**4. Synthesis of Heterocyclic Peptides**

Despite of lot of challenges associated with synthesizing complex peptide molecules [132–135], syntheses of diverse aromatic/heteroaromatic peptides were accomplished by several research groups employing diverse techniques of peptide synthesis including solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS), and a mixed solid-phase/solution synthesis strategy, irrespective of whether these congeners belong to linear analogues [136–151] or are cyclic in nature [152–169]. Literature is enriched with reports involving synthesis of various heterocyclic cyclopolypeptides bearing thiazole/thiazoline/tryptophan/histidine moieties viz. cyclodidemnamide B [42], dolastatin 3 [90], aeruginazole A [170], didmolamide B (**29**) [171], dolastatin 10 (**20**) [172], scleritodermin A (**10**) [173], obyanamide (**8**) [174,175], marthiapeptide A [176], diandrine C [177], diandrine A [178], sarcodactylamide [179], segetalin C [180], segetalin E [181], annomuricatin B [182], and gypsin D [183].

The first total synthesis of thiazole and methyloxazoline-containing cyclohexapeptides didmolamides A and B was accomplished by the solid phase assembly of thiazole-containing amino acids and Fmoc-protected α-amino acids. The synthesis of thiazole-containing didmolamide B (**29**) was also achieved using solution phase peptide synthesis. The crucial thiazole amino acid was synthesized by MnO2 oxidation of a thiazoline prepared from an Ala-Cys dipeptide using bis(triphenyl)oxodiphosphonium trifluoromethanesulfonate. The final macrolactamization was accomplished efficiently by benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 4-dimethylaminopyridine (DMAP) [171].

A practical approach to asymmetric synthesis of dolastatin 10 (**20**) was found to involve SmI2-induced cross-coupling and asymmetric addition of chiral N-sulfinyl imine [172].

The synthesis of the C1–N15 fragment of the marine natural product scleritodermin A (**10**) was accomplished through a short and stereocontrolled sequence. The highlights of this route included synthesis of a novel conjugated thiazole moiety 2-(1-amino-2-*p*-hydroxyphenylethane)-4- (4-carboxy-2,4-dimethyl-2Z,4E-propadiene)-thiazole (ACT) fragment and the formation of the α-keto amide linkage by the use of a highly activated α,β-ketonitrile [173]. The total synthesis of a cytotoxic *N*-methylated thiazole-containing cyclic depsipeptide obyanamide (**8**) was accomplished that included the preparation of two protected fragments before macrocyclization, starting from material (S)-2-aminobutyric acid. The synthesis has led to a reassignment of the C-3 configuration in β-amino acid residue. As a result, the configuration at C-3 position has been amended as *R* [174,175].

The cytotoxic polythiazole-containing cyclopeptide marthiapeptide A having a linked trithiazole−thiazoline system was synthesized via two routes. The initial strategy involved a macrocyclization of the linear precursor via a peptide-coupling reaction between the amine on the alanine residue and the carboxylic acid end of isoleucine. However, the cyclization was not successful, which was attributed to the closing point being too close to the rigid heterocyclic thiazole moiety. The second strategy involved closing between the thiazoline and peptide in which successful cyclization can be attributed to the flexibility of the thiazoline, which allows a connection between the molecule's head and tail [176].

#### **5. Structural Activity Relationships**

Structural activity relationships (SAR) are prime keys to diverse aspects of drug discovery, ranging from primary screening to extensive lead optimization. SAR can be used to predict bioactivity from the molecular structure. This powerful technology is used in drug discovery to guide the acquisition or synthesis of desirable new compounds as well as to further characterize existing molecules. The principle of structure–activity relationship indicated that there is a relationship between molecular structures and their biological activity and solely depends on the recognition of which structural characteristics correlate with chemical and biological reactivity.

The lissoclinamides, heterocyclic peptides isolated from sea squirt Lissoclinum patella, are derived from a cyclic heptapeptide in which a threonine has been cyclised to an oxazoline and two cysteines have been cyclised to give a thiazole or thiazoline. While comparing natural and synthetic lissoclinamides, it was found that the replacement of thiazoline rings with oxazolines decreased activity to a greater extent than replacement of oxazoline rings with thiazolines [184]. This study further showed that it was not the individual components of the macrocycle that conferred high activity, but rather, the overall conformation of this molecule was responsible for the bioactivity. While comparing structures of lissoclinamides 4 and 5, it was observed that these compounds differ only in the oxidation state of a single thiazole unit but that this difference makes lissoclinamide 5 two orders of magnitude less cytotoxic than lissoclinamide 4 against bladder carcinoma (T24) cells [55].

In raocyclamides (**42**,**43**), the presence of oxazoline moiety was found to be essential for cytotoxicity against sea urchin embryos. The cyanobacterium-derived cyclopolypeptides raocyclamide A and B (**42**,**43**) possessed thiazole and oxazoline rings in their composition, but raocyclamide A (**42**) contained an additional oxazoline moiety in its structure. This structural change results in a lot of variation in the biological response. While comparing the bioeffects of these cyclopolypeptides, it was found that raocyclamide A (**42**) inhibited the division of embryos of *Paracentrotus lividus* with an effective dose for 100% inhibition (ED100) of 30 μg/mL, whereas raocyclamide B (**43**) was inactive even at the concentrations of 250 μg/mL [32].

Replacement of d-valine moiety with d-methionine adjacent to one of the thiazole rings in the structure of macrocyclic thiazole and methyloxazole-containing allelochemical nostocyclamide resulted in cyanobacterial cyclopeptide nostocyclamide M (**54**) with inactivity toward grazers, but this structural modification does not affect the allelopathic activity against anabaena 7120 [36].

The reduction of isoleucylthiazole (Ile-Tzl) residue of a thiazole- and methyloxazoline-containing cyclooligopeptide of cyanobacterial origin, aerucyclamide B, to an isoleucylthiazoline (Ile-Tzn) residue resulted in a close analogue aerucyclamide A. From this one structural modification, the antiplasmodial activity was found to decrease by 1 order of magnitude. Further, the cyclohexapeptide aerucyclamide C underwent hydrolysis reaction using trifluoroacetic acid to form ring-opened products microcyclamide 7806A and microcyclamide 7806B. This change in structure from rigid, disk-like cyclamides to methyloxazoline (mOzn) ring-opened hydrolysis products resulted in loss of antimicrobial and cytotoxic activities [38]. In comparison, aerucyclamide B was the most active antiplasmodial compound among aerucyclamides against chloroquine-resistant strain K1 of *P. falciparum*, with selectivity against a rat myoblast cell line, whereas against parasite T. brucei rhodesiense, the most active compound was aerucyclamide C.

The cyclic structure of oxazole-rich, thiazole-containing polypeptide mechercharmycin A was found to be essential for its strong antitumor activity against human lung cancer and leukemia cells. The cyclic ring opening of mechercharmycin A resulted in linear peptide mechercharmycin B which did not displayed any inhibitory activity toward any of the cell lines [79].

The ascidian-derived cytotoxic cyclic hexapeptides, bistratamides A and B, differed from each other only by the presence or absence of one double bond. The conversion of one thiazoline in bistratamide A to a thiazole in bistratamide B, i.e., oxidation of thiazoline to thiazole, resulted in a less toxic compound. For example, comparing bioactivities of bistratamides A and B, the former has an IC50 value of about 50 μg/mL and latter has an IC50 value greater than 100 μg/mL against human cell lines including fibroblasts and bladder carcinoma cells [60].

Replacement of the alanine unit adjacent to the thiazole ring by a threonine unit in cyanobacterium-derived modified cyclohexapeptide venturamide A (**31**) resulted in a related cyclic hexapeptide venturamide B (**32**). This structural change reflected an increase in antimalarial activity against *Plasmodium falciparum* and cytotoxic activity toward mammalian Vero cells. However, with this modification, a decrease in bioactivity against *Trypanasoma cruzi* and MCF-7 cancer cells was observed [34].

The lyngbyabellin family of thiazole-containing peptolides are known to exhibit moderate to potent cytotoxicity against a number of different cancer cell types through the promotion of actin polymerization. In the HCT116 colon cancer cell line assay, reproducible IC50 values (40.9 ± 3.3 nM) were obtained for lyngbyabellin N, confirming the potent cytotoxic effect of this new member of the lyngbyabellin class and suggesting that the side chain of lyngbyabellin N was an essential structural feature for this potent activity. However, this trend was not entirely consistent within this structure class as other lyngbyabellin analogs lacking the side chain were found to exhibit bioactivity against HT29 and HeLa cells [29]. When compared to lyngbyabellin A, lyngbyabellin J displayed slightly less bioactivity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells. The cytoskeletal actin-disrupting lyngbyabellin 27-deoxylyngbyabellin A was found to be more potent than lyngbyabellin A against HT29 and HeLa carcinoma cell lines (IC50 values: 27-deoxylyngbyabellin A, 0.012 and 0.0073 μM; lyngbyabellin A, 0.047 and 0.022 μM), indicating the importance of hydroxylation at the C-27 position. However, lyngbyabellin A, its 27-deoxy analog, and lyngbyabellin J exhibited more cytotoxic activity against the two cell lines when compared to peptolide lyngbyabellin B (IC50 values: 1.1 and 0.71 μM). The configuration of the hydroxy acid-derived unit esterified to the 7,7-dichloro-3-acyloxy-2-methyloctanoic acid residue (here, Dhmpa) was not found to have a profound effect on the activity. Furthermore, close analysis of bioactivity data indicated that the cytotoxicity of cyclic and acyclic lyngbyabellins appeared to be similar [30].

The antithrombin cyclopolypeptides and cyclotheonellazoles had structural features similar to another *Theonella* sponge-derived peptide oriamide (**9**) in having nonproteinogenic amino acids like 4-propenoyl-2-tyrosylthiazole and 3-amino-4-methyl-2-oxohexanoic acid and showed potent inhibitory activity against the serine protease enzymes chymotrypsin and elastase. Cyclotheonellazole complexes with elastase/chymotrypsin exhibit a tetrahedral transition state involving the keto group of Amoha and Ser195 of elastase, while the side chain of Amoha fits in the enzyme S1 pocket. Cyclotheonellazole A, which contains a 2-aminopentanoic acid residue, was found to be the most potent inhibitor. This was probably due to a better compatibility with the enzyme S2 subsite. Cyclotheonellazoles B and C contained the amino acids leucine and homoalanine, and it appeared that the length and the branching of the aliphatic chain influenced the bioactivity. Further, these cyclopeptides were inactive against the malaria parasite plasmodium falciparum at IC50 values of greater than 20 μg/mL [68].

Ulongamides (**1**–**3**) are cyanobacterium-derived β-amino acid- and thiazole-containing cyclic peptides with weak cytotoxic properties. In cyclodepsipeptide ulongamide F (**3**), the lack of an aromatic amino acid or the N-methyl group adjacent to the hydroxyl acid (N-methylphenylalanine/N-methyl tyrosine in ulongapeptides A–E and l-valine in ulongapeptide F) was found to be detrimental to bioactivity. This was evident from the observation that ulongamide F (**3**) was inactive at <10 μM against KB and LoVo cells in comparison to ulongapeptides A (**1**) and D (**2**), which displayed cytotoxicity against both cell lines [13].

#### **6. Biological Activity**

Although thiazole-containing cyclopolypeptides of marine origin are associated with a number of bioactivities including antitubercular, antibacterial, antifungal, and inhibitory activity against serine protease enzymes chymotrypsin and elastase; anti-HIV activity; antiproliferative activity; antimalarial activity; and inhibitory activity against the transcription factor activator protein-1, the majority of them were found to exhibit anticancer activity. Various pharmacological activity-associated marine-derived Tzl-containing cyclopolypeptides along with susceptible cell line/organism with minimum inhibitory concentration are tabulated in Table 2.


**Table 2.** Heterocyclic Tzl-based peptides (TBPs) with diverse pharmacological activities.


#### **Table 2.** *Cont.*


**Table 2.** *Cont.*

<sup>a</sup> MIC—minimum inhibitory concentration, <sup>b</sup> CNS—central nervous system, <sup>c</sup> KB—ubiquitous KERATIN-forming tumor cell subline, <sup>d</sup> ED100—effective dose for 100% inhibition.

#### **7. Mechanism of Action**

Heterocyclic thiazole-based peptides act by a variety of mechanisms including inhibiting microtubule assembly/mitosis, arresting nuclear division, inducing tumor cell apoptosis, causing microtubule depolymerization, inhibiting the protein secretory pathway through preventing cotranslational translocation, inducing G1 cell cycle arrest and an apoptotic cascade, inhibiting the phosphorylation of ERK and Akt, disrupting the cellular actin microfilament network, overproducing 1,3-β-D-glucan, activating the caspase-3 protein expression and decrease in B-cell lymphoma 2 (Bcl-2) levels, inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) luciferase and nitrite production, etc.

Dolastatin 10 (**20**) is a pentapeptide with potential antineoplastic activity, derived from marine mollusk Dolabella auricularia. Its mechanism of action involves the inhibition of tubulin polymerization, tubulin-dependent guanosine triphosphate hydrolysis, and nucleotide exchange, and it is a potent noncompetitive inhibitor of vincristine binding to tubulin. Binding to tubulin, dolastatin 10 (**20**) inhibits microtubule assembly, resulting in the formation of tubulin aggregates and inhibition of mitosis. This thiazole-containing linear peptide also induces tumor cell apoptosis through a mechanism involving bcl-2, an oncoprotein that is overexpressed in some cancers. Microtubule inhibitors from several chemical classes can block the growth and development of malarial parasites, reflecting the importance of microtubules in various essential parasite functions. Dolastatin 10 (**20**) was a more potent inhibitor of *P. falciparum* than any other microtubule inhibitor like dolastatin 15. Dolastatin 10 (**20**) caused arrested nuclear division and apparent disassembly of mitotic microtubular structures in the parasite, indicating that compounds binding in the "Vinca domain" of tubulin can be highly potent antimalarial agents [185].

Symplostatin 1 (**21**), an analog of dolastatin 10 (**20**), is a potent antimitotic with antiproliferative effects that act by causing microtubule depolymerization, formation of abnormal mitotic spindles that lead to mitotic arrest, and initiation of apoptosis involving the phosphorylation of the anti-apoptotic protein Bcl-2. Symplostatin 1 (**21**) inhibited the polymerization of tubulin in vitro, consistent with its mechanism of action in cells and suggesting that tubulin may be its intracellular target. Additionally, symplostatin 1 (**21**) was found to inhibit the proliferation and migration of endothelial cells, suggesting that it may have antiangiogenic activity [186].

Largazole is a cyclic peptide with thiazole/thiazoline residues, including a number of unusual structural features, including a 3-hydroxy-7-mercaptohept-4-enoic acid unit and a 16-membered macrocyclic cyclodepsipeptide skeleton. Largazole showed potent and highly selective inhibitory activities against class I HDACs (histone deacetylases) and displayed superior anticancer properties. Largazole was found to strongly stimulate histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth and induced apoptosis in the tumor. This effect is likely mediated by modulation of levels of cell cycle regulators, by antagonism of the AKT pathway through IRS-1 downregulation, and by reduction of epidermal growth factor receptor levels [187].

Lyngbyabellins are hectochlorin-related peptides with thiazole moieties that are associated with actin polymerization activity. These lipopeptides were found to induce perceptible thickening of the cytoskeletal elements with a relatable increase in binucleated cells. Lyngbyabellin A was found to disrupt the cellular actin microfilament network in A10 and, accordingly, disrupted cytokinesis in colon carcinoma cells, causing the formation of apoptotic bodies. Lyngbyabellin E exhibited actin polymerization ability and was found to completely block the cellular microfilaments, forming binucleated cells [188].

Scleritodermin A (**10**) is a cytotoxic cyclic peptide with an unusual N-sulfated side chain and a novel conjugated thiazole moiety as well as an α-ketoamide group. Scleritodermin A (**10**) has significant in vitro cytotoxicity against a panel of human tumor cells lines, and this depsipeptide acts through inhibition of tubulin polymerization and the resulting disruption of microtubules, which is the target of a number of clinically useful natural product anticancer drugs [64].

Theonellamides are sponge-derived antifungal and cytotoxic bicyclic dodecapeptides with a histidine-alanine bridge. Specific binding of these peptides to 3β-hydroxysterols resulted in overproduction of 1,3-β-D-glucan and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A because of its direct interaction with 3β-hydroxyl groups of sterols. Membrane action of theonellamide A proceeds via binding to the membrane surface through direct interaction with sterols and modification of the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. Theonellamides represents a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-beta-D-glucan synthesis [189].

Phalloidin is a tryptophan containing bicyclic phallotoxin, which functions by binding and stabilizing filamentous actin (F-actin) and effectively prevents the depolymerization of actin fibers. Due to its tight and selective binding to F-actin, derivatives of phalloidin-containing fluorescent tags are used widely in microscopy to visualize F-actin in biomedical research. Though phallotoxins are

highly toxic to liver cells, they add little to the toxicity of ingested death cap, as they are not absorbed through the gut [190].

Jaspamide (Jasplakinolide) is a cytotoxic cyclodepsipeptide with bromotryptophan moiety that induces apoptosis in human leukemia cell lines and brain tumor Jurkat T cels by activation of caspase-3 protein expression and decrease in Bcl-2 levels. Apoptosis induced by Jaspamide was associated with caspase-3 activation, decreased Bcl-2 protein expression, and increased Bax levels, suggesting that jaspamide induced a caspase-independent cell death pathway for cytosolic and membrane changes in apoptosis cells and a caspase-dependent cell death pathway for poly (ADP-ribose) polymerase (PARP) protein degradation [191].

Azonazine is a unique peptide with a macrocyclic heterocyclic core of the benzofuro indole ring system with diketopiperazine residue. This hexacyclic dipeptide displayed anti-inflammatory activity and was found to act by inhibiting NF-κB luciferase and nitrite production [192].

#### **8. Issues Associated with Marine Peptides in Drug Development**

Marine peptides are fascinating therapeutic candidates due to their diverse bioactivities. They demonstrate significant chemical and biological diversity for drug development including minimized drug–drug interaction, less tissue accumulation, and low toxicity. Approximately 40% of existing small molecules and 70% of new candidates under development pipelines suffer from the low solubility problem, which is a major reason for their suboptimal drug delivery as well as failures in their development process. Approaches such as cyclodextrin complexation and solid dispersions have been employed to address this challenge and recommend the better formulation over their existing dosage forms [193–198]. Likewise, peptides, being biomacromolecules, also exhibit various challenges such as limited water solubility, stability aspects, as well as structural and synthesis complexities, limiting their full exploitation in drug development [199,200]. Table 3 portrays various issues associated with peptide drug development. Amidst the major challenges, difficulty in optimization of the required peptide length to achieve pharmacologically useful levels for receptor activation accounts for the hindered drug development of marine-based peptides. The optimization depends on variables including the size, accessibility, and fit of ligand-binding surfaces, ligand stability, and receptor residency time. Further, the high proteolytic instability of peptide-based therapeutics can be conquered by alteration of the side chains and amide bonds, which in turn makes the peptide resistant to proteolytic degradation [201]. The challenges of low bioavailability and short half-life can be overpowered by three approaches: (i) modification of the peptide backbone through the introduction of D-amino acids or unnatural amino acids, (ii) alteration of the peptide bonds with reduced amide bonds or β-amino acids, and (iii) attachment of a fatty acid. Approaches (i) and (ii) drive the peptide backbone through introducing cyclization, reduced flexibility, and enzyme digestion. Approach (iii) could lead to more specific binding to the target leading to enhanced half-life and bioavailability with fewer side effects [202]. Intracellular delivery of peptides has been a subject of interest due to their membrane-binding ability to exert action on the cell surface. Also, involving the protein transduction domain allows intracellular peptide delivery. Although the liposomal and nanoparticle drug delivery takes advantage of fusing the peptides for intracellular drug delivery, they also face the problem of low encapsulation efficiency [202]. During the process development of peptide synthesis, it is difficult to identify the critical process parameters to achieve expected purity and yield. In addition, the peptide synthesis process also depends on the specifications or requirements and targeted volumes. However, the establishment of acceptable standards and proven ranges may be lacking, which in turn accelerates their manufacturing costs during drug development.


**Table 3.** Issues associated with marine peptide drug development.
