Crotofolane Diterpenoids: Structural Diversity, Natural Sources, and Therapeutic Potential

Abstract

Crotofolanes are an unusual class of diterpenoid family, which are distinguished by a unique fused tricyclic 5/6/7-ring skeleton, primarily reported from Croton genus (Euphorbiaceae). This work comprehensively summarizes the reported crotofolanes, including their sources, structural classifications, biosynthesis, and biological activities. A total of 62 crotofolane derivatives have been isolated from 11 Croton species in the period from 1975 to April 2025. Some crotofolanes possess notable biological properties such as anti-HIV, antimalarial, cytotoxic, and immunosuppressive. The review also emphasizes the distinct oxidative changes and rearrangements that result in structural variation among crotofolanes.

INTRODUCTION

The genus Croton (Euphorbiaceae) includes about 1300 species of herbs, shrubs, and trees in tropical and subtropical regions globally. Croton species are traditionally used for the treatment of diverse conditions such as cancer, malaria, fever, inflammation, sexually transmitted infections, stomachache, abscesses, urticaria, leprosy, and psoriasis, and possess hypoglycemic, antihypertensive, and antioxidant activities.[1] These are distributed across a wide range of countries in Africa, Asia, North America, South America, and Australia. The plants of the genus are reported to yield different types of diterpenoids, including crotofolanes.[2,3,4]

Crotofolanes are a rare class of diterpenoids that have fused tricyclic core skeletons of five-, six-, and seven-membered rings. The first diterpenoid, crotofolin A, was reported from Jamaican Croton species, C. corylifolius in 1975.[5] It is noteworthy that 62 crotofolanes have been identified from 1975 till June 2025, and all are from the Croton genus.[2,3,4] These diterpenoids have been isolated from different parts of 11 Croton species, including C. corylifolius L. (Jamaica)[5]Formatting.; C. cascarilloides Räuschel (Japan)[3,6,7,8,9,10]; C. caracasanus Pittier (Venezuela),[11] C. dichogamus Pax. (Kenya),[12] C. megalocarpus Hutch. (Kenya)[13]; C. kilwae Radcl.-Sm. (Tanzania),[14] C. insularis Baill. (Australia)[15]; C. argyrophyllus Mull. (Brazil)[16]; C. kongensis Gagnep. (China)[17]; C. argyratus Blume (Malaysia)[18]; and C. Haumanianus J. Léonard (Congo).[19] Crotofolane diterpenoids are classified into crotofolane, crotofolane alkaloids, nor-crotofolane, isocrotofolane, and seco-crotofolane according to their structures. The current work aimed to comprehensively review the reported crotofolane diterpenoids, including their sources and bio-activities.

SEARCH METHODOLOGY

A comprehensive search was carried out across different databases and publishers, including Google Scholar, Web of Science, PubMed, SciFinder, Scopus, Wiley Online Library, American Chemical Society, SpringerLink, J-STAGE, SAGE Journals, and Taylor and Francis. The keywords used for the search were “crotofolane diterpenes” and “crotofolane biological activity.” The relevant studies related to chemistry, sources, and biological activities of crotofolane diterpenoids published up to the beginning of June 2025 were included.

BIOSYNTHESIS, ISOLATION, AND IDENTIFICATION

Crotofolane diterpenoids are biosynthesized from cembranes via casbane and lathyrane through cross-annular cyclization.[2,3,4]

This metabolite isolated either from the petroleum ether, CH₂CH₂, or CHCl₃ fraction of MeOH or EtOH extract using SiO₂, Sephadex LH-20, and RP-18 CC, and finally purified with HPLC.[14] The structures were assigned and confirmed by HR-MS and NMR, in addition to their stereochemistry, which was confirmed by X-ray, ECD, NOESY, and chiral HPLC, as well as chemical methods.[3,6,7,8,9,10]

CROTOFOLANE DITERPENOID CLASSES AND BIOLOGY ACTIVITIES

Crotofolane-type diterpenoids

Crotofolin A (1) is the first derivative with a unique carbon skeleton that was isolated from Croton corylifolius benzene extract.[5] Its structure features a fused cyclopentenone, cyclohexene, and seven-membered ring with an exocyclic methylene and a butenolide moiety.[5] Bruke et al.[20] reported 3, a novel and highly functionalized crotofolane diterpene from Jamaican Croton corylifolius, along with 1, 2, and 4. Its structure also features a rare pseudo acid moiety, unusual among diterpenes. Compound 4 (IC₅₀: 10-30 μg/mL) showed moderate cytotoxicity against HeLa, HT29, MCF-7, MM96, and NFF cell lines.[15] The new crotofolane diterpenoids, 6 and 7 were isolated from the African shrub Croton dichogamus leaves CH₂Cl₂ fraction.[21] Compound 6 possesses a crotofolane skeleton with a fused tricyclic core, an epoxide moiety, and a unique furan ring instead of the common butenolide group in the related diterpenes.[21] While 7 is structurally related to 6 but differs by containing an exocyclic methylene and an acetoxy group.[21] Compound 8, a crotofolane diterpene was isolated from Croton Haumanianus trunk barks pet ether extract. It features two epoxide rings, a furan ring, and an exocyclic methylene group.[19] The undescribed crotofolane diterpenoids 9 and 14, along with 8 were isolated from the Kenyan Croton dichogamus root. Compound 9 is structurally similar to 8, with a substituting C-1β acetate group with a ketone group in 9, while 14 has an aromatic ring. These compounds had no activity on NCI-59 and Caco-2 cell lines.[22] The new crotofolane diterpenes with epoxide group: 10-13 were isolated from Croton argyrophyllus stems and roots. Compounds 12 and 13 are C-2 epimers of 10 and 11, respectively. Their skeleton features fused tetracyclic rings with furan ring, as well as epoxide groups between C-5 and C-ringsll compounds showed no cytotoxic properties against HL-60, CEM, MCF-7, HCT-8, and B16.[16] Compounds 15-18, new crotofolane-type diterpenoids along with 3, were separated from Croton caracasanus stems CH₂Cl₂ fraction. These compounds have a fused tricyclic 5/6/7 ring system of crotofolane diterpenoids, with different substitution patterns [Figure 1]. They demonstrated no cytotoxic potential against PC-3, HeLa, and MCF-7 in the MTT method.[11]

Figure 1.

Structures of crotofolane diterpenoid 1-23

Kawakami et al. reported 19-26, structurally rare crotofolane diterpenoids were reported from Croton cascarilloides stems collected in the Okinawa Islands. Compounds 19 and 20 possessed 2S-methylbutyric acid, while compounds 24-26 featured isobutanoic acid moieties.[6,7] Kawakami et al. further investigated Croton cascarilloides leaves BuOH fraction, resulting in isolation of new crotofolane diterpenoids: 27-29. Crotocascarin K's absolute configuration was confirmed by X-ray crystallography and the modified Mosher’s method.[8] Compound 31 a monochlorinated rare crotofolane diterpenoid, and the new 32, along with 29 were isolated from Croton megalocarpus bark. Compounds 31 and 29 (CC₅₀s 28 and 5.5 nM, respectively) were found to prohibit HIV-1EIIIB replication in vitro. Also, 31 and 29 suppressed HIV-1 protease (%inhibition 63 and 75%, respectively). However, 31 and 29 had no cytotoxic potential against MT-4 and FM-55-M1 cell lines.[13]

On the other side, 30 and 33-37 were separated from Croton cascarilloides stems CH₂Cl₂ fraction.[9] Kawakami et al. reported 38-42 from Croton cascarilloides leaves EtOAc fraction. Compounds 38-41 possessed isobutyric acid moiety, 39 has an acetyl group, whereas 42 is a C₉-hydroxylated crotocascarin K [Figure 2].[10] The new crotofolane diterpenoid, 43 isolated from Kenyan Croton dichogamus, demonstrated potent anti-HIV activity through suppressing viral replication (IC₅₀ 0.002 µg/mL), compared to zidovudine, abacavir, and tenofovir (IC₅₀s 0.002, 0.05, and 0.04 ± 0.01 µg/mL, respectively). Its anti-HIV activity contributed to HIV-1 protease inhibition based on in-silico modeling.[12] Besides, it showed cytotoxic potential (IC₅₀ 84 µM) versus MT-4 cells.[12] In a study by Mahambo et al., new crotofolane diterpenoids, 44-49 were isolated from Croton kilwae leaves and stem bark.[14] Among them, 44-46 displayed potent anti-Plasmodium activity against chloroquine-resistant Plasmodium falciparum Dd2 cells (%growth inhibition 80-100%; Conc. 50 μM), whereas 47-49 controlled parasitemia (26, 42, and 60%, respectively).[14]

Figure 2.

Structures of crotofolane diterpenoid 24-49

nor-Crotofalane diterpenoids

Compounds 51 and 52 rearranged nor-diterpenoids with 2S-methylbutyric acid moiety were separated Croton cascarilloides stems. These compounds were proposed to be biosynthesized from 20 through several reactions, such as decarboxylation, oxidation, C–C bond migration.[6,7] Also, 50 was obtained from Croton cascarilloides stems. Its biosynthetic pathway involves oxidative cleavage and crotofolane precursor ring rearrangement.[9] The nor-crotofolane: compound 54 was isolated from Croton cascarilloides leaves. It is similar to crotocascarin β, except for lacking the 2-methylbutyric acid moiety.[8] Compound 53 is an 8S-configured trinor rearranged crotofolane with a C-8 tertiary hemiketal functional group isolated from Croton cascarilloides leaves.[10]

Seco-crotofolane diterpenoids

Also, two 1,14-seco-crotofolane derivatives: 55 and 56 were isolated from Croton insularis stem. They originated from 57 and 58 through an unprecedented homo-Baeyer-Villiger rearrangement [Figure 3]. Only, 56 (IC₅₀ 12 µg/mL) was active against the MM96L cell line.[15]

Figure 3.

Structures of nor-crotofalane (50-52), seco-crotofolane (55 and 56), crotofolane endoperoxides (57 and 58), isocrotofolane (59), and crotofolane diterpenoid alkaloids (60-62)

Crotofolane endoperoxides

In 2014, Maslovskaya et al. isolated new oxidative endoperoxides crotofolane derivatives, 57 and 58 from Croton insularis stem. These compounds exhibited unusual peroxide bridges, which were proposed to be derived from 4 via a singlet oxygen [4 + 2] cycloaddition.[15] Compound 57 displayed efficacy against HeLa and MM96L cells (IC₅₀s 10 and 6 μg/mL, respectively) in the MTS assay.[15]

Isocrotofolane diterpenoids

Isocrotofolane glucoside (59) possessed a new rearranged carbon framework with a β-glucopyranoside moiety at C-6 that was obtained from the leaves of Croton cascarilloides.[8]

Crotofolane diterpenoids alkaloids

In 2018, Gao et al. isolated and identified diterpenoid alkaloids: 60-62 from Croton cascarilloides twigs and leaves. These compounds have crotofolane diterpene tricyclic core fused with α, β-unsaturated γ-lactam.[23] Compound 60 features 5,6- and 4,14-epoxide moieties, with 2R/4S/5S/6S/7S/9S/13S/14R configuration based on X-ray. Compound 61 is a stereoisomer of 60 with different 4,14-epoxide configuration, while 62 has oxygenated C-9 to form hemiketal amine. These compounds originated from crotofolane diterpenoids, through aminolysis and oxidative modifications. Compounds 61 and 62 demonstrated moderate immunosuppressive activities by inhibiting ConA-caused T-lymphocyte cell proliferation and LPS-induced B lymphocyte cell proliferation (IC₅₀s 16.27 and 10.29 μM, respectively), compared to cyclosporin A, indicating the C-9 hemiketal amine group has a crucial role in the immunosuppressive activity.[23]

CONCLUSION

Crotofolane diterpenoids, a structurally distinct and physiologically varied class of natural metabolites, are only found in the Croton genus. Their chemotaxonomic significance is reflected in their prevalence among geographically different Croton species, especially C. cascarilloides. They have a wide variety of structural diversity from the normal crotofolanes to rearranged nor- and seco-forms, endoperoxides, and alkaloid derivatives. Some crotofolanes demonstrated moderate cytotoxic potential, while others had substantial anti-HIV and antimalarial properties, as well as immunosuppressive potential. Further researches are needed to explore their mechanisms of action and to assess other possible bioactivity and their safety profiles.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

The Deanship of Scientific Research (DSR) at King Abdulaziz University (KAU), Jeddah, Saudi Arabia has funded this project, under grant no. (IPP: 25-166-2025).