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LIMONOIDS FROM A7’ALANTZA ZEYLANICA

RAYMOND D. BENNETT, SHIN HASEGAWA and ROSALIND Y. WONG*

USDA, ARS, Fruit and Vegetable Chemistry Laboratory, 263 South Chester Avenue, Pasadena, CA 91106, U.S.A.; *USDA, ARS,

Western Regional Research Center, Albany, CA 94710, U.S.A.

(Receiced in recked form 25 October 1993)

Key Word Index--Atulantia zeylanica; Rutaceae; limonoids; limonoid glucosides; cycloatalantin; dehydrocycloatalantin; isocycloatalantin 17-p-D-glucopyranoside; propellanes.

Abstract-A limonoid glucoside and two aglycones, structurally related to the unusual propellane-type limonoid cycloepiatalantin, were isolated from Atalantia zeylanica seeds. The molecular conformation of cycloepiatalantin was determined by X-ray crystallographic analysis. The glucoside, isocycloatalantin 17-b-D-glucopyranoside, is a 6/?- hydroxy-7-keto analogue of cycloepiatalantin, and the aglycones, cycloatalantin and dehydrocycloatalantin, are the 7-epimer and the 7-ketone, respectively, of the latter.

INTRODUCHON

Although limonoids have been known as constituents of citrus fruits for over I50 years, it was only recently that limonoid glucosides were found to be present in high concentrations in citrus. Since our initial reports of the structures of 10 limonoid glucosides from grapefruit (Citrus parudisii) seeds [l, 23, we have been looking for such compounds in other citrus species and hybrids and also in other members of the Rutaceae. These investiga- tions have resulted in the finding of several new limonoid glucosides [3-61. Here we report that Atalantia zeylanica

Oliv. contains a limonoid glucoside structurally related to a unique limonoid previously isolated from another Atalantia species, as well as two aglycones of the same

type.

RESULTS AND DISCUSSION

Previously four limonoids were isolated from Atalantia

monophylla and their structures were determined. Limon- oids of the Rutaceae may be classified into two groups based on the state of oxidation of C-19: either oxygenated as in limonin (1) or methyl. One of the A. monophyh

limonoids, atalantolide [7], belongs to the latter cat- egory, while the other three, atalantin (2), dehydroatalan- tin (3) and cycloepiatalantin (4) [8], contain a five- membered cyclic ether linking C-19 to C-4. The last compound is unique in that it contains two five-mem- bered rings fused to the 5,10-bond of the B-ring, in a propellane system.

An extract of A. zeylanica seeds was partitioned be- tween methylene chloride and water. The methylene chloride extract contained two compounds giving an Ehrlich-positive reaction on TLC which is characteristic of limonoids [9], and one such compound was present in

0 0

/&-yfo 2j-$y$o 3 4 R=a.OH

5 R=B-OH

the aqueous extract. The three limonoids were isolated by column chromatography. The ‘H NMR spectrum of the first aglycone showed typical limonoid furan, H-17 and H-15 signals. In addition, only four C-methyl reson- ances were observed, thus indicating that C-19 was oxy- genated. Other features of the spectrum included a down- field, widely separated pair of AB doublets ascribable to a double bond conjugated to a carbonyl group. The small coupling constant (5 Hz) suggested that this system was in a five-membered ring. The expected AB quartet for the

163

164 R. D. BENNETT et al.

19 protons was located around 4 ppm, with a coupling constant (10 Hz) consistent with their location in a tive- membered ether ring. These two AB systems were very similar to those observed previously in 4. Although most of the proton resonances of the aglycone were consistent with those of 4, the carbinol signal was much further downfield (64.07 vs 3.46), which suggested that this compound might be the 7-epimer of 4. Based upon the NMR evidence, we had assigned a boat conformation to the B-ring of the A. monophylla limonoids [g]. This evidence was convincing in the case of 2, since a long- range coupling observed between H-5 and H-7 requires that these protons be equatorial, which is only possible if the B-ring has the boat conformation. The evidence for such a conformation in the case of 4 was more equivocal, however, being largely based upon the upfield position of the H-7 signal. We have now reinvestigated this question using 2D NMR methods not available at the time of the original structure determination, and we have been able to assign the methyl resonances in the proton spectrum of 4. The 4/?-methyl signal was the furthest downfield, at 6 1.49, but with a boat B-ring this group would be directly above the 6-ketone and should be strongly shielded. On the other hand, with a chair B-ring the 4/&methyl group would be m the deshielding region of the 6-ketone, which would account for the downtield shift observed. Since the NMR data were contradictory and it was important to know the conformation of the B-ring for the structure determination of the aglycone, we undertook an X-ray crystallographic analysis of 4. (We were unable to do this with the limonoids isolated in the present work because suitable crystals could not be obtained.)

The X-ray analysis confirmed the basic structure of 4 previously deduced from the NMR data. The perspective view of the molecule is shown in Fig. 1. The B-ring assumes a flattened chair conformation with C-8, the

Fig. 1. Perspective view of cycloeplatalantin (4) with crystallo- graphic numbering scheme. Open bonds represent double bonds; solid bonds represent single bonds; solid thermal ellip

soids, drawn at the 50% probability level. represent oxygen

atoms; an arbitrary radius of0.2 A is assigned to carbon atoms.

apex, and C-5, the foot, being out of plane by 0.70 and -0.23 A, respectively. The C-ring has a normal boat conformation, with C-9 and C-13 displaced by 0.65 and 0.62 A, respectively, from the ring plane. The D-ring assumes a flattened boat conformation, with C-13 and C- 16 displaced by 0.35 and 0.18 i(, respectively. from its mean ring plane. The A’-ring is slightly puckered, with the oxygen atom and C-19 displaced by -0.25 and 0.26 A, respectively, from the mean ring plane. The A-ring and the furan ring arc essentially planar.

A molecular model based on the X-ray data shows that the 7,Kproton of 4 is in a position to be shielded by the epoxide oxygen, which accounts for the upfield shift of its resonance. If the aglycone from A. zeylunica was the 7- epimer of 4, the carbinol proton would be well-removed from the epoxide oxygen and thus its resonance should be located downfield from that of 4, as is observed. The ‘-‘CNMR spectrum of the aglycone showed signals in accord with all of the functional groups of 4. The major differences were for the C-7 and C-9 resonances, which were about 5 and 7 ppm, respectively, downfield from the corresponding resonances of 4. This supports a 7/I-hy- droxyl configuration for the aglycone; the axial 7a-hy- droxyl of 4 shields C-9 via a y-yuuchr interaction with the axial 9-proton, but this effect would not occur with an equatorial 7/I?-hydroxyl. The hydroxyl was shown un- equivocably to be in the 7-position by the observation of a three-bond long range coupling between the carbinol carbon and the b-methyl protons in a COLOC NMR spectrum. All of the other NMR data were consistent with structure 5 for the aglycone, and therefore we have named it cycloatalantin.

The ‘H NMR spectrum of the second aglycone was similar to that of 5 and showed the presence of the propellane ring system. However, no carbinol proton signal was present. The ’ %I NMR spectrum showed three ketone carbonyl resonances instead of two as in 5. Thus, it seemed most likely that this compound is the 7- keto analogue of 5. In the case of compounds 2 and 3, conversion of a 7/I-hydroxyl to a ketone caused the expected downfield shift of the C-8 resonance, but in addition downfield shifts for C-5, C- 1 I and C-l 2 and an upfield shift of C-14, apparently due to a change in conformation of the ring system on forming the ketone. The same shifts were observed in comparing the 13C NMR spectra of S and the second aglycone (Table 1). and accordingly we have assigned the structure 6 to this compound and named it dehydrocycloatalantin. Unless water was carefully excluded, 6 readily formed a hydrate and signals for this derivative were usually present to some extent in the NMR spectra of 6. The resonances in the “CNMR spectrum of the hydrate were closer to those of 5 than of 6 (Table I), and the position of hydration was shown to be C-7 by the observation of a long range coupling between the hydrated carbon (at 96 ppm) and the g-methyl protons in a COLOC NMR spectrum, thus leading to the structure 7 for the hydrate.

The ‘H NMR spectrum of the limonoid from the aqueous extract showed signals characteristic of limon- oid l7-glucosides (H-l 5 and glucose H- 1) and also of the

Limonoids from Atolantia reylanica 165

Table 1. 13C NMR spectra of Atolantia limonoids

C s 6 I 8

1

2

3

4

5

6 7

8

9

10

11

12

13 14

15 16

17

19

20

21

22 23

4a-Me

4/3-Me 8-Me 13-Me

D-G~c- 1

LSGIC-2

o-Glc-3 D-Glc-4

D-Glc-5

D-Glc-6

171.6 166.5 171.1 129.6 130.2 129.4 200.1. 197.8 200.6

85.4 86.2 85.4 74.1 77.5 71.6

202.1. 188.5 199.9 81.3 191.3 95.9 44.1 49.5 46.5 39.7 38.4 35.8 61.1 60.6 60.1 17.4 19.0 16.7 27.7 30.9 25.9

37.8 38.9 36.8 69.3 65.0 69.1 53.6 52.0 56.2

167.3 167.0 167.9 77.3 77.7 77.4

68.4 66.6 69.4 120.2 120.8 120.2

143.3 143.6 143.6 110.2 110.5 110.5 141.7 141.9 142.1 28.0 25.5 28.8

25.3 27.3 25.1 13.1 10.9 14.5 19.1 19.5 18.0

._

-

-

167.0

132.2

207.8

84.4

63.2

73.2

210.1 49.3

36.7

62.3

18.2

27.4

44.0

70.3

57.2 168.7

77.6

66.1

125.3

141.4

112.5 140.9

23.8

27.4

16.9

25.1

104.6

74.0

76.8 70.3

76.2

61.3

Chemical shifts are in d units. All spectra were run

in DMSO-d, at 67.8 MHq compounds 5-7 at 35” and compound 8 at 50”.

*Assignments may be reversed.

propellane ring system (H-l, H-2 and H-19). The pre- sence of a downfield carbinol proton resonance suggested that this compound might be the glucoside derivative of 5. However, the ’ %Z NMR spectrum did not support this assignment. In particular, the C-5 resonance was 8 ppm upfield from that of 5, while the C-8 resonance was 5 ppm downfield (Table 1). These shifts are consistent with a 6-

hydroxy-7-ketone system. This was confirmed by a COLOC NMR spectrum, in which a long range coupling was observed between one of the carbonyl carbons and the I-methyl protons. Such a coupling is only possible for a 7-ketone. For a 68-hydroxyl configuration the carbinol proton would be equatorial and thus deshielded by the 7- carbonyl group, which would account for its downfield position in the ‘H NMR spectrum. The sugar resonances were almost identical to those observed previously for limonoid glucosides, and thus the sugar is D-gkose.. It

was shown to be attached at the 17-position, as is the case for all of the known limonoid glucosides, by a strong

cross-peak between H-17 and glucose H-l in the 2D NOESY spectrum. The coupling constant of the glucose H-l resonance (7.3 Hz) established a B-linkage. Thus, on the basis of the NMR data we assigned structure 8 to this glucoside and named it isocycloatalantin 17$-D- glucopyranoside.

The citrus limonoids show little variation in the substi- tution pattern of the B-ring. With the exception of three 7a-hydroxy compounds present in low concentration in grapefruit [lo], all of the known citrus limonoids contain a 7-keto group, with the 6-position unsubstituted. Other members of the Rutaceae, on the other hand, contain various combinations of 6- and 7-oxygenated substitu- ents, as well as the unusual H-S/l configuration in some cases [ll]. It appears that Atalantio may represent a particularly diversified genus. The three limonoids isolated in this work are different from the four obtained previously from A. monophylla. Furthermore, the glucoside has a different B-ring structure from the two aglycones. This suggests a divergent biosynthetic path- way for the glucoside.

EXPERIMENTAL

General. NMR spectral assignments were made on the basis of COSY, NOESY, DEPT, HETCOR and COLOC spectra, as well as by comparison with data from limonoids of known structure.

Isolation oj limonoids. Fruits from an A. zeylanica tree (CRC accession # 3725) in the Citrus Varietal Collection of the Citrus Research Center and Agricultural Research Experimental Station, University of California, River- side, CA, were harvested in November 1992. The seeds were ground in 0.5 M Tris buffer at pH 8.0. After 20 hr of incubation the mixt. was centrifuged and the supernatant was filtered. The soln was acidified to pH 3.0 and ex- tracted with CH,CI,. The limonoid glucoside in the aq. soln was isolated by successive chromatography on DEAE-Sephacel (Pharmacia), Dowex 50, XAD-2 and C- 18 reverse phase columns by the procedures described previously [12]. Final purification of 8 was achieved by gel filtration chromatography on BioGel P-2 (Biorad), eluting with H20.

The CH,Cl, extract was chromatographed on a silica gel column and the limonoids were eluted with increasing concns of EtOH in CH,CI,. Considerable decomposi- tion of these compounds occurred during chromato- graphy, so subsequent purification was by crystallization.

166 R. D. BENNFX et al.

Compound 5 was crystallized from EtOAc and com- pound 6 from Me&O.

C~c~our~~u~fi~ (5). This compound partially decom- posed during chromatography and crystallization, so that a pure sample could not be obtained. ‘HNMR (270 MHz, DMSO-d,, 35”): 60.97 (3H, s, Me-Q, 1.07 (3H, s, MC-~%), 1.14 (3H, s, Me-13), 1.25 (3H, s, Me-4/j), 3.84 (lH, d, J=lOHz, H-19), 3.96 (fH, d, J= lOHz, H-19), 4.07(1H,s,H-7),4.60(1H,s,H-f5),5.51(1H,s,H-f7),6.26 [lH, d, 3=5.6 Hz, H-2). 6.50(1H, d, J= 1 Hz, H-22), 7.66 (IH, s, H-23). 7.70 (IH, d, J= 1 Hz, H-21), 8.18 (IH, d, J = 5.6 Hz, H-i).

Dehydrocyoloatalantin (6). Mp 245-247’. ‘H NMR (270 MHz, DMSO-d,, 35^): itf.00 (3H. s, Me-13), 1.09 (3H,s, Me-4r), 1.22 (3H, s, Me-4/i), 1.23(3H, s, Me-@, 3.77 (lH,~,~=fO.SHz,H-l9),3.8?(fH,s,H-f5),4.23~fH,d~~ =10.5 Hz, H-19), 5.47 (IH, s, H-17), 6.15 (IH. d, J = 5.6 Hz. H-2). 6.49 (1 H, d, J = 1 Hz, H-22). 7.62 (1 H. s, H- 23), 7.69(1H,d,J= 1 Hz, H-21),8.03(fH,d,J=5.6 Hz. H- 1).

Dehydro~~~ctoataiunti hydrate (7). ‘H NMR (270 MHz, DMSO-d,, 35”): CT 1.02 (3H, s, Me-81, 1.10 (3H, s, Me&), 1.13 (3H, s, Me-131, 1.24 (3H, s, Me-481, 3.83 (IH, d, J

=9.8Hz.H-f9),3.92(fH,d,J=9.8Hz,H-f9),4.72(fH,s, H-f5),5.52(fH,s,H-17),6.03(1H,s,7-OH),6.25(fH,d,J = 5.4 Hz, H-2), 6.50 (t H, d, J = I Hz, H-22), 6.64 (1 H, s, 7- OH), 7.66(fH.s, H-23). 7.?O(fH,d,J=f Hz, H-21), 8.05 (IH, J, J=5.4Hz, H-f).

isc~ycioutaianrin 1 %P_wglucopyranoside (8). ‘H NMR (270 MHz, DMSO-d,, 50 ): 60.93 (3H, s, Me-81, 1.04 (6H, s, Me-41, Me-4/I), 1.30 (3H, s, Me- 13). 2.75 (I H, s, H- 15). 3.66(fH,d,~~fO.5Hz,H-f9),3.82(iH,d,~=fO.SH~,H- f9),4.06(1H,d,J=7.3 Hz,GlcH-1),4,61 (fH,s,H-6),5.11 (lH,s, H-l7),6.24(lH,d,~=5.6Hz, H-2),6.54(lH,d,J = 1 Hz, H-22),7.49 (1 H, s, H-23),7.53 (1 H, d, J = 1 Hz, H- 21), 7.91 (fH, d, J=5.6 Hz, H-f).

X-Ruy cr~sru~lo~ruph~c analysis o~c~rl~~e~~aru~u~rin (4). C26H280H, M, 468.6, orthorhombic, space group P2,2,21, a= 11.419(3), h= f1.635(3), c= 16.919(3) A, U =2247.9 A”, D,== 1.38cm-‘, Z=4, F(000)=992, and p (CuKsc)= 8.60 cm” I. The empirical formula is

C,,H&,, M, 468.6; the calculated density is 1.38 gcrn-. I. Intensity data were measured on a Nicofet R3 diffractometer with graphite monochromatized CuKr radiation (iv= 1.5418 A) by the f?- 20 scan tcch- nique with variable scan speed (4-30” min ‘) at room temp. The intensity data were corrected for background and Lorentz-pofarization effects [ 133, but not for absorption. In the final cycles of refinement, a secondary extinction correction (0.005) was included to minimize the dis- crepancy between IF,1 and IF,1 of the most intense reflec- tions; this fed to a significant improvement in the dis- crepancy index (R value). The crystal structure was solved by direct methods. Atomic coordinates, thermal parameters. and scale factors were refined by a ‘blocked- cascade’ full-matrix least-squares procedure with the SHELXTL Cl43 program package. The function mini- mized was &u(lF,I--/F,l)*, where o=Ca2 IF,,I +O.OOl IF,1 ’ ] - ‘. Scattering factors were from ref. [ 15 3; those of

oxygen were corrected for anomalous dispersion. Posi- tions of all non-hydrogen atoms were refined anisotropi- tally, and all hydrogen positions were estimated but verified in subsequent difference Fourier maps and in- cluded at invariant idealized values in the respective structure-factor calculation. The final least-squares structure refinement converged at R=0.039 and R, = 0.048 (309 parameters for 2459 unique reflections with IF,! 2 3alF,I in the range 3’ < 20 I 114’). The average parameter shift is &- O.la, and difference Fourier syn- thesis excursions are within + 0.4 eA - j. The absolute configuration of the structure was determined by com- paring the R-values for the two enantiomeric structures; according to Hamilton’s statistical criteria [ 161, the en- antiomer with the lower R, value has a probability of being correct to a significance level better than 0.5%.

Supp~eme~rury p~~~icu~j~~. For 4, a complete list of final atomic co-ordinates, table of bond lengths and bond angles, and anisotropic thermal parameters for the non- hydrogen atoms has been deposited at the Cambridge Crystallographic Data Centre.

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