Shashikumar K Paknikar1*, Rukmal Ratnayake2 and Poul Erik Hansen3
Received: June 25, 2024; Published: July 02, 2024
*Corresponding author: Shashikumar K Paknikar, skpakni@yahoo.co.in (SKP), rurat@ou.ac.lk (RR), poulerik@ruc.dk (PEH)
†Dedicated to Professor Dr. Sukh Dev on his 100th Birthday.
DOI: 10.26717/BJSTR.2024.57.008981
The structure of 2, a tetracyclic sesquiterpene hydrocarbon isolated from the methylene chloride extract of Hortonia angustifolia has been revised based on the results from Density Functional Theory (DFT) calculations and is proved to be identical with (-)-ishwarane 1.
Keywords: Genus Hortonia; Tetracyclic Sesquiterpene; DFT Calculations; Structure Revision; Identity; (-)–Ishwarane
The genus Hortonia (family Monimiaceae) is endemic to Sri Lanka and only three species H. angustifolia (Trimen), H. floribuda (Wight ex Arn) and H. ovalifolia (Wight) are found at different geographical locations and have been found to exhibit mosquito larvicidal as well as antifungal activities [1]. During the past fifteen years all three species have been subjected to phytochemical investigation and observed that while butenolides are responsible for mosquito larvicidal activity [2] the sesquiterpene fraction exhibits antifungal activity [1]. The previously known teracyclic sesqiterpene hydrocarbon, ishwarane 1, [3] was isolated from the dichloromethane extract of all the species of the genus Hortonia and shown to have antifungal activity against Clodosporium codosporiods [4]. Ishwarane 1 has been isolated from different sources apparently unrelated to the family Monimiaceae [4]. As a part of the investigation on the bioactive compounds from the genus Hortonia, another tetracyclic sesquiterpene hydrocarbon was isolated from the dichloromethane root extract of Hortonia angustifolia, [α]D -64.41o (CHCl3) [5]. The assignment of structure including relative stereochemistry was based on extensive spectral analysis (1D, 2D NMR, HR-ESI-MS) and was assigned the 1,5,12-trimethyltetracyclo- [6.3.0.0.3,504,8] dodecane skeleton 2 (Figure 1). Since (-)-ishwarane 1 was isolated from the same source, the newly isolated hydrocarbon 2 looked in all probability structurally related to 1. Based on the known chemical interrelationship among 1 and the other ishwarane based sesquiterpenes [6] a putative structure 2a was considered for Hortonia tetracyclic sesquiterpene. (-)-Ishwarane 1 contains a tricyclo- [3.2.1.02,7] octane system 3 and the putative structure 2a possesses tricyclo [3.2.1.02,8] octane part structure as shown in 4 (Figure 1). A close examination of molecular models of 1 and 2a revealed that ring-A is common in both the structures. Comparison of the chemical shifts of the ring A protons (at C1, C2 and C3 methylene, C4 methine and C14, C15 methyl protons) and the corresponding 13C NMR chemical shifts (of C1, C2, C3, C4, C5, C10, C14 and C15 carbons) were found to be nearly identical [4].
The difference lies in the arrangement of a rings B, C and D. The tricyclo [3.2.1.02,7] octane ring system 3 of (-)-ishwarane 1 has undergone an acid catalysed molecular rearrangement to produce tricyclo [3.2.1.02,8] octane system 4 present in hortoisoisharane 2a (Figure 1). In recent years, comparison of 13C-NMR chemical shifts experimentally observed on natural product and those calculated by density functional theory (DFT) [7-11] has become an unambiguous method for checking the correctness of structure and stereochemical assignments of natural products [12] based on 1D and 2D NMR spectral analysis and HR-ESI-MS data. In an attempt to determine the stereochemistry of structure 2a by Density Functional Theory (DFT) calculations, some problems surfaced and a reassignment of the structure became essential. The results of the study are presented here.
The isolation of a tetracyclic sesquiterpene hydrocarbon from H. angustifolia was reported and was assigned structure 2 based on an extensive 1D and 2D NMR spectral analysis together with HR-ESI-MS. One important feature of natural products belonging to isoprenoids is the stereochemistry. 13C NMR has recently been shown to be a useful tool in establishing this [13]. Analysis of compound 2 (Figure 1) turned out to be difficult as it is a hydrocarbon, so the spread in both the 1H and the 13C chemical shifts is narrow even after changing the NMR solvent to C6D6 from CDCl3, making the analysis of even 2D NMR spectra complicated. While most of the structural assignments were the same as reported previously [5], a reassignment of the resonances based on 1H/13C/COSY/ HMBC correlations revealed that besides from the cyclohexane ring, two CH2 groups were not neighbours (Table 1). In addition, an important feature was the coupling constant of 11.9 Hz observed as a geminal coupling according to the COSY spectrum indicating that both H-9 and H-12 should be a part of the proposed five-membered rings. The full analysis led to the assignment as included in Table 1. An attempt to determine the absolute structure of the molecule from H. angustifolia by 13C NMR showed that a comparison of experimental and calculated nuclear shielding by Density Functional Theory (DFT) calculations (GIAO approach) gave a very poor fit, indicating that the assigned structure probably needed revision (Figure 2). However, plotting the experimental reassigned 13C chemical shifts from Table 2 were very close to those of (-)-ishwarane vs. calculated nuclear shielding, and gave a very good correlation (Figure 3). Therefore, it was concluded that the compound isolated from H. angustifolia was as a matter of fact (-)-ishwarane 1. The stereochemistry is confirmed by the 13C NMR calculations.
Table 1: 1H, 13C chemical shifts and correlations obtained from DEPT, COSY, HMQC and HMBC spectra for 2a.
Note:
a. The HMQC spectrum confirmed that all CH2 groups had two different
1H chemical shifts
b. A coupling constant of 11.9 Hz could be measured.
Note:
a. B3LYP/6-31G(d) (see experimental).
The DFT calculations have been found to be extremely helpful in conclusively showing that the laevorotatory tetracyclic sesquiterpene isolated from the methylene chloride root extract of H. angustifolia possesses neither structure 2 nor 2a but is identical with (-)-ishwarane 1.
Calculations
Structures were subjected to calculation of carbon chemical shifts using the Gaussian 16 program package [14] using the B3LYP/ 6-31G(d) functional and Pople basis set. [15]. The nuclear shielding was calculated using the GIAO software [16,17].
The authors declare that they have no conflict of interest.
We thank Professor Shailesh R Shah (The Maharaja Sayajirao University of Baroda) and Dr. Kamlesh Fondekar (Head, R & D, Godrej Agrovat, Mumbai) for their interest and help in manuscript preparation. We also thank Mr. Ramakant Harlalka, Managing Director, Nishant Aromas, Palghar, for his interest and support.
1. Isolation of natural products from H. angustifolia.
2. Use of DFT calculations to predict correct structure of complex
fused ring compounds.
3. Correction of published structures.