Chapter 6 Device Command Set
6.4 Run Control
6.5.7 Hardware Breakpoint Query: Retrieve a list of supported hardware breakpoint types
Isolation and Biological Activities of Marine Sulfated Steroids
Francisca Carvalhala, Marta Correia-da-Silvaa,c*, Emília Sousaa,c, Madalena Pinto,a,c Anake Kijjoa b,c
a
Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas,
Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228,
4050-313 Porto, Portugal
b
Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge
Viterbo Ferreira, 228, 4050-313 Porto, Portugal
c
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de
Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos,
Portugal
*corresponding author at Laboratório de Química Orgânica e Farmacêutica, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; e- mail: [email protected].
Abstract
Marine world is rich in unique molecules and can be an inspiring source of novel drugs. Currently, seven marine-derived drugs are in the market with FDA approval and several more are in the clinical pipeline. Many secondary metabolites, with a large structural diversity, have been isolated from the marine world, namely sulfated steroids. In this work, the biological activities described so far for marine sulfated steroids, namely antimicrobial, antitumor, cardiovascular, osteoblastic proliferation, and antifouling were compiled and discussed. Structure-activity relationship concerning the diverse steroid scaffolds, the number and position of sulfate groups is indicated. This work puts forward the potential of marine sulfated steroids for both pharmaceutical and chemical industries.
Introduction
Sulfur, in the form of sulfate salt, is the third most abundant element in seawater. 78 So, it is
not a surprise that sulfated compounds are extensively distributed in marine organisms. 79 Sulfated
compounds are found in a wide variety of organisms, from prokaryotes to multicellular species, being flavonoids the phenolic small molecules with more sulfated derivatives described from plants and sea grass. 59 In marine biota several sulfated secondary metabolites of different chemical group
(e.g., terpenoids, carotenoids, steroids, alkaloids, etc.) have been identified, 79 but they are not
evenly distributed among the different phyla, as few are found in microorganisms and marine macroalgae, in contrast to the most part discovered in marine invertebrates. 79-80 Sulfated sterols
constitute one of the most numerous groups of these secondary metabolites, but biological functions of sulfated steroids are still poorly understood. 36 However, some are known for their use
in chemical protection against predators, 81 pathogenic or fouling microorganisms. 33 Others,
structurally close to bile acids and alcohols of higher animals, are capable of facilitating food absorption and digestion in some invertebrates. 82
Nearly 300 sulfated steroids were isolated from various marine organisms, but mostly in two large phyla of marine invertebrates, echinoderms (Echindermata) and sponges (Porifera) (Figure 1). However sulfated steroids were also found in other marine organisms, like in the bile acid of a marine teleost Mola mola L. (sunfish), 83 a marine microalga, Hymenomonas sp., 84 the
marine annelid Arenicola cristata (lugworm), 85 the urine of teleost, like female Atlantic salmon 86
and the bile of the salmon shark, Lamna ditropis. 87
Figure 1. Marine invertebrates from where sulfated steroids were mostly isolated.
Marine sulfated steroids can be divided into mono-, di-, and trisulfated steroids, depending on the number of sulfate groups and most part belong to sterols class. Exceptionally, it was reported the isolation of some steroids dimers with more than three sulfate groups (Figure 2). Amaroxocane B, with two sulfated cores bridged by an oxocane, was isolated from sponge Phorbas amaranthus. 81 Fibrosterol sulfate A and C, with four sulfate groups, and fibrosterol
Echinodermata
Porifera
sulfate B, with five sulfate groups, were obtained from Lissodendoryx (Acanthodoryx) fibrosa, and are composed of two cholestane monomers, with differing configuration at C-3 and oxygenation at C-22 in only one monomer. 88 At least manadosterol A and B with a substituted ciclopentane ring
in the connecting side chain like fibrosterol A and B, were isolates from the marine sponge Lissodendryx fibrosa. 89 Regarding the referred derivatives, their biological activities will be
detailed in the next section.
Figure 2. Sulfated steroids dimers with more than three sulfate groups.
From literature, it is evident that monosulfated steroids are more abundant than disulfated, and disulfated steroids more than trisulfated.
Monosulfated steroids were mostly isolated from echinoderms, particularly starfish, 90-101
although di- and trisulfates were mainly found in sponges. 102-113 The disulfates were also found in
starfishes, 97 and in brittle stars, 114 and the trisulfated steroids in starfishes. 115 Monosulfated
steroids, besides having higher diversity among sulfated steroids (Figure 3), are more widespread in the marine species, since it was found in microalgae, 84 teleost, 83 annelied, 85 among others.
Most part of the isolated monosulfated steroids possesses the sulfate group in the ring system and only a few in the side chain. Different positions for the sulfate group in the ring system were found, but the most usual position is at carbon 3 on ring A (Figure 3.a.). 37, 90, 93-95, 98-100, 113, 115- 138 In the same ring, sulfate group can be in position C-2 (Figure 3.b.) 109, 139-140; the second most
common position is the sulfate group in C-6, in the ring B (Figure 3.c.)92, 97, 125, 141-147; or it can be
Figure 3. Different positions found in the isolated monosulfated steroids, in which sulfate group is present in the main chain. Highlighted is the most common position of the sulfate group.
All monosulfated steroids are simultaneously hydroxylated and sulfated (Figure 4.a.), except for hymenosulfate, isolated from Haptophyte Hymenomonas sp., that is persulfated. 84
Among the monosulfates exist a group of glycosylated steroids (Figure 4.b.), mostly found in starfish, with the carbohydrate moieties in the ring system 91 or in the side chain 92 and few with the
sugar moieties in both main and side chains. 101 Some of these steroidal glycosides sulfates are
considered classical asterosaponins, with the sulfate group in C-3 and the carbohydrate chain attached to the O group in C-6. 98 Seven compounds were also isolated carrying sulfate and
phosphate groups simultaneously in the ring system (Figure 4.c.). 100, 129
Figure 4. Types of monosulfated steroids found in marine biota.
The sulfate groups in disulfated steroids might be present in the in the ring system, and mostly, both in the ring system and side chain (Figure 5.a.). Steroids that have one sulfate group in the ring system and the other in the side chain have one common structure: a sulfate group in C-3,
150 with the exceptions of three sterols, which have a hydroxyl group in C-3 and a sulfate group at
position C-21 of the lateral chain. 102, 151 Disulfated steroids that have both sulfate groups in the ring
system can have both sulfate groups in ring A, 102-103 one sulfate in ring A and other in ring B, 97
and one sulfate in ring A and a sulfonamide in ring D (Figure 5.b.). 106, 152
Disulfated steroids are mainly simultaneously hydroxylated and sulfated (only few are persulfated - halistanol sulfate, 153 halistanol B disulfated, 104 lembehsterol B, 107 and sulfated
stanols 154. Contrary, most steroid trisulfates are persulfated. 115, 118 Among disulfated compounds,
Figure 5. Different positions found in the isolated disulfated steroids, in which the sulfate groups are present in the main chain. Highlighted is the most common positions of the sulfate groups.
Trisulfated steroids, as it happens in disulfated steroids, might have all the sulfate groups in the cyclic moiety or simultaneously in the ring system and side chain. In the case of trisulfated steroids, the three sulfate groups appear more frequently together in the ring system (Figure 6). 111- 112 Only three steroids were found with one sulfate group in the side chain. 115
Figure 6. Different positions found in the marine trisulfated steroids, in which sulfate groups are present in the main chain. Highlighted is the most common position of the sulfate groups.
The study of the biological/pharmacological activities of referred secondary metabolites can bring novel structures for the treatment of important pathologies, principally chemotherapeutic agents. In this work the biological activities of marine sulfated steroids, isolated between 1978 and 2015 were organized. Two nonsystematic studies were found concerning sulfated steroids: a review on marine polar steroids from 2001, which included a topic devoted to marine sulfated steroids, 36
and a book chapter from 2008, which included a topic concerning sulfated steroids but not necessarily steroids from marine origin. 155
Some of the most important pharmacological activities found in the sulfated steroids can be divided in: antimicrobial (antibacterial, antifungal, antiviral), antitumor, and cardiovascular (antihypertension, antiplatelet, hemolytic). Other activity was also described, namely the osteoblastic proliferation inhibition. Some sulfated steroids were also described with antifouling activity, showing their potential for different applications, other than therapeutic drugs. In this section, the most promising products are highlighted concerning their biological activities. Some
structure-activity relationships (SAR) considerations were tentatively established because very few derivatives were investigated and the studies are sparse.
Antimicrobial activity
Antibacterial
Several reasons contribute to the imperative need for new antibiotic agents as the fact that infectious diseases represent the second major cause of death in the world, the rapid development resistance by bacteria to therapeutic agents available, and the low turn on investiment, which is intrinsic to the lower numbers of new therapeutic agents approved by FDA. 156-157
Carboxylated sterol sulfates (compounds 1-3, Figure 7), isolated from the sponge Toxadocia zumi, showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus (at 100 μg/disk and 50 μg/disk, respectively). 37 In this study, the corresponding hydroxy-acids
were also tested and no activity was showed, indicating that both 19-carboxylic acid and 3β-sulfate groups were required for the activity. 37 Compound 4 (Figure 7), isolated from the starfish Luidia clathrata, also inhibited the growth of the bacterial species S. aureus and B. subtilus at a concentration of 50 μg/disk 33 and polymastiamide A (compound 5, Figure 7), extracted from the
marine sponge Polymastia boletiformis showed in vitro activity against S. aureus (100 μg/disk). 120
Polymastiamide A is the first example of a marine natural product with an uncommon side chain containing an aide bond linking the steroid part to a non-proteinaceous amino acid. 120 Annasterol
sulfate (compound 6, Figure 7), a marine sulfated steroid from the sponge Poecillastra laminaris, is an inhibitor of endo-β-1, 3-glucanase. The microbial cell wall of bacteria is composed principally of β-glucanases and the hydrolytic action of enzymes endo-β-1, 3-glucanase is detrimental to bacteria. Therefore, annasterol sulfate (6) showed activity against B. subtilus and Proteus vulgaris inhibiting endo-β-1, 3-glucanase at 1.0 and 2.8 μg/0.02 unit of activity. 124 Halistanol sulfate
(compound 7, Figure 7) is an antimicrobial steroid sulfate from the marine sponge Halicondria cf. moorei Bergquist, that inhibited the growth of Gram-positive and Gram-negative bacteria. 110 In
1981, the acid hydrolysis of halistanol sulfate was afforded and no longer antimicrobial activity was found. 110 Topsentiasterol sulfates A-E (compounds 8-12, Figure 7), isolated from the sponge Topsentia sp., are the first examples of isolated polysulfated steroids possessing a butenolide or a furan functionality at the end of the side chain. 158 These steroids were active against Pseudomonas aeruginosa and E. coli at 10 μg/ disk. 158
Figure 7. Sulfated steroids that showed antibacterial activity.
From the aforementioned studies, it is possible to highlight the large spectrum of action of steroid sulfates against bacteria and the constant presence of the sulfate group in C-3 position, and in the case of compound 4 in position C-15. Interesting to note, all the steroids with antibacterial activity were isolated from sponges, except compound 4 that was isolated from a starfish.
Antifungal
Antifungal sulfated steroids require particular attention as there is a constant need for new agents that would be effective against opportunistic fungal infections and could provide an alternative to chemical fungicides used to fight against pathogens. The discovery of new antifungal drug is necessary. 135
Polymastiamide A (compound 5, Figure 8) exhibited in vitro activity against Candida albicans and Pythium ultimum (75 and 25 μg/disk, respectively). 120 Like Polymastiamide A (40),
two minor sulfated steroid-amino acid conjugates, compounds 13 and 14 (Figure 8), were isolated from the sponge Polymastia boletiformis. 135 These compounds demonstrated moderate inhibitory
effects on C. cucumerinum at 60 and 30 μg/disc respectively (inhibition zones of 8.0 and 10.1 mm, respectively). Compound 14 also showed significant activity against Candida albicans at 100 μg/disc (inhibition zone diameter of 9.8 mm). 135
Ten new steroidal sulfates, acanthosterol A-J, were isolated from a marine sponge, Acanthodendrilla sp. 146 Among them, six were the first polyhydroxysteroids with an acetoxyl
group. Antiyeast tests were carried out only for acanthosterols I and J (compounds 15 and 16, respectively, Figure 8) that showed growth inhibitory activity against Saccharomyces cerevisae, strain A364A and its mutants. 146 Acanthosterol I (15) had an inhibitory zone of 7 mm against the
strain A364A and Acanthosterol J (16) showed 11 mm effect. Both showed a higher inhibitory zone against the mutant than wild-type strains. 146 Anasteroside A and B (compounds 17 and 18,
from each other for the lack of hydroxyl group at C-20 in anasteroside A (17) that showed to be the most active compound against the plant pathogenic fungus Cladosporium cucumerinum during this study (inhibition zone of approximately 12 mm at concentration value of 10 μg/spot), while anasteroside B (18), with a shorter side chain, was inactive at all tested concentrations. 130
Versicoside A (19, Figure 8) was the major saponin isolated from A. minuta and is structurally similar to anasteroside A and B, containing the same hexasaccharide chain, but different side chain of the aglycon. To evaluate the influence of the oligosaccharide moiety in the antifungal activity, versicoside A was enzymatically hydrollysed to give the triglycosyde forberoside H and the pentaglycoside thornasteroside A. While versicoside A and thornasteroside A were active (inhibition zone of approximately 8 mm and 5 mm at concentration value of 10 μg/spot, respectively), forberoside H showed no antifungal activity in all concentration range. 130 The results
obtained in this experimental work suggested that the side chain in the steroidal aglycon moiety, the structure of the sugar portion, and the presence of a sulfate group at the C-3, might play an important role in the antifungal activity of sulfate steroidal saponins. 130 Another three compounds
were isolated from the starfish Anasterias minuta two new steroidal xylosides, minutosides A and B (20 and 21, respectively, Figure 8), together with the known pycnopodioside B (22), 95 isolated
for the first time from the starfish Pycnopodia helianthoides. 94 Minutoside A (20) and pycnoposide
(22, Figure 8) exhibited antifungal activity against Cladosporium cucumerinum (inhibition zones of 7-10 mm at concentrations of 10-60 μg/spot). 95 125These compounds were also moderately
active against Aspergillus flavus with inhibition zones of 5-7,5 mm at concentrations of 5-10 μg/spot. Minutoside B (21), the first example of a steroidal xyloside containing an amide function in the aglycon from a natural source, showed activity only to the higgest concentration (20-60 μg/spot) against C. cucumerinum and A. flavus (inhibition zones of 3-4 mm and 5-10 mm, respectively). The desulfated analogues of compounds 22 and 20 were inactive against both fungi at all the tested concentrations. In the article was concluded that the presence of a sulfate group in the aglycon moiety could play an important role in the antifungal activity of the monoglycosides. 95
Topsentiasterol sulfates D and E (11 and 12, respectively, Figure 8) showed activity against C. albicans and Mortierella remannianus (at 10 μg/disc). 158
Figure 8. Sulfated steroids that showed antifungal activity.
From these studies, it is possible to conclude that sulfated steroids have a wide spectrum of action against fungus. These compounds have in common the sulfate group in position C-3 and in the majority a carbohydrate attached to the ring system.
Antiviral
In the area of antiviral research, the majority of studies performed are related with the human immunodeficiency virus (HIV), against which sulfated compounds have showed interesting inhibitory effects, 113 like weinbersterol disulfate A (23, Figure 9), from the sponge Petrosia weinbergi, that exhibited inhibitory activity with EC50 (half maximum effective concentration) values around 1.0 μg/mL. 103 Halistanol sulfate (7, Figure 9), ibisterol sulfate (24, Figure 9), 26-
methylhalistanol sulfate (25, Figure 9) and 25-demethylhalistanol sulfate (26, Figure 9), originally isolated from Halichondria cf. moorei (steroid 7), 110 Topsentia sp. (steroid 24) and Pseudaxinyssa digitate (steroids 25-26), showed essentially complete protection against the cytopathic effects of HIV-1 infection in the NCI primary screen (IC50, half maximum inhibitory concentrationvalues of
41 μg/mL for compound 7, > 128 μg/mL for compounds 24, 17 μg/mL for compound 25, and 56 μg/mL for compound 26). 111 These sterols were also tested against a strain of HIV-2 and showed
protection up to 70 or 80% (IC50 values of 41, 51, 94 and 56 μg/mL, for compounds 7, 24, 25 and
26), at a point that there was also observable toxicity toward the uninfected control. All these sterols share a common 2α, 3β, and 6α-trisulfate substitution pattern. 111 In 1994, several other
sterols were tested against HIV-1 and HIV-2 and in general sterols active against HIV-1 were also active against HIV-2. Sterols with sulfate groups on the A and B rings were the most active, although sterols with oxygenation in the D ring did not showed activity. 111
Haplosamates A and B (27 and 28), like Ibisterol sulfate B and C (29 and 30), were isolated from a Philippine sponge Xestospongia sp., and were shown to inhibit HIV type 1 integrase. 106113 Haplosamates A and B (27 and 28) represent the first examples of marine sterols
with the sulfamate functionality and an unprecedent six-membered ether ring. The IC50 values obtained were higher (50 μg/mL and 15 μg/mL, respectively) 106 than of compounds 29 and 30 (2.3 μg/mL and 1.8 μg/mL, respectively). 113 Clathsterol (31), collected from the Red Sea
sponge
Clathria sp., exhibited activity against HIV-1 reverse transcriptase at a concentration of 10 μg/mL. 159Activities against other viruses were also studied. The two new tetrahydroxylated steroid disulfates, weinbersterol disulfates A and B (compounds 23 and 32), isolated from the sponge Petrosia weinbergi, showed activity in vitro against feline leukemia virus (FeLV) (EC50 of 4.0 and 5.2 μg/mL respectively). These compounds possess an unprecedented cyclopropane-containing side chain, showing one more example for the diversity in the side chain structures of sponge sterols. 103 Compound 33, isolated from ophiuroid (or brittle star) Ophioplocus januarii, and
compounds 34-36, previously isolated from the Pacific ophiuroids Ophiocoma dentata, Ophiarthrum elegans, and Ophiarachna incrassate, 160 were tested for their inhibitory effect on the
replication of RNA (polio virus, PV; Junin virus, JV; respiratory syncytial virus, RSV) and DNA (Herpes simplex virus, HSV-1) viruses. Compounds 33 and 35 showed a marked RSV inhibitory activity (65% and 68% inhibition of RSV replication at 40 μg/mL, respectively) and proved to be active against PV, with a percentage reduction of cytopathic effect of 75 at 40 μg/mL. Compound 36 inhibited 54% of JV replication. HSV-1 was less sensitive to the sulfated sterols. Taking in account the results obtained in the antiviral tests and the fact that these four compounds only differ in the side chain, the authors indicate the importance of the ∆22 double bond (compounds 33 and 35) for the inhibitory effect on the replication of RSV also the ∆24(28) double bond (compound 36) on JV replication. 150 Compound 37, was the most effective inhibitor of HSV-1 (19.51.1
g/mL), HSV-2 (17.90.4 g/mL) and pseudorabies virus (PrV, 17.20.6 g/mL), from a series of steroids compounds. Sulfation was proved to be required for antiviral activity, since non sulfated parent compounds were not active. 161
Figure 9. Compounds tested for antiviral activity.
It is interesting to highlight that all the marine antiviral sulfated steroids were isolated from sponges and the majority were trisulfated or presented a common 2α, 3β-disulfate substitution pattern.
Antitumor activity
The National Cancer Institute found that bioassays with marine organism extracts were much more likely to yield anticancer drugs than terrestrial sources. 162
Cytoxicity
Echinoclasterol sulfate (38, Figure 10), contains an uncommon OH at C-17 and a unique phenethylamonium ion as counter ion, which was the first example in marine natural products. 139
It was isolated from the marine sponge Echinoclathria subhispida, and inhibited PC-9 human lung cancer cell line with an IC50 of 600 μM. 139 Compound 39 (Figure 10) showed cytotoxic activity
against the cell lines of murine fibro sarcoma, WEHI 164 (IC50 value of 50.4 μg/mL) and murine
Luidiaquinoside and psilasteroside (compounds 40 and 41, Figure 10), isolated from the starfish Luidia quinaria and Psilaster cassiope, showed marginal cytotoxicity in vitro against cell lines of rat basophilic leukemia (RBL-2H3) (IC50 of 31.3 and 5.4 μg/mL, respectively). 131
Compound 40 contains a novel pentasacharide chain composed of D-glucose, as the branching point, D-quinovose and D-frucose. Compound 41 (Figure 10) contains a hexassacaride side chain in which arabinose residue is detectable in the furanose form. 131
Asterasterol A (compound 43) was isolated from an Antarctic starfish of the family Asteriidae and represents the first marine naturally occurring steroid with a seven-membered lactone B ring. Two corresponding 6-oxo steroids were also isolated, asterasterol B and asterasterol C (42) that differ from each other for the presence of a ∆22 double bond in 42. All three were tested against human broncopulmonary non- small-cell-lung-carcinoma cells (NSCLC-N6), but only asterasterol C (42) showed moderate activity with IC50 higher than 30 μg/mL. 163 Arenicolsterol A
(compound 43), is an enolic sulfated sterol isolated from a marine annelid Arenicola cristata, that showed inhibitory effect on the growth of human cervix cancer cell line (Hela) and human non- small cell lung cancer cell line (NCI-h6), with IC50 of 3.1 μg/mL and 7.6 μg/mL and inhibition rate