Cytotoxic Effects of Diterpenoid Alkaloids Against Human Cancer Cells

Diterpenoid alkaloids are isolated from plants of the genera Aconitum, Delphinium, and Garrya (Ranunculaceae) and classified according to their chemical structures as C18-, C19- or C20-diterpenoid alkaloids. The extreme toxicity of certain compounds, e.g., aconitine, has prompted a thorough investigation of how structural features affect their bioactivities. Therefore, natural diterpenoid alkaloids and semi-synthetic alkaloid derivatives were evaluated for cytotoxic effects against human tumor cells [A549 (lung carcinoma), DU145 (prostate carcinoma), MDA-MB-231 (triple-negative breast cancer), MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), KB (identical to cervical carcinoma HeLa derived AV-3 cell line), and multidrug-resistant (MDR) subline KB-VIN]. Among the tested alkaloids, C19-diterpenoid (e.g., lipojesaconitine, delcosine and delpheline derivatives) and C20-diterpenoid (e.g., kobusine and pseudokobusine derivatives) alkaloids exhibited significant cytotoxic activity and, thus, provide promising new leads for further development as antitumor agents. Notably, several diterpenoid alkaloids were more potent against MDR subline KB-VIN cells than the parental drug-sensitive KB cells.

Numerous diterpenoid alkaloids have been isolated from various Aconitum, Delphinium, and Garrya (Family Ranunculaceae) species and are classified according to their chemical structures as C 18 -, C 19 or C 20 -diterpenoid alkaloids ( Figure 1) [10,11]. The C 19 -diterpenoid alkaloids may be divided into six types: aconitine, lycoctonine, pyro (C 8 =C 15 or C 15 =O), lactone (δ-valerolactone rather than cyclopentyl C-ring), 7,17-seco, and rearranged ones [10,11]. Most of the isolated C 19 -diterpenoid alkaloids are aconitine-and lycoctonine-types and include aconitine, mesaconitine, hypaconitine and jesaconitine, all of which are extremely toxic. The C 20 -diterpenoid alkaloids may be divided into ten types: atisine, denudatine, hetidine, hetisine, vakognavine, napelline, kusnezoline, racemulosine, arcutine, and tricalysiamide [10,11]. Most of the isolated C 20 -diterpenoid alkaloids are atisine-, hetisine-, and napelline-types and include atisine, kobusine, pseudokobusine and lucidusculine, which are far less toxic [12]. The pharmacological properties of the C19-diterpenoid alkaloids have been studied extensively and reviewed [12]. Aconitine is a toxin that exhibits activity both centrally and peripherally, acting predominantly on the cardiovascular and respiratory systems by preventing the normal closing of sodium channels [12]. This extreme toxicity resulted in the use of Aconitum extracts as poisons in hunting and warfare [13], although extracts were also used as traditional medicines by oral and topical routes. For example, the roots of Aconitum plants have been used as "bushi", an herbal drug in some prescriptions of traditional Japanese medicine for the treatment of hypometabolism, dysuria, cardiac weakness, chills, neuralgia, gout, and certain rheumatic diseases [14]. However, proper processing is essential to reduce the content of toxic alkaloids and avoid inadvertent poisoning [15][16][17]. Such obstacles encourage a good understanding of the relationships between structure and cytotoxic activity of aconitine and related compounds before they can be considered for modification and development as chemotherapeutic agents.
Our previous study demonstrated the effects of various naturally occurring and semi-synthetic C19and C20-diterpenoid alkaloids on the growth of the A172 human malignant glioma cell line [18]. Antitumor properties and radiation-sensitizing effects of various types of novel derivatives prepared from C19-and C20-diterpenoid alkaloids were also investigated [19]. Two novel hetisine-type C20diterpenoid derivatives showed significant suppressive effects against the Raji non-Hodgkin's lymphoma cell line [20]. In addition, the effects of various semi-synthetic novel hetisine-type C20diterpenoid alkaloids on the growth of the A549 human lung cancer cell line were examined and subsequent structure-activity relationships for the antiproliferative effects against A549 cells were considered [21]. Since 2012, several diterpenoid alkaloid components and their derivatives exhibited antiproliferative activity against human tumor cell lines, including A549 (lung carcinoma), DU145 (prostate carcinoma), MDA-MB-231 (estrogen and progesterone receptor-negative & HER2-negative triple-negative breast cancer), MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), KB (identical to cervical carcinoma HeLa derived AV-3 cell line), and multidrug-resistant (MDR) subline KB-VIN [P-glycoprotein (P-gp) overexpressing vincristine-resistant KB subline]. Among such alkaloids, C19-diterpenoid (e.g., lipojesaconitine, delpheline, and delcosine derivative) and C20diterpenoid (e.g., kobusine and pseudokobusine derivatives) alkaloids have shown significant antiproliferative activity, as well as provided promising new leads for further development as antitumor agents.
The pharmacological properties of the C 19 -diterpenoid alkaloids have been studied extensively and reviewed [12]. Aconitine is a toxin that exhibits activity both centrally and peripherally, acting predominantly on the cardiovascular and respiratory systems by preventing the normal closing of sodium channels [12]. This extreme toxicity resulted in the use of Aconitum extracts as poisons in hunting and warfare [13], although extracts were also used as traditional medicines by oral and topical routes. For example, the roots of Aconitum plants have been used as "bushi", an herbal drug in some prescriptions of traditional Japanese medicine for the treatment of hypometabolism, dysuria, cardiac weakness, chills, neuralgia, gout, and certain rheumatic diseases [14]. However, proper processing is essential to reduce the content of toxic alkaloids and avoid inadvertent poisoning [15][16][17]. Such obstacles encourage a good understanding of the relationships between structure and cytotoxic activity of aconitine and related compounds before they can be considered for modification and development as chemotherapeutic agents.
Our previous study demonstrated the effects of various naturally occurring and semi-synthetic C 19 -and C 20 -diterpenoid alkaloids on the growth of the A172 human malignant glioma cell line [18]. Antitumor properties and radiation-sensitizing effects of various types of novel derivatives prepared from C 19 -and C 20 -diterpenoid alkaloids were also investigated [19]. Two novel hetisine-type C 20 -diterpenoid derivatives showed significant suppressive effects against the Raji non-Hodgkin's lymphoma cell line [20]. In addition, the effects of various semi-synthetic novel hetisine-type C 20 -diterpenoid alkaloids on the growth of the A549 human lung cancer cell line were examined and subsequent structure-activity relationships for the antiproliferative effects against A549 cells were considered [21]. Since 2012, several diterpenoid alkaloid components and their derivatives exhibited antiproliferative activity against human tumor cell lines, including A549 (lung carcinoma), DU145 (prostate carcinoma), MDA-MB-231 (estrogen and progesterone receptor-negative & HER2-negative triple-negative breast cancer), MCF-7 (estrogen receptor-positive, HER2-negative breast cancer), KB (identical to cervical carcinoma HeLa derived AV-3 cell line), and multidrug-resistant (MDR) subline KB-VIN [P-glycoprotein (P-gp) overexpressing vincristine-resistant KB subline]. Among such alkaloids, C 19 -diterpenoid (e.g., lipojesaconitine, delpheline, and delcosine derivative) and C 20 -diterpenoid (e.g., kobusine and pseudokobusine derivatives) alkaloids have shown significant antiproliferative activity, as well as provided promising new leads for further development as antitumor agents.

34-23
Striking observations from the data in Table 2 were the consistent identities of the most potent alkaloids . Alkaloids 34-8, 34-10, 34-19, and 34 Interestingly, the active alkaloids were generally effective against P-gp overexpressing MDR subline KB-VIN, while alkaloids such as vincristine and paclitaxel are ineffective due to excretion from the MDR cells by P-gp. These results suggest that these diterpenoids are not substrates for P-gp.
The identity of the substituent on the C-6 acyl group affected the cytotoxic potency. For instance, the alkaloids with the highest potency against the KB-VIN cell line contained chloro (37-1), fluoro (37- 12, 37-18, and 37-19), trifluoromethyl (37-9, 37-13, and 37-18), ethoxy , or benzyloxy  substituents on the acyl group. Against the KB-VIN cell line, alkaloids 37-18 and 37-19 with both fluoro and trifluoromethyl/methyl groups were more potent than 37-9 with only a single trifluoromethyl group and even more potent than 37-2 with a single fluoro group. Similarly, alkaloid 37-13 showed increased cytotoxic activity against most cell lines compared with the related fluorinated alkaloids 37-14 and 37-15. Moreover, alkaloids with nitro, methoxy, phenyl, trifluoromethoxy, trifluoromethythio, and methyl carboxylate groups on a C-6 benzoate ester were generally less potent.

37-13
showed increased cytotoxic activity against most cell lines compared with the related fluorinated alkaloids 37-14 and 37-15. Moreover, alkaloids with nitro, methoxy, phenyl, trifluoromethoxy, trifluoromethythio, and methyl carboxylate groups on a C-6 benzoate ester were generally less potent.
In mechanism of action studies on selected diterpenoid alkaloids, the hetisine-type C 20 -diterpenoid alkaloid derivatives 68-7 and 68-22 showed important suppressive effects against Raji cells. Further study indicated that 68-22 inhibited extracellular signal-regulated kinase phosphorylation but induced enhanced phosphoinositide 3 kinase phosphorylation, leading to accumulation of Raji cells in the G1 or sub G1 phase [20]. More investigation is certainly warranted.
Furthermore, previously our study, Antitumor properties and radiation-sensitizing effects of various types of novel derivatives prepared from C19and C20-diterpenoid alkaloids were also investigated [19]. Two novel hetisine-type C20-diterpenoid derivatives (68-7 and 68-20) showed significant suppressive effects against the Raji non-Hodgkin's lymphoma cell line [20].  Among the aconitine-type C 19 -diterpenoid alkaloids, the fatty acid ester at C-8 and the anisoyl group at C-14 found in 17 may be important to the cytotoxic activity. Compounds without the fatty acid ester at C-8 were inactive, and compounds with an unsubstituted benzoyl group at C-14 were less potent.
Funding: This study was supported in part by NIH grant CA177584 from the National Cancer Institute awarded to K.H.L. as well as the Eshelman Institute for Innovation, Chapel Hill, North Carolina, awarded to M.G.