Phytane- and labdane-type diterpenoids from the liverwort Haplomitrium mnioides

Phytochemistry, Vol.29, No. Printedin Great Britain.

2, pp. 585-589, 1990.

003l-9422/90$3.00f 0.00 0 1990PergamonPressplc

PHYTANE- AND LABDANE-TYPE DITERPENOIDS FROM THE LIVERWORT HAPLOMITRIUM MNIOIDES* YOSHINORIASAKAWA,MASAOTOYOTAand TORU MASUYA Faculty of Pharmaceutical Sciences,Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan (Received 30 June 1989)

Key Word Index-ffaplomitrium mnioides; Calobryales; phytane-

and labdane-type

diterpenoids;

Hepaticae; chemosystematics.

haplomitrenone;

haplomitrenolides

A, Band

C;

Abstract-Haplomitrenone, a novel phytane-type and haplomitrenolides A, B and C, three novel labdane-type diterpenoids have been isolated from the liverwort, Haplomitrium mnioides and their structures determined by ‘H and 13C NMR spectra. Haplonitrium mnioides is not so close to Takakia as previously suggested.

INTRODUCTION

RESULTSAND

The modern liverworts universally synthesize lipophilic mono-, sesqui- and di-terpenoids. Furthermore, some liverworts produce characteristic aromatic compounds including flavonoids [2]. It has been gradually recognized that these chemical constituents are significant taxonomic markers in Hepaticae [2-lo]. Haplomitrium mnioides Schust. belongs to the order Calobryales which is considered to be a very primitive taxon. The Calobryales are divided in the suborder Calobryineae (including H. mnioides and H. gibbsiae) and the suborder Takakiineae (including Takakia lepidozioides and T. ceratophylla) [ll]. These species grow in limited places, thus it is quite difficult to collect them in large amounts. Recently we undertook the analysis of crude extracts obtained from fresh cultured and herbarium specimens (13-498 mg) of Takakia and of fresh cultured H. mnioides (210 mg) by TLC, GC and GC-MS. This led to the detection of many unidentified sesquiterpenoids with one known eudesmanolide and triterpenoids in T. lepidozioides and two sesquiterpene hydrocarbons, a sesquiterpene alcohol and a few diterpenoids in H. mnioides and suggested that there is no chemical affinity between Takakia and Haplomitrium species [12]. Markham and Porter [13] reported the presence of flavonoid glycosides in the New Zealand H. gibbsiae and suggested no affinities of flavonoids between Takakia and Haplomitriwn. In the course of the collection of liverworts, we found a large mat of H. mnioides in the Tokushima Prefecture and obtained 203 g of fresh material. In order to clarify the taxonomical relationship between Haplomitrium and Takakia, we re-examined the chemical constituents of the natural H. mnioides and isolated four novel diterpenoids. The present paper describes their structural characterization and chemotaxonomic significance.

*Part 33 in the series ‘Chemosystematics part 32, see ref. [l]. Pwm

29:2-o

of Bryophytes’

For

585

DISCUSSION

A combination of CC on silica gel and Sephadex LH20 of the ether extract of H. mnioides resulted in the isolation of four novel diterpenoids, haplomitrenone (l), and haplomitrenolide A (3), B (4) and C (5). Haplomitrenone (1)

The molecular formula of 1 was determined to be C,,H,,O, (HRMS). The UV and IR spectra indicated the presence of an a&unsaturated ketone group (237 nm; 1688 cm- ‘; 6, 197.6). The ‘H NMR spectrum (Table 1) contained the signals of three vinyl methyls, a tertiary methyl group on a carbon-bearing oxygen atom, a conjugated vinyl group, two ttisubstituted olefinic protons, an exomethylene, an isolated methylene and one proton on a carbon-bearing oxygen atom. One of the two oxygen atoms in 1 was an ether oxygen as no hydroxyl absorption band was observed in the IR spectrum. The 13CNMR spectrum (Table 2) showed the presence of 20 carbon signals and the chemical shifts of six sp2 carbons, two methylene carbons and one methyl carbon were quite similar to those of I-myrcene (2) (Table 2), except for one trans-vinyl methyl group, indicating that 1 possessed the partial structure A (Fig. 1). The remaining partial structures B and C were confirmed by the following spectral evidence. The mass spectrum of 1 showed the base peak at m/z 83 (Fig. 1). The ‘H-13C long range COSY NMR spectrum (Table 3) indicated that the carbonyl carbon (C5) at S 197.6 was correlated with the olefinic proton (H-4) at 66.11 which was further correlated with two vinyl methyls (C-l and C-3) at 627.8 and 20.9. Moreover, the ‘H NMR and mass spectral data of the partial structure B resembled those of a furanosesquiterpene isolated from Phymaspermum paruifolium [14]. In ‘H-13CNMR long range COSY NMR spectrum, the carbonyl carbon was further correlated with the isolated methylene protons (H-6) which have further cross peaks with the tertiary methyl group (C-7). This methyl group was further long range coupled with the carbon bearing the ether oxygen.

5.88 br s

2.30d (15.6) 2.63 d (15.6)

1.29s 2.69 d (6.0) 1.63 q (6.0)

2.15-2.22

1.45 s 5.23 m 2.15-2.22 m 2.1552.22 m

4.99 6.36 4.99 5.20

6.11 br s

2.43 d (15.6)// 2.80 t (15.6)11

1.32s

2.78 t (6.3) 1.68 m

2.20 m

I .63 s

5.23 M 2.20 m 2.20 m

5.00 d (8.8) 6.37 dd (17.6, 10.8) 5.06 d (10.8) 5.24d (17.6)

m dd (17.6, 10.7) m d (I 7.6)

m

2.08 s

1.50S

12

2.16s

1.90s

It

Table

1.38 3 1.24 s

COSY and difference

br s br d (2.0) hr s 5

NOE experiments.

1.68 s 1.39 s

6.03 hr s 7.06 dd (2.0, 1.5) 7.16 br s 1.23s

br s br d br s s 1.73 s 1.42 .s

6.56 7.65 7.72 1.67

(I .5)

2.70 dd (14.7, 8.8, 8.3) 2.33 ddd (14.7, 5.8, 2.0) 5.92 dd (8.8. 5.8)

1.97 br dd (13.7, 8.3) 1.73 br dd (13.7, 5.4) 5.11 dd (8.3, 5.4)

2.76 br dd (12.7, 7.8) 2.34 hr dd (12.7, 4.9) 5.72 dd (7.8, 4.9) 6.34 7.42 7.38 1.70

2.84 br d (8.3)

5.91 s

m

m m

m

1.93 br s

5.69 s

2.31 s

1.4221.55 2.55 m 1.42-1.55 1.42-1.55 1.23 m 1.42-1.55

0.97 ddd (13.2, 13.2, 3.4)f 1.95 ddd (13.2, 3.4, 3.4) I .30 ddd (13.2, 3.4, 3.4) 1.45 dddd (13.2, 13.2, 3.4, 3.4) 1.02 ddd (13.1, 13.2, 3.4) 1.95 ddd (13.2, 3.4, 3.4) 1.80 s

%

1, 3-5 (400 MHz, TMS as int. standard) -

3f

data for compounds

2.80 m

5.12 s

2.13 s

1.49-1.64 m 2.52 br d (13.2) 1.49-1.64 m 1.49-l.64 m 1.499164 m 1.49-l.64 m

3-I

I. ‘H NMR spectral

‘H-‘H *All assignments were confirmed by spin decoupling, tchloroform-d,. : Benzene-d,. $Pyridine-d,. / Figures in parentheses are coupling constants in Hz. “Awgnments may be interchangeable.

llfl 12 13 14 15 16 17 18 19 20

68 I 8 9 10 lla

38 4 5 6a

2P 3a

1B 2a

lcl

H

-

m m m m

m

1.84s 1.39 s

6.59 br s 7.67 hr d (2.0) 7.76 br s 1.76s

2.97 dd (14.7, 7.7) 2.83 dd (14.7, 4.4) 6.00 dd (7.7, 4.4)

5.96 s

3.33 s

1.27-1.65 2.38 m 1.27-1.65 1.27-1.65 1.27-1.65 1.27-1.65

45

m (13.2) m m m m

I .4)

3.70 s

br s dd (2.4, br s s 2.06 s COOMe

6.58 7.67 7.76 1.67

2.71 ddd (14.7, 8.0, 8.0) 2.39 ddd (14.7, 4.9, 2.0) 5.97 dd (8.0, 4.9)

2.93 br d (8.0)

5.97 s

3.62 s

1.55-1.79 2.60 br d 1.55-1.79 1.55-1.79 1.55-1.79 1.55-1.79

%

z &

rc > 5: 2 s

Diterpenoids

Table

2. i3CNMR

spectral data of compounds (100 MHz, CDCl,)

Haplomitrenone C

(I)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

27.8 156.4 20.9 123.8 197.6 53.7 58.3 17.3 63.1 27.3 36.1 134.4 16.0 124.7 26.6 31.3 146.3 115.8 138.9 113.8

1 and

/I-Myrcene C

587

from Hnplomitrium mnioides 2

(2)

Cl51

Moreover, the methyl group (C-13) at 6 1.64 was correlated with C-11 (636.1), H-10 with C-9 (663.1), and H-11 with C-10 (627.3), respectively. Thus, the three partial structures could be connected and the gross structure of 1 was characterized. The stereochemistry at C-7 and C-9 was determined by difference NOE experiments. Haplomitrenone (1) showed the NOE between C-8-Me and H-10. Irradiation of the triplet signal at 62.78 (H-9) caused the quartet at 61.68 (H-10) to collapse to a doublet, indicating that the epoxy ring was trans. Thus, the relative stereostructure of haplomitrenone was established to be 1. Haplomitrenolide A (3)

1 2 3 4 5 6 7 8 9 10

27.7 131.5 17.7 124.4 27.0 31.7 146.3 112.9 139.2 115.6

*All assignments were confirmed by INEPT, ‘H-l% and lH-13C long range COSY NMR spectroscopy.

COSY

The compound 3 had the molecular formula C,,H,,O, ([Ml’ at m/z 328.1684) and its spectral data suggested the presence of a b-substituted conjugated ketone (1232 nm; 1679 cm-‘; 6, 5.72; 6, 196.Q a flsubstituted furane ring (6, 6.34, 7.42; 6, 108.0, 127.0, 139.2, 143.9) and a Uactone group (1745 cm-i; 6cl71.8). The ‘HNMR spectrum (Table 1) also contained one vinyl methyl and one proton on a carbon bearing an ester oxygen. The 13C NMR spectrum (Table 4) contained the signals of three methyls, trisubstituted olefinic carbons, a methine carbon bearing an oxygen atom, four methylenes, two methines and two quaternary carbons. Thus compound 3 was a tricyclic diterpene with a P-substituted furane ring. The locations of the functional groups were confirmed by the ‘H-‘H and ‘H-i3C long range COSY NMR spectra and decoupling experiments. ‘H-‘H correlations were observed between (i) H-l 1 and H-12, (ii) H-9

Table 4. 13CNMR spectral data for haplomitrenolides A (3) B (4) and C (5) (100 MHz, TMS as int. standard) C

Fig. 1.

Table

3. ‘H-i3C correlation pounds 1 and 3 1

for com-

3

‘H

13C

‘H

13C

4

1 3 5 5 7 8 9 10 11 13 16 19 17

18 5

5 4 6 10 18 19 18 9 3 4 5 20 3 4 5 19

6

10 11 14 18 20

9 17 19

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CO,Me

3* 34.2 19.1 43.1 32.8 60.9 196.5 130.0 155.0 49.2 50.4 27.9 72.4 127.0 108.0 143.9 139.2 22.3 171.8 21.8 34.4

3t 34.0 19.4 43.4 33.1 61.0 195.1 130.2 154.6 48.8 50.9 27.7 72.0 127.5 108.2 143.9 139.0 22.8 171.4 21.5 34.5

4

3: 34.0 19.6 43.4 33.1 60.7 196.0 129.8 156.5 49.1 50.9 28.3 72.4 127.9 109.0 144.5 139.9 23.1 172.4 21.8 34.3

5

35.7 19.1 42.8 33.1 53.1 196.6 130.0 155.9 73.7 56.9 29.9 70. I 127.8 108.9 144.4 139.7 19.8 173.5 23.1 34.4

*Chloroform-d,. TBenzene-d,. 1 Pyrdine-d,. All assignments were confirmed by INEPT, and ‘H-i% long range COSY NMR spectra.

‘H-l%

32.7 19.2 38.3 43.9 57.2 194.6 128.8 158.2 49.1 50.3 28.2 72.3 127.5 109.0 144.6 139.9 22.2 172.3 18.6 179.0 52.3

COSY

Y. ASAKAWA et al.

3 4 5

RI= H.R’= Me R’= OH,R’= Me R’ = H, R’ = COOMe

(1736 cm- ‘; 6, 3.70, 3H, s; 6, 179.0, 52.3). The identity of the coupling pattern in the ‘H NMR spectrum (Table 1) of 5 with that of 3 were easily confirmed by spin decoupling experiments and the ‘H-‘H COSY NMR spectrum. In the ‘H NMR spectrum of 5, the signal of one of the two methyl groups found in 3 was replaced by a signal of one methoxycarbonyl group and H-5 was largely lower shifted than that of 3, showing that the equatorial methyl group (C-20) at C-4 of 3 was replaced by a methoxycarbonyl group. This was further supported by the presence of the higher chemical shifts of C-3, C-5 and C-19 (y-effect) and the lower chemical shift of C-4 ([1effect) than those of 3 in the ‘“CNMR spectrum of 5 (Table 4). Thus, the structure of haplomitrenolide established to be 5. The absolute configuration

3

Fig. 2.

and H-11/j, (iii) H-7 and H-llr, and H-7 and H-17. Irradiation of the multiplet signal at 65.72 (H-12) caused the broad singlet at 66.34 (H-14) to collapse to a sharp singlet indicating that the /&substituted furane ring was attached to C-l 1. The ‘Hpi3C long range COSY spectrum showed the cross peaks as shown in Table 3. From the above spectral data, the partial structure A (Fig. 2) was characterized. The remaining partial structure B (three methylene groups in ring A) was also confirmed by extensive spin decoupling experiments of 3 measured in CDCI, and C,D,. Moreover, the chemical shifts of C-l, C-2, C-3 and C-4 (ring-A) in the 13C NMR spectrum of 3 were similar to those of labdaneand kaurane-type diterpenes possessing no oxygen function in ring-A [15]. The stereochemistry of 3 was established by the NOES observed between (i) H-5 and H-20, (ii) H-5 and H-9, (iii) H-l (eq) and H-12, and (iv) H-19 and H-20. The lower chemical shift (6, 2.52, br d) of H-l (eq) was due to the influence of the lactone carbonyl at C-10 and the ketone group at C-6. On the basis of the above evidence, the structure of haplomitrenolide A was established to be 3. Haplomitrenolide

B (4)

The IR spectrum of compound (4), C,,H,,O, ([Ml’ at m/z 344.1597), indicated the presence of a hydroxyl group (3370 cm- ‘). The ‘H NMR spectral pattern of 4 were quite similar to that of 3, except for the presence of the lower chemical shift at 63.33 (H-5) and the appearance of the double doublet signals (H-11~ and H-11/l) indicating that 4 was a compound possessing one axial tertiary hydroxyl group at C-9 in 3. This assumption was further confirmed by the presence of the higher chemical shifts of C-5 and C-12 (t-effect), and the lower chemical shifts of C-9 (r-effect) and C-l 1 (p-effect) than those found in 3 (Table 3). Thus, the structure of haplomitrenolide B was established to be 4. Haplomitrenolide The last 372.1622),

C (5)

compound contained

(5), CZ,HZ406 ([Ml+ a methoxycarbonyl

at m/z group

C was

of the above new diterpenoids remain to be clarified. Previously, the crude extract of the cultured H. mnioides was analysed by TLC, GC and CC-MS [12]. The TLC, GC, GC-MS patterns between the cultured and the natural H. mnioides are almost identical. Haplomitrium mnioides produces the complex diterpenoids described above and chemically speaking it is a more advanced species than those of the Isotachidaceae and the Herbertaceae families belonging to the Jungermanniales [ 151. In Takakia lepidozioides and T. cerutophylla, neither haplomitrenone nor haplomitrenolides have been detected even by GC-MS [12]. In this study, it is further clarified that Haplomitrium and Takakia genera are absolutely different and the Takakiales is regarded as a proper order. EXPERIMENTAL

Mps: uncorr. The solvents used for spectral determinations were TMS-CDCI,, C,D, or pyridine-d, [‘HNMR (400 MHz); i3CNMR (lOOMHz)]; CHCI, (IR: Lr]u); EtOH (UV; CD) unless otherwise stated. MeOH-CHCI, (1: 1) was used for Sephadex LH-20 CC. TLC. CC and GCMS were carried out as previously reported [ 161. Plant material. Haphnitrium mnioides Schust. was collected in Kaifu-cho, Tokushima Prefecture in May 1987 and identified by Dr M. Mizutani. A vsoucher specimen was deposited in the Institute of Pharmacognosy, Tokushima Bunri University. Extraction und isolation. The fresh material (203.3 g) was airdried for two days and ground mechanically. The powder was extracted with Et,0 for 3 months. After filtration and evapn of the solvent, the crude extract (1.054 g) was obtained as a dark green oil. A small amount of the crude extract was analysed by TLC, CC and GCMS to detect the same unknown sesquiterpenoids as those found in the cultured M. mnioides and the phytosterols, campe-, stigma- and sito-stem1 [ 121. The remaining crude extract (1.042 g) was chromatographed on Sephadex LH-20 to give an oil (499 mg), which was further chromatographed on silica gel using a C,H,--EtOAc gradient to give 4 fractions. Fr. 1 (C,H,)(lO mg) contained a mixture of unidentified sesquiterpene hydrocarbons. Fr. 2 (9: 1) (48 mg) was further purified by prep. TLC to afford haplomitrenone (I) (28 mg) as a pale yellow oil: [a]n +7.8 (c 1.0); UVi,,, nm (log a): 237 (4.27); IR I’,,, cm-i: 2890, 2840, 1688, 1620, 1610, 890; ‘H and “CNMR: Tables 1 and 2; HRMS: [M]’ 302.2224; C,,H,,O, requires: 302.2245; EIMS m/z (rel. in(.): 302 [Ml’ (O.l), 284(4), 149 (50) X3 (100), 55 (19). Fr. 3 (4: 1) (63 mg) was purified by prep. TLC to give haplomitrenolide A (3) (15 mg) as needles: mp 1788180’; [%]o +76.7” (c 0.5); UV i.,,, nm (loga): 221 (4.06), 232 (4.07); IR “Ina. cm -‘: 3140, 2910, 2850, 1745, 1679, 1135, 1130. 865, 590; CD +36000; ‘H and ‘jCNMR: Tables 1 and 4; HRMS: co1244nm [M]’ 328.1684; C,,,H,,O, requires: 328.1675. EIMS nt,‘z (rel.

Diterpenoids

from Haplomitrium mnioides

int.): 328 [Ml’ (37) 313(21),234(64). 189(48), 147(50), 134(100), 122 (45) 122 (45), 94 (55). Fr. 4 (7: 3) (29 mg) was purified by prep. TLC to afford haplomitrenolide B (4) (4 mg) and haplomitrenolide C (5) (6 mg) as needles: 4: mp 194-196” (decomp.); [x]n -37.5’ (pyridine; c 0.2); IR r!“,f: cm- ‘: 3370, 3130, 2915, 1718, 1662, 870; ‘H and ‘jCNMR: Tables 1 and 4; HRMS: [M]’ 344.1597; C,,H,,O, requires 344.1624; EIMS m/z (rel. int.): 344/M/+ (IO), 326 (7) 250 (100) 205 (29), 137 (68), 95 (32). 82 (27), 69 (25) 55 (21), 41 (27). Compound 5: 195-196”; [SC& + 121.2” (c, 0.25 C,H,N); IR ~,“,f: cm-‘: 3140, 2950, 1736, 1722, 1687, 865; ‘Hand ‘-‘CNMR: Tables 1 and 4; HRMS: 372.1622; C,,H,,O, requires 372.1573; EIMS m/z (rel. int.): 372 [M]’ (7), 357 (12), 313(14),278(100),246(30),202(86), 175(76), 174(87), 147(39),94 (31). Acknowledgements-We thank Dr M. Mizutani (The Hattori Botanical Laboratory, Nichinan, Miyazaki, Japan) for his identification of the liverwort.

REFERENCES

1. Hashimoto, T., Tori, M. and Asakawa, Y. (1989) Phytochemistry 28, 3377. 2. Asakawa, Y. (1982) in Progress in the Chemistry of Organic Natural Products (Herz, W., Grisebach, H. and Kirby, G. W. eds), Vol. 42, p. 1. Springer, Wien. 3. Asakawa, Y. (1982) J. Hattori Eat. Lab. 53, 283. 4. Asakawa, Y., Toyota, M., Bischler, H., Campbell, E. 0. and Hattori, S. (1984) J. Hattori Bat. Lab. 57, 383.

589

5. Gradstein, S. R., Matsuda, R. and Asakawa, Y. (1985) in Contributions to a Monograph of the Hepaticae Subfamily Ptychanthoideae (Gradstein, S. R. ed.), p. 63. J. Cramer, Vaduz. 6. Asakawa, Y. and Inoue, H. (1984) in Studies on Cryptogams in Southern Chile (Inoue, H., ed.), p. 109. Kenseisha, Tokyo. 7. Asakawa, Y. and Inoue, H. (1984) in Studies on Cryptogams in Southern Chile (Inoue, H., ed.), p. 177. 8. Asakawa, Y. and Inoue, H. (1987) in Studies on Cryptogams in Southern Peru (Inoue, H., ed.), p. 107. Tokai University Press, Tokyo. 9. Asakawa, Y. and Inoue, H. (1987) in Studies on Cryptogams in Southern Peru (Inoue, H., ed.), p. 119. Tokai University Press, Tokyo. 10. Gradstein, S. R., Asakawa, Y., Mues, R. and Klein, R. (1988) J. Hattori Bot. Lab. 64, 159. 11. Schuster, R. (1979) in Bryophytes Systematics (Clarke, G. C. S. and Duckett, J. G., eds), p. 41. Academic Press, New York. 12. Asakawa, Y., Hattori, S., Mizutani, M., Tokunaga, N. and Takemoto, T. (1979) J. Hattori Bot. Lab. 45, 77. 13. Markham, K. R. and Porter, L. J. (1979) Phytochemistry 18, 611. 14. Bohlmann, F. and Zdero, C. (1972) Tetrahedron Letters 9, 851. 15. Wehrli, F. W. and Nishida, T. (1979) in Progress in the Chemistry of Organic Natural Products (Herz, W., Grisebach, H. and Kirby, G. W., eds), Vol. 36, p. 1. Springer, Wien. 16. Asakawa, Y., Tori, M., Takikawa, K:, Krishnamurty, H. and Kar, S. K. (1987) Phytochemistry 26, 1811.