Occurrence and Properties of Thiosilvatins

marine drugs

Review Occurrence and Properties of Thiosilvatins

Maria Michela Salvatore 1 , Rosario Nicoletti 2,3 , Marina DellaGreca 1 and Anna Andolﬁ 1,*

1 Department of Chemical Sciences, University of Naples ‘Federico II’, 80126 Naples, Italy; [email protected] (M.M.S.); [email protected] (M.D.) 2 Council for Agricultural Research and Economics, Research Centre for Olive, Citrus and Tree Fruit, 81100 Caserta, Italy; [email protected] 3 Department of Agriculture, University of Naples ‘Federico II’, 80055 Portici, Italy * Correspondence: andolﬁ@unina.it; Tel.: +39-081-2539179

Received: 27 September 2019; Accepted: 21 November 2019; Published: 26 November 2019

Abstract: The spread of studies on biodiversity in different environmental contexts is particularly fruitful for natural product discovery, with the finding of novel secondary metabolites and structural models, which are sometimes specific to certain organisms. Within the large class of the epipolythiodioxopiperazines, which are typical of fungi, thiosilvatins represent a homogeneous family that, so far, has been reported in low frequency in both marine and terrestrial contexts. However, recent observations indicate that these compounds have been possibly neglected in the metabolomic characterization of fungi, particularly from marine sources. Aspects concerning occurrence, bioactivities, structural, and biosynthetic properties of thiosilvatins are reviewed in this paper.

Keywords: thiosilvatins; secondary metabolites; diketopiperazines; epipolythiodioxopiperazines; fungi; biological activities

1. Introduction A huge chapter on research on biodiversity is represented by studies concerning the biochemical properties of the manifold organisms which are part of natural ecosystems. Novel secondary metabolites are continuously discovered, disclosing a surprising chemodiversity in terms of both structural and biosynthetic aspects. Although most classes of compounds are spread throughout the several kingdoms of nature, a certain specificity results in some cases. An example in this respect is represented by the epipolythiodioxopiperazines (ETPs), so far only reported from fungi [1]. ETPs are a large and structurally diverse class of bioactive secondary metabolites originating from diketopiperazines and characterized by the presence of a disulfide bridge or a polysulfide dioxopiperazine six-membered ring. Due to their bioactivities, ETPs are receiving attention in recent years [2]. This review is focused on thiosilvatins, a specific family of ETPs resulting from the enzymatic assemblage of two amino acids (i.e., l-tyrosine/l-phenylalanine and glycine), generally integrated with two methylated sulfur atoms. Unlike a related family including hyalodendrin, gliovictin, and their analogues, in this homogeneous group of compounds, the pivotal nitrogen deriving from the aromatic amino acid is not engaged in structural modifications other than methylation (Figure1).

Mar. Drugs 2019, 17, 664; doi:10.3390/md17120664 www.mdpi.com/journal/marinedrugs Mar.Mar. Drugs Drugs 20192019, 17, 17, x, FOR 664 2 of2 of13 14 Mar. Drugs 2019, 17, x FOR 2 of 13

Figure 1. Basic structure of thiosilvatins. Figure 1. Basic structure of thiosilvatins. The finding of natural products displaying this kind of molecular structure only started in the The ﬁnding of natural products displaying this kind of molecular structure only started in the 1980s [3],The and finding even of nowadays natural products reports aredisplaying quite infrequent. this kind Theof molecular present paper structure provides only starteda review in ofthe 1980s [3], and even nowadays reports are quite infrequent. The present paper provides a review of the the1980s current [3], andknowledge even nowadays concerning repo occurrence,rts are quite bioact infrequent.ivities, structural,The present and paper biosynthetic provides aspectsa review of of current knowledge concerning occurrence, bioactivities, structural, and biosynthetic aspects of these thesethe currentcompounds. knowledge To ensure concerning a comparative occurrence, examinat bioactionivities, of the severalstructural, structures and biosynthetic reviewed herewith, aspects of compounds. To ensure a comparative examination of the several structures reviewed herewith, the thethese name compounds. of some compounds To ensure wasa comparative adapted to examinat conformion to previouslyof the several characterized structures reviewed analogs. herewith, namethe name of some of some compounds compounds was was adapted adapted to conform to conform to previously to previously characterized characterized analogs. analogs. 2. Structures and Chemical Properties 2. Structures and Chemical Properties (3R,6R)-1,4-Dimethyl-3-(4-(3-methyl-2-butenyloxy)benzyl)-3,6-bis(methylthio)piperazine-2,5- (3R,6,6RR)-1,4-Dimethyl-3-(4-(3-methyl-2-butenyloxy)benzyl)-3,6-bis(methylthio)piperazine-2,5-dione)-1,4-Dimethyl-3-(4-(3-methyl-2-butenyloxy)benzyl)-3,6-bis(methylthio)piperazine-2,5- dione(1), the (1), founder the founder product product of this of compound this compound series, series was, isolated was isolated for the for ﬁrst the time first in time 1981 in along 1981 withalong its withdione its (deprenyl1), the founder analogue product (16) [3] of. thisSubsequently, compound 1 series was named, was isolated cis-bis(methylthio)silvatin for the first time in when1981 along the deprenylwith its deprenyl analogue analogue (16)[3]. Subsequently, (16) [3]. Subsequently,1 was named 1 wascis -bis(methylthio)silvatinnamed cis-bis(methylthio)silvatin when the only when sulfur the onlybridged sulfur thiosilvatin, bridged thiosilvatin, dithiosilvatin dithiosilvatin (2), was characterized (2), was characterized and submitted and tosubmitted a reductive to a methylation reductive methylationonly sulfur giving bridged 1 and thiosilvatin, its epimer dithiosilvatin in C-6 (3) [4] ((Figure2), was 2).characterized and submitted to a reductive givingmethylation1 and giving its epimer 1 and in its C-6 epimer (3)[4] in (Figure C-6 (23).) [4] (Figure 2).

1 2 3 1 2 3 Figure 2. Structures of cis-bis(methylthio)silvatin (1, NM = 408 U), dithiosilvatin (2, NM = 378 U), Figuretrans-bis(methylthio)silvatin 2. Structures of cis-bis(methylthio)silvatin (3, NM = 408 U). (1, NM = 408 U), dithiosilvatin (2, NM = 378 U), transFigure-bis(methylthio)silvatin 2. Structures of cis-bis(methylthio)silvatin (3, NM = 408 U). (1, NM = 408 U), dithiosilvatin (2, NM = 378 U), trans-bis(methylthio)silvatin (3, NM = 408 U). The most relevant structural modiﬁcations observed in this class of compounds are in the numberThe most of sulfurs, relevant the structural degree ofmodifications methylation obse of heteroatoms,rved in this class and theof compounds presence of are a dimethyl in the The most relevant structural modifications observed in this class of compounds are in the numberallylic of chain sulfurs, (Table the3 ).degree A controversial of methylation issue of he concerningteroatoms, thiosilvatinsand the presence could of bea dimethyl represented allylic by number of sulfurs, the degree of methylation of heteroatoms, and the presence of a dimethyl allylic chainnomenclature. (Table 1). A controversial In fact, several issue compounds concerning werethiosilvatins inconsistently could be designatedrepresented with by nomenclature. trivial names, chain (Table 1). A controversial issue concerning thiosilvatins could be represented by nomenclature. Inabbreviations, fact, several compounds or according were to inconsistently the IUPAC standards, designated which with trivial generally names, are abbreviations, not linked to or the In fact, several compounds were inconsistently designated with trivial names, abbreviations, or accordingname of to the the founder IUPAC compound.standards, which For generally instance, are Sch not 54794 linked (4) to and the Sch name 54796 of the (5) founder have the according to the IUPAC standards, which generally are not linked to the name of the founder compound.same structures For instance, and stereostructures Sch 54794 ( of4) 1andand Sch3, but 54796 their (5 amino) have functions the same are structures not methylated; and compound. For instance, Sch 54794 (4) and Sch 54796 (5) have the same structures and stereostructuresconsequently, theyof 1 and could 3, but be their respectively amino functions named cis are- andnot metransthylated;-dinor-bis(methylthio)silvatin consequently, they could [5 ]. stereostructures of 1 and 3, but their amino functions are not methylated; consequently, they could beLikewise, respectivelycis-3-(4-hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)-2,5-piperazinedione named cis- and trans-dinor-bis(methylthio)silvatin [5]. Likewise, cis (16-3-(4-) and be respectively named cis- and trans-dinor-bis(methylthio)silvatin [5]. Likewise, cis-3-(4- hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)-2,5-piperazinedionetrans-6-(4-hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)piperazine-2,5-dione (16) (17and) were namedtrans-6-(4- in the hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)-2,5-piperazinedione (16) and trans-6-(4- hydroxybenzyl)-1,4-dimethyl-3,6-bis(original manuscripts according to IUPACmethylthio)piperazine-2,5-dione standards [3,6], but they could (17 be) easilywere namednamed respectively in the hydroxybenzyl)-1,4-dimethyl-3,6-bis(methylthio)piperazine-2,5-dione (17) were named in the originalcis- and manuscriptstrans-deprenyl-bis(methylthio)silvatin. according to IUPAC standards [3,6], but they could be easily named respectivelyoriginal manuscripts cis- and trans according-deprenyl-bis(methylthio)silvatin. to IUPAC standards [3,6], but they could be easily named respectivelyMany difficulties cis- and trans in -deprenyl-bis(methylthio)silvatin.nomenclature derive from the stereostructural aspects. Concerning compoundsMany 25difficulties–27, they were in nomenclaturenot named in thederive original from manu thescript stereostructural [7]; however, aspects. a trivial Concerningname can becompounds assigned according 25–27, they to weretheir structurallynot named in relate the originald compound manu bilainscript [7];B. However, however, thea trivial absence name of cana completebe assigned stereostructure according todeterminatio their structurallyn prevents relate clarifyingd compound if compound bilain B. 25 However, is actually the its absence epimer ofor a rathercomplete its diasteroisomer. stereostructure determination prevents clarifying if compound 25 is actually its epimer or ratherIn recent its diasteroisomer. years, names of some new compounds, such as saroclazines, fusaperazines, and bilains, were assignedIn recent consideringyears, names their of some sources, new compounds,rather than referring such as saroclazines, to their structural fusaperazines, relationships. and bilains, For thewere reasons assigned explained considering above, theirin Table sources, 1 some rather comp thanounds referring have also to their been structuralrenamed accordingrelationships. to the For founderthe reasons compound. explained above, in Table 1 some compounds have also been renamed according to the founder compound.

Formula, Code Compound Structure nominalFormula, mass (U) Mar.Code Drugs 2019, 17, 664 Compound Structure 3 of 14 N-Demethyl analogues nominalFormula, mass (U) Code Compound Structure N-Demethyl analogues nominalFormula, mass (U) Code Compound Structure Formula, Code Compound N-Demethyl analogues Structure nominal mass (U) Table 1. Thiosilvatins reported as natural products. Compound names proposed nominal in this mass review (U) N-Demethyl analogues Formula, Codeare underlined. CompoundSch 54794; Structure C18H24N2O3S2 4 N-Demethyl analogues nominalFormula, mass (U) Code cis-dinor-bis(methylthio)silvatinCompoundSch 54794; Structure C18H24380N2O 3S2 4 N-Demethyl analogues cis-dinor-bis(methylthio)silvatin Formula, Nominal nominalFormula,380 mass Mass (U) CodeCode SchCompound 54794; Compound Structure Structure C18H24N2O3S2 4 N-Demethyl analogues nominal(U) mass (U) cis-dinor-bis(methylthio)silvatinSch 54794; C18H24380N2 O3S2 4 Sch 54794; N-Demethyl analogues C18H24N2O3S2 4 cis-dinor-bis(methylthio)silvatin N-Demethyl analogues 380 cis-dinor-bis(methylthio)silvatinSch 54794; C18H38024N 2O3S2 4 cis-dinor-bis(methylthio)silvatinSch 54794; C18H24380N2 O3S2 4 cis-dinor-bis(methylthio)silvatinSch 54796; C18H38024N2 O3S2 5 Sch 54794;Sch 54794; C18H24NC218OH324SN2 2O3S2 4 4 trans-dinor-bis(methylthio)silvatinSch 54796; C18H24380N2O 3S2 5 cis-dinor-bis(methylthio)silvatincis-dinor-bis(methylthio)silvatin 380 380 trans-dinor-bis(methylthio)silvatinSch 54796; C18H24380N2 O3S2 5 trans-dinor-bis(methylthio)silvatinSch 54796; C18H24380N2 O3S2 5 Sch 54796; C18H24N2O3S2 5 trans-dinor-bis(methylthio)silvatin 380

trans-dinor-bis(methylthio)silvatinSch 54796; C18H38024N 2O3S2 5 trans-dinor-bis(methylthio)silvatin 380 Sch 54796;Sch 54796; C18H24CN182HO324SN2 2O3S2 5 5 19 26 2 3 2 trans C H N O S 6 trans-dinor-bis(methylthio)silvatinSaroclazine-dinor-bis(methylthio)silvatinSch 54796; A 380C18H38024N 2O3S2 5 C19H26394N2O 3S2 6 trans-dinor-bis(methylthio)silvatinSaroclazine A 380 C19H26394N2 O3S2 6 Saroclazine A C19H26394N2 O3S2

6 Saroclazine A C19H26N2O3S2 6 Saroclazine A 394 394 C19H26NC192OH326SN2 2O3S2 6 6 SaroclazineSaroclazine A A 394C19H26394N2 O3S2 6 Saroclazine A C19H26N2O3S2 7 Saroclazine B C19H39426N 2O3S2 6 Saroclazine A C19H26394N2O 3S2 7 Saroclazine B 394 C19H26394N2 O3S2 7 Saroclazine B C19H26394N2 O3S2 7 Saroclazine B C19H26N2O3S2 C19H26N2O3943S2 7 7 SaroclazineSaroclazine B B Dethio analogues 394C19H39426N 2O3S2 7 Saroclazine B Dethio analogues C19H26394N2 O3S2 7 Saroclazine B Dethio analogues

Dethio analogues C19H39426N 2O3S2 7 SaroclazineSilvathione B Dethio analogues C18H22N2O3S 8 Dethio analogues Silvathione C18H22346394N2 O 3S 8 Dethio analogues Silvathione C18H34622N 2O3S 8 Dethio analogues Silvathione C18H22CN18H2O223463NS2 O3S 8 8 SilvathioneSilvathione C18H22N2O3S 8 Dethio analogues 346 346 346 Silvathione C18H22N2O3S 8 3-(4-(3-Methyl-2- Silvathione C18H34622N 2O3S 8 butenyloxy)benzyl)-3-(methylthio)-3-(4-(3-Methyl-2- C17H22N2O3S 9 Silvathione C18H34622N 2O3S 8 butenyloxy)benzyl)-3-(methylthio)-2,5-piperazinedione; C17H22334N2 O3S 9 3-(4-(3-Methyl-2-3-(4-(3-Methyl-2- 346 butenyloxy)benzyl)-3-(methylthio)-dinor-methylthiosilvatin2,5-piperazinedione;butenyloxy)benzyl)-3-(methylthio)-3-(4-(3-Methyl-2- C17H22CN172HO334223NS 2O3S 9 9 3-(4-(3-Methyl-2- dinor-methylthiosilvatin2,5-piperazinedione; 33417 22 2 3 butenyloxy)benzyl)-3-(methylthio)-2,5-piperazinedione; C H334N O S 9 butenyloxy)benzyl)-3-(methylthio)-3-(4-(3-Methyl-2-dinor-methylthiosilvatin C17H22N2O3S 9 dinor-methylthiosilvatin2,5-piperazinedione; 334 butenyloxy)benzyl)-3-(methylthio)-dinor-methylthiosilvatin2,5-piperazinedione;3-(4-(3-Methyl-2- C17H33422N 2O3S 9 6-Hydroxy-3-(4-(3-methyl-2-dinor-methylthiosilvatin butenyloxy)benzyl)-3-(methylthio)-2,5-piperazinedione; C17H33422N 2O3S 9 3-(4-(3-Methyl-2- 6-Hydroxy-3-(4-(3-methyl-2-dinor-methylthiosilvatinbutenyloxy)benzyl)-3-6-Hydroxy-3-(4-(3-methyl-2- C17H22N2O4S 10 butenyloxy)benzyl)-3-(methylthio)-2,5-piperazinedione; C17H33422N 2O3S 9 (methylthio)piperazine-2,5-dione;6-Hydroxy-3-(4-(3-methyl-2-butenyloxy)benzyl)-3-butenyloxy)benzyl)-3- C17H22CN172HO223504NS 2 O4S 10 10 dinor-methylthiosilvatin2,5-piperazinedione; 334 (methylthio)piperazine-2,5-dione;dinor-hydroxy-methylthiosilvatin6-Hydroxy-3-(4-(3-methyl-2-butenyloxy)benzyl)-3-(methylthio)piperazine-2,5-dione; 350C17H35022N 2O4S dinor-methylthiosilvatin 10 dinor-hydroxy-methylthiosilvatin6-Hydroxy-3-(4-(3-methyl-2-dinor-hydroxy-methylthiosilvatin (methylthio)piperazine-2,5-dione;butenyloxy)benzyl)-3- C17H22350N2 O4S 10 butenyloxy)benzyl)-3- C17H22N2O4S (methylthio)piperazine-2,5-dione;dinor-hydroxy-methylthiosilvatin6-Hydroxy-3-(4-(3-methyl-2- 350 10 (methylthio)piperazine-2,5-dione;butenyloxy)benzyl)-3- C17H35022N 2O4S dinor-hydroxy-methylthiosilvatin6-Hydroxy-3-(4-(3-methyl-2- 10 dinor-hydroxy-methylthiosilvatin (methylthio)piperazine-2,5-dione;6-Hydroxy-3-(4-(3-methyl-2-butenyloxy)benzyl)-3- C17H35022N 2O4S 10 FusaperazineFusaperazine B (relative B (relative C H CN18HO24SN2O4S 11 dinor-hydroxy-methylthiosilvatin 18 24 2 4 11 (methylthio)piperazine-2,5-dione;butenyloxy)benzyl)-3- C17H35022N 2O4S 10 Fusaperazinestereochemistry)stereochemistry) B (relative 364C18H24364N2 O4S 11 dinor-hydroxy-methylthiosilvatin(methylthio)piperazine-2,5-dione; 350 Fusaperazinestereochemistry) B (relative C18H36424N 2O4S 11 dinor-hydroxy-methylthiosilvatin1,4-Dimethyl-6-(4-(3-methyl-2- Fusaperazinestereochemistry) B (relative C18H24364N2 O4S

11 1,4-Dimethyl-6-(4-(3-methyl-2-Fusaperazinebutenyloxy)benzyl)-6- B (relative C18H24N2O4S 11 stereochemistry)1,4-Dimethyl-6-(4-(3-methyl-2- 19 36424 2 4 C H N O S 12 methylsulfanyl-piperazine-2,3,5-Fusaperazinebutenyloxy)benzyl)-6-stereochemistry) B (relative C18H36424N 2O4S butenyloxy)benzyl)-6- 376 11 1 C19H24CN192HO244SN2O4S 12 12 methylsulfanyl-piperazine-2,3,5-Fusaperazinemethylsulfanyl-piperazine-2,3,5stereochemistry)trione; B (relative C18H36424N 2O4S 11 1 376 376 6-oxo-methylthiosilvatintrione; -trione; Fusaperazinestereochemistry) B (relative C18H36424N 2O4S 11 6-oxo-methylthiosilvatin 1 6-oxo-methylthiosilvatinstereochemistry) 364 1 6-(4-(3-Methyl-2- 1 6-(4-(3-Methyl-2- butenyloxy)benzyl)-6-6-(4-(3-Methyl-2-butenyloxy)benzyl)-6- 1 C H CN17HO20NS 2O4S 13 13 methylsulfanyl-piperazine-2,3,5-methylsulfanyl-piperazine-2,3,5 17 20 2 4 butenyloxy)benzyl)-6- 1 348 -trione; C17H34820N 2O4S 13 methylsulfanyl-piperazine-2,3,5-trione; dinor-6-oxo-methylthiosilvatin 1 348 dinor-6-oxo-methylthiosilvatintrione;

dinor-6-oxo-methylthiosilvatin

14 Fusaperazine E C19H24N 2O3S 14 Fusaperazine E C19H36024N 2O3S 360

C19H24N2O3S 15 Fusaperazine F C19H36024N 2O3S 15 Fusaperazine F 360

Deprenyl analogues Deprenyl analogues cis-3-(4-Hydroxybenzyl)-1,4- dimethyl-3,6-bis(methylthio)-2,5-cis-3-(4-Hydroxybenzyl)-1,4- C15H20N2O3S2 16 dimethyl-3,6-bis(methylthio)-2,5-piperazinedione; C15H34020N2 O3S2 16 cis-deprenyl-bis(methylthio)silvatin piperazinedione; 340 cis-deprenyl-bis(methylthio)silvatin

trans-6-(4-Hydroxybenzyl)-1,4- trans-6-(4-Hydroxybenzyl)-1,4-dimethyl-3,6- bis(methylthio)piperazine-2,5-dimethyl-3,6- C15H20N2O3S2 17 bis(methylthio)piperazine-2,5-dione; C15H34020N2 O3S2 17 trans-deprenyl- dione; 340 bis(methylthio)silvatintrans-deprenyl-

bis(methylthio)silvatin

C13H16N2O3S2 18 Fusaperazine A C13H31216N2 O3S2 18 Fusaperazine A 312

C13H16N2O2S2 19 Citriperazine A C13H29616N2 O2S2 19 Citriperazine A 296

C13H16N2O2S2 20 Citriperazine B C13H29616N2 O2S2 20 Citriperazine B 296

2 2

1,4-Dimethyl-6-(4-(3-methyl-2- 1,4-Dimethyl-6-(4-(3-methyl-2-butenyloxy)benzyl)-6- C19H24N2O4S 12 methylsulfanyl-piperazine-2,3,5-1,4-Dimethyl-6-(4-(3-methyl-2-butenyloxy)benzyl)-6- C19H37624N 2O4S 12 methylsulfanyl-piperazine-2,3,5-1,4-Dimethyl-6-(4-(3-methyl-2-butenyloxy)benzyl)-6-trione; 1,4-Dimethyl-6-(4-(3-methyl-2- C19H37624N 2O4S 12 methylsulfanyl-piperazine-2,3,5-6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6-trione; C19H37624N2 O4S 1,4-Dimethyl-6-(4-(3-methyl-2-6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6- 12 methylsulfanyl-piperazine-2,3,5-trione; C19H24N2O4S 12 methylsulfanyl-piperazine-2,3,5- 376 6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6-trione; 376 6-(4-(3-Methyl-2- C19H24N2O4S 12 methylsulfanyl-piperazine-2,3,5-6-oxo-methylthiosilvatintrione; butenyloxy)benzyl)-6-6-(4-(3-Methyl-2- 376 6-oxo-methylthiosilvatintrione; C17H20N2O4S 13 methylsulfanyl-piperazine-2,3,5-butenyloxy)benzyl)-6-6-(4-(3-Methyl-2- 6-oxo-methylthiosilvatin C17H34820N 2O4S 13 methylsulfanyl-piperazine-2,3,5-butenyloxy)benzyl)-6-6-(4-(3-Methyl-2-trione; 6-(4-(3-Methyl-2- C17H34820N 2O4S 13 methylsulfanyl-piperazine-2,3,5-dinor-6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6-trione; Mar. Drugs 2019, 17, 664 C17H34820N2 O4S 4 of 14 dinor-6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6-6-(4-(3-Methyl-2- 13 methylsulfanyl-piperazine-2,3,5-trione; C17H20N2O4S 13 methylsulfanyl-piperazine-2,3,5- 348 dinor-6-oxo-methylthiosilvatinbutenyloxy)benzyl)-6-trione; 348 C17H20N2O4S 13 methylsulfanyl-piperazine-2,3,5-dinor-6-oxo-methylthiosilvatintrione; Table 2. Cont. 348 dinor-6-oxo-methylthiosilvatintrione; 14 Fusaperazine E C19H24N 2O3S

dinor-6-oxo-methylthiosilvatin Formula, Nominal360 Mass 14 CodeFusaperazine Compound E Structure C19H24N 2O3S (U) 14 Fusaperazine E C19H36024 N 2O3S

14 Fusaperazine E C19H36024N2 O3S

14 Fusaperazine E C19H36024N 2O3S C19H24N2O3S 14 14 FusaperazineFusaperazine E E C19H36024N 2O3S 360 360

C19H24N2O3S 15 Fusaperazine F C19H36024N 2O3S 15 Fusaperazine F C19H36024N 2O3S 15 Fusaperazine F C19H36024N2 O3S 15 Fusaperazine F C19H24CN19H2O360243NS 2O3S 15 15 FusaperazineFusaperazine F F 360C19H36024N 2O3S 15 Fusaperazine F Deprenyl analogues 360

Deprenyl analogues cis-3-(4-Hydroxybenzyl)-1,4- Deprenyl analogues

dimethyl-3,6-bis(methylthio)-2,5-cis-3-(4-Hydroxybenzyl)-1,4- Deprenyl analogues C15H20N2O3S2 16 DeprenylDeprenyl analogues analogues dimethyl-3,6-bis(methylthio)-2,5-cis-3-(4-Hydroxybenzyl)-1,4-piperazinedione; C15H34020N 2O3S2 16 cis-deprenyl-bis(methylthio)silvatin Deprenyl analogues dimethyl-3,6-bis(methylthio)-2,5-cis-3-(4-Hydroxybenzyl)-1,4-piperazinedione;cis-3-(4-Hydroxybenzyl)-1,4- C15H34020N 2O3S2 16 cis-3-(4-Hydroxybenzyl)-1,4- cisdimethyl-3,6-bis(methylthio)-2,5--deprenyl-bis(methylthio)silvatindimethyl-3,6-bis(methylthio)-2,5-piperazinedione; C H CN15HO20340NS 2 O3S2 16 16 15 20 2 3 2 cisdimethyl-3,6-bis(methylthio)-2,5--deprenyl-bis(methylthio)silvatincis-3-(4-Hydroxybenzyl)-1,4-piperazinedione; 340C15H20N2O3S2 16 trans-6-(4-Hydroxybenzyl)-1,4-piperazinedione; 340 cisdimethyl-3,6-bis(methylthio)-2,5--deprenyl-bis(methylthio)silvatincispiperazinedione;-deprenyl-bis(methylthio)silvatin C15H34020N 2O3S2 16 trans-6-(4-Hydroxybenzyl)-1,4-dimethyl-3,6- cis-deprenyl-bis(methylthio)silvatinpiperazinedione; 340 bis(methylthio)piperazine-2,5-trans-6-(4-Hydroxybenzyl)-1,4-dimethyl-3,6- C15H20N2O3S2 17 cis-deprenyl-bis(methylthio)silvatintrans-6-(4-Hydroxybenzyl)-1,4- transbis(methylthio)piperazine-2,5--6-(4-Hydroxybenzyl)-1,4-dimethyl-3,6-dione; C15H34020N 2O3S2 17 dimethyl-3,6- trans-6-(4-Hydroxybenzyl)-1,4-transdione;-deprenyl- 340 bis(methylthio)piperazine-2,5-dimethyl-3,6- C15H20CN152HO203NS22O3S2 17 17 bis(methylthio)piperazine-2,5-dimethyl-3,6- bis(methylthio)piperazine-2,5-transbis(methylthio)silvatin-6-(4-Hydroxybenzyl)-1,4-trans-deprenyl-dione; 340C15H20340N2 O3S2 17 dione; bis(methylthio)piperazine-2,5-bis(methylthio)silvatintransdimethyl-3,6-dione;-deprenyl- C15H20340N2 O3S2 17 trans-deprenyl-bis(methylthio)silvatin bis(methylthio)piperazine-2,5-bis(methylthio)silvatintransdione;-deprenyl- C15H34020N 2O3S2 17 trans-deprenyl-dione; 340 bis(methylthio)silvatin C13H16N2O3S2 18 bis(methylthio)silvatinFusaperazinetrans-deprenyl- A C13H16CN13H2O312163NS 2O3S2 18 18 bis(methylthio)silvatinFusaperazineFusaperazine A A 312C13H31216N 2O3S2 18 Fusaperazine A C13H16312N2 O3S2 18 Fusaperazine A C13H16N2O3S2 18 Fusaperazine A 312 C13H31216N 2O3S2 18 Fusaperazine A 312 C H CN13HO16NS2O2S2 19 19 CitriperazineCitriperazine A A 13 16 2 2 2 296C13H29616N 2O2S2 19 Citriperazine A C13H29616N 2O2S2 19 Citriperazine A C13H16296N2 O2S2 19 Citriperazine A C13H16N2O2S2 C13H16296N2 O2S2 2019 Citriperazine BA 13 29616 2 2 2 CC13HH29616NN 2OO2SS2 19 Citriperazine A C13H16N2O2S2 20 20 CitriperazineCitriperazine B B 296 296C13H29616N 2O2S2 20 Citriperazine B C13H16296N2 O2S2 20 Citriperazine B Prenyl chain modified analogues 2 C13H16296N2 O2S2 20 Citriperazine B Prenyl chain modified analogues Prenyl chain2 modiﬁed analogues C13H29616N 2O2S2 20 cis-3-(4-(4-Hydroxy-3-methyl-2-Citriperazine B Prenyl chain modified analogues 2 296 butenyl)oxy)benzyl)-1,4-dimethyl-cis-3-(4-(4-Hydroxy-3-methyl-2-cis-3-(4-(4-Hydroxy-3-methyl-2- 2 C20H28N2O4S2 21 3,6-bis(methylthio)piperazine-2,5-butenyl)oxy)benzyl)-1,4-dimethyl-cis-3-(4-(4-Hydroxy-3-methyl-2-butenyl)oxy)benzyl)-1,4-dimethyl- 2 C H CN20HO42428SN 2O4S2 21 21butenyl)oxy)benzyl)-1,4-dimethyl-3,6-bis(methylthio)piperazine-2,5-3,6-bis(methylthio)piperazine-2,5-dione; 20 28 2 4 2 2 424C20H28424N2O 4S2 21 3,6-bis(methylthio)piperazine-2,5-dione; bis-(methylthio)silvatinoldione; 424 bis-(methylthio)silvatinolbis-(methylthio)silvatinoldione; bis-(methylthio)silvatinol

C H CN20HO26NS2O5S2 22 22 Bilain ABilain A 20 26 2 5 2 438C20H43826N 2O5S2 22 Bilain A C20H26438N2O 5S2 22 Bilain A 438

C20H30N2O5S2 23 Bilain B C20H44230N 2O5S2 23 Bilain B C20H30442N2O 5S2 23 Bilain B 442

C23H31N3O6S2 24 Bilain C C23H50931N 3O6S2 24 Bilain C C23H31N3O6S2 24 Bilain C 509 509

C20H30N2O5S2 25 Bilain D C20H44230N 2O5S2 25 Bilain D C20H30N2O5S2 25 Bilain D 442 442

C21H32N2O5S2 26 Bilain E C21H45632N 2O5S2 26 Bilain E C21H32456N2O 5S2 26 Bilain E 456

C21H32N2O5S2 27 Bilain F C21H45632N 2O5S2 27 Bilain F C21H32N2O5S2 27 Bilain F 456 456

3 3 3

Prenyl chain modified analogues Prenyl chain modified analogues cis-3-(4-(4-Hydroxy-3-methyl-2- Prenyl chain modified analogues butenyl)oxy)benzyl)-1,4-dimethyl-cis-3-(4-(4-Hydroxy-3-methyl-2- Prenyl chain modified analogues C20H28N2O4S2 21 3,6-bis(methylthio)piperazine-2,5-butenyl)oxy)benzyl)-1,4-dimethyl-cis-3-(4-(4-Hydroxy-3-methyl-2- Prenyl chain modified analogues C20H42428N 2O4S2 21 3,6-bis(methylthio)piperazine-2,5-butenyl)oxy)benzyl)-1,4-dimethyl-cis-3-(4-(4-Hydroxy-3-methyl-2-dione; 20 42428 2 4 2 C H N O S 21 butenyl)oxy)benzyl)-1,4-dimethyl-cis3,6-bis(methylthio)piperazine-2,5--3-(4-(4-Hydroxy-3-methyl-2-bis-(methylthio)silvatinoldione; C20H28424N2O 4S2 21 butenyl)oxy)benzyl)-1,4-dimethyl-3,6-bis(methylthio)piperazine-2,5-bis-(methylthio)silvatinoldione; C20H28424N2O 4S2 21 3,6-bis(methylthio)piperazine-2,5-bis-(methylthio)silvatinoldione; 424 bis-(methylthio)silvatinoldione; C20H26N2O5S2 22 bis-(methylthio)silvatinolBilain A C20H43826N 2O5S2 22 Bilain A C20H43826N 2O5S2 Mar. Drugs22 2019, 17, 664 Bilain A 5 of 14 C20H26438N2O 5S2 22 Bilain A C20H26438N2O 5S2 22 Bilain A Table 3. Cont. 438 C20H30N2O5S2 23 Bilain B Formula, Nominal Mass Code Compound Structure C20H44230N 2O5S2 23 Bilain B (U) C20H44230N 2O5S2 23 Bilain B C20H30442N2O 5S2 23 Bilain B C H CN20HO30442NS2O 5S2 23 23 Bilain B Bilain B 20 30 2 5 2 442 442

C23H31N3O6S2 24 Bilain C C23H50931N 3O6S2 24 Bilain C C23H50931N 3O6S2 24 Bilain C 509 C23H31CN233HO316SN23O6S2 24 24 Bilain CBilain C 509C23H31509N3O 6S2 24 Bilain C 509

C20H30N2O5S2 25 Bilain D C20H44230N 2O5S2 25 Bilain D C H NC20OH44230SN 2O5S2 25 25 Bilain BilainD D 20 30 2 5 2 442C20H30442N2O 5S2 25 Bilain D

C20H30442N2O 5S2 25 Bilain D 442 C21H32N2O5S2 26 Bilain E C H NC21OH45632SN 2O5S2 26 26 Bilain EBilain E 21 32 2 5 2 456C21H45632N 2O5S2 26 Bilain E C21H32456N2O 5S2 26 Bilain E C21H32456N2O 5S2 26 Bilain E 456 C H CN21HO32SN2O5S2 27 27 Bilain FBilain F 21 32 2 5 2 456C21H45632N 2O5S2 27 Bilain F C21H45632N 2O5S2 27 Bilain F C21H32456N2O 5S2 27 Bilain F C21H32456N2O 5S2 27 Bilain F Many diﬃculties in nomenclature derive from the stereostructural aspects. Concerning456 compounds

25–27, they were not named in the original manuscript [7]; however, a trivial name can be assigned according to their structurally related compound bilain B. However, the absence of a complete stereostructure determination prevents clarifying if compound 25 is actually its epimer or rather its diasteroisomer. 3 In recent years, names of some new compounds,3 such as saroclazines, fusaperazines, and bilains, were assigned considering their sources, rather than3 referring to their structural relationships. For the reasons explained above, in Table3 some compounds3 have also been renamed according to the founder compound. 3 In general, the biosynthesis of secondary metabolites is stereospecific. In fact, the stereochemistry of chiral carbons in the dioxopiperazinic ring of thiosilvatins is essentially 3R,6R, even if these compounds display some structural differences. This is observed in strains belonging to unrelated species, such as Fusarium chlamydosporum [8], Penicillium waksmanii [9], Penicillium brevicompactum [10], Trichoderma virens [3,11], all producing thiosilvatins with the same stereostructure [i.e., cis-bis(methylthio)silvatin (1) and its deprenyl analogue (16)] (Tables3–5). On the other hand, this is not a common trend in compounds belonging to the ETPs class [2]. In fact, both stereoisomers (i.e., 3R,6R and 3S,6R) were reported for compounds in the hyalodendrin/gliovictin family, deriving from the amino acids l-phenylalanine and l-serine [12–14] (Figure3). Interestingly, this family also includes a compound named vertihemiptellide A, representing the first dimer resulting from the formation of disulfide bridges between two hyalodendrin units [15]. Mar. Drugs 2019, 17, x FOR 5 of 13

C21H32N2O5S2 26 Bilain E 456

C21H32N2O5S2 27 Bilain F 456

In general, the biosynthesis of secondary metabolites is stereospecific. In fact, the stereochemistry of chiral carbons in the dioxopiperazinic ring of thiosilvatins is essentially 3R,6R, even if these compounds display some structural differences. This is observed in strains belonging to unrelated species, such as Fusarium chlamydosporum [8], Penicillium waksmanii [9], Penicillium brevicompactum [10], Trichoderma virens [3,11], all producing thiosilvatins with the same stereostructure [i.e., cis-bis(methylthio)silvatin (1) and its deprenyl analogue (16)] (Tables 1–3). On the other hand, this is not a common trend in compounds belonging to the ETPs class [2]. In fact, both stereoisomers (i.e., 3R,6R and 3S,6R) were reported for compounds in the hyalodendrin/gliovictin family, deriving from the amino acids L-phenylalanine and L-serine [12–14] (Figure 3). Interestingly, this family also includes a compound named vertihemiptellide A, representingMar. Drugs 2019 the, 17 first, 664 dimer resulting from the formation of disulfide bridges between two6 of 14 hyalodendrin units [15].

(3S,6S)-Hyalodendrin A26771A or (3R,6R)-Hyalodendrin

(3R,6R)- bisdethiodi(Methylthio) (3S,6S)-bisdethiodi(Methylthio) (Z)-6-Benzylidene-3- hydroxymethyl-1,4-dimethyl-3- hyalodendrin or A26771E or (3R,6R)-gliovictin hyalodendrin ethylsulfanylpiperazine-2,5-dione

(3S,6S)-bis-N-Norgliovictin (3R,6R)-bis-N-Norgliovictin Figure 3. Hyalodendrins and gliovictins.Figure 3. Hyalodendrins and gliovictins. 3. Fungal Sources 3. Fungal Sources As introduced above, so far ETPs have been only reported from fungi. More specifically, As introduced above, so far ETPs have been only reported from fungi. More specifically, thiosilvatins have been detected as secondary metabolites of 22 strains belonging to 17 taxa that thiosilvatins have been detected as secondary metabolites of 22 strains belonging to 17 taxa that occupy different geographic and climatic zones, terrestrial and marine habitats, and are associated with occupy different geographic and climatic zones, terrestrial and marine habitats, and are associated different substrates/hosts (Tables4 and5). With the exception of a single taxon in the Basidiomycota, with different substrates/hosts (Tables 2–3). With the exception of a single taxon in the Basidiomycota, that is Coriolus (=Irpex) consors, all the other strains are representative of taxa in the Ascomycota. that is Coriolus (=Irpex) consors, all the other strains are representative of taxa in the Ascomycota. Particularly, they belong to the Sordariomycetes (order Hypocreales, 7 strains/6 taxa; order Xylariales, Particularly, they belong to the Sordariomycetes (order Hypocreales, 7 strains/6 taxa; order 1 strain/taxon), and to the Eurotiomycetes (14 strains/9 taxa, all of them in the Eurotiales). Twelve Xylariales, 1 strain/taxon), and to the Eurotiomycetes (14 strains/9 taxa, all of them in the Eurotiales). strains, that is more than half of the total number, belong to the genus Penicillium, well known for Twelve strains, that is more than half of the total number, belong to the genus Penicillium, well known its widespread occurrence in every ecological context including the sea [16]. The species Penicillium for its widespread occurrence in every ecological context including the sea [16]. The species crustosum and T. virens include strains from both kind of sources. Penicillium crustosum and T. virens include strains from both kind of sources.

Mar. Drugs 2019, 17, 664 7 of 14

Table 4. Marine-derived fungal strains producing thiosilvatins.

Species (Strain) Source Geographic Origin Compound Code Ref. Cordyceps javanicus 1 Jaspis cf. coriacea (sponge) Fiji 1, 3 [17] (961331) Fusarium chlamydosporum Carpopeltis aﬃnis (red alga) Japan 1, 4, 5, 11, 16, 18 [8] (OUPS-N124) Similan Islands Nigrospora sp. (PSU-F12) Annella sp. (gorgonian) 5 [18] (Thailand) Penicillium bilaiae Huon estuary, Boat ramp 1, 22, 23, 24 [19] (MST-MF667) Tasmania (Australia) Penicillium commune Muricella abnormalis Danzhou, Hainan 1 [20] (518) (gorgonian) (China) Penicillium crustosum Sediment Prydz Bay (Antarctica) 1, 3, 15 [21] (HDN153086) Penicillium sp. (KMM Padina sp. (brown alga) Vietnam 19, 20 [22] 4672) Penicillium sp. (2556) Mangrove plant China 4, 5 [23] Penicillium waksmanii Sargassum ringgoldianum Japan 1, 16, 21 [9] (OUPS-N133) (brown alga) Rhizosphere soil of Sarocladium kiliense Thespesia populnea Guangxi (China) 1, 6, 7, 12 [24] (HDN11-84) (mangrove) Gracilaria vermiculophylla Trichoderma virens (Y13-3) Yangma Island (China) 16, 17 [6] (red alga) 1 This strain identiﬁed with the older species name of Paecilomyces cf. javanica in the original report.

Table 5. Fungal strains from non-marine sources producing of thiosilvatins.

Species (Strain) Source Geographic Origin Compound Code Ref. Aspergillus silvaticus Soil Tafo (Ghana) 8, 2 [4] (IFO8173) Coriolus (=Irpex) consors ATCC collection 1, 3 [25] (ATCC11574) Penicillium amphipolaria Quartermain Soil 14 [26] (DAOM695760) Mountains (Antarctica) Contaminant in culture of Penicillium Ceratocystis ulmi (plant Edmonton (Canada) 1, 9, 10, 16 [10] brevicompactum pathogenic fungus) Penicillium crustosum Viguiera robusta (plant) Brazil 1, 3, 14 [27] (VR4) Fruiting body of Isaria Penicillium crustosum Sichuan province cicadae (entomopathogenic 1, 13, 25, 26, 27 [7] (MK285663) (China) fungus) Penicillium crustosum Red soil Yunnan (China) 12, 13 [28] (YN-HT-15) Penicillium roqueforti Blue cheese USA 1 [29] (ATCC10110) Endophytic in Pinellia Penicillium sp. Nanjing (China) 1 [30] ternata (plant) Tolypocladium sp. Quercus virginiana (plant) Tamalupas (Mexico) 1, 4, 5, 9 [5] Trichoderma virens 1 Soil California, USA 1, 16 [3,11] (CMI101525) 1 This strain identiﬁed with the older species name of Gliocladium deliquescens in the original report. Mar. Drugs 2019, 17, 664 8 of 14

With reference to the specific compounds, 1 undoubtedly represents the most common product of Mar.this Drugs family, 2019 having, 17, x FOR been reported as a secondary metabolite of about 2/3 of the strains, while its7 transof 13 stereoisomer has been detected in just four of these strains, both marine and terrestrial. Compounds 4, Compounds 4, 5, 12, 14 and 16 were also obtained from strains from both environments. Among the 5, 12, 14 and 16 were also obtained from strains from both environments. Among the rest, compounds rest, compounds 6, 7, 11, 15 and 17–24 have been reported from just a single strain of marine origin, 6, 7, 11, 15 and 17–24 have been reported from just a single strain of marine origin, while compounds while compounds 2, 8–10, 13, and 25–27 have been only found in terrestrial strains. These data could 2, 8–10, 13, and 25–27 have been only found in terrestrial strains. These data could be indicative of be indicative of a relatively higher chemodiversity characterizing marine strains, also considering a relatively higher chemodiversity characterizing marine strains, also considering that reports from that reports from marine sources only started in 1998 when there were already four strains and eight marine sources only started in 1998 when there were already four strains and eight products known products known from terrestrial sources (Figure 4). Since 1998, the new products discovered from from terrestrial sources (Figure4). Since 1998, the new products discovered from marine fungi more marine fungi more than doubled those obtained from non-marine strains. Moreover, in the last two than doubled those obtained from non-marine strains. Moreover, in the last two years there were years there were five reports concerning new thiosilvatins-producing strains from marine sources five reports concerning new thiosilvatins-producing strains from marine sources compared to two compared to two from terrestrial sources, which might imply that a more widespread occurrence at from terrestrial sources, which might imply that a more widespread occurrence at sea is likely to be sea is likely to be disclosed as investigations concerning marine fungal strains progress. Finally, no disclosed as investigations concerning marine fungal strains progress. Finally, no comparison can be comparison can be made between strains of the same species (P. crustosum and T. virens) obtained made between strains of the same species (P. crustosum and T. virens) obtained from both marine and from both marine and terrestrial sources, whose secondary metabolite profiles do not match, or share terrestrial sources, whose secondary metabolite profiles do not match, or share single compounds. single compounds. This could be interpreted not only in terms of intraspecific variation, but also as This could be interpreted not only in terms of intraspecific variation, but also as a consequence of the a consequence of the different culturing and extraction procedures. Moreover, it must also be different culturing and extraction procedures. Moreover, it must also be considered that detection of considered that detection of some compounds is often impaired by their presence in low quantities, some compounds is often impaired by their presence in low quantities, or by inherent difficulties in the or by inherent difficulties in the identification depending on their infrequent occurrence. However, identification depending on their infrequent occurrence. However, the finding of two species from both the finding of two species from both marine and terrestrial sources within such a limited strain marine and terrestrial sources within such a limited strain sample supports a recently-consolidated sample supports a recently-consolidated inference that most fungal species are able to thrive in inference that most fungal species are able to thrive in different environmental conditions, obliterating different environmental conditions, obliterating the old misconception that the occurrence of the old misconception that the occurrence of specialized taxa occurs in either marine or non-marine specialized taxa occurs in either marine or non-marine contexts [16,31,32]. contexts [16,31,32].

10 Non-marine sources 8 Marine sources

Numberof reports 2

Numberof Products 8 1980-1989 1990-1999 2000-2009 2010-2019

FigureFigure 4. 4. HeadHead to to tail tail comparison comparison of of number number of of reports reports dealing dealing with with thiosilvatin thiosilvatinss and and number number of of new new products obtained from marine and non-marinenon-marine sources. 4. Proposed Biosynthetic Pathways for Thiosilvatins 4. Proposed Biosynthetic Pathways for Thiosilvatins The biosynthesis of ETPs involves non-ribosomal peptide synthetases (NRPSs), multi-domain The biosynthesis of ETPs involves non-ribosomal peptide synthetases (NRPSs), multi-domain enzymes controlling all activities required to incorporate constituents into their products, and a range of enzymes controlling all activities required to incorporate constituents into their products, and a range associated enzymes [33,34]. In fact, the non-ribosomal pathway is frequently used by microorganisms of associated enzymes [33,34]. In fact, the non-ribosomal pathway is frequently used by to produce a wide range of structurally diverse secondary metabolites [35]. microorganisms to produce a wide range of structurally diverse secondary metabolites [35]. In general, the genes that encode enzymes for secondary metabolite biosynthesis are clustered in the fungal genome [36]. Some ETP gene clusters, such as the ones involved in sirodesmin and gliotoxin biosynthesis, were identified by generating mutations in these genes and analyzing secondary metabolite profiles of the resultant mutants. In fact, the gene deletions may result in abrogation of the biosynthetic pathway. The comparative analysis of many fungal genome sequences

Mar. Drugs 2019, 17, 664 9 of 14

In general, the genes that encode enzymes for secondary metabolite biosynthesis are clustered in the fungal genome [36]. Some ETP gene clusters, such as the ones involved in sirodesmin and gliotoxin biosynthesis, were identified by generating mutations in these genes and analyzing secondary metabolite profiles of the resultant mutants. In fact, the gene deletions may result in abrogation of the biosynthetic pathway. The comparative analysis of many fungal genome sequences has displayed similarities between the gliotoxin and sirodesmin clusters, proving the conservation of the main biosynthetic genes in the ETP clusters. It is thus likely that similar core enzymes are responsible for the biosynthesisMar. Drugs 2019 of, 17 the, x FOR ETP backbone, but the structural diversity depends on other genes that appear8 onlyof 13 in some clusters, many of them remaining to be fully identified [37–41]. BasedBased on on these these pieces pieces of of evidence, evidence, the the thiosilvatin thiosilvat biosynthesisin biosynthesis was was predicted predicted according according to the to one the reportedone reported for gliotoxin for gliotoxin [1,42,43 ].[1,42,43]. In fact, similarIn fact, to simi otherlar ETPs, to other thiosilvatins ETPs, thiosilvatins derive from thederive condensation from the ofcondensation two amino acidsof two which amino can acids be further which altered can be by further epimerization, altered by methylation, epimerization, or cyclization. methylation, The or origincyclization. and mechanism The origin of and incorporation mechanism of theof incorporation sulfur atoms into of the dipeptidesulfur atoms are unclear,into the anddipeptide according are tounclear, different and hypotheses according they to coulddifferent be derived hypotheses from methionine,they could be cysteine, derived sodium from sulfate,methionine, or glutathione. cysteine, Particularly,sodium sulfate, the formationor glutathione. of a diiminium Particularly, intermediate the formation followed of a bydiiminium nucleophilic intermediate attack of thefollowed cysteine by thiolatenucleophilic residue attack of glutathione of the cysteine is possibly thiolate involvedresidue of (Figure glutathione5)[ 2 ,is39 possibly]. In order involved to justify (Figure the presence 5) [2,44]. ofIn C-6order epimers to justify on the piperazinepresence of ring,C-6 epimers two possible on the mechanisms piperazine ring, of nucleophilic two possible attack mechanisms have been of 1 proposednucleophilic (Figure attack5, reactionhave been mechanism proposed a (Figure). 5, reaction mechanism a1).

FigureFigure 5.5.Proposed Proposed biosyntheticbiosynthetic pathwayspathways forfor thiosilvatins.thiosilvatins. S-GS-G representsrepresents glutathione.glutathione.

An alternative biosynthetic pathway, even if less credited, has been proposed for the sulfurization of diketopiperazines. Due to the slow rate of dipeptide cyclization, the sulfur insertions and further chemical transformations might occur while the linear dipeptide is still covalently bound to the NRPS [45]. A different biosynthetic pathway could be possible for monosulfurate compounds, which represent an extensive group in the thiosilvatin compounds series, as exemplified in the reaction mechanisms a2, a3, and b in Figure 5. Sulfur insertion could happen on C-3 or C-6 of the hydroxypiperazine ring in the iminium intermediate followed by hydroxyl oxidation or water elimination to obtain precursors of 12, 13 and 14, 15. Silvathione (8) might have a different biosynthetic pathway with monoimmyl intermediate which involves C-6 and 1N (Figure 5, reaction mechanism c).

Mar. Drugs 2019, 17, 664 10 of 14

An alternative biosynthetic pathway, even if less credited, has been proposed for the sulfurization of diketopiperazines. Due to the slow rate of dipeptide cyclization, the sulfur insertions and further chemical transformations might occur while the linear dipeptide is still covalently bound to the NRPS [44]. A different biosynthetic pathway could be possible for monosulfurate compounds, which represent an extensive group in the thiosilvatin compounds series, as exemplified in the reaction mechanisms a2, a3, and b in Figure5. Sulfur insertion could happen on C-3 or C-6 of the hydroxypiperazine ring in the iminium intermediate followed by hydroxyl oxidation or water elimination to obtain precursors of 12, 13 and 14, 15. Silvathione (8) might have a different biosynthetic pathway with monoimmyl intermediate which involves C-6 and 1N (Figure5, reaction mechanism c). Further chemical transformations (e.g., methylation, oxidation) possibly occur on the backbone of thiosilvatins in order to obtain an ample variety of natural products. In fact, nitrogen and oxygen atoms can be methylated, while the phenolic hydroxyls are frequently prenylated (i.e., 1–15, 21–24).

5. Biological Activities Although no conclusive demonstration has been obtained yet, the opinion is prevalent that ETPs are important for the producing strains in the interaction with other organisms. These compounds have been reported for a wide array of bioactive properties, including antibiotic, antiviral, cytotoxic and anti-inflammatory effects. Bioactivities basically depend on thiol-disulphide exchange reactions, and the relative effects of the single compounds are considered to be somehow related to the oxidation/reduction status of the sulfurs [1]. Unlike the homologue hyalodendrin/gliovictin family, a few members of which have been more extensively investigated with reference to their antibiotic and antiproliferative properties, and mechanisms of action [15,45,46], for thiosilvatins the available data are still quite preliminary for drawing a clear judgment concerning their biological activity and opportunities for pharmaceutical exploitation. No antifungal properties could be evidenced in assays carried out with cis-bis(methylthio)silvatin on Parastagonospora (Septoria) nodorum [19], and yeast strains of Candida albicans [30] and Saccharomyces cerevisiae [29]. Also, this compound and bilain A did not display antihelmintic activity against the barber’s pole worm (Haemonchus contortus), a common parasitic nematode of ruminants [19], while cis-deprenyl-bis(methylthio)silvatin (16) and trans-deprenyl-bis(methylthio)silvatin (17) did not show toxic effects on Artemia salina and four marine phytoplankton species (Chattonella marina, Heterosigma 1 akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense) at a concentration of 100 µg mL− [6]. Assays concerning antibacterial activity mostly provided negative results, too. In fact, 16 and 17 were inactive against five marine-derived pathogenic Gram-negative bacteria (Vibrio parahaemolyticus, V. anguillarum, V. harveyi, V. splendidus, and Pseudoalteromonas citrea) in an agar disk-diffusion assay at a dose of 20 µg/disk [6]. No effects were observed for 1 against Escherichia coli and Bacillus subtilis [19] and, together with its trans stereoisomer (3), fusaperazine E (14) and trans-dinor-bis(methylthio)silvatin (5), against Enterococcus faecalis [27]. However, more recently, some extent of antibacterial properties by 1 cis-bis(methylthio)silvatin have been reported against Staphylococcus aureus (MIC 43.4 µg mL− )[30], E. 1 coli and B. subtilis (IC50 30.0 µg mL− )[29]. In line with the recent trend to screen natural products in the aim of finding new anticancer compounds, more circumstantial data are available with reference to the antiproliferative activity against tumor cell lines. In this respect, fusaperazine A (18) and 1 exhibited weak cytotoxic activities against 1 P388 murine lymphocytic leukaemia cells (ED50 22.8 and 7.7 µg mL− , respectively) [8], confirming previous findings concerning the latter compound [9]. In another study cis-bis(methylthio)silvatin was cytotoxic (0.15 µM) on NS-1 mouse myeloma cells, while bilain A (22) was inactive [19]. Again on 1 P388 cells, cis-dinor-bis(methylthio)silvatin (4) exhibited weak cytotoxic activity (ED50 21.5 µg mL− ), whereas its analogue 5 was inactive along with fusaperazine B (11) and 16 [8]. Afterwards, 4 and 5 were found to remarkably inhibit the growth of two human cell lines HEp2 (larynx carcinoma) and HepG2 Mar. Drugs 2019, 17, 664 11 of 14

(liver carcinoma) [22]. Citriperazines A and B (19, 20) did not exhibit cytotoxic activity against three human prostate cancer cells (22Rv1, PC-3 and LNCaP) at concentrations up to 100 µM, also without any significant effect on cell cycle progression [22]. Cytotoxic effects have been also reported for saroclazine B(7) against HeLa (cervyx uteri carcinoma) cells (IC50 4.2 µM) [24], and fusaperazine F (15) against the K562 (chronic myelogenous leukemia) cell line (IC50 12.7 µM) [21]. In a quite peculiar assay carried out on zebrafish larvae, 1 and 6-oxo-methylthiosilvatin (12) promoted gastrointestinal motility via acting on the cholinergic nervous system, while bilains D–F (25–27) lacking the double bond in the lateral chain were inactive [7]. Finally, on account of the platelet-activating factor (PAF) inhibitory effects also known for other diketopiperazines [47], a weak activity was displayed by compound 5 in the PAF assay (IC50 50 µM), while the related 4 was inactive [5].

6. Conclusions As introduced above, literature concerning the occurrence and properties of thiosilvatins is not extensive. Although half of the reports refer to strains of Penicillium, the available data show that biosynthetic aptitude for these compounds can be found in distantly related fungal species, in line with what is known for the homologue hyalodendrin/gliovictin family, other ETPs, and many mycotoxins. Actually, this biological phenomenon, known under the name of synapomorphy, is quite difficult to explain in phylogenetic terms, since it would imply that the genetic base encoding for biosynthesis of these secondary metabolites was acquired or lost many times along with the separation of lineages during the evolution of fungi. However, as the work on genome sequencing of fungi progresses, the evidence is accumulating that biosynthesis of many classes of mycotoxins is controlled by clustered genes. And the discovery that fungi may exchange gene clusters through the so-called horizontal gene transfer (HGT) has disclosed a more reasonable biological explanation, according to which fungal species thriving in the same ecological niche or sharing the same substrate may somehow establish a successful interaction at the genetic level resulting in modification of their metabolome [48,49]. In this respect, the occurrence in clusters of genes involved in the biosynthesis of ETPs has been demonstrated in the case of some major members of this class, such as gliotoxin [50], sirodesmin [37], and verticillin [51]. Moreover, gene clusters with all eight genes encoding for the common ETP moiety have been found in several unrelated ascomycetes species [1]. Following assumptions in comparative genomics, more recent evidence indicates that such a cluster may be present even in fungal species which so far have not been reported for production of these compounds [52]. The accumulation of data concerning metabolomics of fungal strains/species is fundamental in order to provide more circumstantial support to this theory and to shed light on the circumstances which make HGT possible. In this regard, thiosilvatins appear to represent a meaningful group of compounds, characterized by a uniform molecular model, possibly reflecting a definite biosynthetic scheme. More prompts for developing investigations on the biological properties of thiosilvatins derive from the availability of more refined bioassays able to elucidate the effects of compounds displaying low levels of cytotoxicity. An interesting example in this respect is provided by the finding of a diketopiperazine derivative inhibiting prion replication in the micromolar range, which introduces these compounds as a promising lead scaffold in the search of products against these problematic disease determinants [53]. Finally, the very recent finding from a strain of Penicillium roqueforti from blue cheese [29] introduces the opportunity to better investigate the effects deriving from a dietary intake of cis-bis(methylthio)silvatin, also in association with the roquefortines and other bioactive products reported from this species of biotechnological relevance [54,55].

Author Contributions: Conceptualization, R.N. and A.A.; writing—review and editing, M.M.S., R.N., M.D. and A.A. Funding: Financial support was provided by Ministero Italiano dell’Istruzione, dell’Università e della Ricerca (MIUR) through Finanziamento delle Attività Base della Ricerca (FFABR) 2017. Conﬂicts of Interest: The authors declare no conﬂict of interest. Mar. Drugs 2019, 17, 664 12 of 14

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