Bioactive Natural Spirolactone-Type 6,7-seco-ent-Kaurane Diterpenoids and Synthetic Derivatives

Diterpenoids are widely distributed natural products and have caused considerable interest because of their unique skeletons and antibacterial and antitumor activities and so on. In light of recent discoveries, ent-kaurane diterpenoids, which exhibit a wide variety of biological activities, such as anticancer and anti-inflammatory activities, pose enormous potential to serve as a promising candidate for drug development. Among them, spirolactone-type 6,7-seco-ent-kaurane diterpenoids, with interesting molecular skeleton, complex oxidation patterns, and bond formation, exhibit attractive activities. Furthermore, spirolactone-type diterpenoids have many modifiable sites, which allows for linking to various substituents, suitable for further medicinal study. Hence, some structurally modified derivatives with improved cytotoxicity activities are also achieved. In this review, natural bioactive spirolactone-type diterpenoids and their synthetic derivatives were summarized.


Natural Bioactive Spirolactone-Type Diterpenoids
By the end of October 2018, 105 spirolactone-type diterpenoids have been isolated from Isodon species. Several exhibited biological activities and are summarized below.
In 1995, loxothyrin A (7, Figure 2) was isolated by Sun's Group from the leaves of I. loxothyrsa [66]. It showed cytotoxicity effects toward hormone-dependent human prostatic LNCaP and breast ZR-75-1 cancer cell lines with ED50 values of 13.5 and 7.2 μg/mL, respectively.
Four new ent-kaurane diterpenoids were isolated from the I. enanderianus in the same year by and coworkers [69]. Among which, a new spirolactone-type diterpenoid was named 6epiangustifolin (10, Figure 2), and tested for its cytotoxicity toward K562 cells. The results showed that 10 exhibited inhibitory activity with an IC50 value of 0.0865 μg/mL against the K562 cell line, which was stronger than cis-platin, the positive reference.

Natural Bioactive Spirolactone-Type Diterpenoids
By the end of October 2018, 105 spirolactone-type diterpenoids have been isolated from Isodon species. Several exhibited biological activities and are summarized below.
In 1995, loxothyrin A (7, Figure 2) was isolated by Sun's Group from the leaves of I. loxothyrsa [66]. It showed cytotoxicity effects toward hormone-dependent human prostatic LNCaP and breast ZR-75-1 cancer cell lines with ED 50 values of 13.5 and 7.2 µg/mL, respectively.
Four new ent-kaurane diterpenoids were isolated from the I. enanderianus in the same year by and coworkers [69]. Among which, a new spirolactone-type diterpenoid was named 6-epiangustifolin (10, Figure 2), and tested for its cytotoxicity toward K562 cells. The results showed that 10 exhibited inhibitory activity with an IC 50 value of 0.0865 µg/mL against the K562 cell line, which was stronger than cis-platin, the positive reference.
Han et al. isolated five new and eight known spirolactone-type diterpenoids from I. rubescens var. lushiensis in 2003. The cytotoxicity of most isolates were tested against K562 cell line. Among which, ludongnin J, guidongnin A, angustifolin, and ludongnin A (15-18, Figure 2) showed significant inhibitory effects with IC 50 values from 0.18 to 0.83 µg/mL. Furthermore, compound 15 also exhibited inhibitory activities against liver cancer CA and uterine cervix cancer Hela cell lines with IC 50 values below 0.70 µg/mL [71]. Moreover, in 2010, Luo et al. also found that compound 18 exhibited cytotoxicity against promyelocytic leukemia HL-60 cells with an IC 50 value of 3.1 µM [72].
In the same year, Han et al. also isolated two new and four known spirolactone-type diterpenoids from I. rubescens var. lushiensis [73]. All isolates were tested for their cytotoxic effects against K562, human breast cancer Bcap37, CA, human nasopharyngeal cancer CNE, human cystic cancer BIU87, human stomach cancer BGC823, and Hela cell lines. Lushanrubescensin H, isodonoiol, isodonal, and rabdosin B (19-22, Figure 2) displayed cytotoxic activities with IC 50 values from 2.29 to 28.64 µg/mL.
Han et al. isolated five new and eight known spirolactone-type diterpenoids from I. rubescens var. lushiensis in 2003. The cytotoxicity of most isolates were tested against K562 cell line. Among which, ludongnin J, guidongnin A, angustifolin, and ludongnin A (15-18, Figure 2) showed significant inhibitory effects with IC50 values from 0.18 to 0.83 μg/mL. Furthermore, compound 15 also exhibited inhibitory activities against liver cancer CA and uterine cervix cancer Hela cell lines with IC50 values below 0.70 μg/mL [71]. Moreover, in 2010, Luo et al. also found that compound 18 exhibited cytotoxicity against promyelocytic leukemia HL-60 cells with an IC50 value of 3.1 μM [72].
In the same year, Han et al. also isolated two new and four known spirolactone-type diterpenoids from I. rubescens var. lushiensis [73]. All isolates were tested for their cytotoxic effects against K562, human breast cancer Bcap37, CA, human nasopharyngeal cancer CNE, human cystic cancer BIU87, human stomach cancer BGC823, and Hela cell lines. Lushanrubescensin H, isodonoiol, isodonal, and rabdosin B (19-22, Figure 2) displayed cytotoxic activities with IC50 values from 2.29 to 28.64 μg/mL.
Four new spirolactone-type diterpenoids were identified by Li et al. from the aerial parts of I. sculponeatus in the 2010 [78]. Among which, sculponeatin N and sculponeatin O (29 and 30, Figure 3) displayed strong inhibitory activities (IC 50 ) on K562 and HepG2 cell lines between 0.21 and 0.39 µM.
Three new spirolactone-type diterpenoids were isolated by Tanaka and coworkers from I. japonicus in the same year [83]. Their antifungal activities were evaluated. Particularly, hikiokoshin A (39, Figure 3) displayed antifungal activities against Cryptococcus neoformans and Aspergillus niger with IC 50 values of 16 µg/mL each.
In 2014, eighteen new spirolactone-type diterpenoids were isolated and determined by Wang and coworkers from I. eriocalyx var. laxiflora [84]. The cytotoxic effects of all isolates were tested against A-549, SMMC-7721, MCF-7, HL-60, and SW-480 cells. Laxiflorolide C and laxiflorin B (40 and 41, Figure 3) exhibited selective cytotoxic activities with IC 50 s between 0.6 and 18.8 µM. Moreover, laxiflorolide C and laxiflorin B also showed inhibitory effects on LPS stimulated NO production in RAW264.7 cells, with IC 50 s of 1.5 and 0.5 µM, respectively.

Synthetic Spirolactone-Type Diterpenoid Derivatives
Though spirolactone-type diterpenoids exhibited cytotoxic effects with interesting molecular skeletons, the amount of spirolactone-type diterpenoids extracted from natural sources could not meet the needs of drug development. In order to achieve large scale compound supply, convenient methods have been built up. Lead tetraacetate was used as oxidation to finish C-6 and C-7 bond cleavage of commercially available oridonin to produce spirolactone-type core structure. The synthesis routine is illustrated in Scheme 1. Based on this core, diverse spirolactone-type derived compounds could be obtained [85].

Synthetic Spirolactone-Type Diterpenoid Derivatives
Though spirolactone-type diterpenoids exhibited cytotoxic effects with interesting molecular skeletons, the amount of spirolactone-type diterpenoids extracted from natural sources could not meet the needs of drug development. In order to achieve large scale compound supply, convenient methods have been built up. Lead tetraacetate was used as oxidation to finish C-6 and C-7 bond cleavage of commercially available oridonin to produce spirolactone-type core structure. The synthesis routine is illustrated in Scheme 1. Based on this core, diverse spirolactone-type derived compounds could be obtained [85]. The results showed that they exhibited stronger cytotoxicity than 43. Among them, 51 (Table 1) exhibited the strongest cytotoxicity with IC50 values of 1.27, 2.24, 1.05, and 1.54 μM, respectively.  Li and coworkers linked several acids to spirolactone-type core structure with ether bond (Scheme 3) [86]. The antiproliferative activities were tested against the above four cancer cell lines. Target derivatives were also more potent than parent compound oridonin 68 (Table 1) showed the most potent inhibitory activities with IC50s below 1.39 μM. The structure-activity relationships (SARs) were also disclosed. When R were alkyl groups (59)(60)(61), with the increased length of R groups, stronger cytotoxicity could be observed in MGC-803 cell line. Furthermore, when R were aromatic groups (62)(63)(64)(65)(66)(67)(68), their activities were stronger than those of alkyl groups, particularly, when substituted by chloro. The most potent 68 was selected to explore antiproliferative mechanism. The results indicated that 68 could induce apoptosis in a dose-dependent fashion and arrest cell accumulation at G2/M phase in Bel-7402 cells. The results showed that they exhibited stronger cytotoxicity than 43. Among them, 51 (Table 1) exhibited the strongest cytotoxicity with IC50 values of 1.27, 2.24, 1.05, and 1.54 μM, respectively.  Li and coworkers linked several acids to spirolactone-type core structure with ether bond (Scheme 3) [86]. The antiproliferative activities were tested against the above four cancer cell lines. Target derivatives were also more potent than parent compound oridonin 68 (Table 1) showed the most potent inhibitory activities with IC50s below 1.39 μM. The structure-activity relationships (SARs) were also disclosed. When R were alkyl groups (59)(60)(61), with the increased length of R groups, stronger cytotoxicity could be observed in MGC-803 cell line. Furthermore, when R were aromatic groups (62-68), their activities were stronger than those of alkyl groups, particularly, when substituted by chloro. The most potent 68 was selected to explore antiproliferative mechanism. The results indicated that 68 could induce apoptosis in a dose-dependent fashion and arrest cell accumulation at G2/M phase in Bel-7402 cells.  Li and coworkers linked several acids to spirolactone-type core structure with ether bond (Scheme 3) [86]. The antiproliferative activities were tested against the above four cancer cell lines. Target derivatives were also more potent than parent compound oridonin 68 (Table 1) showed the most potent inhibitory activities with IC 50 s below 1.39 µM. The structure-activity relationships (SARs) were also disclosed. When R were alkyl groups (59)(60)(61), with the increased length of R groups, stronger cytotoxicity could be observed in MGC-803 cell line. Furthermore, when R were aromatic groups (62)(63)(64)(65)(66)(67)(68), their activities were stronger than those of alkyl groups, particularly, when substituted by chloro. The most potent 68 was selected to explore antiproliferative mechanism. The results indicated that 68 could induce apoptosis in a dose-dependent fashion and arrest cell accumulation at G2/M phase in Bel-7402 cells.  linkers (75b, 75d, 75f, 76b, 76d, and 76f), the antiproliferative effects were better than those of alkyl linkers (75a, 75c, 75e, 76a, 76c, and 76e). Particularly, compound 76d (Table 1) showed the most potent IC50s between 0.86 and 3.75 μM against MGC-803, K562, Bel-7402, and CaEs-17 cells. The NO-releasing properties were evaluated by Griess assay. The results showed that all derivatives more than 15 μM released NO in 1 h which would contribute to their antiproliferative activities. Furthermore, a further mechanism of 76d was studied in Bel-7402 cells. They found that 76d could induce cell cycle arrest at the S phase. It was also found that 76d could decline the mitochondrial membrane potentials which indicated that 76d induced apoptosis through intrinsic pathways. In order to discover more bioactive spirolactone-type diterpenoid derivatives, two series of novel derivatives with various substituents at 14-OH were designed and synthesized by Xu (Table 1) exhibited IC50s between 0.68 and 2.2 μM, which was the strongest derivatives of this series [88]. The mechanism of action of 82 was also investigated. After treatment with 82, the mitochondrial membrane potential In 2016, Xu's Group synthesized several furozan-based NO-donating derivatives (Scheme 4) [87]. Compared with parent compounds 43 and 47, all the synthetic target molecules showed improved antiproliferative activities, especially toward Bel-7402 cell line. The SARs revealed when R 2 was aromatic linkers (75b, 75d, 75f, 76b, 76d, and 76f), the antiproliferative effects were better than those of alkyl linkers (75a, 75c, 75e, 76a, 76c, and 76e). Particularly, compound 76d (Table 1) showed the most potent IC 50 s between 0.86 and 3.75 µM against MGC-803, K562, Bel-7402, and CaEs-17 cells. The NO-releasing properties were evaluated by Griess assay. The results showed that all derivatives more than 15 µM released NO in 1 h which would contribute to their antiproliferative activities. Furthermore, a further mechanism of 76d was studied in Bel-7402 cells. They found that 76d could induce cell cycle arrest at the S phase. It was also found that 76d could decline the mitochondrial membrane potentials which indicated that 76d induced apoptosis through intrinsic pathways.  linkers (75b, 75d, 75f, 76b, 76d, and 76f), the antiproliferative effects were better than those of alkyl linkers (75a, 75c, 75e, 76a, 76c, and 76e). Particularly, compound 76d (Table 1) showed the most potent IC50s between 0.86 and 3.75 μM against MGC-803, K562, Bel-7402, and CaEs-17 cells. The NO-releasing properties were evaluated by Griess assay. The results showed that all derivatives more than 15 μM released NO in 1 h which would contribute to their antiproliferative activities. Furthermore, a further mechanism of 76d was studied in Bel-7402 cells. They found that 76d could induce cell cycle arrest at the S phase. It was also found that 76d could decline the mitochondrial membrane potentials which indicated that 76d induced apoptosis through intrinsic pathways. In order to discover more bioactive spirolactone-type diterpenoid derivatives, two series of novel derivatives with various substituents at 14-OH were designed and synthesized by Xu et al. in 2017 (Schemes 5 and 6). The antiproliferative activities of all derivatives were evaluated against four human cancer cell lines (MGC-803, MCF-7, Bel-7402, and K562). Compound 82 (Table 1) exhibited IC50s between 0.68 and 2.2 μM, which was the strongest derivatives of this series [88]. The mechanism of action of 82 was also investigated. After treatment with 82, the mitochondrial membrane potential In order to discover more bioactive spirolactone-type diterpenoid derivatives, two series of novel derivatives with various substituents at 14-OH were designed and synthesized by Xu et al. in 2017 (Schemes 5 and 6). The antiproliferative activities of all derivatives were evaluated against four human cancer cell lines (MGC-803, MCF-7, Bel-7402, and K562). Compound 82 (Table 1) exhibited IC 50 s between 0.68 and 2.2 µM, which was the strongest derivatives of this series [88]. The mechanism of action of 82 was also investigated. After treatment with 82, the mitochondrial membrane potential in MCF-7 cell declined. Western blot results showed that 82 could increase the levels of p-ERK, Bax and caspase 3, and reduced the expression of P53 and Bcl-2. 82 also induced cell accumulated at the G2/M phase. In short, these results illustrate that derivative 82 induced apoptosis through a mitochondria-related pathway. in MCF-7 cell declined. Western blot results showed that 82 could increase the levels of p-ERK, Bax and caspase 3, and reduced the expression of P53 and Bcl-2. 82 also induced cell accumulated at the G2/M phase. In short, these results illustrate that derivative 82 induced apoptosis through a mitochondria-related pathway.

Conclusions
In summary, natural spirolactone-type diterpenoids exhibited cytotoxic effects. Its synthetic derivatives showed more potent antiproliferative effects than the corresponding parent compounds. Hence, spirolactone-type diterpenoids are worthy of further research. However, there are few indepth pharmacological reports on spirolactone-type diterpenoids so far. We hold the view that, for drug exploration, further studies should firstly focus on the detailed mechanism study. Based on these, spirolactone-type diterpenoid derivatives with clear target should be explored. We hope this review can provide useful information in the field of bioactive natural and synthetic spirolactonetype diterpenoids. in MCF-7 cell declined. Western blot results showed that 82 could increase the levels of p-ERK, Bax and caspase 3, and reduced the expression of P53 and Bcl-2. 82 also induced cell accumulated at the G2/M phase. In short, these results illustrate that derivative 82 induced apoptosis through a mitochondria-related pathway.

Conclusions
In summary, natural spirolactone-type diterpenoids exhibited cytotoxic effects. Its synthetic derivatives showed more potent antiproliferative effects than the corresponding parent compounds. Hence, spirolactone-type diterpenoids are worthy of further research. However, there are few indepth pharmacological reports on spirolactone-type diterpenoids so far. We hold the view that, for drug exploration, further studies should firstly focus on the detailed mechanism study. Based on these, spirolactone-type diterpenoid derivatives with clear target should be explored. We hope this review can provide useful information in the field of bioactive natural and synthetic spirolactonetype diterpenoids.

Conclusions
In summary, natural spirolactone-type diterpenoids exhibited cytotoxic effects. Its synthetic derivatives showed more potent antiproliferative effects than the corresponding parent compounds. Hence, spirolactone-type diterpenoids are worthy of further research. However, there are few in-depth pharmacological reports on spirolactone-type diterpenoids so far. We hold the view that, for drug exploration, further studies should firstly focus on the detailed mechanism study. Based on these, spirolactone-type diterpenoid derivatives with clear target should be explored. We hope this review can provide useful information in the field of bioactive natural and synthetic spirolactone-type diterpenoids.

Conflicts of Interest:
The authors declare no conflicts of interest.