Antitumor Profile of Carbon-Bridged Steroids (CBS) and Triterpenoids

This review focuses on the rare group of carbon-bridged steroids (CBS) and triterpenoids found in various natural sources such as green, yellow-green, and red algae, marine sponges, soft corals, ascidians, starfish, and other marine invertebrates. In addition, this group of rare lipids is found in amoebas, fungi, fungal endophytes, and plants. For convenience, the presented CBS and triterpenoids are divided into four groups, which include: (a) CBS and triterpenoids containing a cyclopropane group; (b) CBS and triterpenoids with cyclopropane ring in the side chain; (c) CBS and triterpenoids containing a cyclobutane group; (d) CBS and triterpenoids containing cyclopentane, cyclohexane or cycloheptane moieties. For the comparative characterization of the antitumor profile, we have added several semi- and synthetic CBS and triterpenoids, with various additional rings, to identify possible promising sources for pharmacologists and the pharmaceutical industry. About 300 CBS and triterpenoids are presented in this review, which demonstrate a wide range of biological activities, but the most pronounced antitumor profile. The review summarizes biological activities both determined experimentally and estimated using the well-known PASS software. According to the data obtained, two-thirds of CBS and triterpenoids show moderate activity levels with a confidence level of 70 to 90%; however, one third of these lipids demonstrate strong antitumor activity with a confidence level exceeding 90%. Several CBS and triterpenoids, from different lipid groups, demonstrate selective action on different types of tumor cells such as renal cancer, sarcoma, pancreatic cancer, prostate cancer, lymphocytic leukemia, myeloid leukemia, liver cancer, and genitourinary cancer with varying degrees of confidence. In addition, the review presents graphical images of the antitumor profile of both individual CBS and triterpenoids groups and individual compounds.


Introduction
In both natural and synthetic steroids, when an additional ring is formed within the steroid skeleton, through a direct bond between any two carbon atoms (or more) of the steroid ring system or an attached side chain, such steroids (or triterpenoids) are called carbon-bridged steroids [1,2]. Analyzing the literature data from 1920, we concluded that the first mention of cyclopropane-containing hormones appeared in the mid-1930s of the twentieth century [2][3][4]. Steroids containing a cyclopropane ring in the side chain, such as gorgosterol, were first isolated from marine organisms in the early 1940s [4][5][6], and other 22,23-cyclopropyl sterols, such as dimethyl-gorgosterol, acanthasterol, demethylacanthasterol, acanthastanol, and 9,11-secogorgosterol, all of which have 22R, 23R and 24R configurations, have been isolated from marine sources [7][8][9][10][11][12]. Natural triterpenes containing a cyclopropane ring, and called cycloartanes, were first found in the early 1950s [13][14][15]. Table 1. Biological activities of cyclopropane-containing carbon-bridged steroids.
The Parthenium argentatum (commonly known as guayule) extract contained a cytotoxic steroid named argentatin A (36), which showed a cytotoxic effect against the human colon cancer cell lines (HCT15, HCT116, and SW620) and normal epidermal keratinocytes cell line [80].
A hexane extract of the wood of Dysoxylum muelleri has a yielded triterpenoid called dysoxin 3b (72), and dysoxylic acid A (73) was isolated from the hexane extract of the wood and bark of Dysoxylum pettigrewianum [102,103]. Dichapetalins are a small group of triterpenoids found primarily in the Dichapetalaceae and Euphorbiaceae. Thus, bioactive dichapetalins A (74), C (75), E (76), and G (77) were found in extracts of the roots of Dichapetalum madagascariense, and dichapetalin A (74) showed a strong and selective cytotoxic activity [104,105]. The aerial parts of Phyllanthus acutissima contained in CH 2 Cl 2 extracts of several dichapetalin-type triterpenoids, acutissimatriterpenes A (78, chemical structures 78-89 are shown in Figure 6, and their biological activity is shown in Table 6), B (79), C (80), D (81), and E (82). The obtained compounds were demonstrated cytotoxic and anti-HIV-1 activities [106]. The 90% MeOH-soluble fraction of the leaves of Dysoxylum cumingianum led to the isolation of triterpenoids (84 and 85), which showed significant enhanced cytotoxicity in the presence of colchicine, indicating that they might have some MDR-reversal effect [107].      Natural ecdysteroids are found in marine invertebrates, insects, or plants, and they provide a remarkable resource of insect hormone analogues that influence insect development and metamorphosis and thus play a significant role in the chemical interactions between some marine invertebrates and insects [108]. Rare 14-deoxy-14,18-cyclo-20-hydroxyecdysone (86) was obtained by photochemical transformation of 20-hydroxyecdysone [109].
A pentacyclic 3α,5α-cyclopregnane-type framework steroids represent a small group of natural lipids related to carbon-bridged steroids. These steroids have been found in both marine invertebrates and some terrestrial species. Two cytotoxic steroids, vladimuliecins A (90) and B (91), were isolated from the rhizome of Vladimiria muliensis. Both steroids demonstrated the cytotoxicity against cancer cell lines, including human leukemia cell (HL-60), human hepatoma cell (SMMC-7721), and human cervical carcinoma cell (HeLa) lines [115]. Chemical structures 90-102 are shown in Figure 7, and their biological activity is shown in Table 7.     An unusual steroid, named withawrightolide (92), was detected and isolated from the aerial parts of Datura wrightii (family Solanaceae). Isolated steroid showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cancer cell lines [116].
A rare steroid named calysterol (129), the minor sterol component of the sponge Calyx niceaensis and Petrosia ficiformis, possessing the unique feature of a cyclopropene ring bridging C23,24 [145][146][147], and isocalysterol (130), was detected in the same sponge [148].   The dichloromethane-2-propanol (1:1) extract of the Indonesian marine sponge Strepsichordaia aliena yielded 20,24-bishomoscalarane sesterterpenes named honulactones A (131), B (132), E (133), F (134), and G (135). Honulactones A and B exhibited cytotoxicity against P-388, A-549, HT-29, and MEL-28 (at IC 50 1 µg/mL) human tumor cell lines [149], and honu'enone (136) [150]. Chemical structures 131-142 are shown in Figure 10, and their biological activity is shown in Table 10.    It is known that human skin is responsible for the production of vitamin D. When exposed to ultraviolet radiation, which penetrates the epidermis and photolysis provitamin D3 to previtamin D3, and is photolyzed to 5,6-transvitamin D3 and two cyclopropanecontaining derivatives of vitamin D3, suprasterol I (137) and suprasterol II (138). The resulting photolysis products are used for the treatment and prevention of psoriasis [151]. Mushrooms are a rich source of ergosterol, which is a precursor to vitamin D2. Wild-grown mushrooms have been shown to contain small amounts of vitamin D2. In addition, it is known that the content of vitamin D2 and its derivatives such as suprasterol I and II in cultivated mushrooms increases when exposed to artificial ultraviolet radiation. In addition, vitamin D2 and its derivatives suprasterol I and II have been found in mushrooms Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes, as well as several mushroom powders, Pleurotus eryngii, and Agaricus bisporus [152]. When studying the photosynthesis of vitamin D, using the modelling of non-adiabatic molecular dynamics, another cyclopropane-containing metabolite (139) was identified [153].
It is known that carbon-bridged steroids are a rare group of synthetic lipids that are interesting, both in the beauty of the chemical structure, and show a wide range of biological activities. We have selected several carbon-bridged steroids containing a cyclopropane ring in the molecule that are not found in nature (149-164, chemical structures 149-164 are shown in Figure 12, and their biological activity is shown in Table 11). This is done to compare the biological activities of natural and synthetic steroids [18].     Thus, 6β-hydroxy-3α,5-cyclo-5α-androstan-17-one (149), and other analogues (150, 151 and 158) were synthesized as steroidal blood pressure-lowering hormones [160,161]. Cyclosteroids (152 and 153), which show an anabolic effect, were synthesized from 19-nor steroids, and would be of great interest for sports medicine as representatives of anabolic steroids [162,163], although other cyclosteroids (154)(155)(156)(157) were synthesized as potential agents with antitumor properties [164][165][166].

Cyclobutane Containing Steroids and Triterpenoids
The cyclobutane unit is found as a basic structural element in a wide range of naturally occurring compounds in bacteria, fungi, plants, and marine invertebrates [18,19,[169][170][171][172][173][174]. The chemistry and biochemistry of cyclobutanes is widely described in the scientific literature and is of great interest to chemists and pharmacologists, since many representatives of this class of compounds demonstrate a wide range of biological activities [18,19,73,[175][176][177][178].
Scalarane sesterterpenoids 20,24-bishomoscalaranes, carteriofenones Е(184), F (185), G (186), and H (187) were obtained from the marine sponge Carteriospongia foliascens, collected from the South China Sea. These compounds represented rare, naturally occurring scalaranes with a cyclobutane ring [193]. Chemical structures 184-196 are shown in Figure 14, and their biological activity is shown in Table 13.    The shrub Phyllanthus hainanensis, which is endemic to the island of Hainan province of China, has been used in traditional Chinese medicine for over 1000 years, has great pharmaceutical potential to treat diseases such as cancer and diabetes, and is also used to prevent, and treat, chronic hepatitis B virus infection [194,195]. Several highly modified triterpenoids, with a new carbon skeleton by incorporating two unique motifs of a 4,5and a 5,5-spirocyclic systems and containing cyclopropane and cyclobutene fragments, named phainanoids A (188), B (189), C (190), D (191), E (192), F (193), G (194), H (195), and I (196), have been determined in the extracts of the Phyllanthus hainanensis [196,197]. All compounds exhibited exceptionally potent immunosuppressive activities in vitro against the proliferation of T and B lymphocytes. The most potent one, phainanoid F, showed activities against the proliferation of T cells with IC 50 value of 2 nM (positive control CsA = 14 nM) and B cells with IC 50 value of <1.6 nM (CsA = 352.8 nM), which is about 7 and 221 times as active as CsA, respectively.

No. Antitumor & Related Activity, (Pa) * Lipid Metabolism Regulators, (Pa) * Additional Predicted
Trichoside B (197, chemical structures 197-212 are shown in Figure 15, and their biological activity is shown in Table 14), withanolide glucoside, has been isolated from the n-butanolic fraction of the 75% methanolic extract of aerial parts of Tricholepis eburnea [198], and other unusual cyclobutene, containing secosteroid (198), was detected in oil from a pineal tropical plant Sida cordata (family Malvaceae), which is used to treat various diseases and ailments in many complementary and alternative medicine systems [199]. Studying the photoproducts obtained by photochemical processes of vitamin D, cyclobutane, containing vitamin D (199), was identified [200]. Toxisterol (200), as a minor transformation product of vitamin D2, has been found in various mushrooms [152].      A unique non-olefinic product containing a cyclobutane fragment (201) was obtained from 5,10-seco steroid containing ∆ 1(10) -and ∆ 5(6) -double bonds in the AB ring during photochemical transformation [201]. The steroid altrenogest, a progestin of the 19-nortestosterone group, which is widely used in veterinary medicine to suppress or synchronize estrus in horses and pigs, using photolysis experiments gives two photoproducts: (202) and (203) [202].

No. Antitumor & Related Activity, (Pa) * Lipid Metabolism Regulators, (Pa) * Additional Predicted
In the chemistry of steroid hormones, the modification of the skeleton of natural steroids is used to obtain compounds with a narrower and more targeted spectrum of biological action, which makes it possible for their practical application. Among the many types of such transformed steroids, compounds containing an additional carbocycle are of great interest [203][204][205].

Comparison of Biological Activities of Natural and Synthetic CBS and Triterpenoids
It is known that the chemical structure of both natural and synthetic molecules predetermines biological activity, which makes it possible to analyze the structure-activity relationships (SAR). Such a wise idea was first proposed by Brown and Fraser more than 150 years ago, in 1868 [242]; although, according to other sources, SAR originates from the field of toxicology, according to which Cros, in 1863, determined the relationship between the toxicity of primary aliphatic alcohols and their solubility in water [243]. More than 30 years later, Richet in 1893 [244], Meyer in 1899 [245], and Overton in 1901 [246] separately found a linear correlation between lipophilicity and biological effects. By 1935, Hammett [247,248] presented a method of accounting for the effect of substituents on reaction mechanisms using an equation that considered two parameters, namely the substituent constant and the reaction constant. Complementing Hammett's model, Taft proposed, in 1956, an approach for separating the polar, steric, and resonance effects of substituents in aliphatic compounds [249]. Combining all previous developments, Hansch and Fujita laid out the mechanistic basis for the development of the QSAR method [250], and the linear Hansch equation, and Hammett's electronic constants, are detailed in the book by Hansch and Leo published in 1995 [251].
Some well-known computer programs can, with some degree of reliability, estimate the pharmacological activity of organic molecules isolated from natural sources or synthesized compounds [252][253][254]. It is known that classical SAR methods are based on the analysis of (quantitative) structure-activity relationships for one or more biological activities, using organic compounds belonging to the same chemical series as the training set [255].
Computer program PASS, which has been continuously updating and improving for the past thirty years [256], is based on the analysis of a heterogeneous training set included information about more than 1.3 million known biologically active compounds with data on ca. 10,000 biological activities [257,258]. Chemical descriptors implemented in PASS, which reflect the peculiarities of ligand-target interactions, and the original realization of the Bayesian approach for elucidation of structure-activity relationships provides the average accuracy, and predictivity, for several thousand biological activities equal to about 96% [259,260]. In several comparative studies, it was shown that PASS outperforms, in predictivity, some other recently developed methods for the estimation of biological activity profiles [261][262][263]. Freely available via the Internet, PASS Online web-service [264] is used by more than thirty thousand researchers from almost a hundred countries to determine the most promising biological activities for both natural and synthetic compounds [258][259][260]265]. To reveal the hidden pharmacological potential of the natural substances, we are successfully using PASS for the past fifteen years [266][267][268][269][270].
In the current study, we obtained PASS predictions for about three hundred steroids and triterpenoids produced by different living organisms. PASS estimates are presented as Pa values, which correspond to the probability of belonging to a class of "actives" for each predicted biological activity. The higher the Pa value is, the higher the confidence that the experiment will confirm the predicted biological activity [260].

Antitumor Activity of Cyclopropane-Containing CBS and Triterpenoids
Analyzing the data obtained using the PASS of natural cyclopropane containing steroids and triterpenoids, it can be stated that, out of 102 lipid molecules (1-102, see Figures 1-7 and Tables 1-7), only 27 showed antitumor activity with a reliability of more than 90 percent, with two steroidal glycosides, (25) and (41), showed antitumor activity with more than 99% confidence. Thus, PASS has confirmed the cytotoxic properties of these steroids, which have been determined experimentally. Other sterols and triterpenoids, with a cyclopropane ring, demonstrated weak to moderate antitumor activity with 70 to 90 percent confidence.
Triterpenoid saponins, (146,98.7%), (147,98.0%), and (148, 96.9%), containing the cyclopropane ring at position 15:27, were isolated from the leaves and flowers extracts of Verbesina virginica, demonstrating the highest degree of confidence-more than 96%. A 3D graph of the predicted antitumor and related activities is shown in Figure 23.  : 146, 147, and 148) showing the highest degree of confidence, more than 96%, which were isolated from the leaves and flowers extracts of Verbesina virginica, and can be used in clinical medicine as potential agents with strong antitumor activity. showing the highest degree of confidence, more than 91%. These steroids derived from marine sponges Petrosia weinbergi, Xestospongia sp., Poecillastra compressa, and Tethya sp., and can be used in clinical medicine as potential agents with strong antitumor activity.
Triterpenoid saponins, (146,98.7%), (147,98.0%), and (148, 96.9%), containing the cyclopropane ring at position 15:27, were isolated from the leaves and flowers extracts of Verbesina virginica, demonstrating the highest degree of confidence-more than 96%. A 3D graph of the predicted antitumor and related activities is shown in Figure 23.  : 146, 147, and 148) showing the highest degree of confidence, more than 96%, which were isolated from the leaves and flowers extracts of Verbesina virginica, and can be used in clinical medicine as potential agents with strong antitumor activity.

Antitumor Activity of Cyclobutane-Containing CBS and Triterpenoids
Cyclobutane containing steroids and triterpenoids , isolated from natural sources as well as semi-and synthetic compounds, were also analyzed using PASS. Most of these lipid molecules showed moderate antitumor activity with 70 to 90 percent confidence, and only three, (197,92.9%), (206,90.8%), and (214, 90.9%), steroids showed antitumor activity with more than 90% confidence. A 3D graph of the predicted antitumor and related activities is shown in Figure 24.

Antitumor Activity of Cyclobutane-Containing CBS and Triterpenoids
Cyclobutane containing steroids and triterpenoids , isolated from natural sources as well as semi-and synthetic compounds, were also analyzed using PASS. Most of these lipid molecules showed moderate antitumor activity with 70 to 90 percent confidence, and only three, (197,92.9%), (206,90.8%), and (214, 90.9%), steroids showed antitumor activity with more than 90% confidence. A 3D graph of the predicted antitumor and related activities is shown in Figure 24. The withanolide glucoside named trichoside B (197) is of type A-nor-sterols, and was isolated from the methanolic extract of aerial parts of Tricholepis eburnea, which is native to Afghanistan, compound (206) is a testosterone derivative dimer, and the steroid (214) contains a cyclobutane ring in ring A of the steroid.

Miscellaneous Cyclosteroids and Triterpenoids
Miscellaneous cyclosteroids and triterpenoids (222-276, see , and Tables 16-18) make up one-fifth of all compounds presented in this work. Two-thirds of lipid molecules demonstrate moderate activity, and seventeen compounds show strong antitumor activity with a confidence level of more than 90%, and the triterpenoid called taccalonolide Q (271) has the widest spectrum of biological activities among antitumor agents. A 3D graph of the predicted antitumor activities is shown in Figure 25. The data we obtained using PASS are supported by the data just published by Peng and colleagues, which shows a wide range of biological activities of taccalonolides [271]. The withanolide glucoside named trichoside B (197) is of type A-nor-sterols, and was isolated from the methanolic extract of aerial parts of Tricholepis eburnea, which is native to Afghanistan, compound (206) is a testosterone derivative dimer, and the steroid (214) contains a cyclobutane ring in ring A of the steroid.

Miscellaneous Cyclosteroids and Triterpenoids
Miscellaneous cyclosteroids and triterpenoids (222- 276, see Figures 17-21, and  Tables 16-18) make up one-fifth of all compounds presented in this work. Two-thirds of lipid molecules demonstrate moderate activity, and seventeen compounds show strong antitumor activity with a confidence level of more than 90%, and the triterpenoid called taccalonolide Q (271) has the widest spectrum of biological activities among antitumor agents. A 3D graph of the predicted antitumor activities is shown in Figure 25. The data we obtained using PASS are supported by the data just published by Peng and colleagues, which shows a wide range of biological activities of taccalonolides [271].
1 Figure 25. The 3D graph shows the predicted and calculated pharmacological activities of taccalonolide Q (271). Taccalonolide Q, similar to other taccalonolides, is a class of highly acetoxylated pentacyclic steroids containing 28 carbons, known microtubule stabilizing cytotoxic agents isolated from the genus Tacca that have selective anti-cancer properties. Taccalonolide Q has a C2-C3 epoxide group and an enol-γ-lactone fused with the unique E ring. In addition to total antineoplastic activity with a high confidence level of 93%, taccalonolide Q demonstrates selective activity against renal cancer, sarcoma, pancreatic cancer, lymphocytic leukemia, myeloid leukemia, and genitourinary cancer.

Conclusions
This review focuses on a rare group of carbon-bridged steroids (CBS) and triterpenoids found in lipid extracts from various natural sources such as green, yellow-green, and red algae, sea sponges, soft corals, ascidians, starfish, and other marine invertebrates. These compounds are also found in amoebas, fungi, fungal endophytes, and plants. There are 276 steroids and triterpenoids presented in this review, which demonstrate a wide range of biological activities, but the most pronounced antitumor profile. This review summarizes biological activities as experimentally obtained and published in the open press, as well as by using the extensive PASS program. We must state that two-thirds of carbon-bridged steroids and triterpenoids show moderate activity levels with 70 to 90% confidence, and only one-third of these lipids show strong antitumor activity with more than 90% confidence. All lipid material presented is divided into four groups, which include: (a) CBS and triterpenoids containing a cyclopropane moiety; (b) CBS and triterpenoids with cyclopropane ring in the side chain; (c) CBS and triterpenoids containing a cyclobutane moiety; (d) CBS and triterpenoids containing cyclopentane, cyclohexane, or cycloheptane moieties. The most important conclusion shows that some CBS and triterpenoids from different lipid groups demonstrate selective action on different types of tumor cells, such as renal cancer, sarcoma, pancreatic cancer, prostate cancer, lymphocytic leukemia, myeloid leukemia, liver cancer, and genitourinary cancer with different degree of reliability.