Sulfated and Sulfur-Containing Steroids and Their Pharmacological Profile

The review focuses on sulfated steroids that have been isolated from seaweeds, marine sponges, soft corals, ascidians, starfish, and other marine invertebrates. Sulfur-containing steroids and triterpenoids are sourced from sedentary marine coelenterates, plants, marine sediments, crude oil, and other geological deposits. The review presents the pharmacological profile of sulfated steroids, sulfur-containing steroids, and triterpenoids, which is based on data obtained using the PASS program. In addition, several semi-synthetic and synthetic epithio steroids, which represent a rare group of bioactive lipids that have not yet been found in nature, but possess a high level of antitumor activity, were included in this review for the comparative pharmacological characterization of this class of compounds. About 140 steroids and triterpenoids are presented in this review, which demonstrate a wide range of biological activities. Therefore, out of 71 sulfated steroids, thirteen show strong antitumor activity with a confidence level of more than 90%, out of 50 sulfur-containing steroids, only four show strong antitumor activity with a confidence level of more than 93%, and out of eighteen epithio steroids, thirteen steroids show strong antitumor activity with a confidence level of 91% to 97.4%.

Studies of the last decades of marine sediments have shown that extracts from these geological formations contain a wide variety of steroids, terpenoids, and other lipids [22][23][24][25][26][27][28][29]. These findings indicate that such a variety of lipids is of organic origin, and, apparently,  Table 1.   Table 1. Several sulfated steroids have been found and isolated from the marine starfish. Thus, sulfated polyhydroxysteroid named microdiscusol G (3) was isolated from the Arctic starfish Asterias microdiscus. The 28-sulfooxy-24-methylcholestane side chain in (3) has been found among starfish steroid metabolites for the first time [48]. Two steroids (4 and 5) and an unusual glucoside (6) were isolated from the starfish Archaster typicus collected in shallow waters of the Quang Ninh province (Vietnam). Isolated compounds showed moderate toxic effects in the sperm and 8-blastomere tests on embryonal development of the sea urchin Strongylocentrotus intermedius [49,50].
Polyoxygenated steroids belonging to a new structural group of sponge steroids called gracilosulfates B (7) and D (8) were isolated from the marine sponge Haliclona gracilis. Both compounds inhibited the expression of prostate-specific antigen in a human prostate carcinoma epithelial tumor cell lines (22Rv1) [51]. Steroid glycoside, granulatosides D (9), belonging to the group of bi-and monoglycosides of polyhydroxysteroids, respectively, was isolated from the ethanolic extract of the starfish Choriaster granulatus. The obtained compound showed immunomodulatory properties, increasing the intracellular ROS (reactive oxygen species) level in peritoneal murine macrophages by 20% and decreasing intracellular ROS level by 21% in pre-treated with endotoxic lipopolysaccharide from E. coli peritoneal macrophages [52].
Cholesterol sulphate (14, structure see Figure 2 and activities see Table 2) has been isolated from the starfish Asterias rubens [55,56], and compound (14) and cholestanol sulphate (15) were identified as the major components among the 34 different sterol sulfates of the sea cucumber Eupentacta fraudatrix [57].
An Australian marine sponge Stilopus australis produced a sulfated steroid with the pregnane skeleton (30) [63], and annasterol sulfate (31) was isolated from the Pacific deep-water sponge Poecilastra laminaris, and this compound showed a β-1,3-glucanase inhibitor [64]. Polyhydroxy-steroid monosulphate (32, for structure see Figure 3 and for for activities see Table 3) was found in the extract of the sponge Toxadocia zumi [65]. Rare mono sulfate steroid with the sulfate group in 2β-position (33) was isolated from the sponge Echinoclathria suhispida collected from the Japan Sea near the coast of Japan [66]. The Malaysian sponge Haliclona sp. from an Indo-Pacific has yielded haliclostanone sulphate (34) [67]. Cytotoxic steroid (35) containing a sulfate group in 6α-position was found in the sponge Dysidea fragilis collected from the lagoon of Venice, Italy [68]. A sulfated steroid called apheloketotriol (36) was isolated from a Far Eastern sponge Aphelasterias japonica [69], and acanthosterol E (37) was found in the sponge Acanthodendrilla sp. containing sulfate group in 6-position [70].

Di-and Poly-Sulfated Steroids Derived from Marine Sources
Natural di-and poly-sulfates of steroids represent a rare group of bioactive lipids. Their total content in marine organisms is two to three times less than that of steroids containing one sulfate group [2,16,18,43]. The antiviral orthoesterol B (55) showed antiviral activity was found in the marine sponge Petrosia weinbergi [82]. Weinbersterol B (56), a sulphated tetrahydroxy steroid with an unprecedented cyclopropane-containing side chain, was isolated from the sponge Petrosia weinbergi. This compound is active in vitro against feline leukemia virus and active in vitro against HIV [83].
A polyhydroxylated sterol derivative called topsensterol B (65) has been isolated from a marine sponge Topsentia sp. collected from the South China Sea. The isolated compound exhibited cytotoxicity against human gastric carcinoma cell line SGC-7901 with an IC 50 value of 8.0 µM [98], and topsentiasterol sulphate E (66) was found in extracts of the sponge Spheciospongia sp., collected in the Philippines. This compound inhibited PKCzeta with an IC 50 value of 1.21 µM, and in a cell-based assay also inhibited NF-kappa B activation with EC 50 value of 12 µM [99].
Until now, there has been no evidence that microorganisms do not participate in the formation of sulfur-containing steroids, but nevertheless, these lipid markers are of great interest for biochemistry, geochemistry, and pharmacology [119,[120][121][122]. More than 3000 terpenoids and other lipid molecules have been isolated from sedimentary rocks and marine sediments-this is an established fact [28,30.33,39-42,111-114]. Sulfur-containing  Table 4.
An unusual pregnane-type steroid called krempene A (72, structures are shown in Figure 6 and activity showed in Table 5) was isolated from the marine soft coral Cladiella krempfi. The isolated compound contains a very unusual structural motif, with a hexacyclic oxadithiino unit fused to the steroidal ring A [128].  . Sulfur-containing steroids and triterpenoids derived from marine invertebrates, sediments, crude oil, and other sources, and their pharmacological profile is shown in Table 5.
Many authors have attempted to investigate the organic matter generated in hypersaline environments, including both sea and salt seas, and a warm eutrophic salt lake in Southern California, where sulfur-containing compounds have been found and isolated, including unusual thiosterols (81)(82)(83)(84)(85)(86)(87), and their unusual chemical structures have been established [113,114,[124][125][126][127]. Interesting data were obtained by Chinese scientists who studied the sulfur-rich heavy oils in Jinxian Sag, Bohai Bay Basin, northern China, and found high abundances of organic sulfur compounds, including a series of short-chain steranes ( Tables 5 and 6). The authors believe that the occurrence of abundant sulfur-containing steroids was the result of extensive sulfurization during early diagenetic stages, because many more double bonds, hydroxyl groups, and carbonyl groups exist in sterols and steranes, which are prone to attack by inorganic sulfur [141].  Tables 5 and 6). The authors believe that the occurrence of abundant sulfur-containing steroids was the result of extensive sulfurization during early diagenetic stages, because many more double bonds, hydroxyl groups, and carbonyl groups exist in sterols and steranes, which are prone to attack by inorganic sulfur [141].   Table 6. Several series of organic sulfur compounds have been identified in several oils and sediment extracts including Rozel Point Oil (Box Elder County, UT, USA, Miocene). Series of isoprenoid thiophenes, isoprenoid thiolanes, isoprenoid bithiophenes, isoprenoid thienylthiolanes, isoprenoid benzothiophenes, 2,5-di-n-alkylthiolanes, 2,6-di-n-alkylthianes and 2,4-di-n-alkyl-benzo(b)thiophenes, and several thiophene and thiolane steranes (84-86, 92, 93 and 95) have been identified [142]. A sulfur-containing sterane, 4α,7α-epithio-5βcholestane (96), has been identified in a sediment extract from the Miocene Northern Apennines marl (Italy) [143]. Another 3α,7α-epithio-24-Me-5β-cholestane (97) was isolated from the polar fractions of sediment extracts and a crude oil taken from a Jurfed Darawish oil shale Jordan [144]. C27-C29 homologs have been detected in sediment extracts of three different formations and in one petroleum sample. These sulfur-containing steroids are probably formed by an intramolecular reaction of inorganic sulfides with early diagenetic products of ∆5,7-sterols.
Several sulfur-containing compounds (86, 92, 94, 97, 99, 102, and 104) were isolated from water-methanol extracts of the organic phase of solutions obtained from treatment with microbial mats, which were deposited in a Lagoona setting, revealing three lithofacie, and limestones with fine-scale parallel laminations, limestones with undulated (stromatolite-type) laminations, and massive limestones. These different lithofacies refer to The Upper Jurassic Calcaires en plaquettes Formation, which is located on the southern Jura in France [145]. The triterpenoid thiane (105) was identified from organic matter in the Holocene and latest Pleistocene sediments of the Cariaco Basin, Venezuela [146].
A series of sulfur-containing sterols (98-101, 103, 107, and 108, for structure see Figure 8 and for activities see Table 7) have been characterized in a wide range of sediments, and they were isolated from a sample from Sémecourt (Paris basin, France) [147], and compound (109) was detected in petroleum from Alberta province, Western Canada [148]. A series of sulfur-containing sterols (98-101, 103, 107, and 108, for structure see Figure 8 and for activities see Table 7) have been characterized in a wide range of sediments, and they were isolated from a sample from Sémecourt (Paris basin, France) [147], and compound (109) was detected in petroleum from Alberta province, Western Canada [148].  Table 7. Table 7. Biological activities of sulfur-containing steroids.  Table 7.
A series of thiophenes with C-3 alkylated steroid carbon skeletons (114)(115)(116) have been identified in sediments of the Miocene Monterey Formation (California, USA) and in the Turonian Tarfaya basin (Morocco). Their carbon skeletons were unusual in the sense that the alkyl sidechains at C-3 are almost exclusively isopentyl, 3-methylpentyl, and 2,3dimethylbutyl moieties, whilst n-alkyl (pentyl or hexyl) moieties are almost absent [144].

Epithio Steroids
Semi-synthetic and synthetic epithio steroids represent a rare group of bioactive lipids, since they are hydrophobic molecules insoluble in water, which were not found in nature. Epithio steroids have been reported to possess a variety of cytotoxic activities, and they are widely used as anticancer agents. The thiirane group is an important substance and shows some promising biological activities.

Comparison of Biological Activities of Sulfated and Sulfur-Containing Steroids
The concept, which was formed more than 150 years ago, that the biological activity of natural and synthetic compounds depends on their chemical structure has been confirmed at the current time [165]. Using this concept, it is generally accepted that the biological activity of both natural and synthetic compounds depends on their chemical structure [166,167]. Apart from the sharp jumps in biological activity that are observed for some medicinal compounds [168], this can be considered a violation of this rule; however, for most chemical compounds, the structure-activity ratio (SAR) is widely used in medicinal chemistry and pharmacology to search for and optimize new pharmacological agents [169].
Software PASS is the first software for in silico estimation of biological activity profiles [170], of which the development was started more than 30 years ago [171]. Its current implementation predicts about 10,000 pharmacological effects, molecular mechanisms of action, pharmacological effects, toxicity, side effects, anti-targets, transporters-related interactions, gene expression regulation, and metabolic terms [166]. Due to the utilization of chemical descriptors that reflect the essential features of ligand-target interactions and a robust mathematical approach for analysis of structure-activity relationships, the average accuracy of PASS predictions was 96% [172]. Based on the PASS predictions provided by the appropriate web-service [173], over 29,000 researchers from 105 countries selected the most promising virtually designed molecules for synthesis and determined the optimal directions for testing their biological activity [174][175][176][177].
In this study, PASS predictions were used to estimate the general pharmacological potential for the analyzed natural, semi-synthetic, and synthetic sulfated and sulfurcontaining steroids, and triterpenoids. For about ten thousand pharmacological effects and molecular mechanisms of action, probabilities of belonging to the class of "active" Pa, varied from zero to one, were estimated. 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 probability of confirming the predicted activity in the experiment. On the other hand, estimated Pa values might be relatively small for some activities if the analyzed molecule is not like the active compounds from the PASS training set. Pa values are indicated for all steroids and triterpenoids presented in this article in Tables 1-8.
Since the end of March 2021, a new version of the PASS program has been used, with an increased amount of both natural and synthetic compounds, and, accordingly, the database of biological activities has increased.

Antitumor Activity of Natural Mono-, Di-, and Poly-Sulfated Steroids
Currently, about 7000 articles have been published covering various aspects of marine and terrestrial sulfated steroids and triterpenoids. Analyzing the data obtained using PASS compounds presented in this review, it can be stated that out of 71 marine sulfated steroids and triterpenoids, the activity is estimated with Pa from 68.6% to 94.8%, and only thirteen marine sulfated steroids demonstrate strong antitumor activity with a reliability of 90% to 94.8% (see Tables 1-4 Figures 1-5). However, most sulfated steroids exhibited moderate antitumor activity with 68% to 90% confidence. A 3D graph of the predicted antitumor and related activities is shown in Figure 10. In addition, many sulfated steroids exhibit moderate anti-hypercholesterolemic activity, and some steroids exhibit strong anti-hypercholesterolemic activity with a confidence level greater than 90% (see Figure 11). An interesting finding was that some sulfated steroids show wound-healing properties with more than 90% confidence, and this data is presented in Figure 12. Several sulfated steroids such as 1 (93.1%), 2 (92.7%), and 21 (92.8%) exhibit hemostatic properties with more than 92% confidence, and other steroids 29 (93.  Tables 1-4).

Biological Activity of Sulfur-Containing and Epithio Steroids
Sulfur-containing steroids are an interesting class of natural lipids, but most of these compounds exhibit weak or moderate antitumor activity. Some steroids have no antitumor activity, and only four steroids have strong antitumor activity. Figure 13 demonstrates the activity of these steroids. In addition, a sulfur-containing steroid called spironolactone (73) excreted from human urine is of interest. According to the PASS data, this steroid is multifunctional and demonstrates seventeen different activities. Figure 14 shows the pharmacological profile of this steroid. One feature should be noted for all sulfur-containing steroids: most of these compounds exhibit dermatological properties such as weak or moderate anti-eczematic and anti-psoriatic activity.   (43), and P- (52). Steroids that belong to this group, according to the data obtained by the PASS, have confirmed more than 90% of their biological activity.   (43), and P- (52). Steroids that belong to this group, according to the data obtained by the PASS, have confirmed more than 90% of their biological activity.  (43), and P- (52). Steroids that belong to this group, according to the data obtained by the PASS, have confirmed more than 90% of their biological activity.

Biological Activity of Sulfur-Containing and Epithio Steroids
Sulfur-containing steroids are an interesting class of natural lipids, but most of these compounds exhibit weak or moderate antitumor activity. Some steroids have no antitumor activity, and only four steroids have strong antitumor activity. Figure 13 demonstrates the activity of these steroids. In addition, a sulfur-containing steroid called spironolactone (73) excreted from human urine is of interest. According to the PASS data, this steroid is multifunctional and demonstrates seventeen different activities. Figure 14 shows the pharmacological profile of this steroid. One feature should be noted for all sulfur-containing steroids: most of these compounds exhibit dermatological properties such as weak or moderate anti-eczematic and anti-psoriatic activity.   These steroids show the highest degree of confidence, more than 92%. Figure 14. The 3D Graph (X and Y views) predicted and calculated activities of the spironolactone, which was isolated from the human urine extracts. According to PASS data, this steroid demonstrated seventeen different activities, with five activities having confidence of more than 90%. The main pharmacological properties and activities of spironolactone (73) are diuretic (99.1%), anti-hyperaldosteronism (96.3%), anti-hypertensive (94%), renal disease treatment (93.4%), and heart failure treatment (91.2%). As shown in numerous publications, spironolactone demonstrates properties as a potential diuretic, antihypertensive agent, or agent for the treatment of renal failure and heart failure, as well as an agent in the treatment of hyperaldosteronism (including Conn's syndrome) and female hirsutism (due to additional antiandrogenic actions). All these properties and activities are confirmed by the PASS data as shown in this figure and shown in Table 5. Figure 14. The 3D Graph (X and Y views) predicted and calculated activities of the spironolactone, which was isolated from the human urine extracts. According to PASS data, this steroid demonstrated seventeen different activities, with five activities having confidence of more than 90%. The main pharmacological properties and activities of spironolactone (73) are diuretic (99.1%), anti-hyperaldosteronism (96.3%), anti-hypertensive (94%), renal disease treatment (93.4%), and heart failure treatment (91.2%). As shown in numerous publications, spironolactone demonstrates properties as a potential diuretic, antihypertensive agent, or agent for the treatment of renal failure and heart failure, as well as an agent in the treatment of hyperaldosteronism (including Conn's syndrome) and female hirsutism (due to additional antiandrogenic actions). All these properties and activities are confirmed by the PASS data as shown in this figure and shown in Table 5.
Most epithio steroids are classified as anabolic steroids and are used in bodybuilding diets, as well as stimulants by athletes or a specific category of people during physical or physiological stress [152,[178][179][180].
Several epithio steroids shown in Table 7 show strong antitumor activity, and Figure 15 shows the activity of these steroids. In addition, some epithio steroids exhibit antisecretory activity with more than 90% confidence and can be classified as antisecretory drugs. It is known that the first antisecretory drugs (antimuscarinic) were available in the 1950s, and it was not until the 1970s that the first histamine antagonist appeared. This seems to be a watershed moment in the history of acid peptic ulcer treatment. Later in the 1980s, sulfur-containing ranitidine, omeprazole, lansoprazole, rabeprazole and other proton pump inhibitors, pantoprazole, esomeprazole, and dexlanzoprazole were discovered. Their effectiveness in reducing acid secretion has been associated with side effects such as osteoporosis, malabsorption of iron and vitamin B12, hypomagnesemia, acute and chronic kidney disease, dementia, acute myocardial infarction, Clostridium difficile infection, and others [181][182][183]. Figure 16 demonstrates the properties of some epithio steroids as potential agents with antisecretory activity.
As shown by the PASS data, the four epithio steroids 122, 123, 129, and 138 exhibit properties to increase the efficiency and improve the contraction of the heart muscle, i.e., are typical cardiotonic agents with a Pa confidence of 70% to 93.6%.
1980s, sulfur-containing ranitidine, omeprazole, lansoprazole, rabeprazole and other proton pump inhibitors, pantoprazole, esomeprazole, and dexlanzoprazole were discovered. Their effectiveness in reducing acid secretion has been associated with side effects such as osteoporosis, malabsorption of iron and vitamin B12, hypomagnesemia, acute and chronic kidney disease, dementia, acute myocardial infarction, Clostridium difficile infection, and others [181][182][183]. Figure 16 demonstrates the properties of some epithio steroids as potential agents with antisecretory activity.  As shown by the PASS data, the four epithio steroids 122, 123, 129, and 138 exhibit properties to increase the efficiency and improve the contraction of the heart muscle, i.e., are typical cardiotonic agents with a Pa confidence of 70% to 93.6%.

Conclusions
The review focuses on the interesting topic of sulfated steroids, which have been isolated from algae, sea sponges, soft corals, ascidians, starfish, and other marine invertebrates. In addition, sulfur-containing steroids and triterpenoids that are isolated from sedentary marine coelenterates, plants, marine sediments, crude oil, and other geological deposits are also presented in this review. Additionally, several semi-synthetic and synthetic epithiosteroids, which are a rare group of bioactive lipids that have not yet been found in nature but have a high level of antitumor activity, were included in this review for the comparative pharmacological characterization of this class of compounds.
This review presents 139 steroids and triterpenoids that exhibit a broad spectrum of biological activity. The data obtained with the PASS program show that thirteen sulfated steroids exhibit strong antitumor activity with a confidence level of more than 90%. Among the sulfur-containing steroids, only four show strong antitumor activity with a confidence level of more than 93%, although most of the presented epitosteroids show strong antitumor activity with a confidence level of 91% to 97.4%. These data are of great interest to pharmacologists and clinical physicians.