2.1. Aconitine-Type C19-Diterpenoid Alkaloids
The tested aconitine-type C
19-diterpenoid alkaloids included 21 natural alkaloids, aconitine (
1), deoxyaconitine (
2), jesaconitine (
3), deoxyjesaconitine (
4), aljesaconitine A (
5), secojesaconitine (
6), mesaconitine (
8), hypaconitine (
9), hokbusine A (
10), 14-anisoyllasianine (
12),
N-deethylaljesaconitine A (
13), aconine (
14), lipomesaconitine (
15), lipoaconitine (
16), lipojesaconitine (
17), neolinine (
18), neoline (
19), 14-benzoylneoline (
20), isotalatizidine (
21), karacoline (
22), and 3-hydroxykaracoline (
23), isolated from the rhizoma of
Aconitum japonicum THUNB. subsp.
subcuneatum (NAKAI) KADOTA [
22,
23,
24,
25,
26,
27,
28] (
Figure 2). Two synthetic aconitine-type C
19-diterpenoid alkaloids, 3,15-diacetyljesaconitine (
7) [
26] and 3-acetylmesaconitine (
11) [
29] prepared from secojesaconitine (
6) and mesaconitine (
8), respectively (
Figure 2), were also tested.
Eighteen of the 23 tested aconitine-type C
19-diterpenoid alkaloids, both natural alkaloids (
1~
6,
8~
10,
12~
14,
18~
23) and synthetic analogs (
7 and
11), were inactive (IC
50 > 20 or 40 μM) [
27,
28,
30] (
Table 1). Three natural diterpenoid alkaloids (
15~
17) exhibited cytotoxic activity against five human tumor cell lines (A549, MDA-MB-231, MCF-7, KB, and MDR KB subline KB-VIN) (
Table 1). Lipojesaconitine (
17) showed significant cytotoxicity against four tested cell lines with IC
50 values of 6.0 to 7.3 μM, but weak cytotoxicity against KB-VIN (IC
50 = 18.6 μM) [
28]. Lipomesaconitine (
15) showed moderate cytotoxicity against the KB cell line (IC
50 = 9.9 μM), but weak cytotoxicity against the other four human tumor cell lines (IC
50 =17.2 ~ 21.5 μM) [
27]. Lipoaconitine (
16) was weakly cytotoxic (IC
50 = 13.7 ~ 20.3 μM) against all five human tumor cell lines [
28]. Based on the results, the fatty acid ester at C-8 and the anisoyl group at C-14 found in
17 may be important to the cytotoxic activity of aconitine-type C
19-diterpenoid alkaloids.
2.2. Lycoctonine-Type (7,8-diol) C19-Diterpenoid Alkaloids
The tested lycoctonine-type (7,8-diol) C
19-diterpenoid alkaloid group included 12 natural alkaloids, namely nevadensine (
24),
N-deethylnevadensine (
25), and virescenine (
27), purified from rhizoma of
Aconitum japonicum subsp.
subcuneatum [
27], and 18-methoxygadesine (
26), delphinifoline (
28), delcosine (
34), 14-acetyldelcosine (
34–43), and 14-acetylbrowniine (
35), purified from root of
Aconitum yesoense var.
macroyesoense (NAKAI) TAMURA [
31,
32,
33,
34], and andersonidine (
30), pacifiline (
31), pacifinine (
32), and pacifidine (
33), purified from seeds of
Delphinium elatum cv. Pacific Giant [
35] (
Figure 3). The remaining tested C
19-diterpenoid alkaloids from this subtype were synthetic alkaloids,
N-deethyldelsoline (
29) [
18], 1-acetyldelcosine (
34-1) [
36], 1,14-diacetyldelcosine (
34-2) [
37], 1-(4-trifluoromethylbenzoyl)delcosine (
34-24) [
30], delsoline (
34-42) [
37], 1,14-di-(4-nitrobenzoyl)-delcosine (
34-45) [
30], 14-acetyl-1-(4-nitrobenzoyl)delcosine (
34-46) [
30], and 1-acyl or 1,14-diacyldelcosine derivatives (
34-3~
34-23,
34-25~
34-41,
34-44, and
34-47) [
38], prepared from delcosine (
34) or delsoline (
34-42) (
Figure 3). These 42 C
19-diterpenoid alkaloids were evaluated for antiproliferative activity against four to five human tumor cell lines (A549, DU145, MDA-MB-231, MCF-7, KB, and KB-VIN) [
30,
38] (
Table 2). Several tested lycoctonine-type (7,8-diol) C
19-diterpenoid alkaloids, both natural alkaloids (
24~
28,
30~
33) and a synthetic alkaloid (
29), were inactive (IC
50 > 20 or 40 μM). All tested delcosine derivatives that contain an acetyl or methoxy group, both natural alkaloids (
34,
34-43,
35) and synthetic analogs (
34-1,
34-2,
34-42), were inactive (IC
50 > 20 μM). However, acylation, except with an acetyl group, of the C-1 and/or C-14 hydroxy group of
34 led to various degrees of antiproliferative activity. Among the C-1 esterified alkaloids, the synthetic derivatives
34-6,
34-8,
34-10, and
34-18 exhibited significant potency against all cell lines (average IC
50 9.3, 5.3, 5.0, and 6.9 µM, respectively). Also, alkaloids
34-3,
34-16,
34-17,
34-21,
34-25,
34-27,
34-31,
34-32,
34-38, and
34-40 showed moderate potency toward all cell lines (average IC
50 12.7−20.7 µM). While alkaloid
34-32 displayed good antiproliferative activity (IC
50 8.7 µM) against KB cells, it was much less potent against A549, MDA-MB-231, and KB-VIN cells. Alkaloids
34-5,
34-13,
34-15,
34-29,
34-35,
34-37, and
34-41 exhibited only weak potency against all cell lines (average IC
50 22.0−26.5 µM). Finally, alkaloids
34-24,
34-30, and
34-34 were inactive against all five human tumor cell lines, while
34-12,
34-33, and
34-39 showed limited potency.
Among the derivatives esterified at both C-1 and -14, alkaloids 34-19 and 34-20 exhibited significant potency against all five tested cell lines (average IC50 4.9 and 5.0 µM, respectively). Alkaloid 34-9 (average IC50 11.9 µM) showed significant antiproliferative activity against MDA-MB- 231 and KB cells (IC50 4.7 and 5.8 µM, respectively) comparable with 34-19 and 34-20, but was less potent against MCF-7 and A549 (IC50 12.2 and 24.8 µM, respectively) and inactive against KB-VIN. Alkaloid 34-23 exhibited only weak potency toward all cell lines (average IC50 23.7 µM). Alkaloids 34-4, 34-7, 34-11, 34-14, 34-26, 34-36, 34-45, 34-46, and 34-47 were inactive against all five human tumor cell lines, while 34-22 and 34-28 showed limited potency.
Particularly, C-1 monoacylated delcosine derivatives (34-3, 34-6, 34-8, 34-10, 34-13, 34-21, 34-25, 34-27, and 34-35) were significantly more potent compared with corresponding C-1,14 diacylated delcosine derivatives (34-4, 34-7, 34-9, 34-11, 34-14, 34-22, 34-23, 34-26, 34-28 and 34-36). Thus, a C-1 acyloxy group and C-14 hydroxy group are crucial for enhanced antiproliferative activity of 1-derivatives. Regarding alkaloids 34-18 (pentafluorobenzoate at C-1, hydroxy at C-14), 34-19 (pentafluorobenzoate at C-1 and C-14), and 34-20 (pentafluorobenzoate at C-1, acetate at C-14), all three alkaloids were essentially equipotent against three of the five tumor cell lines, while 34-18 was somewhat less potent than the diacylated alkaloids against MCF-7 and KB-VIN cells.
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
34-20 exhibited the highest potency against all five tested tumor cell lines with IC
50 values ranging from 4.3 to 6.0 µM. The same range of potency was found with alkaloid
34-18 against A549 cells, with alkaloids
34-9 and
34-18 against MDA-MB-231 cells, and with
34-6,
34-9, and
34-18 against KB cells. The potencies of
34-6 and
34-17 (IC
50 5.6−11.8 µM) generally ranked somewhat below those of the most potent alkaloids, except against the MCF-7 cell line, where they were even less active.
The identity of the substituent(s) on the acyl group affected the antiproliferative potency. Notably, among the 1,14-diacyl and 1-acyl-14-acetyl derivatives, only alkaloids 34-19 and 34-20 with one or two pentafluorinated benzoyl esters, respectively, showed significant potency against all five tested cell lines. Alkaloid 34-9 with two 3-nitro-4-chlorobenzoyl groups showed good potency against certain cell lines. Similarly, the 1-monoacylated alkaloids with the highest potency against the five tumor cell lines contained 3-nitro-4-chloro- (34-8) and pentafluoro- (34-18) as well as 4-dichloro-methyl- (34-10) benzoyl esters. The chlorinated alkaloids 34-8 and 34-10 as well as 34-6, which has 3,5-dichloro substitution on the benzoate ester, were more potent than 34-5 with only a single chloro group or 34-13 with chloro and fluoro groups. Similarly, alkaloid 34-18 showed increased antiproliferative activity against the five tumor cell lines compared with other fluorinated alkaloids 34-13~34-17, 34-21~34-27. Moreover, with some exceptions against certain cell lines, alkaloids with bromo (34-3 and 34-4), dimethylamino (34-12), dimethoxy (34-29), trimethoxy (34-30), diethoxy (34-31), benzyloxy (34-32), cyano (34-33), methylenedioxy (34-34 and 34-35), nitro (34-45 and 34-46), and ethoxy (34-47) substituted benzoate esters or phenylacetyl (34-37), cinnamoyl (34-38 and 34-39), 1-naphthoyl (34-40), and anthraquinone-2-carbonyl (34-41) esters were less potent or inactive.
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.
2.3. Lycoctonine-Type (7,8-methylenedioxy) C19-Diterpenoid Alkaloids
The tested lycoctonine-type (7,8-methylenedioxy) C
19-diterpenoid alkaloids included 19 natural alkaloids, delcorine (
36), delpheline (
37), pacinine (
38), yunnadelphinine (
39), melpheline (
40), bonvalotidine C (
41),
N-deethyl-
N-formylpaciline (
42),
N-deethyl-
N-formylpacinine (
43), isodel-pheline (
44), pacidine (
45), eladine (
46),
N-formyl-4,19-secopacinine (
47),
N-formyl-4,19-secoyunna-delphinine (
48), iminoisodelpheline (
49), iminodelpheline (
50), laxicyminine (
51),
N-deethyl-19-oxo-isodelpheline (
52),
N-deethyl-19-oxodelpheline (
53), and 19-oxoisodelpheline (
54), purified from seeds of
Delphinium elatum cv. Pacific Giant [
35,
39,
40,
41,
42] (
Figure 4). The remaining 22 tested C
19-diterpenoids were synthetic derivatives (
37-1~
37-22) [
43] prepared from
37 (
Figure 4).
All tested lycoctonine-type (7,8-methylenedioxy) C
19-diterpenoid alkaloids were evaluated for antiproliferative activity against human tumor cell lines [
30,
40,
41,
42,
43] (
Table 3). The lycoctonine-type (7,8-methylenedioxy) C
19-diterpenoid alkaloids, both the natural alkaloids (
36~
54) and synthetic analogs that did not contain a C-6 ester group (
37-20 and
37-22), were inactive (IC
50 > 20 or 40 μM). Among the C-6 esterified alkaloids,
37-1,
37-17, and
37-18 exhibited the highest average potency toward four tested cell lines (A549, DU145, KB and KB-VIN; average IC
50 9.83, 9.57, and 9.41 μM, respectively). Alkaloids
37-3,
37-5~
37-7,
37-9,
37-10,
37-12,
37-13,
37-16, and
37-19 showed moderate potency against all tested cell lines (average IC
50 13.9−20.8 µM). However, alkaloid
37-13 showed significantly increased cytotoxic activity (IC
50 10.2 μM) against A549 cells compared with
37-1,
37-17, and
37-18, but was generally less potent against DU145 and KB cells. While alkaloids
37-12,
37-13,
37-16, and
37-19 displayed good antiproliferative activity (IC
50 6.8, 9.1, 6.5, and 4.7 µM, respectively) against KB-VIN cells, they were much less potent against A549, DU145, and KB cells. Alkaloids
37-4 and
37-21 were inactive against all tested cancer cell lines, while
37-2,
37-8,
37-11, and
37-14 exhibited only weak potency toward all cell lines (average IC
50 23.0−29.2 µM).
The most noticeable observations from the data in
Table 3 were the degree and relative ratio of KB/KB-VIN potency. Among the four cancer cell lines tested, the highest potency was found against the KB-VIN cell line by alkaloids
37-17~
37-19 (IC
50 4.22, 4.40, and 4.71 μM, respectively), followed by alkaloids
37-16,
37-12,
37-1,
37-13, and
37-9 (IC
50 6.50, 6.80, 8.27, 9.10, and 11.9 μM, respectively). Generally, all active alkaloids showed the highest potency against the KB-VIN cell line compared with the other three tested cancer cell lines. Moreover, alkaloids
37-12,
37-16,
37-13, and
37-19 showed over two-fold selectivity between the two cell lines (ratio of IC
50 KB/IC
50 KB-VIN: 2.15, 2.28, 2.31, and 2.57, respectively). Alkaloids
37-2,
37-5, and
37-17 displayed weak selectivity between the KB and KB-VIN cell lines (ratio of IC
50 KB/IC
50 KB-VIN: 1.55, 1.36, and 1.62, respectively). Finally, alkaloids
37-1,
37-3,
37-6~
37-9,
37-11,
37-14,
37-15, and
37-18 displayed similar potency against the KB and KB-VIN cell lines (ratio of IC
50 KB/IC
50 KB-VIN: 1.07, 1.17, 1.06, 1.21, 1.04, 1.25, 1.07, 1.07, 1.17, and 1.23, respectively).
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 (37-16), or benzyloxy (37-17) 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.