Isoflavones, a sesquiterpene lactone-monoterpene adduct and other constituents of Gaillardia species

Phyrochemlstrq,

00319422/91 %3.00+0.00 ((’ 1991Pergamon Press plc

Vol. 30, No. 4. pp 1273 1279. 1991

Printedin GreatBntain.

ISOFLAVONES, A SESQUITERPENE LACTONE-MONOTERPENE ADDUCT AND OTHER CONSTITUENTS OF GAZLLARDZA SPECIES WERNER HERZ, KIMBERLY D. PETHTEL and DANIEL RAULAIS Department

of Chemistry,

The Florida State University, Tallahassee, FL 32306, U.S.A. (Received

Key Word Index -Gaillardia aestioalis; phenethyl

G. suacis;

17 July 1990)

Compositae; Heliantheae; isoflavones; nerolidol

glucoside; sesquiterpene lactone-monoterpene

glucosides;

adduct.

parts of Gaillardia suaais gave orobol 7,3’-dimethyl ether and the new isoflavones 7,3’-dimethoxy5,6,4’-trihydroxyisoflavone and 7-methoxy-5,6,4’-trihydroxyisoflavone. Gaillardia aestioalis from central Texas gave the 2’-acetylglucopyranosides of 12-hydroxynerolidol and 10,l I-dihydro-12-hydroxynerolidol and the 2’,6’diacetylglucopyranoside of 2-phenethyl alcohol as well as some common plant constituents, while the main constituent of several other G. aestiualis collections was a Diels-Alder adduct of multigilin and I-hydroxy-z-phellandrene. Abstract-Aerial

INTRODUCTION

While Gaillardia species (Heliantheae, subtribe Gaillardiinae) typically elaborate sesquiterpene lactones, generally of the helenanolide type [l-7], we reported a number of years ago [8] that two collections of G. suaois (Gray and Engelm.) Britton and Rusby, a member of section Ayassizia, appeared to be devoid of such lactones and suggested that this might be due to the lack of epidermal resiniferous glands whose presence is characteristic of many other Gaillardiinae [Stoutamire, W. P., personal communication]. Investigation of a new collection of G. suuuis from central Texas has now confirmed the absence of sesquiterpene lactones, but has also led to the isolation of three isoflavones lb, 2a and 3a, the last two of which appear to be new. The only substances which could be identified in the complex extract of a small central Texas collection of Gaillardia aestiualis (Walt.) Rock, a member of section Hokmdia, were hispidulin (6-methoxy-5,7,4’-trihydroxyflavone) and the glycosides 4, Sa and Sb. We were not successful in reisolating the main sesquiterpene lactone constituent of several G. aestiualis collections examined by us in the late 1950s and middle 1960s which the analytical tools then at our disposal had prevented us from identifying. While the few remaining crystals of this substance were unfortunately not suitable for an X-ray analysis their examination using high field NMR spectrometry has now permitted formulation of the unknown as the Diels-Alder adduct 6~.

RESULTS AND DlSCU!WON

Mass and ‘HNMR spectral evidence (see Experimental) indicated that one of the isoflavones from G. suauis, mp 179-180”, is a dimethyl ether of orobol (la). One of the two methoxyls is located at C-7 of the isoflavone skeleton because of the presence in the NMR spectrum of a singlet at d 12.85 (5-OH) exchangeable with D,O and the presence in the mass spectrum of prominent

ions at m/z 167 [A, + H] and 148 [B] [9]. The second methoxyl is therefore either at C-3’ as in lb or at C-4’ as in lc. Formula lc has been claimed for a dimethyl ether, mp 170-172”, of orobol from Simsia foetido [IO]; however, the data presented in ref. [lo] would apply equally well to an isoflavone of formula 1b. Formula 1b has been assigned to an isoflavone from several WJ>ethia species [1 l-133 on the basis of the benzene-induced solvent shifts of the methoxy resonances; no mp was specified for this substance. That our isoflavone of mp 179-180” had formula lb was shown conclusively by NOE spectrometry. Irradiation at the frequency of the methoxyl signal at 63.88 produced a 12.2% enhancement in the combined intensities of the mutually coupled narrowly split H-6 and H-8 signals while irradiation at the frequency of the second methoxyl signal at 63.94 enhanced the intensity of the narrowly split H-2’ signal at 67.14 by 6.3% and not that of the H-5’ doublet at 66.99. Consequently the second methoxyl group was at C-3’ of ring B and the isoflavone from G. suauis is orobol 7,3’-dimethyl ether. The “CNMR spectrum of lb is listed in Table 1. A second isoflavone Za, mp 216”. is similar to lb but contains one additional hydroxyl group in ring A on the basis of mass and NMR spectral evidence (see Experimental). One of the two ring A hydroxyls is on C-5 because of the presence in the ‘H NMR spectrum of an exchangeable singlet at 612.38. In the 13CNMR spectrum (Table 1) the signals originating from ring B duplicated those of lb; consequently the ring B methoxyl is on C-3’ and the ring B hydroxyl on C-4’. This was further demonstrated by NOE spectrometry; irradiation at the frequency of the methoxyl signal at 63.91 enhanced the intensity of the narrowly split H-2’ signal at 67.10 by 6.5%, but not that of the H-5’ signal. Irradiation at the frequency of the second methoxyl signal at 63.95 enhanced the ring A singlet at 66.44 by 6.6%. This and the fact that both methoxy carbons resonated near 656 [ 143 excludes 5,7-dihydroxy-6-methoxy or 5,7-dihydroxy-8methoxy substitution for ring A, but does not discriminate between 5,6-dihydroxy-7-methoxy and 5,8-

1273

1274

W. HERZ et al.

R’“~~R3 OH

la b

0 2a Rl, R3=Me, R2, R4=H

R’P2R’=H

b R’, R3=H, R2, R4=Me

R’, R2 = Me, R3=H

c R’, R4sH, R2. R3=Me

c R’. R3=Me, R2=H

3a R’=Me, R2=H h R’=H. R’=Me

13

15

14

H.

OAc 9*

5a b 10, li-dihydxo

6a

R=H

8”

OH

h R=Ac

// Q Me

0

OH

7a R=Sen

n

b R=Ang

dihydroxy-7-methoxy substitution. However, presence of a substituent on C-g is excluded when the 13CNMR spectra of lb and 2a (Table 1) are compared. The observed chemical shift changes on going from 1b to h are

compatible only with introduction of a new oxygen substituent at C-6. Hence the new isoflavone is 7.3’dimethoxy-5,6,~-trihydroxyisoflavone. Its properties differentiate it from two isomeric previously known isoflav-

Constituents of Gaillardia Table 1. ‘“CNMR

spectra of compounds (67.89 MHz, Me,CO-d,)*

lb

C 2 3 4 5 6 7 8 9 10 1’ 2 3’ 4 5 6 -0Me

154.43 d: 124.01 s 181.47 s 163.30 s 98.57 dj 166.40 s 92.69 dj 158.61 s 106.52 s 122.95 s 113.61 d 147.62 s 147.91 s 115.53d 122.49d$ 56.16q 55.30 q

24. 154.13 d: 122.40 s 181.27 s 154.28 s 130.31 s 154.28 s 95.72 d: 157.88 s 105.10 s 122.26 s 113.72dJ 147.27 s 147.65 s 115.5OdJ 122.00 d:

56.31 4 55.91 4

lb,

2a and

3a

3P 154.37 d: 122.40 s 181.27 s 154.28 s 130.31 s 154.28 s 95.72 d$ 157.88 s 105.10 5 121.73 s 130.31 d: 115.31 df 157.88 s 115.37dS 130.31 df 56.31 q

*Multiplicities by DEPT pulse sequence. tThree drops DMSO-d, added. IAssignments by single frequency heteronuclear decouplmg.

one dimethyl ethers with the same substitution pattern, compounds 2b (iristectorigenin B) [ 15,163and 2c (iristectorigenin A) [lS].* A third isoflavone isolated from G. suauis contains a hydroxyl at C-4’ of ring B and two hydroxyls, one of which is at C-5, and a methoxy group on ring A on the basis of the mass and ‘HNMR spectra. The 13CNMR spectrum listed in Table 1 shows that the chemical shifts of the ring A carbon atoms essentially coincide with those of Za. Hence the third isoflavone is 7-methoxy-5,6,4’trihydroxyisoflavone (3~). an isomer of tectorigenin (3b). The structure of 4 from the central Texas collection of G. aestioalis was deduced from the 5OOMHz ‘HNMR spectrum and extensive decoupling (see Experimental). The location of the two acetates on C-2’ and C-6’ of the glucopyranose portion of the molecule was apparent from the paramagnetic shifts of H-2’ and H-6’a,b as well as from the chemical shifts of C-l’ to C-6’ in the 13C NMR spectrum while b-orientation at C-l’ was dictated by the magnitude of J,,.Z. = 7.8 Hz. A second fraction from the Texas collection of G. aestiualis was an inseparable 1: 1 mixture of the 3-0-2’acetyl-j?-D-glucopyranosides of 12-hydroxynerolidol Sa and its 10,l 1-dihydro analogue 5b. As in the case of 411 mass spectrometry, both electron impact and chemical ionization, did not give the molecular ions, but provided

*The literature on these two substances is confusing. Structures 2b and 2c originally assigned [17-193 to iristectorigenin A and iristectorigenin B, respectively, have had to be interchanged in the light of [ 15, 161. The nature of another dimethoxytrihydroxyisoflavone obtained by hydrolysis of an isoflavone diglucoside from Juniperus mucropoda and claimed to be 2b in spite of its very low mp of 93” [20] remains to be established.

1275

evidence for the presence of the carbohydrate and the two sesquiterpene moieties, while ‘H and “CNMR spectra and extensive decoupling (see Experimental) indicated that the C-2’ hydroxyl of both constituents of the mixture carries the acetate residue and that the glycosidic linkage is p. That the 10,l l-double bond of 5a is E could be deduced from the chemical shifts of C-IO, C-12 and C-13 and the shifts of the protons attached to these carbons. The results described in the preceding two paragraphs provided an impetus for reexamining the remaining small sample of a complex sesquiterpene lactone obtained consistently in addition to hispidulin and 2,6-dimethoxybenzoquinone from several G. aestiualis collections in 1959 and 1965. That the substance which we have called aestivalin contains a unit similar to or identical with the fastigilins from G. fastigiata [21, 223, e.g. fastigilin (7a), but with an angelate esterifying the hydroxyl group on C-6 as in 7b (multigilin [23]), was already apparent from spectroscopic evidence gathered 25 years ago as well as from the formation of a monoacetate which resulted in a paramagnetic shift of the signal attributable to H-9 and the disappearance of the characteristic cyclopentenone and angelate absorptions on catalytic hydrogenation of the acetate. However, aestivalin clearly contains an additional unit linked to C-l 1 and/or C-13 of the sesquiterpene lactone skeleton although neither combustion nor mass spectrometry permitted derivation of a satisfactory empirical formula at that time. ‘H NMR spectrometry at 500 MHz (Table 2, assignments by extensive decoupling in CDCI, and in C,D, solution where signal separation was somewhat improved) together with the *‘C NMR spectrum (Table 3) has now demonstrated that the empirical formula is C30H,,0, (C32H,20, for the acetate) and that aestivalin is 6a, the product of a biological Diels-Alder reaction between 7b and the apparently unknown %hydroxy-aphellandrene (8). Vinylic H-2” is coupled vicinally to H-3” and allylically to the vinyl methyl hydrogens of the menthene moiety as well as to H-6” which in turn is coupled to the protons of two methylene units (H-13a,b and H-S”a,b) while H-3” is additionally coupled to only one other proton (H-4”) which marks the point of attachment of the isopropyl radical to the bicycle-(2,2,0)-octene system. The presence of three methyl singlets at 60.92, 0.96 and 1.05 (H-15, H-9” and H-10”) two of which experience significant paramagnetic shifts on addition of trichloroacetylisocyanate (most of the other shifts evident from Table 2 are due to reaction of the /I-orientated 9hydroxyl group with the reagent) as well as a C-O singlet at 672.83 demonstrate attachment of a tertiary hydroxyl to C-8”. Combination of 7b with the monoterpene unit also converts the H-6 signal from the broadened singlet characteristic of helenalin stereochemistry at C-6 to a sharp singlet. Significant peaks in the mass spectra can now be accounted for by facile loss of the elements of water and angelic acid and by a retro-Diels-Alder reaction regenerating the sesquiterpene and the monoterpene unit. The latter constitutes the base peak in the mass spectrum of the tetrahydro derivative of the acetate 6b, a 1: 1 mixture of the C-2’-epimers formed by saturation of the cyclopentenone and angelate double bonds. Of the eight structural and stereoisomers theoretically possible for a product from a Diels-Alder reaction between 7b and 8, we prefer formula 6 for the following reasons: (1) transition states which place the C-4 substitu-

1276

W. HE.RZ er al.

Table

2. ‘H NMR

H

6~ (CDCI,)

6

I

8

3.00ddd (1 I. 3, 1.9) 7.70 dd (6.1, 1.9) 6.05 dd (6.1. 3) 5.52 s 2.62 d (7) 5.25 dd (6.9. 2.2)

Y

3.49 dd (9.9, 2.2)

IO

2.28 ddq (9.9, 2, 6.6)

13a 14t

2.23 dt (14.9, 1.5) 1.94dd(l5, 1.5) 1.37 d (6.6)

1st

0.91 s

spectra of compounds

6~ ((‘,,D,

(C,D,)

3”

3.02 dd (6.8. 1)

4’

2.13 hr dd (9, 7)

2.85 ddd 6.94 dd 5.67 dd 5.16 5 3.45 d (6.8) 5.99 dd (6.8. 2.0) 3.23 dd (10. 2) 1.95 ddq (10. 2. 68) 2.22 dr l.‘4dd(14, 15) 1.05 d (6.X) 0.90 s 5.58 qq I 84 dy I .59 yuinr 5.42 d quint 2.96 dd 2.36 ddd (9, 7, 1)

5”a

1.x7 m:

1.78ddd (13. 9. 3)

5”h

l.14ddd: 2.63 m 1.93 d (1.5)

0.94:

2 3 6 7

13b

3’

5.98 qq (7.1, 1.4)

4’

1.89 dq (7.2, 1.6)

5t

I .67 quint

2”

5.72dqubir

6’ 7”

( I .5) (6.5. I)

2.27 hr I .92 d

9”t

1.12 5

1.OO .x

10”t

0.96 s

0.Y4 5

6a and b (500 MHz) +TAI)

5.22 hr d (10.5)

3.12 7.67 6.06 5.54 2.65 4.93 5.00

2.49 ddq

2.54 m

2.13 Jr (14, 1.5)

2.21 m

1.95 dt (14. 1.5)

1.94 In

3.12 ddd 6.94 dd 5.71 dd 5.75 5 2.73 d (7) 5.34 hr d (7)

dd (10.5.

1.19 d (6.7)

tl.YO s

0.95 s

5.69 qq

6.02 qq

I .90 dq

1.91 dq

I .68 quinr 5.32 d qumt 3.17 dd 2.68 ddd . 1.70 0.72 ddd (I 3. 7, 2) 2.27 hr I.89 d 1.32 5 1.20 5

I .9)

I .70 yuinr 5.71 d quinr 2.XY dd

2.2 m 1.87ddd(l3.9,

3)

obsc.

2.6 m: I 93 d I.11 s O.Y6 s 2.20 5

8.78 s. 8.05 s

NH *Run

on less than

thrtensity :Partly

I mg

of remaining

sample

3 protons.

obscured.

ent of the hydroxyphellandrene partner in close proximity to the lactone carbonyl or the angelate ester of the dienophile would be disfavoured; (2) the signals of the methyl and vinyl protons of the angelate are found at higher held than in multigilin or fastigilin C [21 -231, apparently because of shielding by the double bond of the bicycle-[2,2,2]-octene moiety; (3) the methyl singlets of the C-4” substituent exhibit widely different shifts but at higher held than is to be expected for methyls on carbon carrying a hydroxyl group. This indicates shielding by the bicycle-[2,2,2]-octene double bond; (4) the easily identiliable H-3” signal is coupled only to H-2” (6.3 Hz) and H4” (1 Hz), but not to the mutually coupled methylene protons on C-13 and/or C-5”. The last datum eliminates the C-l 1 stereochemistry shown in formula 9 which is that established or postulated for several naturally

*Identification

[29J of the source of these adducts as roots of

Helenium autumnaL

supplied by a German

harvested

in China

is suspect.

American

species, could conceivably

While

firm from material

If. autumn&.

of If. autumn&

never resulted in the isolation of alantolactone tone. On the other available

isoalantolactonc.

cultivated

in

have

or isoalantolac-

hand roots of the similarly

helenium from plant material

a North

have been cultivated

previous extensive investigations

mercially

ddd dd dd s d dd (6.9, 1.9)

0.96 d (6.6)

Act

China,

6h (CDCls)*

named

in the Orient

Inula

are com-

and are good sources of alantolactone

and

occurring Diels-Alder adducts involving the r,b unsaturated lactone function of one sesquiterpene lactone molecule with a lactone ring closed to C-8 and the diene function of another [2427] or the C-l I stereochemistry of the adducts resulting from reaction of diplophyllin or diplophyllide as the dienophile with a fusicoccin derivative as the diene 12111.It is, however, analogous to the stereochemistry assigned to two naturally occurring adducts arising from zingiberene as the diene and alantolactone or isoalantoalactone as the dienophiles and to that of a comparable adduct synthesized from isoalanotlactone and R( -)-r-phellandrene [29].* The suggestion has been made [30] that G. aesrivalis and G..jir.stiyiarcr are conspecific and that G.fasri@aru is no more than a variety of G. aestivolis if it is to be given taxonomic recognition at all. Our earlier work on G. jastigiata [2l] which differs from the results on G. aestivalis described here neither proves nor disproves this contention as our collections of G..fastigiato and G. aesrivalis came from different sources and locales and since techniques for isolation and identification of plant constituents have become considerably more sophisticated. Clearly the matter deserves further investigation. The discovery of isoflavones in G. suuvis is noteworthy as isoflavonoids are rare in Compositae [31]. Aside from the previously cited report on Simsia foetido [lo] isoflavones have so far been found only in Wyethia species [ I1 13, 32-341 while coumestans have been reported only from Eclipra alha and Wedelia calendulacea [SS, 361.

Constituents Table

3. 13CNMR

spectrum of compound 67.89 MHz)+

6a

C

49.60 d t 161.46dt 129.32 di 209.15 s 54.71 s 70.83 d t 53.10 dt 83.76 d? 77.96 dt 32.07 d 55.86 s 179.87 s 30.20 r : 15.56 q 17.37 q

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 *Multiplicity ZAssignments tAssignments

6a

c 1’ 2’ 3’ 4 5 !, 1 2” 3” 4”

I, 5 6” 7” 8” 9” lo” 1

(CDCI,.

6a 164.85 s 127.16 s 138.43 dt 15.41 qt 20.14 qt 147.44 s 121.24dt 38.31 dt 42.88 d t 29.36 t $ 36.79 dt 19.86 qt 72.83 s 29.21 q 25.99 qq

established by DEPT pulse method. by heteronuclear decoupling. interchangeable.

EXPERIMENTAL Extraction ojGaillardia suavis. Above ground parts (0.9 kg) of G. suaois (Gray and Engelm.) Britt. and Rusby, collected by Dr M. W. Bierner on 29 April 1989 along Highway 16 3.1 miles northeast of Highway 290 junction in Fredricksburg, Gillespie County, Texas (Bierner No. 89-9 on deposit in the herbarium of the University of Texas) were extracted with CHCI,. The material was worked-up in the usual fashion [37] to give 2.8 g of crude extract which was adsorbed on 10 g of silica gel (7&230 mesh) and chromatographed over 140 g of the same adsorbent, 25 ml frs being collected as follows: Frs l-8 (C,H,), 9-16 (C,H,-EtOAc. 9: I), 17-24 (C,H,-EtOAc, 4: 1). 25-32 (C,H,-EtOAc, 7:3), 3340 (C,H,-EtOAc, 3:2). 4148 (C,H,-EtOAc, 1: 1). 49-56 (C,H,-EtOAc, 2: 3). 574 (C,H,-EtOAc, 3:7), 65-72 (C,H,-EtOAc, 1:4), 73-80 (C,H,-EtOAc, 1:9), 81-88 (EtOAc), 89-104 (EtOAc-MeOH, 1: 1). Frs were monitored by TLC and ‘H NMR spectrometry. Frs l-20 were discarded. Frs 27-28 were purified over Scp hadex LH-20-100 (CH,Cl,) followed by PTLC (CH,Cl,-Et,O, 19: 1) to give 10 mgorobol7,3’-dimethyl ether(lb), mp 179-180”. (ref. [12] records no mp); EIMS (m/z (rel. int., %) 315 ([M + I]‘, 22.9), 314([M]+, 100). 313 (20), 299(6.8), 271 (9.4). 167 (A, +H, 34). 148 (B, 12.4); ‘H NMR (500 MHz, CDCI,) 6 12.85 (s, S-OH, exch D,O), 7.87 (s, H-2). 7.14 (d. J= 1.5 Hz, H-2’). 6.99 (d, I = 8 Hz, H-5’). 6.95 (dd, J = 8, 1.5, H-6’). 6.41 (d, J = 2.3 Hz, H-8). 6.37 (d, J=2.3 Hz H-6), 5.51 (s, 4’-OH, exch D,O), 3.94 (s, 3’OMe) and 3.89 (s, 7-OMe); ‘H NMR (270 MHz, Me&O-d,) 68.23 (s, H-2), 7.23 (d, J = 2 Hz, H-2’), 7.05 (dd, J = 8, 2 Hz, H-6’) 6.87 (d. J=O Hz, H-S), 6.53 (d, .I=2 Hz, H-8). 6.34 (d, 5=2 Hz, H-6), 3.90 and 3.87 (both s, two *Me); ‘jC NMR: Table I. Frs 29-32 (80 mg) and 3340 (45 mg) were discarded. Frs 4143 (8.5 mg) deposited 2a on standing, mp 216”: ElMS m/z (rel. int.,%)33l([M+l]*,l5.9)330([M]‘,l00),315(8.7),300(21.6), 287 (19.8), 183 (A, +H, 6.6). 182 (A,. 7.5). 149 (B+ H, 17.1). 139 (16.4), 136 (17.9); ‘H NMR (500 MHz, CDCI,) 6 12.38 (s, 5-OH), 7.91 (s, H-2),7.lO(d.J= 1.5 HL H-2’),6.95(d.5=8 Hz,H-5’),6.92 (dd, J=8, 1.5 Hz, H-6’), 6.44 (s, H-8). 5.32 (s, 4’-OH), 3.95 (s. OMe-7) and 3.91 (s, OMe-3’); ‘H NMR (270 MHz, Me&O-d,) s8.24(s.H-2),7.26(d.J=2 Hz, H-2’),7.07(dd,J=8.5,2 HI H-6’)

of Goillardia

1277

6.89 (d, J=8.5 Hz, H-5’), 6.52 (s, H-8), 3.97 and 3.89 (both s, -Me); “C NMR: Table 1. Frs 29-41 were complex mixts and were discarded. Frs 4447 on standing deposited solid material (19 mg) which was identified as a 2: I mixt. of 2.a and 3a. The residue (165 mg) was rechromatographed over silica gel (eluent C,H,-EtOAc, 4: 1) to give 45 mg linoleic acid and 7 mg 7-methoxy-5,6,4’trihydroxyisoflavone (3s) as a yellow somewhat impure (by NMR criteria) amorphous solid; EIMS mjz (rel. int., %) 301 ([M+ I]‘. 23.4). 300 ([Ml+, 100). 299 (12.8), 285 (11.4), 257 (15.6). 188 (13.8). 182 (A, 14.0). 167 (30.4). 153 (16.8). 139 (149.9), 136(37.9). 118 (B, 25.5); ‘H NMR (500 MHz. CDCl,)d7.93 (s, H2). 7.40 (2~. d, J = 9 HI H-2’ and H-6’). 6.90 (2~. d, J = 9 Hz, H-3’ and H-5’). 6.49 (s, H-8) and 3.97 (s, -0Me); ‘H NMR (270 MHz, Me,CO-d,) 68.19 (s, H-2). 7.45 (2p, d, J = 9 Hz, H-2’ and H-6’). 6.90 (2~. d, J=9 HI H-3’ and H-S), 6.50 (s, H-8) and 3.96 (s, -0Me); “CNMR: Table 1. Extraction ojG. aestivalis. (A) Above ground parts (0.6 kg) of Gaillardia aesriualis (Walt.) Rock var. oeslir;alis, collected by Dr M. W. Bierner on 1 June 1989 along Highway 2336 4.0 miles south of the junction with Highway 290 near McDade, Bastrop County, Texas (Voucher Bierner No. 89-30 on deposit in Herbarium of the University of Texas), were extracted with CHCI, and worked-up in the usual fashion. The crude extract (8 g) was adsorbed on 30 g of silica gel and chromatographed over 300 g of the same adsorbent (dry column), 50 ml frs being collected as follows: Frs l-8 (C,H,), 9-18 (C,H,-Me&O, 9: I), 19-28 (C,H,-Me,CO, 4: I), 29-38 (C,H,-Me,CO, 7:3), 3948 (C,H,-Me&O, 3:2), 49-58 (C,H,-Me,CO, 1: I), 5966 (C,H,--Me&O, 2:3) and 67.-74 (Me,CO). Frs 6-9 contained only tracesof material. Frs 19-26 were combined and filtered over Sephadex LH-20 (CHCI,); ‘H NMR analysis indicated this to be a complex mixt. whose further purification could not be achieved; manipulation of frs 27-30 gave similar results. Filtration offrs 31-34 over Sephadex LH-20 (CHCI,) followed by radial chromatography (1 mm, silica gel plate, n-hexane-EtOAc gradient, flow rate 3 mlmin-‘) gave 80 mg of slightly impure 4. Frs 35-39 (412 mg) were rechromatographed (slhca gel. dry column, C,H, and C,H,-Me&O mixts, 10 ml frs). Frs 8-l 1 of the rechromatogram (C,H,-Me&O, 4: 1 and 3: 1) and filtration over Sephadex (MeOH) gave 4.5 mg of hispidulin. Frs 17-19 (C,H,-Me,CO, 2:1) and 2:3) gave 52.5 mg of pure 4. Purification of frs 4047 of the original chromatogram by filtration over Sephadex (CHCI, and CHCl,-MeOH) followed by repeated chromatography over silica gel was not successful. Rechromatography of Frs 4847 (combined wt 600mg) over silica gel (CHCI,-MeOH, 19:1), 20 mlfrsfurnished 510 mgofamixt.of3compoundsinfrs I l-15 which on further purification on Sephadex furnished 53 mg arctiin and 35 mg of a 1: 1 mixt. of Sa and 5b. (B) Above ground parts (0.3 kg) of G. aestiualis grown at the Cranbrook Institute of Science. Bloomfield Hills, Michigan, from seed collected 14 miles north of Oberlin, Louisiana, and supplied to us in October 1959 by Dr W. P. Stoutamire (then at the Cranbrook Institute) (W. P. S. No. 1357) as G. lanceolata Michx (for correct name see ref. [38]) were extracted with CHCI, in November 1959 and worked-up in the usual fashion. The crude gum, wt 3.5 g, was chromatographed over 30 g of alumina using C,H,XHCl, as eluent (1: 1). 50 ml frs being collected. Frs 4 and 5 which appeared to contain solid material crystallized from C,H,-petrol and were repeatedly Me,CO-petrol to afford ca 0.15 g of crystalline aestivalin (6~). (C) Above ground parts (12.5 kg) of G. aestiualis collected in late summer 1965 south of Tallahassee, Leon County, Florida, and identified by Professor R. K. Godfrey (voucher number no longer available) were extracted with CHCI, in 1965 and

1278

W. HFRZ et al.

worked-up m the usual fashion. The crude gum, wt. 45.1 g, was chromatographed over 780 g of silicic acid (Mallinckrodt 100 mesh), 300 ml frs being collected and monitored by TLC. Frs I IO (eluent C,H&HCl,, 1: 1, 1.2 g of gum) were complex mixts and not further investigated. Frs 11.-15 (1 g of solid material) were combined, recrystallized from C,H,-hexane and identified as 2,6-dimcthoxybenzoquinone, mp 252-253 ‘. by comparison with an authentic sample. Frs l&25 (eluent C,H, CHCI,. 1 : I. 1.3 g) were mlxts containing phenolic matcrial. Frs 26-U) (C,H,-CHCI,. I :2, l.6g) and frs 41-70 (C,H,-CHCI,. 1: 1. 1.3 g) were complex mixts. Frs 71-101 (CHCI,. 1. I5 g) on rcchromatography over 20 g of acid- washed alumma and then over silica gel afTorded 6a. Frs 102-120 (CHCI,-MeOH, 99:1 and 49:1) afforded 6a and frs 120 130 (CHCI, MeOH) gave hispldulin identified by comparison with an authentic sample. AddItional amounts of6a were obtamed by repeated chromatography of the more polar fr.. giving a total of c.a 2.5 g of aestivahn. 2-Phm~lethonol B-r,-2’.6’-diacety!yl~~~pyranoside (4). Gum; PCIMS m z (rcl. int.. ?/,) 247 (LC,,H,,O:+HJ. 100). 229 (3.9), 187 (1.7); ‘H NMR (CDCII, 500 MHz)d7.28 (m, 3p) and 7.2O(m, 2p.aromaticHs).4.13(ddd,J=9.5.6.8.59Hz.H-la).3.67(ddd,J -9.5. 7. 7 Hz, H-lb). 2.91 (ddd, J=6.8. 7. 14.5 Hz. H-Za), 2.87 (ddd. J = 5.9, 7. 14.5 Hz, H-Zb), 4.44 (d, J = 7.8 Hz, H-l), 4.78 (dd. J-7.8. 9.5 Hz H-2’). 3.58 (dd. J=9.5. 9.5 Hz. H-3’). 3.43 (m, 2p, centrc of AB part of ABXY system, H-4’ and H-5’). 4.50 (hr dd. J =3.7. 12 HZ_ H-6’a).4.29 (dd, J= I?, 1.2 Hz, H-6’b), 2.13 and 1.98 (each sand 3p, AC): “C NMR (CDCI,. 67.89 MHz_ multiphcitics determined by DEPT pulx sequence, aliphatlc multiplets assigned by heteronuclcar decoupling) aromatic Cs----138.57 s (Cl), 126.16 d. (C-2. C-6). 128.86 d (C-3, C-5), 128.24 d (C-4); aliphatlc Cs 70.26 I (C-l ), 35.25 t. (C-2). 100.74 d (C-l’), 73.58 d (C-2’). 75.06 d (C-3’). 70.64 d (C-4’). 73.02 d (C-5’). 63.25 f (C-6’). 171.49 .s. 170.66.~ (-acetate C=O). 20.71 q, 20.71 q (acetate Me). 12-ffydroxynerolidol und 10.1 I -dihydro- I2-hydroxynerolidol 3-0-/~-(D)-2’occ~ty!y~ucopvranoside (5~ and S&J. Gum; EIMS m;‘; (rcl. mt.. %) 223 (X.3), 222 (14.3). 205 (37.6). 203 (14.2). 189 (8.8). 181 (22.0). 163 (9.6). 161 (13.6). 149 (11.1). 147 (18.X), 145 (27.7). 139 (12.1). 137 (1X.0). 135 (35.2). 134 (35.6). 133 (16.2). 126 (31.1). 123(21.7), 122(21.5), 121(67.4). 120(12.5), 119(24.3).97(31.5).96 (17.3). 95 (60.9), 94 (43.3). 93 (100); PCIMS m/: (rel. int., %) 223 (39.7. CsH,,O;+H, C,,H*,O and C,,H,,O+H), 221 (13.4. C,,H,,O), 205 (100). 203 (51.5). 193 (11.0,; ‘HNMR (CDCI,, 5OOMHz)65.l9(hrd.J=l7Hz,H-laofboth5aand5b,5.23(dd. J = 11.1, I.0 HI. H-lb of both), 5.74 and 5.73 (each dd, J= 17.1, 11.1, H-2 of both), _ 1.6 (m, H-4a of both), s 1.5 (tn. H-4b of both), 1.95 (m, H-5a.b of both), 5.06 (tq, J = 7, 1 Hr H-6 of both), 2.0 (m. H-8a.b of Sa), 1.95 (m, H-8a.b of Sb), 2.1 (tn. H-9a.b of 5a), l.9(m,H-9a,bofSb),5.35(tq,J-7.l,1.2Hz.H-lOofk). 1.35(m, H-lOaof5b),l.04(m,H-IObof5b),-l.6(m.H-lIof5b),3.96(hr, H-l2a. bof5a). 3.47(dd,J = 10.6. 5.9 Hz, H-I2a ofSb), 3.4O(dd.J = 10.6.6.2 Hz.H-l2bof5b),1.64(hr,3p,H-l3of5a),0.9O(d,3p,J =7Hz,H-13ofSb). 1.55and 1,57(eachhr,3p,H-14ofboth). 1.33 (s, 3~. H-l 5 of both), 4.53 (d. J = 8.1 Hz, H-l’ of both). 4.75 (dd. ./ -X.9, 8.1 Hz. H-2’ of both). 3.59 and 3.60 (each dd, J=9.1, 8.1 Hz. H-3’ of both), 3.55 (r, J = 9.1 Hz, H-4’ of both), 3.28 (hr dd. J = 9.1. 3.5, 2.5 Hz, H-5’ of both), 3.83 (hr dd. J = 12.1. 2.5 Hz. H6’a of both), 3.79 (dd, J = 12.1, 2.5 Hz. H-6’b of both). 2.12 (s. 3p. AC); “CNMR (CDCI,. 67.89 MHc multiplici(les by DEPT pulse sequence, tdcnotes multiplets assigned by heteronuclear decoupling) 6115.63 I (C-lt). 141.89 d (C-?t), 80.36 s (C-3). 41.551 (C-4). 22.161 (C-5t). 124.48d and l24.lOd (C-6t), 135.23 s (C-7). 39.67 I and 39.04 t (C-8). 25.99 t and 25.08 I (C-9). 125.81 d (C-10 of Sat), 36.62 t (C-IO of Sb), 134.71 s (C-l 1 of Sa). 35.52 d(C-I 1 ofSbt), 68.68 t (C-12 of Sat), 68.15 (1. C-12 ofSbt), 13.58q(C-13ofSat). 16.52q(C-13ofSbt). 15.84qand 15.78q

(C-14).23.32 y(C-l5t).96.01 d(C-l’t) 73.98 d(C-2’t). 75.31 d(C3’t). 70.64 d (C-4’+). 75.31 d (C-5’+). 62.02 I (C-6’). 170.54 s and 20.94 4 (AC). Aestiwlin (6a). Mp 203.5 -204’ after repeated crystallization Me,CO-diisopropyl ether and from Me,CO-hexane, UV i,, 222 nm (E 12 700); ether: C,H,+iiisopropyl ,R ,.$$I! cm - 1: 3560. 3400 br, 1770, 1715 (strong). 1640. 1620, 1575, 1450. 1438. 1375, 1340, 1310, 1225. 1180, 1135. 1103, 1075, 1055, 1035, 1015, 1000. 985. 940, 900, 865, 830, ‘HNMR: in Table 2; 13CNMR: Table 3. In the high resolution EIMS the molecular *on was not observed. The peak at highest mjz (5.1%) corresponded to C,,H,,O, due to loss ofangelic acid and H,O. (Calc. 394.2143. Found 394.2151). Peaks at m/z 361 (10.6%). 261 (35.8%), 243 (lO.O%), 215 (5.8%). 197 (3.6%) and 83 (100) (retro-DIeIs-Alder reaction), corresponded to CLOH2sOh C, sH, .O, (loss of angelic acid and retro-Diels-Alder). C,,H ,5 and CSHTO (angelate ion). respectlvcly; PCIMS nvz 495 ([M + 1 - H,O]+, 100%). 480, 437, 403. 395 and 361. C. H, 0 analyses were not wlthin accepted limits for C,,H,,O,. apparently due IO stubborn retention of solvent. Acctylation of 0.2 g of 6a m 1 ml of dry pyridinc and 2 ml of Ac,O. work-up m the usual fashion and repeated recrystallization from Me,CO-hexane afforded 0.15 g of 6b. mp 173 175 ‘; UVi.,,, 222 nm(c 12000). IR vL!z” cm-‘: 3400 br, 1750,17OO(v. strong), 1620, 1470, 1230, 1170. 1150. 1130, 1080, 1050, 1030, 1015. loo0. 980. 945: ‘H NMR: m Table 2. (Calc. for C 32H 420 8: C, 69.29: H. 7.63: 0. 23.08. Found: C. 69.69; H. 7.43; 0. 23.54). Hydrogenatton of 0.455 g of 6b in EtOH with prcreduced PtO, at slight pressure resulted in the uptake of two mol-eqvts of H,. After filtration and removal ofsolvent the residue was chromatographed over silica gel using C,H, CHCI, (1: I). CHCl, and CHCl,--MeOH (49: I) as cluents. The material eluted with the more polar solvents solidified: recrystallization from Mc,CO .hexane furnished a tetrahydro derivative. mp I l&l 11”; IR I!:‘$~ cm-‘: 3500, 3400 hr. 1775, 175O(v. strong), 1650. 1460. 1400. 1375, 1360, 1290. 1240, 1180, 1160, 1140, 1110. 1090. 1070, 1060. 1040, 1020. 975. 945, 908. 890, X70: ‘H NMR (300 MHG CDCI,)65.68(dquint, J=6.?. 1 Hz, H-2”), 5.37(s.H-6). 5.08(d, J = I I, I 5 Hz. H-9). 4.84 (dd, J = 7. 1.5 Hz. H-8). 2.85 (dd. J -6.3, I Hz. H-3”),2.6(m, H-6”).2.56(d. J=7 Hz, H-7).2.35-2.5(m.Zp), 2.18-2.35 (tn. 2~. H-IO. H-13a). 2.17 (5. 3p, AC), 2.0 2.17 (m. 3~). 1.96(m, lp), 1.91 (hr,3p, H-7”). 1.8.-1.9(m,Zp), l.SS(m, IH). 1.4(m, Ip), l.25(m, 2~). l.10.s.0.98 .s.0.72s(each 3p. H-15, H-9”and HIO”), 1.02 (two superimposed ds. 3p. J = 7 Hz, H-5’ of two C-2’cpimers), 0.86 (two superimposed IS, 3p, J = 7 HZ H-l’s of two C2’-eptmcrs) superimposed on 2p multiplet; PCIMS mi; (rcl. int., %) 541 ([M + 1 - H,O]’ 41.3). 407 (retro-Diels-Alder + 1. 100). 393(11.8),347(19.6),305(20.7).263(19.8),245(11.0), 135(15.8), 134 (13.5). (Calc. for Cj2H,,0s: C. 68.79; H, 8.30; 0. 22.91. Found: C. 68.87; H. 8.34: 0. 22.76).

Acknowledgements We wish to thank and Mr Richard C. Rosanske for the ments. K.M.P. thanks the Florida State stipend in the Undergraduate Research ment of Chemistry.

Dr Alicia B. GutiCrrez spectroscopic measurellniverslty for a summer Program of the Depart-

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