**4. Miscellaneous**

Among the known marine pyrroles, there are also complex architectural frameworks containing macrocyclic ring systems, not only one or more sugar residues, but also multiple amide bonds forming peptides or even cyclopeptides. Therefore, in the following section, structures and classes are presented that could not be classified in the previous chapters due to their mostly complex and intriguing scaffolds.

In 2019, a scalarane sesterterpenoid featuring a 6/6/6/6/5-pentacyclic core was isolated from the sponge *Scalarispongia* sp. The fused pyrrole **412** represents the first pyrrole derivative in the rare class of N-heterocyclic scalaranes (Figure 35). MNP **412** was found to show moderate inhibition against six human cancer cell lines in bioactivity assays (GI50 values ranging between 14.9 μM and 26.2 μM) [313].

The bispyrrole curvulamine (**413**) originates from the fungus *Curvularia* sp. IFB-Z10, produced in a symbiontic way with the host, the White Croaker (*Argyrosomus argentatus*) (Figure 58) [314]. In the course of structure elucidation and determining the crystal structure of the unprecedented framework of curvulamine A, the authors also made efforts to elucidate the biosynthetic pathway using NMR-based 13C labeling experiments. Curvulamine (**413**) possesses antibacterial activity in the sub-micromolar range [314], whereas the biogenetic related trispyrrole curindolizine (**414**) lacks these bioactivities. However, antiinflammatory activities in lipopolyssacharide (LPS)-stimulated RAW 264.7 macrophages (IC50 = 5.31 μM ± 0.21 μM) could be observed. Surprisingly, as a by-product of reisolating curvulamine (**413**), curindolizine (**414**) was discovered in 2016, two years after the initial isolation of curvulamine (**413**) from the same fungus (Figure 58). On this basis, it is also assumed that curindolizine (**414**) represents the product of an in vivo Michael addition of the metabolites curvulamine (**413**) and the elimination product derived from procuramine (**125**) (cf. Figure 16) [125].

**Figure 58.** Polycyclic, complex molecular frameworks of condensed pyrrole MNPs **412**–**416**.

Another complex polycyclic scaffold is displayed by the densanins A (**415**) and B (**416**) (Figure 58) [315]. After extensive NMR studies, including the application of the Mosher ester method, the 3D structure featuring seven stereogenic centers and a 1-azabicyclo[3.2.1] octane core was determined to be biosynthetically derived from 3-alkylpyridines. The hexacyclic diamines **415** and **416**, isolated from the sponge *Haliclona densaspicula* in 2012, showed no cytotoxicity but promising inhibition of the NO production in LPS-induced BV2 microglial cells (IC50 values of 1.05 μM and 2.14 μM, respectively) [315].

Their promising bioactivity and challenging structures have inspired organic chemists ever since to develop a successful total synthesis of these MNPs [316–318]. The group of Maimone published the first successful synthesis of (−)-curvulamine (**413a**) in 2012, which was only feasible after extensive reconnaissance and several failures (Scheme 25) [319,320]. Starting from commercially available chemicals, they employed a feasible 10 step sequence to (−)-curvulamine (**413a**). The first key step was the coupling of racemic cyanohydrin **417**, as a masked acyl anion, with pyrroloazepinone **418**. This regioselective process was mediated by NaHMDS, followed by quenching the resulting enolate with NIS. After extensive investigation, the iodide was found to undergo cyclization under simple irradiation conditions in MeOH.

In this way, compound **419** was prepared in a 30% yield over two steps. After addition of lithiated ethyl vinyl ether, subsequent epimerization to the favored diastereomer **421**, and activation of the secondary alcohol with ClCSOPh, the thiocarbonate epimers (1*R*/1*S*)-**422** could be separated. The desired isomer **422** was reduced by deoxygenation and hydrolysis of the enol ether. The final step involves a diastereoselective reduction of the racemic ketone under CBS reduction conditions, yielding a 1:1 epimeric mixture of alcohols **413a** and **413b** that was readily separated into the enantiopure MNPs (Scheme 25) [319].

**Scheme 25.** A linear ten-step sequence yielding the natural bispyrrole (−)-curvulamine **413a**.

Syntheses such as the one shown by Maimone et al. play a significant in the development of potential active pharmaceutical ingredients as marine organisms often cannot be easily cultivated for mass production [319]. Further synthetic attempts, e.g., to prepare densanins, were undertaken by Yang and co-workers in 2016, whereas only the BCD tricyclic core could be achieved [321].

#### *4.1. Pyrroloiminoquinone and Related Analogs*

The pyrroloiminoquinones feature a central core in a broad variety of MNPs, divided into subclasses of iso-/batzellins, damirons, discorhabdins, epinardins, makaluvamines, prianosins, tsitsikammamins, wakayins, and veiutamines [322–324]. Among them, a new subclass of the heteroatom-rich macrophilones was established in 2017. Macrophilone A (**423**), isolated from the *Macrorhynchia philippina*, represents a rare example of the underexplored group of hydroids (Figure 59) [325]. Macrophilone A (**423**), together with a synthetic derivative prepared in the same study, was able to block the conjugation cascade of small ubiquitin-like modifier (SUMO). The SUMO conjugation to protein substrates occurs through an enzymatic cascade and is critical for the regulation of various cellular processes. It is often disrupted in diseases, including cancer, resulting in the disturbance of the protein balance [325].

**Figure 59.** Members of the macrophilones group **423**–**429**.

Once more, the group of Gustafson and co-workers published the isolation of six further macrophilones B–G (**424**–**429**) from the same source one year later (Figure 59) [326]. Just as its related congener macrophilone A (**423**), compounds **424**–**429** showed moderate to weak inhibition effects of SUMO conjugation cascade (IC50 values ranging between 11.9 μM and >100 μM). Furthermore, they exhibited significant toxicity against several cancer cell lines (no values given) [326].

To investigate their bioactivity potential, the first isolation of macrophilone A (**423**) was accompanied by its synthesis [325]. The authors started their ingeniously short approach from commercial formylindole **430**, which was nitrated and the aldehyde functionality reduced subsequently to furnish compound **431**. Oxidation by Fremy's salt yielded the iminoquinone, which, after the introduction of the thioether group by sodium methanethiolate, furnished the natural product **423** in just 4% yield over three steps (Scheme 26) [325].

**Scheme 26.** Synthesis of macrophilone A (**423**) in a linear sequence of 5 total steps.

In 2019, makaluvamine Q (**432**) was discovered, marking the first time a makaluvamine derivative was isolated from a marine *Tsitsikamma* sponge within the Latrunculiidae family (Figure 60). Besides the shown DNA intercalation and topoisomerase I inhibition (27% inhibition of DNA nicking), makaluvamine Q (**432**) was found to be most active against HeLa cells in cell viability assays (14.7% ± 0.5% metabolic activity at 10 μM). In addition, the authors showed possible biosynthetic relationships between the isolated subclasses [327].

**Figure 60.** Pyrroloiminoquinones and related derivatives **432**–**436** isolated from natural sources, which share a similar biosynthetic pathway.

Only a few months later, the Keyzers lab isolated makaluvamine W (**433**) and 6- bromodamirone B (**434**) from the sponge *Strongylodesma tongaensis* (Figure 60). Both isolated pyrrole derivatives **433** and **434** lacked cytotoxic activity against the leukemia cell

line HL-60, highlighting the importance of an iminoquinone scaffold in bioactivity considerations [328].

The benzoxazole moiety in makaluvamine W (**433**) is also found in citharoxazole (**435**), isolated from the sponge *Latrunculia (Biannulata) citharistae* in 2011 (Figure 60). The latter compound represented the first oxazole derivative in this family at that time [329].

In 2013, the Hamann laboratory isolated a complex heptacyclic pyrroloiminoquinone **436** containing seven stereogenic centers together with five different heterocycles (Figure 60).The TFA salt of atkamine (**436**) was isolated from the sponge *Latrunculia* sp. The structure elucidation of this complex framework was guided by spectroscopic methods, including ECD spectroscopy to analyze the absolute configuration. Furthermore, preparative olefin metathesis was used to localize the (*E*)-configured double bond [330].

Due to their promising bioactivity, a large number of synthetic studies have been conducted on these pyrrole alkaloids (e.g., makaluvamines [331,332], damirones [333,334], batzellines [335,336]). The first synthesis of makaluvone was completed by the Tokuyama group in 2012 [337]. Starting with 4-methoxy-2-nitroaniline and using a procedure reported by the Buchwald group [338], the 4-iodoindoline **437** was prepared in a 22% yield over nine steps. Subsequent construction of the quinoline scaffold using a benzyne intermediate generated by LiTMP and trapping of the carbanion by a bromine donor resulted in the formation of tricyclic system **438**. DDQ-oxidation to form the indole, removal of the Nprotecting groups, and oxidation of the aromatic core yielded the iminoquinone **439**. The last two steps included the methylation of the pyrrole nitrogen, methyl ether cleavage, and isomerization to makaluvone **442** (Scheme 27) [337].

**Scheme 27.** Two different routes which target pyrroloquinolines **442**, **443**, and **444**. The first route favors the formation of the quinoline followed by pyrrole aromatization, while the second one uses a biomimetic approach with a late-stage quinoline ring closure.

A shorter and more efficient synthetic sequence to several aminoquinolines was reported five years later by the Spiteller group (Scheme 27) [339]. Using vanillin as starting material, the indole **440** was prepared in 23% yield over seven steps. Vilsmeier formylation, Henry reaction, and LiAlH4 reduction of the nitroolefin then furnished the tryptamine **441**. Removal of the benzylic protecting groups under hydrogenolytic conditions, oxidation of the prepared hydroquinone followed by biomimetic intramolecular Michael addition

and aerobic reoxidation then gave the targeted pyrroloquinoline. The last step involved halogenation to obtain makaluvamine O (**443**) and batzelline D (**444**), respectively [339].

A new member of the tsitsikammamines, namely 16,17-dehydrotsitsikammamine A (**445**), was identified from the Antarctic sponge *Latrunculia biformis* in 2018 (Figure 61). The crude extract of bis-pyrroloiminoquinone **445** showed promising anticancer activity against seven cancer cell lines (inhibition percentage >90% each at 200 μg/mL) [340].

**Figure 61.** Pyrroloiminoquinones **445** and **446** as well as pyrroloquinones **447**–**450**.

The new tsitsikammamine C (**446**) was isolated as the TFA-salt from *Zyzzya* sp. in 2012 and represents the 18-methyl derivative of tsitsikammamine B (Figure 61). In biological assays, a potent growth inhibition of *Plasmodium falciparum* chloroquine-sensitive (3D7, IC50 value of 13 nM) and chloroquine-resistant (Dd2, IC50 value of 18 nM) cell lines was observed [341].

Thiazine-derived metabolites were discovered in the Australian marine sponge *Plakortis lita* in 2013 and given the names thiaplakortones A–D (**447**–**450**) (Figure 61) [342]. The structures were determined by using NMR and MS analytics as well as comparing chiroptical data to literature values to confirm the absolute configuration of the 2- methylaminopropanoic acid side chain of thiaplakortone C (**449**) and D (**450**). This substituent also suggests the biosynthesis from L-tryptophan and cysteine to yield the tricyclic framework. As the aforementioned tsitsikammamine C (**446**), all tested thiaplakortones **447**–**450** display significant antimalarial activity against chloroquine-sensitive (3D7, IC50 values ranging between 51 nM and 650 nM) and chloroquine-resistant (Dd2, IC50 values ranging between 6.6 nM and 171 nM) *Plasmodium falciparum* cell lines [342].

In 2014, the first synthesis of thiaplakortone A (**447**) was realized by the Quinn laboratory (Scheme 28) [343]. Starting from commercially available 4-hydroxyindole (**451**), indole **452** was obtained in 54% yield over five steps. Benzyl-deprotection, oxidation, and treatment with 2-aminoethanesulfinic acid, generated an intermediary dihydrothiazine, which, upon saponification and final deprotection, led to the formation of thiaplakortone A (**447**) (Scheme 28) [343].

**Scheme 28.** Facile total synthesis of thiaplakortone A (**447**) in a nine-step approach.

Another subclass of biologically active pyrrole alkaloids is the zyzzyanones, merging the *bis*-pyrrolo functionality together with a pyrroloquinone scaffold. The known zyzzyanones A–D (**457**–**460**), isolated in 1996, were synthesized for the first time by Velu and co-workers in 2013 (Scheme 29) [344]. The authors developed a modular approach that provides access to all four zyzzyanones A–D (**457**–**460**). Starting with the known tosylprotected indole-4,7-dione (**453**) [345], treatment with benzylamine resulted in amination. The bispyrroloquinone framework was constructed by ring-closing procedure with diethyl acetal **454** and Mn(OAc)3. After methylation with MeI, the expected monomethylated amine **455** was obtained alongside the unexpected demethylated amine **456**. Both intermediates **455** and **456** were converted in a series of deprotection and/or formylation reactions to generate the zyzzyanones A–D (**457**–**460**) [344].

**Scheme 29.** A divergent modular approach providing access to known zyzzyanones A–D (**457**–**460**).

The discorhabdin journey started with the isolation of the first member of the class, discorhabdin C (its congeners A and B were reported later), in 1986 [346]. In the following years, a dozen more family members were isolated, biologically evaluated, and synthesized. In the decade 2010–2020, 12 further members were identified (Figures 62 and 63). The representatives of this diverse subclass featuring promising bioactivities contain a tetracyclic pyrroloiminoquinone core with a spirocyclic cyclohexadienone moiety. The discorhabdins are thought to be biosynthetically derived from makaluvamines, formed by the coupling of tyramine derivatives with the biosynthetic key precursor of simple pyrroloiminoquinones. In addition, these intermediates also give access to many further subclasses already mentioned [323].

**Figure 62.** Discorhabdins **461**–**465** resulted from the sponge *Latrunculia* sp. collected in Alaskan and New Zealandian oceans.

**Figure 63.** Further discorhabdins **466**–**471**, including a new complex pyrroloiminoquinone **472**.

An interesting and at the same time cautionary discovery was made in 2010 when discorhabdin A was isolated for the first time from *Latrunculia oparinae*. In addition to the strong dependence of the color of the solution on the solvent when ethanol (red) and methanol (green) were used, the optical rotation also changed its sign in this solvent switch [347].

Similarly, the Hamann laboratory published the isolation of two new compounds, dihydrodiscorhabdin B (**461**) and discorhabdin Y (**462**) from the Alaskan sponge *Latrunculia* sp. (Figure 62) [348]. Upon structure elucidation using CD and optical rotation, pyrrole **461** showed decomposition, therefore only the absolute stereoinformation of discorhabdin Y (**462**) could be assigned. The azepine derivative **463** was also identified in the same sponge for the first time as a natural product (Figure 62) [348]. Previously, it was only known as a semisynthetic compound, prepared by reduction of natural discorhabdin C and treatment of the resulting dienol with sulfuric acid, initiating an alkenyl (C-20) migration to form discorhabdin benzene derivative (**463**) [349].

Two new diastereomers of discorhabdin H and K, namely discorhabdin H2 (**464**) and K2 (**465**) were isolated from different sponge populations of *Latrunculia* sp. in 2010

(Figure 62) [350]. Combined structure elucidation was performed by NMR, MS, and extensive ECD-spectroscopy, allowing the assignment of the absolute configuration of the known discorhabdins 2-hydroxy-D, D, H, N, and Q by comparing the recorded with experimental ECD spectra. Furthermore, natural (+)-(6*S*,8*S*)-discorhabdin B was used as a starting point for semi-synthesis to establish the absolute configurations of discorhabdins S, T, and U [350].

The synthetically known didebromodiscorhabdin C (**466**) [351], along with two new discorhabdin derivatives **467** and **468** were isolated for the first time from the sponge *Sceptrella* sp. (Figure 63) [352]. Following previous studies, the absolute configuration was solved by a combination of optical rotation and ECD spectroscopy. In bioactivity studies, average to striking effects were observed against Gram-positive and Gram-negative bacteria (MIC values ranging between 25 μg/mL and >100 μg/mL), as well as against the K562 leukemia cell line and sortase A (IC50 values ranging between 2.1 μM and 127.4 μM), with the hemiaminal **468** remarkably showing a more than tenfold higher inhibition than *p*-(hydroxymercury)benzoic acid sodium salt as a positive control [352].

Promising anticancer activity against six cell lines was observed by bioactivity-guided isolation (IC50 values of crude extract ranging between 4.0 and 56.2 μg/mL) of three new discorhabdins **469**–**471** from *Latrunculia biformis* (Figure 63) [353]. Discorhabdins **470** and **471** are the first derivatives bearing an ester moiety, containing a simple acetyl group or a C28-fatty acid. In the publication, the binding affinity of discorhabdins to anticancer targets (topoisomerase I–II, indoleamine 2,3-dioxygenase) was also determined [353].

Aleutianamine (**472**), the first member of a new class of pyrroloiminoquinone alkaloids, is characterized by a highly fused and multiply bridged heptacyclic ring system and was isolated from the North Pacific sponge *Latrunculia austini* Samaai (Figure 63) [354]. The elucidation of the structure required the combination of preparative spectroscopic methods and advanced computational approaches. It has been supposed that this complex molecular framework is derived from two proteinogenic amino acids, tryptophan, and tyrosine. The authors mentioned that makaluvamine F or discorhabdin A might be the precursors of aleutianamine (**472**), which exhibits promising activity against pancreatic cancer cell lines (IC50 values between 25 nM and 1 μM) [354].
