Marine Isonitriles and Their Related Compounds

Marine isonitriles represent the largest group of natural products carrying the remarkable isocyanide moiety. Together with marine isothiocyanates and formamides, which originate from the same biosynthetic pathways, they offer diverse biological activities and in spite of their exotic nature they may constitute potential lead structures for pharmaceutical development. Among other biological activities, several marine isonitriles show antimalarial, antitubercular, antifouling and antiplasmodial effects. In contrast to terrestrial isonitriles, which are mostly derived from α-amino acids, the vast majority of marine representatives are of terpenoid origin. An overview of all known marine isonitriles and their congeners will be given and their biological and chemical aspects will be discussed.

The first review about isonitrile natural products was published by Edenborough and Herbert in 1988 [7] while the first review on marine isonitriles and related compounds by Chang and Scheuer appeared five years later [8]. Chang also published a review on marine and terrestrial isonitriles along with their congeners in 2000 [9]. In the same year Garson et al. published a review about the structures, biosynthesis and ecology of marine isonitriles [10]. Reviews about the biosynthesis of marine isonitriles were published by Garson in 1989 [11], by Chang and Scheuer in 1990 [12], and by Garson in 1993 [13]. An update of Garson's 1988 review appeared in 2004 [14]. A review about isolation, biological activity and chemical synthesis of isocyanoterpenes was published in 2015 by Schnermann and Shenvi [15].
Since the number of known marine-derived isonitrile natural products has increased significantly since 2000, we intend to give the reader a complete overview of all members of this compound class known to date. In addition, aspects about biosynthesis and bioactivity will be discussed.

Marine Isonitriles and Related Compounds
Most marine isonitriles and their formamide and isothiocyanate congeners are either sesquiterpenoids (Section 2.1) or diterpenoids (Section 2.2). The sesquiterpenoids have only one nitrogenous functional group whereas the diterpenes bear up to three functional groups. Carbonimidic dichlorides (Section 2.3), which originate from the corresponding isonitriles or isothiocyanates, are also known. Finally, there are some miscellaneous structures (Section 2.4) which can not be classified in the previous sections.
Since the number of known marine-derived isonitrile natural products has increased significantly since 2000, we intend to give the reader a complete overview of all members of this compound class known to date. In addition, aspects about biosynthesis and bioactivity will be discussed.

Marine Isonitriles and Related Compounds
Most marine isonitriles and their formamide and isothiocyanate congeners are either sesquiterpenoids (Section 2.1) or diterpenoids (Section 2.2). The sesquiterpenoids have only one nitrogenous functional group whereas the diterpenes bear up to three functional groups. Carbonimidic dichlorides (Section 2.3), which originate from the corresponding isonitriles or isothiocyanates, are also known. Finally, there are some miscellaneous structures (Section 2.4) which can not be classified in the previous sections.

Eudesmanes
The first eudesmane-type isonitrile, acanthellin-1 (Eu2), was isolated from the sponge Acanthella acuta by Minale et al., collected in the Bay of Naples, Italy, in 1974 (  In 1993, Burgoyne et al. reported the isolation of acanthene B (Eu12) from an Acanthella sponge and acanthene C (Eu13) from the nudibranch Cadlina luteomarginata but they were unable to isolate the parent isonitrile Eu11 [23]. However, this compound, halichonadin C (Eu11), was obtained from the sponge Halichondria sp. from Unten Port, Okinawa, Japan [24]. In this sample, Ishiyama et al. also found halichonadin B (Eu14) with a carbamate group and halichonadin A (Eu15), a dimer with two eudesmane skeletons bridged by a urea linkage [24].
In 2008, Zubia et al. investigated specimens of the marine sponge Axinyssa sp. and were able to isolate as many as twelve new cadinane-type sesquiterpenoids, the axinisothiocyanates A-L [51]. For the non-oxygenated axinisothiocyanate K (Ca18), vide supra. Axinisothiocyanate J (Ca23) carries a hydroxy group at C-4. In contrast, axinisothiocyanates A (Ca24), B (Ca25) and C (Ca26) have two hydroxy groups at C-4 and C-5 and differ in their relative configuration. The same applies to axinisothiocyanate D (Ca27), axinisothiocyanate E (Ca28) and axinisothiocyanate F (Ca29) with the hydroxy groups at C-4 and C-7.
Another dihydroxylated isonitriloid, this time oxygenated at C-4 and at C-1, is axinisothiocyanate G (Ca30). Furthermore, two isothiocyanates with one hydroxy group at C-4 and one hydroperoxy group at C-1, axinisothiocyanates H (Ca31) and I (Ca32) were isolated from the same source. The last member of the series, axinisothiocyanate L (Ca33), is characterized by a keto group at C-3.
In contrast to the above-mentioned spiroaxanes Sp2-Sp12, the isonitrile Sp13 and the isothiocyanate Sp14 both carry the nitrogenous functional group at C-1. Both spiroaxanes were isolated from the marine sponge Acanthella acuta [62].

Aromadendranes
Another type of sesquiterpene isonitriles is based on the aromadendrane-skeleton (Ar1) (Figure 8). The first example of this class was isolated directly in the early days of the discovery of marine isonitriles shortly after axisonitrile-1 (Ax2). It was found in the Mediterranean sponge Axinella In contrast to the above-mentioned spiroaxanes Sp2-Sp12, the isonitrile Sp13 and the isothiocyanate Sp14 both carry the nitrogenous functional group at C-1. Both spiroaxanes were isolated from the marine sponge Acanthella acuta [62].
An isothiocyanate with an opposite stereochemistry with the exception of C-10, (1R,4S,5S,6R,7S,10R)-(+)-isothiocyanatoalloaromadendrane (Ar9), was found in 1996 by Hirota et al. in an Acanthella cavernosa from Hachijo-jima Island (Japan) [20]. The absolute configuration was elucidated by comparison with the corresponding alcohols synthesized from (−)-alloaromadendrene. The compound was isolated again in 2010 by Lyakhova et al. from the Vietnamese nudibranch Phylidiella pustulosa [27]. The enantiomer Ar10 of this compound was prepared synthetically in 1994 by da Silva et al. and allowed the determination of the absolute configuration [65]. Ar9 and axisothiocyanate-2 (Ar4) were found to display potent antifouling activity against cyprid larvae of the acorn barnacle Balanus amphitrite [20].  In 1988, Breakman reported the isolation of an isonitrile, an isothiocyanate, and an isocyanate from the sponge Acanthella acuta (Banyuls, France) bearing the nitrogen functionality at C-1 instead of C-10 of the aromadendrane skeleton. Due to a misassigned reference substance, a wrong relative configuration was initially published [9,57], which was corrected two years later [67]. The first two compounds were found in parallel in an Acanthella acuta sponge collected in the Bay of Naples (Italy) by Mayol who elucidated the correct relative stereochemistry of Ar13 and Ar14 [62].
The formamide Ar16 was discovered in 2007 by Zhang et al. in the Spanish dancer nudibranch Hexabrandies sanguinens collected in the South China Sea [68]. The isolation of the corresponding isocyanate Ar15 was reported in 2007 by Jumaryatno from an Australian specimen of Acanthella cavernosa (Coral gardens, Gneerings reef, Mooloolaba) [32]. In 1992, He and Faulkner were able to identify the C1/C10-epimer Ar12 of isothiocyanate Ar14 in a sponge Axinyssa aplysinoides collected at Ant Atoll (Pohnpei/Micronesia) [69].

Epimaalianes
The epimaaliane sesquiterpenoids have a similar skeleton to the eudesmanes with a methyl group in C-4 and C-10 position but they possess an additional bond between C-6 and C-11, creating a cyclopropane ring (Ep1, Figure 9).
In 1988, Breakman reported the isolation of an isonitrile, an isothiocyanate, and an isocyanate from the sponge Acanthella acuta (Banyuls, France) bearing the nitrogen functionality at C-1 instead of C-10 of the aromadendrane skeleton. Due to a misassigned reference substance, a wrong relative configuration was initially published [9,57], which was corrected two years later [67]. The first two compounds were found in parallel in an Acanthella acuta sponge collected in the Bay of Naples (Italy) by Mayol who elucidated the correct relative stereochemistry of Ar13 and Ar14 [62].
The formamide Ar16 was discovered in 2007 by Zhang et al. in the Spanish dancer nudibranch Hexabrandies sanguinens collected in the South China Sea [68]. The isolation of the corresponding isocyanate Ar15 was reported in 2007 by Jumaryatno from an Australian specimen of Acanthella cavernosa (Coral gardens, Gneerings reef, Mooloolaba) [32]. In 1992, He and Faulkner were able to identify the C1/C10-epimer Ar12 of isothiocyanate Ar14 in a sponge Axinyssa aplysinoides collected at Ant Atoll (Pohnpei/Micronesia) [69].

Pupukeananes
The isonitrile Ep7, the isothiocyanate Ep8 and the formamide Ep9 with the functional group attached to C-1 instead of C-4 were found during further investigations of extracts of the sponge Axinella cannabina by Ciminiello et al. [72].
In addition to the C-9 functionalized pupukeananes, some C-2 and C-5 functionalized representatives of this class ar known as well. 2-Isocyanopupukeanane (Pu7) was obtained by Hagadone et al. from the nudibranch Phyllidia varicosa together with isonitrile Pu2 [77]. He et al. isolated the corresponding 2-thiocyanatopupukeanane (Pu8) from the sponge Axinyssa aplysinoides [69]. Recently the corresponding formamide Pu9 was isolated from Phyllidia coelestis Bergh, collected near the Koh-Ha Islets, Thailand [78]. Furthermore, one pupukeanane with an isothiocyanate group at C-5 (Pu10) was obtained by Marcus et al. from a sponge of the genus Axinyssa [43].
The thiocyanate axinythiocyanate A (Bi16) was obtained from the sponge Axinyssa isabela, collected in the Gulf of California, Mexico [33].
In 2014, Cheng et al. reported the isolation of ten oxygenated formamidobisabolanes (Bi26-Bi35) from a Chinese sponge (Axinyssa sp.) [92]. Axinyssine C (Bi26) has two hydroxy groups at C-10 and C-11 and a double bond in 8-position. Axinyssine D (Bi27) has the same structure as Bi26 but bears a methoxy group at C-10. Axinyssines E and F (Bi28 and Bi29) have the same functional groups as Bi26/Bi27 but the double bond is located at C-7, one hydroxy group is found at C-10 and the other hydroxy/methoxy group is attached to C-9. However, axinyssine G (Bi30) has one hydroxy group at C-9 and an epoxy group at C-10. NOE experiments revealed a 7E geometry and the coupling constant J H´9{H´10 = 8.0 Hz suggests a threo-orientation of the two stereocenters in the side chain. The absolute configuration of C-9 was determined by ECD spectra of the Rh 2 (OCOCF 3 ) 4 complex. Axinyssine H (Bi31) has a hydroxy group at C-7 and a keto group at C-10. Axinyssine I (Bi32) is the methoxylated analogue of Axinyssine H (Bi31). The compounds Bi33-Bi36 are the reduced compounds of axinyssine H (Bi31) and axinyssine I (Bi32). Axinyssine J (Bi33) has two hydroxy groups and the hydroxy-group-bearing C-10 has S-configuration. In contrast, axinyssine K (Bi34) has R-configuration at C-10. Axinyssine L (Bi35) carries a methoxy group at C-7 and a hydroxy group at the S-configured 10-position. The corresponding 3-formamido-8-methoxybisabolan-9-en-10-ol (Bi36) was isolated earlier [90].

Guaianes
Guaiane-type sesquiterpenoids have a similar skeleton to the aromadendranes but are devoid of the cyclopropane ring (Gu1, Figure 13). Tada et al. reported the first isolation of guaiane isonitriloids in 1988. The isonitrile Gu2, the isothiocyanate Gu3 and the formamide Gu4 were obtained from an unidentified sponge [93]. However, the authors speculated that the formamide Gu4 was not produced by the sponge but rather was an isolation artifact originating from hydrolysis of Gu2 during column chromatography [93]. The isothiocyanate Gu3 was also isolated from the tropical marine sponge Acanthella cavernosa wich was followed by a more detailed characterization but the orientation of the isothiocyanato group could not be elucidated [32]. Tada et al. deduced the relative configuration of the other congeners based on X-ray crystallography of the isonitrile Gu2 [93].
obtained from an unidentified sponge [93]. However, the authors speculated that the formamide Gu4 was not produced by the sponge but rather was an isolation artifact originating from hydrolysis of Gu2 during column chromatography [93]. The isothiocyanate Gu3 was also isolated from the tropical marine sponge Acanthella cavernosa wich was followed by a more detailed characterization but the orientation of the isothiocyanato group could not be elucidated [32]. Tada et al. deduced the relative configuration of the other congeners based on X-ray crystallography of the isonitrile Gu2 [93]. (1S*,4S*,5R*,10S*)-10-Isothiocyanatoguaia-6-ene (Gu5) was isolated from the sponge Trachyopsis aplysinoides collected in Palau [52]. The corresponding isonitrile Gu6 was isolated in 2004 from the nudibranch Pthyllidiella pustulosa from the South Chinese Sea [35].
In 2008, Li et al. reported the isolation of 4,5-epi-10-isothiocyanatoisodauc-6-ene (Gu8) from the Hainan marine sponge Axinyssa sp. [84]. In contrast to the other guaianes (Gu2-Gu6), one of its methyl groups is not located at C-10 but at C-1 and the isopropyl group and the methyl group at C-8 and C-4 are interchanged. The corresponding isonitrile (Gu7) was obtained recently by Wright et al. from the nudibranch Phyllidia ocellata [94].
In 2008, Li et al. reported the isolation of 4,5-epi-10-isothiocyanatoisodauc-6-ene (Gu8) from the Hainan marine sponge Axinyssa sp. [84]. In contrast to the other guaianes (Gu2-Gu6), one of its methyl groups is not located at C-10 but at C-1 and the isopropyl group and the methyl group at C-8 and C-4 are interchanged. The corresponding isonitrile (Gu7) was obtained recently by Wright et al. from the nudibranch Phyllidia ocellata [94].
Another unusual isonitrile/isothiocyanate pair, Gu9/Gu10, was isolated in 1987 by Mayol et al. from the sponge Acanthella acuta [62]. Similar to the isonitrile Gu7 and the isothiocyanate Gu8, the methyl group is located at C-1 and the isopropyl group and the methyl group are interchanged but the nitrogenous functional group is attached to the exocyclic allylic carbon.

Diterpenoids
Beside the cyclic sesquiterpenoid compounds already presented in this review, a large fraction of marine isonitriles and isothiocyanates were found to have diterpenoid backbones. They can be categorized into three classes: Acyclic diterpenes, kalihinanes, and amphilectanes.

Acyclic
The smallest class, the acyclic diterpenoids, is represented by a isonitriloid triad consisting of isocyanate Acy1, isothiocyanate Acy2 and formamide Acy3, which were the only representatives found until 2006 ( Figure 15). The triad was found in 1974 by Burreson et al. along with cyclic sesquiterpenes in an unidentified Halichondria species [39,40].

Diterpenoids
Beside the cyclic sesquiterpenoid compounds already presented in this review, a large fraction of marine isonitriles and isothiocyanates were found to have diterpenoid backbones. They can be categorized into three classes: Acyclic diterpenes, kalihinanes, and amphilectanes.

Acyclic
The smallest class, the acyclic diterpenoids, is represented by a isonitriloid triad consisting of isocyanate Acy1, isothiocyanate Acy2 and formamide Acy3, which were the only representatives found until 2006 ( Figure 15). The triad was found in 1974 by Burreson et al. along with cyclic sesquiterpenes in an unidentified Halichondria species [39,40].  [98]. Despite the occurrence of these three new members this class of acyclic compounds still appears as an exception among the other diterpenoid isonitriles which are usually cyclic.

Kalihinanes
Common to all kalihinanes is the decalin ring system which, apart from some exceptions (cavernenes) which will be discussed later on, either bears a tetrahydofuran, a tetrahydropyran or a dihydropyran moiety linked to C-7. All three rings can bear a large variety of functional groups including isocyano-, isothiocyanato-and hydroxy groups or chlorine. The kalihinanes can be further classified into kalihinols, kalihinenes, kalihipyranes and cavernenes. Most kalihinanes were found in A. cavernosa whereas also a few examples exist that were isolated from the sponge Phakellia pulcherrima.

Kalihinols
Characteristic for the kalihinols is the tertiary alcohol function at C-4 of a trans-decalin system

Kalihinanes
Common to all kalihinanes is the decalin ring system which, apart from some exceptions (cavernenes) which will be discussed later on, either bears a tetrahydofuran, a tetrahydropyran or a dihydropyran moiety linked to C-7. All three rings can bear a large variety of functional groups including isocyano-, isothiocyanato-and hydroxy groups or chlorine. The kalihinanes can be further classified into kalihinols, kalihinenes, kalihipyranes and cavernenes. Most kalihinanes were found in A. cavernosa whereas also a few examples exist that were isolated from the sponge Phakellia pulcherrima.

Kalihinols
Characteristic for the kalihinols is the tertiary alcohol function at C-4 of a trans-decalin system being part of a bifloran skeleton ( Figure 17). They can be further subdevided into two groups according to their substitution with a tetrahydropyran or a tetrahydrofuran moiety at C-7. The first eleven kalihinols were found by the Scheuer group of Hawaii in 1987 in two sponges of Acanthella sp. (later indentified as A. cavernosa) [99] and were published in a series of communications [100][101][102]. The kalihinols A-H were isolated from an Acanthella sp. collected at Guam, whereas examination of a Fijian Acanthella species revealed kalihinols X (Kol4), Y (Kol9) and Z (Kol3). Kalihinols A (Kol1), E (Kol6), X (Kol4), Y (Kol9) and Z (Kol3) are all linked at C-7 to a C-14 chlorinated tetrahydropyran moiety and bear an isonitrile function at C-5. Kalihinol E (Kol6) represents the C-14-epimer of kalihinol A (Kol1) just having another orientation of the chlorine substituent. The formamide derivatives of A and E, 10β-formamido kalihinol A (Kol2) and 10β-formamido kalihinol E (Kol7) were isolated in 1996 by Hirota et al. from A. cavernosa while the isothiocyanato derivative, found in the same producer by Xu et al. in 2004, was named kalihinol O (Kol8) [20]. Kalihinol Z (Kol3) on the other hand represents the C-10-epimer of kalihinol A (Kol1), whereas kalihinol X (Kol4) is the 10-isothiocyanato derivative of kalihinol Z (Kol3). Further investigation of A. cavernosa by Sun et al. in 2009 revealed a kalihinol with the opposite stereochemistry at C-10, 10-epi-kalihinol X (Kol5) [103]. Unlike the other tetrahydropyran kalihinols, kalihinol Y (Kol9) bears an exocyclic methylene group at C-10. In 1998, Wolf was able to isolate along with kalihinol Y its double bond isomer ∆ 9 -kalihinol Y (Kol10) from the sponge Phakellia pulcherrima, which was the first time that kalihinanes were found in sponge other than A. cavernosa [104]. Independently from Wolf, Miyaoka also discovered the same kalihinol in A. cavernosa in the same year [105]. In antihelmintic screenings, kalihinol Y (Kol9) displayed extremely high activity against the rodent-pathogenic roundworm Nippostongylus brasiliensis. The kalihinols A (Kol2), X (Kol4) and Z (Kol3) also showed high activity [106].
Kalihinol F (Kol21), a representative of the tetrahydrofuran-type kalihinols, is substituted with three isonitrile groups at C-5, C-10 and C-15. It was shown to be a topoisomerase I inhibitor in Asterina pectinifera, inhibiting the chromosome separation in fertilized starfish eggs [108] and to have antimicrobial activity against Bacillus subtilis, Staphylococcus aureus and Candida albicans [101]. Kalihinol B (Kol20) differs from Kalihinol F (Kol21) just in the functional group at C-15, which is a chlorine instead of an isocyano group. In 2004, Bugni was able to isolate the formamide derivatives 10-formamido kalihinol F (Kol22) and 15-formamido kalihinol F (Kol23) from two Philippine specimens of A. cavernosa [109]. The C-15 isothiocyanate derivative of kalihinols B and F was named kalihinol G (Kol24), while kalihinol H (Kol26) represents the C-10-isothiocyanato derivative of kalihinol F [102]. The first representatives of the tetrahydrofuran subclass of kalihinols were kalihinols B (Kol20), C (Kol28), D (Kol30), F (Kol21), G (Kol24) and H (Kol26) discovered by the Scheuer group in the 1980s ( Figure 18) [101,102]. The stereochemical and functional variations occur at C-5 and C-10 as in the tetrahydropyran-type kalihinols. Additionally, the tetrahydrofuran moiety can be functionalized at C-15 with isonitrile, isothiocyanate, formamide or chlorine. Examples bearing an isopropenyl side chain are also known.
Kalihinol F (Kol21), a representative of the tetrahydrofuran-type kalihinols, is substituted with three isonitrile groups at C-5, C-10 and C-15. It was shown to be a topoisomerase I inhibitor in Asterina pectinifera, inhibiting the chromosome separation in fertilized starfish eggs [108] and to have antimicrobial activity against Bacillus subtilis, Staphylococcus aureus and Candida albicans [101]. Kalihinol B (Kol20) differs from Kalihinol F (Kol21) just in the functional group at C-15, which is a chlorine instead of an isocyano group. In 2004, Bugni was able to isolate the formamide derivatives 10-formamido kalihinol F (Kol22) and 15-formamido kalihinol F (Kol23) from two Philippine specimens of A. cavernosa [109]. The C-15 isothiocyanate derivative of kalihinols B and F was named kalihinol G (Kol24), while kalihinol H (Kol26) represents the C-10-isothiocyanato derivative of kalihinol F [102]. The first member of the class bearing an isopropenyl side chain was kalihinol C (Kol28), which is functionalized at C-5 and C-10 with two isonitrile groups. Kalihinol D (Kol30) represents the 5-Clderivative of kalihinol F (Kol21) [102]. Its C-15-isothiocyanato derivative, kalihinol T (Kol31), was isolated by Xu in 2012 from A. cavernosa [107].
Already in 1988, Omar et al. were able to isolate a variant of the kalihinols with swapped positions of the hydroxy and isonitrile groups at C-4 and C-5 ( Figure 19) [106]. The stereochemistry of both groups is opposite compared to the normal kalihinols, presumably due to a different opening of the intermediate epoxide in the biosynthetic pathway. The first example of this so-called "isokalihinols" was isokalihinol F (Kol36) which, except for C-4 and C-5, showed the same configuration and functionalization as kalihinol F (Kol21). 8-OH-Isokalihinol F (Kol41), a rather unusual isokalihinol bearing an additional hydroxy group at C-8, was isolated by Clark et al. in 2000 from A. cavernosa collected at Heron Island (Great Barrier Reef, Australia) [31]. The first member of the class bearing an isopropenyl side chain was kalihinol C (Kol28), which is functionalized at C-5 and C-10 with two isonitrile groups. Kalihinol D (Kol30) represents the 5-Cl-derivative of kalihinol F (Kol21) [102]. Its C-15-isothiocyanato derivative, kalihinol T (Kol31), was isolated by Xu in 2012 from A. cavernosa [107].
In 1998, the Miyaoka group was able to isolate the tris-isothiocyanato compound 5,10-bisisothiocyanatokalihinol G (Kol34) [105]. Further tetrahydrofuran-type kalihinols were presented in the same year by Wolf et al.
Already in 1988, Omar et al. were able to isolate a variant of the kalihinols with swapped positions of the hydroxy and isonitrile groups at C-4 and C-5 ( Figure 19) [106]. The stereochemistry of both groups is opposite compared to the normal kalihinols, presumably due to a different opening of the intermediate epoxide in the biosynthetic pathway. The first example of this so-called "isokalihinols" was isokalihinol F (Kol36) which, except for C-4 and C-5, showed the same configuration and functionalization as kalihinol F (Kol21). 8-OH-Isokalihinol F (Kol41), a rather unusual isokalihinol bearing an additional hydroxy group at C- Three further isokalihinols with an NC-function at C-4 are known to date: Isokalihinol B (Kol35), being related to kalihinol B (Kol20), was found in 1990 by Fusetani in a marine sponge collected in Kuchihoerabu Island of the Satsunan Archipelago (Japan) which was identified as Acanthella klethra [110]. Rodriguez and Crews revised this assignment afterwards based on voucher samples to be actually A. cavernosa [99]. Isokalihinol B (Kol35) and kalihinene (Ken1) demonstrated besides antifungal activities against Mortierella romannicus and Penicillium chrysogenum also cytotoxic potency against P388 murine leukemia cells (IC50 = 0.8 μg/mL and 1.2 μg/mL), respectively [110].
In 2012, Xu et al. reported two novel C-4 formamido representatives of isokalihinols in a South China Sea specimen of A. cavernosa, kalihinol M (Kol39) bearing an isothiocyanato group at C-10 and a chlorine atom at C-15 and kalihinol N (Kol40), the 4-formamido-iso-derivative of kalihinol O (Kol8), representing the first example of isokalihinols of the tetrahydropyran type [107].

Kalihinenes
Common to all kalihinenes is the trisubstituted Δ 4 -double bond and a heterocyclic C8-substituent at C-7 equal to the kalihinols. However, unlike the kalihinols, which to date exclusively featured trans-decalin skeletons, the kalihinenes also exist with cis-decalin backbones ( Figure 20).
In 2012, Xu et al. reported two novel C-4 formamido representatives of isokalihinols in a South China Sea specimen of A. cavernosa, kalihinol M (Kol39) bearing an isothiocyanato group at C-10 and a chlorine atom at C-15 and kalihinol N (Kol40), the 4-formamido-iso-derivative of kalihinol O (Kol8), representing the first example of isokalihinols of the tetrahydropyran type [107].

Kalihinenes
Common to all kalihinenes is the trisubstituted ∆ 4 -double bond and a heterocyclic C 8 -substituent at C-7 equal to the kalihinols. However, unlike the kalihinols, which to date exclusively featured trans-decalin skeletons, the kalihinenes also exist with cis-decalin backbones ( Figure 20).
Three kalihinenes with a trans-decalin framework were reported in 1994 by Faulkner et al. [112]. Besides kalihinene A (also named diepi-kalihinene) (Ken9) which is, apart from the double bond, identical to kalihinol F (Kol21), the group could also isolate kalihinene B (or 1-epi-kalihinene) (Ken10) and 15-isothiocyanato-1-epi-kalihinene (Ken11). In contrast to the twelve isolated members of the tetrahydrofuran-substituted kalihinene series, only four tetrahydropyran derivatives are known to date. The first three examples were the kalihinenes X (Ken14), Y (Ken15), and Z (Ken16) isolated in 1995 by Okino et al. from A. cavernosa [111]. All of them bear a formamide function at C-10 and a chlorine at C-14. Differences only consist in the stereochemistry at C-1 and C-14. In 2012, the Xu group added the missing fourth diastereomer kalihinene E (Ken13) also found in the same producer [113]. Its absolute configuration was elucidated by X-ray crystallography.
Three kalihinenes with a trans-decalin framework were reported in 1994 by Faulkner et al. [112]. Besides kalihinene A (also named diepi-kalihinene) (Ken9) which is, apart from the double bond, identical to kalihinol F (Kol21), the group could also isolate kalihinene B (or 1-epi-kalihinene) (Ken10) and 15-isothiocyanato-1-epi-kalihinene (Ken11). In contrast to the twelve isolated members of the tetrahydrofuran-substituted kalihinene series, only four tetrahydropyran derivatives are known to date. The first three examples were the kalihinenes X (Ken14), Y (Ken15), and Z (Ken16) isolated in 1995 by Okino et al. from A. cavernosa [111]. All of them bear a formamide function at C-10 and a chlorine at C-14. Differences only consist in the stereochemistry at C-1 and C-14. In 2012, the Xu group added the missing fourth diastereomer kalihinene E (Ken13) also found in the same producer [113]. Its absolute configuration was elucidated by X-ray crystallography.
Kalihinene (Ken1), kalihinol A (Kol1), and kalihinol E (Kol6) were also found by Manzo et al. in 2004 in Phylidiella pustulosa, a nudibranch from the South China Sea [35]. The dietary origin of these diterpenoids is strongly supported by several investigations.
Besides the tetrahydropyran and tetrahydrofuran series of kalihinenes, a third group of kalihinenes with a different pendant C 8 -unit exists. The first member of this class, kalihipyran (Kpy1), was discovered by Faulkner et al. in 1994 in A. cavernosa (Figure 21) [112]. It shows a kalihinene type trans-decalin system with an isonitrile function at C-10 and is connected at C-7 to a dihydropyran with isopropenyl side chain. Its C-10 formamide derivative kalihipyran A (Kpy2) was isolated in 2012 by Xu et al. along with the cis-decalin derivative kalihipyran C (Kpy4) [113]. Kalihipyran B (Kpy3) isolated by Fusetani, again features a trans-decalin backbone, but contains a chlorine in the side chain [114]. The configuration at C-14 remains unknown. Besides the tetrahydropyran and tetrahydrofuran series of kalihinenes, a third group of kalihinenes with a different pendant C8-unit exists. The first member of this class, kalihipyran (Kpy1), was discovered by Faulkner et al. in 1994 in A. cavernosa (Figure 21) [112]. It shows a kalihinene type trans-decalin system with an isonitrile function at C-10 and is connected at C-7 to a dihydropyran with isopropenyl side chain. Its C-10 formamide derivative kalihipyran A (Kpy2) was isolated in 2012 by Xu et al. along with the cis-decalin derivative kalihipyran C (Kpy4) [113]. Kalihipyran B (Kpy3) isolated by Fusetani, again features a trans-decalin backbone, but contains a chlorine in the side chain [114]. The configuration at C-14 remains unknown.

Cavernenes and Other Intermediates
Over the years, a couple of potential intermediates in the biosynthetic pathway of kalihinanes have been identified ( Figure 22). In 1996, the first example of a new class of kalihinane diterpenes with an open-chained substituent at C-7 and a kalihinene cis-decalin framework was discovered in Cymbastella hooperi by König et al. who determined the relative configuration to correspond to that of (1S*,6R*,7R*,10S*,11R*)-10-isothiocyanatobiflora-1,14-diene (Int5) ([α] D = +45˝(CHCl 3 , c = 0.26)) [115]. Besides the tetrahydropyran and tetrahydrofuran series of kalihinenes, a third group of kalihinenes with a different pendant C8-unit exists. The first member of this class, kalihipyran (Kpy1), was discovered by Faulkner et al. in 1994 in A. cavernosa (Figure 21) [112]. It shows a kalihinene type trans-decalin system with an isonitrile function at C-10 and is connected at C-7 to a dihydropyran with isopropenyl side chain. Its C-10 formamide derivative kalihipyran A (Kpy2) was isolated in 2012 by Xu et al. along with the cis-decalin derivative kalihipyran C (Kpy4) [113]. Kalihipyran B (Kpy3) isolated by Fusetani, again features a trans-decalin backbone, but contains a chlorine in the side chain [114]. The configuration at C-14 remains unknown.    [116]. Its relative configuration was not elucidated.
From the same sponge, four further members of this class of intermediates were reported by Xu et al. in 2012, which all bear a formamide group at C-10 and differ just in the nature of the side chain linked to C-7 and the cisor trans-configuration of the decalin moiety. Cavernene A (Int1), a trans-decalin, is connected to an isoprenoid unit. In contrast to cavernene A (Int1), cavernene B (Int2) shows a monoolefinic isoprenoid side chain, while cavernene C (Int3) is identical to cavernene B (Int2) apart from the cis-decalin moiety. Cavernene D (Int4) possesses a trisubstituted epoxide in the side chain and the same trans-decalin unit as cavernenes A and B and represents the C-10-formamido derivative of Int7 [113].
The missing link in the kalihinol synthesis was discovered by Wolf et al. in 1998 in a sample of Phylidiella pulcherrima and was named pulcherrimol (Int6) [104]. In contrast to the other intermediates, it shows the decalin framework of kalihinol A, which makes its role as an intermediate in the biosynthesis of kalihinols plausible.
For the proposed role of the intermediates in the kalihinane synthesis see the Chapter 3 on biosynthesis.

Amphilectanes
Besides the diterpenoid class of highly substituted decalins, the kalihinanes, which are found almost exclusively in A. cavernosa and apart from the small group of acyclic diterpenes, a third class of naturally occurring diterpenoid isonitriles with tricyclic or tetracyclic structures is known.
These so-called amphilectanes are predominantly found in Amphimedon sp., Hymenacidon amphilecta and Halichondria sponges and can furthermore be subdivided based on their carbon frameworks into amphilectanes, cycloamphilectanes, isocycloamphilectanes, neoamphilectanes and isoneoamphilectanes ( Figure 23). Recently the assignment of the species Hymeniacidon amphilecta has been revised to Pseudoaxinella amphilecta and will be referred to in the latter way hereinafter [117].  [116]. Its relative configuration was not elucidated. Examination of an A. cavernosa sponge, collected at Heron Island (Great Barrier Reef, Australia) by Clark et al. in 2000 resulted in the isolation of two oxirane-derivatives, 11,12-epoxy-10-isocyano-4,14-bifloradiene (Int7) possessing a trisubstituted epoxide in the side chain and a trans-decalin framework, and 11,18-epoxy-10-isocyano-4,14-bifloradiene (Int8) featuring a terminal epoxide group. The authors suggest the latter one to be a precursor to the kalihipyran ring system [31].
From the same sponge, four further members of this class of intermediates were reported by Xu et al. in 2012, which all bear a formamide group at C-10 and differ just in the nature of the side chain linked to C-7 and the cis-or trans-configuration of the decalin moiety. Cavernene A (Int1), a transdecalin, is connected to an isoprenoid unit. In contrast to cavernene A (Int1), cavernene B (Int2) shows a monoolefinic isoprenoid side chain, while cavernene C (Int3) is identical to cavernene B (Int2) apart from the cis-decalin moiety. Cavernene D (Int4) possesses a trisubstituted epoxide in the side chain and the same trans-decalin unit as cavernenes A and B and represents the C-10-formamido derivative of Int7 [113].
The missing link in the kalihinol synthesis was discovered by Wolf et al. in 1998 in a sample of Phylidiella pulcherrima and was named pulcherrimol (Int6) [104]. In contrast to the other intermediates, it shows the decalin framework of kalihinol A, which makes its role as an intermediate in the biosynthesis of kalihinols plausible.
For the proposed role of the intermediates in the kalihinane synthesis see the Chapter 3 on biosynthesis.

Amphilectanes
Besides the diterpenoid class of highly substituted decalins, the kalihinanes, which are found almost exclusively in A. cavernosa and apart from the small group of acyclic diterpenes, a third class of naturally occurring diterpenoid isonitriles with tricyclic or tetracyclic structures is known. These so-called amphilectanes are predominantly found in Amphimedon sp., Hymenacidon amphilecta and Halichondria sponges and can furthermore be subdivided based on their carbon frameworks into amphilectanes, cycloamphilectanes, isocycloamphilectanes, neoamphilectanes and isoneoamphilectanes ( Figure 23). Recently the assignment of the species Hymeniacidon amphilecta has been revised to Pseudoaxinella amphilecta and will be referred to in the latter way hereinafter [117].  [118]. In the same source, the goup around Wratten was also able to isolate its 15formamide derivative Amp2 [119].
In 2011, Lamoral-Theys et al. were able to reisolate compounds Amp1, Amp23 and Amp25 from the Caribbean sponge Pseudoaxinella flava from the Grand Bahamas along with a new representative with cis-junction Amp26. Amphilectenes Amp25 and Amp26 showed cytotoxic effects against the apoptosis-sensitive human PC3 prostate cancer cell line whereas Amp1 and Amp23 displayed cytostatic effects [128].
A putative precursor Amp27 of the amphilectene compounds with a biflora-4,9,15-triene skeleton was isolated by Hirota in 1996 [20]. The absolute configuration and a plausible biosynthetic pathway to the amphilectanes were reported in 2005 by Ciavatta [120].

Neoamphilectene
In 1992, a compound with an unusual amphilectane-framework in which ring C is spirocyclic to the cyclohexene ring was found by Sharma in a sponge of the Adociae family, collected off the island of Miyako (Japan, Figure 27). To date 7-isocyanoneoamphilecta-11,15-diene (Neo1) remains the only known representative of its class [116]. One example of an isocycloamphilectane with a different substitution pattern at C-15 and C-20 is known, the diisocyano compound 7,15-diisocyanoadociane (Ica7), isolated in 1980 by Kazlauskas [126,130].

Neoamphilectene
In 1992, a compound with an unusual amphilectane-framework in which ring C is spirocyclic to the cyclohexene ring was found by Sharma in a sponge of the Adociae family, collected off the island of Miyako (Japan, Figure 27). To date 7-isocyanoneoamphilecta-11,15-diene (Neo1) remains the only known representative of its class [116]. One example of an isocycloamphilectane with a different substitution pattern at C-15 and C-20 is known, the diisocyano compound 7,15-diisocyanoadociane (Ica7), isolated in 1980 by Kazlauskas [126,130].

Neoamphilectene
In 1992, a compound with an unusual amphilectane-framework in which ring C is spirocyclic to the cyclohexene ring was found by Sharma in a sponge of the Adociae family, collected off the island of Miyako (Japan, Figure 27). To date 7-isocyanoneoamphilecta-11,15-diene (Neo1) remains the only known representative of its class [116].

Carbonimidic Dichlorides
Carbonimidic dichlorides or dichloroimines represent a rare group of natural products of which to date only 16 representatives are known which are all of marine origin (Figure 29 and Figure 30). They have been isolated from four marine sponges (Pseudaxinyssa pitys, Axinyssa sp., Stylotella aurantium and Ulosa spongia) and from the nudibranch Reticulidia fungia presumably feeding on these sponges. Carbonimidic dichlorides exhibit a strong IR absorption at ~1650 cm −1 and a low intensity 13 C signal at δ ≈ 127 ppm and can therefore be unequivocally identified.
The first examples of this class of natural products were isolated in 1977 from the Indo-pacific

Carbonimidic Dichlorides
Carbonimidic dichlorides or dichloroimines represent a rare group of natural products of which to date only 16 representatives are known which are all of marine origin (Figures 29 and 30). They have been isolated from four marine sponges (Pseudaxinyssa pitys, Axinyssa sp., Stylotella aurantium and Ulosa spongia) and from the nudibranch Reticulidia fungia presumably feeding on these sponges. Carbonimidic dichlorides exhibit a strong IR absorption at~1650 cm´1 and a low intensity 13 C signal at δ « 127 ppm and can therefore be unequivocally identified.
The first examples of this class of natural products were isolated in 1977 from the Indo-pacific marine sponge Pseudoaxinyssa pitys by Wratten, who identified the trans linear isoprenoid derivative Dcl2 containing three chlorines [134]. The substance was isolated again in 1997 by Simpson from Stylotella aurantium, a tropical marine sponge collected at Heron Island (Great Barrier Reef, Australia), and was named stylotellane B (Dcl2) [135]. The sponge also contained farnesyl isothiocyanate (Mis1) and Stylotellane A (Dcl1), a similar sesquiterpene without the chlorine substituent at C-2. Both stylotellanes were testet against tumor cell line P-388, however only a weak activity was observed for stylotellane B (Dcl 2).
Investigation     Examination of a marine sponge of the genus Axinyssa collected off Hachijo-jima Island (Japan) by Hirota et al. in 1998 led to the isolation of three new oxygenated sesquiterpenoid carbonimidic dichlorides [53]. Axinyssimide A (Dcl5) represents the 10,11-epoxy derivative of stylotellane B while axinyssimides B (Dcl6) and C (Dcl7) turned out to be the 10,11-dihydroxy derivatives resulting from a ring-opening of the oxirane moiety. Axinyssimides B and C, differing in magnitude and sign of their optical rotation, are diastereomers. All three compounds demonstrated potent antifouling activities against the cypris larvae of the acorn barnacle Balanus amphitrite with an IC 50 value of 1.2 µg/mL for axinyssimide A, 70% inhibition at a concentration of 0.5 µg/mL for axinyssimide B and 90% inhibition at the same concentration for axinyssimide C respectively [53].
Another monocyclic carbonimidic dichloride (Dcl10) was isolated in 2001 by Musman et al. from Stylotella aurantium collected from a coral reef of Iriomate Island (Okinawa, Japan) [137]. Both monocyclic carbon skeletons can be derived from Stylotellane B by an enzymatic chlorination including a formal "chloronium ion" initiated cyclization [139].
Further examination of the Pseudoaxinyssa pitys saple of Wratten in 1978 also revealed four examples of bicyclic sesquiterpenes with carbonimidic dichloride functionality [140]. Compound Dcl11, its ∆ 7,8 -double bond isomer Dcl12 and the non-hydroxylated derivative Dcl13 all show a Z-configurated ∆ 14 -double bond in the side chain. The fourth isolated isomer, isoreticulidin B (Dcl17) showing a ∆ 7,8 -14(E)-diene system, was also found in the nudibranch Reticulidia fungia collected at Irabu Island (Okinawa) in 1999 by Tanaka et al. besides a monocyclic (Dcl8) and two bicyclic compounds that were named reticulidins A (Dcl15) and B (Dcl16) [141]. In 2001, Musman et al. were able to determine the absolute configuration of reticulidin A by a modification of Mosher's method to be 2R,3R,5S,10S [137]. The same group also revealed the structure of the non-hydroxylated derivative Dcl14 of reticulidin A and isoreticulidin B isolated from Stylotella aurantium.

Other Marine Isonitriles and Related Compounds (Miscellaneous Structures)
Among the isonitriles, isothiocyanates, and formamides of marine origin there are compounds that cannot be categorized in the structural families already presented ( Figure 31).
Irabu Island (Okinawa) in 1999 by Tanaka et al. besides a monocyclic (Dcl8) and two bicyclic compounds that were named reticulidins A (Dcl15) and B (Dcl16) [141]. In 2001, Musman et al. were able to determine the absolute configuration of reticulidin A by a modification of Mosher's method to be 2R,3R,5S,10S [137]. The same group also revealed the structure of the non-hydroxylated derivative Dcl14 of reticulidin A and isoreticulidin B isolated from Stylotella aurantium.

Other Marine Isonitriles and Related Compounds (Miscellaneous Structures)
Among the isonitriles, isothiocyanates, and formamides of marine origin there are compounds that cannot be categorized in the structural families already presented ( Figure 31).  [135,140]. Its corresponding formamide Mis2 and isofarnesyl formamide (Mis4) were found in 2008 in Axinyssa sp. collected in Sanya, Hainan Province, China by Li [84]. Until now, farnesyl isocyanide (Mis3) remains elusive which might be caused by the instability of this compound.
Despite from the huge variety of marine isothiocyanates just a few examples of structures bearing a thiocyanate functionality are known ( Figure 35). Among these is thiocyanatin A (Mis33) that was isolated from Oceanapia sp. (collected off the northern Rottnest shelf, Australia) in 2001 by Capon et al. together with its Δ 8 -and Δ 7 -elimination products thiocyanatins B (Mis34) and C (Mis35) [146]. Thiocyanatin A was reported to possess in vitro nematicidal activities against the barber pole worm Haemonchus contortus whereas the non-hydroxylated derivatives thiocyanatins B and C showed no activity at all.  Figure 34) [92]. The corresponding isonitrile of Mis32 1-acetyl-4-isocyano-4-methylcyclohexane had already been discovered in the nudibranch Phyllidia sp. by Gulavita et al. in 1986 [74]. These four compounds could represent catabolic derivatives of the axinyssine sesquiterpenoids presented earlier.
Despite from the huge variety of marine isothiocyanates just a few examples of structures bearing a thiocyanate functionality are known ( Figure 35). Among these is thiocyanatin A (Mis33) that was isolated from Oceanapia sp. (collected off the northern Rottnest shelf, Australia) in 2001 by Capon et al. together with its ∆ 8 -and ∆ 7 -elimination products thiocyanatins B (Mis34) and C (Mis35) [146]. Thiocyanatin A was reported to possess in vitro nematicidal activities against the barber pole worm Haemonchus contortus whereas the non-hydroxylated derivatives thiocyanatins B and C showed no activity at all.  Three other thiocyanates with a unique carbon skeleton were found in the ascidian Clavelina cylindrica collected from the Bay of Islands (South Bruny Island, Tasmania) by Li et al. in 1994 (Figure 36) [147]. Cylindricines F (Mis40) and G (Mis41) show the same carbonyl skeleton as cylindricin A (Mis36) which was the first natural pyrrolo[2,1-j]quinoline to be identified. The isolation of cylindricine H (Mis30), the C-4-acetoxy-derivative of cylindricin G, was reported together with its corresponding isothiocyanate cylindricin I (Mis43) and the pyrido[2,1-j]quinoline isothiocyanate cylindricin J (Mis45), which shows the cylindricin B ring system (Mis44) [148]. Li also reported the interconvertibility of both ring systems.  Three other thiocyanates with a unique carbon skeleton were found in the ascidian Clavelina cylindrica collected from the Bay of Islands (South Bruny Island, Tasmania) by Li et al. in 1994 (Figure 36) [147]. Cylindricines F (Mis40) and G (Mis41) show the same carbonyl skeleton as cylindricin A (Mis36) which was the first natural pyrrolo[2,1-j]quinoline to be identified. The isolation of cylindricine H (Mis30), the C-4-acetoxy-derivative of cylindricin G, was reported together with its corresponding isothiocyanate cylindricin I (Mis43) and the pyrido[2,1-j]quinoline isothiocyanate cylindricin J (Mis45), which shows the cylindricin B ring system (Mis44) [148]. Li also reported the interconvertibility of both ring systems. Three other thiocyanates with a unique carbon skeleton were found in the ascidian Clavelina cylindrica collected from the Bay of Islands (South Bruny Island, Tasmania) by Li et al. in 1994 (Figure 36) [147]. Cylindricines F (Mis40) and G (Mis41) show the same carbonyl skeleton as cylindricin A (Mis36) which was the first natural pyrrolo[2,1-j]quinoline to be identified. The isolation of cylindricine H (Mis30), the C-4-acetoxy-derivative of cylindricin G, was reported together with its corresponding isothiocyanate cylindricin I (Mis43) and the pyrido[2,1-j]quinoline isothiocyanate cylindricin J (Mis45), which shows the cylindricin B ring system (Mis44) [148]. Li also reported the interconvertibility of both ring systems.
Three other thiocyanates with a unique carbon skeleton were found in the ascidian Clavelina cylindrica collected from the Bay of Islands (South Bruny Island, Tasmania) by Li et al. in 1994 (Figure 36) [147]. Cylindricines F (Mis40) and G (Mis41) show the same carbonyl skeleton as cylindricin A (Mis36) which was the first natural pyrrolo[2,1-j]quinoline to be identified. The isolation of cylindricine H (Mis30), the C-4-acetoxy-derivative of cylindricin G, was reported together with its corresponding isothiocyanate cylindricin I (Mis43) and the pyrido[2,1-j]quinoline isothiocyanate cylindricin J (Mis45), which shows the cylindricin B ring system (Mis44) [148]. Li also reported the interconvertibility of both ring systems.

Biosynthesis
Two years after the first isolation of a marine isonitrile (Ax2) in 1973, Fattorusso et al. and Burreson et al. observed that the isonitriles are often occurred associated with the corresponding formamido and isothiocyanato analogues. Therefore, they speculated that the formamide function was the precursor for the isonitrile function which itself was the precursor for the isothiocyanate function [5,6].
Iengo et al. fed the sponge Axinella cannabina with [ 14 C]-labeled axamide-1 (Ax4) over five days in well aerated sea water. No incorporation of the labeled formamide was observed and therefore the authors assumed that the formamide function is not the precursor for the isonitrile function. Nevertheless the authors questioned their own results because of several factors such as an insufficient incorporation rate [151].
Hagadone et al. performed experiments with the sponge Hymeniacidon sp. in its natural habitat. They synthesized [ 13 C]-labeled 2-formamidopupukeanane (Pu9) and 2-isothiocyanatopupukeanane and fed the sponges with these compounds. However, no [ 13 C]-labeled 2-isocyanopupukeanane (Pu7) was isolated after usual workup. Thereby neither the formamide function nor the isothiocyanato function appear to be the precursor for the isonitrile. Additionally, they observed, that [ 13 C]-formate

Biosynthesis
Two years after the first isolation of a marine isonitrile (Ax2) in 1973, Fattorusso et al. and Burreson et al. observed that the isonitriles are often occurred associated with the corresponding formamido and isothiocyanato analogues. Therefore, they speculated that the formamide function was the precursor for the isonitrile function which itself was the precursor for the isothiocyanate function [5,6].
Iengo et al. fed the sponge Axinella cannabina with [ 14 C]-labeled axamide-1 (Ax4) over five days in well aerated sea water. No incorporation of the labeled formamide was observed and therefore the authors assumed that the formamide function is not the precursor for the isonitrile function. Nevertheless the authors questioned their own results because of several factors such as an insufficient incorporation rate [151].
Hagadone et al. performed experiments with the sponge Hymeniacidon sp. in its natural habitat. They synthesized [ 13 C]-labeled 2-formamidopupukeanane (Pu9) and 2-isothiocyanatopupukeanane and fed the sponges with these compounds. However, no [ 13 C]-labeled 2-isocyanopupukeanane (Pu7) was isolated after usual workup. Thereby neither the formamide function nor the isothiocyanato function appear to be the precursor for the isonitrile. Additionally, they observed, that [ 13 C]-formate is not a component in the biosynthesis of marine isonitriles. Finally, the sponge Hymeniacidon sp. was fed with [ 13 C]-labeled isocyanopupukeanane (Pu7) and appearance of labeled 2-formamidopupukeanane (Pu9) and 2-isothiocyanatopupukeanane, detected by GC-MS, demonstrated that the isonitrile function is the precursor for the formamido function as well as the isothiocyanato function [152].
Experiments with doubly labeled [ 13 C, 15 N]-cyanide were performed by Karuso and Scheuer with the sponge Ciocalypta sp. and showed that also the nitrogen in the isonitrile has its origin in cyanide. Moreover [ 14 C]-cyanide was also incorporated in the sponge Acanthella sp. which produces the diterpene kalihinol F (Kol21). Some evidence exists that several natural products isolated from sponges may actually have been synthesized by microrganisms associated with these sponges [154]. Thereby the biosynthesis of marine isonitriles differs distinctly from that in terrestrial microorganisms [155].

Sesquiterpenoids
All sesquiterpenoids are produced from farnesyl pyrophosphate (I) and by different cyclizations and Wagner-Meerwein rearrangements the various skeletons will be formed ( Figure 38).
Additionally, farnesyl diphosphate (I) can convert to the bicycle VI with a cyclobutane ring and after 1,6-cyclization the tricyclic carbocation VIII is available which is the precursor for the epicaryolanes (So8) [94]. Furthermore, the carbenium ion VIII can transform by a 1,2-alkyl shift to the cation IX, which is the precursor for isocyanoclovane (So13) and isocyanoclovene (So12) [94].
In addition to the 1,6-cyclization to XII, XI can also transform to cation XV via 1,6-cyclization. Firstly, this carbocation XV is the precursor for the cadinanes (Ca2-Ca34) [44]. Secondly, after 1,2-H shift to the cation XVI and formation of a cyclopropane ring, the precursor XVII for the cubebanes (Fu9-Fu11) is formed [44]. Moreover, after a Wagner-Meerwein rearrangement of XVII to XVIII, the precursor for the spiroaxanes is obtained [44]. Finally, the cation XV can cyclize to the tricyclic system XX, which has two options for a 1,2-alkyl shift. The first option leads to cation XXI which is the precursor for the pupukeananes (Pu2-Pu10). The second option is a 1,2-alkyl shift to cation XXII which is the precursor for the neopupukeananes (Pu11-Pu13) [79]. Mar. Drugs 2016, 14, x 38 of 76 Figure 38. Proposed biosynthesis to sesquiterpenoids. Figure 38. Proposed biosynthesis to sesquiterpenoids.

Diterpenoids
Rodriguez et al. proposed a possible biosynthetic pathway for diterpenoids in 1994 and postulated the ring closure by transand cis-cyclases [99]. Garson et al. extended the biosynthetic pathway to further structures in 2004 [14].
All Diterpenes are formed from the pyrophosphate XXIII ( Figure 39). After integration of an additional double bond, the substrate XXIV can cyclize with a cis-cyclase to the (4S,8S)-hexahydro naphthalene system XXV or with a trans-cyclase to the (4R,8S)-hexahydronaphthalene system XXVII.

Carbonimidic Dichlorides
Along with the discovery of the carbonimidic dichlorides in 1977, Wratten already proposed the hypothesis that this uncommon functionality may result from enzymatic chlorination of the corresponding isonitriles or isothiocyanates ( Figure 40) [134]. Feeding experiments using radioactive inorganic cyanide in 1997 by Simpson et al. proved this hypothesis to be correct [135].
Starting from farnesyl pyrophosphate (XXXVI), they were able to show the presence of radioactive stylotellane A in Stylotella aurantium fed with potassium [ 14 C]-cyanide or isothiocyanate. It could be biosynthesized from farnesyl isocyanide (XXXVII) as well as from the corresponding farnesyl isothiocyanate (Mis1). Through enzymatic chlorination, stylotellane A can be converted into stylotellane B which is the precursor for both monocyclic carbon skeletons as well as for the bicyclic sesquiterpenoid derivatives, which all result from an enzyme catalyzed chlorinating cyclization. Hydroxylation could either occur from allylic oxidation of stylotellane B at C-9 or after the cyclization. After addition of hydrogen cyanide and epoxidation of XXV to XXVI, a nucleophile can attack the epoxide under ring opening and the resulting alcohol can attack at the double bond under formation of the tetrahydrofuran ring in the THF-kalihinenes (Ken1-Ken12) [14,99].
For the other marine diterpenes (4R,8S)-hexahydronaphthalene system XXVII is the origin. After addition of hydrogen cyanide, a double bond migration and an epoxidation form the epoxide XXVIII. In further steps, the hydroxylated epoxide XXIX is formed and after nucleophilic attack of the hydroxy group at the epoxide and subsequent elimination of water, the biogenesis of the kalihipyrans (Kpy1-Kpy4) is achieved [14,100].
After addition of hydrogen cyanide and epoxidation of XXVII without double bond migration, epoxide XXX is generated. Garson and Simpson postulated the attack of a nucleophile at both positions of the epoxide XXX and after attack of the oxyanion at the double bond under ring closure either the THP-kalihinols (Kol1-Kol19) or the THF-kalihinols (Kol20-Kol34) or isokalihinols (Kol35-Kol41), respectively, should be formed [14,99]. The hydroxy and isonitrile function at C-4 or C-5, respectively, are established by epoxidation of the double bond and following nucleophilic attack at C-4 or C-5.
A further option for the (4R,8S)-hexahydronaphthalene system XXVII is an additional cyclization and subsequent addition of hydrogen cyanide to the dodecahydrophenalene system XXXIV which represents the precursor for the amphilectanes (Amp1-Amp26). Moreover, a further cyclization of XXXIV to the tetradecahydropyrene system XXXV provides the precursor for the cycloamphilectanes (Cam1-Cam10) [14,99].

Carbonimidic Dichlorides
Along with the discovery of the carbonimidic dichlorides in 1977, Wratten already proposed the hypothesis that this uncommon functionality may result from enzymatic chlorination of the corresponding isonitriles or isothiocyanates ( Figure 40) [134]. Feeding experiments using radioactive inorganic cyanide in 1997 by Simpson et al. proved this hypothesis to be correct [135].

Biological Activity
In general, marine isonitriles have a weak to moderate activity in mammalian cell cytotoxicity assays (10-100 μM) against a broad range of cell lines [15]. Toxicity studies of simple isonitriles revealed surprisingly low toxicity with oral and subcutaneous toxic doses up to LD50 = 5 g/kg [159]. Starting from farnesyl pyrophosphate (XXXVI), they were able to show the presence of radioactive stylotellane A in Stylotella aurantium fed with potassium [ 14 C]-cyanide or isothiocyanate. It could be biosynthesized from farnesyl isocyanide (XXXVII) as well as from the corresponding farnesyl isothiocyanate (Mis1). Through enzymatic chlorination, stylotellane A can be converted into stylotellane B which is the precursor for both monocyclic carbon skeletons as well as for the bicyclic sesquiterpenoid derivatives, which all result from an enzyme catalyzed chlorinating cyclization.
Hydroxylation could either occur from allylic oxidation of stylotellane B at C-9 or after the cyclization. Investigations of the biosynthetic pathway to the cyclic carbonimidic dichlorides were performed by Simpson et al. in 2004 using further feeding experiments [139].

Biological Activity
In general, marine isonitriles have a weak to moderate activity in mammalian cell cytotoxicity assays (10-100 µM) against a broad range of cell lines [15]. Toxicity studies of simple isonitriles revealed surprisingly low toxicity with oral and subcutaneous toxic doses up to LD 50 = 5 g/kg [159].
Ireland and coworkers reported on the antibacterial activity of kalihinols A (Kol1), F (Kol21), G (Kol24), J (Kol13), X (Kol4), Y (Kol9), 10-formamido-kalihinol F (Kol22), 15-formamido-kalihinol F (Kol23) and kalihinene (Ken1) on the basis of inhibition of the bacterial folate biosynthesis as well as on the growth inhibition of Bacillus subtilis [109]. The most potent substances tested were the pyranyl-type kalihinols Y (Kol9) and X (Kol4), each showing an MIC of 1.56 µg/mL. Nevertheless, negative results of selectivity tests for the folate biosynthesis inhibition mechanism strongly suggest an additional mechanism of action for the pyranyl-bearing derivatives. The furanyl type kalihinols F (Kol21) and G (Kol24) and kalihinene (Ken1) on the other hand are more selective inhibitors than the pyranyl type kalihinols. The substitution pattern at C-10 seems to be important for the potency which becomes apparent in the loss of activity for kalihinol A (Kol1) which just differs in the orientation of the isonitrile group at C-10 from the highly active kalihinols Y (Kol9) and X (Kol4) which bear an exo-methylene and an isothiocyanate group, respectively. Finally, the presence of a formamido-substituent, irrespective of its position, is accompanied by a significiant decrease of activity which may be caused by a diminished cellular uptake [109].
The most promising biological activity of marine isonitriles and their related compounds is their antimalarial activity. A large part of the humanity lives in malaria endangered regions. In 1996, Wright and König gave an overview of the antimalarial effects [115].
Isocycloamphilectenes Ica 1, 2, 3, 4 and 9, cycloamphilectenes Cam 5 and 7, amphilectenes Amp 13, 15, 16, 18, 20 and 22, isoneoamphilectene Ina1, eudesmanes Eu17 and Eu21 and axisonitrile-3 (Sp2) all demonstrated significiant in vitro activity against the malaria parasite Plasmodium falciparum [115,162]. Investigations by Wright and König revealed the isocycloamphilectenes diisocyanoadociane (Ica1) and (Ica4) to display the highest antimalarial activity and selectivity with IC 50 values of 4 nM and 9 nM, respectively. Structure-activity relationship studies for the amphilectane derivatives using computer-based molecular modeling resulted in the conclusion that the orientation of the C-4 side chain and the negative electrostatic potential in the region of C-7 have a major impact on the activity of these compounds. An α-orientation of the C-4 side chain as for amphilectenes Amp16, 18 and 22 results in a decrease of activity due to unfavourable steric influences, which also occur with the OH-group of compound Amp20. An isonitrile group at C-7 shows best results for the antimalarial activity. Another functional group at this center or a migration of the ∆ 11,20 -exo-double bond of Amp13 to ∆ 11,12 (Amp15) causes a distinct loss of antiplasmodial activity, whereas the KB cytotoxicity stays almost unaffected. On the other hand, the nature of the C-20-functionality seems to have no influence on the activity [162]. In agreement with these findings are the results of Chanthathamrongsiri et al. who proved the C-8 isonitrile Amp2 to have a tenfold higher antiplasmodial activity than the isocyanate (Amp8) and the isothiocyanate Amp9. The C-7-formamide Amp17 was inactive [123].
In a series of investigations Wright et al. showed that the high antiplasmodial activity of Ica1 (IC 50 = 4 nM), Amp13 (IC 50 = 47 nM) and Cam5 (IC 50 = 24 nM) may arise from the inhibition of detoxification processes in P. falciparum. Ica1 and, to a lesser degree, Amp13 and Cam5, inhibit the crystallization of free-heme (ferriprotoporphyrin IX) to hemozoin which may cause a colloidal osmotic instability in P. falciparum. Additionally, a concomitant impairment of H 2 O 2 detoxification processes may arise [41,161,163].
Both effects are attributed to an interaction of the marine isonitriles with hemoglobin in a cavity close to heme [41]. Initial studies proposed a direct binding to the free heme iron [161].
In 2015, Daub et al. proved kalihinol B (Kol) to exhibit antiplasmodial activity (IC 50 = 8.4 nM for the wild type of the 3D7 strain of P. falciparum and 4.6 nM for chloroquine-resistant Dd2 strain) in a similar range as kalihinol A (Kol1). The group developed a twelve-step synthesis towards kalihinol B (Kol20), thus establishing a synthetic route to other kalihinol derivatives for further antiplasmodial testing [164].
As is obvious from the promising activities against P. falciparum, the marine isonitriles may well be viewed as attractive lead structures for new drugs against malaria.

Conclusions
Since the last review on marine isonitriles in 2000, the number of isolated marine isonitriles and their related compounds has continued to increase steadily ( Figure 41). Today, the marine isonitriles and their derivatives are still very interesting molecules in terms of their structure, their biosynthesis, and their potential application in the biomedical field. Although it can safely be assumed that the malaria parasite Plasmodium falciparum and the isonitrile-bearing marine organisms have never met in recent evolution, the pronounced antimalarial activity of natural isonitriles is one of several reasons to continue research on these odd, yet fascinating molecular gemstones from the oceans.

Overview about All Marine Isonitriles and Their Related Compounds
All known marine isonitriloids are listed in the following table (Table 1). Psammaplin B (Mis47) Psammaplysilla purpurea 1991 [150]