380 Planta Med. 57(1991)

Cytotoxic Clerodane Diterpenes from Polyalthia ion gfoiia GengxianZhao', JeeH. Jung2, DavidL. Smith2, Karl V. Wood2, and JerryL. McLaughlin23 Visiting scholar from Nanjing College of Traditional Chinese Medicine, WHO. Nanjing Collaborative Center of Traditional Medicine, Nanjing, 210005, People's Republic of China 2 Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana 47907, U.S.A. Address for correspondence

Abstract

O 1516 OR

of the stem bark of Polyalthia longifolia Thw. (Annonaceae) has led to a novel clerodane diterpene, 16-

oxo-cleroda-3, 13(1 4)Z-dien- 1 5-oic acid, which was named polyalthialdoic acid (3). The bioassays also led to the previously known related diterpenes, kolavenic acid (2) which has not been reported as a constituent of this plant, and 1 6a-hydroxy-cleroda-3,13(1 4)Z-dien- 15,16olide (1) which is previously known to be in this plant. These structures were identified by chemical and spectroscopic methods. All three compounds (1—3) were significantly bioactive in the brine shrimp bioassay; they strongly inhibited the growth of crown gall tumors on

potato discs; and they were cytotoxic in three human tumor cell lines. These activities suggest potential antitumor applications. Compound 3 was the most active (ED50 values ca. 6 x 10_I ig/ml in the human tumor cell culture systems).

Key words

Polyalthia longfolia, clerodane diterpene, polyalthialdoic acid, kolavenic acid, cytotoxic agents.

Introduction In pursuing our interest in the antitumor constituents of the Annonaceae, we have examined the stem bark of Polyalthia longifolia Thw. (Annonaceae) for bioactive components. This plant is a commonly cultivated evergreen tree in India. Previous phytochemical work on Polyalthia species has led to the isolation of polyalthic acid (1), a labdane diterpene acid; aitholactone (2), a cytotoxic

phenyltetrahydrofurano-2-pyrone; proanthocyanidin tnmer (3); two zinc-containing compounds, zincpolyanemine and 2-mercaptopyridine N-oxide Zn salt, which showed antimalarial activity against Plasmodium berghei (4, 5); tetrahydroprotoberbenines (6); various aporphine alkaloids (6—11); a number of indolosesquiterpenes (7, 8, 12—18); and a few diterpenes based on the clerodane skeleton (19—

220

ii

Bioassay-directed chemical investigation 2

,CH3

1

10

9

8

H3

17

HCCH3

H3C

1 RH

2 R1

la R Ac

2a R'

3

R1

3a R

3

CH3,

R2 CH3 R2 CH3 R2 CHO

CH3, R2

CHO

21). We report the activity-directed isolation and identification of 1 6-oxo-cleroda-3, 13(1 4)Z-dien-1 5-oic acid (3), a novel clerodane diterpene which is named polyalthialdoic acid, in addition to two known related diterpenes kolavenic acid (2) and 1 6a-hydroxy-cleroda-3, 13(1 4)Z-dien-1 5,16olide (1). All of these compounds (1—3) exhibited significant inhibition of crown gall tumors and cytotoxicities against human tumor cell lines of A-549 (human lung carcinoma),

MCF-7 (human breast carcinoma), and HT-29 (human colon adenocarcinoma).

Materials and Methods The stem bark of Polyalthia longfolia Thw. (Annonaceae) was collected and dried in February—March, 1988; it was supplied commercially by United Chemical and Allied Products, 10 Clive Row, Calcutta-i, India.

instruments Melting points: Mettler FP5. uncorrected; optical rotation: Perkin Elmer 241 polarimeter; UV: Beckman DU-7 spectrometer; IR: Perkin Elmer 1600 FTIR spectrometer; 1H- and 13CNMR Varian VXR-500S and Chemagnetics A-200 spectrometers; low resolution MS: Finnigan 4000; high resolution MS: Kratos MS 50.

Bioassays The extracts, fractions and isolated pure compounds were routinely evaluated in a test for lethality to brine shrimp larvae (BST) (22) and assayed for inhibition of crown gall tumors on potato discs (23). In vitro cytotoxicity tests were per-

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Received: August 27, 1990

Planta Med. 57(1991) 381

Cytotoxic Clerodane Diterpenes from Polyalthia longfolia

formed at the Purdue Cell Culture Laboratory, Purdue Cancer Center, against A-549 (human lung carcinoma), MCF-7 (human breast carcinoma), and HT-29 (human colon adenocarcinoma) with adriamycin as a positive control.

Polyalthialdoic acid (3) White powder; m.p. 167—170 °C, [aI?: — 36.7° (C 0.0003 g/ml) abs. EtOH); UV A 268 nm (s 7591); JR (KBr) max cm1 3430 (OH), 2935, 2725, 1697 (C=O), 1636 (C=C). 1587, 1429,

1381, 1328, 1112; CIMS (isobutane) m/z (%): 319.2253 (50, MH,

Dried stem bark of P. longfo1ia (4.6 kg) was pulverized and extracted with 95% EtOH. The syrupy extract (702 g) (FOOl) was partitioned between CHC13/H20; the water solubles and the insoluble interface residue were labelled F002 (153 g) and F004 (144 g), respectively. The CHC13 soluble fraction (F003, 406 g) was then partitioned between 90% MeOH/hexane. The MeOH fraction was vacuum evaporated to give 347 g of dark residue (P005). The residue of the hexane extracts (F006) weighed 55g.

P005 (108 g), which was the more active of the two fractions derived from F003 (Table 1), was chromatographed on a silica gel column and eluted with a CHC13-MeOH gradient to give 57 fractions from which 10 pools were made on the basis of TLC analysis (silica gel, MeOH-CHC13, 1 : 8, sprayed with 2.5% phosphomolybdic acid in ethanol). The pools (5—8) most active in the brine shrimp assay (Table 2) were combined (total 38 g) and re-

calc. for: C20H3103: 319.2273), 301 (100, MH — H20), 289(1, M — CHO), 273 (1.4, M — COOH), 219 (1, M — C4H303), 205 (3, M — C5H503), 191 (7, M — C6H7O3), 117 (11); 99(36, C4H3O). ElMS m/z (%): 190 (4, C14H2), 121 (6). 107 (23). 95 (30), 93 (34), 91 (30), 79 (39), 67 (41), 55 (100), 53 (63); 1H-NMR (500MHz), CDC13); see Table 3; '3C-NMR (50MHz, CHCI3); see Table 4.

Lactonization of 3 Compound 3(6mg) in pyridine (2 ml) was treated with Ac20 (2 ml) and left at room temperature for 24 hours. The reaction mixture was poured into 2 ml of cold water and extracted with CHC13. The organic layer, after washing with H20, was dried to give a gummy residue, which was purified by chromatography over silica gel using hexane — CHC13 (1 : 2) to yield a lactonized com-

CHC13-MeOH gradients. Compound 1 (4.9 g) was crystallized from

pound. The lactonized derivative of 3 was identical with compound 1 on TLC [silica gel; CHCJ3-MeOH (95.5) and CHCI3-EtOAc (8 : 2); sprayed with 2.5% of phosphomolybdic acid in ethanol]. Spectral data confirmed this identification: IR (KBr) 'max cm1: 3343 (OH),

with hexane-CHCI3 (5 : 5) gave a gummy compound 2(35mg). Com-

(200MHz, CDC13) a: 5.99 (s, IH-16), 5.85 (s, 1H-14), 5.19 (s, 1H-3),

chromatographed over a silica gel column with hexane-CHC13,

fractions eluted with hexane-CHCJ3 (1.5:8.5). Fractions eluted pound 3 (50 mg) was obtained as a white powder from the CHC13MeOH (9: 1) fractions.

2920, 1738 (C=O), 1644 (C=C), 1444, 1133, 944; 1H-NMR 1.59 (s, 3H-18), 1.01 (s, 3H-19), 0.82 (d, 3H-17, J= 6 Hz), 0.78 (S. 3H-20).

Methylation of 3

Compound I

Treatment of 3 with CH2N2 yielded the methyl

Colorless needles, m.p. 100—101 °C, [a]2: —48° (c 0.0005 glml, abs. EtOH), UV A 211.5 nm (s = 3889); JR (KBr) Vmax cm1: 3323 (OH), 2923, 2872, 1728 (C=O), 1646 (C=C), 1446,

ester 3a as a colorless gum: JR (KBr) Vmax cm1: 2957, 2725 (CHO),

1385, 1333, 1190,1133,944; CIMS (isobutane): m/z(%) 319.2232 (22.6, MH, calc. for C20H3103: 319.2273), 301 (100, MH — H20), 283 (55), 219 (2, M* — C4H303), 205 (M — C5H5O3), 191 (24, M' —

CH3),

C6H703),

1728 (C=O), 1698 (C=O), 1639 (C=C), 1436, 1382, 1253, 1209, 1098, 1017, 876, 798; ElMS m/z (%): 332 (62, Mi, 317 (53, M — 301 (100, M — OCH3); 1H-NMR (200 Hz, CDCI3): see Table 3.

Results and Discussion

117 (15.8), 163 (5), 149 (8), 137 (13), 123 (17.6), 109

(10.6), 95 (34.7); 1H-NMR (500MHz CDC13): see Table 3; '3C-NMR (125 MHz, C6D6) see Table 4.

Solvent partitions (FOO2—F006) of the ethanol extract (FOOl) of the stem bark of P. long.folia were

Acetylation of I

tested for brine shrimp lethality, and the results are shown

A sample of 1 (20mg) was treated with Ac20/

in Table 1. Active fraction (FOO5) was subjected to silica gel

pyridine (1 : 1 at room temperature overnight. Workup in the usual

way afforded acetate la colorless needles; m.p. 171—172°C; JR

Table 1 BST bioassay results of partitions of the EtOH extract of P. longifolia.

(KBr)vmaxcm1: 2695, 1762 (C=O), 1648 (C=C), 1450.1375,1210, 982, 881; ElMS m/z(%): 360 (0.7, Mt), 301 (23, M — CH3COO), 300

Fraction No.

(32), 285 (100), 267 (9), 205 (2, M — C8H904),

C7H704),

189 (37), 175(10), 119(19), 107 (34), 95 (37).

Compound 2 EtOH); IV A

BST LC50 (ppm)

95% Confidence Intervals

46.5

29.8—73.3

191 (10, M —

Colorless gum; [a12: — 63.3° (c 0.0003 gJml abs. 237.5 nm (s = 3532.5); JR (Cd4) Vmax cm1: 2500—

FOOl (EtOH extract) F002 (H20 partition) F003 (CHCI3 partition) F004 (interface) F005 (aq. MeOH partition) F006 (hexane partition)

>1000

———

36

21.7—56.8

>1000

———

47.2

31.2—69.2

259

3500 (broad band, OH), 2932, 1690 (C=O), 1638 (C=C), 1436, 1382, 1252, 1171, 870, 787; CIMS (isobutane) nilz (%): 305.2456 (100, MH, calc. for C20H3302: 305.2481), 287 (32, MH — H20), 259 (21, M — COOH), 205(37, M — C5H502), 191 (12, M — C6H702). 155

Table 2 BST bioassay results of column fraction pools from the 90% MeOR extract (F005).

(20), 137 (30), 123 (19), 109 (25), 95 (36); 1H-NMR (500MHz,

Pools

BST LC50 (ppm)

95% Confidence Intervals

120.7

96.3—3562

CDCJ3): see Table 3; 13C-NMR (125 MHz, C6D6): see Table 4.

Met hylation of 2

1 2 3

>200

Treatment of 2 with CH2N2 yielded the methyl

4

25.0

ester 2a as a colorless gum; JR Vm (CCI4) cm1: 2928, 1720 (C=O), 1647 (C—C), 1456, 1438, 1379, 1224, 1149; ElMS m/z(%): 318(3, M*), 287(2, M — OCH3). 189(54, M — C7H1102), 149(11), 135(22), 119 (23), 107 (57), 95 (100); 1H-NMR (500MHz, CDC13): see Table

5

10.6 14.1

3.

13.9

6 7 8 9

31.9

10

>200

9.0 15.5

———

0.8—75.4 6.8—55.9 0—59.4 0.2—77.6 0.7—25.2 0.003—71.8 6.1—82.5 ———

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Isolation of diterpenes

382 Planta Med. 57(1991)

Gengxian Zhao et al.

Table 3 'H-NMR spectral assignments (ô H) of compounds 1, la, 2, 2a, 3, 3a. 1

la

2

2a

3

3a

2.05(m) 1.95(m) 5.20(br.)

2.09(m) 2.05(m) 5.20(br.) 1.98(m) 1.91(m) 5.69 (q,J= 1 Hz) 2.17 (d,J= 1Hz)

0.81 (d,J=7 Hz) 1.59 (d,J= 1Hz)

0.80 (d,J=7Hz) 1.57 (d,J= 1Hz)

0.81 (d,J=7Hz)

19

1.0 (s) 0.78(s)

0.98(5) 0.75(s)

1.0 (s)

20

2.06(m) 2.02(m) 5.19(br.) 1.98(m) 1.89(m) 5.64 (q,J= 1 Hz) 2.14(d,J= 1 Hz) 0.79(d,J—7Hz) 1.56 (d,J= 1Hz) 0.97(s)

2.07(m) 2.07(m) 5.18(br.) 2.54(m) 2.54(m) 6.44(s)

6.01 (s)

2.03(m) 1.92(m) 5.17(br.) 2.27(m) 2.07(m) 5.95(s) 6.82(s)

0.73(s)

0.71 (s)

2.12(m) 2.12(m) 5.20(br.) 2.52(m) 2.52(m) 6.40(s) 9.49(s) 0.84 (d,J=7 Hz) 1.52 (d,J= 1Hz) 0.98(s) 0.66(s) 3.79(s)

H

2

2' 3 12

12' 14 16 17

18

2.3 (m) 2.09(m) 5.84(s)

1.58(d,J— 1Hz)

-Me

3.66(s)

-OAc

9.51 (s)

0.80 (d,J=7 Hz) 1.56 (d,J= 1Hz) 0.97(s) 0.68(s)

2.16(s)

Proton signals for H-i, H-6, H-7, H.8, H-b and H-il are in the methylene envelope at = 1.2—1.8 ppm.

further supported by the typical fragments at n-lIz 219

'3C-NMR spectral assignments ( C) of compounds 1—3.

2

1

C 1

18.50

18.64

19.40

2 3 4 5 6 7 8 9 10

21.30

27.18

20.86

121.02 143.88

120.97 143.96 38.44 27.77 36.52 36.49 38.92 46.62 35.14 37.11 160.06 128.40 164.42 19.49 16.13 18.34 18.48 20.17

120.54 144.06 38.19

38.36 27.12 36.90 36.43 38.73 46.60

11

27.64

12 13 14 15 16 17 18 19

34.84 170.90 117.03 172.50 99.78 116.09 18.26 18.32 20.08

20

(C16H7), 205 (C15H5) and 191 (C14H) in their mass spectra which were formed by cleavages between the C-12/C-13, C11/C-12, and C-9/C-11 bonds, respectively.

3

Compound 1 (C0H300) was isolated as a major bioactive component from the stem bark. On the basis of 1H- and 1C-NMR analysis of I and its acetate la (see Tables 3 and 4), compound 1 was identified as 16a-

26.90 36.51 37.12 39.29 46.61 27.57 36.85 153.28 138.93 174.51 194.83

hydroxy-cleroda-3, 13(1 4)Z-dien-1 5,1 6-olide, which was previously reported from the leaves of the same plant and its structure was established by X-ray crystallography (19). The identity of compound 2 was established by comparison of its spectral data with those of compound 1, and the spectral data (Tables 3 and 4) of its methyl ester 2a with those described in the literature (24). Compound 2 has already been reported as kolavenic acid (16-methylcleroda-3,13(14)E-15-carboxylic acid) isolated from Hardwickia pinnata (24),

16.19 18.18

18.05 19.98

column chromatography and afforded ten pools. The BST bioassay (see Table 2) indicated that pools 5—8 possessed significant bioactivity, and a combination of these fractions

yielded the three clerodane diterpenes (1—3) upon rechromatography, directing each step with the BST bioassay. The 1H- and 13C-NMR spectra of these three compounds (Tables 3 and 4) were characterized by two tertiary, one secondary, and one olefinic methyl signals and the signals of a f3-substituted butene in the lowfield region,

suggesting the clerodane type of diterpenes. This was Bioassay Tests BSTLC50(ppm) (95% confidence interval) Potato disc bioassay (% inhibition of tumors) A-549ED50(i&g/ml) MCF-7ED50(.sgJmI) HT-29ED50(.tg/ml)

ment of the C-16 methyl group in compound 2 by an al-

dehyde in compound 3. The molecular formula of 3 (C20H0O) was deduced by peak matching for the MH in high resolution CIMS. The presence of the aldehyde group was indicated by the proton signal at = 9.51 ppm (16-H, 1H, singlet) and was confirmed by the carbon signal at ô 194.83 ppm (16-C). TheIR absorption band of the aldehyde at 2725 cm1 was also observed. When compound 3 was treated with acetic anhydride in pyridine, the product was identical with corn-

1

la

2

2a

3

4.9

NT

9.1

NT

6x101

(1.3—9.7)

3a Adriamycin NT

NT

NT

NT

(2 x 10_I —1.2)

(3.9—16.5)

83.2

NT

81.6

NT

2.97 2.15

>10

4.18

3.01 2.63

1.91

8.65

3.34 1.81 1.39

For descriptions of bioassays, see text.

NT not tested.

The structure of polyalthialdoic acid (3) was very close to that of 2. The only difference was the replace-

5.2x 10_i

95.2

6.84x10' 3.07 3.15x102 5.52x10' 3.43 7.44x102 7.53x 10_i

2.73 4.33x 10-2

Table 5 Cytotoxicities of compounds 1, la, 2, 2a, 3, 3a, and adriamycin (reference).

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Table 4

Planta Med. 57(1991) 383

Cytotoxic Clerodane Diterpenes from Polyalthia longfo1ia

References

pound 1 on TLC [silica gel; CHC13-MeOH 95 : 5, CHC13-EtOAc

(8 2); sprayed with 2.5% phosphomolybdic acid in ethanoll and in its IR and 1H-NMR spectra clearly indicating

that a lactonization had taken place in the a,f3-substituted butene moiety; this led us to deduce that a carboxyl group located at c-i 5 had reacted with the aldehyde group to form a lactone.

The presence of the carboxyl group was confirmed by treating compound 3 with CH2N2 to yield the

methyl ester, 3a. The 1H-NMR spectrum of 3a showed a characteristic methoxy peak at ó = 3.79 ppm (singlet, 3H). This was further supported by the IR spectrum of 3a in which the carboxyl OH absorption band disappeared, and

its carbonyl peak was hypsochromically shifted to 1698 cm1.

Gopinath, K. W., Govindachari, T. R., Parthasarathy, P. C., Viswanathan, N. (1961) Helv. Chim. Acta 44, 1040. Loder, J. W., Nearn, R. H. (1977) Heterocycles 7, 113. Agrawal, S., Misra, K. (1979) Curr. Sci. 48, 141. Han, K. Y., Hsu, P. H., Huang, Fl. P., Liu, M. C., Hsu. H. Y., Meng, L. N., Chen, C. L., Chu, T. Y. (1980) K'O Hsueh Tung Pao 25, 285. I-Ian, K. Y., Xu, B. X., Wang, X. P., Liu, M. Z., Xu, X. Y., Meng, L. N., Chen, Z. L., Zhu, D. Y. (1981) Huaxue Huebao 39, 433. 6 Guinaudeau, H., Ramahatra, A., Leboeuf, M., Cave, A. (1978) P1. Med. Phytoth. 12, 166. Hamonniére, M., Leboeuf, M., Cave, A. (1977) Phytochemistry 16, 1029. 2

Cave, A., Guinaudeau, H., Leboeuf, M., Ramahatra, A., Razafindrazaka, J. (1978) Planta Med. 33, 243. M. H. A., Shamma, M. (1982) J. Nat. Prod. 45,471. ° Zarga, Hamonnière, M., Leboeuf, M., Cave, A. (1971) C. R. Acad. Sci. Paris,

The compound 1 6-oxo-cleroda-3, 13(1 4)E-

dien-i5-oic acid was previously isolated and identified

from P. longfolia; and this E-isomer failed to be lactonized by chemical reaction (19). Considering that the bioactive compound, polyalthialdoic acid (3), isolated in our work, can successfully be lactonized, we assigned to compound 3 the Z-configuration at the C-13(14) double bond. From the above data, it was evident that compound 3 possessed the structure of 1 6-oxo-cleroda-3, 13(1 4)Z-dien-1 5-oic-acid and is a new product, isomeric to the known E-isomer.

Activities

12

13

14 15 16 17 18 19

No cytotoxic activities have been previously reported regarding the Polyalthia diterpenes, although antifeedant activity against castorlooper (A chaea janata) has been noted (19). As shown in Table 5, significant bioactivities were observed for compounds 1—3 in the brine shrimp lethality test, in inhibiting crown gall tumors on potato discs, and in the human tumor cell lines of A-549

20

(human lung carcinoma), MCF-7 (human breast car-

24

cinoma), and T-29 (human colon adenocarcinoma). The de-

rivatives (Ia, 2a, and 3a) were somewhat less active, suggesting that the hydroxy and carboxyl groups are important factors for their cytotoxicities. Compound 3 is the most active, showing nonselective cytotoxicities at about onetenth the potency of reference adriamycin.

Acknowledgements This investigation was supported by funds from the National Cancer Institute, NIH (Grant No. ROl CA 30909). The authors are grateful to the United Chemical & Allied Products Company. Calcutta, India for the collection of the plant material. Thanks are due to the Cell Culture Laboratory, Purdue Cancer Center, for the cytotoxicity data, and to Jon E. Anderson and Yu-hua Hui for determining 13C-NMR spectra.

21

22 23

Chem. 23, 423. Riche, C., Chiaroni, A., Dubois, G., Hocquemiller, R., Kunesh, N., Leboeuf, M.. Cave, A. (1980) Planta Med. 39, 206. Leboeuf, M., Hammonière, M., Cave, A., Kunesch, M., Wenkert, E. (1976) Tetrahedron Left. 355. Okorie, D. A. (1980) Tetrahedron 36, 2005. Okorie, D. A. (1981) Phytochemistry 20, 2575. Hasan, C. M.. Healey, T. M., Waterman, P.C., Schwalbe, C. H. (1982) J. Chem. Soc., Perkin Trans. 1,2807. Hoquemiller, R., Dubois, G., Leboeuf, M., Cave, A., Kunesch, N., fiche, C., Chiaroni, A. (1981) Tetrahedron Lett. 22, 5057. Kunesch, N., Cave, A., Leoeuf, M., Hoquemiller, R., Dubois, G., Gui!let, E., Lallemand, J.-Y. (1985) Tetrahedron Lett. 26,4937. Phadnis, A. P., Patwardhan, S. A., Dhaneshwar, N. D., Tavale, S. T., Guru Row, 1. N. (1988) Phytochemistry 27, 2899. Kijjoa, A., Pinto, M. M., Herz, W. (1989) Planta Med. 55, 209. Pinto, M. M., Kijjoa, A., Tantisewie, B., Herz, W. (1990) Proceedings of the 17th IUPAC International Symposium on the Chemistry of Natural Products, India, p. 288. Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E., McLaughlin, J. L. (1982) Planta Med. 45, 31. Ferrigni, N. H., Putnam, J. E., Anderson, B., Jacobsen, L. B., Nichols, D. E., Moore, D. S., McLaughlin, J. L. (1982) J. Nat. Prod. 45, 679. Misra, R., Pandey, H. C., Dev, 5. (1964) Tetrahedron Lett. 3751.

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Sèr. C. 278, 921. Johns, S. 11., Lamberton, J. A., Li, C. S., Sioumis, A. A. (1970) Aust. J.

Cytotoxic clerodane diterpenes from Polyalthia longifolia.

Bioassay-directed chemical investigation of the stem bark of Polyalthia longifolia Thw. (Annonaceae) has led to a novel clerodane diterpene, 16-oxo-cl...
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