*2.5. Structural Features of Bridged Heterocyclic Peptide Bicycles*

Bicyclic peptides form one of the promising platforms for drug development owing to their biocompatibility and chemical diversity to proteins. Bioactive bicyclic peptides exist as disulfide-bridged peptide bicycles (e.g., ulithiacyclamide A, B, E, F, and G), histidino-tyrosine bridged peptide bicycles (e.g., aciculitins A–C), histidino-alanine bridged peptide bicycles (e.g., Theonellamides A, B, C, F, and G and Theogrenamide) and are derived from marine sponges/tunicates, plants, and mushrooms.

Ulithiacyclamide A is a strong cytotoxic disulfide-bridged peptide bicycle characterized by a symmetrical dimeric structure consisting of oxazoline and thiazole rings in addition to a transannular disulfide isolated from marine tunicate/ascidian *Lissoclinum patella*. The structure of ulithiacyclamide B

closely resembled the structure of ulithiacyclamide with the exception of the replacement of one of the two d-leucine units with d-phenylalanine residue, resulting in an asymmetrical dimeric structure. Because the configuration of both leucine and phenylalanine was d, both thiazole amino acids possessed *R* configurations in ulithiacyclamide. The structures of ulithiacyclamides E, F, and G are related in structure to ulithiacyclamide B but with either both (in the case of ulithiacyclamide E) or just one of the two (in the cases of ulithiacyclamides F and G) oxazoline rings existing as their hydrolyzed l-threonine counterpart. Ulithiacyclamides F and G were found to be isomers and contained one oxazoline including one "free" threonine unit and were anhydro forms of ulithiacyclamide E. Ulithiacyclamide and ulithiacyclamide B exhibited cytotoxicity against the KB cell line with IC50 values of 35 and 17 ng/mL, respectively [51,53,56,57,117].

Aciculitins A–C are cytotoxic and antifungal glycopeptidolipids from the lithistid sponge *Aciculites orientalis*. They consist of a bicyclic peptide structure that contains a histidine-tyrosine bridge, with an unusual combination of tyrosine and histidine residues joined through the 3- -position of tyrosine and the 5- -position of histidine [118]. Theonegramide is a peculiar antifungal peptide that presents an intra-cycle histidine-alanine bridge in which the imidazole ring is substituted by a d-arabinose moiety. The alanine portion of histidinoalanine was found to have the (*R*)-configuration while the histidine portion with the (*S*)-configuration [119]. Theonellamides (TNMs) are members of a distinctive family of sterol-binding bioactive bicyclic dodecapeptides, with theonellamide F being a novel antifungal bicyclic dodecapeptide with an unprecedented histidinoalanine bridge composed of unusual amino acid residues like τ-l-histidino-d-alanine, (2*S*,4*R*)-2-amino-4-hydroxyadipic acid (Ahad), and (3*S*,4*S*,5*E*,7*E*)-3-amino-4-hydroxy-6-methyl-8- (*p*-bromophenyl)-5,7-octadienoic acid (Aboa). Theonellamide F was found to be a useful agent for investigating membrane structures in cells and inhibited growth of various pathogenic fungi including *Candida* sp., *Trichophyton* sp., and *Aspergillus* sp. [120,121].

Moroidin is a unique bicyclic peptide bearing residues like histidine, tryptophan, arginine, and β-leucine, isolated from the seeds of the Chinese herb *Celosia argentea* (Amaranthaceae), that remarkably inhibited the polymerization of tubulin [122]. Celogentins are unique cyclopolypeptides containing a bicyclic ring system; an unusual C–N bond formed by Trp and His residues; and an unusual amino acid, β-substituted Leu, isolated from the seeds of *Celosia argentea*. Celogentins A–C inhibited the polymerization of tubulin, and celogentin C was found to be 4 times more potent than moroidin in the inhibitory activity [123]. Phalloidin is a rigid bicyclic peptide containing an unusual cysteine-tryptophan linkage, isolated from the death cap mushroom *Amanita phalloides*. This cycloheptapeptide is commonly used in imaging applications to selectively label F-actin in fixed cells, permeabilized cells, and cell-free experiments [124]. α-Amanitin is a highly toxic hydrophobic bicyclic octapeptide found in a genus of mushrooms known as Amanita, including *Amanita phalloides*, *Amanita verna*, and *Amanita virosa*. The cytotoxicity found in amanitin is the result of inhibition of RNA polymerases, in particular RNA polymerase II, which precludes mRNA synthesis [124].

#### *2.6. Structural Features of Other Heterocyclic Peptides from Marine Resources*

Azonazine is a unique anti-inflammatory peptide with a macrocyclic heterocyclic core of the benzofuro indole ring system with diketopiperazine residue and possesses structural similarity with diazonamide A. The absolute configuration of this marine sediment-derived fungus-originated complex peptide was established as 2*R*,10*R*,11*S*,19*R*. The first total synthesis of hexacyclic dipeptide ent-(−)-azonazine was accomplished using a hypervalent iodine-mediated biomimetic oxidative cyclization to construct the highly strained core [125].

The pyridine ring (in the form of 3-hydroxypicolinic acid, 3HyPic) also forms part of cyclopeptide structures such as fijimycins and etamycin. Fijimycins A–C are cyclic depsipeptides from a marine-derived *Streptomyces* sp. which possessed in vitro antibacterial activity against three methicillin-resistant *Staphylococcus aureus* (MRSA) strains. The depsipeptide fijimycin A was found to contain eight subunits including α-phenylsarcosine (l-PhSar), *N*,β-dimethylleucine (l-DiMeLeu), sarcosine (Sar), 4-hydroxyproline (d-Hyp), and 3-hydroxypicolinic acid (3HyPic). Fijimycin A was defined as a stereoisomer of etamycin A containing d-α-phenylsarcosine. While comparing the structure of fijimycin B with fijimycin A, there was disappearance of α-phenylsarcosine (PhSar) and the existence of an *N*-methylleucine (l-*N*MeLeu) residue. Comparison of structures of fijimycins C and A suggested that the alanine (Ala) moiety in fijimycin A was replaced by a serine (Ser) unit. Etamycin A, also called virifogrisein I, was isolated from cultures of a terrestrial *Streptomyces* species which exhibited considerable activity against Gram-positive bacteria as well as *Mycobacterium tuberculosis*.

Fijimycins A and C and etamycin A exhibited strong antibiotic activities against the three MRSA strains (ATCC33591, Sanger 252, UAMS1182). However, fijimycin B showed weak inhibition against both ATCC33591 and UAMS1182, which indicated that the α-phenylsarcosine unit might be vital for significant antibacterial activity. The similar antimicrobial activities of the stereoisomers fijimycin A and etamycin A suggested that substituting d- for l-α-phenylsarcosine had little effect on the anti-MRSA activities [126].

Jaspamide P is a sponge-derived modified jaspamide derivative possessing antimicrofilament activity and characterised by a modification of the *N*-methylabrine (*N*-methyl-2-bromotrypthophan) residue. Structural analysis of this cyclopeptide indicated the presence of a 4-methoxy-1,3-benzoxazine-2-one heteroaromatic system. Jaspamide P was found to exhibit cytotoxic activity against HT-29 and MCF-7 tumour cell lines. Modifications of the methylabrine residue, claimed as essential for the observed biological activity, appeared to have little influence on the observed antiproliferative effect [127].

Wainunuamide is an unusual histidine containing cycloheptapeptide, containing three proline units. There were adjacent *cis* and *trans* proline residues in the structure of wainunuamide. Similar patterns were also found in cyclooligopeptide phakellistatin 8 and were found to be powerful β-turn inducers. The stereochemistry of all residues including histidine, phenylalanine, and leucine was found to be l. Wainunuamide exhibited weak cytotoxic activity in A2780 ovarian tumor and K562 leukemia cancer cells [128].

Ohmyungsamycins A and B are marine bacterium-derived cytotoxic and antimicrobial cyclic depsipeptides composed of 12 amino acid residues, including unusual amino acids such as *N*-methyl-4-methoxy-l-tryptophan, β-hydroxy-l-phenylalanine, and *N,N*-dimethylvaline. Ohmyungsamycins A and B showed significant inhibitory activities against diverse cancer cells as well as antibacterial effects against both Gram-positive and Gram-negative bacteria. Sungsanpin is a serine-rich lasso peptide containing 15 amino acid units from a deep-sea streptomycete in which eight amino acids form a cyclic peptide and the remaining seven amino acids including l-tryptophan unit form a tail that loops through the ring. It is the first example of a lasso peptide from a marine-derived microorganism and displays inhibitory activity with the human lung cancer cell line A549 in a cell invasion assay [129].

Desotamide and destolamide B are l-tryptophan containing bioactive peptides from marine microbe *Streptomyces scopuliridis* SCSIO ZJ46. These cyclohexapeptides displayed good antibacterial activities against *Streptococcus pnuemoniae*, *Staphylococcus aureus*, and *methicillin-resistant Staphylococcus epidermidis* (MRSE). In a complementary fashion, the antibacterial activities of destolamides revealed the "Tryptophan" moiety to be essential, thereby highlighting a critical structural element to this advancing antibacterial scaffold [130].

#### **3. Stereochemical Aspects**

Stereochemistry includes the study of the relative arrangement of atoms or groups in a molecule in three-dimensional space and its understanding is crucial for the study of complex molecules like heterocyclic peptides, which are of paramount biological significance.

*cis,cis*- and *trans,trans*-ceratospongamides (**44**,**45**) are new bioactive thiazole-containing cyclic heptapeptides from the marine red alga *Ceratodictyon spongiosum* and symbiotic sponge *Sigmadocia symbiotica*. The structures of ceratospongamides (**44**,**45**) contained two l-phenylalanine residues, one (l-isoleucine)-l-methyloxazoline residue, one l-proline residue, and one (l-proline)thiazole residue and were found to be proline amide conformers. The change in conformation of a cyclooligopeptide ceratospongamide from "*trans*" to "*cis*" resulted in complete loss of bioactivity, e.g., *trans, trans*-isomer of ceratospongamide (**45**) was found to be a potent inhibitor of the expression of a key enzyme in the inflammatory cascade, secreted phospholipase A2 (sPLA2), with an ED50 of 32 nM in a cell-based model for anti-inflammation, whereas *cis,cis*-isomer (**44**) was inactive [77] (Figure 15).

**Figure 15.** Structures of *cis,cis*-ceratospongamide (**44**) and *trans,trans*-ceratospongamide (**45**) with Pro-Tzl residues (\*change in stereochemistry at C-24 and C-47 carbonyls).

Ulithiacyclamides are thiazole-containing cyclopolypeptides, isolated from the ascidian *Lissoclinum patella*. Bicyclic isomeric ulithiacyclamides F and G contained one oxazoline and one "free" threonine and were found to be anhydro forms of ulithiacyclamide E. Ulithiacyclamides F and G exhibited anti-multiple drug resistant (MDR) activity against vinblastine-resistant CCRF-CEM human leukemic lymphoblasts [51].

Lissoclinamides 4, 5, 7, and 8 are all cyclic heptapeptides derived from sea squirt *Lissoclinum patella* that have the same sequence of amino acids around the ring and differ from one another only in their stereochemistry or the number of thiazole and thiazoline rings. For lissoclinamide 8, the valine residue was at position 31, the same sequence that occurs in lissoclinamide 4. Therefore, the only difference between lissoclinamides 4 and 8 resided in the stereochemistry of one or two of the amino acids. The d configuration was assigned to "Phe-Tzl" and the l-configuration was assigned to "Val-Tzn" moiety in lissoclinamide 4. However, both lissoclinamides 4 and 8 contained similar residues like l-Pro-mOzn and l-Phe. Further, there was similarity in the structural components of lissoclinamides 2 and 3; the only difference was in the stereochemistry around Ala-Tzl moiety, d in the case of the former and l in the latter [55,56].

Lyngbyabellins are thiazole-containing halogenated peptolides derived from cyanobacteria, possessing cytotoxic properties. The configurations at C-15 and C-16 in lyngbyabellin A were found to be 15*S* and 16*S*. Further, C-26 and C-3 in the peptolide has the *S* configuration. The stereochemical assignments of lyngbyabellins E and H were found to be 2*S*, 3*S*, 14*R*, 20*S*, 26*R*, and 27*S*. The stereoconfigurations assigned to lyngbyabellin N was 2*S*, 3*S*, 14*R*, and 20*S*. The absolute configuration of the *N,N*-dimethylvaline (DiMeVal) residue in lyngbyabellin N was found to be l, whereas the absolute configurations of the leucine statine were determined to be 3*R* and 4*S*. The absolute configurations of lyngbyabellin J were found to be 2*S*, 3*S*, 14*R*, 20*R*, 21*S*, 27*R*, and 28*S*. An overall cyclic constitution was not required for potent cytotoxic properties in lyngbyabellins as acyclic peptides like lyngbyabellins F and I also exhibited significant cytotoxic properties [27–30].

The cyclopolypeptides bistratamides M and N (**46**,**47**) were found to be isomers of each other and differed in the configuration of alanine residue attached to the thiazole ring. The configuration was l in bistratamide M (**46**) and was found to be d in bistratamide N (**47**). Bistratamide M (**46**) was found to be slightly more cytotoxic against lung, breast, and pancreatic carcinoma cells in comparison to bistratamide N (**47**). Similarly, bistratamides K and L (**50**,**51**) are isomers, differing in the configuration of alanine residue attached to the thiazole ring. The configuration was d in bistratamide K (**50**) and was found to be l in bistratamide L (**51**). Further, bistratamide G (**39**) was found to be *O*-isostere of bistratamide H (**40**) and bistratamide J was found to be *S*-isostere of bistratamide I (**41**). The compounds containing two thiazole rings were found to be more active than those containing a thiazole ring and an oxazole ring [50,61]. Moreover, the gross structure of cytotoxic cyclopeptide keramamide G (**49**) was found to be almost the same as that of keramamide F (**48**), the only change being the different stereochemistry at C-13 of the α-keto-β-amino acid (Figure 16).

**Figure 16.** Structures of bistratamide M (**46**) with configuration l at C-20, bistratamide N (**47**) with configuration d at C-20, keramamide F (**48**) with stereochemistry *R* at C-13, keramamide G (**49**) with stereochemistry *S* at C-13, bistratamide K (**50**) with configuration d at C-26, and bistratamide l (**51**) with configuration l at C-26.

Grassypeptolides D and E are diasteromeric cyclic peptides from a red sea *Leptolyngbya* cyanobacterium. These cyclodepsipeptides were found to contain two aromatic residues, phenyllactic acid (Pla), *N*-methylphenylalanine (*N*-Me-Phe); β-amino acid residue 2-methyl-3-aminobutyric acid (Maba); and 2-aminobutyric acid (Aba) residue. Further, structural analysis indicated the presence of a 2-methylthiazoline carboxylic acid derived from *N*-methylphenylalanine (*N*-Me-Phe-4-Me-thn-ca) and an Aba-thn-ca unit. Grassypeptolides D and E showed significant cytotoxicity to HeLa (IC50: 335 and 192 nM) and mouse neuro-2a blastoma cells (IC50: 599 and 407 nM). These depsipeptides were found to be threonine/N-methylleucine diastereomers and possesssed different configurations for both l-Thr and *N*-Me-l-Leu in grassypeptolide E (**53**) relative to grassypeptolide D (**52**). Grassypeptolide D (7*R*,11*R*; d-*allo*-Thr and *N*-Me-d-Leu) (**52**) was found to be approximately 1.5-fold less cytotoxic to HeLa cervical carcinoma and neuro-2a mouse blastoma cells than grassypeptolide E (7*S*,11*S*; l-Thr and *N*-Me-l-Leu) (**53**). Moreover, grassypeptolides A and C were found to be the *N*-methylphenylalanine epimers with stereochemistry (7*R*,11*R*,25*R*,29*R*) and (7*R*,11*R*,25*R*,29*S*), respectively. Grassypeptolide C showed 16–23-fold greater potency than grassypeptolide A against colorectal adenocarcinoma HT29 and cervical carcinoma HeLa cells [25] (Figure 17).

**Figure 17.** Structures of grassypeptolide D (**52**) with stereochemistry *R* at C-7 and C-11 of d-*allo*-Thr and *N*-Me-d-Leu residues and grassypeptolide E (**53**) with stereochemistry *S* at C-7 and C-11 of l-Thr and *N*-Me-l-Leu residues.

Nostocyclamide M(**54**) and tenuecyclamide C (**55**) were found to be diasteromers. Nostocyclamide M (**54**) has the same constitution as tenuecyclamide C (**55**) but differs in the configuration of methionine in the structure. Adjacent to one of thiazole ring, d-methionine was present in cyclic hexapeptide nostocyclamide M (**54**) wheresas there was l-methionine in cyclic hexapeptide tenuecyclamide C (**55**). Nostocyclamide M (**54**) displayed allelopathic activity like nostocyclamide but was inactive against grazers unlike the latter [36] (Figure 18).

**Figure 18.** Structures of nostocyclamide M (**54**) with Gly-Tzl and Met-Tzl residues, having methionine configuration d at C-12, and tenuecyclamide C (**55**) with Gly-Tzl and Met-Tzl residues, having methionine configuration l at C-12.

Ulongamides (**1**–**3**) are thiazole-containing cytotoxic cyclic depsipeptides with a novel β-amino acid, 3-amino-2-methylhexanoic acid (Amha), stereochemistry which differentiates ulongamides A–C from ulongamides D–F. The former has the Amha residue in 2*R*,3*R* configuration, while the latter contains an Amha unit in 2*S*,3*R* configuration. The 2-hydroxy-3-methylpentanoic acid (Hmpa) residue was found to be part of ulongamide E and F (**3**) structures, and the configuration of the residue was 2*S*,3*S*. Furthermore, stereochemistry of the 2-hdroxyisovaleric acid (Hiva) unit present in ulongamide d (**2**) was found to be *S* [13].

Calyxamides A and B (**56**,**57**) are cyclic peptides containing 5-hydroxytryptophan (Htrp), isolated from the marine sponge *Discodermia calyx*. These peptides contained residues like 2,3-diaminopropionic acid (Dpr) in addition to (*O*-methylseryl)thiazole moiety. Calyxamides A and B (**56**,**57**) possessed the same planar structure but are isomeric at the 3-position of the 3-amino-2-keto-4-methylhexanoic acid (AKMH) residue like keramamides F and G (13*S* and 13*R*). Structures of calyxamides differ in stereochemistry on isoleucine moiety adjacent to (*O*-methylseryl)thiazole moiety. Calyxamide B (**57**) was found to be the diastereomer of calyxamide A (**56**) and displayed more cytotoxicity against P388 murine leukemia cells, with an IC50 value of 0.9 μM, in comparison to calyxamide A (IC50: 3.9 μM) (**56**) [110] (Figure 19).

**Figure 19.** Structures of calyxamide A (**56**) with *O*-Me-Ser-Tzl moiety, having stereochemistry *S* at the 3-position of 3-amino-2-keto-4-methylhexanoic acid (AKMH) residue, and Calyxamide B (**57**) with *O*-Me-Ser-Tzl moiety, having stereochemistry *R* at the 3-position of AKMH residue.

Aciculitamides A and B are bicyclic *E* and *Z* isomeric peptides obtained from the lithistid sponge *Aciculites orientalis* and result from oxidation of the imidazole ring of aciculitins A–C, bicycles containing an unusual histidino-tyrosine bridge. Aciculitamide A did not show any cytotoxicity against HCT-116 and/or antifungal activity [118].

Sclerotides A and B are cyclopolypeptides from marine-derived fungus, *Aspergillus sclerotiorum* PT06-1. These cyclic hexapeptides contained amino acid residues like l-threonine, l-alanine, phenylalanine, serine, anthranilic acid (AA), and dehydrotryptophan (Δ-Trp). Sclerotides A and B were found to be *Z* and *E* isomers and differed in stereochemistry of dehydrotryptophan. Sclerotide B showed more antifungal activity against *Candida albicans* with MIC values of 3.5 μM in comparison to sclerotide A (MIC: 7 μM). In addition, sclerotide B exhibited weak cytotoxic activity against the HL-60 cell line (IC50: 56.1 μM) and selective antibacterial activity against *Pseudomonas aeruginosa* (MIC: 35.3 μM) [131].
