Spirocyclic Motifs in Natural Products

Spirocyclic motifs are emerging privileged structures for drug discovery. They are also omnipresent in the natural products domain. However, until today, no attempt to analyze the structural diversity of various spirocyclic motifs occurring in natural products and their relative populations with unique compounds reported in the literature has been undertaken. This review aims to fill that void and analyze the diversity of structurally unique natural products containing spirocyclic moieties of various sizes.


Introduction
Natural products play the central role in drug discovery [1] due to their inherent biological activity and because have a wide span of structural diversity. Spirocyclic compounds have also occupied a special place in medicinal chemistry [2]. Spirocycles are thought to possess a good balance of conformational rigidity and flexibility to be, on one hand, free from absorption and permeability issues characteristic of conformationally more flexible, linear scaffolds. On the other hand, spirocycles are more conformationally flexible compared to, for example, flat aromatic heterocycles and can adapt to many proteins as biological targets; thus, increasing the chances of finding bioactive hits [3]. Spirocycles are distinctly three-dimensional and initial hits can be further optimized via manipulation of the molecular periphery whose three-dimensional positioning is well defined [4]. We thought it worthwhile to gain insight into the structural diversity of naturally-occurring spirocyclic compounds in relation to the information of their biological activity which would provide a new angle for designing novel bioactive, druglike compounds. Modern literature features a limited number of reviews devoted to total syntheses of spirocyclic natural products [5], including one for spirolactones [6] and one for spirooxyindoles [7]. Illustrative examples of approved natural-product drugs containing a spirocyclic motif include antifungal drug griseofulvin (1) and diuretic drug spironolactone (2). Interesting related compounds that have not achieved clinical approval include isochromanquinone antibiotic griseusin B (3) [8,9] and spirotriprostatin (4) [10] (Figure 1).
For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity of structurally unique and well characterized (i.e., those whose structures were assigned using modern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder databases, or the Dictionary of Natural Products (DNP). The occurrence of various ring combinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is presented in Table 1. For the purpose of the analysis presented in this review, we considered the chemical diversity f structurally unique and well characterized (i.e., those whose structures were assigned using odern analytical techniques) spirocyclic compounds registered in the ChemBL or SciFinder atabases, or the Dictionary of Natural Products (DNP). The occurrence of various ring mbinations (A = any atom, mostly carbon or oxygen) selected for discussion in this review is resented in Table 1. pose of the analysis presented in this review, we considered the chemical diversity nique and well characterized (i.e., those whose structures were assigned using cal techniques) spirocyclic compounds registered in the ChemBL or SciFinder the Dictionary of Natural Products (DNP). The occurrence of various ring = any atom, mostly carbon or oxygen) selected for discussion in this review is le 1. ysis presented in this review, we considered the chemical diversity l characterized (i.e., those whose structures were assigned using spirocyclic compounds registered in the ChemBL or SciFinder of Natural Products (DNP). The occurrence of various ring ostly carbon or oxygen) selected for discussion in this review is ring combinations in the spirocyclic natural products analyzed in this this review, we considered the chemical diversity .e., those whose structures were assigned using ounds registered in the ChemBL or SciFinder ucts (DNP). The occurrence of various ring xygen) selected for discussion in this review is Considering the uneven distribution of the ring combination occurrence statistics (Table 1), the present review is structured according to the size of the [x.y.0] spirocyclic system. The review aims to cover either rare representatives of the spirocyclic systems that seldom occur in the natural product realm or only structurally-unique, representative compounds for those spirocyclic systems that are more widely populated with natural products reported in the literature, with an emphasis on their associated biological activities and the solid structure assignment techniques employed (structures assigned solely based on mass-spectrometric measurements are not taken into account).

[2.4.0] Spirocyclic System
Spirocyclic motifs containing a cyclopropane unit were found in some sesquiterpenes (5-7) which were isolated from the essential oils of South-American Schinus terebinthifolius fruit [11] (Figure 2). Considering the uneven distribution of the ring combination occurrence statistics (Table 1), the present review is structured according to the size of the [x.y.0] spirocyclic system. The review aims to cover either rare representatives of the spirocyclic systems that seldom occur in the natural product realm or only structurally-unique, representative compounds for those spirocyclic systems that are more widely populated with natural products reported in the literature, with an emphasis on their associated biological activities and the solid structure assignment techniques employed (structures assigned solely based on mass-spectrometric measurements are not taken into account).

[2.4.0] Spirocyclic System
Spirocyclic motifs containing a cyclopropane unit were found in some sesquiterpenes (5-7) which were isolated from the essential oils of South-American Schinus terebinthifolius fruit [11] ( Figure 2). In 2017, a novel condensed [2.4.0] spirocycle (8) was reported [12]. It was isolated and characterized among the secondary metabolites of the Helminthosporium velutinum plant and was named cyclohelminthol X ( Figure 3). This compound was shown to inhibit the growth of a human colon adenocarcinoma (COLO201) cell line with moderate potency (IC50 = 16 μM), and, much more potently (IC50 = 0.35 μM)-leukemia HL60 cell line [12]. In 2017, a novel condensed [2.4.0] spirocycle (8) was reported [12]. It was isolated and characterized among the secondary metabolites of the Helminthosporium velutinum plant and was named cyclohelminthol X ( Figure 3). This compound was shown to inhibit the growth of a human colon adenocarcinoma (COLO201) cell line with moderate potency (IC 50 = 16 µM), and, much more potently (IC50 = 0.35 µM)-leukemia HL60 cell line [12].  Bioassay-guided separation of Valerianae Radix plant extract led to the isolation and characterization of valtrate (9), which inhibited Rev protein mediated transport of HIV-1 from the nucleus to cytoplasm (Figure 4). This compound also inhibited p-24 production of HIV-1 virus without any notable cytotoxicity displayed against MT-4 cells. The presence of the chemically labile oxirane ring as part of the generalized [2.4.0] spirocyclic system is likely critical for the observed inhibition, as 9 was shown to covalently interact with cysteine [13]. Bioassay-guided separation of Valerianae Radix plant extract led to the isolation and characterization of valtrate (9), which inhibited Rev protein mediated transport of HIV-1 from the nucleus to cytoplasm ( Figure 4). This compound also inhibited p-24 production of HIV-1 virus without any notable cytotoxicity displayed against MT-4 cells. The presence of the chemically labile oxirane ring as part of the generalized [2.4.0] spirocyclic system is likely critical for the observed inhibition, as 9 was shown to covalently interact with cysteine [13].
Additional two compounds (31 and 32) containing this and another ([4.4.0]) spirocyclic system are discussed in Section 7 of this review. Bioassay-guided separation of Valerianae Radix plant extract led to the isolation and characterization of valtrate (9), which inhibited Rev protein mediated transport of HIV-1 from the nucleus to cytoplasm (Figure 4). This compound also inhibited p-24 production of HIV-1 virus without any notable cytotoxicity displayed against MT-4 cells. The presence of the chemically labile oxirane ring as part of the generalized [2.4.0] spirocyclic system is likely critical for the observed inhibition, as 9 was shown to covalently interact with cysteine [13]. Additional two compounds (31 and 32) containing this and another ([4.4.0]) spirocyclic system are discussed in Section 7 of this review.

[2.5.0] Spirocyclic System
This group of spirocyclic natural products is represented by sesquiterpenoids illudins M and S (10 and 11, respectively) isolated from fungi, including the highly poisonous Jack-o′-lantern mushroom Omphalotus illudens. Compound 11 is currently in Phase II clinical trials against ovarian, prostate, and gastrointestinal cancers ( Figure 5).

[2.5.0] Spirocyclic System
This group of spirocyclic natural products is represented by sesquiterpenoids illudins M and S (10 and 11, respectively) isolated from fungi, including the highly poisonous Jack-o -lantern mushroom Omphalotus illudens. Compound 11 is currently in Phase II clinical trials against ovarian, prostate, and gastrointestinal cancers ( Figure 5). nucleus to cytoplasm (Figure 4). This compound also inhibited p-24 production of HIV-1 virus without any notable cytotoxicity displayed against MT-4 cells. The presence of the chemically labile oxirane ring as part of the generalized [2.4.0] spirocyclic system is likely critical for the observed inhibition, as 9 was shown to covalently interact with cysteine [13]. Additional two compounds (31 and 32) containing this and another ([4.4.0]) spirocyclic system are discussed in Section 7 of this review.

[3.4.0] Spirocyclic System
This is an exceedingly rare type of spirocyclic motif encountered among natural products. The only compound reported in the literature to date containing such a spirocyclic system presented as a combination of a β-lactone and a pyrrolidine ring (19) was isolated from marine-derived Streptomyces strain collected in the southern area of the Korean Jeju Island [19] (Figure 9). This structurally intriguing compound displayed antibacterial activity.

[3.4.0] Spirocyclic System
This is an exceedingly rare type of spirocyclic motif encountered among natural products. The only compound reported in the literature to date containing such a spirocyclic system presented as a combination of a β-lactone and a pyrrolidine ring (19) was isolated from marine-derived Streptomyces strain collected in the southern area of the Korean Jeju Island [19] (Figure 9). This structurally intriguing compound displayed antibacterial activity.  (15) and fumagillin (16).

[3.4.0] Spirocyclic System
This is an exceedingly rare type of spirocyclic motif encountered among natural products. The only compound reported in the literature to date containing such a spirocyclic system presented as a combination of a β-lactone and a pyrrolidine ring (19) was isolated from marine-derived Streptomyces strain collected in the southern area of the Korean Jeju Island [19] (Figure 9). This structurally intriguing compound displayed antibacterial activity.

[3.7.0] Spirocyclic System
This intriguing spirocyclic combination of four and eight-membered rings is represented in only four closely-related sesquiterpene bis-lactones, 21-24 ( Figure 11), isolated from poisonous plants in the Illicium genus grown in China [21]. These structures could also be viewed as possessing a [3.5.0] spirocyclic motif.

[3.7.0] Spirocyclic System
This intriguing spirocyclic combination of four and eight-membered rings is represented in only four closely-related sesquiterpene bis-lactones, 21-24 ( Figure 11), isolated from poisonous plants in the Illicium genus grown in China [21]. These structures could also be viewed as possessing a [3.5.0] spirocyclic motif.

[3.7.0] Spirocyclic System
This intriguing spirocyclic combination of four and eight-membered rings is represented in only four closely-related sesquiterpene bis-lactones, 21-24 ( Figure 11), isolated from poisonous plants in the Illicium genus grown in China [21]. These structures could also be viewed as possessing a [3.5.0] spirocyclic motif.
A [4.4.0] spirocyclic lactone moiety is found (in combination with a [2.4.0] spirocyclic oxirane) in limonoids 33-34, which were recently isolated from Trichilia connaroides ( Figure 13). For these compounds, some insights into a possible biosynthetic pathway have been provided. Likewise, these compounds were screened for various types of bioactivity and have been shown to inhibit NO production in a cellular model of inflammation (induced in RAW264.7 cell line with LPS) by 25.89% and 37.13% at 25 and 50 µM, respectively [32].
Rather   Figure 13). For these compounds, some insights into a possible biosynthetic pathway have been provided. Likewise, these compounds were screened for various types of bioactivity and have been shown to inhibit NO production in a cellular model of inflammation (induced in RAW264.7 cell line with LPS) by 25.89% and 37.13% at 25 and 50 μM, respectively [32]. Studies of secondary metabolite structures in endophyte fungus Penicillium purpurogenum unveiled a series of unique sesquiterpene lactone compounds (35)(36)(37) containing spirocyclic combinations of three five-membered rings ( Figure 14). All three compounds were screened against several cancer cell lines (melanoma M14, colon cancer HCT-116, glioma U87, ovary cancer A2780, stomach cancer BG-823, hepatoma Bel-7402, and lung cancer A549) and several pathogenic microorganisms (Mycobacterium spegmatis (ATCC70084), Staphylococcus aureus (ATCC25923), and Staphylococcus epidermidis (ATC12228)); however, no activity was detected at 50 μM [33].  Studies of secondary metabolite structures in endophyte fungus Penicillium purpurogenum unveiled a series of unique sesquiterpene lactone compounds (35)(36)(37) containing spirocyclic combinations of three five-membered rings ( Figure 14). All three compounds were screened against several cancer cell lines (melanoma M14, colon cancer HCT-116, glioma U87, ovary cancer A2780, stomach cancer BG-823, hepatoma Bel-7402, and lung cancer A549) and several pathogenic microorganisms (Mycobacterium spegmatis (ATCC70084), Staphylococcus aureus (ATCC25923), and Staphylococcus epidermidis (ATC12228)); however, no activity was detected at 50 μM [33].  In the course of the thorough structural investigation of a series of iridoid glycosides isolated from the Morinda citrifolia plant, a revised structure was assigned. In particular, dehydromethoxygaertneroside (42), dehydroepoxymethoxygaertnoside (43), and citrifolinoside A (44) were shown to be structurally distinct compounds, all of which, however, possessed a   In the course of the thorough structural investigation of a series of iridoid glycosides isolated from the Morinda citrifolia plant, a revised structure was assigned. In particular, dehydromethoxygaertneroside (42), dehydroepoxymethoxygaertnoside (43), and citrifolinoside A (44) were shown to be structurally distinct compounds, all of which, however, possessed a [4.4.0] spirocyclic lactone moiety ( Figure 16) [35].  In the course of the thorough structural investigation of a series of iridoid glycosides isolated from the Morinda citrifolia plant, a revised structure was assigned. In particular, dehydromethoxygaertneroside (42), dehydroepoxymethoxygaertnoside (43), and citrifolinoside A (44) were shown to be structurally distinct compounds, all of which, however, possessed a        Rather intriguing and unique is the structure of spirocyclic hydantoins possessing a furanose unit. One of the first representatives of these natural products (hydantocidine 52) was isolated from Streptomyces hygroscopicus ( Figure 20). Hydantocidine displayed herbicidal properties which were linked to its ability to inhibit adenylate succinate synthase [39].  Rather intriguing and unique is the structure of spirocyclic hydantoins possessing a furanose unit. One of the first representatives of these natural products (hydantocidine 52) was isolated from Streptomyces hygroscopicus ( Figure 20). Hydantocidine displayed herbicidal properties which were linked to its ability to inhibit adenylate succinate synthase [39]. Rather intriguing and unique is the structure of spirocyclic hydantoins possessing a furanose unit. One of the first representatives of these natural products (hydantocidine 52) was isolated from Streptomyces hygroscopicus ( Figure 20). Hydantocidine displayed herbicidal properties which were linked to its ability to inhibit adenylate succinate synthase [39].
Secondary metabolite investigation of the liquid culture of entomogenous fungus Isaria cateniannulata led to the identification of a new spirocyclic compound 74 containing a 1,6-dioxaspiro [4.4]nonane moiety ( Figure 27). The compound showed weak inhibitory activity against the HeLa cancer cell line [53].  Mycotoxins related to tryptoquialanine A (71) were isolated from Penicillium spp. and Aspergillus clavatus [51]. For tryptoquialanines, the biosynthetic pathway has been recently elucidated [25]. Another spirooxyindole lactone lactam compound 73 isolated from Coix lachryma-jobi L. has been recently reported and shown to possess activity against human lung cancer (A549) and colon carcinoma (HT-29 and COLO205) cell lines [52].
Secondary metabolite investigation of the liquid culture of entomogenous fungus Isaria cateniannulata led to the identification of a new spirocyclic compound 74 containing a 1,6-dioxaspiro [4.4]nonane moiety ( Figure 27). The compound showed weak inhibitory activity against the HeLa cancer cell line [53].
Spirocyclic [4.4.0] tetrahydrofurans are featured in a series of twelve natural products 75a-l dubbed bipolaricins ( Figure 28). These compounds are ophiobolin-type tetracyclic sesterterpenes from a phytopathogenic Bipolaris sp. fungus. They were tested for HMGCoA reductase inhibition as well as anti-inflammatory and cytotoxic activities. The biological activity discovered provided the basis for considering these compounds as leads for antiinflammation and antihyperglycemic therapy developments [54]. elucidated [25]. Another spirooxyindole lactone lactam compound 73 isolated from Coix lachryma-jobi L. has been recently reported and shown to possess activity against human lung cancer (A549) and colon carcinoma (HT-29 and COLO205) cell lines [52].
Secondary metabolite investigation of the liquid culture of entomogenous fungus Isaria cateniannulata led to the identification of a new spirocyclic compound 74 containing a 1,6-dioxaspiro [4.4]nonane moiety ( Figure 27). The compound showed weak inhibitory activity against the HeLa cancer cell line [53]. Spirocyclic [4.4.0] tetrahydrofurans are featured in a series of twelve natural products 75a-l dubbed bipolaricins ( Figure 28). These compounds are ophiobolin-type tetracyclic sesterterpenes from a phytopathogenic Bipolaris sp. fungus. They were tested for HMGCoA reductase inhibition as well as anti-inflammatory and cytotoxic activities. The biological activity discovered provided the basis for considering these compounds as leads for antiinflammation and antihyperglycemic therapy developments [54].     In terms of biological activity, the current data are mostly limited to cytostatic and antibacterial properties. The natural products isolated within the last 1-2 years are poorly investigated with regard to their biological properties.

[4.5.0] Spirocyclic System
Secondary metabolite investigation of Teucrium viscidum led to the identification of a [4.5.0] spirocyclic compound (77) possessing a unique skeleton [57]. A skeleton of similar complexity had only been featured once in the literature three decades before that [58] (Figure 31).   (77) possessing a unique skeleton [57]. A skeleton of similar complexity had only been featured once in the literature three decades before that [58] (Figure 31).  (77) possessing a unique skeleton [57]. A skeleton of similar complexity had only been featured once in the literature three decades before that [58] (Figure 31).   Another example of an all-carbon [4.5.0] spirocyclic system is provided by spirocarolitone (80), recently isolated from Ruptiliocarpon caracolito [60] (Figure 33).    Another example of an all-carbon [4.5.0] spirocyclic system is provided by spirocarolitone (80), recently isolated from Ruptiliocarpon caracolito [60] (Figure 33).    Another example of an all-carbon [4.5.0] spirocyclic system is provided by spirocarolitone (80), recently isolated from Ruptiliocarpon caracolito [60] (Figure 33).    New biologically active sesquiterpenoids 83-85 possessing an all-carbon [4.5.0] spirocyclic system were isolated from rhizomes of Acorus calamus (Figure 35). Compound 83 exhibited weak hepatoprotective activities against APAP-induced HepG2 cell damage [62].  Perhaps the most clinically advanced natural spirocyclic compound-spirocyclic benzofuran griseofulvin (88) isolated from Penicillium griseofulvum has been employed in clinical practice for therapy against ring worms [64] and was marketed by GlaxoSmithKline under the trade name Grisovin TM [65] (Figure 37).  More examples of bioactive [4.5.0] spirocyclic lactones are provided by abyssomicins (92a-c, Figure 39), which were isolated from Actinobacteria and shown to inhibit p-aminobenzoate biosynthesis [69]. Perhaps the most clinically advanced natural spirocyclic compound-spirocyclic benzofuran griseofulvin (88) isolated from Penicillium griseofulvum has been employed in clinical practice for therapy against ring worms [64] and was marketed by GlaxoSmithKline under the trade name Grisovin TM [65] ( Figure 37).  Perhaps the most clinically advanced natural spirocyclic compound-spirocyclic benzofuran griseofulvin (88) isolated from Penicillium griseofulvum has been employed in clinical practice for therapy against ring worms [64] and was marketed by GlaxoSmithKline under the trade name Grisovin TM [65] (Figure 37).  More examples of bioactive [4.5.0] spirocyclic lactones are provided by abyssomicins (92a-c, Figure 39), which were isolated from Actinobacteria and shown to inhibit p-aminobenzoate biosynthesis [69].  Perhaps the most clinically advanced natural spirocyclic compound-spirocyclic benzofuran griseofulvin (88) isolated from Penicillium griseofulvum has been employed in clinical practice for therapy against ring worms [64] and was marketed by GlaxoSmithKline under the trade name Grisovin TM [65] (Figure 37).  More examples of bioactive [4.5.0] spirocyclic lactones are provided by abyssomicins (92a-c, Figure 39), which were isolated from Actinobacteria and shown to inhibit p-aminobenzoate biosynthesis [69]. More examples of bioactive [4.5.0] spirocyclic lactones are provided by abyssomicins (92a-c, Figure 39), which were isolated from Actinobacteria and shown to inhibit p-aminobenzoate biosynthesis [69].
Antibacterial and antitumor compound lactonamycin Z (93) was isolated from Streptomyces sanglieri [70] and is an example of a [4.5.0] spirocyclic lactone embedded in a complex polycyclic system ( Figure 40).
Perenniporide A (102) was the only spirocyclic lactone derivative of the naphthalenone family of natural products perenniporides A-D isolated from solid cultures of a fungus Perenniporia sp. inhabiting the larva of Euops chinesis, a phytophagous weevil with high host specificity to the medicinal plant Fallopia japonica (Figure 44) [75].    A rather unique [4.5.0] spirocyclic lactone moiety was identified in sesquiterpene abiespiroside A (104), which was isolated from Chinese tree Abies dalavayi ( Figure 46). For this compound, anti-inflammatory activity was discovered [78].    A rather unique [4.5.0] spirocyclic lactone moiety was identified in sesquiterpene abiespiroside A (104), which was isolated from Chinese tree Abies dalavayi ( Figure 46). For this compound, anti-inflammatory activity was discovered [78].     A rather unique [4.5.0] spirocyclic lactone moiety was identified in sesquiterpene abiespiroside A (104), which was isolated from Chinese tree Abies dalavayi ( Figure 46). For this compound, anti-inflammatory activity was discovered [78].     A rather unique [4.5.0] spirocyclic lactone moiety was identified in sesquiterpene abiespiroside A (104), which was isolated from Chinese tree Abies dalavayi ( Figure 46). For this compound, anti-inflammatory activity was discovered [78]. A rather unique [4.5.0] spirocyclic lactone moiety was identified in sesquiterpene abiespiroside A (104), which was isolated from Chinese tree Abies dalavayi ( Figure 46). For this compound, anti-inflammatory activity was discovered [78].    A whole series of spirolactones containing a terpenoid carane system (106)(107)(108)(109)(110) was reported as synthesized in enantioselective fashion ( Figure 48). For these compounds, insect-feeding deterrent activity was reported [80].   A whole series of spirolactones containing a terpenoid carane system (106)(107)(108)(109)(110) was reported as synthesized in enantioselective fashion ( Figure 48). For these compounds, insect-feeding deterrent activity was reported [80].  A whole series of spirolactones containing a terpenoid carane system (106)(107)(108)(109)(110) was reported as synthesized in enantioselective fashion ( Figure 48). For these compounds, insect-feeding deterrent activity was reported [80].     A whole series of spirolactones containing a terpenoid carane system (106)(107)(108)(109)(110) was reported as synthesized in enantioselective fashion ( Figure 48). For these compounds, insect-feeding deterrent activity was reported [80].   The structures of these compounds are reminiscent of spirocyclic dihydrofuran 8,9-dehydrotheaspirone, both enantiomers of which (113a-b) have been reported as volatile constituents of nectarines [82]. Their presence in the fruit was connected to some specific organoleptic properties of some kinds of nectarines ( Figure 50 constituents of nectarines [82]. Their presence in the fruit was connected to some specific organoleptic properties of some kinds of nectarines ( Figure 50)    Rather unique is the structure of heliespirone 115 isolated from highly polar fractions of Helianthus annuus L. extract [84]. In this natural product, tetrahydrofuran forms a spirocyclic motif with a quinone-like moiety ( Figure 52).  Rather unique is the structure of heliespirone 115 isolated from highly polar fractions of Helianthus annuus L. extract [84]. In this natural product, tetrahydrofuran forms a spirocyclic motif with a quinone-like moiety ( Figure 52).  Rather unique is the structure of heliespirone 115 isolated from highly polar fractions of Helianthus annuus L. extract [84]. In this natural product, tetrahydrofuran forms a spirocyclic motif with a quinone-like moiety ( Figure 52).  Another example of similarly polyoxygenated [4.5.0] spirocyclic tetrahydrofuran is provided by quinochalcone 117, named saffloquinoside A, isolated from Carthamus tinctorius (Figure 54) [86]. Compound 117 was evaluated in vitro for the inhibitory effect on the release of β-glucuronidase from rat polymorphonuclear neutrophils (PMNs) induced by the platelet-activating factor (PAF). It exhibited anti-inflammatory activity and the inhibitory rate was 54.3% (at 10 ⁻5 mol/L concentration). Another example of similarly polyoxygenated [4.5.0] spirocyclic tetrahydrofuran is provided by quinochalcone 117, named saffloquinoside A, isolated from Carthamus tinctorius (Figure 54) [86]. Compound 117 was evaluated in vitro for the inhibitory effect on the release of β-glucuronidase from rat polymorphonuclear neutrophils (PMNs) induced by the platelet-activating factor (PAF). It exhibited anti-inflammatory activity and the inhibitory rate was 54.3% (at 10 −5 mol/L concentration).
Another example of similarly polyoxygenated [4.5.0] spirocyclic tetrahydrofuran is provided by quinochalcone 117, named saffloquinoside A, isolated from Carthamus tinctorius (Figure 54) [86]. Compound 117 was evaluated in vitro for the inhibitory effect on the release of β-glucuronidase from rat polymorphonuclear neutrophils (PMNs) induced by the platelet-activating factor (PAF). It exhibited anti-inflammatory activity and the inhibitory rate was 54.3% (at 10 ⁻5 mol/L concentration).  Another example of nitrogen-containing [4.5.0] spirocyclic system is provided by surugatoxin (119) isolated from the toxic Japanese ivory shell (Babylonica japonica) ( Figure 56). This toxin suppresses the presynaptic nervous system [88]. Its total synthesis, in the racemic form, was achieved in 1994 by the Inoue group [89].  Another example of similarly polyoxygenated [4.5.0] spirocyclic tetrahydrofuran is provided by quinochalcone 117, named saffloquinoside A, isolated from Carthamus tinctorius (Figure 54) [86]. Compound 117 was evaluated in vitro for the inhibitory effect on the release of β-glucuronidase from rat polymorphonuclear neutrophils (PMNs) induced by the platelet-activating factor (PAF). It exhibited anti-inflammatory activity and the inhibitory rate was 54.3% (at 10 ⁻5 mol/L concentration).  Another example of nitrogen-containing [4.5.0] spirocyclic system is provided by surugatoxin (119) isolated from the toxic Japanese ivory shell (Babylonica japonica) ( Figure 56). This toxin suppresses the presynaptic nervous system [88]. Its total synthesis, in the racemic form, was achieved in 1994 by the Inoue group [89]. Another example of nitrogen-containing [4.5.0] spirocyclic system is provided by surugatoxin (119) isolated from the toxic Japanese ivory shell (Babylonica japonica) (Figure 56). This toxin suppresses the presynaptic nervous system [88]. Its total synthesis, in the racemic form, was achieved in 1994 by the Inoue group [89]. A [4.5.0] spirocyclic system is recognizable in spirostaphylotrichins which are spirocyclic γ-lactams mainly produced by several endophytic fungal strains of Curvularia, Pyrenophora, and Staphylotrichum. These are exemplified by spirostaphylotrichin X (120), characterized as an antiinfluenza agent targeting RNA polymerase PB2 [90], and spirostaphylotrichin W (121), investigated as a potential mycoherbicide for cheatgrass (Bromus tectorum) biocontrol [91] ( Figure  57). A [4.5.0] spirocyclic system is recognizable in spirostaphylotrichins which are spirocyclic γ-lactams mainly produced by several endophytic fungal strains of Curvularia, Pyrenophora, and Staphylotrichum. These are exemplified by spirostaphylotrichin X (120), characterized as an antiinfluenza agent targeting RNA polymerase PB2 [90], and spirostaphylotrichin W (121), investigated as a potential mycoherbicide for cheatgrass (Bromus tectorum) biocontrol [91] (Figure 57). A [4.5.0] spirocyclic system is recognizable in spirostaphylotrichins which are spirocyclic γ-lactams mainly produced by several endophytic fungal strains of Curvularia, Pyrenophora, and Staphylotrichum. These are exemplified by spirostaphylotrichin X (120), characterized as an antiinfluenza agent targeting RNA polymerase PB2 [90], and spirostaphylotrichin W (121), investigated as a potential mycoherbicide for cheatgrass (Bromus tectorum) biocontrol [91] (Figure  57). Summarizing this Section, the scaffold diversity stemming from the general [4.5.0] spirocyclic framework is comparable to that of the [4.4.0] spirocyclic system discussed earlier ( Figure 30) and is shown in Figure 58. Summarizing this Section, the scaffold diversity stemming from the general [4.5.0] spirocyclic framework is comparable to that of the [4.4.0] spirocyclic system discussed earlier ( Figure 30) and is shown in Figure 58.
In 2003, investigation of the neutral ether extracts of the fungus Fomes cajanderi led to the isolation of three novel ketal lactones named fomlactones A (125), B (126), and C (127) (Figure 60). The compounds clearly possess a [4.6.0] spirocyclic lactone moiety. However, their biological potential remains to be investigated [93]. A very unique spirocyclic [4.6.0] framework formed by a spiro[benzofuranonebenzazepine] skeleton is featured in natural products (±)-juglanaloid A (128a-b) and (±)-juglanaloid B (129a-b). These benzazepine alkaloids were isolated from the bark of Juglans mandshurica. Remarkably, both racemic natural products were successfully resolved by chiral separation and absolute configurations were unambiguously assigned ( Figure 61). These enantiopure versions were screened for their in vitro inhibitory activities against self-induced Aβ1-42 aggregation using the Thioflavin T (Th-T) assay using curcumin as a reference compound. The compounds demonstrated promise acting as inhibitors of amyloid β aggregation [94].  A very unique spirocyclic [4.6.0] framework formed by a spiro[benzofuranonebenzazepine] skeleton is featured in natural products (±)-juglanaloid A (128a-b) and (±)-juglanaloid B (129a-b). These benzazepine alkaloids were isolated from the bark of Juglans mandshurica. Remarkably, both racemic natural products were successfully resolved by chiral separation and absolute configurations were unambiguously assigned ( Figure 61). These enantiopure versions were screened for their in vitro inhibitory activities against self-induced Aβ 1-42 aggregation using the Thioflavin T (Th-T) assay using curcumin as a reference compound. The compounds demonstrated promise acting as inhibitors of amyloid β aggregation [94]. A very unique spirocyclic [4.6.0] framework formed by a spiro[benzofuranonebenzazepine] skeleton is featured in natural products (±)-juglanaloid A (128a-b) and (±)-juglanaloid B (129a-b). These benzazepine alkaloids were isolated from the bark of Juglans mandshurica. Remarkably, both racemic natural products were successfully resolved by chiral separation and absolute configurations were unambiguously assigned ( Figure 61). These enantiopure versions were screened for their in vitro inhibitory activities against self-induced Aβ1-42 aggregation using the Thioflavin T (Th-T) assay using curcumin as a reference compound. The compounds demonstrated promise acting as inhibitors of amyloid β aggregation [94].  Another recent example (reported in 2019) of a [4.6.0] spirocyclic system is provided by grayanane diterpenoid auriculatol A (131) isolated from leaves of Rhododendron auriculatum ( Figure 63). This compound is the first example of a 5,20-epoxygrayanane diterpenoid bearing a 7-oxabicyclo[4.2.1]nonane motif and a trans/cis/cis/cis-fused 5/5/7/6/5 pentacyclic ring system. Auriculatol A showed analgesic activity in the acetic acid-induced writhing test [96].

Figure 70. Thielavialides A−E (148-152) and pestafolide A (153).
A very similar [5.5.0] spirocyclic moiety can be found in the structure of pteridic acids C and F (154 and 155, respectively) isolated in 2017 from a culture broth of the marine-derived actinomycete Streptomyces sp. SCSGAA 0027 ( Figure 71). While these compounds were seen as potential leads for antibacterial drug discovery, their extensive testing for antimicrobial activity against two gorgonian pathogenic fungal strains Aspergullus versicolor SCSGAF 0096 and Aspergullus sydowii SCSGAF 0035; a human pathogenic fungal strain Candida albicans SC5314; and two bacterial strains Escherichia coli and Bacillus subtilis, showed that the compounds had only a weak antimicrobial activity [106]. A unique [5.5.0] spirocyclic skeleton formed by a hexahydropyran and a pyrrolo[2,1-c]morpholine moieties is found in pollenopyrroside A (156) and B (157) isolated from bee-collected Brassica campestris pollen ( Figure 72). The Chinese team who reported these natural products in 2010 also proposed a biosynthetic pathway that involves a reaction of 3-deoxy-D-fructose and 5-oxymethyl-2-formyl-pyrrole as the key step. Biological testing of these aldehyde compounds using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method revealed that they possess no cytotoxicity against A549, Bel7420, BGC-823, HCT-8, and A2780 cancerous cell lines at 10 μg/mL [107]. A very similar [5.5.0] spirocyclic moiety can be found in the structure of pteridic acids C and F (154 and 155, respectively) isolated in 2017 from a culture broth of the marine-derived actinomycete Streptomyces sp. SCSGAA 0027 ( Figure 71). While these compounds were seen as potential leads for antibacterial drug discovery, their extensive testing for antimicrobial activity against two gorgonian pathogenic fungal strains Aspergullus versicolor SCSGAF 0096 and Aspergullus sydowii SCSGAF 0035; a human pathogenic fungal strain Candida albicans SC5314; and two bacterial strains Escherichia coli and Bacillus subtilis, showed that the compounds had only a weak antimicrobial activity [106]. together with bis-spiro-azaphilone pestafolide A (153) ( Figure 70). All these compounds were isolated from the endophytic fungal strain, Thielavia sp. PA0001, occurring in the healthy leaf tissue of aeroponically grown Physalis alkekengi [105].

[5.6.0] Spirocyclic System
An interesting group of natural products representative of this spirocyclic system are periplosides (164), a spiro-orthoester group-containing pregnane-type glycosides discovered in the course of phytochemical investigation of the root bark of Periploca sepium (Figure 77). The [5.6.0] spirocyclic orthoester core is distinctly modified with a steroid unit on one hand (R 1 ) and with an oligosaccharide moiety on the other (R 2 ). The compounds were evaluated for their inhibitory activities against the proliferation of T-lymphocytes. As a result, one specific compound (periploside C), the most abundant glycoside containing a spiro-orthoester moiety found in the plant, exhibited the most favorite selective index value (SI = 82.5). The inhibitory activity and the SI value appear to depend on the constitution of the saccharide chain [112].
Molecules 2018, 23, x 31 of 39 spirocyclic orthoester core is distinctly modified with a steroid unit on one hand (R 1 ) and with an oligosaccharide moiety on the other (R 2 ). The compounds were evaluated for their inhibitory activities against the proliferation of T-lymphocytes. As a result, one specific compound (periploside C), the most abundant glycoside containing a spiro-orthoester moiety found in the plant, exhibited the most favorite selective index value (SI = 82.5). The inhibitory activity and the SI value appear to depend on the constitution of the saccharide chain [112]. The remarkable, from a structural perspective, spirolide G (165), was isolated from Danish strains of toxigenic dinoflagellate Alexandrium ostenfeldii. The toxicological profile of this compound was evaluated [113]. Interestingly, in addition to the spirocyclic [5.  Referring back to the chemical investigation of the marine-derived fungus Eurotium sp. SCSIO F452 discussed above in connection with compounds belonging to the [5.5.0] spirocyclic system, an intriguing [5.6.0] spirocyclic compound 166 ( Figure 79) was also isolated from the same species [109]. This is a case of one species giving rise to a diversity of spirocyclic frameworks, underscoring the significance of spirocycles in the natural product realm. One particular example of such spirocycle diversity derived from a single organism is discussed in Section 13 below.  The remarkable, from a structural perspective, spirolide G (165), was isolated from Danish strains of toxigenic dinoflagellate Alexandrium ostenfeldii. The toxicological profile of this compound was evaluated [113]. Interestingly, in addition to the spirocyclic [5. spirocyclic orthoester core is distinctly modified with a steroid unit on one hand (R 1 ) and with an oligosaccharide moiety on the other (R 2 ). The compounds were evaluated for their inhibitory activities against the proliferation of T-lymphocytes. As a result, one specific compound (periploside C), the most abundant glycoside containing a spiro-orthoester moiety found in the plant, exhibited the most favorite selective index value (SI = 82.5). The inhibitory activity and the SI value appear to depend on the constitution of the saccharide chain [112]. The remarkable, from a structural perspective, spirolide G (165), was isolated from Danish strains of toxigenic dinoflagellate Alexandrium ostenfeldii. The toxicological profile of this compound was evaluated [113]. Interestingly, in addition to the spirocyclic [5.  Referring back to the chemical investigation of the marine-derived fungus Eurotium sp. SCSIO F452 discussed above in connection with compounds belonging to the [5.5.0] spirocyclic system, an intriguing [5.6.0] spirocyclic compound 166 ( Figure 79) was also isolated from the same species [109]. This is a case of one species giving rise to a diversity of spirocyclic frameworks, underscoring the significance of spirocycles in the natural product realm. One particular example of such spirocycle diversity derived from a single organism is discussed in Section 13 below.  Referring back to the chemical investigation of the marine-derived fungus Eurotium sp. SCSIO F452 discussed above in connection with compounds belonging to the [5.5.0] spirocyclic system, an intriguing [5.6.0] spirocyclic compound 166 ( Figure 79) was also isolated from the same species [109]. This is a case of one species giving rise to a diversity of spirocyclic frameworks, underscoring the significance of spirocycles in the natural product realm. One particular example of such spirocycle diversity derived from a single organism is discussed in Section 13 below.
Molecules 2018, 23, x 31 of 39 spirocyclic orthoester core is distinctly modified with a steroid unit on one hand (R 1 ) and with an oligosaccharide moiety on the other (R 2 ). The compounds were evaluated for their inhibitory activities against the proliferation of T-lymphocytes. As a result, one specific compound (periploside C), the most abundant glycoside containing a spiro-orthoester moiety found in the plant, exhibited the most favorite selective index value (SI = 82.5). The inhibitory activity and the SI value appear to depend on the constitution of the saccharide chain [112]. The remarkable, from a structural perspective, spirolide G (165), was isolated from Danish strains of toxigenic dinoflagellate Alexandrium ostenfeldii. The toxicological profile of this compound was evaluated [113]. Interestingly, in addition to the spirocyclic [5.  Referring back to the chemical investigation of the marine-derived fungus Eurotium sp. SCSIO F452 discussed above in connection with compounds belonging to the [5.5.0] spirocyclic system, an intriguing [5.6.0] spirocyclic compound 166 ( Figure 79) was also isolated from the same species [109]. This is a case of one species giving rise to a diversity of spirocyclic frameworks, underscoring the significance of spirocycles in the natural product realm. One particular example of such spirocycle diversity derived from a single organism is discussed in Section 13 below.   (Figure 80). This compound showed considerable cytotoxicity against DU145 cells with an IC50 value of 9.5 μM [114].

[6.6.0] Spirocyclic System
This type of spirocyclic framework is exceedingly rare in the natural product domain, with only one example of unique 1-oxaspiro[6.6]tridecane 168, a spirocyclic nortriterpenoid Spiroschincarin A isolated from the fruit of Schisandra incarnate ( Figure 81) [115].

Plant Species Distinctly Rich in Diverse Spirocyclic Natural Products
Some cases when the same plant or microorganism gave rise to secondary metabolites with several structurally-diverse spirocyclic frameworks were discussed above. However, one recent example published in 2019, describing a chemical investigation of monoterpenoid indole alkaloids isolated from the roots of Gelsemium elegans (also briefly discussed in Section 8 of this review), stands out from the standpoint of hitherto unprecedented skeletal diversity [116]. In particular, the following spirocyclic frameworks were encountered among the natural products isolated from this species:

[6.6.0] Spirocyclic System
This type of spirocyclic framework is exceedingly rare in the natural product domain, with only one example of unique 1-oxaspiro[6.6]tridecane 168, a spirocyclic nortriterpenoid Spiroschincarin A isolated from the fruit of Schisandra incarnate ( Figure 81) [115].

Plant Species Distinctly Rich in Diverse Spirocyclic Natural Products
Some cases when the same plant or microorganism gave rise to secondary metabolites with several structurally-diverse spirocyclic frameworks were discussed above. However, one recent example published in 2019, describing a chemical investigation of monoterpenoid indole alkaloids isolated from the roots of Gelsemium elegans (also briefly discussed in Section 8 of this review), stands out from the standpoint of hitherto unprecedented skeletal diversity [116]. In particular, the following spirocyclic frameworks were encountered among the natural products isolated from this species:

Plant Species Distinctly Rich in Diverse Spirocyclic Natural Products
Some cases when the same plant or microorganism gave rise to secondary metabolites with several structurally-diverse spirocyclic frameworks were discussed above. However, one recent example published in 2019, describing a chemical investigation of monoterpenoid indole alkaloids isolated from the roots of Gelsemium elegans (also briefly discussed in Section 8 of this review), stands out from the standpoint of hitherto unprecedented skeletal diversity [116]. In particular, the following spirocyclic frameworks were encountered among the natural products isolated from this species:

Conclusions and Perspectives
Spirocyclic scaffolds are omnipresent in the natural products domain. By analyzing the diversity of spirocyclic systems reported for natural products in the literature, one can appreciate an uneven distribution of such motifs according to the spirocycle type: certain motifs are more abundant than others and some are rather scarce, exemplified by only a handful of naturally occurring compounds. The most widespread are the [5.5.0], [4.5.0], and [4.4.0] spirocycles. In terms of associated bioactivities discovered and reported for spirocyclic products, these are mostly limited to the usual profiling in the context of antiproliferative, anti-inflammatory, and antimicrobial activities. However, the strong connections of spirocyclic frameworks to the natural product domain and their emerging privileged motif status in the synthetic drug discovery argues in favor of the need for more thorough panel profiling of all newly-discovered natural products, as novel and hitherto unprecedented bioactivity leads could be discovered. Certain scarcely-populated areas of the spirocyclic natural product space can be specifically developed into synthetic libraries and investigated for bioactivity. More spirocycles appear to have been discovered in the last 5-10 years, with an apparent advent of plant species giving rise to several types of spirocyclic frameworks in the course of their chemical investigation. The spirocyclic natural product discovery, therefore, appears to be on the rise and is likely to inspire new scaffolds for drug design and screening library development.

Conclusions and Perspectives
Spirocyclic scaffolds are omnipresent in the natural products domain. By analyzing the diversity of spirocyclic systems reported for natural products in the literature, one can appreciate an uneven distribution of such motifs according to the spirocycle type: certain motifs are more abundant than others and some are rather scarce, exemplified by only a handful of naturally occurring compounds. The most widespread are the [5.5.0], [4.5.0], and [4.4.0] spirocycles. In terms of associated bioactivities discovered and reported for spirocyclic products, these are mostly limited to the usual profiling in the context of antiproliferative, anti-inflammatory, and antimicrobial activities. However, the strong connections of spirocyclic frameworks to the natural product domain and their emerging privileged motif status in the synthetic drug discovery argues in favor of the need for more thorough panel profiling of all newly-discovered natural products, as novel and hitherto unprecedented bioactivity leads could be discovered. Certain scarcely-populated areas of the spirocyclic natural product space can be specifically developed into synthetic libraries and investigated for bioactivity. More spirocycles appear to have been discovered in the last 5-10 years, with an apparent advent of plant species giving rise to several types of spirocyclic frameworks in the course of their chemical investigation. The spirocyclic natural product discovery, therefore, appears to be on the rise and is likely to inspire new scaffolds for drug design and screening library development.