Novel Epoxy Butenolides from the Genus Hortonia

This article is published under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract: Two new epoxy butenolides, (4S)-4-methyl-2-(9epoxy-11-dodecynyl)-2-butenolide and (4S)-4-methyl-2-((9Z)11-epoxy-9-dodecenyl)-2-butenolide were isolated from the dichloromethane extract of Hortonia angustifolia. They were characterized by means of Mass and NMR spectroscopic analysis. The latter compound showed moderate mosquito larvicidal activity against Aedes aegypti.


INTRODUCTION
Sri Lanka is a plant biodiversity hot spot with 25 % of its flowering plants being endemic.The independent diversity of Sri Lankan flora in comparison to peninsular India has led to speculation that during the continental drift, Sri Lanka may have experienced a higher degree of impoverishment, which would have contributed to the facilitation of speciation of new taxa on the other hand.Among the lower plants particularly among lichens, the recent reports of new species being discovered indicate that their diversity may be as prominent as the higher plants (Orange et al., 2001).Sri Lankan plants have been tested for biological activity with promising results (Hewage et al., 1997).In addition, the structural diversity among Sri Lankan higher and lower plants are typified by the discovery of, antioxidant alkaloids, (Puvenendran et al., 2008), compounds with iron sequestering ability (Kathirgmanathar et al., 2006;Karunaratne et al., 1992;Karunaratne et al., 2005;Tezuka et al., 1994;Bandara et al., 1990), and aromatic acids and ketones (Thadhani et al., 2012), all exhibiting a variety of biological activities.
The genus Hortonia is endemic to Sri Lanka and Dassanayake (1996) records three distinct species (H.floribunda Wight ex Arn., H. angustifolia (Thw.)Trimen and H. ovalifolia Wight).Some phytogeographers assume the genus Hortonia to have begun in Gondwanaland about 100-200 million years ago (Jayasekara, 1994).A noteworthy aspect of the chemistry of the genus Hortonia is the presence of compounds with five-membered rings highlighting their importance in natural products in nature (Piers and Karunaratne, 1984).We have previously reported the isolation of several biologically active and unique butenolide and hydrazulenone natural products from the three species of genus Hortonia (Ratnayake et al., 2001;Ratnayake et al., 2008a;Ratnayake et al., 2008b;Ratnayake et al., 2008c;Carr et al., 2012).

MATERIALS AND METHODS
The plant material H. angustifolia was collected from Kanneliya and was identified by Dr. Siril Wijesundara, former Director Department of Botanic Gardens.

RESULTS AND DISCUSSION
Compound 1 gave [M + ] at 276.1730 in its HREIMS suggesting that molecular formula of the compound 1 was C 17 H 24 O 3 .The IR spectrum of compound 1 showed an absorption at 2110 cm -1 indicating the presence of an ethynyl moiety, an a,b-unsaturated g-lactone carbonyl at 1755cm -1 and unsaturation at 1680 cm -1 .The absorption at 1270 cm -1 and 950 cm -1 indicated the presence of an epoxide moiety.The UV spectrum of compound 1 showed absorption at l max 231 nm indicating that an a,b-unsaturated g-lactone moiety was present.
Its 1 H NMR spectrum showed a broad singlet for twelve aliphatic methylene protons at d 1.31 (H-8, 9, 10, 11, 12, 13).The multiplet at d 1.53 and the broad triplet at d 2.25 (J = 7.3 Hz) were assigned to H-7 and H-6, respectively.The down field 1 H and 13 C NMR resonances were assigned to the conjugated double bond in the g-lactone ring [d H 6.99, 1H, d, J = 1.7 Hz, H-3, (d C 148.9 and 134.2)], an aliphatic methine residue attached to an oxygen atom [d H 4.99, H-4, dq, J = 6.8, 1.7 Hz, (d C 60.3)] and a g-lactone carbonyl (d 173.7)The H-H COSY spectrum of compound 1 showed coupling between d 3.08 (H-15) and (H-14) of the epoxide ring.In addition the H-H COSY spectrum showed both H-15 (d 3.08, dd) and H-14 (d 2.33, dt) HMBC correlation with H-17 (d 1.68, broad s) indicating that epoxide moiety was attached to the ethynyl group.The coupling observed between C-5 methyl doublet (d 1.40, d, J = 6.8 Hz) and H-4 (d 4.99, dq, J = 6.8, 1.7 Hz) established that C-5 methyl is attached to C-4 of the g-lactone ring.The C-6 methylene protons which coupled to H-3 (d 6.99, d, J = 1.7 Hz) also correlated to H-7 (d 1.53, m) suggesting that 12 C atoms containing side chain was situated at C-2.With a negative optical rotation literature precedent indicated that the absolute configuration of compound 1 at C-4 was S (Martinez et al., 1981).

99
Compound 1 was found to be inactive against the 2 nd instar larvae of Aedes aegypti at 10 ppm (Bandara et al., 2000).
Molecular formula of C 17 H 26 O 3 was established by HREIMS and was fully consistent with the number of carbon centers detected in the 13 C NMR spectrum.Four of the five degrees of unsaturation present were accounted for by the ester carbonyl, a double bond, an a, b-unsaturated g-lactone ring.The remaining degree of unsaturation was assigned to an epoxide ring.
Its 1 H NMR spectrum showed broad singlet for twelve aliphatic methylene protons at d~ 1.38 (H-7, 8, 9, 10, 11 and 12).A doublet of a tripletat d 2.38 was assigned to H-13 (2H, J = 7.5, 1.2 Hz) and the HMBC spectrum showed coupling to the adjacent methylene group H-12 (J = 7.5 Hz), and allylic coupling to the olefinic protons H-14 (J = 7.5 Hz) and H-15 (J = 1.2 Hz).The appearance of a multiplet at d 2.80 was due to H-6. 1 H and 13 C NMR resonances could be assigned to a terminal epoxide moiety [d H 3.17,2H,d,J = 2.2 Hz;4.72,1H,m,(d C 81.2 and71.8)].This epoxide in turn was attached to a cis-double bond 15) [d H 5.50,dq,J = 10.8,1.2 Hz,d H 6.05,dt,J = 10.8,7.5 Hz,) and secondary methyl group [d H 1.43,d,J = 6.6 Hz,(d C 14.5)].The presence of a cis-double bond in compound 2 was further substantiated by the absence of a 700-900 cm -1 IR absorption that is characteristic of trans double bonds.There is ample literature precedent for the occurrence of cis-double bonds in acetogenin side chains.Similar to previously reported butenolides (Ratnayake et al., 2001)  The H-H COSY spectrum of compound 2 showed coupling between d 3.17 (H-17) and d 5.50 (H-15) in addition to correlation between d 5.50 (H-15) and d 6.05 (H-14) indicating that epoxide moiety was attached to a double bond moiety.The presence of only two olefinic protons along with three epoxy protons confirmed that it was the epoxide group which was terminal and not the double bond.The coupling observed between H-15 (d 5.50) and H-13 (d 2.38), H-14 (d6.05) and H-13 (d 2.38) indicated that the cis-double bond was attached to C-13.The correlation between C-5 methyl doublet (d 1.43) and H-4 (d 4.60, m) established that C-5 methyl is attached to C-4 of the g-lactone ring.The COSY spectrum further showed that C-6 methylene protons (d 2.80, m) were coupled to H-3 (d 6.62) suggesting that C 12 side chain was situated at C-2.
With a negative optical rotation literature precedent indicated that the absolute configuration of compound 2 at C-4 was S (Martinez et al., 1981).Compound 2 exhibited moderate mosquito larvicidal activity at 10 ppm (LC 50 = 7.87).

Table 1 :
and one methyl(d 19.2).The presence of a carbonyl group (d 173.9) and oxygenated methine carbon (d 60.3) suggested an ester function.The molecular formula of C 17 H 24 O 3 was established by HRMS and was fully consistent with the number of carbon centers detected in the 13 C NMR spectrum.NMR spectroscopic data of epoxy butenolides1 and 2.