**1. Introduction**

The oceans cover more than 70% of the earth's surface and comprise around 95% of the volume of the biosphere. This impressive size of the marine habitat and its biological diversity known to date lead to the assumption of an enormous, ye<sup>t</sup> still largely unexplored world, carrying an unused potential for research areas such as pharmacology, medicine, crop protection, or food technology. Furthermore, the uniqueness of marine life is reflected by the fact that only a small fraction of the 30,000 marine natural products (MNPs) known at present can also be found in terrestrial sources [1]. Additionally, the isolation and investigation of MNPs is a rapidly expanding field of research at the interface of biology and chemistry [2–10]. Looking back to 2009, when only 20,000 MNPs were known, an impressive increase of 50% has been achieved in the past 11 years, which highlights the importance of the marine habitat in this context [11].

Among the marine alkaloids, which are largely composed of nitrogen-containing heterocycles, the pyrroles form a large group of intriguing natural products which occur in marine organisms ranging from microbes over algae and sponges to animals. Their structural diversity including terpenoid-, polyketide-, carbohydrate-, lipid-, and peptideframeworks [7,12] accompanied by attractive biological properties, has spurred a considerable interest of chemists [6,13–19].

This review focuses on marine pyrrole alkaloids containing at least one pyrrole moiety, which were discovered during the decade of 2010 to 2020. The number of newly discovered pyrrole MNPs surged in this decade and many structural revisions resulted in a deeper knowledge of their biogenetic origin and structural relations.

In addition to the reported structures and their biological sources, known biological activities and, where applicable, the first total syntheses of these compounds will be shown. Furthermore, this review is subdivided by structural subclasses based on the substitution pattern of the pyrrole core. As a delineation, only MNPs with intact pyrrole functionality are described, whereas indole alkaloids [20], the saturated heterocycles pyrroline and pyrrolidine [21], as well as other fused systems (e.g., carbazoles) and pyrrole derivatives lacking a genuine pyrrole core [22–25], will not be covered. Several other specific overviews

**Citation:** Seipp, K.; Geske, L.; Opatz, T. Marine Pyrrole Alkaloids. *Mar. Drugs* **2021**, *19*, 514. https:// doi.org/10.3390/md19090514

Academic Editor: Asunción Barbero

Received: 20 August 2021 Accepted: 7 September 2021 Published: 10 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

focusing on subclasses such as bromopyrroles [26,27] and pyrrole-imidazole alkaloids (PIA) [13,14,28] or with the focus on the isolation source [14,25,27], have been published. In contrast, we intend to provide the reader with an impression of the multiple facets of pyrrole alkaloids in the marine environment.

The five-membered planar 6 π heteroaromatic pyrrole core with its high electron density is a reactive and privileged structural motif found in many biomolecules. It can provide stacking interactions, coordinate metal ions, or form hydrogen bonds when devoid of a substituent in the 1-position. Probably, the most well-known pyrrole derivatives in nature possess a tetrapyrrole skeleton, which can, e.g., be found in heme, chlorophyll, and several other porphyrinoid cofactors [29,30]. However, pyrroles possessing much simpler architectures have also attracted considerable interest, e.g., as promising lead structures in medicinal chemistry [15]. The biggest-selling drug of all time, the blood cholesterol lowering HMG-CoA reductase inhibitor atorvastatin (Lipitor®), is a pyrrole derivative. Not surprisingly, many pyrrole MNPs have also been associated with various pharmacological activities, such as cytotoxic [31,32], anti-bacterial [33,34], anti-fungal [35], and anti-cancer properties [6,36,37].

#### **2. Non-Halogenated Marine Pyrrole Alkaloids**

The alkaloids presented in this chapter are identified by a non-halogenated pyrrole core. Despite their structural diversity, the biosynthetic origin of these alkaloids can be traced back to a small number of possible biosynthetic pathways. According to the stunning logic of nature, only a few building blocks such as the amino acids glycine, serine, tryptophan, and proline are necessary to construct their pyrrole units.

A well-known pathway involves δ-aminolevulinic acid (ALA) as a key intermediate, which is produced from glycine and succinyl-CoA. An enzyme-catalyzed Knorr-type condensation–cyclization reaction of two molecules of δ-aminolevulinate yields porphobilinogen as a central intermediate, from which the trialkyl-substituted pyrroles are derived. Porphobilinogen is prone to self-condensation under acidic conditions and can further react to polypyrrolic systems, most notably the tetrapyrroles. Another major biosynthetic pathway is the dehydrogenation of proline to the common pyrrole-2-carboxylate unit. The activation of proline is suggested to involve a peptidyl carrier protein (PCP) forming a thioester linkage. In the next step, a controlled four-electron oxidation process with a flavoprotein desaturase occurs. These two C −N desaturation steps of the prolyl-*S*-PCP and subsequent tautomerization lead to the desired pyrrolyl-2-carboxyl-*S*-PCP product. Starting from this activated intermediate, a broad spectrum of reactions such as enzymatic transfer to nucleophiles or enzymatic halogenations can occur to create the world of marine pyrrole alkaloids [25,30,38,39].

## *2.1. Simple Pyrroles*

The pyrrole derivative 1-(4-benzyl-1 *<sup>H</sup>*-pyrrol-3-yl)ethanone (**1**) was found in a coculture of the marine-derived fungi *Aspergillus sclerotiorum* and *Penicillium citrinum* in 2017 (Figure 1). The acylated pyrrole **1** shows only medium toxicity against brine shrimp (LC50 values of 46.2 μM) and oppositely increases the growth of *Staphylococcus aureus* at 100 μg/mL [40].

**Figure 1.** Simple pyrrole alkaloids **1**–**3** isolated from different marine organisms.

Investigation of an endophytic strain of *Fusarium incarnatum* yielded another acylated pyrrole, fusarine (**2**), isolated from the marine mangrove fruit *Aegiceras corniculatum* in 2012 (Figure 1). Alkaloid **2** is expected to be formed biosynthetically via a Paal–Knorr cyclization of a primary amine and a 1,3-dicarbonyl, but showed neither antiproliferative nor cytotoxic potential against HUVEC, K-562, and HeLa human cell lines [41].

Another simple pyrrole is represented by geranylpyrrol A (**3**), which is counted among the small class of pyrrolomonoterpenoids and derives from pyrrolostatin (Figure 1). It was isolated from a mutant strain of *Streptomyces* sp. CHQ-64 in 2017 but did not display any toxicity against eight tested human cancer cell lines [42].

The pyrroloterpenoid glaciapyrrol A (**10b**) was already isolated along with its congeners glaciapyrrols B and C in 2005. Despite extensive investigations, the relative configuration of C-11 and the overall absolute configuration could not be determined at this time [43]. Through the first total synthesis of its four diastereomers by Dickschat in 2011, the relative configuration of the three stereocenters could be unequivocally established [44]. The authors devised an enantioselective synthesis starting from geraniol (**4**) using a Sharpless epoxidation to furnish alcohol **5**. Protection of the alcohol functionality and subsequent Sharpless dihydroxylation followed by intramolecular cyclization served as the key step and stereoselectively generated compound **6**. After several steps including a protection/deprotection sequence followed by oxidation and Horner–Wadsworth–Emmons (HWE) reaction using phosphonate **7**, ester **8** was obtained in 64% over four steps. Saponification, the addition of pyrrolyl Grignard **9,** and final TBS-deprotection finally produced *ent*-(−)-glaciapyrrol A (**10a**) showing the opposite optical rotation as the original publication from 2005. The authors, therefore, identified the natural product as (+)-glaciapyrrol A (**10b**) (Scheme 1) [44].

**Scheme 1.** Enantioselective approach towards the total synthesis of pyrrolosesquiterpenoid **10b** by a Sharpless epoxidation/dihydroxylation sequence, leading to the unnatural *ent*-(−)-glaciapyrrol A (**10a**).

The bromotyrosine-derived pyrrole alkaloid pseudocerolide A (**11**), was isolated from a marine sponge (*Pseudoceratina* sp.) from the South China Sea in 2020 and its proposed structure could be confirmed by X-ray crystallography (Figure 2). Unfortunately, compound **11** exhibited no activities against methicillin-resistant *Staphylococcus aureus*, *Escheriachia coli*, or *Candida albicans* [45].

**Figure 2.** Pseudocerolide A (**11**) and quinolinone alkaloids **12**–**17** isolated from marine origin.

The unusual pyrrolyl 1-isoquinolone alkaloids **12** and **13** were discovered from a habitat in the South China Sea within a co-culture of two mangrove endophytic fungi (strain No. 1924 and 3893) in 2006 [46]. It took until 2011, when König and co-workers isolated methyl marinamide (**15**) from the marine sponge (*Ircinia variabilis*) and reported a revised structure of **15**, in which the previously assumed 1-isoquinolone of **13** was reassigned as a 4-quinolinone unit on the basis of X-ray crystallography. Unfortunately, **15** showed only weak or no effects in the biological evaluation on cannabinoid receptors [47]. In accordance with the findings of König, Zhu and Chen, chemically modified the previously isolated compound **14** in 2013, which also led to the revision of the structure **12** to **14** for marinamide in the same fashion, further confirming the revision of marinamide by König and co-workers [48]. However, one year before the report of König, the Lin laboratory isolated the same compound **14**, but referred to it as penicinoline (Figure 2) [49]. Both compounds **14** and **15** display promising in vitro cytotoxicity towards 95-D and HepG2 cell lines (IC50 values of 0.57 μg/mL and 6.5 μg/mL, respectively) as well as insecticidal activity against *Aphis gossypii* (100% mortality at 1000 ppm) [48,49].

The related congener penicinoline E (**16**) was isolated from an endophytic fungus *Penicillium* sp. ghq208 in 2012 alongside quinolactacide (**17**), which was isolated from a marine source for the first time [50,51]. In biological assays, moderate cytotoxicity against HepG2 was exclusively attributed to 4-quinolinones **14** and **15** (IC50 values of 11.3 μg/mL and 13.2 μg/mL, respectively), indicating the importance of the free carboxy function at C3 (Figure 2) [51].

Based on the auspicious pharmacological activities of penicinoline E (**16**), marinamide (**14**), and methyl marinamide (**15**), the Nagarajan group established their total synthesis in 2017 for further biological testing [52]. They achieved a two- to three-step approach, characterized by a Suzuki–Miyaura coupling and subsequent dearomatization as key steps from their starting materials **18**, **19**, and **20**. They were also able to unambiguously confirm the structure of penicinoline E (**16**) by X-ray crystallography (Scheme 2) [52].

**Scheme 2.** A high-yield sequence towards pyrrolyl 4-quinolinones **14**, **15**, and **16** starting from 2-chloroquinoline precursors **18** and **19** by Nagarajan et al.

Furthermore, the antimalarial properties against the 3D7 strain of *Plasmodium falciparum* were evaluated and the decarboxylated derivative **16**, as well as the methyl ester **15**, showed significant activity (IC50 value of 1.56 μM for both). These results have been confirmed by binding mode studies of the synthesized ligands **14**, **15**, and **16** to the CYTB protein of *Plasmodium falciparum* [52].

Another pharmacologically interesting compound class is the indanomycins, which possess a variety of biological activities such as antibacterial [53], insecticidal [54], and antiprotozoal [55] properties. In 2011, the group of Kelly and co-workers published a study on the biosynthesis of indanomyincs, including an intramolecular Diels–Alder cyclization of a tetraene as the key step [56]. Two years later, researchers isolated three new representatives of these pyrrole ethers from the culture broth of a marine *Streptomyces anibioticus* strain PTZ0016 which possess in vitro activity against *Staphylococccus aureus* (MIC values between 4.0 and 8.0 μg/mL). Based on their previous derivatives and on the α- or β-orientation of the pyran ring, they were named 16-deethylindanomycins. The relative and absolute configurations of iso-16-deethylindanomycin (**23**), iso-16-deethylindanomycin methyl ester (**24**), and 16-deethylindanomycin methyl ester (**25**) were established by extensive NMR and CD spectroscopy (Figure 3) [57].

**Figure 3.** Three new members **23**–**25** of the indanomycin-group, discovered in 2013.

Another important source of bioactive MNPs is represented by the genus *Agelas* (family Agelasidae), which provides a wide diversity of glycolipids [58,59], diterpene alkaloids [60–62], and pyrrole alkaloids [63–66]. To date, more than 130 pyrrole alkaloids have been isolated from over 20 *Agelas* species, all of which share a unique bromo- or debromopyrrole-2-carboxamide moiety alongside several linear side chains, anellated ring systems, or dimeric structural units [67].

In 2017, Li et al. reported the isolation of the nakamurines A–C (**26**–**28**) from the South China Sea sponge *Agelas nakamurai*. They only differ in the side chain of the carboxamide unit, however, no activity could be observed for any of the compounds in cytotoxicity tests and antiviral assays. In antimicrobial assays, only nakamurine B (**27**) showed weak inhibitory effects against *Candida albicans* (MIC = 60 μg/mL, Figure 4) [67].

**Figure 4.** Isolation of five pyrrole-2-carboxamides (**26**–**30**) from the sea sponge *Agelas nakamurai*.

A few weeks later, the same group published the extraction of two non-brominated pyrroles, **29** and **30**, from the same sponge *Agelas nakamurai* [68]. For structure elucidation, the racemic pairs were resolved by chiral HPLC with the absolute stereochemistries determined by quantum chemical calculations and measurements of molar rotations. The carboxamide **30** was listed in *SciFinder Scholar* with no associated reference at that time, but the analytical data were reported for the first time. In cytotoxicity and antimicrobial tests, no activity could be observed for any of the enantiomers of nakamurine D (**29**) or for compound **30** (Figure 4) [68].

In 2017, Li and co-workers were able to isolate a new class of racemic pyrroles, the nemoechines A–C (**31**, **32**, and **124**), from the species *Agelas* aff. *nemoechinata* (Figure 5) [69]. Nemoechine A (**31**) differs from the two related congeners **32** and **124** by its unusual bicyclic cyclopentane-fused imidazole skeleton, whereas nemoechine B (**124**) features a fused pyrrole core and is therefore specified in Section 2.4. Nemoechine C (**32**), with its butyric acid ester side chain, shows structural similarity to pyrrole **30** and differs only by an additional methylene group. Unfortunately, nemoechine A (**31**) and C (**32**) did not show any promising activities which complies with the inactivity of the structurally related pyrroles **29** and **30** [69].

**Figure 5.** Isolation of nemoechine A (**31**) and C (**32**), debromokeramadine (**33**), and clathrirole B (**34**).

The isolation of pyrrole-2-aminoimidazole (P-2-AI) debromokeramadine (**33**) from the marine sponge *Agelas* cf. *mauritiana* was reported alongside the first total syntheses of **33** and keramadine (**41**) in 2015. Interestingly, **33** and the previously isolated derivative keramadine (**41**), feature a (*Z*)-configuration at the C=C double bond, which is in contrast to the well-known natural key-precursor oroidin featuring an (*E*)-configured double bond (Figure 5) [70,71].

Clathrirole B (**34**), extracted from the marine sponge *Clathria prolifera*, represents another P-2-AI alkaloid. The carboxylic acid ester **34** is a C-11 epimer of manzacidin D (**35**), which was isolated from the marine sponge *Astrosclera willeyana* back in 1997 (Figure 5) [72]. Interestingly, compound **34** completely lacks antifungal activity against *Saccharomyces cerevisiae*, whereas diastereomer **35** and derivatives thereof proved to be potent antifungals

against this yeas<sup>t</sup> [35]. Thus, the authors concluded that the absolute configurations at both C-9 and C-11 may have a massive influence on the antifungal activity of this compound class [73].

The authors applied a one-pot approach with a regioselective oxidative addition in which partially brominated *<sup>N</sup>*-acylpyrrole-1,2-dihydropyridines **36** and **37** were reacted with guanidine **38** in a double nucleophilic substitution to generate the aminoimidazoline moiety. Finally, the cyclic aminal structure is ring-opened by TFA, resulting in the MNPs **33** and **41** (Scheme 3) [71].

**Scheme 3.** Synthesis of keramadines **33** and **41**, including a regioselective oxidative addition followed by acid mediated bond cleavage of the aminal.

In the previously reported isolation of MNPs from *Agelas* aff. *nemoechinata* and *nakamurai*, the class of nakamurines and nemoechines were presented [68,69]. It should be mentioned that the group of Li isolated several structurally related pyrrole alkaloids from marine sources and identified them as known compounds that had been synthesized but not isolated from natural sources before. Therefore, carboxamides **42**–**47**, isolated from marine sources for the first time, are grouped together in Figure 6. The *<sup>N</sup>*-acylglycine methyl ester **42** identified in both sponges is related to nakamurine C (**28**) but carries an additional methylene group [68,69]. The synthetically known pyrrole **43** bearing two more methylene groups in the side chain, was isolated from *Agelas nakamurai* [68,74,75].

**Figure 6.** Synthetically known pyrrole-2-carboxamides **42**–**47**, isolated for the first time from marine origin.

Some reduction products of the methyl esters and an amine derivative are represented by compounds **44**–**46**, of which **45** occurs in both sponges, whereas **44** and **46** were exclusively isolated from the *Nemoechinata* sp. [68,69,76,77]. The carboxamide **47** is a debromo analog of mukanadin B and is present in *Agelas nakamurai* [68,78,79]. Compounds **42**–**47** described show neither cytotoxicity nor antimicrobial activity.

The Arctic hydrozoan *Thuiaria breitfussi* (family Sertulariidae) produces a class of indole-oxazole-pyrrole MNPs named breitfussins. Biosynthetically, the breitfussins may share a similar biogenesis as the phorbazoles (cf. Figure 33), arising from the dipeptides Pro-Trp or Pro-Tyr. In the first isolation and analysis of breitfussin A (**48**) in 2012, high-resolution

mass spectrometry indicated a ratio of non-hydrogen atoms to hydrogen of 2:1 which makes the structural elucidation by spectroscopic methods challenging [80]. The authors, however, could identify a brominated 4-methoxyindole moiety, a 2-substituted pyrrole core as well as an unresolved C3NO fragment suggestive of an oxazole core, which finally prevented the unambiguous determination of the entire structure. By applying a combined approach of atomic force microscopy (AFM), computer-aided structure elucidation (CASE) and calculation of 13C-NMR shifts through density functional theory (DFT), the structure of breitfussin A (**48**) could be unequivocally determined (Figure 7) [80]. A recently published article describes the isolation of further non-halogenated congeners, namely breitfussins C (**49**), D (**50**), and F (**51**), of which structures **49** and **50** could also be confirmed by total syntheses (Figure 7) [81].

**Figure 7.** Molecular structures of breitfussins **48**–**51** isolated from the marine hydrozoan *Thuiaria breitfussi*.

Given the promising cytotoxic activities of the breitfussins C (**49**) and D (**50**) against several cancer cell lines with IC50 values below 10 μM, extensive research on the breitfussin scaffold in search for selective kinase inhibitors has been performed [81]. Due to their promising bioactivity but extremely challenging heteroaromatic core in terms of structure elucidation, the breitfussins are attractive starting points for ongoing synthetic work [82].

The first total synthesis and hence the structure validation of breitfussin A (**48**) was published by the Bayer group in 2015 [83]. They used an approach involving two Suzuki couplings in which the oxazole and pyrrole moieties were installed sequentially. First, indole **52** was converted with oxazole **54** into coupling product **55**, followed by double lithiation of the oxazole core. Coupling with *N*-Boc-2-pyrrole boronic acid (**20**) furnished pyrrole **57**, which, after removal of all protection groups, resulted in the formation of breitfussin A (**48**) [83]. Alongside the isolation of additional breitfussins in 2019, the Bayer laboratory employed the same approach as in their previous publication for the synthesis of breitfussin C (**49**) and D (**50**). Here, only the penultimate step varied by acid-mediated Boc-deprotection, since deiodination of the oxazole core was required (Scheme 4) [81].

Bisindole pyrroles represent a class of MNPs having similar biological activities. The lynamicins F (**59**) and G (**60**) were isolated from a marine-derived *Streptomyces* sp. SCSIO 03032 [84], extending the lynamicin family, of which lynamicins A–E have been isolated back in 2008 (Figure 8) [85]. Unfortunately, no antimicrobial or cytotoxic activities were observed for **59** and **60** against several indicator strains or cancer cell lines. In 2017, the first total synthesis of the antimicrobial lynamicin D (**72**) was achieved, thereby enabling the implementation of further biological assays (Scheme 5). It turned out that lynamicin D (**72**) influenced the splicing of pre-mRNAs by upregulating the level of the key kinase SRPK1, which is involved in both constitutive and alternative splicing [86].

**Scheme 4.** Total synthesis of the three breitfussins A (**48**), C (**49**), and D (**50**) by introducing the oxazole and pyrrole functionalities via two consecutive Suzuki coupling reactions.

**Figure 8.** Structures of lynamicins F (**59**) and G (**60**), indimicins A–E (**61**–**65**), dichlorochromopyrrolic acid derivative **66**, and isohalitulin (**67**).

**Scheme 5.** Key step of the synthesis of lynamicin D (**72**) by a Suzuki coupling.

In addition to the alkaloids **59** and **60**, a new family of MNPs consisting of a unique 1,3-dimethyl-2-hydroindole motif, the indimicins (IDMs) A–E (**61**–**65**), were discovered in 2015 (Figure 8) [84]. Besides the usual spectroscopic data, an X-ray structure of indimicin A (**61**) could be obtained, which allowed determining the absolute configuration of the hydroindole moiety. Of compounds **61**–**65**, only indimicin B (**62**) was active against the breast cancer cell line MCF-7 (IC50 value of 10.0 μM ± 0.3 μM), whereas all seven alkaloids **61**–**65** did not show any antimicrobial or cytotoxic activities against several indicator strains or cancer cell lines [84].

Very recently, the *Streptomyces* sp. SCSIO 11791 revealed another bisindolylpyrrole (**66**)**,** displaying moderate cytotoxicity against a human breast cancer cell line (MDA-MB-435, IC50 value of 19.4 μM), while no antibacterial properties could be observed (Figure 8) [87].

In isohalitulin (**67**), isolated from the marine sponge *haliclona tulearensis* in 2010, the structure is dominated by a bis-dihydroxyquinoline functionality (Figure 8) [88]. Compound **67** exhibits a detectable toxicity to brine shrimp (*Artemia salina*, LD50 value of 0.9 mM). It is also worth mentioning that minute amounts and instability of isohalitulin (**67**) prevented the unequivocal determination of its structure. However, **67** shows very similar analytical data to its congener halitulin and should differ only in the position of the two phenolic OH groups (Figure 8). Although no experiments were performed to deduce the stereochemistry of **67**, the authors mentioned that, on the grounds of common biogenetic precursors, it most probably has the same absolute configuration as halitulin [88].

The total synthesis of lynamicin D (**72**) commenced with the synthesis of the coupling partners **69** and **71**, prepared from commercially available precursors **68** and **70**. Dibrominated pyrrole **69** was obtained by a Vilsmeier–Haack reaction, followed by oxidation, esterification, and final bromination. On the other side, 5-chloro-1*H*-indole (**70**) was first iodinated and Boc-protected and the introduction of the pinacol moiety on the basis of Pd-catalysis resulted in the formation of indole precursor **71**. Building blocks **69** and **71** were then subjected to the key Suzuki coupling. Final removal of the Boc-group gave lynamicin D (**72**) in 73% yield over two steps (Scheme 5) [86].

The suberitamides and denigrins constitute another family of highly substituted pyrrole alkaloids. The symmetrical, nearly planar suberitamide B (**73**) was isolated from the marine sponge *Pseudosuberties* sp. in 2020 and bears a fully substituted pyrrole core. This storniamide-related compound inhibits the enzymatic activity of Cb1-b (E3 ubiquitin ligase) with an IC50 value of 11 μM, which, according to the authors, is caused by the rigid, highly substituted pyrrole scaffold (Figure 9) [89].

**Figure 9.** Highly substituted 3,4-diarrylpyrroles suberitamide B (**73**) and denigrin E (**74**).

In 2020, denigrin E (**74**) was isolated from a new *Dactylia* sp. along with several members of the pyrrolone family. Unfortunately, no inhibitory activity against PAX3-FOXO1 luciferase expression was observed in biological assays (Figure 9) [90]. By considering the substitution pattern of these 3,4-diarylpyrroles **73** and **74**, a close relationship as potential precursors of lamellarins (see Section 2.4.1) in a biosynthetic context can be suggested.

Among the huge variety of marine alkaloids, aromatic polyketides (APK) represent another large class of MNPs and pyrrole-containing representatives have been described. The group of Zhang and co-workers isolated the decaketide pyrrole SEK43F (**75**) generated from pathway crosstalk of the host *Streptomyces albus* J1074 and the heterologous fls-gene cluster from *Micromonospora rosaria* SCSIO N160 (Figure 10) [91]. It should be mentioned that the configuration of the double bond in **75** could not be unequivocally determined. The same group also isolated another tri-methylated bis-pyrrole **76** (Figure 10) [91], which has only been known as a synthetic product before [92,93]. Both compounds **75** and **76** displayed negligible antibacterial activity, whereas the APK **75** showed weak to moderate cytotoxicity against four human cancer cell lines (SF-268, MCF-7, NCI-H460, and HePG-2, with IC50 values of 56.46 μM ± 0.87 μM, 35.73 μM ± 1.45 μM, 44.62 μM ± 2.49 μM, and 39.22 μM ± 3.00 μM, respectively, Figure 10).

**Figure 10.** Representation of an APK (**75**) and three pyrroles **76**–**78** including the important class of tambjamines.

The family of tambjamines consisting of a central bi-pyrrole unit is counted among the 4-methoxypyrrolic natural products. In 2010, tambjamine K (**77**) was isolated as the main secondary metabolite from the Azorean nudibranch mollusk *Tambja ceutae* and in minute amounts from the bryozoan *Bugula dentata* (Figure 10) [94]. Just as its family members, tambjamine K (**77**) exhibited remarkable to moderate antiproliferative activity against tumor and non-tumor mammalian cells with IC50 values between 3.5 nM and 19 μM. It is suspected that the strong activity is caused by the bipyrrolic structure with its DNA-targeting properties and by the ability to form ion complexes [94].

The macrocyclic tambjamine MYP1 (**78**) is produced by the marine bacterium *Pseudoalteromonas citrea* and was isolated in 2019 (Figure 10) [95]. The authors highlighted the

important differences of the α- and β-rotamers in the tambjamine conformations, which are thought to play an essential role in their bioactivity. Moreover, the group provides an X-ray structure by co-crystallization of **78** with formic acid, unequivocally confirming the proposed structure of compound **78** [95].

Based on the promising bioactivity of compound **77**, Lindsley et al. were prompted to publish their first three-step total synthesis of tambjamine K (**77**) four months after its initial isolation [96]. The first step involved a Vilsmeier–Haack haloformylation which generated enamine **80** in 59% yield. A Suzuki coupling with Boc-1*H*-pyrrol-2-ylboronic acid (**20**) followed by acid-mediated condensation of isopentylamine resulted in the formation of tambjamine K (**77**) in 31% over two steps (Scheme 6) [96]. In addition to the natural product synthesis, a series of unnatural derivatives were synthesized followed by biological assays to evaluate basic structure–activity relationships (SAR). However, the natural product **77** showed moderate activity (IC50 values of 13.7 μM and 15.3 μM against HCT116 and MBA231, respectively), whereas the unnatural analogs were more potent in inhibiting the viability, proliferation, and invasion of HCT116, MBA231, SW 620, and H520 NSCLC cancer cell lines (IC50 values between 146 nM and 10 μM) [96].

**Scheme 6.** A linear 3-step sequence to tambjamine K (**77**).

In addition to the tambjamines which consist of a bipyrrole core functionalized with various imines, the functionalization with an additional pyrrole moiety in the prodiginine structures represents another well-studied family. With the isolation of the marineosins A (**85a**) and B (**86**) in 2008, this prodiginine-related family opened up a new field of research with several new contributions being made in the last decade [97]. In 2014, the Reynolds laboratory focused on the final steps of the marineosin biosynthesis, by exploring the biosynthetic gene cluster *mar* which can produce marineosins by a heterologous expression in a *Streptomyces venezuelae* derived JND2 strain. They replaced the *marA* and *marG* gene with the spectinomycin resistance *aadA* gene which led to the isolation and elucidation of 16-ketopremarineosin A (**83**) and premarineosin A (**84**) as well as 23-hydroxyundecylprodiginine (HUPG) (**81**) and its oxidized derivative **82**, respectively (Figure 11). As marineosin production was not observed, the authors concluded that both genes, *marA* and *marG*, are essential for the biosynthesis of marineosins [98]. Three years later, the Reynolds group reported another gene (*marH*) from the same cluster which has the ability to catalyze the condensation of a methoxybipyrrole carbaldehyde (MBC) and 2-undecylpyrrole (UP) to generate undecylprodiginine (UPG). The gene also hydroxylates the C-23 position of UPG to construct HUPG (**81**) and hence is essential for the biosynthetic pathway of marineosins [99].

**Figure 11.** Different prodiginine-based pyrrole alkaloids **81** and **82** together with marineosin-type spiroaminals **83**–**86**.

Not only the biosynthetic pathway but also the stereoselective synthesis of marineosins, their substructures, and derivatives have attracted much attention. In 2014, the Reynolds laboratory followed up on their previous publications regarding marineosins and reported the first total synthesis of HUPG (**81**) and premarineosin A (**84**). To this end, a divergent synthetic approach of nine steps in total stereospecifically provided 23- hydroxyundecylprodiginine (**81**). The final cyclization forming the spiro-tetrahydropyranaminal unit of the premarineosin A (**84**) was then achieved by a biosynthetic approach via the Rieske oxygenase MarG (Scheme 7) [100]. This strategy yields several other prodiginine derivatives and premarineosin analogs that show promising cytotoxic and antimalarial activities [100].

**Scheme 7.** Divergent synthesis of premarineosin A (**84**) including a bioinspired MarG catalyzed spirocyclization as the final step.

Based on unsuccessful synthetic attempts (with the exception of individual key motifs) of several research groups [101–106], Shi and co-workers presented the first total synthesis of marineosin A (**85a**) in 2016 [107]. The synthesis commenced with the commercially available (S)-pyrone **89**, which was converted into key fragment **90** in 10% yield over 14 steps. Lewis acid-mediated spirocyclization and ring-closing metathesis followed by hydrogenation furnished spiro lactam **91** in 37% yield over three steps. The last two steps consisted of a Paal–Knorr reaction and a Vilsmeier–Haack reaction, not only allowing for the preparation of the sensitive pyrrole moieties in a late-stage procedure but also directly giving access to marineosin A- (Scheme 8). It is also worth mentioning that five X-ray structures of important intermediates could be obtained, underpinning the validity of the synthesis. However, the NMR spectra, appearance, and optical rotation of the resulting marineosin A- (**85a**) exhibited some deviations when compared to the isolated

natural product, suggesting that the natural and synthetic compounds likely differ in their stereochemistry [107].

**Scheme 8.** The first total synthesis of 7-*epi*-marineosin A (**85a**) by Shi and co-workers in a linear 19 step sequence and the structural reassignment of C7-OMe from (*R*) to (*S*) by the Harran laboratory using a chromophore disruption approach.

It was however not until 2019, that the Harran group solved the puzzle by a total synthesis and concomitant reassignment of C7-(*R*) in **85a** to C7-(*S*) resulting in the structure **85b** for (+)-marineosin A [108]. To this end, a bioinspired approach with reversed fragment polarity was applied, starting from the previously prepared bipyrrole **92** and cyclic ketone **93**. Condensation product **94** was stabilized by quenching with NaOMe, generating a novel but still unstable premarineosin **95**. After exposure to acidic conditions, a prodiginine chromophore was formed, which, after 6-exo trig cyclization mediated by acidic MnO2, was converted to a premarineosin derivative. The formed vinylogous imidate was hydrogenated from the less hindered face, resulting in the formation of (+)-marineosin A (**85b**), whose spectroscopic data are in full agreemen<sup>t</sup> with those reported for the isolated natural product **85b** (Scheme 8) [108].
