*2.2. Structural Features of Tzl-Containing Linear Peptides*

In addition to cyclopolypeptides, heterocyclic thiazole ring-based linear peptides are also obtained from marine organisms. Micromide (**17**), apramides (**18**,**19**), dolastatin 10 (**20**), symplostatin 1 (**21**), dolastatin 18 (**22**), lyngbyapeptins A and C (**23**,**24**), and lyngbyabellin F (**25**) and I (**26**) are the best examples of linear peptides containing thiazole rings.

Micromide (**17**) is a highly *N*-methylated linear peptide containing structural features common to many cyanobacterial metabolites, including a d-amino acid, a modified cysteine unit in the form of a thiazole ring and *N*-methylated amino acids. The structrural components of this peptide included moieties like 3-methoxyhexanoic acid, *N*-Me-Gly-thiazole, and other *N*-methylated amino acids viz. *N*-Me-Phe, *N*-Me-Ile, *N*-Me-Val, etc. Micromide (**17**) was found to exhibit cytotoxicity against KB cells [92]. On the other hand, the cyanobacterium-derived apramides A–G are linear lipopeptides containing a thiazole-containing modified amino acid unit. Structural analysis of apramide A (**18**) suggested the presence of a 2-methyl-7-octynoic acid moiety (Moya) and six amino acid residues (*N*-Me-Ala, Pro, *N*,*O*-diMe-Tyr, and 3 units of *N*-Me-Val) and a C-terminally modified amino acid unit (*N*-Me-Gly-thz). Structures of apramide B and apramide C (**19**) differed from apramide A (**18**) in having the presence of a 7-octynoic acid unit (Oya) and 2-methyl-7-octenoic acid moiety (Moea) in lieu of the Moya moiety of apramide A (**18**). Apramides D–F differed from apramide A (**18**), B, and C (**19**), only by bearing a Pro-Tzl unit instead of the *N*-Me-Gly-Tzl residue, which had caused a drastic impact on the conformational behavior. The lipopeptide apramide A (**18**) was found to enhance elastase activity [93] (Figure 7).

**Figure 7.** Structures of micromide (**17**), apramide A (**18**), and apramide C (**19**) with terminal *N*-Me-Gly-Tzl residues.

The dolastatins are sea hare- and marine cyanobacterium-derived compounds that exhibit cytotoxic properties. Dolastatin 10 (**20**) is a linear thiazole-containing heterocyclic peptide bearing *N*,*N*-dimethylvaline, (3*R*,4*S*,5*S*)-dolaisoleucine, (2*R*,3*R*,4*S*)-dolaproine, and (*S*)-dolaphenine [94]. Like dolastatin 10 (**20**), cyanobacterium-derived symplostatin 1 (**21**) is a potent microtubule inhibitor. Symplostatin 1 (**21**) differed from dolastatin 10 (**20**) by the replacement of the *iso*-propyl group by a *sec*-butyl group on the first *N*-dimethylated amino acid. Symplostatin 1 (**21**) is a very potent cytotoxin but not as potent as dolastatin 10 (**20**), whereas synthetic analogues lacking the *N,N*-dimethylamino acid residue were reported to be markedly less cytotoxic. The structure of symplostatin 1 (**21**) differed from dolastatin 10 (**20**) by only one additional CH2 unit in the *N*-terminal residue. The absolute configuration of the stereocenter at C-26 in symplostatin 1 (**21**) was found to be 26*S*. The biological evaluation of symplostatin 1 (**21**) revealed that it is highly active against certain tumors and comparable in its activity with isodolastatin H. Both dolastain 10 (**20**) as well as its methyl analog, symplostatin 1 (**21**) were found to be potent microtubule depolymerizers [95,96].

Dolastatin 18 (**22**) is another cancer cell growth inhibitory linear peptide bearing thiazole moiety from the sea hare, the structure of which is derived from two α-amino acids (Leu and MePhe), a dolaphenine (Doe) unit, and the new carboxylic acid 2,2-dimethyl-3-oxohexanoic acid (dolahexanoic acid, Dhex). Dolastatin 18 (**22**) was found to significantly inhibit growth of human cancer cell lines [97] (Figure 8).

**Figure 8.** Structures of dolastatin 10 (**20**), symplostatin 1 (**21**), and dolastatin 18 (**22**) with terminal Phe-Tzl residues.

Lyngbyapeptins are thiazole-containing lipopeptides with a rare 3-methoxy-2-butenoyl moiety with a high level of *N*-methylation. The cyanobacterium-derived lyngbyapeptin A (**23**) is a linear modified peptide with a 2-substituted thiazole ring. In comparison to lyngbyapeptin A (**23**), lyngbyapeptin B and C possess the same/similar characteristic C- and N-terminal modification and differed by containing other amino acid units in between. Structural analysis of lyngbyapeptin B indicated the presence of two *N*,*O*-dimethyltyrosine residues, an *N*-methylvaline unit, a thiazole-containing modified alanine (Ala-thz) unit, and a 3-methoxy-2-butenoic acid (Mba) moiety with the absolute stereochemistry *S* for the methylated amino acids. The structure of lyngbyapeptin C (**24**) differed from that of lyngbyapeptin B in having the presence of an *N*-terminal unit and 3-methoxy-2-pentenoic acid (Mpa) residue. The structure of lyngbyapeptin D (**27**) differed from that of lyngbyapeptin A (**23**) in having *N*-Me-Val residue instead of *N*-Me-Ile in addition to *N*-Me-Leu, a thiazole-containing modified proline (Pro-thz) unit and *N*,*O*-dimethyltyrosine (*N*,*O*-diMe-Tyr) [98,99]. Lyngbyabellin F (**25**) and I (**26**) are linear dichlorinated lipopeptides that showed the presence of two 2,4-disubstituted thiazole rings. Lyngbyabellin I (**26**) and F (**25**) were found to be cytotoxic to human lung tumor and neuro-2a mouse neuroblastoma cells [100] (Figure 9).

**Figure 9.** *Cont*.

**Figure 9.** Structures of lyngbyapeptin A (**23**) with Pro-Tzl moiety, lyngbyapeptin C (**24**) withAla-Tzl moiety, lyngbyabellin F (**25**) with α,β-dihydroxyisovaleric acid (DHIV)-Tzl residue, lyngbyabellin I (**26**) with Val-Tzl moiety, and lyngbyapeptin D (**27**) with Pro-Tzl moiety.

### *2.3. Structural Features of Thiazole (Tzl)- and Oxazole (Ozl)-Containing Cyclopeptides*

In addition to cyclic peptides with thiazole/thiazoline rings, mixed heterocyclic ring-based cyclopeptides are also derived from marine resources. Comoramide A, didmolamides A–C (**28**–**30**), vemturamides (**31**,**32**), dolastatins E and I (**34**,**35**), microcyclamide (**36**), bistratamides (**37**–**41**), raocyclamides (**42**,**43**), tenuecyclamides, patellamides, and lissoclinamides are bioactive cyclooligopeptides containing thiazole and oxazole rings.

Comoramides are cyanobactins that contained prenylated amino acids. The ascidian-derived cyclopeptide comoramide A was isolated with threonine heterocyclized in position 5 and prenylated in position 3 and was found to contain six amino acids in its structure, including two amino acids that existed as a 5-methyloxazoline (mOzn) heterocycle and as a thiazoline ring (Tzn). The additional amino acid moieties present were l-alanine, l-phenylalanine, and l-isoleucine. Like patellin, trunkamide A, mollamide, and hexamollamide, comoramide A was found to be a unique type of peptide that contained threonine residue for which the side chain is modified as dimethylallyl ether. This cyclohexapeptide exhibited structural similarilty with another ascidian-derived cycloheptapeptide mollamide in two amino acids viz. Ile-Tzn and Phe-Thr. Comoramide A was found to be cytotoxic against the A549, HT29, and MEL-28 tumor cell lines [45].

Didmolamides A and B (**28**,**29**) are ascidian-derived cyclohexapeptides that contained two l-alanylthiazole residues and one l-phenylalanine moiety in common but didmolamide A (**28**) contained 5-methyloxazoline (mOzn) heterocycle in addition, which is replaced by l-threonine moiety in didmolamide B (**29**). Morover, didmolamide C (**30**) differs from didmolamides A and B (**28**,**29**) in the oxidation state of the heterocyclic rings, having two thiazoline rings (instead of thiazoles) in didmolamide C (**30**). Additionally, didmolamide C (**30**) was found to contain a methyloxazole ring instead of a methyloxazoline ring of didmolamide A (**28**). Didmolamide A (**28**) displayed mild cytotoxicity against the A549, HT29, and MEL28 tumor cell lines [48,101] (Figure 10).

**Figure 10.** Structures of didmolamide A (**28**) with Ala-Tzl moieties, didmolamide B (**29**) with Ala-Tzl moieties, and didmolamide C (**30**) with Ala-Tzn moieties.

Venturamides (**31**,**32**) are cyanobacterium-derived thiazole- and methyloxazole-containing cyclohexapeptides that exhibited antimalarial and cytotoxic activities. Structural analysis of venturamide B (**32**) indicated the presence of d-alanine, d-valine, and d-*allo*-threonine in addition to three heteroaromatic moieties. The polypeptide venturamide B (**32**) was identified as cyclo-d-*allo*-Thr-Tzl-d-Val-Tzl-d-Ala-mOzl. The cyclic hexapeptide venturamide B (**32**) differed from venturamide A (**31**) in having a d-threonine unit in place of the d-alanine adjacent to the thiazole ring. There was a close similarity between the structures of venturamide A (**31**) and blue-green alga-derived cyclopeptide dendroamide A (**33**): however, d-valine and d-alanine are exchanged with each other, adjacent to two thizaole heterocycles at C-12 and C-20. Venturamides (**31**,**32**) showed strong in vitro activity against *Plasmodium falciparum*, with only mild cytotoxicity to mammalian Vero cells. Also, mild activity against *Trypanasoma cruzi*, *Leishmania donovani*, and MCF-7 cancer cells was also reported for venturamides [34] (Figure 11).

**Figure 11.** Structures of venturamide A (**31**) with Ala-Tzl and Val-Tzl residues, venturamide B (**32**) with Thr-Tzl and Val-Tzl residues, and dendroamide A (**33**) with Val-Tzl and Ala-Tzl residues.

The sea hare-derived cyclopolypeptides dolastatins E and I (**34**,**35**) were found to contain three kinds of five-membered heterocycles viz. oxazole/methyloxazole (Ozl/mOzl), thiazole (Tzl), and thiazoline/oxazoline (Tzn/Ozn), in addition to one residue each of d-alanine and l-alanine and one residue of d-isoleucine in dolastatin E (**34**) while one residue each of l-alanine, l-valine, and l-isoleucine in the case of dolastatin I (**35**). Although both of these cyclic hexapeptides displayed cytotoxicity against HeLa S3 cells, in comparison, dolastatin I (**35**) was found to be more cytotoxic than dolastatin E [75,76]. On the other hand, in addition to two thiazole (Tzl) and one methyloxazole (mOzl) rings, the cyanobacterium-derived cyclopeptide microcyclamide (**36**) contained two usual amino acids, l-isoleucine and l-alanine, and one *N*-methylhistidine residue. Overall, the hexapeptidic structure was composed of three units viz. thiazole-methylhistidinyl, thiazole-isoleucinyl, and methyloxazole-alanyl units. This cyclic hexapeptide displayed a moderate cytotoxic activity against P388 murine leukemia cells [35] (Figure 12).

**Figure 12.** Structures of dolastatin E (**34**) with Ile-Tzl moiety, dolastatin I (**35**) with Ala-Tzl moiety, and microcyclamide (**36**) with Ile-Tzl and *N*-Me-His-Tzl residues.

The ascidian-derived bistratamide A and B contained heteroaromatic rings viz. methyloxazoline (mOzn) and thiazoline (Tzn) rings in common in addition to one residue each of alanine, phenylalanine, and l-valine. However, bistratamide A differed from bistratamide B only in the conversion of one thiazoline ring to a thiazole, i.e., these hexapeptides differed only by the the presence or absence of one double bond. Both these cyclohexapeptides displayed activity toward human cell lines viz. MRC5CV1 fibroblasts and T24 bladder carcinoma cells. Bistratamides C and D (**37**,**38**) possessed one thiazole ring in common in addition to two l-valine residues. However, bistratamide C (**37**) differed from bistratamide D (**38**) in having an l-alanine moiety instead of additional l-valine. Moreover, the other two heteroaromatic rings in bistratamide D (**38**) were methyloxazoline and oxazole, whereas in bistratamide C (**37**), oxazole and thiazole rings were present. Bistratamides E and F were found to contain three residues of l-valine in addition to thiazole and methyloxazoline rings. Bistratamide F differed from bistratamide E in having an additional oxazoline ring instead of a second thiazole ring in bistratamide E. Similarily, bistratamides G and H (**39**,**40**) were found to contain three residues of l-valine in addition to thiazole and methyloxazole rings. Bistratamide G (**39**) differed from bistratamide H (**40**) in having an additional oxazole ring instead of a second thiazole ring in bistratamide H (**40**). Further, bistratamide I (**41**) contained three residues of l-valine in addition to one thiazole and one oxazole ring. The ascidian-derived bistratamides M and N (**46**,**47**) are oxazole-thiazole-containing cyclic hexapeptides that displayed moderate cytotoxicity against four human tumor cell lines including NSLC A-549 human lung carcinoma cells, MDA-MB-231 human breast adenocarcinoma cells, HT-29 human colorectal carcinoma cells, and PSN1 human pancreatic carcinoma cells. Moreover, bistratamides G-I (**39**–**41**) and J showed weak to moderate activity against the HCT-116 human colon tumor cell line [50,59–61] (Figure 13).

**Figure 13.** Structures of bistratamide C (**37**) with Val-Tzl and Ala-Tzl residues, bistratamide D (**38**) with Val-Tzl moiety, bistratamide G (**39**) with Val-Tzl moiety, bistratamide H (**40**) with two Val-Tzl residues, and bistratamide I (**41**) with Val-Tzl moiety.

Raocyclamides (**42**,**43**) are cyclooligopeptides in which the ring system contains amide links only, and they contain three heteroaromatic rings symmetrically arranged in a peptide chain with different connected aliphatic amino acids providing structural diversity. Raocyclamides A and B (**42**,**43**) are cyanobacterium-derived oxazole- and thiazole-containing cyclic hexapeptides with cytotoxic

properties. Raocyclamide A (**42**) contained three standard amino acid residues viz. d-isoleucine, l-alanine, and d-phenylalanine and three modified amino acids viz. thiazole, oxazole, and oxazoline. In comparison, raocyclamide B (**43**) contained four standard amino acid residues viz. d-isoleucine, l-alanine, d-phenylalanine, and d-serine and two modified amino acids viz. thiazole and oxazole. Raocyclamide A (**42**) differed from raocyclamide B (**43**) in having an additional heterocyclic ring "oxazoline" with a d-configuration instead of a d-serine residue. Raocyclamide A (**42**) was found to be moderately cytotoxic against sea urchin embryos [32] (Figure 14).

**Figure 14.** Structures of raocyclamide A (**42**) and raocyclamide B (**43**) with d-Ile-Tzl residues.

The ascidian-derived lissoclinamides 1–10 and cyanobacterium-derived tenuecyclamide A and B are other cyclopolypeptides containing thiazole, thiazoline, methyloxazole, and methyloxazoline rings which displayed cytotoxicity against SV40 transformed fibroblasts and transitional bladder carcinoma cells as well as inhibited the division of sea urchin embryos [102–105].

Various heterocyclic marine-derived thiazole-based cyclopolypeptides including those having thiazoline (Tzn), oxazole (Ozl), oxazoline (Ozn), 5-methyloxazole (mOzl), 5-methyloxazoline (mOzn), 5-hydroxytryptophan (Htrp), N-methylimidazole (mImz), histidine (His), tryptophan (Trp), 2-bromo-5-hydroxytryptophan (Bhtrp), and N-methyltryptophan (Metrp) rings in addition to thiazole, together with their molecular formulas and composition, are tabulated in Table 1.


**Table 1.** Heterocyclic thiazole-based cyclopolypeptides from marine resources.


**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*

\* Tzl: Thiazole, Tzn: Thiazoline, Ozl: Oxazole, Ozn: Oxazoline, mOzl: 5-methyloxazole, mOzn: 5-methyloxazoline, Htrp: 5-hydroxytryptophan, mImz: N-methylimidazole, His: histidine, Trp: tryptophan, Bhtrp: 2-bromo-5-hydroxytryptophan, Metrp: N-methyltryptophan.

#### *2.4. Structural Features of Thiopeptide Antibiotics*

Thiopeptides are a novel family of antibiotics which are associated with a lot of pharmacological properties including immunosuppressive, antineoplastic, antimalarial, and potent antimicrobial activity against Gram-positive bacteria. Due to their interesting structures and bioprofile against bacteria, thiopeptides have attracted the attention of researchers and scientists as a new class of emerging antibiotics. The most important characteristic feature of the thiopeptides is the central nitrogen-containing six-membered ring with diverse oxidation states. On the basis of different oxidation states of the central ring of thiopeptides, they can belong to the "a series" with a totally reduced central piperidine, the "b series" with a 1,2-dehydropiperidine ring, and the "c series" with a piperidine ring fused with imidazoline. All members of series a, b, and c have a macrocycle which contains a quinaldic acid moiety. The d series shows a trisubstituted pyridine ring, and the e series is known for the hydroxyl group in the central tetrasubstituted pyridine ring. The e series also presents a macrocycle formed by a modified 3,4-dimethylindolic acid moiety. The central ring in thiopeptides serves as a scaffold to at least one macrocycle and a tail, containing different thiazoles and oxazoles which are developed by dehydration/dehydrosulfanylation of amino acid like serine, cysteine, etc. TP-1161, YM-266183, YM-266184, kocurin, baringolin, geninthiocin, Ala-geninthiocin, and Val-geninthiocin are examples of thiopeptides from marine resources [111].

TP-1161 belongs to the "d series" of thiopeptide antibiotics, produced by a marine sediment-derived *Nocardiopsis* sp. Structural features of this thiopeptide include the three 2,4-disubstituted thiazoles and one 2,4-disubstituted oxazole moiety in addition to the presence of a trisubstitued pyridine (Pyr) functional unit and an unusual aminoacetone moiety. TP-1161 displayed good activity against a panel of Gram-positive bacteria including *Staphylococcus aureus*, *Staphylococcus haemolyticus*, *Staphylococcus epidermidis*, *Enterococcus faecium*, and *Enterococcus faecalis* [112].

YM-266183 and YM-266184 are novel thiopeptide antibiotics produced by *Bacillus cereus* isolated from a marine sponge and structurally related to a known family of antibiotics that include thiocillins and micrococcins. Structural analysis of these thiopeptides indicated the presence of several unusual amino acids with heteroaromatic moieties, including the six thiazole rings, a 2,3,6-trisubstituted pyridine residue to which three of thiazole units are attached, a 2-amino-2-butanoic acid unit with an aminoacetone residue, a (*Z*)-2-amino-2-butenoic acid unit attached to a threonine residue, and a 3-hydroxyvaline moiety. There was a close similarity in structures of YM-266183 and YM-266184 except for the presence of a methoxy group (C55) in YM-266184 instead of the hydroxy group of

YM-266183. These new antibacterial substances were found to exhibit activity against drug-resistant bacteria [113].

Kocurin is a new anti-methicillin-resistant *Staphylococcus aureus* (MRSA) bioactive compound, belonging to the thiazolyl peptide family of antibiotics, obtained from sponge-derived *Kocuria* and *Micrococcus* spp. Structural analysis of this thiopeptide indicated the presence of several heteroaromatic moieties, including one thiazoline and four thiazole rings, one methyloxazole ring and a 2,3,6-trisubstituted pyridine residue to which two of thiazole units and one methyloxazole unit are attached, aromatic amino acids like phenylalanine and tyrosine, and two proline units. Kocurin was found to be closely related to two known thiazolyl peptide antibiotics with similar modes of action: GE37468A and GE2270. The antimicrobial activity profile of kocurin indicated the extreme potency against Gram-positive bacteria with minimum inhibitory concentration (MIC) values of 0.25–0.5 μg/mL against methicillin-resistant *Staphylococcus aureus* (MRSA) [114].

Baringolin is a novel thiopeptide of the d series, containing a central 2,3,6-trisubstituted pyridine, derived from fermentation of the marine-derived bacterium *Kucuria* sp. The macrocycle in baringolin contained three thiazoles—a methyloxazole and pyridine ring, a thiazoline ring with an α-chiral center, and a pyrrolidine motif derived from a proline residue—in addition to three natural amino acids viz. tyrosine, phenylalanine, and asparagine. The long peptidic tail was found to be a pentapeptide containing three methylidenes resulting from dehydration of serine that is attached to the pyridine through a fourth thiazole. This thiopeptide displayed important antibacterial activity against *Staphylococcus aureus*, *Micrococcus luteus*, *Propionibacterium acnes*, and *Bacillus subtilis* at nanomolar concentrations [115].

Ala-geninthiocin, geninthiocin, and Val-geninthiocin are new broad-spectrum thiopeptide antibiotics produced from the cultured marine *Streptomyces* sp. Structural analysis of all three thiopeptides indicated the presence of heteroaromatic moieties, including one thiazole and two oxazole rings, one methyloxazole ring, and a 2,3,6-trisubstituted pyridine residue to which two of thiazole units are attached at the 2 and 3 positions, including proteinogenic amino acid viz. l-threonine. The peptide structure of Ala-geninthiocin is largely similar to geninthiocin, the only difference being the presence of an l-Alanine residue instead of dealanine at the C-terminal amide. Further, Val-geninthiocin contained l-valine moiety instead of l-hydroxyvaline of geninthiocin. Ala-geninthiocin was found to exhibit good activity against Gram-positive bacteria including *Staphylococcus aureus*, *Bacillus subtilis*, *Mycobacterium smegmatis*, and *Micrococcus luteus* as well as cytotoxicity against A549 human lung carcinoma cells. When compared to geninthiocin, Ala-geninthiocin displayed better cytotoxicity but antibiotic activity against Gram-positive bacteria was comparatively low. Val-geninthiocin was found to possess more antifungal activity against *Mucor hiemalis* and cytotoxicity against A549 human lung carcinoma cells and L929 murine fibrosarcoma in comparison to geninthiocin. Further, Ala-geninthiocin and Val-geninthiocin displayed weak to moderate antifungal activity against *Candida albicans*, whereas geninthiocin was inactive. Ala-geninthiocin and geninthiocin displayed moderate antibiotic activity against Gram-negative bacteria *Chromobacterium violaceum*, whereas val-geninthiocin was inactive [116].
