Psammaplysins: Insights from Natural Sources, Structural Variations, and Pharmacological Properties

Marine natural products (MNPs) continue to be in the spotlight in the global drug discovery endeavor. Currently, more than 32,000 structurally diverse secondary metabolites from marine sources have been isolated, making MNPs a vital source for researchers to look for novel drug candidates. The marine-derived psammaplysins possess the rare and unique 1,6-dioxa-2-azaspiro [4.6] undecane backbone and are represented by 44 compounds in the literature, mostly from sponges of the order Verongiida. Compounds with 1,6-dioxa-2-azaspiro [4.6] undecane moiety exist in the literature under five names, including psammaplysins, ceratinamides, frondoplysins, ceratinadins, and psammaceratins. These compounds displayed significant biological properties including growth inhibitory, antimalarial, antifouling, protein tyrosine phosphatase inhibition, antiviral, immunosuppressive, and antioxidant effects. In this review, a comprehensive literature survey covering natural occurrence of the psammaplysins and related compounds, methods of isolation, structural differences, the biogenesis, and biological/pharmacological properties, will be presented.


Introduction
The oceans are considered the largest habitat on the Earth. It is reported that the total number of the identified marine species is 240,000 [1], and it is estimated that there are 1.4-1.6 million marine species on Earth [2]. Another, more recent estimate for marine species is about a half lower (0.7-1.0 million) [3]. The reason for the enormous range in the estimated number of marine species lies in the deficiency of the data about the diversity of marine microbes and other microscopic organisms. For example, sufficient data are available on marine mammals and fishes, while enough and satisfied data about the huge microbial diversity and the phytoplankton in the oceans still need to be revealed. Alone in Europe, it is estimated that about 41,000-56,000 species exist with 5000-20,000 species yet to be identified. Annually, there are about 1000-1500 new marine species documented.
Marine invertebrates are the most diverse group of marine life that exist in the oceans with highest biological and chemical diversity. A total of 38,925 marine-derived natural products, published in 38,645 articles, were identified from marine organisms [4]. Such chemical biodiversity is attributed to the fact that these compounds are produced by mostly sessile organisms present in the marine environment. These sessile organisms are extremely susceptible to be attacked by predators. Marine invertebrates, such as sponges, tunicates, bryozoans, gorgonians, and soft corals evolve chemicals as a defense mechanism against highly mobile predators. Marine sponges are one of the most productive phyla of marine invertebrates and considered as an outstanding source of biologically active secondary metabolites [5].

The Chemistry of Psammaplysins
The psammaplysin backbone is composed of two dibrominated moieties, 8,10-dibromo-4-hydroxy-9-methoxy-1,6-dioxa-2-azaspiro[4.6]undeca-2,7,9-triene-3-carboxylic acid (subunit A), and 3-(4-(2-aminoethyl)-2,6-dibromophenoxy)propan-1-amine subunit (subunit B, moloka'iamine), linked together through an amidic linkage between the carboxylic moiety (C-9) of the substituted spirooxepinisoxazoline unit and the terminal amino group at C-10 of the moloka'iamine ( Figure 3). Interestingly, moloka'iamine (subunit B) and its substituted derivatives were reported from several Verongiid sponges, but there is no single report in the literature about the existence or isolation of the separated dibrominated spirooxepinisoxazoline moiety (subunit A). The substituted and dibrominated spirooxepinisoxazoline unit has been always associated with the moloka'iamine moiety via an amidic moiety. This can be explained by the necessity of such combination as a defense tool for sponges' survival against predators [41]. unit B) and its substituted derivatives were reported from several Verongiid sponges, but there is no single report in the literature about the existence or isolation of the separated dibrominated spirooxepinisoxazoline moiety (subunit A). The substituted and dibrominated spirooxepinisoxazoline unit has been always associated with the moloka'iamine moiety via an amidic moiety. This can be explained by the necessity of such combination as a defense tool for sponges' survival against predators [41].

The Absolute Configuration of Psammaplysins
Later in 2015, the absolute configuration of the stereogenic carbons (C-6 and C-7) of psammaplysin A was verified as 6R and 7R (Figure 4), respectively, through using experimental and calculated electronic circular dichroism (ECD) data and NMR analysis of MPA esters prepared from the acetamide derivative of psammaplysin A. Detailed conformational analyses of a truncated model compound of psammaplysin A with an in vacuo method and with the Polarizable Continuum Model (PCM) solvent model for MeOH have identified the major conformers and factors governing the ECD spectrum of psammaplysin A. The correlation of the ECD data of psammaplysin A will allow future configurational assignments of related psammaplysin analogs on the basis of comparison their ECD spectra [42]. Since all reported natural psammaplysins in this review possess a negative sign of optical rotation, we therefore assume that all reported psammaplasyins possess the same biosynthetic pathway and therefore have absolute configuration at C-6 and C-7 as 6R and 7R. Accordingly, all structures in this review have been drawn with the 6R,7R configuration regardless of their original drawings in the original manuscripts.

The Absolute Configuration of Psammaplysins
Later in 2015, the absolute configuration of the stereogenic carbons (C-6 and C-7) of psammaplysin A was verified as 6R and 7R (Figure 4), respectively, through using experimental and calculated electronic circular dichroism (ECD) data and NMR analysis of MPA esters prepared from the acetamide derivative of psammaplysin A. Detailed conformational analyses of a truncated model compound of psammaplysin A with an in vacuo method and with the Polarizable Continuum Model (PCM) solvent model for MeOH have identified the major conformers and factors governing the ECD spectrum of psammaplysin A. The correlation of the ECD data of psammaplysin A will allow future configurational assignments of related psammaplysin analogs on the basis of comparison their ECD spectra [42]. ety via an amidic moiety. This can be explained by the necessity of such combina defense tool for sponges' survival against predators [41].

The Absolute Configuration of Psammaplysins
Later in 2015, the absolute configuration of the stereogenic carbons (C-6 and psammaplysin A was verified as 6R and 7R (Figure 4), respectively, through usin imental and calculated electronic circular dichroism (ECD) data and NMR ana MPA esters prepared from the acetamide derivative of psammaplysin A. Detailed mational analyses of a truncated model compound of psammaplysin A with an method and with the Polarizable Continuum Model (PCM) solvent model for MeO identified the major conformers and factors governing the ECD spectrum of ps lysin A. The correlation of the ECD data of psammaplysin A will allow future co tional assignments of related psammaplysin analogs on the basis of comparison th spectra [42]. Since all reported natural psammaplysins in this review possess a negative optical rotation, we therefore assume that all reported psammaplasyins possess t biosynthetic pathway and therefore have absolute configuration at C-6 and C-7 as 7R. Accordingly, all structures in this review have been drawn with the 6R,7R co tion regardless of their original drawings in the original manuscripts. Since all reported natural psammaplysins in this review possess a negative sign of optical rotation, we therefore assume that all reported psammaplasyins possess the same biosynthetic pathway and therefore have absolute configuration at C-6 and C-7 as 6R and 7R. Accordingly, all structures in this review have been drawn with the 6R,7R configuration regardless of their original drawings in the original manuscripts.
At the beginning, phase I includes prior extraction of the sponge materials with nhexane for the removal of undesired lipophilic materials followed by extraction with desired solvent(s). Otherwise, the materials are directly extracted with one solvent (MeOH), or a mixture of solvents (MeOH-CH2Cl2, MeOH-CHCl3) or sequential extraction with different solvents (MeOH followed by CH2Cl2, etc.) ( Figure 5). The partition phase (II) starts with either solvent-solvent partitioning of the crude extract using different immiscible solvents or mixture of solvents or applying direct flash VLC chromatography on either resin, normal-or reversed-phase silica using a variety of organic and aqueous solvents ( Figure 5). In some cases, phase III starts with targeting specific bioactivity using suitable screen or targeting a specific class of the compounds using LC-MS clusters to target the multi-brominated compounds. Otherwise, direct sub-fractionation of the extracts' fractions on resin, normal-or reversed-phase silica was performed to prepare the targeted  At the beginning, phase I includes prior extraction of the sponge materials with nhexane for the removal of undesired lipophilic materials followed by extraction with desired solvent(s). Otherwise, the materials are directly extracted with one solvent (MeOH), or a mixture of solvents (MeOH-CH 2 Cl 2 , MeOH-CHCl 3 ) or sequential extraction with different solvents (MeOH followed by CH 2 Cl 2 , etc.) ( Figure 5). The partition phase (II) starts with either solvent-solvent partitioning of the crude extract using different immiscible solvents or mixture of solvents or applying direct flash VLC chromatography on either resin, normalor reversed-phase silica using a variety of organic and aqueous solvents ( Figure 5). In some cases, phase III starts with targeting specific bioactivity using suitable screen or targeting a specific class of the compounds using LC-MS clusters to target the multi-brominated compounds. Otherwise, direct sub-fractionation of the extracts' fractions on resin, normalor reversed-phase silica was performed to prepare the targeted subfractions for the final purification ( Figure 5). The final purification of the targeted compounds was usually achieved by MPLC or HPLC on normal-, reversed-phase silica, or CN columns using a variety of eluting solvents ( Figure 5).

Natural Occurrence
As mentioned above, in 1983 Kashman's group reported the isolation of psammaplysins A (1) and B (2) (Table 1) from the Red Sea sponge Psammaplysilla purpurea [39]. Almost a decade later, psammaplysin C (3) (Table 1), an N-methylated derivative of psammaplysin B, was identified in 1992 from the sponge Druinella (=Psammaplsilla) purpurea, family Druinellae (Aplysinellidae), order Verongida which was collected from the shallow reef waters off Makaluva Island of the Fiji Island Group in the South Pacific [43]. In 1993, the new analogs psammaplysins D (4) and E (5) (Table 1), along with psammaplysin A (1), were purified from a new species of the sponge Aplysinella (Family Aplysinellidae) collected from a vertical coral wall at Pingelap Atoll, Micronesia [44]. subfractions for the final purification ( Figure 5). The final purification of the targeted compounds was usually achieved by MPLC or HPLC on normal-, reversed-phase silica, or CN columns using a variety of eluting solvents ( Figure 5).

Natural Occurrence
As mentioned above, in 1983 Kashman's group reported the isolation of psammaplysins A (1) and B (2) (Table 1) from the Red Sea sponge Psammaplysilla purpurea [39]. Almost a decade later, psammaplysin C (3) (Table 1), an N-methylated derivative of psammaplysin B, was identified in 1992 from the sponge Druinella (=Psammaplsilla) purpurea, family Druinellae (Aplysinellidae), order Verongida which was collected from the shallow reef waters off Makaluva Island of the Fiji Island Group in the South Pacific [43]. In 1993, the new analogs psammaplysins D (4) and E (5) (Table 1), along with psammaplysin A (1), were purified from a new species of the sponge Aplysinella (Family Aplysinellidae) collected from a vertical coral wall at Pingelap Atoll, Micronesia [44].

Natural Occurrence
As mentioned above, in 1983 Kashman's group reported the isolation of psammaplysins A (1) and B (2) (Table 1) from the Red Sea sponge Psammaplysilla purpurea [39]. Almost a decade later, psammaplysin C (3) (Table 1), an N-methylated derivative of psammaplysin B, was identified in 1992 from the sponge Druinella (=Psammaplsilla) purpurea, family Druinellae (Aplysinellidae), order Verongida which was collected from the shallow reef waters off Makaluva Island of the Fiji Island Group in the South Pacific [43]. In 1993, the new analogs psammaplysins D (4) and E (5) (Table 1), along with psammaplysin A (1), were purified from a new species of the sponge Aplysinella (Family Aplysinellidae) collected from a vertical coral wall at Pingelap Atoll, Micronesia [44]. Table 1. Chemical structures and natural sources of psammaplyins.

Natural Occurrence
As mentioned above, in 1983 Kashman's group reported the isolation of psammaplysins A (1) and B (2) (Table 1) from the Red Sea sponge Psammaplysilla purpurea [39]. Almost a decade later, psammaplysin C (3) (Table 1), an N-methylated derivative of psammaplysin B, was identified in 1992 from the sponge Druinella (=Psammaplsilla) purpurea, family Druinellae (Aplysinellidae), order Verongida which was collected from the shallow reef waters off Makaluva Island of the Fiji Island Group in the South Pacific [43]. In 1993, the new analogs psammaplysins D (4) and E (5) (Table 1), along with psammaplysin A (1), were purified from a new species of the sponge Aplysinella (Family Aplysinellidae) collected from a vertical coral wall at Pingelap Atoll, Micronesia [44]. Table 1. Chemical structures and natural sources of psammaplyins.

Natural Occurrence
As mentioned above, in 1983 Kashman's group reported the isolation of psammaplysins A (1) and B (2) (Table 1) from the Red Sea sponge Psammaplysilla purpurea [39]. Almost a decade later, psammaplysin C (3) (Table 1), an N-methylated derivative of psammaplysin B, was identified in 1992 from the sponge Druinella (=Psammaplsilla) purpurea, family Druinellae (Aplysinellidae), order Verongida which was collected from the shallow reef waters off Makaluva Island of the Fiji Island Group in the South Pacific [43]. In 1993, the new analogs psammaplysins D (4) and E (5) ( Table 1), along with psammaplysin A (1), were purified from a new species of the sponge Aplysinella (Family Aplysinellidae) collected from a vertical coral wall at Pingelap Atoll, Micronesia [44]. Table 1. Chemical structures and natural sources of psammaplyins.
Finally, in 2021, the name "psammaceratin" was given to the first psammaplysin dimer in the series. Psammaceratin A (44) ( Table 1) was reported from the Red Sea sponge Pseudoceratina arabica collected in the Red Sea [54].

Structural Variations
Comparing to psammaplysin A, psammaplysin C (3) represents the first N-methylated derivative of the psammaplysin series. In addition to an OH moiety at C-19, psammaplysin D (4) possesses an isopentadecanoyl residue at the terminal amine of the compound through an amidic linkage. Furthermore, psammaplysin E (5) possesses an unprecedented cyclopentene-dione backbone linked to the terminal amine of the compound [44].
Interestingly, ceratinamide A (7) is the first compound with an N-formyl functionality at the terminal amine, while ceratinamide B (8) contains a 13-methyltetradecanoic acid moiety through an amidic linkage with the terminal amino group of the compound [45].
Psammaplysin F (10) represents the second N-methylated psammaplysin analog in this series [46]. In addition to a terminal N-methyl moiety, psammaplysin G (11) possesses an urea moiety at the terminal amino group. Further, psammaplysin G is the first psammaplysin analog with a tertiary amine in this series.
Psammaplysin H (12) possesses an N-trimethyl substitution, being the first quaternary psammaplysin analog in this series.
Surprisingly, psammaplysins I (13) and J (14) are lacking the bromine atom at C-18 of the moloka'iamine part of the molecule, being the first psammaplysin analogs with only three bromine atoms.
Psammaplysins K (15), K dimethoxy acetal (16), L (17), M (18), and 19hydroxypsammaplysin E (6) display discrepancy in the structural unit attached to C-16 of the aromatic moiety or the terminal amine of the compound. For example, psammaplysin K possesses an aldehydic moiety at C-16 instead of the ethylamine part, while a dimethoxy acetal moiety exists at C-16 in psammaplysin K dimethoxy acetal. Further, psammaplysin L contains a 2-oxazolidinone moiety at C-16, while a glycolamide moiety appears at the terminal amine in psammalysin M. Finally, 19-hydroxypsammaplysin E contains the previously reported cyclopentene-dione moiety in psammaplysin E in addition to an OH at C-19 [50].
On the contrary from psammaplysin A, ceratinadins E (40) and F (41) possess two and three moloka'iamine units, respectively. Thus, ceratinadins E and F possess a total of six and eight bromine atoms, respectively. Another feature in both compounds is the connectivity of all moloka'iamine moieties through N-methylated urea and the Nmethylation of all terminal amines of the moloka'iamines' moieties [52]. In addition to the C-19 OH moiety, frondoplysins A (42) and B (43) possess an unprecedented bioconjugates of a meroterpene moiety attached to the terminal amine of the psammaplysin backbone making these compounds the first example in this group with a terpene backbone via "N-C" linkage. Finally, psammaceratin A (44) is composed of two units of psammaplysin A connected together via an unprecedented (2Z,3Z)-2,3-bis(aminomethylene)succinamide moiety, thus representing the first dimer among this class [54].
On the contrary from psammaplysin A, ceratinadins E (40) and F (41) possess two and three moloka'iamine units, respectively. Thus, ceratinadins E and F possess a total of six and eight bromine atoms, respectively. Another feature in both compounds is the connectivity of all moloka'iamine moieties through N-methylated urea and the N-methylation of all terminal amines of the moloka'iamines' moieties [52]. In addition to the C-19 OH moiety, frondoplysins A (42) and B (43) possess an unprecedented bioconjugates of a meroterpene moiety attached to the terminal amine of the psammaplysin backbone making these compounds the first example in this group with a terpene backbone via "N-C" linkage. Finally, psammaceratin A (44) is composed of two units of psammaplysin A connected together via an unprecedented (2Z,3Z)-2,3-bis(aminomethylene)succinamide moiety, thus representing the first dimer among this class [54].

Compounds Antimicrobial Activity Reference
Psammaplysin A (1) In vitro activity against gram positive bacteria and E. coli Antibacterial activity against
From the above results, it could be concluded that the growth inhibition of the psammaplysins towards cancerous cell lines suggests that the spirooxepinisoxazoline ring system is an essential element for the activity. The N-terminal substitution with a cyclopentene-dione moiety, as in psammaplsyins E (5) and 19-hydroxypsammaplysin E (6), or 4-chloro-2-methylenecyclopentane-1,3-dione moiety, as in psammaplysin X (35) and 19hydroxypsammaplysin X (36), increases the activity. Further, introduction of 19-OH group, as in psammaplysin B (2) versus psammaplysin A (1), diminishes the activity (Table 3).
On the contrary, psammaplysin D (4), however, lacked activity (GI 50 > 10 µM), which might be explained by its high lipophilicity. Furthermore, the existence of a terminal N-methyl group, as in psammaplysin F (10), or an urea moiety, as in psammaplysin Z (38), diminished the growth inhibition effect (Table 3).
Finally, ceratinadins E (40) and F (41) were reported to show antiplasmodial activities against the drug-resistant (K1) and drug-sensitive (FCR3) strains of P. falciparum. Moreover, ceratinadin E (40) was found to display higher selectivity indices (SI) than ceratinadin F (41) [52] (Table 4). The antimalarial evaluation of 13 psammaplysins analogs clearly shows that psammaplysin F (10) is the most potent active compound against Dd2 strain, while ceratinadin E (40) possesses a greater antimalarial activity towards K1 strain and a better selectivity index than psammaplysins F (10). The addition of a terminal N-methyl, as in psammaplysin F (10), enhances the activity. However, ceratinadin F (41), which possesses several N-methyls, did not show significant antimalarial activity, which could be attributed to the high lipophilicity of the compound. Though the antimalarial activity against drugresistant strains of P. falciparum is unknown, psammaplysin H (12), a quaternary analog with a trimethylamino group instead of a methylamino group, possesses a potent antimalarial activity and better selectivity against a drug-sensitive strain of P. falciparum than psammaplysins F (10) without any significant cytotoxicity against the HEK293 384 cell line [47,48,50] (Table 4).
Comparing the activities of psammaplysins F (10), G (11), and H (12) towards two mammalian cell lines (HEK293 and HepG2), psammaplysin H was found to display a minimal toxicity at the highest concentration tested (40 µM), giving this compound a parasite-specific selectivity index (SI) of >97. In contrast, psammaplysins G and F display higher toxicity to these cell lines with IC 50 values between 3.71 and 18.96 µM, respectively. These preliminary structure-activity data suggest that full methyl-substitution of the terminal amine (N-quaternization) is essential for optimal antimalarial activity and better selectivity [48].

Compounds with Antifouling Activities
When evaluated for their antifouling activity, psammaplsyins A (1), E (5), ceratinamides A (7) and B (8) have reported to inhibit the metamorphosis and settlement of the barnacle B. Amphitrite [45] (Table 5). The highest activities of psammaplysin A (1) and ceratinamide A (7) suggests the importance of a terminal amine or an N-formyl moiety for a maximum antifouling activity. Furthermore, ceratinamide A (7) was found to induce a larval metamorphosis of the ascidian Halocynthia roretzi [45] (Table 5).

Compounds with Other Reported Activities
Psammaplysin D (4) was reported to display anti-HIV towards the Haitian RF strain of HIV-I [44] (Table 5). Recently, psammaplysin F (10) was reported to increase the efficacy of the antitumor drugs bortezomib and sorafenib through regulation of the synthesis of stress granules [59] (Table 6). Psammaplysin F (10) and its urea semisynthetic analogs 45, 51, 53 and 54 Reduce the mitochondrial membrane potential (MMP) [55] Frondoplysins A (42) and B (43) were described to inhibit protein-tyrosine phosphatase IB (PTP1B). The compounds were found to have a higher activity than the positive control oleanolic acid [53] and thiazolidinediones [60] and were similar to benzofuran and benzothiophene biphenyls [61] (Table 6). Further, frondoplysin A was found to possess in vivo antioxidant activity in transgenic fluorescent zebrafish over five times stronger than that of vitamin C [53] without any cytotoxicity [53] (Table 6).
It has been described that psamaplysin F (10) andits urea semisynthetic analogs (45, 51, 53 and 54) strongly reduce the mitochondrial membrane potential (MMP). Further, it was found that psammaplysin F strongly affects the mitochondrial morphology and reduced the number of end points and branch points within the tubular structure of individual mitochondria, leading to visible fragmentation of the mitochondrial tubular network. These findings provide a strong rationale for more detailed mechanistic studies of psammaplysin F and derivatives as novel mitochondrial poisons [55].

Summary
Since the first report of psammaplysins A and B in 1983, additional 42 compounds have been reported until now from 12 marine sponge species, including 10 Verongiid and two non-Verongiid sponges. The field was most active in the years 2012 (21 compounds), Reduce the mitochondrial membrane potential (MMP) [55]

Summary
Since the first report of psammaplysins A and B in 1983, additional 42 compounds have been reported until now from 12 marine sponge species, including 10 Verongiid and two non-Verongiid sponges. The field was most active in the years 2012 (21 compounds   The majority of the psammaplysins (41 compounds, 93%) come mainly from four Verongiid genera, including Aplysinella (26 compounds), Pseudoceratina (six compounds), Suberea (six compounds), and Psammaplysilla (three compounds) and non-Verongiid (three compounds) genera, including Dysidea (two compounds) and Hyattella (one compound) (Figures 8 and 9). The majority of the psammaplysins (41 compounds, 93%) come mainly from four Verongiid genera, including Aplysinella (26 compounds), Pseudoceratina (six compounds), Suberea (six compounds), and Psammaplysilla (three compounds) and non-Verongiid (three compounds) genera, including Dysidea (two compounds) and Hyattella (one compound) (Figures 8 and 9).    As shown above, reported compounds with 1,6-dioxa-2-azaspiro[4.6]undecane moiety vary mainly in the presence of substituents at C-19 and/or the terminal amine, which greatly affected the biological properties of the compounds. From those, only 29 compounds have been found to possess variable bioactivities, such as cancer cell growth inhi- As shown above, reported compounds with 1,6-dioxa-2-azaspiro[4.6]undecane moiety vary mainly in the presence of substituents at C-19 and/or the terminal amine, which greatly affected the biological properties of the compounds. From those, only 29 compounds have been found to possess variable bioactivities, such as cancer cell growth inhibition (12 compounds), antimalarial (6 compounds), antifouling (four compounds), antimicrobial (three compounds), and other activities (four compounds). The remaining 15 compounds are either inactive in one or more screens or are not evaluated at all ( Figure 10).  In conclusion and from the data discussed before, some candidates with 1,6-dioxa-2azaspiro[4.6]undecane skeleton exhibited significant antimalarial activity and growth inhibitory effects towards several human cancerous cell line making them attractive scaffolds for the development of potent antimalarial and antitumor leads. In conclusion and from the data discussed before, some candidates with 1,6-dioxa-2-azaspiro[4.6]undecane skeleton exhibited significant antimalarial activity and growth inhibitory effects towards several human cancerous cell line making them attractive scaffolds for the development of potent antimalarial and antitumor leads.