Perovskone, a potential antiprotozoal hit compound, quantification and derivatization

Abstract

Perovskone is a structurally fascinating and intricate isoprenoid with significant antiprotozoal activities. This study quantified perovskone in various parts of its primary source, Salvia hydrangea, collected from two distinct climatic regions over three years. Its quantity was also assessed in three other Salvia species that are taxonomically close to S. hydrangea. Moreover, semi-synthetic derivatives (3–5) were synthesized from perovskoneto improve its antiparasitic capabilities. 1D and 2D NMR and HRMS were used to identify the structures, and perovskone to then the compounds were evaluated in vitro against Leishmania donovani, Trypanosoma brucei, Plasmodium falciparum, and Trypanosoma cruzi and cytotoxicity was assessed in rat L6 skeletal myoblasts. Perovskone was the highest in S. hydrangea leaves and stems at 0.053% dry weight (1.5% dry extract), followed by flowers at 0.04% of dry weight. It was below the limit of quantification (LOQ) in roots and in other Salvia species. No significant difference was observed between samples of different years. Derivative 3 demonstrated significant efficacy against P. falciparum (IC₅₀ 0.08 µM, selectivity index 80), indicating a two-fold increase in activity compared to perovskone and matching the effectiveness of artemisinin and chloroquine. Given the results, S. hydrangea is a viable source for extracting perovskone and developing a new class of antiprotozoal lead compounds.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49277-6.

Introduction

Tropical diseases encompass a diverse range of infectious illnesses, disproportionately affecting populations in low- and middle-income countries. Parasitic diseases, caused by protozoan, helminthic, and ectoparasitic organisms, are a significant subset of tropical illnesses. The emergence of drug-resistant parasites further complicates the fight against these diseases, highlighting the urgent need for novel therapeutic strategies¹. Malaria remains one of the most serious parasitic diseases worldwide. According to the latest World Health Organization (WHO) report, approximately 263 million malaria cases and 597,000 deaths were recorded globally in 2023, with the African Region accounting for the vast majority of cases and deaths and children under five years of age representing most malaria-related fatalities². The disease is primarily caused by Plasmodium falciparum, the most virulent human malaria parasite. While artemisinin-based combination therapies are still generally effective, signs of partial resistance to artemisinin derivatives in Southeast Asia and more recently in Africa poses a serious threat to malaria control and elimination efforts³. Similarly, leishmaniasis is a neglected tropical disease caused by intracellular protozoan parasites from around 20 species of the genus Leishmania, which are classified into the subgenera Leishmania, Viannia, and Mundinia. This disease occurs in nearly 100 countries, predominantly in tropical and subtropical regions, and impacts over 12 million people globally⁴. Current treatments, including pentavalent antimonials, amphotericin B, and miltefosine, are associated with high toxicity, prolonged treatment regimens, increasing resistance, and high costs⁵. African trypanosomiasis (sleeping sickness) and Chagas disease, caused by Trypanosoma cruzi, also remain important public health concerns with limited and often toxic therapeutic options⁶. Although incidence of trypanosomiasis has declined substantially due to control programs, the disease is fatal without treatment⁷. These limitations collectively highlight the pressing need for safer, more effective, and affordable antiprotozoal agents.

In the search for new treatments, natural products have historically played a pivotal role in antimalarial drug discovery. Notable examples include quinine, isolated from Cinchona species, which became the first effective treatment for malaria and laid the groundwork for modern antimalarial chemotherapy. Another discovery is artemisinin, derived from Artemisia annua, which revolutionized malaria treatment. Plants have evolved an impressive repertoire of bioactive compounds, many of which possess potent medicinal properties. The genus Salvia, encompassing over 1000 species, boasts a rich history of traditional medicinal use worldwide. Interestingly, Salvia species are found in some regions where tropical diseases are endemic⁸. While traditionally used for ailments like fever and sweating, a few studies have reported their ethnomedicinal use against malaria, trypanosomiasis, and leishmaniasis⁹. Chemical analyses reveal the presence of terpenoids and phenolics in Salvia, some of which have shown promise as antiprotozoal agents in research¹⁰.

Salvia hydrangea DC. ex Benth., commonly known as “Gol-e Arooneh” in Farsi, is a widely distributed aromatic plant native to Iran, Anatolia, and Transcaucasia¹¹. S. hydrangea has been employed in traditional medicine for various ailments, including leishmaniasis¹². The aerial parts of S. hydrangea have been traditionally used in Iranian folk medicine as anti-worm and anti-leishmanial remedies^13,14. Our research group has been conducting a comprehensive study on the biological activities of Iran’s native and endemic plants^15–17. As part of this ongoing project, the n-hexane extract from S. hydrangea exhibited promising activity against P. falciparum, the parasite responsible for malaria, with an impressive IC₅₀ value of 3.2 µg/mL¹⁸. Encouraged by these initial findings, our research focused on the isolation and purification of bioactive compounds from the plant with antiplasmodial, antileishmanial, and antitrypanosomal properties. Throughout the project, several novel isoprenoid compounds with unique structures were isolated and identified in small quantities from this plant^13,14,18. The main component, perovskone (1), was isolated from the n-hexane extract and purified, resulting in 4.6 g from 20 kg of dried plant material.

Perovskone (1) possesses a fascinating structure. This natural compound contains a complex array of six fused or bridged rings and eight asymmetric centers. Unlike typical triterpenoids derived from squalene, perovskone is classified as a pseudo-triterpenoid due to its biogenesis from the coupling of monoterpene and diterpene units¹⁹. This unique structure has attracted significant research interest, with several attempts undertaken to achieve the total synthesis of perovskone and its related derivatives²⁰.

In our recent work, we investigated the antiplasmodial properties of perovskone (1) as a promising hit compound²¹. We also synthesized a series of semi-synthetic derivatives and evaluated their antiprotozoal activity. Notably, some of them showed activities comparable to or surpassing those of the current treatments. This prompted us to synthesize a series of additional derivatives of this compound.

Driven by the unique structural features of perovskone (1) and its potential as a lead compound, we initiated a thorough investigation into its presence within plant sources. S. hydrangea emerged as a prime candidate due to its initial promise. To gain a deeper understanding of perovskone’s content within this plant, we meticulously quantified its levels across various parts (roots, stems and leaves, and flowers) and across its two primary growth regions over multiple years. We further extended our exploration by analyzing the presence of perovskone in Perovskia abrotanoides—recently renamed Salvia abrotanoides—the original source from which perovskone was isolated. Additionally, two other Salvia species, S. multicaulis and S. verticillata, which share morphological similarities with S. hydrangea, were also examined. Moreover, in order to increase the number of semi-synthetic derivatives of this compound, three epoxide products were synthesized from perovskone (1) and their antiplasmodial effects were evaluated.

Results and discussion

This study investigated the presence and quantity of perovskone in n-hexane extracts of different Perovskia and Salvia species. Perovskone was first isolated and structurally identified from the n-hexane extract of P. abrotanoides in 1992, yielding 90 mg from 18 kg of dried plant material¹⁹. In 2011, our research team subsequently isolated it as the major component of the aerial parts of the n-hexane extract of S. hydrangea, and the isolated perovskone and its derivatives as well as analogues demonstrated significant antiparasitic activities. Following these observations, we decided to investigate and quantify the amount of perovskone in various S. hydrangea populations, as well as in other plants exhibiting similar external characteristics to S. hydrangea (Table 1). To achieve this objective, a chromatographic method was employed for the measurement of perovskone quantity in the samples. The method utilized a calibration curve established within the concentration range of 30–500 µg/mL. The limit of quantification (LOQ) and limit of detection (LOD) for the method were determined to be 30 µg/mL and 6 µg/mL, respectively. The calibration curve demonstrated a high degree of linearity with a correlation coefficient (R²) of 0.99 (Figure S22). All samples were subjected to analysis using the established chromatographic method.

Table 1.

Plant material used for perovskone quantification.

Plant name	Collecting time	City-Province	Voucher number	Altitude (m)	Latitude (N)	Longitude (E)	Part of the plant (sample number)
Salvia hydrangea DC. ex Benth.	June-2014	Kohin- Qazvin	MPH-2493	1530	36.34	49.56	Leaves -Stems (1)
							Flowers (2)
	June-2015	Kohin- Qazvin	MPH-2615	1530	36.34	49.56	Leaves -Stems (3)
							Flower (4)
	June-2016	Kohin- Qazvin	MPH-2722	1530	36.34	49.56	Leaves -Stems (5)
							Flowers (6)
							Roots (7)
	June-2016	Abadeh- Fars	MPH-2723	1890	31.13	52.54	Leaves -Stems (8)
							Flowers (9)
Perovskia abrotanoides Karel.	July − 2016	Ghamsar- Isfahan	KBGH-813	1920	51.32	33.64	Leaves -Stems (10)
							Flowers (11)
Salvia multicaulis Vahl.	June-2016	Kohin- Qazvin	MPH-2724	1530	36.34	49.56	Aerial parts (12)
Salvia verticillata (L.) P. Beauv.	June-2016	Kohin- Qazvin	MPH-2725	1530	36.34	49.56	Aerial parts (13)

Perovskone content analysis

The HPLC chromatogram of one of the samples is shown in Fig. 1. The results demonstrated that the perovskone content in various parts of the P. abrotanoides samples collected from Javitan was below the LOQ (30 µg/mL). This indicates that perovskone was not quantifiable in these samples. Furthermore, perovskone was not detected in other Salvia species resembling S. hydrangea. In contrast, perovskone was successfully detected in the leaves and stems of S. hydrangea collected from Kohin and Abadeh. However, the perovskone content in the roots of this plant was below the LOQ. The quantified perovskone content in different parts of S. hydrangea (leaves and stems, flowers, and roots) is detailed in Table 2, expressed as the amount per gram of dried plant material.

Fig. 1.

Chromatogram of the leaves and stems extract of S. hydrangea collected from Kohin.

Table 2.

Perovskone content (mg/g dry weight of the plant).

Plant	Sample	Perovskone content
S. hydrangea	2016, Qazvin, Leaves -Stems (1)	0.53
	2016, Qazvin, Flowers (2)	0.39
	2015, Qazvin,, Leaves -Stems (3)	0.45
	2015, Qazvin, Flowers (4)	0.40
	2013, Qazvin,, Leaves -Stems (5)	0.49
	2013, Qazvin, Flowers (6)	0.35
	2016, Abadeh,, Leaves -Stems (7)	0.40
	2016, Abadeh, Flowers (8)	0.30

As shown in Table 2, the content of perovskone was found to be higher in the leaves and stems than in the flowers of S. hydrangea. This suggests that these parts of the plant may be a better source of this compound for medicinal purposes. There was no significant difference in the content of perovskone between samples collected in different years, suggesting that the concentration of the compound is relatively stable in the plant. The effect of the growth region on the content of perovskone was also found to be negligible.

Semi-synthetic derivatives of Perovskone

Since in our recent report²¹, semisynthetic derivatives containing polar functional groups demonstrated notable antiplasmodial and antileishmanial activities, we extended our investigation to the design of new derivatives incorporating additional polar functionalities, such as an epoxide group, in order to further explore their biological potential.

In our initial approach, the reaction of perovskone in the presence of selenium dioxide and tert-butyl hydroperoxide led to the formation of compounds 2 and 3 in 28% and 40% yields, respectively (Fig. 2). Compound 2 arose from allylic oxidation of perovskone (1). It seems that after dehydration of 2, a conjugated diene was produced, which subsequently underwent epoxidation to yield compound 3.

Fig. 2.

Synthetic pathway for compounds 2 and 3.

The identification of compound 2 by 1D and 2D NMR analyses, as well as by single-crystal X-ray crystallography, was thoroughly described in our previous report²¹.

Diagnostic differences in the NMR data of compound 3 compared with those of perovskone included the presence of two oxygenated methines [δ_H 3.49 (d, J = 3.6 Hz, H-21; δ_C 62.2), δ_H 3.04 (m, H-22), δ_C 53.6] and two oxygen-bearing tertiary carbons [δ_C 57.0 and 66.5] as well as the absence of the Δ²²⁽²³⁾ double bond. In comparison to perovskone, the molecular formula of compound 3 contained two additional oxygen atoms. Given that 11 indices of hydrogen deficiency were required, compound 3 was expected to contain two epoxide moieties. Key HMBC correlations from H₃-30 (δ_H 1.23), H_α-25 (δ_H 1.64), and H_β-25 (δ_H 1.73) to both C-23 (δ_C 57.0) and C-24 (δ_C 66.5) confirmed the location of one epoxide ring at C-23 and C-24. A COSY correlation between H-21 and H-22, along with HMBC correlations from H₃-30 to C-22, from H_α-7 (δ_H 2.32) and H_β-7 (δ_H 1.80) to C-21, and from H-21 to C-8 (δ_C 48.7) and C-9 (δ_C 52.5) confirmed the position of the second epoxide group at C-21 and C-22. Diagnostic NOESY cross-peaks between H₃-30 and H_β-25 confirmed their cofacial orientation and suggested an α-orientation for the epoxide ring at C-23 and C-24. Moreover, NOE correlations between H₃-30/H-22 and H-21/H_β-7 established their respective proximities and confirmed the α-orientation of the second epoxide ring.

Aiming for direct monoepoxidation of perovskone at C₂₂-C₂₃, we employed a milder oxidizing agent, m-CPBA. This approach yielded two stereoisomeric epoxides, compounds 4 and 5 (Fig. 3).

Fig. 3.

Synthetic pathway for compounds 4 and 5.

Careful inspection of the NMR and HRMS data indicated that compounds 4 and 5 had the same planar structure. In both compounds, the perovskone double bond was replaced by an epoxide group. This inference was confirmed by the HMBC correlations from H₃-30 (δ_H 1.17 in 4 and 1.14 in 5) to C-22 (δ_C 60.3 in 4 and 58.8 in 5), C-23 (δ_C 57.8 in 4 and 57.9 in 5), and C-24 (δ_C 48.2 in 4 and 47.2 in 5), and from H_β-21 (δ_H 1.53 in 4 and 0.98 in 5) and H_α-21 (δ_H 2.94 in 4 and 2.81 in 5) to C-22 and C-23. The structural difference between compounds 4 and 5 lies in their relative configuration. The NOESY spectrum supported the relative configurations of the stereocenters. In compound 4, diagnostic NOE cross-peaks were observed between H_α-24/H₃-30 and between H_α-21/H₃-30, which corroborated that the epoxide bridge was β-oriented. In contrast, in compound 5, NOE correlations of H-15/H₃-30 and H_β-25/H₃-30 indicated that H₃-30 was β-oriented; therefore, an α-orientation was proposed for the epoxide ring.

The antiprotozoal activity of compounds 3˗5 was evaluated in vitro against several protozoan parasites, including T. brucei rhodesiense trypomastigotes, T. cruzi intracellular amastigotes, P. falciparum intraerythrocytic forms, and axenically grown L. donovani amastigotes. Their cytotoxicity was measured in rat skeletal L6 myoblast cells (Table 3). The antiprotozoal activity of compound 2 was reported in our previous report²¹.

Table 3.

In vitro activity of compounds 3–5 against T. b. rhodesiense (STIB900), T. cruzi (Tulahuen C2C4), L. donovani (MHOM-ET-67/L82), and P. falciparum (NF54), and cytotoxicity in L6 cells.

Compound	T.b. rhodesiense trypomastigotes IC50 ± MAD[a]	T. cruzi intracellular amastigotes IC50 ± MAD[a]	L. donovani axenically grown amastigotes IC50 ± MAD[a]	P. falciparum intraerythrocytic forms IC50 ± MAD[a]	L6 cells IC50 ± MAD[a]
1	13.3 ± 0.2 (1.0)	0.89 ± 0.22 (14.9)	7.1 ± 1.8 (1.9)	0.19 ± 0.01 (70.0)	13.2 ± 0.1
3	9.7 ± 0.5 (0.7)	8.1 ± 0.7 (0.8)	5.8 ± 1.7 (1.2)	0.08 ± 0.0 (83.0)	6.7 ± 1.5
4	61.9 ± 11.8 (0.2)	4.5 ± 1.1 (2.8)	7.1 ± 0.9 (1.8)	0.86 ± 0.02 (14.9)	12.8 ± 0.1
5	90.0 ± 2.5 (0.2)	9.9 ± 2.1 (1.7)	11.8 ± 0.9 (1.4)	1.9 ± 0.2 (8.6)	16.7 ± 2.4
Positive controls	0.01[b]	2.61[c]	0.51[d]	0.018[e], 0.014[f]	0.017[g]

[a] Values are means ± MAD (mean absolute deviation) of two independent determinations expressed in µM. Selectivity index (SI) indicated in brackets: IC₅₀ in L6 cells divided by IC₅₀ in the titled parasitic strain. [b] Melarsoprol. [c] Benznidazole. [d] Miltefosine. [e] Chloroquine. [f] Artemisinin. [g] Phodophyllotoxin.

Compound 3 exhibited exceptional activity against P. falciparum with IC₅₀ values of 0.08 µM and good selectivity index (SI) of 83, indicating a favorable balance between potency and cytotoxicity. In contrast, compound 4 exhibited reduced but still significant antiplasmodial activity against P. falciparum (IC₅₀ = 0.86 µM). The difference in potency between compounds 3, 4, and 5 indicated that the number, position and stereochemistry of the epoxide moieties may modulate interactions with parasite-specific molecular targets, thereby affecting binding affinity and biological activity²². Small stereochemical changes are frequently associated with substantial differences in antimalarial potency due to altered target binding affinity and spatial complementarity²³.

Compound 4 also displayed substantial effectiveness against T. cruzi with IC₅₀ value of 4.5 µM, suggesting that the structural modification broadens its antiparasitic spectrum. Differences in activity across protozoan species may reflect variation in molecular targets, redox metabolism, or cellular uptake mechanisms, which are known to differ substantially between Plasmodium, Trypanosoma, and Leishmania parasites^24,25.

Moderate antileishmanial activity against L. donovani was observed for both compounds 3 and 4, with IC₅₀ values of 5.8 and 7.1 µM, respectively. However, their SIs remained relatively low (1.2 and 1.8, respectively), raising concerns about their safety profile. None of the tested compounds showeed significant activity against T. b. rhodesiense.

Conclusion

Perovskone was identified as a major component of the extract of S. hydrangea. The content of perovskone was quantitatively determined in various parts of the plant collected over different years. Additionally, the effect of the growth region on the content of perovskone was investigated. Furthermore, we synthesized and characterized a novel family of complex isoprenoids exhibiting promising antiparasitic activities. Identification of perovskone as the parent scaffold for this family of active isoprenoids holds significant promise for overcoming a major hurdle in natural product drug development. Given the high perovskone content in the extract of the aerial parts of S. hydrangea and the plant’s widespread distribution, this species can be considered a reliable source for perovskone extraction. Further research, including the domestication of the plant, is recommended. Despite the unknown specific mechanism of action, the unique chemical structures of perovskone and its derivatives may indicate varied modes of action compared to existing drugs and could overcome drug resistance. Undoubtedly, a thorough understanding of their mechanisms of action will be invaluable for evaluating the future potential of this class of compounds.

Experimental section

General

Materials and solvents were purchased from Merck, Sigma-Aldrich, and Kimia Exir chemical companies and used as received without additional purification. Silica gel 60 F254 plates (Merck) were used in analytical thin-layer chromatography and the bands were detected under UV light. For column chromatography, silica gel 70–230 mesh and 230–400 mesh (Merck) were employed. HPLC analysis were conducted on a set-up consisting of an Agilent 1100 binary pump (G1312A) incorporating a degasser (G1379A), an autosampler (G1313A), and a PDA detector, using a Knauer C₁₈ column (4.6 mm × 250 mm, 5 µm). NMR spectra, including one- and two-dimensional experiments, were acquired on a Bruker Avance II 600 spectrometer (Bruker) operating at 600.19 MHz (¹H) and 150.91 MHz (¹³C) at 300 K. Chemical shifts (δ) are reported in ppm, and coupling constants (J) are hiben in Hz. Deuterated chloroform (CDCl₃) containing 0.03% TMS was used as the solvent. High-resolution mass spectrometry dara were obtained using a Thermo Vanquish UHPLC system coupled to a Thermo Exploris 120 Orbitrap mass spectrometer equipped with an electrospray ion source (ESI).

Plant materials

The plants were collected at full flowering stage from their natural habitats (Table 1). The samples of S. hydrangea, S. multicaulis, and S. verticillata were identified and authenticated by the plant taxonomist Prof. Ali Sonboli and voucher specimens were deposited at the herbarium of Medicinal Plants and Drugs Research Institute (MPH), Shahid Beheshti University, Tehran, Iran (Table 1). P. abrotanoides was identified by Dr. Hossien Batooli, and a voucher specimen (KBGH-813) was deposited at the Herbarium of Kashan Botanical Garden, Research Institute of Forests and Rangelands, Kashan, Iran. These common, widespread plants are not endangered plants, and the collection of samples with the permission of the university was done only for academic study by observing the necessary guidelines for collecting plants (IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora).

Extraction of Perovskone for content analysis

Five grams of homogenized powder of the 13 samples listed in Table 1 were subjected to n-hexane extraction via maceration at room temperature. The extraction was performed three consecutive times, each time using 50 mL of the solvent. The extracts were concentrated under reduced pressure to dryness and then stored in sealed vials in the dark, at 4 °C until use.

Preparation of calibration curve

A stock solution of perovskone standard (≥ 98% purity) was prepared in dimethyl sulfoxide (DMSO) at a concentration of 1000 µg/mL. From this stock, working solutions were prepared in the concentration range of 30–500 µg/mL by dilution with DMSO. The injection volume for all samples was 10 µL. An analytical chromatographic column was employed for the analysis, utilizing a mobile phase consisting of water (solvent A) and acetonitrile (solvent B), both containing 1% formic acid. Gradient elution was implemented with the following program: 90% A to 100% B over 0–20 min, followed by holding at 100% B for 20–35 min. The flow rate was set at 0.4 mL/min. The wavelength of maximum absorption (267 nm) (Figure S23) was selected for detection, and all analyses were performed in triplicate. The same chromatographic conditions were applied to the analysis of plant extracts.

Method validation

Linearity of measurement

The linearity of the analytical method was evaluated by establishing a relationship between the analyte concentration (perovskone) and the detector response. The linear range was determined by plotting the response versus concentration and confirmed through calculation of the regression coefficient. Linear dynamic range was evaluated from 30 (LOQ) to 500 µg/mL.

Limit of detection (LOD)

The LOD represents the minimum detectable amount of perovskone in the sample. In this study, the LOD was determined using a signal-to-noise ratio (S/N) of 3:1.

Limit of quantification (LOQ)

The LOQ represents the lowest quantifiable concentration of perovskone that could be quantitfied with acceptable precision and accuracy. Here, the LOQ was established using an S/N of 10:1.

Purification of Perovskone in gram-scale

Air-dried aerial parts of S. hydrangea (20 Kg) were ground and extracted via maceration with n-hexane (100 L × 3 times, each 6 h) at 40 °C. The combined extracts were concentrated under vacuum to afford a residue (330 g). This residue was fractionated using column chromatography over silica gel, eluted with a stepwise gradient of n-hexane-ethyl aetate. Fraction 2 (95 g) was recrystallized from methanol to yield perovskone (1) (4.6 g; 1.4% of the dry extract). This purified compound was used both as the analytical standard for calibration curve and as the starting material for the semisynthetic reactions.

Synthetic procedures

Synthesis of compounds 2 and 3

tert-Butylhydroperoxide (0.250 mmol, 2.5 eq.) was added to a mixture of perovskone (0.100 mmol, 1 eq.) and selenium dioxide (0.050 mmol, 0.5 eq.) in DCM (2 mL). The reaction mixture was stirred at room temperature for 2 h, andprogress was monitored by TLC. The reaction was stopped after 2 h. After completion, the solvent was evaporated under reduced pressure. The residue was extracted with ethyl acetate (3 × 20 mL), and the combined organic extracts were dried over Na₂SO₄, followed by solvent removal under reduced pressure. The crude mixture was purified by preparative TLC on silica gel [chloroform/hexane (1:9)] to afford 13.0 mg of compound 2 (28%), and 19.1 mg of compound 3 (40%).

Compound 3: 40% yield, white solid; [α]_D²⁵ +72.3 (c 0.1, CHCl₃); ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 481.2948 [M + H]⁺ (exact mass calcd. for C₃₀H₄₁O₅ 481.2949).

Table 4.

A ¹H and ¹³C NMR spectroscopic data of compounds 3–5 (δ in ppm).

Position	3		4		5
	δH mult. (J in Hz)	δC, type	δH mult. (J in Hz)	δC, type	δH mult. (J in Hz)	δC, type
1 a 1 b	1.10c 1.29c	41.7, CH2	1.10c 1.32c	42.1, CH2	1.13c 1.34c	41.9, CH2
2 a 2 b	1.36, m 1.61c	19.1, CH2	1.40c 1.69c	19.7, CH2	1.45c 1.72c	19.4, CH2
3 a 3 b	1.52c 1.74c	42.0, CH2	1.55c 1.72c	42.8, CH2	1.57, m 1.80, br d (14.2)	42.4, CH2
4	-	33.7, C	-	33.7, C	-	33.6, C
5	0.83, dd (11.8, 3.6)	53.5, CH	0.79c	53.9, CH	0.87, d (11.4)	53.9, CH
6 α 6 β	1.32c 1.55c	21.6, CH2	1.34c 1.42c	21.1, CH2	1.42c 1.50, m	21.6, CH2
7 α 7 β	2.32,dd (14.3, 7.8) 1.80c	37.8, CH2	1.93, dd (14.2, 8.0) 1.25c	41.3, CH2	2.02, dd (14.9, 8.2) 1.25, t (13.9)	41.1, CH2
8	-	48.7, C	-	46.0, C	-	48.6, C
9	-	52.5, C	-	52.3, C	-	52.5, C
10	-	89.6, C	-	88.4, C	-	88.5, C
11	-	96.4, C	-	96.9, C	-	96.8, C
12	-	169.0, C	-	167.0, C	-	170.0, C
13	-	122.8, C	-	121.6, C	-	123.2, C
14	-	201.2, C	-	200.6, C	-	200.0, C
15	3.07, sept (7.1)	24.2, CH	3.14, sept (6.5)	24.3, CH	3.12, sept (6.7)	24.3, CH
16	1.07, d (7.1)	20.2, CH3	1.14, d (6.5)	19.5, CH3	1.09, d (6.7)	20.0, CH3
17	1.02, d (7.1)	19.5, CH3	1.18, d (6.5)	20.8, CH3	1.15, d (6.7)	20.3, CH3
18	0.77, s	31.9, CH3	0.81, s	31.1, CH3	0.83, s	31.9, CH3
19	0.72, s	21.8, CH3	0.78, s	21.9, CH3	0.80, s	21.9, CH3
20 α 20 β	2.25, d (14.5) 2.03, d (14.5)	47.3, CH2	2.53, d (13.5) 1.64, d (13.5)	54.8, CH2	2.42, d (14.0) 1.63, d (14.0)	52.6, CH2
21	3.49, d (3.6)	62.2, CH	2.94c 1.53c	35.7, CH2	2.81, dd (14.5, 6.7) 0.98, br d (14.5)	37.3, CH2
22	3.04c	53.6, CH	2.91c	60.3, CH	2.90, br t (3.5)	58.8, CH
23	-	57.0, C	-	57.8, C	-	57.9, C
24	-	66.5, C	2.27c	48.2, CH	2.29, t (9.3)	47.2, CH
25 α 25 β	1.64c 1.73c	31.9, CH2	1.54c 2.13, m	31.6, CH2	1.59c 2.20, m	29.7, CH2
26	2.53, dd (12.0, 8.6)	52.3, CH	2.25c	53.3, CH	2.39, d (10.0)	53.7, CH
27	-	90.0, C	-	88.9, C		90.5, C
28	1.60, s	27.3, CH3	1.65, s	27.4, CH3	1.67, s	27.6, CH3
29	1.28, s	24.5, CH3	1.36, s	24.4, CH3	1.41, s	24.4, CH2
30	1.23, s	17.8, CH3	1.17, s	21.2, CH3	1.14, s	22.0, CH3

^COverlapping signals.

Synthesis of compounds 4 and 5

Perovskone (0.221 mmol, 1 eq.) was dissolved in dry DCM (3 mL) and cooled to 0 °C,.meta-Chloroperoxybenzoic acid (mCPBA) (0.221 mmol, 1 eq.) was added, and the reaction mixture was allowed to warmed to room temperature. After 1 h, the solvent was evaporated under reduced pressure and the residue was extracted with chloroform (3. The organic layer was dried over Na₂SO₄ and concentrated. The crude material was purified by preparative TLC (ethyl acetate/hexane, 1:5 as eluent) to obtain 56.7 mg of compound 4 (R_f = 0.26, 55%) and 34.0 mg of compound 5 (R_f = 0.52, 33%) as white solids.

Compound 4: 55% yield, white solid; [α]_D²⁵ +88.8 (c 0.15, CHCl₃); ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 467.3164 [M + H]⁺ (exact mass calcd. for C₃₀H₄₃O₄, 467.3155).

Compound 5: 33% yield, white solid; [α]_D²⁵ +81.9 (c 0.27, CHCl₃); ¹H and ¹³C NMR data, see Table 4; HRESIMS m/z 467.3167 [M + H]⁺ (exact mass calcd. for C₃₀H₄₃O₄, 467.3155).

In vitro antiprotozoal assays

In vitro antiprotozoal activities of the synthesized compounds were evaluated against T. b. rhodesiense (STIB900) trypomastigotes, T. cruzi (Tulahuen C4) amastigotes, L. donovani (MHOM-ET-67/L82) axenic amastigotes, and P. falciparum (NF54) intraerythrocytic forms. Cytotoxicity was evaluated using L6 rat skeletal myoblasts cells. Assays were performed according to established protocols previously described by one of us²⁶. The selectivity index was calculated as the ratio of IC₅₀ in L6 cells to the IC₅₀ value for each parasite.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

M.M.F. and P.S. designed and coordinated the project. F.A. isolated and purified perovskone. M.K.Z.Z. optimized and executed the chemical syntheses and experiments. M.A. provided the instrumental facilities and performed the NMR and HRMS experiments. M.K. performed the antiprotozoal and cytotoxicity assays. MT helped with the experimental procedures. The manuscript was written by M.K.Z.Z., M.T., M.O., and M.M.F. with input and suggestions from P.S. and M.K. All authors contributed to the editing of the manuscript.

Funding

This research did not receive funding.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on request.

Competing interests

The authors declare no competing interests.