The reported investigations revealed the purification of various classes of sesquiterpenes that are substituted by isonitrile or isothiocyanate functionalities, including mono-, bi-, and tri-cyclic skeletons with 3-, 5-, 6-, and/or 7-membered rings (Figure 1 and Table 2). Frequently, formamide derivatives were reported along with both isothiocyanate and/or isonitrile moieties. Isonitrile-containing metabolites have been reported from some species belonging to Penicillium and Axinella genera [30]. Several reports stated their characterization from Acanthella. It was reported that A. cavernosa (Dendy, 1922) can convert cyanide and thiocyanate for isocyanide and isothiocyanate biosynthesis, which could be attributed to the presence of rhodanese or the equivalent enzyme [31]. Therefore, thiocyanate was postulated to be the precursor for the isothiocyanate moiety in terpenes by direct utilization or oxidative desulphurization of cyanide, conversion to isocyanide terpenes, and reinsertion of sulfur [31].

In 1987, l-isocyanoaromadendrane (3) was reported as a novel isonitrile sesquiterpene from the fish toxic CH₂Cl₂ fraction of A. acuta using SiO₂CC (silica gel column chromatography), assigned by spectral and chemical methods [34].

Furthermore, 5 and 9 were isolated from Japanese A. cavernosa by SiO₂ CC and RP-HPLC and identified by different spectroscopic methods [40]. Ximaocavernosin O (11) was isolated from an A. cavernosa Et₂O fraction using silica gel/MCI/Sephadex LH-20/(RP)-HPLC/chiral-phase HPLC and was characterized by spectroscopy, X-ray diffraction, and QM-NMR analyses as well as Mosher’s method and TDDFT-ECD calculations [33]. Compound 11 is similar to 2 and was formerly reported from nudibranch Hexabranchus sanguineus with a C-10 phenyl urea fragment instead of a C-10 formamide in 2 [33]. New aromadendrane-type sesquiterpenoids, 16 and 12, were purified from the Hainan A. cavernosa using SiO₂/Sephadex LH-20 CC/chiral-phase HPLC by Shen et al.. Their configurations were elucidated based on spectral, TDDFT-ECD, and X-ray analyses and optical rotation measurements. Compound 16 with a 2S/4S/5R/6S/7S configuration ([α]_D +169.6]) is identical to 2β-hydroxyaromadendr-1(10)-en-9-one ([α]_D ^_186]) except for the optical rotation (Figure 2) [33].

Spiroaxane skeletons containing sesquiterpenes are of rare natural occurrence. Compound 19, a new sesquiterpene isocyanide with a spiroaxane (spiro [5,6] decane) skeleton was obtained from Chinese Acanthella sp., which is a 3-oxo derivative of 17 [41]. Additionally, 23 is a spiroaxane sesquiterpene with a C-6 isocyanate and was purified and characterized by Jumaryatno et al. from A. cavernosa specimens collected from Coral gardens/Gneerings reef/Mooloolaba/Australia and from A. klethra collected from Pelorus Island, Queensland, in addition to 17 [19,37].

Additionally, 23–30 were isolated from A. cavernosa Et₂O fractions using silica gel/MCI/Sephadex LH-20/(RP)-HPLC/chiral-phase HPLC and characterized by spectral, X-ray diffraction, and QM-NMR analyses as well as Mosher’s method and TDDFT-ECD calculations [33]. Compounds 23–30 are spiroaxane derivatives involving compounds with C-6 isothiocyanate (e.g., 23–25) and formamide or a 1-phenethyl urea fragment (e.g., 26–30) [33] (Figure 3). In 2019, Wu et al. reported the purification of axamide-3 (32) from the acetone fraction of A. cavernosa collected from Xidao Island (Hainan Province, China) which was characterized by NMR spectral data and optical rotation [48].

Acanthellin-1 (34) is a bicyclic sesquiterpene with isopropylidene and isonitrile moieties. It was separated as an optically active oil from the ether fraction of the acetone extract of A. acuta collected from the Bay of Naples using SiO₂CC, and was characterized by NMR and chemical methods, as well as optical rotation [30] (Figure 4). A chromatographic investigation of A. klethra collected from Pelorus Island, Queensland, yielded sesquiterpenoids with isothiocyanate and isonitrile groups, i.e., 42, 44, and 45, that were assigned by spectral and X-ray analyses. Compounds 42, 44, and 45 are of eudesmane-type and are related to 34. Compounds 45 and 44 are different in stereo-configuration at C-7 [19,44]. Additionally, 39 and 43 are in the bicyclic cis-eudesmane class of sesquiterpenes, possessing isocyanate and isothiocyanate functionalities, respectively, and were purified and specified from A. acuta [47], whereas 35 is a stereoisomer of 5 [46].

Axiriabiline A (38) was obtained from the acetone fraction of A. cavernosa collected from Xidao Island (Hainan Province, China) and characterized by NMR spectral data and optical rotation [48]. Burgoyne et al. (1993) purified two new sesquiterpenoid acanthenes B and C (35 and 37) along with 40 and 42–44 from the hexane fraction of unidentified Acantbella species using SiO₂ flash CC/HPLC. The compounds were characterized by spectral analyses [46].

In 2000, Clark et al. isolated a new sesquiterpene, 46, that has a 1R/6R/7S/10R configuration and C-10 isothiocyanato functionality and [α]_D +3 [45]. In addition, Nogata et al. purified a new sesquiterpene, 65, and the known 67 from A. cavernosa EtOH extracts utilizing SiO₂/Sephadex LH-20/ODS HPLC. These compounds were assigned based on spectral data and chemical transformations [49] (Figure 5). Compound 65 has a C-10 formamido functionality instead of the C-10-OH in 67 [49].

New cadinane-type sesquiterpenoids, ximaocavernosins A–G (49–56), were isolated from A. cavernosa Et₂O fractions using SiO₂/MCI/Sephadex LH-20/(RP)-HPLC/chiral-phase HPLC and characterized by spectroscopy, X-ray diffraction, and QM-NMR analyses as well as Mosher’s method and TDDFT-ECD calculations [33]. Compounds 49–56 have cadinane frameworks with a Δ^5,6 double bond and a C-10 isothiocyanate but differ in stereochemistry and oxidation patterns [33]. In 2019, Wu et al. reported the purification of 10-formamido-4-cadinene (65) from the acetone fraction of A. cavernosa collected from Xidao Island (Hainan Province, China), which was characterized by NMR spectral data and optical rotation [48]. New cadinane-type sesquiterpenoids, 49 and 68–73, were purified from Hainan A. cavernosa using SiO₂/Sephadex LH-20 CC/chiral-phase HPLC (Figure 6). Their configurations were elucidated based on spectral, TDDFT-ECD, and X-ray analyses and optical rotation measurements. Maninsigin D and ximaocavernosin Q were obtained as racemic forms, which were separated into their enantiomers [(+)-68/(−)-69 and (+)-70/(−)-71] using chiral-phase HPLC [43].

New axane sesquiterpenoids, 74 and 75, in addition to 77, were separated from the antifungal hexane fraction of A. cavernosa collected from the Hachijo-Jima Islands using flash CC/sephadex LH-20/HPLC. They were elucidated based on spectral data [23]. Compound 74 is a rare oxygenated tricyclic sesquiterpene cyanide belonging to axane-type sesquiterpenes [23]. Furthermore, 66, 75, 76, and 85 were isolated by SiO₂ CC and RP-HPLC and identified by alpha-D, spectral data, and chemical methods from Japanese A. cavernosa [40]. Additionally, the new epimaaliane sesquiterpene 79, along with 78, were specified from the antimicrobial acetone extracts of A. pulcherrima using spectral and optical rotation measurements. Compound 79 is an enantiomer of 78 with an opposite [α]_D value and differs at the ring junction [35]. Burgoyne et al. purified epimaaliane-type sesquiterpenes 80 and 81 from the hexane fraction of an unidentified Acantbella species using SiO₂ flash CC and HPLC. The compounds were characterized by spectral analyses [46] (Figure 7).

Notably, Shen et al. proposed that 12, 16, 49, 68–73, and 84 originate from E,E-farnesyl diphosphate (E,E-FPP), as illustrated in Scheme 1 [43].

Diterpenoids are among the common metabolites reported from various Acanthella species. These compounds are characterized by the existence of nitrogenous functionalities such as isothiocyanato, isocyano, and/or formamido groups. These diterpenes are classified into two major classes, kalihinanes and biflorane derivatives, according to the 8C side chain (Figure 8 and Table 3). Kalihinanes have a decalin frame structure with C-7-attached dihydropyran, tetrahydropyran, or tetrahydofuran moiety. Additionally, these rings may carry various substituents such as OH, Cl, isothiocyanato, isocyano, and formamido groups or chlorine. They include kalihinenes, kalihinols, and kalihipyranes. Kalihinols are spilt into two main categories, tetrahydrofuran (I) and tetrahydropyran (II) groups, according to the C-7 substitution. Commonly, they have trans-decalin framework with a C-4 or C-5 tertiary alcohol and an isocyanate moiety at C-10 and/or C-5. The first group has a tetrahydrofuran moiety featuring NCS, NC, or Cl at C-15, or the gem-dimethyl is substituted by an isopropenyl moiety, whereas the tetrahydropyran group possesses Cl atom at C-14. Kalihinenes have a Δ⁴-trisubstituted double bond and possess similar structural features to kalihinols, while biflorane diterpenoids are a class of kalihinane diterpenes featuring a linear eight-carbon open chain substituent at C-7. Biosynthetically, these metabolites are proposed to result from the cyclization of the biflorane skeleton (trans or cis form) and geranylgeranyl pyrophosphate [51]. Their stereochemistry has been determined using spectroscopic, X-ray and/or CD analyses, as well as chemical and computational methods.

Kalihinol A (88) was the first reported member of this diterpenoid class in 1984 [52] (Table 3). It is a tricyclic diterpenoid belonging to group II, containing isocyano, hydroxyl, and chlorine moieties. It was separated from the CCl₄ extract of Acanthella sp. and characterized by NMR and X-ray analyses [52] and its configuration was assigned as 1S/4R/5R/6S/7S/10S/11R/14S using the CD exciton chirality method [65] (Figure 9).

In 1987, Chang et al. purified and characterized isocyano-diterpenoids kalihinols A–H (88, 94, 96–98, 100, 108, and 111) and X–Z (125, 127, and 130) from Acanthella sp. obtained from Guam and Fiji using spectral and X-ray analyses. They differ in the C-7 attached moiety; the tetrahydropyran with C-14 chlorine (e.g., 25, 88, 98, 130, and 127); or the tetrahydro-furanyl moiety, with 15-NC (e.g., 97 and 100), 15-NCS (e.g., 108), 15-C1 (e.g., 94), or isopropenyl replacing gem-dimethyl (e.g., 96) [22] (Figure 10).

Additionally, 101 was isolated as colorless needles from A. carvenosa by flash chromatography and HPLC. It resembles 100 with a difference in the substitution at C₄ and C₅ [53]. Additionally, new members of the kalihinols family, 114 and 116, along with 125 and 127, were purified from A.cavernosa collected from Thailand by SiO₂ CC and HPLC and identified by extensive NMR data. Compound 114 is similar to 125 with a C-5-N-formyl instead of the isothiocyanoate moiety in 125 [18]. Compounds 117 and 118 are two novel C-4 formamido analogs of isokalihinols reported from the South China Sea specimen of A. cavernosa. They have a trans-decalin ring at C-7 of the tetrahydrofuran and tetrahydropyran rings, respectively. Compound 117 possesses a C-15 chlorine atom and a C-10 isothiocyanato group, whereas 118 is an example of tetrahydropyran-type isokalihinol. They have 1S/4S/5S/6S/7S/10S/11R/14S and 1S/4S/5S/6S/7S/10S/11R/14R configurations, respectively [16]. From Okinawan Acanthella sp., new members of kalihinane-type diterpenes, 110, 115, and 129, were purified from the EtOAc fraction using SiO₂ CC and HPLC. Compound 129 is of tetrahydropyran type and is closely similar to 127, with a trisubstituted olefinic bond in 129 instead of the exo-methylene group in 127, while 110 and 115 have three and two isothiocyano groups, respectively [57] (Figure 11).

In 1994, Trimurtulu et al. reported new diterpene isonitriles, 103 and 113, from A. carvenosa collected from The Seychelles. Compound 103 differs from 101 in the trans-decalin ring system configuration, whereas 113 has an isothiocyanate group instead of one of the C-10 isonitrile groups of 103 [59]. Besides, Xu et al. reported new diterpenoids, kalihinols O–T (119–124), together with 88, 98, 109, 115, and 126, from A. cavernosa in the South China Sea using SiO₂ and Sephadex LH-20 CC [16]. Their structures and stereo-structures were determined by NMR/CD/X-ray analyses [16]. Compound 119 is structurally similar to 98, with a C-10 isothiocyanate instead of a C-10 isonitrile in 98, whereas 120 is an isocyanato analog of 88 and 123 is a C-5 formamide analog of 126. Furthermore, 121 and 122 are the C-14 epimers of 120 and 115, respectively, and 124 is the C-15-isothiocyanato analog of 97 [16]. Clark et al. in 2000, purified a new kalihinol-type diterpenoid, 8-OH-isokalihinol F (102), from A. cavernosa obtained from Heron Island, Great Barrier Reef, Australia, which was structurally similar to 101 with an additional C-8 OH [45]. In addition, new formamide analogs, 104 and 106, were purified by Bugni et al. in 2004 from two Philippine A. cavernosa specimens. They featured formamide moieties at C-10 and C-15, respectively, instead of the isonitrile in 100 [21]. Furthermore, a new kalihinol diterpene, 126, was isolated from Hainan Acanthella sp. by SiO₂ and Sephadex LH-20 CC and was assigned as a C-10 epimer of 127 [42]. Additionally, new α-acyloxy-amide-substituted diterpenoids, kalihiacyloxyamides A–H (131–138), were separated from South China Sea A. cavernosa EtOAc fractions using SiO₂/Rp-18 CC/RP-HPLC that were elucidated based on spectral, X-ray, and CD analyses (Figure 12). These metabolites featured isobutyl amide (e.g., 131 and 132), iso-amyl ester (e.g., 133, 134, 137, and 138), and phenethyl ester (e.g., 135 and 136) groups [62].

The first member of this group is kalihinene (139), which was purified from an A. klethra EtOH extract using SiO₂ CC/Develosil ODS-5 CC/HPLC and assigned by NMR and X-ray analyses [23]. Furthermore, compounds 143 and 144 were reported as novel monounsaturated kalihinane class diterpenes derived from A. Cavernosa toxic CH₂Cl₂ extracts against Artemia salina and Lebistes reticulatus using VLC/Flash/Rp-18 CC. These two compounds are diastereoisomers of 139 that feature a trans-decalin skeleton instead of the trans-decalin skeleton of kalihinene. On the other hand, 143 and 144 are epimers at C-10 [54]. Additionally, 145 and 146 are tertrahydropyran/trans-decalin and tetrahydrofuran/cis-decalin analogs bearing formamido groups at C-10 and C-15, respectively (Figure 13). Kalihinene E (145) is a C-14 epimer of 148 with 1S/6S/7S/10S/11R/14R configuration [63]. In 1994, Rodríguez et al. purified new diterpenoids belonging to kalihinene and 6-OH kalihinene groups (148 and 150–156), along with 139 from A. cavernosa collected from a Fijian location. These compounds were characterized based on spectral and X-ray analyses as well as biogenetic evidence [51]. Additionally, 140–142 are new metabolites reported from A. carvenosa collected from The Seychelles [59]. Compound 140 is a C-1 isomer of 139, 141 has C-15 isothiocyanate instead of C-15 isonitrile in 140, and 142 is an isomer of 140 and 139 [59].

Kalihipyran (164) is a tricyclic kalihinene-type diterpene with a C-7 isopropenyl-containing dihydropyran moiety and a C-10 isonitrile [59]. Compound 167 is an isomer of 165 with cis-decalin [63], while 166 has a C-15 chlorine atom [55,56]. In 2022, Wang et al. purified new kalihinane diterpenoids, kalihioxepanes A–G (157–163), from South China Sea A. cavernosa by the means of SiO₂/Sephadex LH-20/HPLC. The structures were elucidated by spectral and X-ray analyses, in addition to quantum chemical calculation methods [64] (Figure 14).

These metabolites possess a rare C-7-attached oxepane ring with a C-14 Cl atom. Compounds 157–160 have a trans decalin skeleton; however, 161–163 have a cis-decalin skeleton with C-10 isonitrile (e.g., 157 and 158) and formamide (e.g., 159–163) groups [64]. Wang et al. proposed that 157–163 are biosynthesized using geranylgeraniol as a precursor (Scheme 2). The latter undergoes a series of reactions, including cyclization, double-bond migration, nucleophilic addition, and oxidation reactions, to give epoxide biflorane (A, the key intermediate). Nucleophilic substitution of biflorane forms 157 and 158. After that, hydration generates 159–161 which are then dehydrated to give 162 and 163, respectively [64].

From the Japanese A. cavernosa, biflora-4,9,15-triene (168) was separated, which is a rare biflorane diterpene related to 66, by replacing the methyl hydrogen of the isopropyl group of 66 with a prenyl group [40]. In 2012, Xu et al. reported 169–172 from CH₂Cl₂ extracts of South China Sea A. cavernosa, bearing a C-10 formamide group that varied in the decalin moiety (cis or trans) configuration and nature of C-7-linked side chain [63] (Figure 15). Their structures were assigned by spectral and X-ray analyses. Compounds 169, 170, and 172 are trans-decalin derivatives, with a C-7 isoprenoid unit, a mono-olefinic isoprenoid sidechain, and a trisubstituted epoxide in the side chain, respectively. In contrast, 171 had a cis-decalin moiety [63].

Investigation of A. cavernosa DCM/MeOH extracts led to the separation of two oxirane analogs with a trans-decalin framework, 173 and 174, featuring a trisubstituted epoxide and a terminal epoxide group in the side chain, respectively. Compound 174 was suggested to be a precursor of the kalihipyran skeleton [45]. Clark et al. proposed that the biosynthesis of pyranyl and furanyl kalihinols involves epoxidation of the bifloradiene precursor’s terminal double bond by a nucleophilic attack at either epoxide end by a cyanide ion to form a hydroxyisocyanide. The latter initiates cyclisation to afford a bicyclic system (Scheme 3). Compounds 173 and 174 are alternative epoxidation products. Compound 174 was suggested to be a precursor of the kalihipyran skeleton [45].

Several reports have stated the isolation of different classes of alkaloids from this genus. It is noteworthy that bromopyrrole alkaloids are the dominant type reported from the species of this genus. Oroidin 177 is the first member of pyrrole 2-aminoimidazole alkaloids. These alkaloids were reported to have significant bioactivities, as well as chemical defense against predator fish.

In 2010, Hammami et al. purified a novel bromopyrolimidazole analog, 178, along with 177 from Tunisian A. acuta diethyl ether extracts [25]. Four bromo-pyrrole alkaloids, including novel alkaloid hanishin (179) in addition to 175–177, were isolated from A. carteri collected from the northern coast of Hanish Island, Yemen, South Red Sea, by Mancini et al. Compounds 177 and 179 are members of the oroidin family of alkaloids that are considered condensation products of prolines. Compound 179 was proposed to be derived from aminoimidazolinone (I) or amino acid (II) intermediates through 1N-C9 cyclization with subsequent side-chain oxidative breakdown [66] (Scheme 4).

Mattia et al. purified 180 as a brominated alkaloid from Red Sea A. Aurantiaca BuOH extracts. The compound features an aminooxodihydroimidazole ring linked to a pyrroloazepine group via a double bond (Figure 16) [68]. Compounds 180 and 181 were obtained from A. aurantiaca BuOH extracts using Sephadex LH-20 and crystallization and were characterized by spectral and X-ray analyses [67]. In 2014, Macabeo and Guce reported the bromopyrrole-imidazole alkaloids 182–184 from CH₂Cl₂-MeOH extracts of A. carteri from The Philippines [17], while 185 is a pyrrole alkaloid isolated from the n-BuOH fraction of Acanthella sp. using Sephadex LH-20/Rp-18 CC [69].

A series of synthetic reactions including Suzuki–Miyaura coupling and debromination resulted in natural analogs 186 and 187, in addition to new synthetic derivatives (−)-4-bromo-5-phenylphakellin and (−)-4,5-diphenylphakellin. It was found that the C-5 Br substitution with phenyl or H led to a loss in activity, revealing that the C-5 Br is important for α2B adrenoceptor agonistic activity (Scheme 5) [70].

Furthermore, 190 was purified from A. carteri using Sephadex LH-20/SiO₂ CC, giving a bright-orange color with a diazotized benzidine. The compound was characterized by NMR and X-ray analyses, as well as chemical methods. Compound 190 is a 6R/10S brominated alkaloid with a fused C-C pyrrole linkage to the cyclic guanidine core belonging to the 189 series [71].

In 2002, Wiese and his group reported the synthesis of 190 using dihydrooroidin that is converted to 188 (Scheme 6). Then, thermal rearrangement of 188 in the presence of K₂CO₃ produces 190 [73].

Additionally, Grkovic et al. were able to separate tricyclic-guanidine-containing alkaloids, including a new analog mirabilin K (192), along with 191 and 193, from A. cavernosa collected in Southwestern Australia using diol flash chromatography/Rp-18/HPLC. The compounds were characterized by spectroscopic analyses and optical rotation measurements. Compound 192 has a 4S*/7S*/9R*/11S*/12R* configuration, which differs from 191 in the C9-CH₃ group and with the presence of a N-substituted methine group (Figure 17) [24]. Furthermore, 194 and 195 were obtained by Fan et al. from the acetone extracts of A. cavernosa collected from the South China Sea [50].

Diketopiperazines, including the rare cyclo(L-Phe-L-Thr) and cyclo(L-Tyr-L-Ile) (196–202), along with decarboxylated amino acid 207 and deoxyribonucleotides 203–206, were reported and characterized from Fijian A. cavernosa (Figure 18). Their L-L absolute configuration was assigned based on an NMR and CD comparison with synthetic L-L analogs, as well as optical rotation measurements [72].

In 2008, Qui et al. reported the purification of three new nor-steroids, 208–210, along with the known steroids 211–214 from the petroleum ether fraction of A. cavernosa obtained from Hainan Island, China, using SiO₂ CC/HPLC. The new steroids are related to A-ring-contracted steroid analogs featuring carbonyl and ketone groups located at C-3 and C-4; they differ in their C-17 side chains [74] (Figure 19). In addition, 215 was obtained from the acetone extract of the same sponge collected from the South China Sea [50].

Compound 216 was separated from A. vulgata acetone extracts using an MgO column and crystallization from petroleum ether. The compound belongs to carotenoids, as it has a polyene chain with terminal aromatic moieties on both ends [75] (Figure 20). Mancini et al. were able to purify and characterize 219, a novel methyl-branched glycerol enol ether, and the related linear analog 218 from A. carteri obtained from Southern Red Sea Hanish Islands by utilizing flash CC/HPLC and spectral and chemical methods [76]. Compound 219 has an additional methyl group at C-2 of the sidechain compared to 218, and both have a 2`S configuration [76]. In 2010, Hammami et al. separated the sesterterpene 217 and cerebrosides 220–222 from Tunisian A. acuta diethyl ether extracts [25], whereas 226 was purified from the Chinese A. cavernosa by Fan et al. [50].