*2.4. Annellated Pyrroles*

In contrast to simple substituted pyrrole alkaloids, another structural class comprises compounds with an annellated pyrrole core. The position of fusion thereby can differ between 1,2-, 2,3- or 3,4-, with the fused ring being 6- or 7-membered. Additionally, these alkaloids often share a carbonyl moiety in α-position to the bridgehead atom.

From a series of nemoechines isolated in 2017 (see Figure 5, **31** and **32**), nemoechine B (**124**) stands out with its 1,2-condensed pyrrole unit [69]. The synthetically known compound **124** [124] was originally isolated in racemic form from *Agelas* aff. *nemoechinata* and the enantiomers were separated by chiral HPLC. Like its family members **31** and **32**, a lack of cytotoxicity against HL-60, HeLa, P388, and K562 cell lines was reported for both enantiomers (Figure 16) [69].

**Figure 16.** Structures of 1,2-annellated marine pyrrole alkaloids **124**–**127**.

In 2016, procuramine (**125**) was identified as a co-metabolite during the initial isolation and investigation of the biosynthetic pathway of curindolizine (**414**) from *Curvularia* sp. IFB-Z10 (see Figure 58). Structure elucidation was performed by spectroscopic methods and X-ray crystallography (Figure 16) [125].

A new pyrrolooxazine (**126**) was isolated from the marine mudflat fungus *Paecilomyces formosus*, ye<sup>t</sup> the absolute configuration could not be determined because of decomposition during the isolation process. Formoxazine (**126**) showed potential as a radical scavenger in the DPPH assay with an IC50 value of 0.1 μM and antibacterial activity against MDRSA and MRSA (MIC values of 6.25 μg/mL for both) (Figure 16) [126].

In the course of an investigation of marine-derived *Aspergillus versicolor* and in search for new Bacille Calmette-Guérin-inhibiting antibiotics against tuberculosis, the unknown brevianamide T (**127**) could be isolated in 2012 (Figure 16) [127]. Unfortunately, diketopiperazine **127**, isolated along with other members of the brevianamide family, showed no antibacterial properties against *Staphylococcus aureus* (ATCC 6538), *Bacillus subtilis* (ATCC 6633) (Gram-positive bacteria) or *Pseudomonas aeruginosa* (PAO1), *Escherichia coli* (ATCC 25922) (Gram-negative bacteria) or *Candida albicans* (SC 5314, yeast) [127].

A 2,3-fused pyrrole alkaloid, microindolinone A (**128**), was isolated from the actinomycete *Microbacterium* sp. MCCC 1A12207 from the deep sea in 2017 [128]. This tetrahydroindole represents one of two known saturated indoles of natural origin [129]. The absolute configuration at C5-OH was deduced with CD spectroscopy as 5*R*. No potent inhibition was found in anti-allergic bioactivity tests against RBL-2H3 cells (Figure 17) [128].

**Figure 17.** Various 2,3-fused pyrrole alkaloids **128**–**134** isolated between 2010 and 2020.

The natural product **129** was isolated from the gorgonian coral *Verrucella umbraculum* in 2012 and features a pyrrolopyrimidin scaffold. According to the authors, the biosynthesis of this purine alkaloid is similar to that of caffeine, which was also isolated from the same source (Figure 17) [130].

Another important class of MNPs is comprised of the pyrrolactams, which most probably derive from pyrrole-2-carboxamides. Axinelline A (**130**) was isolated alongside its brominated analog **353** (see Figure 51) from the marine sponge *Axinella* sp. in 2017, however, the absolute stereochemistry was not determined (Figure 17) [131].

The two diastereomers (11*R*)- and (11*S*)-debromodihydrohymenialdisine **131a** and **131b** were isolated from the sponge *Cymbastela cantharella* by the Debitus laboratory in 2011 (Figure 17) [132]. The authors assumed that compounds **131a** and **131b** biogenetically arise from dispacamide derivates. Because of their close relationship to the strong kinase inhibitor hymenialdisine, (11*R*)- and (11*S*)-debromodihydrohymenialdisine **131a** and **131b** were tested for Polo-Like-Kinase-1 (PLK-1) inhibition. Unfortunately, but in analogy to the bromo derivatives **386a** and **386b** (see Figure 55), a complete lack of activity was observed, demonstrating the importance of the conjugation at C-10 and C-11 of the unique cyclic system of hymenialdisine [132].

In 2018, the structurally related seven-membered pyrroloazepine stylisine F (**132**) was isolated alongside several other MNPs from the marine sponge *Stylissa massa*. However, the authors mentioned that stylisine F (**132**) most probably occurred as an artifact generated from the corresponding acid upon EtOH extraction. In basic biological investigations, weak or no inhibition against a variety of bacteria was detected (MIC ≥ 128 μg/mL, Figure 17) [133].

In 2015, Fenical and co-workers reported a culture-dependent technique in a nutrientpoor medium combined with long incubation times, which facilitated the cultivation of several marine bacteria able to produce secondary metabolites. The organic extract from strain CNX-216<sup>T</sup> of a cultivated bacterium belonging to the Mooreiaceae family showed activity against *Pontibacillus* sp. and the authors were able to isolate the alkaloids marinoazepinones A (**133**) and B (**134**) from this extract [134]. Besides the incorporation of the unusual amino acid 4-hydroxyphenylglycine, the marinoazepinones **133** and **134** represent the first natural products featuring a rare azepin-3-one framework. CD spectroscopy, X-ray crystallography, and optical rotation were used to elucidate the absolute stereochemistry at C2, but no definite conclusions could be drawn. In bioactivity assays, marinoazepinone B (**134**) exhibited antibacterial activity against the Gram-positive *Pontibacillus* strain CNJ-912 (16 mm inhibition zone), whereas no activity was observed against the Gram-negative *Vibrio shiloi* strain CUA-364 (Figure 17) [134].

The rigidins represent another prominent class of 2,3-fused pyrrole alkaloids, sharing a pyrrolo [2,3-*d*]pyrimidine scaffold [135]. With the first rigidin isolated back in 1990 by Kobayashi and co-workers [136], many MNPs belonging to this family have been isolated until today [137,138]. Although several total syntheses of rigidins are known [139–143], we

want to mention the one-pot multicomponent reaction reported by the Magedov laboratory in 2011, which provides synthetical access to tetrasubstituted 2-aminopyrroles in only four steps and includes the first total syntheses of rigidins B–D (**147**–**149**) [144]. In a first step, *N*-(methanesulfonamido)acetophenones **140** and **141** were prepared from starting materials **135** and **136**, respectively. The multicomponent reaction was then realized by combining either **140** or **141** with aldehydes **138** or **139** under the addition of cyanoacetamide (**137**). The resulting 2-aminopyrroles **142**–**145**, isolated in 83–86% yield, were then converted into pyrimidinediones and after final deprotection, the rigidins A–D (**146**–**149**) could be obtained in four steps at an overall yield of 53–61% (Scheme 12) [144].

**Scheme 12.** The so-far shortest synthetic approach towards rigidin A (**146**), including the first syntheses of rigidins B–D (**147**–**149**) in a one-pot multicomponent reaction.

The annellated pyrrole alkaloids shown so far largely consist of a fused lactone or lactam structure, whereas 3,4-fused pyrroles often share a quinone system. This motif can be found in albumycin (**150**), a novel MNP isolated by heterologous expression from *Micromonospora rosaria* SCSIO N160 genes in *Streptomyces albus* J1074 (Figure 18). In antibacterial tests, only weak activities against several indicator strains were encountered (MIC values >64 μg/mL) [145].

**Figure 18.** Series of isolated isopyrrolo-*p*-benzoquinone **150** and isopyrrolo-1,4-naphthoquinones **151**–**154**.

In 2016, another fused *p*-quinone, biscogniauxone (**151**), was isolated from the marine fungus *Biscogniauxia mediterranea* and belongs to the rare family of isopyrrolonaphthoquinones (Figure 18) [146]. It should be mentioned that the authors assumed the existence

of further derivatives of compound **151**, as metabolites with similar UV spectra were detected in the extracts, albeit without isolation. Significant inhibition of glycogen synthase kinase (GSK-3β, IC50 value 8.04 μM ± 0.28 μM) was observed for biscogniauxone (**151**), while weak inhibition of *Staphylococcus epidermidis* and *Staphylococcus aureus* was found (IC50 values in the range of 100 μM) [146].The nitricquinomycins A–C (**152**–**154**), isolated from *Streptomyces* sp. ZS-A45, complete the selection of isopyrrolonaphthoquinones (Figure 18) [147]. By comparing the spectroscopic data with those of previously reported naphthoquinones bearing a pyrrole core and using NOE experiments for the determination of the relative configuration, as well as ECD spectroscopy for the determination of the absolute configuration, the structure could be determined as indicated. Of compounds **152**–**154**, nitricquinomycin C (**154**) exhibited significant cytotoxicity against the human ovarian cancer cell line A2780 (IC50 value 4.77 μM ± 0.03 μM) but weak antibacterial potential against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* (MIC values > 40 μM) [147].

Another 3,4-fused pyrrole family are the spiroindimicins (SPMs), which contain a remarkable spirocyclic bisindole framework and are highly related congeners of the bisindole pyrroles **59**–**66** (cf. Figure 8). Spiroindimicins A–D (**155**–**158**) were isolated from *Streptomyces* sp. SCSIO 03032 in 2012 [148]. The molecular structures were resolved by spectroscopic methods, with the 3D structures of spiroindimicin A (**155**) and B (**156**) being unambiguously confirmed by X-ray crystallography (Figure 19). Spiroindimicin A (**155**) consists of a [5.6] spirocyclic core, whereas congeners B–D **156**–**158** contain a [5.5] spirocyclic core. This structural difference also influences the bioactivity, which in the case of [5.5] spirocyclic pyrroles **156**–**158** results in good to moderate antitumor activities against various cancer cell lines with IC50 values ranging between 5 μg/mL and 22 μg/mL. Biosynthetic studies sugges<sup>t</sup> the formation of spiroindimicins are proposed to derive from lynamicin by an aryl-aryl coupling of C-3- and C-5-- or by an aryl-aryl coupling of C-3- and C-2--, furnishing the [5.6] or [5.5] spiro-cyclic alkaloids, respectively [148].

**Figure 19.** Structures of spiroindimicins A–H (**155**–**162**) isolated from marine actinobacteria.

The family of spiroindimicins was extended in 2017 by the monochlorinated compounds **159** and **160**, which were isolated from *Streptomyces* sp. MP131-18 (Figure 19) [149]. Spiroindimicins E (**159**) and F (**160**) did not show any activity against Gram-negative test cultures, being in line with the biological properties of their biosynthetic lynamicin-type precursors. In both cases, the antibacterial activity appears to increase with an increasing degree of chlorination on the bisindole backbone [149]. In addition to studies on the biosynthetic gene cluster of *Streptomyces* SCSIO 03032 [150], the group of Zhang, responsible for the isolation of spiroindimicins A–D (**155**–**158**), discovered the halogenase SpmH involved in the biosynthesis of SPMs and IDMs.

In 2019, inactivation of the encoding gene *spmH* then led to the isolation of spiroindimicins G (**161**) and H (**162**), which displayed moderate cytotoxicity against four cancer cell lines (IC50 values between 10.28 μM and 33.02 μM), comparable to their chlorinated congeners **155**–**160** (Figure 19) [151].

The first syntheses of these compounds were achieved by Sperry and co-workers in 2016 [152]. Starting with the alkylation of aniline **163** with bromide **164**, a subsequent Heck reaction and hydrogenation furnished the spirocyclic pentanone **165**. One key step is represented by the Fischer indolization, followed by Boc-protection and radical bromination. After hydrolysis and oxidation, ketone **166** was formed in 50% over five steps. Sequentially, a thioketal and then a vinylsulfone **167** were prepared which allowed for a Montforts pyrrole synthesis. After the final deprotection, (±)-spiroindimicin C (**157**) could be obtained. Additionally, reductive amination furnished (±)-spiroindimicin B (**156**) (Scheme 13) [152].

**Scheme 13.** Total synthesis of spiroindimicins **156**, **157** using the Fischer indolization and Montforts pyrrole synthesis.

Further studies and recent publications highlight the importance of these bisindole alkaloids as promising bioactive compounds and potential new lead structures [153,154].

The structurally remarkable subtipyrrolines A–C (**168**–**170**) incorporating a pyrrolepyrrole-dihydropyridine framework, were isolated from the *Bacillus subtilis* SY2101 strain, derived from sediment samples of the Mariana Trench collected at a depth of 11,000 m (Figure 20) [155]. The structural elucidation was investigated by spectroscopic analysis and supported by X-ray crystallography. Bioactivity assays revealed moderate antiproliferative activities (human glioma U251 and U87MG cells, IC50 values of 36.3 μM and 26.1 μM) as well as moderate antimicrobial potential (*Escherichia coli* and *Candida albicans*, IC50 values between 34 μM and 46 μM, respectively) [155].

**Figure 20.** Subtipyrrolines A–C (**168**–**170**) as novel alkaloids from *Bacillus subtilis* SY2101.

#### 2.4.1. Lamellarins and Related Natural Congeners

To date, more than 65 lamellarins have been discovered since the first isolation of a member of this class by Faulkner et al. in 1985 [156,157]. Divided into type I (with subsections a and b, comprising compounds with a saturated or unsaturated C-5–C-6 unit, respectively) containing a doubly annellated 2,3,4-triarylpyrrole core in form of a 1-aryl-6*H*-chromeno-[4-,3-:4,5]pyrrolo-[2,1-a]isoquinolin-6-one or type II with a simple 3,4-diarylpyrrol-2-carboxylate ring system, the lamellarins comprise a large and prominent class of marine alkaloids. These compounds, derived from sponges, tunicates, and mollusks, exhibit a broad range of often highly potent biological activities, making them interesting targets for synthetic chemists [157,158].

In 2012, Capon and co-workers investigated *Didemnum* sp. and isolated five new lamellarins A1–A5 (**171**–**175**) from the strain CMB-01656 and one further member (A6, **176**) from the strain CMB-02127 (Figure 21) [159]. Together with eight known derivatives, a structure–activity relationship (SAR) study was performed regarding the reversal of multidrug resistance. In the SAR study, the P-glycoprotein (P-gp) inhibition activity was proposed to increase with a higher degree of O-methylation. The synthesis of a permethylated derivative, featuring potential non-cytotoxic P-gp inhibitory activities then confirmed this assumption [159].

**Figure 21.** Members of the lamellarins **171**–**182** (type I) isolated from *Didemnum* sp. in 2012 and 2019.

The lamellarin sulfates represent a small subclass within the lamellarin family. In 2019, the group of Keyzers isolated six new lamellarin sulfates (**177**–**182**) from *Didemnum ternerratum*, a pacific tunicate (Figure 21) [160]. All of them showed similar analytical data to previously reported lamellarins except for the sulfate functional group. The substantial majority of naturally occurring lamellarins show no optical rotation with the exception of lamellarin S (half-life of racemization ≈ 90 days). Surprisingly, the newly isolated sulfates **179**–**182** showed optical activity in ECD analysis, which is due to the hindered rotation of ring F resulting in an axial chirality (atropisomerism). The bioactivity of lamellarins **177**–**182** against human colon carcinoma HCT-116 was investigated, with D-8-sulfate (**182**) showing appreciable cytotoxicity (IC50 = 9.7 μM) [160].

In addition to the representative group of lamellarins [32,156,161–166], further related pyrroles like the polycitons, polycitrins [167], storniamides [168], and denigrins [90,169] as well as the fused alkaloids lukianols [170], dictyodendrins [171], purpurone [172], ningalins [173] and baculiferins can also be included, which extend the family of 3,4- diarylpyrroles. In the molecular backbone, structural variations from fused maleiimide units to highly conjugated carbazole-2,7-diones can be found.

The Capon laboratory isolated the new ningalins E (**183**) and F (**184**) from the species *Didemnum* (CMB-02127), which, according to the authors, share a biosynthetic pathway similar to that of the lamellarins by merging a tyrosine with a defined number of catechols (Figure 22). Only low cytotoxicities against human, bacterial, and fungal cell lines were

observed, whereas the ningalins **183** and **184** showed moderate inhibition of the kinases CK1δ, CDK5, and GSK3β, potential targets for the treatment of neurodegenerative diseases (IC50 values between 1.6 μM and 10.9 μM) [174].

**Figure 22.** Related congeners **183**–**185** of the lamellarins sharing the central fused pyrrole core.

The class of the baculiferins was established by Lin and Bringmann in 2010, yielding pyrrole **185** alongside 14 other new members bearing a carbazole-2,7-dione central core (Figure 22). Baculiferin O (**185**) as a C8 sulfate representative inhibits several tumor cell lines with moderate activity around 33 μM [175].

Because of their promising biological activities such as antiproliferative, multidrug resistance reversal activity, cytotoxicity, and anti-HIV-1 activity, the lamellarin core has served as a potential lead structure for synthetic and medicinal chemists in the past decade [157,158]. The published syntheses of the lamellarins and derivates in the past decade, summarized in Table 1, provide an update of the existing summary by Opatz et al. in 2014 [158] and concentrate the recent review by Iwao et al. in 2020 [157].

**Table 1.** Summary of published synthesis of lamellarins and related analogs in the decade of 2010–2020.



**Table 1.** *Cont.*


**Table 1.** *Cont.*

The longest linear sequence in the synthesis was counted.

This astounding number of syntheses highlights the importance of these pyrrole members of marine origin to many areas of life science. In addition to the constantly increasing number of total syntheses of lamellarins and their natural congeners, the number of synthetic derivatives and biological activity assays has increased similarly [206–214].

#### **3. Halogenated Marine Pyrrole Alkaloids**

This chapter presents the occurrence of halogenated pyrroles which constitute a highly diverse and structurally complex subclass of marine alkaloids. It is considered that at least 25% of organohalogen natural products are halogenated alkaloids, mostly featuring pyrrole, indole, carboline, and other N-heteroaromatic core structures [215,216]. This observation is not too surprising as the marine environment provides both chloride and bromide in virtually unlimited quantities as well as a variety of halogenase enzymes from different organisms, resulting in an excellent environment for biohalogenation of these electronrich substrates [30,217,218]. From a medicinal point of view, the resulting structures are associated with numerous different pharmacological activities such as selective antihistamine [219–221], anti-serotonergic [222], immunosuppressive [223], antibacterial [224], anti-malarial [225], and antiproliferative properties [226]. Therefore, halogenated pyrrole alkaloids can be viewed as potential lead compounds for the development of new, even more potent drugs [15,227].

Given the enormous dimensions and (bio)chemical diversity of marine life and its underexplored nature, it is not surprising that the number of isolated halogenated marine pyrroles is constantly increasing and that countless further halopyrroles are ye<sup>t</sup> to be discovered.

## *3.1. Simple Pyrroles*

i

Ethyl 3,4-dibromo-1*H*-pyrrole-2-carboxylate (**186**) was first isolated from the sponge *Stylissa massa* in 2014 and shows a weak antiproliferative activity against mouse lymphoma cells (L5178Y growth in 27.2% at 10 μg/mL, Figure 23) [228].

**Figure 23.** Simple bromopyrrole alkaloids **186**–**191** isolated from different marine sponges.

A related bromopyrrole **187** was isolated from another sponge (*Agelas cerebrum*) in 2011 and subjected to several antiproliferative tests (Figure 23) [229]. Here, compound **187** and other isolated bromopyrroles did not show any activity against cancer cells (A549 lung cancer cells, HT29 colonic cancer cells, and MDA-MB-231 breast cancer cells). However, when the crude mixture, from which **187** and further bromopyrroles were isolated, was subjected to biological tests, a strong cytotoxic activity (IC50 values around 1 μg/mL) against all three human tumor cell lines could be observed. The authors attributed this effect to the ye<sup>t</sup> underexplored synergism of natural product mixtures containing bromopyrroles [229]. Both compounds **186** and **187** were previously only known as synthetic products [230,231].

Two further simple substituted halopyrroles, **188** and **189**, could be isolated from the South China Sea sponge *Agelas* sp. in 2016. The enantiomers (+)-**188**, (−)-**188**, (+)-**189** and (−)-**189** did not appear to have any antifungal activities using the *Caenorhabditis elegans* candidiasis model (Figure 23) [66]. However, the racemic mixtures of (±)-**188** and (±)-**189** showed effective antifungal activity. Unfortunately, the authors did not provide any values or an explanation of this observation. Despite these results, the authors found out that the corresponding intramolecularly cyclized pyrroloketopiperazine natural products (see Figure 49, **342**–**344**) exhibited significant antifungal activities with survival rates around 50% [66].

Very recently, the corresponding agesasines A (**190**) and B (**191**) featuring the free alcohol functional groups, were isolated from Okinawan marine sponges *Agelas* spp. (Figure 23) [232]. Both compounds were isolated as racemates and, according to the authors, might be artifacts from the extraction process under acidic conditions. In basic antiproliferative tests against human cancer cell lines (HeLa, A549, and MCF7), no cytotoxicity could be observed [232].

In 2012, a new bromopyrrole, 4-bromo-*N*-(butoxymethyl)-1*<sup>H</sup>*-pyrrole-2-carboxamide (**192**), featuring an unusual ether group in its side chain, could be isolated from the marine sponge *Agelas mauritiana* (Figure 24) [233].

**Figure 24.** Simple bromopyrrole alkaloids **192**–**195** and structural similar agelanesins A–D (**196**–**199**).

Further structurally similar halopyrroles **193**–**199** possessing different substituents at their amide side chains were isolated from the Indonesian marine sponges *Agelas linnaei* (Figure 24) [234]. While mauritamide D (**193**), 4-(4,5-dibromo-1-methylpyrrole-2- carboxamido)-butanoic acid (**194**), and agelanin B (**195**) were inactive against L1578Y mouse lymphoma cell lines, the tyramine-unit bearing agelanesins A–D (**196**–**199**) showed prominent to good activity with IC50 values between 9.25 μM and 16.76 μM in this assay. The authors mentioned that the cytotoxicity of the agelanesins **196**–**199** is interconnected with the degree of bromination of the pyrrole ring, resulting in an increased reactivity for the monobrominated agelanesins A (**196**) and B (**197**) compared to **198** and **199** [234].

The tribrominated pyrrole 4--((3,4,5-tribromo-1*H*-pyrrol-2-yl)methyl)phenol (**200**) was isolated from the surface of the coralline alga *Neogoniolithon fosliei* in 2014 and exhibited broad-spectrum antibacterial activity against several *Pseudoalteromonas*, *Vibrio*, and *Staphylo-* *coccus* spp. (inhibition zones > 10 mm, Figure 25). However, no antifungal or antiprotozoal activity was observed by investigating compound **200** [235].

**Figure 25.** Structure of compound **200** and the bromotyrosine-based keronopsamides A–C (**201**–**203**).

A new class of bromopyrrole pigments derived from bromotyrosine were isolated from the marine ciliate *Pseudokeronopsis riccii* in 2010 and were named keronopsamides A–C (**201**–**203**) (Figure 25) [236].

In 2020, pyrrolosine (**204**), a tetrabrominated alkaloid symmetrically dimerized via two amide functionalities, was isolated from *Agelas oroides* [237] and should not be confused with another natural product named pyrrolosine (**206**), the structure of which had been identified as **205** and revised **206** during the 1990s (Figure 26) [238].

**Figure 26.** Molecular structures of bromopyrroles **204**–**211** isolated from sponges and bryozoans.

Further marine bromopyrrole alkaloids **207**–**211** substituted via amide groups were isolated from the Patagonian bryozoan *Aspidostoma giganteum* (Figure 26) [239]. The aspidostomides A–C (**207**–**209**), G (**210**) and H (**211**) bear the well-known bromotyrosine and bromotryptophan structural motifs frequently found in marine natural products [240]. While for aspidostomide A (**207**) the absolute configuration was determined as *R* by a modified Mosher method [241], the configurations of aspidostomides B (**208**) and C (**209**) were assumed to be the same as in compound **207**. The absolute configuration of aspidostomide H (**211**) could not ye<sup>t</sup> be established [239].

In 2019, the first total syntheses of the enantiomeric aspidostomides B (**208**) and C (**209**) were realized by Khan and co-workers (Scheme 14) [242].

**Scheme 14.** First total syntheses of aspidostomides B (**208**) and C (**209**) starting from compound **212**.

Here, compound **212** was reacted in a Wittig olefination and then subjected to bromohydroxylation. Substitution of the bromine with NaN3 followed by reduction furnished amine (±)-**215** in 67% yield over four steps. Amidation of (±)-**215** with either 4,5-dibromopyrrole carboxylic acid (**213**) or 3,4,5-tribromopyrrole carboxylic acid (**214**) delivered products **216** and **217**, respectively. Final demethylation by applying BBr3 then gave the natural products aspidostomides B (**208**) in 67% and C (**209**) in 72% over two steps (Scheme 14) [242].

In 2018, nine new pseudoceratidines (**218**–**226**), of which the tedamides A–D (**223**–**226**) possess an unprecedented 4-bromo-4-methoxy-5-oxo-4,5-dihydro-1*<sup>H</sup>*-pyrrole-2-carboxamide moiety, were isolated from the marine sponge *Tedania brasiliensis*(Figure 27) [243]. It is important to mention that 3-debromopseudoceratidine (**218**) and 20-debromopseudoceratidine (**219**), 4-bromopseudoceratidine (**220**), and 19-bromopseudoceratidine (**221**), tedamides A and B (**223** and **225**), and tedamides C and D (**224** and **226**) have been isolated as pairs of inseparable structural isomers differing in their sites of bromination and oxidation. The inseparable mixture of compounds **218** and **219** showed antiparasitic activity on *Plasmodium falciparum* (EC50 value of 5.8 μM ± 0.5 μM) and displayed weak cytotoxicity in the human liver cancer HepG2 cell line (MDL50 ≥ 400 μM), but with excellent selectivity, as reflected by a dramatically reduced toxicity to healthy cells. The authors also synthesized a number of derivatives that were assayed against several protozoan parasite species, evidencing that the bromine substituents in the pyrrole unit of pseudoceratidine derivatives are inevitable for antiplasmodial activity [243].

**Figure 27.** Nine new pseudoceratidines **218**–**226** from the marine sponge *Tedania brasiliensis*.

Another bromopyrrole alkaloid, clathrirole A (**227**), was isolated from the Myanmarese marine sponge *Clathria prolifera* in 2018 (Figure 28) [73]. It should be noted that the stereogenic centers of the tetrahydropyrimidinium ring of **227** were only assumed to have *R* configuration by comparison of its optical rotation with the enantiomeric *N*-methylmanzadicin C (**228**) which had been isolated and synthesized several years earlier [35,244,245].

**Figure 28.** New bromopyrrole alkaloid **227**. *N*-Methylmanzacidin C (**228**) is shown for comparison.

In this context, the correction of the stereoconfiguration of manzacidin B (**232a**) should also be mentioned. This MNP was synthesized by the Ohfune group in 2007 and its configuration was erroneously determined to match compound **232b [246]**. Three years later, the same group published an alternative synthetic route (Scheme 15) and with the aid of X-ray crystallography, the revised structure of manzacidin B (**232a**) was unambiguously confirmed [247]. Here, aldehyde **229** was transformed into compound **230** using Oppolzer's sultam as a chiral auxiliary, and subsequently generated the *N*-formyl lactone **231** already featuring the stereochemistry of natural manzacidin B (**232a**). Several further steps, including the installation of the pyrrole unit, then delivered the natural product **232a [247]**. Unfortunately, the correction did not provide any information about the experimental section, including reaction conditions and yields.

**Scheme 15.** An alternative synthetic route towards manzacidin B (**232a**) in 2010 revealed that it was incorrectly assigned as compound **232b** in 2007.

In 2015, the group of Köck isolated *N*-methylagelongine (**233**) from the Caribbean sponge *Agelas citrina* (Figure 29) [63].

**Figure 29.** Simple bromopyrrole alkaloids **233**–**236** isolated from the *Agelas* sp.

Two new halopyrroles, nagelamide U and V (**234** and **235**) were isolated from a marine sponge *Agelas* sp. in 2013 and possess a γ-lactam ring with a taurine unit (Figure 29). Here, the relative stereochemistry was examined by ROESY correlations [65].

A related compound, 2-debromonagelamide U (**236**) was isolated from the Okinawan marine sponge *Agelas* sp. two years later. Compound **236** could inhibit the growth of *Trichophyton mentagrophytes* (IC50 value 16 μg/mL), a common fungus causing ringworm in companion animals (Figure 29) [248].

In 2019, three new pyoluteorin analogs, mindapyrroles A–C (**237**–**239**) were isolated from *Pseudomonas aeruginosa* strain 1682U.R.oa.27, a bacterium from the tissue homogenate of the giant shipworm *Kuphus polythalamius* (Figure 30) [249]. The chlorinated pyrrole alkaloids **237** and **239** inhibit the growth of multiple clinically relevant microbial pathogens (MIC values between 2 μg/mL and >32 μg/mL), with mindapyrrole B (**238**) showing the most potent antimicrobial activity (MIC values between 2 μg/mL and 8 μg/mL) and widest selectivity index over mammalian cells [249].

**Figure 30.** Mindapyrroles A–C (**237**–**239**) featuring several central resorcinol-cores.

New diterpene alkaloids, the agelasines O–R (**240**–**243**) bearing a bromopyrrole core, were isolated from the Okinawan marine sponge *Agelas* sp. in 2012 (Figure 31) [61]. The relative stereochemistries of compounds **240**–**243** were elucidated via ROESY-correlations. The agelasines O–R (**240**–**243**) showed good to moderate antimicrobial activities (IC50 values ranging between 8 μg/mL and >32 μg/mL) against a wide range of bacteria, including strains of *Escherichia coli*, *Staphylococcus aureus*, and *Bacillus subtilis*. However, no cytotoxicity against murine leukemia L1210 and human epidermoid carcinoma KB cells was observed [61].

**Figure 31.** Agelasines O–R (**240**–**243**) with a 9- *N*-methyladenine unit from a marine sponge *Agelas* sp.

In 2010, Fenical and co-workers isolated marinopyrroles C–E (**244**–**246**) from the deep ocean actinomycete strain CNQ-418 [250], thereby extending the interesting class of biologically active marinopyrroles, of which marinopyrroles A (**250**) and B (**253**) had been isolated before (Figure 32) [251]. These metabolites contain an unprecedented, highly halogenated 1,3--bipyrrole core which gives them an axis of chirality that, for marinopyrroles A and B as well as C–E (**244**–**246**), results in a stable *M*-configuration at room temperature. Marinopyrrole C (**244**) displayed significant activity against methicillin-resistant *Staphylococcus aureus* with MIC90 values of less than 1 μg/mL. With derivatization experiments, the authors could also show that the presence of the hydrogen-bonding capacity of the salicyloyl hydroxyl groups, the free N–H functionality and the C-5- chlorine substituent were indispensable for the biological activity [250].

**Figure 32.** The unusual structure of marinopyrroles C–E (**244**–**246**) contain a rare 1,3--bispyrrole functionality.

The first total synthesis of a member of the marinopyrrole family was realized by the Li laboratory in 2010 (Scheme 16) [252]. Starting with a TsOH-catalyzed condensation and cyclization of aminopyrrole **247** with α-ketoester **248** furnished an intermediary bi-pyrrole skeleton. After N-protection and transforming the diester to the dialdehyde via a reduction/oxidation sequence, the addition of 2-methoxyphenylmagnesium bromide followed by CrO3 oxidation furnished the diketone **249** in 50% over six steps. After deprotection and chlorination of the pyrrole units with NCS, a final demethylation involving BBr3 gave the natural product, (±)-marinopyrrole A (**250**) in 68% yield over three steps. Unfortunately, selective bromination towards (±)-marinopyrrole B (**253**) under various conditions was unsuccessful [252].

**Scheme 16.** First total synthesis of (±)-marinopyrrole A (**250**) by Li in 2010 and its congener marinopyrrole B (**253**) by Chen in 2013.

Three years later, the Chen laboratory synthesized (±)-marinopyrrole B (**253**) using a similar approach (Scheme 16) [253]. Here, the brominated chloropyrrole **252** was generated over nine steps starting from commercially available pyrrole **251**. The next seven steps were performed almost in the same manner as in the synthesis of marinopyrrole A reported by Li and co-workers, although some reaction conditions were improved. In this way, (±)-marinopyrrole B (**253**) could be obtained in 15% over seven steps [253].

Between 2012 and 2019, several pyrrolyloxazoles belonging to the phorbazole series were isolated from marine organisms. The first study of the Indo-Pacific dorid nudibranch *Aldisa andersoni* resulted in the isolation of 9-chloro-phorbazole D (**254**) and *N*1-methylphorbazole A (Figure 33) (**255**). Both compounds exhibit similar in vitro inhibitory activity against several human cancer lines with IC50 values ranging between 18 μM and 34 μM [254].

**Figure 33.** Phorbazol-based marine bromopyrrole alkaloids **254**–**259**.

A related class of natural bromopyrroles containing the pyrrolyloxazole functionality is the breitfussins. In analogy to breitfussin B (**256**), isolated from the hydrozoan *Thuiaria breitfussi* in 2012 [80], six new breitfussins C–H were discovered in the same producing organism as breitfussins E (**257**), G (**258**), and H (**259**) feature a brominated pyrrole core (Figure 33, for non-halogenated congeners see Figure 7) [81]. Compounds **258** and **259** were isolated as a mixture and thus not evaluated in cytotoxic activity assays, whereas breitfussins **256** and **257** did not show any cytotoxic activity against several tested cancer cell lines [81].

In 2015, breitfussin B (**256**) was synthesized by the Bayer group in the same manner as breitfussin A (**48**) (compare Scheme 4) [83]. In analogy to breitfussin A (**48**), the synthesis commenced with the readily available phenol **260**. After forming the indole building block **261**, iodination and TIPS-protection furnished compound **52**. The oxazole core **54** was installed and carefully iodinated with iodine to ge<sup>t</sup> access to compound **262**. Coupling with Boc-protected pyrrole boronic acid **20** then delivered intermediate **57** possessing the right indole-pyrrolyloxazole functionality. Bromination, protodeiodination, and removal of all protecting groups then furnished breitfussin B (**256**) in 4.3% overall yield (Scheme 17) [83].

**Scheme 17.** Total synthesis of breitfussin B (**256**) starting from phenol **260**.

Simple Pyrrole (Amino)-Imidazole Alkaloids

The pyrrole-imidazole alkaloid (PIA) family comprises a myriad of simple to structurally complex molecules originating from marine organisms. The simplest PIA, oroidin, is believed to be the biogenetic precursor of any natural products belonging to this family and it is considered to be biosynthesized from the fundamental amino acids proline, ornithine, lysine, and/or histidine [13,38,255–257]. However, numerous further considerations on the biogenetical origin of PIAs can be found in the literature so that the biosynthesis of most of these alkaloids still lies in the realm of speculations. Many PIAs are reported to exhibit significant biological activities resulting in a grea<sup>t</sup> interest among synthetic chemists to provide solutions to finally ge<sup>t</sup> access to potent pharmaceutically relevant substances.

In 9-oxethyl-mukanadin F (**263**), isolated in 2016 by the Lin group from a not fully identified sponge *Agelas* sp., the oroidin 2-aminoimidazole moiety is replaced by a hydantoin ring (Figure 34) [66]. Compound **263** was isolated as a racemic mixture and displayed no antifungal activity against *Candida albicans* [66].

**Figure 34.** C-9 functionalized ene-hydantion marine pyrrole alkaloids **263** and **264**.

In 2018, the Barker group published a comprehensive work addressing stereochemical issues of related mukanadin-based alkaloids substituted at C-9 [79]. The publication also describes the total synthesis of (+)- and (−)-mukanadin F (**264a** and **264b**), which finally resulted in the reassignment of its absolute stereochemical configuration and shed light upon many inconsistencies concerning the stereochemistry of C-9-functionalized enehydantoin/imidazole marine natural products published as racemic or scalemic mixtures before (Figure 34 and Scheme 18) [220,258–261].

**Scheme 18.** Total synthesis of (*S*)-mukanadin F (**264b**).

The authors began the synthesis with a selective protection/deprotection sequence of aminodiol (*R*)-**265** producing alcohols (*R*)-**266** and (*R*)-**267**, sequentially. After Swern oxidation and HWE reaction with hydantoin phosphonate **268**, compound (*S*)-**269** could be obtained as a mixture of *E*/*Z* isomers (1:2) in 66% yield over two steps. Simultaneous Boc and PMB deprotection followed by a final C−N coupling step involving trichloroacetyl dibromopyrrole **270** gave (*S*)-mukanadin F ((*S*)-**264b**) as a mixture of *E*/*Z* isomers (1:1.3). The

same procedure starting from (*S*)-**265** delivered ( *R*)-mukanadin F (( *R*)-**264a**) as a mixture of *E*/*Z* isomers (1:2) (Scheme 18) [79].

Successful separation of the *E*/*Z* isomers of ((*S*)-**264b**) and (( *R*)-**246a**) and comparison of NMR spectroscopic data of the synthetic *Z*-configured enantiomers of mukanadin F (**264**) with those reported for the natural product were a match, confirming the alkene geometry [258]. However, new optical rotation measurements revealed that (*S*)-mukanadin F ((*S*)-**264b**) corresponds to the natural product, which is opposite to that proposed for the isolated sample in 2009 [258]. As a last point, the Baker group found out that C-9 functionalized ene-hydantoin/imidazole marine alkaloids are prone to isomerization and racemization with both effects occurring upon light irradiation or under acidic or basic conditions and therefore is likely to occur upon extraction [79]. These findings reveal that compounds of this class most likely exist in nature as pure enantiomers and that other publications concerning their isolation and stereochemical elucidation should be checked carefully.

Recently, *E*-dispacamide (**271**) and slagenin D (**272**) were isolated from the sponge *Agelas oroides* in 2020 (Figure 35). The absolute configuration of compound **272** was established by comparison of its specific rotation with that of synthetic *ent*-slagenin A, indicating its stereogenic centers to be 9*S*, 11*S*, 15*S* configured [237].

**Figure 35.** Related bromopyrrole alkaloids **271**–**274** bearing hydantoin.

A bromopyrrole marine alkaloid **273**, very similar to compound **271**, was isolated from the sponge *Stylissa massa* in 2014 and was given the name dispacamide E (**273**) (Figure 35) [228]. It showed significant inhibitory activities against the kinases GSK-3, DYRK1A, and CK-1 with IC50 values below 19 μM [228]. The reader is advised that careful reading is required to distinguish between the (*E*/*Z*) dispacamides, as the original trivial names relate to the *Z*-configured natural compounds [219,220]. However, new dispacamides possessing *E*-configuration are not consistently given either new trivial names or *E*/*Z*-designated former trivial names.

In nemoechine H (**274**), isolated from the sea sponge *Agelas nemoechinata* in 2019, only the hydantoin core is different compared to compound **273** (Figure 35). Compound **274** exhibited good to moderate cytotoxic activity against K562 and L-02 cell lines with IC50 values of 6.1 μM and 12.3 μM, respectively [262].

Very recently, three new related congeners, 9-hydroxydihydrodispacamide (**275**), 9- hydroxydihydrooroidin (**276**), and 9*E*-keramadine (**277**) were isolated from two different marine sponges *Agelas* spp. (Figure 36). Compounds **275** and **276** were isolated as racemates with the relative configuration of compound **275** still to be deduced [232]. Compound **277** was already known as a synthetic product but was isolated the first time from a natural source [263]. All three compounds **275**–**277** did not show any promising cytotoxicity against human cancer cell lines (HeLa, A549, MCF7) in basic antiproliferative tests [232].

**Figure 36.** Three new PIAs **275**–**277** isolated from the sponge *Agelas* spp. in 2020.

The Berlinck group isolated debromooroidin **278** from a sponge identified as *Dictyonella* sp. in 2018, which displayed proteasome inhibition activity with IC50 values of 27 μM ± 6 μM (Figure 37) [264]. The authors also mentioned that the proteasome inhibitory activity is strongly influenced by the position of the bromine substituent in the pyrrole ring thereby confirming the findings of previous investigations [265,266].

**Figure 37.** Oroidin-derived bromopyrrole alkaloids **278**–**283** bearing imidazole moieties.

In 2009, the acetone/methanol extract of the sponge *Agelas linnaei* permitted the isolation of agelanin A (**279**) and mauritamides B (**280**) and C (**281**) (Figure 37) [234]. The sulfonic acid congeners **280** and **281** contain a taurine unit which is quite a rare structural motif in marine sponge metabolites when combined with a bromopyrrole unit.

Further oroidin-derived pyrrole alkaloids, stylisines B (**282**) and C (**283**), were isolated in 2018 from the sponge *Stylissa massa* (Figure 37). Here, the stereogenic centers could be unambiguously determined via electronic circular dichroism experiments. Unfortunately, compounds **279**–**283** have not shown any promising biological activities so far [133].

In 2010, another new set of halopyrroles, the stylissazoles A–C (**284**–**286**), were isolated from species from the *Stylissa* genus (Figure 38) [267]. No absolute configuration could be determined for the dimeric pyrrole-2-aminoimidazoles **285** and **286** as no optical activity was observed. The authors mentioned that the interconversion of the configurationally unstable chiral carbons C6 and C7 might be the reason for this issue. However, the relative configuration of both stereogenic centers in stylissazole C (**286**) could be determined by NOESY experiments [267].

**Figure 38.** Stylissazoles A–C (**284**–**286**) isolated from the marine sponge *Stylissa carteri*.

The unique bromopyrrole alkaloids agelamadin F (**287**) and tauroacidin E (**288**) were isolated from an Okinawan marine sponge of the genus *Agelas* in 2015 (Figure 39) [64]. Compound **287** is the first example of a bromopyrrole alkaloid bearing an aminoimidazole moiety connected to a pyridinium ring. Tauroacidin E (**288**), possessing an uncommon taurine unit, was isolated as a racemic structure. Both halopyrroles **287** and **288** showed moderate activities against KB and human leukemia K562 cells with IC50 values in the range of 10 μg/mL [64].

**Figure 39.** Unusual aminoimidazole pyrrole alkaloids **287**–**291** with compounds **289**–**291** incorporating a complex contiguous imidazole ring system.

The complex class of massadines was extended by the isolation of three new compounds **289**–**291** from a deep-water sponge of the genus *Axinella* in 2012 (Figure 39) [268]. The eight stereogenic centers of 14- *O*-sulfate massadine (**289**), 14- *O*-methyl massadine (**290**), and 3- *O*-methyl massadine chloride (**94**) were determined by NMR spectroscopy and optical rotation measurements. The generated data confirmed the absolute stereochemistry earlier defined by Köck [269] and Fusetani [270] for related massadines and was also consistent with the data from its enantioselective total synthesis [271]. While compounds **289**–**291** did not show any inhibitory activity against the neurodegenerative disease kinase targets CDK5/p25, CK1δ, and GSK3β, 3- *O*-methyl massadine chloride (**291**) exhibited antibacterial activity against several Gram-positive and -negative bacteria with IC50 values below 5 μM [268].

Three structurally similar alkaloids (**292**–**294**), possessing two or more contiguous ring systems were isolated from the sponge *Stylissa* aff. *carteri* in 2020 (Figure 40) [272]. The absolute stereochemistry of the two new hexacyclic analogs of palau'amine and styloguanidine, debromokonbu'acidin (**292**) and didebromocarteramine (**293**), was determined by comparison of experimental and theoretical ECD spectra. While compound **293** did not show any neuroprotective activity, compound **292** could reduce reactive oxygen species in neuroblastoma SY-SY5Y cells by 35% over a wide range of concentrations [272]. The stereochemistry of futunamine (**294**), featuring a new pyrrolo[1,2-*c*]imidazole core, was

also deduced by ECD analyses. Furthermore, futunamine (**294**) showed neuroprotective effects at 10 μM. Unfortunately, none of the three new compounds **292**–**294** showed any cytotoxic activity [272].

**Figure 40.** Biologically active bromopyrrole imidazole alkaloids **292**–**295** possessing unique structural motifs.

Nagelamide W (**295**), the first monomeric bromopyrrole alkaloid bearing two aminoimidazole moieties, was isolated from a marine sponge *Agelas* sp. by the Kobayashi group in 2013 (Figure 40) [65]. The relative stereochemistry of **295** was elucidated by ROESY correlations and the natural product **295** exhibited inhibitory activity against *Candida albicans* with an IC50 value of 4 μg/mL [65].

In 2014, five new bromopyrrole alkaloids (**296**–**300**) were isolated from an Okinawan marine sponge of the genus *Agelas* (Figure 41) [273]. Tauroacidin C (**298**), tauroacidin D (**299**), and mukanadin G (**300**) were isolated as racemic mixtures. However, the relative stereochemistry of mukanadin G (**300**) was established by ROESY and computational experiments. While compounds **296**–**298** did not show any antimicrobial activity, mukanadin G (**300**) exhibited good to moderate antifungal activity against the human-pathogenic yeas<sup>t</sup> *Candida albicans* and the invasive pathogenic fungus *Cryptococcus neoformans* with IC50 values between 8 and 16 μM [273].

**Figure 41.** Related bromopyrrole alkaloids **296**–**300** and the antifungal mukanadin G (**300**) isolated from *Agelas* sp.

In decarboxyagelamadin C (**301**), isolated from the sponge *Agelas sceptrum* in 2016, a rare morpholine core is located between the pyrrole and imidazole moiety with the relative and absolute stereochemistry being established by NMR and ECD spectroscopy (Figure 42) [274]. Unfortunately, compound **301** did not show any activity in cytotoxicity tests and in antimicrobial assays.

**Figure 42.** Oroidin-based bromopyrrole alkaloids **301**–**303** with nagelamide D (**304**) underwent a reevaluation in 2020.

A new bromopyrrole alkaloid also incorporating a fused 6-membered ring, 2-debromomukanadin G (**302**), was isolated from another *Agelas* sp. alongside 2-debromonagelamide P (**303**) (Figure 42) [248]. While both substances **302** and **303** were isolated as racemates, the relative configuration of compound **302** could be deduced by comparison of its coupling constants with those from mukanadin G (**300**). Compound **303** showed moderate antimicrobial activity against *Trichophyton mentagrophytes* (IC50 value 32 μg/mL), whereas compound **302** exhibited moderate activity against *Cryptococcus neoformans* (IC50 value 32 μg/mL). However, no cytotoxicity was observed against human epidermoid carcinoma KB and murine lymphoma L1210 cells [248].

We also want to mention an inconsistency in the assigned structure for the structurally related nagelamide D (**304**), which was originally isolated in 2004 as a racemate by the Kobayashi group (Figure 42) [275]. Five years later, a total synthesis by the Lovely group [276] revealed that either the assigned structure or the reported NMR data of Kobayashi's work was in error. However, no final evidence was given at this point. A recently published synthetical approach [277] of the same laboratory towards alkaloids belonging to the nagelamide class then corroborated the correctly proposed but incorrectly assigned structure by Kobayashi. In this case, crystallographic measurements [277] unequivocally demonstrated that the assignments for C9, C9-, C10- as well as H9a and H9b were inadvertently switched in the original literature [275].

The Lovely group commenced their synthesis with the iodoimidazoles **305** and **306**, which were transformed into the corresponding coupling partners **307** and **308** over several steps, respectively. A Stille cross-coupling then delivered compound **309**. A reaction sequence involving several protection and deprotection reactions as well as the installation of the azide group via TsN3 furnished diol **310**. Replacing the alcohol functional groups by a pyrrole hydantoin **311**, hydrolysis, and deprotection of the corresponding urea followed by azide hydrogenation finally furnished nagelamide D (**304**) in 32% over four steps (Scheme 19) [277].

**Scheme 19.** A total synthesis of nagelamide D published by the Lovely group led to the correct assignment of nagelamide D (**304**).

A very similar class of compounds, the citrinamines A–D (**312**–**315**), were isolated from the Caribbean sponge *Agelas citrina* in 2015 by the Köck group (Figure 43) [63]. All four compounds **312**–**315** were isolated as racemic mixtures, with the relative configuration of citrinamine C (**314**) being elucidated with the aid of NOESY correlations and comparison of its NMR data with those of nagelamide B, a related congener isolated back in 2004 [275]. It should be mentioned that the same group isolated citrinamines C (**314**) and D (**315**) as a mixture, the separation of which by preparative chromatography failed. Citrinamines B–D (**313**–**315**) showed "considerable" inhibition zones in agar diffusion assays with *Mycobacterium phlei* (no values for the size of the inhibition zones were given). However, all compounds **312**–**315** exhibited no inhibition of cell proliferation of mouse fibroblasts [63]. Here, we would like to mention that the only structural difference between citrinamine A (**312**) and 2-debromonagelamide P (**303**) lies in the additional proton present in compound **303** (Figure 43). As the NMR spectra of both compounds **303** and **312** also appear to be identical, it is highly likely that both compounds **303** and **312** are in fact the same substance, although compound **303** was isolated as a salt and compound **312** as the free base.

**Figure 43.** The dimeric bromopyrrole alkaloids citrinamines A–D (**312**–**315**).

The known class of nagelamides was extended by nagelamides I (**316**) and 2,2-- didebromonagelamide B (**317**), isolated from a marine sponge *Agelas* sp. (Figure 44) [278]. The relative configuration of compound **317** could be deduced by extensive NMR-spectroscopic analysis but the absolute configuration remains unknown. Both compounds **316** and **317** did not show cytotoxicity against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro [278].

**Figure 44.** Five new family members (**316**–**320**) of the nagelamides from *Agelas* sp.

Nagelamides X–Z (**318**–**320**) were isolated from a marine sponge of the genus *Agelas* in 2013 (Figure 44) [279]. Here, the nagelamides X (**318**) and Y (**319**) incorporate a unique tricyclic skeleton consisting of spiro-connected tetrahydrobenzaminoimidazole and aminoimidazolidine moieties. Compounds **318** and **319** were isolated as racemic mixtures with the relative configuration being determined by 2D NMR spectroscopy. Nagelamide Z (**320**) was isolated as an optically active molecule, but its absolute configuration remains

unsolved. Nagelamides X–Z (**318**–**320**) displayed antimicrobial activities against several bacteria and fungi, with IC50 values partly being below 5 μg/mL [279].

In 2012, a new pair of dimeric pyrrole-aminoimidazole alkaloids, (−)-donnazoles A (**321**) and B (**322**), was isolated from the marine sponge *Axinella donnani* (Figure 45). The absolute configurations of **321** and **322** were determined via NOE correlations and ECD measurements [280].

**Figure 45.** Donnazoles A (**321**) and B (**322**) from a marine sponge *Axinella donnani* and further agelamadins C–E (**323**–**325**).

The agelamadins C–E (**323**–**325**), isolated from a marine sponge of the genus *Agelas* in 2014, share the same flat structure but differ in their stereochemistries (Figure 45) [281]. The configurations of compounds **323**–**325** were elucidated by 2D NMR spectroscopy, ECD calculations, and by a phenylglycine methyl ester (PGME) method. To this end, (*R*)- and (*S*)-PGME are condensed with a carboxylic acid functionality, to generate amides enabling the determination of the absolute configuration by means of the diamagnetic anisotropic effect [282]. While agelamadin D (**324**) did not show any antimicrobial activity, agelamadins C (**323**) and E (**325**) displayed moderate inhibitory activity against the human pathogen *Cryptococcus neoformans* with IC50 values of 32 μg/mL each [281].
