Rotenoids and chalcones from Mundulea sericea that inhibit phorbol ester-induced ornithine decarboxylase activity

0031-9422(94)E0153-J

Pergamon

Phytorhemuwy. Vol. 36. No 6. pp. 152%1524 1994 Copyright ‘(,^: 1994 Elscvier Scraa Ltd Pnnkd I,, Great Bnmin. All rights resewed 0031.9422/94 S7lW+O.M

ROTENOIDS AND CHALCONES FROM MUNDULEA SERZCEA THAT INHIBIT PHORBOL ESTER-INDUCED ORNITHINE DECARBOXYLASE ACTIVITY LUMONADIO LUYENGI, IK-SOO LEE, W~~NGCHON

MAR, HARRY

H. S. FONG, JOHN M. PEZZUTO and A. DOUGLAS

KINGHORN* Program for

Collaborative Research in the Pharmaceutical College

of Pharmacy,

University

Sciences,

(Receioed

Key Word hdex-Mundulea

inhibitory

Department

of Illinois at Chicago, I December

of Medicinal

Chicago,

Chemistry

and Pharmacognosy,

IL 60612, U.S.A.

1993)

sericea; Leguminosae; bark; rotenoids; chalcone; omithine decarboxylase

activity.

Abstract-Two novel rotenoids, ( - )- 13a-hydroxydeguelin and ( - )- 13a-hydroxytephrosin, and a new chalcone, munsericin, were isolated from the bark of Mundulea sericea, and their structures were elucidated by spectroscopic methods. Also obtained were the parent rotenoids, deguelin and tephrosin. and the known chalcone, 4-hydroxylonchocarpin. The rotenoids and chalcones exhibited potent inhibitory activity against phorbol ester-induced ornithine decarboxylase activity in cultured mouse 308 epidermal cells.

INTRODUCllON

Mundulea sericea (Willd.) A. Chev. (syn. M. suberosa

Benth.) is a legume shrub that is widely distributed in central and southern tropical Africa and parts of India Cl]. The bark, leaves, seeds and roots of this species are used as a fish poison [2], insecticide [3], and an aphrodisiac 143. Previous phytochemical investigations on this plant have resulted in the isolation of rotenoids [S], flavanones [6], isoflavanones [S], chalcones [63, and an imidazole derivative [7]. As part of our search for novel naturally occurring cancer chemopreventive agents, we have reinvestigated the chemical constituents of M. sericea, since an ethyl acetate-soluble extract of the bark was found to markedly inhibit phorbol ester-induced omithine decarboxylase (ODC) activity in cell culture. Bioassay-guided fractionation of the ethyl acetate extract led to the isolation of several active compounds, of which three are new constituents, (- )- 13a-hydroxydeguelin (1). ( - )- 13a-hydroxytephrosin (t), and a chalcone, munsericin (3). These were accompanied by the structurally related compounds, deguelin (4), tephrosin (5). and 4-hydroxylonchocarpin (6). The present work deals with the structure elucidation of the three new active compounds, l-3. RESULTS AND DISCUSSION ( -)-13z-Hydroxydeguelin (1) was assigned a molecular formula of CZsH,,O, from its HR-mass spectral data

*Author

to whom correspondence

should

be addressed.

(m/z 410.1359). Comparison of its UV, IR, ‘H, “C NMR and EI-mass spectra with those of deguelin (4) [8] indicated that I was a hydroxylated deguelin derivative. The position of the hydroxyl group was deduced as being attached at C-13, since in the ‘HNMR spectrum of 1, there was an absence of the C-13 methylene protons appearing at 64.17 (d, J = 12.0 Hz) and 64.82 (dd, J = 12.0, 3.1 Hz) in 4. Similarly, in the carbon spectrum of 1, there was a lack of the methylene carbon signal expected at about 666 for all rotenoids such as 4 whose C-13 position is unsubstituted [9]. In contrast to 4, additional resonances at 65.81 (d, J = 1.8 Hz) and 690.0 were apparent in the ‘H and i3CNMR spectra of 1. Further evidence for the location of the hydroxyl group at C-13 was obtained in a selective INEPT NMR experiment, in which signal enhancements at chemical shift values corresponding to C-7a (640.4) and C-l la (6 144.5) were observed by irradiation (35cu=6.0 Hz) of H-13 (65.81). This was supported by a *H-‘%Y correlation via a long-range coupling (COLOC) NMR experiment which indicated three-bond couplings between H-13 and C-lla, and between H-13 and C-7a. The small coupling constant of 3.8 Hz between H-7a and H-13a and the similarity of the CD maxima of I to those of 4 supported a cis B/C ring fusion and a (7aS, 13aS) absolute configuration for 1. Moreover, the small coupling constant of 1.8 Hz between H-13 and H-13a suggested a /?-configuration for H- 13 and, therefore, an aconfiguration for the hydroxyl group attached to C-13. Further supportive data were obtained from molecular mechanics calculations. Thus, a preferred energy-minimized structure of 1, generated via computer by a molecular modelling program, indicated a value of 50.67” for the

1523

1524

L. LUYENGI

Rx=Otf

1

R1=H,

2

R1=Rz=OH

4

R,=Rz=H

5

R,=OH,

Rl=H

OH

RI=& Rz=OH 6 R,=OH, R2=H

3

dihedral angle between the two protons H-7a and H-13a, and 58.85” for the angle between H-13 and H-13a, consistent with the small coupling constants of 3.0 and 1.9 Hz, respectively, that were observed in the ‘H NMR spectrum. Compound 1 was. therefore, identified as (-)13~-hydroxydegueIin. A molecular formula of Cr3H,,0s was assigned to a second novel rotenoid (2) from its HR-mass spectral data (m/z 426.1303). Comparison of its UV, IR, ‘H, 13C NMR and EI-mass spectral data with those of 1 indicated that 2 had an additional hydroxyl group. From the ‘H-i3C HETCOR spectrum of 2, it was concluded that the additional hydroxyl group was located at C-7a because ofthe downfield shift of the quaternary C-7a signal in the 13CNMR spectrum (from 640.4 in 1 to 668.4 in 2) and because of the absence of the H-7a signal in the “H NMR spectrum. The similarity of the CD spectrum of 2 to that of 1 suggested a (7aS, 13aS) absolute configuration and a cis B/C ring fusion. The coupling constant of 1.8 Hz between H-13a and H- 13 supported a /3-configuration for H-13 and an x-configuration for the hydroxyl group attached to C-13. Thus, compound 2 was identified as (-)-13a-hydroxytephrosin. Based on its HR-mass spectral data (m/z 322.1217), a molecular formula of C,,H, ,O., was assigned to compound 3. Analysis of the UV, IR, ‘H, “CNMR and EI-

et

al.

mass spectral data of 3 showed some resemblance to the spectral data obtained for the known chalcone. Qhydroxylonchocarpin (6) [lo], which was also isolated from M. sericeu bark in this investigation. The ‘H NMR spectrum of 3 exhibited a singlet integrating for six protons corresponding to two uncoupled methyl groups at 6 1.45, and evidence for a cis-ethylenic moiety at 65.67 and 6.34 (d, J= 10.0 Hz). The two uncoupled methyl groups and the cis-ethylenic moiety represented a gem-dimethyl pyran ring. Also apparent from the ‘HNMR spectrum were protons of an enone functionality occurring at 67.40 and 7.82 (d, J = 13.0 Hz) for H-z and H-8, respectively, as well as a 1,4,6_trisubstituted aromatic ring in which proton signals were observed at 66.43 (2H, m) and 67.82 (lH, d, J =8.1 H~)(ring B). In addition, a 1,3,4-trisubstituted aromatic ring was evident with two ortho-coupled protons at S6.80 and 7.42 (1H, d, J = 8.3 Hz), and a third proton at S 7.26 meta-coupled with the proton resonating at 67.42 (lH, d, J=l.l Hz) (ring A). The ‘%ZNMR spectrum of 3 displayed 20 carbon peaks, composed of three ahphatic and 17 aromatic signals, which included 10 methine and seven quaternary carbon atoms. All proton and carbon correlations were established by means of a ‘H-13C HETCOR NMR experiment. By comparison of the ‘HNMR spectrum of compound 3 to that of 4hydroxylonchoca~in (6), it was apparent that 3 did not have a monosubstituted paru-hydroxybenzyl ring, which was clearly evident in the ‘H NMR spectrum of 4hydroxylonchocarpin (6) from a pair of two protondoublets (J=8.6 Hz at 66.87 and at 7.55). This fact suggested that in 3, C-2’ was unsubstituted and ring B contained two phenolic functions. The downfieid chemical shifts of the two phenolic groups (6 164.0 and 166.2) suggested that they were in ortho- and puru-positions, respectively [93. This was confirmed by the upfield shift of C-l (6 114.2). Moreover, selective INEPT and COLOC NMR experiments supported the position of the two phenolic groups in ring B. Thus, irradiation (3Jcn = 8.0 Hz) of H-5 enhanced the carbon signals at b 166.2 (C-4) and fi 164.0 [C-6) (two-bond coupling), S 108.0 and 114.2 (C-3 and C-l, respectively; three-bond coupling). Similar irradiation t3Jci., =8.0 Hz) of H-P enhanced the carbon peaks at S 192.0 (C-B’) and 6 164.0 (C-6). Also, irradiation (3Jc- = 8.0 Hz) of H- 1” gave enhancements at S 156.3 (C-4’), 678.0 (C-3”) and b 126.7 (C-2’), confirming that the gem-dimethylpyran ring was connected to ring A. A 1H-‘3C COLOC NMR experiment supported this concept, with correlations between C-3’ and the protons H-l”, H-2’ and H-S’ being observed. Therefore. this novel chalcone, munsericin, an isomer of 6, was assigned as structure 3 (3”,3’‘-dimethylpyrano[3‘,4‘]4,6-dihydroxy” chalcone). Compounds 4-6 were identified as the previously known compounds, deguelin, tephrosin, and Chydroxylonchocarpin. respcctiveiy. by comparison of their spectral data with those of corresponding compounds reported in the literature [8-lo]. Unambiguous ‘H and 13C NMR data were obtained for the first time for 4-6 in the present investigation. Compounds 1 6 displayed significant inhibitory activity against phorbol ester-in-

Constituents of Mundulea sericea

duced ornitnine decarboxylase activity in cell culture as exemplified by IC,, (pg ml- ‘) values of 0.004, 0.02, 1.0, 0.0003, 0.001 and 0.7, respectively.

1525

6), 143.9 (s, C-9), 144.5 (s, C-l la), 149.2 (s, C-lo), 157.0 (s, C-14a), 160.6 (s, C-4a), 189.6 (s, C-7). EIMS 70 eV, m/z (rel. int.): 410 CM]’ (33), 392 (13), 377 (14), 363 (12), 208 (lOO), 203 (41), 191 (13), 187 (45), 77 (28). HR-EI-MS found: 410.1359 [Ml+; C,3H,20, requires: 410.1365. EXPERIMENTAL (-)-13a-Hydroxytephrosin (2). Crystals from MeOH, General. Mps: uncorr., CD: CHCI,, ‘H and mp 177-180”. [a];’ - 25” (CHCl,; c 0.10). UV A”,::” nm “C NMR: TMS int. standard. EI-MS: direct inlet system, (log E): 212 (4.30), 235 (4.35), 250 (4.35), 270 (4.40), 300 70eV. TLC: silica gel 60 F,,, plates, sprayed with (4.00), 315 (4.00). CD Ae (CHC13) (nm): -93.7 (332). IR 10% v/v H,SO,; 1lo”, 10 min. Molecular modelling, v!&: cm-‘: 3433,2933,1674,1599,1577,1510,1444,1273, HyperChemRA, Autodesk, Inc. 1113,1087,744. ‘H NMR (300 MHz, CDCI,): 6 1.39 (3H, Plant material. The bark of Mundulea sericea was s, Me-3), 1.45 (3H, s, Me-3), 3.71 (3H, s, OMe-lo), 3.81 collected in Nairobi, Kenya, in 1976. A voucher specimen (3H, s, OMe-9), 4.74 (lH, s, H-13a), 5.57 (lH, d, J has been deposited in the Medicinal Plant Resources = 10.0 Hz, H-2), 5.79 (lH, d, J= 1.8 Hz, H-138), 6.49 (lH, Laboratory, Plant Genetics and Germplasm Institute, d,J=lO.OHz,H-1),6.53(1H,d,J=8.7Hz,H-5),6.55(2H, USDA, Beltsville, Maryland 20705. s, H-8, H-11), 7.74 (lH, d, J=8.7 Hz, H-6). 13CNMR Extraction and isolation. The bark of Mundulea sericea (75.4 MHz, CDCl,): 628.3 (q, Me-3), 28.5 (q, Me-3), 55.9 (5 kg) was exhaustively extracted with MeOH (40 I) and (q, OMe-lo), 56.3 (q, OMe-9), 68.4 (s, C-7a), 75.2 (d, Cthe resultant extract was defatted with petrol (500 ml x 3). 13a), 78.2 (s, C-3), 92.3 (d, C-13), 102.4 (d, C-11), 107.6 (s, This MeOH extract was later partitioned between H,O C-7b), 108.2 (s, C-14b), 109.1 (d, C-5), 111.3 (s, C-6a), 112.3 and EtOAc to give, on evapn in uacuo, 83 g of EtOAc(d, C-8), 115.6 (d, C-l), 128.5 (d, C-6), 129.1 (d, C-2), 144.3 soluble residue. This extract exhibited inhibitory activity (s, C-9), 145.1 (s, C-lla), 151.6 (s, C-lo), 156.1 (s, C-14a), when evaluated against an ornithine decarboxylase 161.5 (s, C-4a), 189.8 (s, C-7). EIMS 70 eV, m/z (rel. int.): bioassay (see below), and was fractionated by CC over 426 [M]’ (26) 224 (99), 209 (11), 203 (77), 181 (98), 180 silica gel (1.5 kg), eluting with CHCl, and increasing (39), 153(20), 143(26). HR-EI-MS found: 426.1303, [Ml’; amounts of MeOH (5-100%). Fractions 3,4,5 and 7 were C,3H,20, requires: 426.1314. the most active, and each was subfractionated by flash Munsericin (3). Yellow crystals from MeOH, mp 160”. CC over silica gel, eluting with different solvent systems UV 1:::” nm (log E):208 (4.45), 230 (4.40), 290 (4.30), 375 to give six active compounds. Thus, flash CC (300 g of (4.50). IR ~2:: cm - ‘: 3200, 2955, 1737, 1631, 1566, 1484, silica gel) of fr. 3 in hexane-CHCl, (19: 1) produced 1460, 1363, 1248, 1226, 1130, 1040. ‘HNMR (300 MHz, deguelin (4) (250 mg, 0.0050% w/w), and tephrosin (5) CDCI,): 6 1.45 (6H, s, Me-3” x 2), 5.67 (lH, d, J = 10 Hz, (220 mg, 0.044% w/w), while flash CC (150 g of silica gel) H-2”), 6.34 (lH, d, J=lOHz, H-l”), 6.43 (lH, d, .I of fr. 4 developed in hexane-CHCl, (9: 1) and further =8.1 Hz, H-3), 6.55 (lH, s, H-5), 6.80 (lH, d, J=8.3 Hz, purification by prep. TLC using the same solvent system H-S), 7.26 (lH, d, J=l.l Hz, H-2’), 7.40 (lH, d, J resulted in the isolation of 4-hydroxylonchocarpin (6) = 13.0 Hz, H-a), 7.42 (lH, dd, J-8.3, 1.1 Hz, H-6’), 7.82 (40mg, 0.0008% w/w). Similar flash CC of fr. 5 using (lH, d, J=8.1 Hz, H-2), 7.82 (lH, d, J=l3.0Hz, H-/I). CHCI, as solvent and purification using prep. TLC in i3CNMR (75.4 MHz, CDCl,): 628.2 (q, Me-3” x 2), 78.0 hexane-EtOAc (19: 1) afforded munsericin (3) (35 mg, (s, C-3”), 103.7 (d, C-5), 108.0 (d, C-3), 114.2 (s, C-l), 116.9 O.o007% w/w). Finally, flash CC (200 g of adsorbent) (d,C-5’), 117.4(d,C-a), 121.4(s,C-3’). 121.6(d,C-1”), 126.7 using CHCI, as solvent and prep. TLC of fr. 7 developed in (d, C-2’), 127.5 (s, C-l’), 130.1 (d, C-6’), 131.4 (d, C-2”), hexane-EtOAc (17: 3) led to the purification of ( - )-132131.9 (d, C-2), 144.7 (d, C-/l), 156.3 (s, C-4’), 164.0 (s, C-6), 166.2 (s, C-4), 192.0 (s, C-p). EI-MS 70 eV, m/z (rel. int.): hydroxydeguelin (1) (60 mg, 0.0012% w/w) and (-)-13~ 322 [M]’ (20), 308 (24). 307 (lOO), 171 (59), 115 (33), 77 hydroxytephrosin (2) (30 mg, 0.0006% w/w). (21). HR-EIMS found: 322.1217 [Ml’; C,oH,80, re(-)-13wHydroxydeguelin (1). Yellowish crystals from quires: 322.1205. MeOH, mp 179”. [a];’ - 14” (CHCI,; ~0.13). UV 1::” Deguelin (4). Yellowish crystals from MeOH, mp nm (log E):210 (4.20), 235 (4.30), 250 (4.30), 270 (4.30), 295 (4.00), 315 (4.00). CD AE (CHCI,) (nm): -69.6 (310). 180-182’, lit. 171” [ll]. [a];’ - 107” (C,H,; ~0.2) [lit. 1597, -97” (ll)]. UV iz:F” nm (log E): 208 (4.50), 219 (4.30), + 30.2 (355). IR v;!: cm - ‘: 3428,2974,2935,1672, 236 (4.33), 270 (4.38), 296 (4.10), 314 (4.00). IR ~2;’ cn- ‘: 1514, 1442, 1394, 1340, 1271, 1215, 1099, 756. ‘HNMR (300 MHz, CDCl,): 6 1.35 (3H, s, Me-3), 1.43 (3H, s, Me- 2935, 1674, 1606, 1512, 1456, 1346, 1273, 1215, 1113. ‘H NMR (300 MHz, CDCI,): 61.36 (3H, s, Me-3), 1.45 3), 3.73 (3H, s, OMe-lo), 3.74(3H, s, OMe-9). 3.95(lH, d,J =3.8 Hz, H-7a), 4.79 (lH, m, H-13a). 5.52 (lH, d, J (3H, s, Me-3), 3.74 (3H, s, OMe-lo), 3.77 (3H, s, OMe-9), 3.83(1H,d,J=4.0Hz,H-7a),4.17(1H,d,J=12.0Hz,H= 10.0 Hz, H-2), 5.81 (lH, d, J= 1.8 Hz, H-13&, 6.42 (lH, 13~),4.82(1H,dd,J=12.0,3.1Hz,H-13a),4.92(1H,brs, s, H-11), 6.43 (lH, d, J=8.7 Hz, H-5), 6.57 (lH, d, J =lO.OHz,H-1),6.77(lH,s, H-8), 7.75(lH,d,J=8.7 Hz, H-13a), 5.55 (lH, d, J=9.8 Hz, H-2), 6.45 (lH, d, J HI H-6). 13C NMR (75.4 MHz, CDCI,): 628.1 (q, Me-3), 28.4 =8.6Hz, H-5), 6.45(lH,s, H-11), 6.64(lH,d,J=9.8 H-l), 6.79 (lH, s, H-8), 7.51 (lH, d, J=8.6Hz, H-6). (q, Me-3), 40.4 (d, C-7a), 55.8 (q, OMe-lo), 56.2 (q, OMe-9). 73.5 (d, C-13a), 78.3 (s, C-3), 90.0 (d, C-13), 101.5 (d, C-l 1). 13CNMR (75.4 MHz, CDCI,): 627.7 (q, Me-3), 28.1 (q, 105.0 (s, C-7b), 109.0 (s, C-14b), 110.0 (d, C-5), 111.5 (d, C- Me-3), 43.9 (d, C-7a), 55.4 (q, OMe-9), 55.9 (q, OMe-lo), 65.9 (t, C-13), 72.0 (d, C-13a). 77.3 (s, C-3), 100.5 (d, C-l 1), 8). 113.4(s,C-6a), 115.5(d, C-l), 128.9(d, C-2), 128.9(d,C-