Abietane diterpene quinones from lepechinia bullata

L. T. JONATHAN

Faculty of Science, Chemistry Department
National University of Lesotho
P.O. Roma 180
LESOTHO

ABSTRACT

Three cytotoxic abietane diterpene quinones, horminone, 7-O-methylhorminone and 6,7-dehydroroyleanone have been isolated for the first time from a methanol (MeOH) extract of Lepechinia bullata (Kunth) Epling (Labiatae). 7-O-methylhorminone is a new natural product, whose structure was unambiguously determined through ¹H-¹³C long range homonuclear correlation (COSY) and heteronuclear correlation (HECTOR) experiments. To date, only a few diterpene quinones have been found to display antitumor activity. In the present study, the three isolates were found to inhibit the growth of P-388 cells although horminone and 7-O-methylhorminone were only marginally active, according to the guidelines of the National Cancer Institute. The compounds did not however, exhibit any significant cytotoxity against KB cells. They represent the first examples of diterpene quinones of the royleanone type to be found cytotoxic against mammalian tumor cells, although horminone has previously been reported to inhibit the growth of Trypanosoma cruzi.

Introduction

Lepechinia bullata (Kunth) Epling (Labiatae), a medicinal plant growing in Colombia, South America, was investigated for antitumour activity. A methanol (MeOH) extract of the above ground parts of the plant was found to be active against P-388 (murine leukaemia) cells (ED₅₀=14.5 mg/ml), but far less sensitive in KB (nasopharyngeal carcinoma) cells (ED₅₀ 40.5 mg/ml).

Phytochemical screening of the bioactive MeOH extract afforded three cytotoxic diterpene quinones, viz., horminone (Fester et al., 1956), 7-O-Methylhorminone (Montes, 1969) and 6,7- dehydroroyleanone (Alpandes et al., 1972). 7-O-methylhorminone is a new natural product whose spectroscopic properties were very similar to those of horminone, thus justifying the royleanone type structure (See also Silver, 1968; Delgado et al., 1986):

Lepechinia bullata has not previously been investigated. Other Lepechinia species such as Lepechinia chalepensia (Fester et al., 1956), Lepechinia floribunda (Montes, 1969), Lepechinia speciosa (Alpandes et al, 1972), Lepechinia salviae (Montes et al., 1983), and Lepechinia graveolens (Riscale and Retamar, 1973) have been analysed for their essential oil content. Diterpenes and triterpenes have also been isolated from Lepechinia chamaedryoides (Silva, 1968) and Lepechinia glomerata (Delgado et al., 1986). The isolation and biological screening of the three abietane diterpene quinones from Lepechinia bullata is reported in this paper.

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Materials and methods

Plant material

The aerial parts of Lepechinia bullata were collected in Colombia in May 1976, by a USDA team. Voucher specimens have been deposited at the National Herbarium, Washington D.C., U.S.A.

Isolation and identification

The crude methanol extract, after being washed with petroleum ether, was partitioned between chloroform (CHCl₃) and aqueous MeOH. The CHCl₃ fraction was chromatographed over silica gel, using CHCl₃ as eluting agent. Fractions (500ml) were collected and combined on the basis of thin layer chromatography (tlc) analysis (see fractionation scheme). Fractions 6-19, on standing in the cold room overnight, deposited an orange precipitate, which was purified by preparative tic and recrystallised from CHCl₃ to give fraction 3 (see Fig. 1). Fractions 47-48, when left overnight in the cold room, deposited a light-green substance which, on repeated chromatography and recrystallisation, gave Fraction 1. Complete spectral analysis (UV, IR, MS ¹H- and ¹³C-NMR) of Fractions 1 and 3, gave data which were in close agreement with those previously reported for horminone (Hensch et al., 1975) and 6,7-dehydroroyleanone (Hensch et al., 1975), respectively.

Flash chromatography, followed by preparative tic of the yellowish- brown solid obtained by evaporation of fractions 31-42, gave the new compound 2 as yellow needles, mp 126-128°C. It was assigned the structure 7-O-methylhorminone, based on its spectroscopic properties. Its mass spectrum displayed a molecular ion at m/z 346, 14 amu higher than that of horminone (m/z 332), shown by the presence of the methoxyl signal, in both the ¹H- and ¹³C-NMR spectra (d3.45 and 57.3 ppm, respectively). The compound was, therefore, most likely a derivative of horminone, with a methoxyl group at either C-7 or C-12. 12-O-methylhorminone has been synthesised and characterised by Hensch et al. (1975). The 7-O-methyl analogue is hitherto unknown.

Further comparison of the ¹H and ¹³C-NMR spectra of 1 and 2 (Table 2 and 3) showed that the most significant difference between them lies in the chemical shift values for H7 and C7. The methoxyl group in 2 caused the H7 signal to move upfield to d4.32, from d4.73 in 1, and concurrently, C7 absorbed downfield at d0.8 from d63.2. The results are consistent with a methoxyl substituent at C7 and not C12 of horminone.

The uv spectra of 2 provides further evidence for a methoxyl substituent at C7. The absorption at 411 nm exhibited a significant bathochromic shift to 524 nm on addition of NaOH, indicating the presence of a quinonoid hydroxyl group at C12. Similar uv shifts have been reported in diterpene quinones bearing a quinonoid hydroxyl function (Lin et al, 1989).

The stereochemistry at C7 was determined from the ¹H-NMR spectrum. Kupchan et al. (1968, 1969) have compared the H-7bH signal of horminone with the H-7ah of taxoquinone, its 7-epimer. They found that at 60 MHz, the H-7bH appeared as a multiplet with W1/2 = 20 Hz, whereas the H-7bH signal was a broad singlet, with W1/2 == 8Hz. In our work, the H7 of 2, measured at 300MHz, was observed as a doublet of doublets with J = 2 and 4Hz, consistent with a b orientation of H7. Compound 2 was, therefore assigned the structure 7-O-methylhorminone.

Biological screening

The MeOH extract and the three isolates were tested for antitumour activity in KB and P-388 cell cultures, according to standard procedures as described previously (Pezzuto et al., 1983, and Arisawa et al, 1984). The results are shown in Table 1. All isolates inhibited the growth of P-388 cells, although 1 and 2 were only marginally active, according to the guidelines of the National Cancer Institute (Geran et al., 1972). They did not, however, show any significant cytotoxicity against KB cells. The three compounds represent the first examples of diterpene quinones of the royleanone type, to be found cytotoxic against mammalian tumour cells. It is worth noting that in both KB and P-388 systems, the unsaturated 6,7-dehydro compound (3) is more active than the 6, 7- saturated structures, leading to speculation that the antitumour activity of these compounds depends on the substitution pattern at the C6-C7 position of these molecules.

Acknowledgements

This work was supported by the Fulbright Program of the United States of America. The Program for Collaborative Research in the Pharmaceutical Sciences (PCRPS), College of Pharmacy, University of Illinois at Chicago, is gratefully acknowledged, for providing the facilities for this investigation. My special thanks go to Dr. Chun-Tao Che, Prof. Harry H.S. Fong, and Prof. Norman R. Farnsworth.

Table 1. Data obtained from pharmacological testing of KB cells (nasopharyngeal carcinoma) and P-388 cells (murine leukemia) with Lepechinia bullata plant extracts and pure compounds

Table 2. Summary of 1H-NMR of Extracts 1 and 2 (300 MHz, CDCI₃)

Table 3. Summary of data of C-13-NMR of Compunds 1 and 2 (90.8 MHz, CDC1₃)

Figure 1: Fractionation scheme for the extracts from Lepechinia bullata