lH, s, C-7 H ) : ms, m , ' e 31' 378 (loo%), 350 ( l i ) , 349 (411, 334 (37), 321 (23), 319 (29), 305 (23), 278 (!l), 249 (17), 206 (19),205 (18), 139 (20), 103 (31) and 89 (10). The compound was identical with an authentic natural sample kindly supplied b y Dr. Suffness. BIOLOGIC.IL acTInTr.-Campt othecin (1,SSC-94600) showed TIC values of 181% and 1759% a t doses of 1.56 and 0.78 mg,'kg, respectively, in t h e P-388 lymphocytic leukemia test system (2) and was cytotoxic in t h e KB and P-388 t e s t systems in cell culture (ED60 0.17 and 0.53 pg/ml respectively).
DISCUSSIOS Camptothecin (l), an alkaloid derived biosynthetically ( 5 , 6) from an indole jE. l l e r c k , Darnistadt, W . Germany. 6Fisher Scientific Co., I t a s c a , 11.
.SEP-OCT
1!3i9]
GUSASEKERA ET
.a.:
PLAST ASTICASCER
AGESTS
4iT
precursor. n-as first isolated from the Cliin e tree Cavzptotheca aciimiiiata Decne. ceae) (4) am1 subsequently from oiiiapotiytes j o e f i d a (Kiglit) Sleunier (Icacinaceae) {(Tjsild Opliiovrhira vzzriigos Linn. (Rubiaceae) (S) . Besides having potent antileukemic and antitumor properties (4.9), caniptothecin also inliibits herpe: (I(!) and other maniiiialian viru*es (9). R-_\lrt!ios~-cam~~tothecin (2, SSC-I i 6 3 _ " 3 ) , fornierlj- obtained from S.joetitla ( i ) \\-as . not further e\-aluntecl biologjcally. but it has previoudJ- shou-n substantial the BIG nielanonia, L-1210 lymphoid leukemia. Len-is L ~ i carcinoma g 1)-mphocytic 1eul;emin trst ryrtenis in :'i:o at doses in the range 0.52 .r! nig kg ( 11 j . This i.5 the fii.t reported isolation of tlii!: alkaloid type from the Apocynaceae. A4CKSOWLEDG31ESTP This n-ork n-as supported by Contract S C I CLI-6TC90 froni t h e Division of Cancer Treatment, S i t i o n n l Cancer Institute, L7.S. Department of H e a l t h , Education, and Welfare. Bethesda, 3ID 20014. The authors ~vouldlike t o t h a n k Drs. Monroe Wall and 3Iakunh K a n i , Research Triangle Institute, for t h e provision of an autlieiiric sample of natural camptothecin, and D r . 11.Suffiiess, S C I , for a sample of 9-niethoxycamptotheciii and provision of biological d a t a pertaining t o this compound.
ReceiL'ed 26
Jli7rcli
1979.
LITERATTRE CITED 1. -1.1.Seida, .i. D. Kinghorn, G . *4.Cordell and S . R . Fanism-orth, J . -1-ot. Prod., 11,
2.
3. 4. 3.
6. 7. 8. 9. 10. 11.
584 (1978). R.L. Geran, S . H. Greenberg, 31.;\I. MacDonald, -4.11. dchuniacher, and B . J . .ihbott, Cuurer Cheiiiofhrr. Rep., 3121, 1 11972). ,S. P. C;iinasekera, G . -4.Cordell and S . 1:. F a r n s n o r t h , Pliyfociieviisiry, in press. 31. E . JTall, 31. C . TJ-ani, C . E . Cook. K . H . Palmer, -4.T . 31cPhail and G . -4.Sini, J . -4vier. Chew. Soc., 88. 3888 11966). G . .I.Cordell) Llojdio, 36, 219 11541. -4.H . Heckendorf and C . 1:. Hiitchinson, Tefroiiedroti L r i f . ,1 9 7 , 4153. T. E . Govindacliari and S . T-isn-annrha~i~ I ~ i d J. . Chain., lo! 453 (19T2) and Piiyioclieiiiis!rj, 11, 3.29 119721. 6 . Tafiir! .J. 11. Selson, D. C. DeLong and G . H . Svohoda! L l o j d i n , 39, 261 f l Y i G I . S. B . Horwitz in "A4ntihiotics. T-01. 111. 3Iechanism of action of antimicrobial and a n t i t n n i u r agents," .J. R.Corroran and F. E . Hahn, Eds., Springer T-erlag -4G, S e w l - o r k >S . l - . 19T5, p . 48. 1.Berker and U. Olshevsky, I s r . J . X c d . S c i . , 9, 1578 (1BT3). 31. Siiffness:.S a t u r a l Prodiicts Branch, S C I . private communication.
