The Bicolorine Group
These types of compounds (such as the parent bicolorine 9) closely resemble trisphaeridine alkaloids but may be differentiated by the presence
of an N-methyl functionality [9]. Like trisphaeridine, they also display flattened architectures, thus considerably
restricting the size of their molecules [9]. 5,6-Dihydrobicolorine (24) was the first of such compounds to be examined for antiproliferative effects [22]. It was isolated together with several other alkaloids from the Spanish plant Narcissus pseudonarcissus subsp. bicolor (L.) Baker. and subjected to a screen involving 2 human cancers (Molt4 T-lymphoma
and HepG2) as well as a normal murine fibroblastic (LMTK) cell line [22]. 5,6-Dihydrobicolorine (24) proved to be highly selective based on its activity against the Molt4 (ED50 0.8 µg/mL) and HepG2
(ED50 > 50 µg/mL) cells (Table 2S, Supporting Information) [22]. This was, however, mitigated by its marked effect on the normal LMTK cell line
(ED50 0.7 µg/mL) as well as the potent activity of the positive control (vincristine sulfate)
against both cancers (ED50s 0.04 and 0.01 µg/mL, respectively) [22]. The investigation of Morikawa et al. (2016), alluded to above, also accounted for
the presence of 5,6-dihydrobicolorine (24) in Narcissus tazetta subsp. chinensis
[20]. This alkaloidʼs biological profile was then fashioned from its ability to inhibit
theophylline-induced melanogenesis effects in B16 4A5 melanoma cells [20]. The best result observed for 5,6-dihydrobicolorine (24) was 61.7% inhibition of melanoma cell growth at a tested dosage of 30 µM, which
closely matched that of the
arbutin standard used (61.5% inhibition at an equimolar concentration) [20].
In addition to the cytotoxic constituents described above in L. radiata
[18], this plant also yielded 2-hydroxy-5,6-dihydrobicolorine (25) [23]. Eight separate tumor cell lines were used to examine this alkaloidʼs cytotoxic
effects, including BEN-MEN-1 benign meningioma, CCF-STTG1 astrocytoma, CHG-5 oligodendroglioma,
SHG-44 astrocytoma, U251 glioblastoma, HL-60, SMMC-7721 hepatocellular carcinoma,
and SW480 colon carcinoma cells [23]. Except for HL-60 cells against which low activity was observed (IC50 81.3 µM), 2-hydroxy-5,6-dihydrobicolorine (25) was inactive (IC50s > 100 µM) against the remaining tumor cells [23]. These measurements were poor compared to the doxorubicin standard, which exhibited
an impressive mean IC50 value of 25.4 nM against all cells screened [23].
Further investigation of the Asian genus Lycoris led to the indigenous Chinese plant Lycoris aurea (LʼHér.) Herb. which yielded 8 compounds, 6 of which were identified as typical Amaryllidaceae
alkaloid structures [24]. One of these was the novel compound 1,2-dihydroxy-5,6-dihydrobicolorine (26), a close relative of 2-hydroxy-5,6-dihydrobicolorine (25) that was identified prior in L. radiata
[23]. No less than 7 human tumor cell lines were engaged for the cytotoxicity evaluation
that accompanied the phytochemical study [24]. These included 4 squamous cells (SCL-1, UMSCC-1, SCC-PKU, TCA-83), 2 epithelial
cells (Hep-2, CAL-27), and a single pharyngeal carcinoma cell line (Detroit-562).
1,2-Dihydroxy-5,6-dihydrobicolorine (26) was shown to be a poor cytotoxic agent against a majority of the cells (IC50s > 100 µM), with low activity
detected only in SCC-PKU squamous carcinoma cells (IC50 87.6 µM) [24]. These measurements were standardized against doxorubicin, which exhibited an impressive
mean IC50 value of 0.023 µM against the 7 cancer cells [24]. Among the 22 alkaloids isolated and identified from L. longituba was the bicolorine-related structure 5,6-dihydrobicolorine (24), subsequently examined for neuroprotective effects modeled on the SH-SY5Y neuroblastoma
cell line [19]. This was estimated via the ability of 5,6-dihydrobicolorine (24) to buffer the cells from injuries induced by H2O2-, Aβ
25 – 35-, or CoCl2
[19]. Accordingly, 5,6-dihydrobicolorine (24) was able to achieve this at the lowest tested concentration (6.25 µM) as shown by
cell viabilities of 65.7%, 70.9%, and 70.9%,
respectively [19]. In the same instance, untreated cells reflected cell viabilities of 59.7%, 59.3%,
and 70.3%, respectively [19].
Bicolorine (9) was identified in P. maritimum among 6 other alkaloids found in its MeOH bulb extract [16]. The accompanying PC-3 cell line examination showed dose-responsive effects at test
concentrations of 5, 10, 20, and 50 µM, with cell viability of 73% observed at 5 µM,
which was markedly reduced to 29% by a 10-fold increase in concentration [16]. Compared to the standard Z-4-ethylmercaptophenylmethylene hydantoin (IC50 37.4 µM), bicolorine (9) with its IC50 of 15 µM was shown to be a better antiproliferative agent [16]. 5,6-Dihydrobicolorine (24) was among the 15 alkaloids described above from Z. candida and screened against HL-60, K562, A549, HepG2, and HT-29 cells [17]. Similar to the results seen for trisphaeridine (8) in the same study, 5,6-dihydrobicolorine (24) was poorly
active against all 5 cell lines (IC50s > 40 µM) compared to the anticancer drug taxol (mean IC50 0.08 µM) (Table 2S, see Supporting Information) [17].
The Cherylline Group
Cherylline-type alkaloids constitute a very small group within the Amaryllidaceae,
which until recently was thought to be comprised of the 2 key representatives, cherylline
(10) and latifine (27) [9]. Their structures harbor the basic isoquinoline motif onto which have been added,
as with most Amaryllidaceae alkaloids, oxygen-related functionalities at the C-6 and
C-7 carbons of ring-A [9]. However, the delineating structural feature of these compounds is α-arylation, which relates to the para-substituted aryl group that is stereoselectively bonded to the benzylic C-4 position
of ring-B [9]. To date, only a single study has been carried out to ascertain the antiproliferative
potential of cherylline and its congeners [25]. Crinum jagus (J.Thomps.) Dandy yielded 9 alkaloid structures, including cherylline (10),
gigantelline (28), and gigantellinine (29), which were then examined for cytotoxic effects against MCF7 adenocarcinoma cells
[25]. The tetrazolium stain XTT showed that cherylline (10), gigantelline (28), and gigantellinine (29) exhibited poor activities (cell viabilities ≥ 80%) at most of the tested dosages
(0.4 – 100 µM) [25]. The best activities were seen at the highest concentration as reflected by cell
viabilities of 15%, 70%, and 80%, respectively [25]. The activity for cherylline (10) at 400 µM closely matched that of lycorine (4) (10% cell viability), which was used as the positive control [25].
The Galanthamine Group
Through the prescription drug galanthamine (1), the parent compound of this alkaloid group, the Amaryllidaceae has been firmly
installed as a biologically and chemically privileged plant family [26]. Structurally, they are tetracyclic compounds consisting of an aryl ring, a carbocyclic
ring, and 2 heterocyclic rings [9]. Ring-A is a 1,2,3,4-tetrasubstituted aryl system, ring-B is a 1,2,3-trisubstituted
heptenyl system, ring-C is a dioxygenated α,β-unsaturated hexyl system, and ring-D is a furanyl system with attachments to each
of the other rings [9]. The distinguishing feature of galanthamine alkaloids is a spiro-junction at the confluence of rings B and C [9]. While most studies of these compounds understandably have focused on their motorneuron-related
functions [26], significant effort as described here has
also gone into probing their cytotoxic properties.
A noteworthy early study to record the antiproliferative effects of the Amaryllidaceae
also had in its number 4 representatives from the galanthamine group of alkaloids
[27]. Intriguingly, this time frame was well before the group became visible in the motorneuron
arena [26]. As such, galanthamine (1), norgalanthamine (30), 3-epigalanthamine (31), and lycoramine (32) ([Fig. 1]) were isolated from Narcissus tazetta L. and examined for cytotoxic effects against HeLa cells [27]. However, none of the compounds were able to significantly inhibit HeLa cell growth
(MICs > 400 µM) relative to the anticancer drug 6-mercaptopurine that was applied
at 1 µM [27]. This study, which incorporated no less than 18 different Amaryllidaceae alkaloid
structures, is also significant in that it was the first to
demonstrate the structure-dependent activities for these alkaloids [27]. Accordingly, while the galanthamine group (and other alkaloids) were inactive or
poorly active on the HeLa cells, narciclasine (2), lycorine (4), and haemanthamine (5), which have since been established as the cytotoxic markers of the family, were
notably active with MICs of 0.1, 6.0, and 4 µM, respectively [27]. A few years later, galanthamine (1) and lycoramine (32) from N. tazetta were examined for their effects on Rauscher leukemia [28]. In the study, NIH/3T3 murine fibroblastic cells were infected with the wild-type
Rauscher leukemia virus, and the MTDs were determined based on the morphology and
density of cells and the pH changes measured for the culture media [28]. These were determined for galanthamine (1) and lycoramine
(32) to be > 200 and 20 µg/mL, respectively (Table 3S, Supporting Information) [28]. The potencies of narciclasine (2) and lycorine (4) over the galanthamine compounds were demonstrated by their respective MTD values
of 0.005 and 0.2 µg/mL [28]. An additional investigation of N. tazetta led to the identification of galanthamine (1), lycoramine (32), and narwedine (33) from its bulb extract [29]. Five human cancers, including MCF7, HeLa, K562, CEM lymphoblastic leukemia, and
G-361 malignant melanoma cells, were selected to probe the cytotoxic effects of the
isolated alkaloids [29]. The only activity detected for the 3 N. tazetta isolates was that of narwedine (33) against CEM cells (IC50 31.6 µM) relative to staurosporine (IC50 0.023 µM), with IC50
values shown to be > 50 µM in all other instances [29]. Despite this poor showing against the cancer cells, galanthamine (1), lycoramine (32), and narwedine (33), when examined for effects against normal cells, were all inactive against BJ fibroblasts
(IC50s > 50 µM) [29].
The study by Weniger et al. (1995) described earlier involved a screen of 25 different
alkaloid structures from various species of the Amaryllidaceae against Molt4, HepG2,
and LMTK cells [22]. Included in the library were the galanthamine representatives galanthamine (1), norgalanthamine (30), and N-formylgalanthamine (34), which were obtained from bulbs of Narcissus confusus Pugsley and Narcissus nivalis Graells [22]. The MTT colorimetric assay indicated good activities for norgalanthamine (30) and N-formylgalanthamine (34) (ED50 s 0.6 and 1.6 µg/mL, respectively) compared to galanthamine (1) (ED50 > 50 µg/mL) against the Molt4 cell line [22]. Only N-formylgalanthamine (34) (ED50 9.0 µg/mL) was active against HepG2 cells, the remaining 2 compounds reflecting
ED50 values that were over 50 µg/mL [22]. By contrast, the reference compound vincristine sulfate exhibited ED50s of 0.04 and 0.01 µg/mL against both of these cell lines, respectively [22]. Despite these cancer cell line activities, all 3 alkaloids were also very active
on the nontumoral LMTK cells (ED50s 0.5, 0.5, and 0.6 µg/mL, respectively) [22]. The poor activity of galanthamine (1) against the HepG2 cell line was later ratified in a separate study [30] where cell viabilities > 85% were detected with all tested concentrations and time
intervals [30]. As many as 23 different alkaloid substances were found in Narcissus cv. Professor Einstein, among which were the galanthamine representatives lycoraminone
(35) and norlycoramine (36) [31]. Nine cell lines were chosen in the antitumor screen using the WST-1 assay to ascertain
the alkaloidsʼ antiproliferative effects [31]. Despite the cell line diversity, lycoraminone (35) and norlycoramine (36) were shown to be inactive, with cell viabilities that were > 90% in all cases relative
to doxorubicin (mean value 26.3%) [31]. The studyʼs positive aspect was that neither compound was active on the normal
fetal lung fibroblast cell line MRC-5, where cell viabilities measured were 96% and
108%, respectively [31].
Studies on Phaedranassa dubia (Kunth) J. F. Macbr. led to the identification of 8 alkaloids, among which were the
galanthamine compounds galanthamine (1), 3-epinorgalanthamine (37), and sanguinine (38) [32]. The attendant cytotoxicity evaluation involved L6 rat myoblasts, where all 3 compounds
exhibited negligible effects (IC50s > 300 µM) [32]. Two galanthamine-type compounds viz. galanthamine (1) and chlidanthine (39) were identified in the Andean representative Chlidanthus fragrans Herb. [33]. HT-29 and Caco-2 colon adenocarcinoma cells from the ATCC were used in the complementary
cytotoxicity evaluation of both alkaloids [33]. In the event, galanthamine (1) and chlidanthine (39) exhibited weak activities against HT-29 (IC50s 49.3 and 52.7 µM, respectively) and Caco-2
(IC50s 61.4 and 72.9 µM, respectively) relative to lycorine (4) (mean IC50 1.1 µM) [33]. However, the study demonstrated that both compounds were relatively nontoxic to
normal FHs-74 intestinal epithelial cells (IC50s 67.6 and 78.1 µM, respectively) [33]. The weak activity for galanthamine (1) against the normal cell line is significant given its usage in Alzheimerʼs disease
chemotherapeutics [26]. Chlidanthine (39) from Chlidanthus fragrans then became part of a library of 22 alkaloids that was subjected to a general cytotoxicity
screen against 16 different cancer cell lines [34]. Also included in the library were lycoramine (32) (from Zephyranthes robusta (Herb. ex Sweet) Baker), narwedine (33) (from Narcissus poeticus L.), and the quaternary salt galanthamine
hydrobromide (40) that was obtained from a commercial source [34]. It was shown that with a few exceptions, the cell viabilities produced by lycoramine
(32), narwedine (33), chlidanthine (39), and galanthamine hydrobromide (40) were mostly > 90% [34]. These estimations were made against doxorubicin as a control for which a mean cell
viability of 28% was determined [34]. This lack of cell line anticancer effects was matched by the performance of the
4 compounds on noncancerous NHDF cells, wherein cell viabilities were unaffected (93%,
103%, 96%, and 98%, respectively) [34].
Phytochemical studies of the Brazilian plant Hippeastrum solandriflorum (Lindl.) Herb. yielded 8 alkaloids, amongst which were identified galanthamine (1), sanguinine (38), and galanthamine-N-oxide (41) [35]. The antiproliferative effects of all isolated compounds were then measured against
4 human cancers including HCT-116 colon carcinoma, HL-60, OVCAR8 ovarian carcinoma,
and SF-295 glioblastoma [35]. Negligible effects (IC50s > 50 µM) were observed for all 3 alkaloids against OVCAR8 and SF-295 cells, as well
as galanthamine (1) and galanthamine-N-oxide (41) against HCT-116 and HL-60 cells [35]. Sanguinine (38) was the only compound of the set that showed activities against the latter 2 of
these cell lines, although at 29.0 and 32.1 µM, respectively, they were mild compared
to the standard doxorubicin (mean
IC50 0.17 µM) [35]. Sanguinine (38) was one of several alkaloid principles identified in a bulb extract of the Brazilian
native Griffinia gardneriana as alluded to earlier in the text [21]. Cytotoxicities against both HepG2 and MCF7 cancer as well as normal L929 cells
were determined via the MTT spectrophotometric method [21]. Intriguingly, sanguinine (38) proved to be twice as active on the normal cell line (IC50 91.1 µM) compared to the tumor cells, where IC50s in each case were > 180 µM [21]. Sanguinine has also been described in Crinum jagus, one of 9 alkaloids to have been so done [25]. These were then exposed to the MCF7 cell line, wherein viabilities were ascertained
by XTT staining [25]. Sanguinine (38) was shown to
be inactive as cell viabilities were seen to be ~ 100% at most of the tested
concentrations [25].
The Japanese plant Lycoris albiflora Koidz. yielded 15 alkaloids, including galanthamine (1), lycoramine (32), and lycoramine-N-oxide (42), as members of the galanthamine group of alkaloids [36]. The biological component of the study utilized HL-60 cells with which to probe
the antiproliferative action of the isolated alkaloids [36]. None of the 3 compounds proved to be active (IC50s > 55 µM) (Table 3S, Supporting Information), with the reference standard etoposide shown to be far superior
(IC50 0.42 µM) [36]. In addition to the phytochemical and biological study described above [24], L. aurea has been pursued further for its cytotoxic alkaloid principles [37]. It yielded 13 alkaloid structures including 4 galanthamine analogs that were identified
as
galanthamine (1), norgalanthamine (30), N-chloromethylgalanthamine (43), and N-chloromethyllycoramine (44) [37]. In the studyʼs cytotoxicity component, the MTT spectrophotometric test was used
to establish the neuroprotectiveness of the isolated compounds from CoCl2- and H2O2-induced injuries in SH-SY5Y cells [37]. Significant protection from CoCl2-induced injury was observed at most tested levels with only galanthamine (1), norgalanthamine (30), and N-chloromethylgalanthamine (43) offering no such effect at the lowest concentration (6.25 µM), although the differences
in these instances were minimal compared to untreated cells [37]. By contrast, all 4 alkaloids were able to significantly protect SH-SY5Y cells from
injuries that were H2O2-based, with
cell viability of 73.3% shown for norgalanthamine (30) at the lowest tested concentration relative to untreated cells (67.3% viable) [37]. Complementing the description of 2-methoxy-1,2,3,4-tetrahydrotrisphaeridine (22) in the study of L. radiata was the isolation of the galanthamine alkaloid 3-epidihydrosanguinine (45) from its EtOH bulb extract [18]. As previously noted, A549 and LoVo cells were used in the adjoining component of
the study to measure the cytotoxic effects of the isolated alkaloids [18]. However, 3-epidihydrosanguinine (45) proved to be weak at such activity, with IC50s in each case > 20 µM relative to doxorubicin (mean IC50 0.53 µM) [18]. Twenty-two alkaloids referred to earlier were identified in bulb extracts of the
Chinese medicinal plant L. longituba, among which were
7 members of the galanthamine group of alkaloids [19]. These were identified by spectroscopic means as galanthamine (1), norgalanthamine (30), sanguinine (38), N-chloromethylgalanthamine (43), N-chloromethyllycoramine (44), 11β-hydroxygalanthamine (46), and 9-O-demethyllycoramine (47) [19]. The cytotoxicity aspect of the study involved a probe to determine if any of these
alkaloids could protect SH-SY5Y neuroblastoma cells from injuries induced by H2O2, Aβ
25 – 35, or CoCl2
[19]. It was seen that all of these alkaloids at 5 dosage levels (6.25, 12.5, 25, 50,
and 100 µM) offered significant neuroprotection from such injuries [19]. Notable responses were observed after cells were treated with galanthamine (1) and
N-chloromethyllycoramine (44) at the lowest tested concentration following exposure to Aβ
25 – 35 and H2O2, respectively [19]. SH-SY5Y cell viabilities in these instances were 80.7% and 82.3% relative to untreated
cells that reflected viabilities of 77.1% and 72.3%, respectively [19].
Commercial grade galanthamine was the substrate employed in additional studies of
the SH-SY5Y neuroblastoma cell line [38]. In this case, the alkaloidʼs protectiveness was estimated via its ability to influence Aβ
1 – 42-mediated toxicity in SH-SY5Y cells [38]. Giesma staining was used to ascertain the clonogenic survival of cells [38], showing a 70% survival rate at the low dosage (0.1 µM) and as high as 95% survival
in cells receiving 10 µM galanthamine [38]. Interestingly, galanthamine alone (i.e., without applying Aβ
1 – 42) at its maximum concentration did not affect the survival of the SH-SY5Y cells [38]. Further studies of neuroprotection of SH-SY5Y cells involved a probe of the parent
compound galanthamine (1) from Phaedranassa lehmannii Regel [39]. Galanthamine (at 3 µg/mL) had little effect on the viability of SH-SY5Y cells (> 90%)
as determined by spectrophotometric means after MTT staining [39]. Also, galanthamine (at 0.3 µg/mL) was able to protect cells from the adverse effects
of rotenone/oligomycin A (3 : 1), in which cell viability of 80% was determined [39]. Similarly, cell viability of 75% was observed when cells treated prior with galanthamine
(0.3 µg/mL) were exposed to okadaic acid (20 nM) [39].
The Montanine Group
Montanine alkaloids are characterized by their B-ring moieties, which are 7-membered
nitrogen-containing heterocycles [9]. A further diagnostic feature of their structures is the presence of a methano-bridge
that straddles ring-B between the nitrogen and C-11 positions [9]. As expected, this feature introduces considerable strain to the molecule, dividing
ring-B into 2 parts [9]. The first part comprising the nitrogen atom, C-6, C-6a, C-10a, and C-11 is nearly
as flat as the aryl A-ring, while the second part made up of C-4a and C-11a (and the
nitrogen atom and C-11) is forced out of the plane to become perpendicular to the
first part (and to ring-A) [9]. Consequently, ring-C adopts a near-perfect boat conformation with C-2 and C-4a
as the bow and stern parts and connectivities to ring-B occurring via C-4a and C-11a [9].
Ring-C usually also incorporates double bond and oxygen-related functionalities
in its structure [9]. The first report on the cytotoxic effects of montanine alkaloids was produced after
a dual phytochemical-biological study of the Ukrainian plant Narcissus angustifolius Curt. [40]. The first part of the study involved the isolation of 7 alkaloids, including the
2 montanine analogs nangustine (48) and pancracine (49) [40]. Normal L6 mouse myoblasts were utilized in the second aspect of the study to measure
the cytotoxicities of the isolated compounds [40]. Encouragingly, neither compound could elicit any response in the myoblast cells
wherein ED50s were > 90 µg/mL, respectively [40]. It has been outlined above that phytochemical investigation of the cultivar Narcissus cv. Professor
Einstein yielded 23 alkaloids [31]. Among these alkaloids identified by their spectroscopic and spectrometric data,
the dihydroxy montanine analog pancracine (49) was one of the plantʼs major constituents [31]. The cytotoxicity probe involved 9 cell lines, one of which was the normal human
fibroblast cell line MRC-5 [31]. Pancracine was shown to be active against all 8 cancer cells, with IC50s of between 2 – 3 µM observed against Molt4, A549, HT-29, MCF7, and SAOS-2 cells
(Table 4S, Supporting Information) [31], while the effects on the remaining cells (Jurkat, A2780, HeLa) were more subdued
(IC50s ~ 5 µM) [31]. However, the overall effect of pancracine (49) (mean IC50 3.5 µM) was over 10-fold less than that observed for the standard doxorubicin (mean
IC50 0.3 µM)
[31]. Nonetheless, pancracine (IC50 5.2 µM) proved to be far less toxic to the normal MRC-5 cell line compared to doxorubicin
(IC50 0.7 µM) [31].
Pancracine (49) was among several alkaloids identified in Pancratium canariense Ker Gawl. [41]. The libraryʼs montanine component was expanded to include a further 2 chloride
analogs (50 and 51) acquired by semisynthetic manipulations [41]. Cytotoxic effects against 4 human tumor cell lines, including A2780 (ovary), SW1573
(lung), T-47D (breast), and WiDr (colon), were determined via the sulforhodamine B assay 48 h after incubation with the respective alkaloids [41]. In the event, pancracine (49) and the chloride analog 50 were significantly active, with mean GI50 values of 7.1 and 5.9 µM, respectively [41]. The best activity seen for pancracine (49) was against SW1573 cells (GI50 4.3 µM), while that for compound 50 was in the A2780 cell line (GI50 3.4 µM) [41]. Compound 51 reflected GI50s > 100 µM against 3 of the cells tested, with weak activity seen against the one
remaining (A2780, GI50 75.2 µM) [41]. The cancer cell line activities for pancracine (49) are noteworthy since it was previously shown to be inactive on nontumoral L6 mouse
myoblasts [40]. In continued attempts to probe the cancer cell line effects of semisynthetic analogs
of montanine (13) ([Fig. 3]) [41], a library of such compounds was assembled via a series of elegant chemical maneuvers [42]. These all emanated from the α-crinane parent compound haemanthamine (5), which, under basic conditions, has been amenable toward transformation into the
montanine skeleton [42]. Interestingly, the natural
compound manthine (52), a C-2 methoxy analog of montanine, was also secured in good yield from haemanthamine
(5) [42]. The 14 other analogs (50, 53 – 65) bore varying degrees of structural diversity that significantly amplified the variability
of the montanine library that was subsequently examined for cytotoxic effects [42]. The panel of cell lines included those that were inherently either resistant (A549
lung carcinoma, SKMEL-28 melanoma, U373 glioblastoma) or sensitive (MCF7 breast adenocarcinoma,
Hs683 oligodendroglioma, B16F10 murine melanoma) to proapoptotic stimuli [42]. Manthine (52) was identified as the pick of the compound library (mean GI50 3.7 µM), with good activities seen for it across both the sensitive and resistant
cell lines [42]. Notable responses were also observed with some of the
semisynthetic analogs such as 53 (mean GI50 11.5 µM), 55 (mean GI50 13.0 µM), 58 (mean GI50 14.5 µM), and 64 (mean GI50 13.8 µM) [42].
Fig. 3 Cytotoxic agents in the montanine, lycosinine, augustamine and cripowellin alkaloid
groups of the Amaryllidaceae.
The study of the Brazilian plant Hippeastrum vittatum (LʼHér.) Herb. is significant because it was the first to unravel the potential of
montanine alkaloids as cytotoxic agents [43]. The fresh bulb EtOH extract yielded 3 known alkaloids, one of which was the parent
compound montanine (13) [43]. The cell lines used for the study included HT-29 (colon adenocarcinoma), H460 (non-small
cell lung carcinoma), RXF393 (renal cell carcinoma), MCF7 (breast cancer), and OVCAR3
(epithelial ovarian cancer) [43]. Montanine (13) exhibited good activities against several Hippeastrum cultivars, including Hippeastrum cv. Ferrari, Hippeastrum cv. Double King, Hippeastrum cv. Pretty Nymph, and Hippeastrum cv. Spartacus [44]. In this instance, the cells screened were the same 9 used above by Breiterova and
co-workers
(2020) [31]. Here, montanine exhibited IC50s < 1.5 µM in most of the cells, except for PANC-1 (IC50 2.3 µM), A2780 (IC50 1.7 µM), and HeLa (IC50 2.0 µM) cells [44].
Lycoris longituba, a Chinese medicinal herb, yielded several alkaloids from its bulb extract, as indicated
earlier [19]. Lycolongirine C (66) was identified from its spectroscopic data as belonging to the montanine group of
alkaloids [19]. In addition to possessing a quaternized nitrogen atom, lycolongirine C is unique
in that the methano-bridge within its structure adopts a β-configuration [19]. Its cytotoxic potential was ascertained via its ability to alleviate injuries to SH-SY5Y neuroblastoma cells induced by H2O2, Aβ
25 – 35, and CoCl2
[19]. Significant protection was observed for lycolongirine C (66) at the lowest tested concentration (6.25 µM) against H2O2-induced injury, with cell viability in this instance shown to be a robust 90.4% relative
to untreated cells (72.6%) [19]. The effect was attenuated following both Aβ
25 – 35 (68.1% versus 58.1%) and CoCl2 (69.7% versus 70.8%) treatments [19]. Montabuphine (67) was a further member of this group found among several alkaloids in the Chinese
plant Lycoris sprengeri Comes ex Baker [45]. Although a significant quantity (40 mg) of this compound was isolated from the
dried bulb material, surprisingly, it was not included, as others were, in the screen
for neuroprotective effects in SH-SY5Y cells after exposure to H2O2, Aβ
25 – 35, and CoCl2
[45].
Several representatives of the Southern African genus Haemanthus are known to be producers of alkaloids belonging to the montanine group of compounds
[4]. Therefore, the finding that 2 of the 4 alkaloids isolated from Haemanthus humilis Jacq. were montanine-type alkaloids was not surprising [46]. After chromatographic and spectroscopic analyses, they were identified as montanine
(13) and coccinine (68) [46]. The cell lines used for the cytotoxicity tests were sourced from the ATCC and included
breast MCF7, Hs578T, MDA-MB-231, colon HCT-15, lung A549, and melanoma SKMEL-28 [46]. As such, montanine (13) was identified as the pick of the 2 compounds, with IC50s < 5 µM in 4 of the 6 cell lines examined [46]. Its best activity was against A549 cells (IC50 1.9 µM), while the
melanoma cell line SKMEL-28 proved to be least susceptible to montanine exposure
(IC50 23.2 µM) [46]. Coccinine (68) exhibited IC50s that were between 5 – 10 µM against A549, MCF7, and Hs578T, while values of 15 – 20 µM
were seen for HCT-15 and MDA-MB-231 cells [46]. Interestingly, coccinine was also poorly active on the SKMEL-28 cell line (IC50 > 50 µM) [46].
The Lycosinine Group
This group of alkaloids, such as lycosinine B (14), are by definition 7-arylindoline entities [9]. In terms of Amaryllidaceae alkaloid chemosystematics, they may be considered as
truncated analogs of the more recognizable lycorine (4) and homolycorine (12) alkaloids of the family [9]. It can be seen that the conversion of lycosinine B (14) into a homolycorine compound is a straightforward operation involving B-ring lactonization
to connect C-6 to C-1 via its carboxylate [9]. Conversion to a lycorane compound would require a 180° rotation about the C-10a/C-10b
bond, N-demethylation, and ring-B closure via imine formation or lactamization [9]. Lycosinine B (14) was amongst 24 alkaloids mentioned above identified in the Chinese plant L. sprengeri
[45]. However, the paucity
of material obtained precluded any further investigation into its neuroprotective
effects in SH-SY5Y cells [45].
By contrast, 22 alkaloids were described from the Chinese medicinal plant L. longituba, among which was the highly functionalized lycosinine-type alkaloid lycolongirine
B (69) [19]. In this instance, all of the isolated compounds were examined for protective effects
on SH-SY5Y neuroblastoma cells that were primed with H2O2 (300 µM), Aβ
25 – 35 (1 µM), or CoCl2 (265 µM) [19]. Lycolongirine B (69) was seen to be most effective in all cases at its lowest tested concentration (6.25 µM)
out of 2-fold serial dilutions starting from 100 µM [19]. The cell viabilities observed for treated and untreated cells were as follows:
after H2O2 exposure (60.9%, 51.5%); after Aβ
25 – 35 exposure (78.3%, 74.4%); after CoCl2 exposure (61.7%, 54.4%) [19].
Apart from these limited studies, a further 3 papers were identified that probed
the mechanistic aspects related to the cytotoxicity of lycosinine alkaloids, as will
be discussed later in the text [12], [13], [47].
The Augustamine Group
These alkaloids have a narrow distribution in the Amaryllidaceae, where they have
to date been located only in the genus Crinum L., notably in Crinum x amabile Donn ex Ker Gawl., Crinum kirkii Baker, and Crinum latifolium L. [48], [49], [50], [51]. Structurally, they are similar to homolycorine alkaloids in that their A, B, and
C rings constitute a phenanthridine system, wherein ring-B (as in the latter) is also
a pyran moiety [9]. However, augustamine alkaloids may be distinguished by the positioning of ring-D,
which is attached to ring-C via C-4a and C-10b, an attachment that occurs via C-4 and C-4a in homolycorine-type compounds [9]. A further marker for these alkaloids is the presence of an ether bridge between
C-2 and C-6, which goes into making up the
highly strained 1,3-dioxolane ring system [9]. Following the description of augustamine (15) in Crinum x augustum Roxb. (synonym Crinum x amabile Donn ex Ker Gawl.) [48], a preliminary screen showed it to be inactive against Molt4 T-lymphoma cells [52]. More recently, interest in the cytotoxic properties of these compounds has been
rekindled [50], [51]. The Asian medicinal plant C. latifolium yielded 11 alkaloid entities from its MeOH leaf extract, one of which was identified
as augustamine (15) [50]. However, it was subsequently shown via the SRB method to be inactive (IC50s > 100 µM) against 5 cancer cells (KB, HepG2, MCF7, SKMEL-2, and LNCaP) that formed
the cytotoxicity component of the study [50]. The following
study involved a phytochemical and biological examination of Crinum x amabile, an Asian representative that was nevertheless sourced from a naturalized population
in Espirito Santo (Brazil) [51]. In the process, 25 alkaloid constituents, including augustamine (15), were isolated from MeOH bulb extracts and identified using spectrometric and spectroscopic
techniques [51]. Examined against normal L6 mouse myoblasts, augustamine (15), encouragingly, exhibited poor activity (IC50 81.6 µg/mL) [51]. By comparison, the standard drug podophyllotoxin was in parallel screening far
more potent (IC50 0.004 µg/mL) [51].
The Cripowellin Group
Cripowellins may be considered as B,C, D-seco-analogs of crinane alkaloids [9]. All compounds described to date have the ethano-bridge positioned in an α-fashion, similar to those seen for the α-crinane series of alkaloids (such as haemanthamine 5) [9]. Interestingly, they have thus far only been seen in the genus Crinum, particularly Crinum x powelli Baker, Crinum latifolium L. and Crinum erubescens L. f. ex Aiton [53], [54], [55]. Although cripowellins A and B (16, 70) were first described over 2 decades ago [53], their re-isolation and biological examination have only ensued in recent years
[54], [55]. Considerable excitement has been generated from such observations that have
pinned these alkaloids as among the most active Amaryllidaceae alkaloids, with
nanomolar level activity against some of the cell lines examined [54], [55]. Dried aerial parts of C. erubescens afforded 5 alkaloids after MeOH extraction, chromatographic separation, and spectroscopic
analysis [54]. Four of these were identified as cripowellins A, B, C, and D (16, 70, 71, 72), which were then tested for antiproliferative effects on the human ovarian cancer
cell line A2780 [54]. In all instances, potent activities were observed as reflected by IC50 values of 0.011, 0.016, 0.025, and 0.028 µM, respectively [54]. Cripowellin A (16), together with 4 other novel cripowellins (73, 74, 75, 76), were recently isolated from the Chinese medicinal plant Crinum latifolium L. collected at Yongning in
the Guanxi Province [55]. While previous studies of C. latifolium yielded alkaloids that were readily identifiable with the Amaryllidaceae [56], the investigation by Chen and co-workers (2018) noteworthily led to the isolation
of the unusual cripowellin compounds presumably due to the exceedingly large amount
of fresh bulb material (400 kg) used in the extraction process [55]. The screen for cytotoxic effects involved 6 cell lines, all of which were lung
cancer variants, including A549, H446, H460, H292, 95-D, and SPCA-1 [55]. Compounds 75 and 76 were the most active in the series (mean IC50s 0.009 µM, respectively) [55]. The best activities observed for compounds 75 and 76 were against H292 and H446 cells (IC50s 0.007 µM, respectively) [55].
All measurements were carried out against the reference standard doxorubicin,
which exhibited a mean IC50 value of 0.023 µM across the 6 cells [55].
Structure-activity Relationships
The impressive array of structural diversity that Amaryllidaceae alkaloids display
has served them well in attempts to understand the features by which their cytotoxic
effects may be modulated [8]. The essential details of the anticancer pharmacophore are reasonably well-understood
based on both semisynthetic modifications and target-orientated total syntheses [8]. However, such attempts have primarily been pursued with phenanthridone, lycorane,
and crinane alkaloids in mind [8]. Nonetheless, the significant number of targets from the minor alkaloid groups already
screened for antiproliferative effects, as shown herein, have provided valuable information
about the features attending their respective pharmacophores. Furthermore, such information
has facilitated a better understanding of the cytotoxic effects associated with the
more recognized alkaloid groups of the family.
Starting with the Amaryllidaceae alkaloid precursor group (the belladine group), it
was seen that none of its representatives (4-O-methylnorbelladine 18, 4-O,N-dimethylnorbelladine 19, or 4-O,N-dimethylnorbelladine-N-oxide 20) were active against KB or HCT116 cells [10], [11]. Although limited in scope, these results are highly informative as they affirm
at the outset that the cytotoxic effects of Amaryllidaceae alkaloids are built upwards
from their belladine precursors in increasing layers of structural, chemical, and
electronic complexity. Furthermore, they highlight that rigid structure formulation,
as found in the phenanthridone, lycorane, and crinane alkaloid groups, is required
for cytotoxicity. These are contrasted against the belladine compounds wherein the
2 aryl rings, since they are benzylic, would experience significant degrees of free
rotation. Since
compounds 18, 19, and 20 are functionalized belladine analogs, it remains to be ascertained what effects the
parent compounds such as belladine (6) and norbelladine (17) might have on cancer cells. Based on the evidence, though, it is fair to assume
that they would be poor cytotoxic agents. Despite this lack of cancer cell line activity,
there are other notable aspects to the anticancer properties of belladine-type compounds,
particularly concerning how they manifest these activities, as is covered in the succeeding
section of the text.
The ismine group was also shown to spawn compounds, such as ismine (7) and norismine (21) ([Fig. 2]), that were devoid of cytotoxic activities in separate studies of HeLa, MCF7, L5178
PAR, L5178 MDR, and PC-3 cells [15], [16]. Furthermore, N-demethylation in the series (as in norismine 21) appears to have no bearing on activity [16]. Ismine compounds are characterized by the installation of a carbon-carbon bond
between rings B and C, a step forward from their belladine precursor [3]. However, this additional structural element is insufficient to potentiate any of
their cytotoxic effects [15], [16]. This notwithstanding, there are mechanistic aspects to the cytotoxicity of ismine
alkaloids worth considering, as is done in the section following
this one.
The formation of ring-B, to afford the isoquinoline motif and attendant adjustments
(such as C-ring oxidations), are the operations that appear to fortify the cytotoxic
responses of Amaryllidaceae alkaloids [8]. The simplest of such a transformation can be envisaged for norismine (21) (via its aldehyde), which could be readily converted to the iminium analog trisphaeridine
(8) via hydrolytic intramolecular cyclization. Similarly, ismine (7) (the N-methylated biphenyl) would afford bicolorine (9) as the cyclized N-methyl iminium ion. The activities observed for trisphaeridine (8) in most cell lines examined were moderate to mild [15], [17], [20]. However, trisphaeridine (8) did notably buffer SH-SY5Y neuroblastoma cells from injuries induced by H2O2,
Aβ
25 – 35, and CoCl2
[19]. Furthermore, there were slight improvements in activities for the partially-saturated
(23) or fully-saturated (22) C-ring trisphaeridine analogs in A549, LoVo, and B16 4A5 melanoma cells [18], [20]. This indicates that the loss of C-ring aromaticity (and concomitant planarity),
together with the introduction of oxygen-related functionalities (as in 22 and 23), are the features that serve to potentiate the effects of the anticancer pharmacophore.
That this is indeed the case may be seen directly through the polyhydroxylated C-ring
variants such as narciclasine (2) and pancratistatin (3), the standout examples of the family in the cancer arena [8]. Some insightful aspects of the cytotoxicity of trisphaeridine (8) from a mechanistic perspective have been
addressed in the literature, as discussed later in this review.
Overall, the bicolorine (9) alkaloids proved to be more active than their close relatives in the trisphaeridine
(8) group of alkaloids. Bicolorine (9) (IC50 15 µM) was more effective than Z-4-ethylmercaptophenylmethylene hydantoin (IC50 37.4 µM) in inhibiting the growth of PC-3 cells [16]. Although 5,6-dihydrobicolorine (24) was inactive (IC50s > 40 µM) against HL-60, K562, A549, HepG2, and HT-29 cells [17], it (61.7%) was able to match arbutin (61.5%) when it came to inhibiting B16 4A5
melanoma cell growth [20]. Its most impressive response was against the Molt4 cell line (ED50 0.8 µg/mL) [22]. Further modifications to the bicolorine skeleton were seen to be at the expense
of activities as seen via 2-hydroxy-5,6-dihydrobicolorine (25) and
1,2-dihydroxy-5,6-dihydrobicolorine (26) in separate exposures to as many as 15 different cancer cell types [23], [24]. Trisphaeridine (8) and bicolorine (9) differ only by the presence of the N-methyl group in the latter, indicating that such an enhancement is beneficial to
the anticancer pharmacophore. Although there were no cell lines available for direct
comparison purposes, the evidence suggests that 5,6-dihydrobicolorine (24) is more active than bicolorine (9), indicating that the flow of charge back to the nitrogen atom as well as a hybridization
shift from sp2 to sp3 at C-6 is advantageous. The case of zephycandidine A (77) is a curious one. While it possesses 2 diagnostic structural elements of bicolorine
(9) viz. a trisubstituted B-ring nitrogen atom as well as a C-6 sp2 center, these have
somewhat capriciously been incorporated into a 1,3-imidazole
ring system [57]. The “externalization” of the imine functionality in zephycandidine A (77), which distinguishes it from bicolorine (9), appears to consolidate the potent cytotoxic effects of this imidazolium compound
[57]. It exhibited a mean IC50 of 5.04 µM in a 5-cell screen involving HL-60, A549, MCF7, SW480, and SMMC-7721 cells
that was superior to that of cis-platin (mean IC50 10.35 µM) [57]. Zephycandidine A (77) (IC50 20.08 µM), as desired, even proved to be less effective than cis-platin (mean IC50 13.30 µM) against normal Beas-2B bronchial epithelial cells [57]. These impressive activities, in hindsight, are perhaps not as surprising given
that phenanthridones such as arolycoricidine (78) and narciprimine (79), with an oxo group at C-6, are known for their
potent cytotoxic effects [5], [8].
The key anticancer alkaloid group of the Amaryllidaceae, as mentioned prior, is the
phenanthridone group, whose members are characterized by A-ring methylenedioxyphenyl,
B-ring lactam, and C-ring polyhydroxy functionalities [5], [8]. The next group in line in terms of potency is the lycorane group, wherein the key
feature is the ethano-tether linking the nitrogen atom to C-4 and thus forming ring-D
[8]. In the crinane alkaloid group, the ethano-bridge is rerouted so that in forging
ring-D, it spans the nitrogen and C-10b positions via the α- or β-face of the molecule [8]. Divergence from the features typifying these 3 alkaloid groups, in general, may
be viewed as detrimental to the anticancer pharmacophore. As seen for the belladine,
ismine, trisphaeridine, and bicolorine groups above, this is also true for the remaining
alkaloid groups of
the Amaryllidaceae. To this extent, the 3 cherylline-related compounds (cherylline
10, gigantelline 28, gigantellinine 29) were all inactive against breast adenocarcinoma MCF7 cells [25]. Compared to phenanthridone, lycorane, and crinane alkaloids, cherylline-type alkaloids
lack A,C-ring connectivity, which in the latter occurs indirectly via the C-4 benzylic carbon [9]. Although good activities were seen for a few representatives of the tazettine (11) and homolycorine (12) alkaloid groups [58], [59], these were outmatched as expected by the phenanthridone, lycorane, and crinane
groups [8]. The lycosinine group is intriguing because its members maintain features that exemplify
both lycorane and homolycorine alkaloids [9]. As such, the activities for
lycosinine-related compounds would be expected to be somewhere in between these
latter 2 groups of alkaloids. Although a wider range of cells still needs to be examined
for this premise to be validated, a single study of lycolongirine B (69) showed that it offered significant protection to SH-SY5Y neuroblastoma cells from
injuries induced by H2O2, Aβ
25 – 35, or CoCl2
[19]. Augustamine-type alkaloids also bear some resemblance to the homolycorine alkaloids
and, as such, would be expected to be poorly cytotoxic [9]. In this regard, augustamine (15) was inactive against as many as 6 different cell lines, including Molt4, KB, HepG2,
MCF7, SKMEL-2, and LNCaP [50], [52]. These results also substantiate the notion that a shift in direction from lycorine
(4) to homolycorine (12) is to the
detriment of the cytotoxic pharmacophore [50], [52].
Given the significance of galanthamine-type alkaloids in the motorneuron disease arena,
it may be construed that their cytotoxic effects should be negligible at best or mild
at worst. However, this is not the case, as seen in the section above for some representatives
of this group. Encouragingly, galanthamine (1) itself proved to be inactive against most cancer cell lines screened. Also appealing
was the fact that it displayed negligible effects on some normal cell lines, such
as BJ fibroblasts and L6 myoblasts [29], [32]. There were some concerns, however, over the fact that galanthamine (1), norgalanthamine (30), and N-formylgalanthamine (34) ([Fig. 1]) displayed potent activities in the evaluation of nontumoral LMTK fibroblast cells
(ED50 s 0.5, 0.5, and 0.6 µg/mL, respectively) [22]. In the instances where
galanthamine (1) displayed significant effects, they were related to protection from injuries to
SH-SY5Y neuroblastoma cells, a coveted response in the motorneuron disease area [19], [37], [38], [39]. O-Demethylation of galanthamine (1) to afford sanguinine (38) (also a natural product) is highly prized in the motorneuron terrain since the latter
is over 10 times more potent than the former in the inhibition of AChE [26]. Although there was some improvement in HCT-116 and HL-60 cells, sanguinine in parallel
screening with galanthamine was mostly inactive [25], [32], [35]. This suggests that the A-ring methoxy to hydroxy transformation, involving a shift
from a hydrogen bond acceptor function to a hydrogen bond
donor-acceptor function, may be slightly beneficial in the cytotoxicity realm.
The effect of ring-B modification has been probed via 4 adjustments: (i) N-demethylation, (ii) quaternization of the nitrogen atom, (iii) N-oxide bond formation, and (iv) hydroxy substitution. Although a limited number of
studies have probed the effects of N-demethylation, the results suggest that such a maneuver is beneficial towards the
cytotoxicity of these alkaloids. Norgalanthamine (30) (ED50 0.6 µg/mL) was among the most active of the 25 Amaryllidaceae alkaloids exposed to
the Molt4 lymphoma cell line, the same instance in which galanthamine (1) displayed an ED50 of > 50 µg/mL [22]. Norgalanthamine (30) was also capable of buffering SH-SY5Y neuroblastoma cells from the deleterious effects
of H2O2, Aβ
25 – 35, and CoCl2
[19], [37]. By removing the N-methyl group, the charge would increase around the nitrogen atom, explaining the
increase in cytotoxicity for norgalanthamine (30). Oxidation of the N-methyl group to an N-formyl (as in N-formylgalanthamine 34) would also tend to pull charge away from the nitrogen atom via delocalization (instead of induction in galanthamine). Hence, N-formylgalanthamine (34) (ED50 1.6 µg/mL) was less active than norgalanthamine (30) (ED50 0.6 µg/mL) but far more potent than galanthamine (1) (ED50 > 50 µg/mL) against Molt4 cells [22]. Quaternization of the nitrogen atom has been achieved via protonation (as in galanthamine hydrobromide 40) and alkylation (as in N-chloromethylgalanthamine 43) [19], [34], [37]. However, only N-chloromethylgalanthamine (43) was able to ameliorate the deleterious effects of H2O2, Aβ
25 – 35, and CoCl2 in SH-SY5Y cells [19], [37]. Since quaternization would not only deplete the charge around the nitrogen atom
but also completely reverse it so that it becomes a cationic species, such a conversion
is cytotoxically disadvantageous. In the formation of an N-oxide (as in galanthamine-N-oxide 41) [35], the bond formed is a dative covalent bond with charge, as expected, shifting from
the nitrogen to the oxygen atom. This, as outlined above and seen via the lack of activity of galanthamine-N-oxide (41) against 4 human cancers (HCT-116, HL-60, OVCAR8, SF-295), appears to be detrimental
to the cytotoxic
pharmacophore [35]. One example involves introducing a substituent at a carbon atom in ring-B, which
is 11β-hydroxygalanthamine (46) [19]. Although this compound is yet to be tested against a wider variety of cell lines,
it did offer SH-SY5Y neuroblastoma cells protection from injuries induced by H2O2, Aβ
25 – 35, and CoCl2
[19].
The modifications probed for ring-C include (i) oxidation of the C-3 hydroxyl, (ii)
substitution at C-3, and (iii) reduction of the double bond. Compared to the former,
it was seen that although narwedine (33) was mildly active against CEM cells (IC50 31.6 µM) [29], it was inactive against as many as 16 different cancer lines as seen via cell viabilities that were mostly > 90% [34]. In narwedine (33), the oxo group is predisposed to hydrogen bond acceptor functions
compared to galanthamine (1), where the hydroxy group would manifest both donor and acceptor functions. Furthermore,
narwedine (33) is characterized by α,β-unsaturation and the consequent flattening of ring-C, which, together with the acceptor
functions of the oxo group, could explain its better activity over galanthamine [29]. The evidence available suggests that C-3
substitution as with chlidanthine (39) offers no benefit as this compound was inactive (or poorly active) against as many
as 18 cancer cells [33], [34]. Both of these studies involved a screen in parallel with galanthamine (1), which, as expected, was also inactive [33], [34]. The C-3 methoxy group in chlidanthine (39) negates the hydrogen bond donor feature linked to the C-3 hydroxyl group. Reduction
of the double bond to afford lycoramine (32) offered no benefits against any of the cell lines examined. This has been verified
separately for norlycoramine (36), which was inactive in a screen of 9 cell lines [31]. Its parent compound, norgalanthamine (30) (with the double bond in the C-4/C-4-a position), by contrast, was identified among
the most potent Amaryllidaceae alkaloids against
the Molt4 lymphoma cell (ED50 0.6 µg/mL) [22]. Given that the furanyl D-ring except for its C-1 methine carbon, is fully substituted,
little can be achieved in modifying its structure; hence all of the compounds examined
reflected an intact D-ring.
Similar to the galanthamine compounds described immediately above, significant effort
has gone into attempts to modify the montanine (13) ([Fig. 3]) nucleus. Given that ring-A is a tetrasubstituted aryl ring system, little can be
achieved by further adjusting its structure [9]. However, there were 2 attempts made to functionalize the vacant C-7 and C-10 positions,
by which the iodo compounds 62 and 63 were accessed [42]. Despite this, none of these compounds were active on a series of cancer cells that
included both apoptosis-resistant and apoptosis-sensitive cells [42]. The adjustments explored for ring-B include C-6 substitution, N-oxide formation, and quaternization of the nitrogen atom. However, oxygen-related
substituents at C-6 (51 and 65) had no effect when exposed to 10 different cancer cell lines [41], [42]. The N-oxide product (60) (mean GI50 > 100 µM) was far less active in a 6-cell screen than its unoxidized precursor (50), which exhibited a mean GI50 value of 18.8 µM [42]. The assumption here, like that observed for the galanthamine oxides above, is that
N-oxidation would have the effect of pulling charge away from the nitrogen atom leading
to a decrease in cytotoxicity [42]. Similarly, quaternization of the nitrogen atom as in compound 61 (mean GI50 > 100 µM) had a gross negative effect on its cytotoxicity against 6 different cancer
cell lines relative to its parent compound (50) (mean GI50 18.8 µM) [42].
Understandably, most SAR-based studies of montanine-type alkaloids have focused on
the 2,3-dihydroxy system constituting the hexyl C-ring. Significantly, a few of these
were themselves natural product entities as opposed to semisynthetic derivatives based
on the montanine scaffold. The parent compound montanine (13) was identified among the most potent montanine-type alkaloids, with submicromolar
level activities against a significant number of cell lines [43], [44], [46]. Interestingly, upon selective C-2 functionalization with various electrophiles,
the resulting products (54–59) to a large extent were able to sustain their activities against 6 cancer cells that
were either susceptible to or resistant to proapoptotic stimuli [42]. This was previously observed for the phenanthridone and lycorine groups, wherein
monosubstitution of the
1,2-dihydroxy system was seen to fortify the anticancer pharmacophore [8]. The understanding here is that such substitutions would negate the hydrogen bond
acceptor capacity of the methoxy group but increase bulk and lipophilicity around
the C-2 region of montanine (13), which may be viewed as beneficial. Furthermore, maintaining the size and polarity
of the C-2 substituent was shown to potentiate the activities of the resultant products
[42]. Compounds 50 and 64 were among the most potent compounds screened by Govindaraju et al. (2018) against
A549, SKMEL-28, U373, MCF7 Hs683, and B16F10 cells [42]. The C-2 halide substituents in compounds 50 and 64 would participate in hydrogen bond acceptor reactions and exert an influence via their innate electronegativities. Interestingly, switching the methoxy and hydroxy
functionalities was also seen to
potentiate the activities of the product (53) in the same 6 cancer cells above [42]. In this instance, hydrogen bond donor-acceptor functions would shift to the C-2
position (compared to C-3 in montanine 13) [42]. Epimerization of the C-2 methoxy group was also a detrimental operation, as montanine
(13) markedly outperformed coccinine (68) in a 6 cancer cell line screen [46]. This is contrasted against the halide compounds 50 and 64, which have opposite configurations at C-3, yet were both able to sustain their activities
[42]. Shifting the substituents from C-2/C-3 (as in montanine 13) to C-3/C-4 (as in nangustine 48) would be expected to be detrimental [40]. Although there is a lack of data suggesting otherwise, nangustine (48) was inactive
against L6 myoblastic cells
[40]. Despite a lack of data to the contrary, it is expected that a radical transformation
where the N-to-C-11 methano-bridge adopts a β-configuration and C-4a becomes R-configured (as in montabuphine 67) would lessen the potency of montanine-type alkaloids [45].
The most potent compounds of the minor alkaloid groups of the Amaryllidaceae are to
be found within the cripowellin group, most of whose members exhibited nanomolar level
cytotoxic activities [54], [55]. Structurally, they are reminiscent of the crinane alkaloids such as haemanthamine
(5), which are characterized by a N-to-C-10b α-ethano bridge [9]. The distinguishing features of cripowellin compounds are the lack of the C-4a/C-10b
bond, with C-4a in the process bearing an oxo group, and the presence of β-glycosylated moieties at C-3 [9]. Therefore, it can be construed that these compoundsʼ potencies must be related
primarily to their close structural similarity to the α-crinane alkaloids [54], [55]. Furthermore, seeing that they are more potent cytotoxic agents than
their α-crinane congeners, this enhancement must be due to the glycosylated functionalities.
Since such functionalities are rare in the Amaryllidaceae, it would be interesting
to establish if glycosylated analogs from the various alkaloid groups in the family
potentiate the effects of their parent alkaloids.
Mechanistic Insight
Since recognizing the phenanthridone alkaloids of the Amaryllidaceae as promising
anticancer drugs, much effort has been diverted to understanding how they manifest
their action [8]. Given the hierarchy of the other minor alkaloid groups in the cancer area, significantly
less information is available on how they might carry out their cytotoxic effects.
Most of the evidence described here suggests a considerable overlap of such modes
of action with those seen for the phenanthridone, lycorane, and crinane alkaloids
[8]. One of the earliest studies to explore this direction is the one carried out by
Jimenez et al. (1976) involving a screen of several Amaryllidaceae alkaloids for their
in vitro protein inhibitory effects in eukaryote cells [27]. Using endomyocarditis viral RNA as a messenger, it was seen that galanthamine (1), norgalanthamine (30), 3-epigalanthamine
(31), or lycoramine (32) (at concentrations as high as 1 mM) were not capable of inhibiting protein synthesis
in S-30 ascites [27]. Narciclasine (2), lycorine (4), and haemanthamine (5), by comparison, were potent inhibitors of this process and at far lower concentrations
[27]. Furthermore, it was demonstrated via the fragment reaction that while narciclasine (2), lycorine (4), and haemanthamine (5) all dose-dependently inhibited peptide bond formation in S-30 ascites ribosomes,
galanthamine (1), norgalanthamine (30), 3-epigalanthamine (31), and lycoramine (32) did not [27]. However, studies carried out some years later found that several Amaryllidaceae
alkaloids could interact with macromolecules [60]. In the event, the minor alkaloid representatives galanthamine
(1) and ismine (7) (at 0.5 mg/mL) exhibited significant binding affinities to calf thymus DNA (63.5%
and 31.5%, respectively) [60]. These activities were all seen to be better than that for lycorine (4) (25.0%) but markedly lower than that observed for haemanthamine (5), which is on record to have among the best binding potencies (99.6%) to calf thymus
DNA [60], [61]. The anticancer drug reference standard vinblastine in the same instance reflected
a binding affinity of 98.2% [60].
In the study of Molt4, HepG2, and LMTK cells above, it was seen that, with some exceptions,
better overall activities were observed for the 25-alkaloid library against the nontumoral
LMTK fibroblast cell line than the cancer cells [22]. The minor alkaloid representatives galanthamine (1), 5,6-dihydrobicolorine (24), norgalanthamine (30), and N-formylgalanthamine (34) exhibited ED50 values that were between 0.5 and 0.7 µg/mL [22]. Since LMTK cells are known to be enriched with cytoskeletal protein entities, it
was suggested that the cytotoxicity of Amaryllidaceae alkaloids might be due to their
detrimental effects on the cytoskeleton [22]. That this could be the case has been ratified in recent times for some key cytotoxic
principles of the Amaryllidaceae [62], [63]. Narciclasine
(2), for example, was shown to impair glioblastoma multiforme proliferation by diminishing
mitotic rates without inducing apoptosis in Hs683 and U373 glioma cells, a diagnostic
feature of which was actin stress fiber formation [63]. Also, narciclasine exhibited potent cytotoxic effects against a series of both
apoptosis-sensitive and apoptosis-resistant cancer cells, a characteristic of which
was its ability to impair the organization of the actin cytoskeleton [62]. The cripowellins, as discussed earlier, were characterized by their interesting
structures and potent antiproliferative activities [54], [55]. Interestingly, cripowellin B (70) (at 10 µM) had prior been shown to manifest its deleterious effects in A375 melanoma,
SW620 colon, and HeLa cervical tumor cells by inhibiting tubulin polymerization [64].
Weniger et al. (1995) observed that most constituents in the diverse 25-alkaloid
library exhibited poor activities against the HepG2 hepatoma cell line [22]. It was suggested that this intrinsic resistance was a consequence of the catabolic
activity of the heme-containing mono-oxygenase enzyme cytochrome P450 (CYP450), which
under metabolic conditions is known to be functional in HepG2 cells [22]. Interestingly, narciclasine (2) (Ki 0.63 µM) was among the most active of the Amaryllidaceae alkaloids tested for CYP3A4
inhibition [65]. Given this observation, it remains to be established whether narciclasine does
attenuate HepG2 cell proliferation. CYP3A4 is responsible for metabolizing roughly
60% of all drugs consumed by humans, thus affording a reliable buffer against drug
overload [66].
Another way that the body can deal with xenobiotic substances (including drugs) is
by expulsive means utilizing ATP-binding proteins, such as the efflux pump protein
P-glycoprotein (P-gp) [67]. P-gp is also expressed in several human cancers where it contributes to chemotherapeutic
resistance [67]. It was observed above that ismine (7) and trisphaeridine (8) exhibited poor activities against both the PAR (L5178 PAR) and MDR (L5178 MDR) forms
of mouse lymphoma [15]. A further aspect of this study was a probe via Rh-123 accumulation to gauge if these alkaloids could reverse multidrug resistance
in L5178 MDR cells via efflux pump inhibition [15]. This was an extension to observations made prior for other alkaloids such as lycorine
(4) and haemanthamine (5), which were seen to harbor moderate efflux pump inhibitory
abilities [61]. However, at 40 µg/mL neither ismine (7) nor trisphaeridine (8) displayed significant efflux pump inhibitory effects as indicated by their fluorescence
activity ratios (R) of < 2, respectively, compared to the standard verapamil (R ~ 3) [15]. The fluorescence activity ratio can be defined as the ratio of the fluorescence
determined for treated versus untreated cells [15]. Thus, these alkaloids were deemed poor substrates for P-gp in the L5178 MDR cell
line [15]. However, at 400 µg/mL, a dramatic spike in the fluorescence ratio was determined
for trisphaeridine (R ~ 50), indicating that its effects on P-gp were possible at higher concentrations
[15]. Apart from this, montanine (13) and coccinine (68) both demonstrated low binding affinity to P-gp as seen
via low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line [68]. While important in the regulation of xenobiotic substances on the one hand [67], these results indicate on the other hand that P-gp efflux will not limit the blood-brain
barrier passage of these or related alkaloids of the family should they realize clinical
fruition [15], [68].
The minor alkaloid group representative trisphaeridine (8) has also been found in Crinum yemense Deflers (synonym Crinum album (Forssk.) Herb.), one of 9 alkaloids to have been done so from MeOH bulb extracts
of the plant [69]. In conjunction with this, the isolated compounds were also examined for inhibitory
effects on nitric oxide (NO) production and induction of inducible nitric oxide synthase
(iNOS) [69]. However, trisphaeridine (8) exhibited no observable effect on NO production in LPS-stimulated mouse peritoneal
macrophages relative to guanidinoethyl disulfide (GED) [69]. It also did not affect the expression and inhibition of iNOS relative to the standard
nonselective inhibitor N-monomethyl-L-arginine (L-NMMA) [69]. By contrast, lycorine (4) (IC50 2.5 µM) was shown to be more potent than GED
(IC50 7.4 µM) at inhibiting NO production in the macrophage cells [69]. iNOS is associated with processes where there is a propensity for the overproduction
of NO and can also be expressed in response to pro-inflammatory elements, such as
interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS) in macrophages, endothelial cells, and smooth muscle
[70]. The role of iNOS in cancer is not clear, but it is understood that high levels
of it can be either cytostatic or cytotoxic to tumor cells. In contrast, low levels
can have the opposite effect and promote tumor growth [70].
Several lines of evidence indicated galanthamine to be a poor cytotoxic agent toward
HepG2 hepatoma cells [21], [22], [30]. For example, all 4 tested galanthamine concentrations (0.1, 1, 10, 100 µM) produced
cell viabilities greater than 85% [30]. Furthermore, in the same study, galanthamine (1) was shown not to affect DNA condensation,
ROS production, generation of MDA, glutathione depletion, redox status, and MMP of
the HepG2 cells [30]. DNA condensation, ROS production, and fluctuations in MMP are significant as markers
in cells undergoing apoptosis, as observed for some representatives of the phenanthridone,
lycorane, and crinane alkaloids of the Amaryllidaceae [8]. Selective apoptosis-inducing effects, for example, have been identified as the
key strategy by which narciclasine (2),
lycorine (4), and haemanthamine (5) manifest their cytotoxicity in cancer cells [8]. The fact that galanthamine (1) could not mediate in such a course of action in HepG2 cells underlines its status
as a reputable Alzheimerʼs drug [30].
Another area probed for the minor alkaloids in the mechanistic arena is their interaction
with the DNA-associated topoisomerases [8]. These enzymes are known for their integral functions in DNA topology regulation,
particularly its overwinding or underwinding [71]. The clinical application of such action is apparent in targeted antibacterial therapy
through the fluoroquinolone class of antibiotics, which are among the most effective
medicines administered as bacterial type II topoisomerase inhibitors [71]. Interestingly, since elevated topoisomerase levels are also detectable in rapidly
dividing cells, such as cancer cells, it has been suggested that targeted blocking
of such activity could be of relevance in cancer chemotherapy [71]. Topoisomerase inhibition has been observed for some lycorane and phenanthridone
class alkaloids of the Amaryllidaceae [8]. The pheanthridones arolycoricidine (78) and narciprimine (79) at relatively low dosages (0.1 – 0.25 µg/mL) were able to markedly inhibit topoisomerase
types I and II [72]. Apart from this, the B-seco alkaloid ismine (7) from Crinum powellii Baker has been probed for topoisomerase inhibitory activity in the yeast Saccharomyces cerevisiae
[73]. In the event, its response was measured against 2 strains of S. cerevisiae, the wild-type strain RAD+ and the mutant strain RAD52Y [73]. Ismine (7) was inactive against both strains with IC12 values in each case shown to be > 2000 µg/mL [73]. Lycorine (4), which was also found in C. powellii, was shown to be over 7 times more active against the mutant RAD52Y strain (IC12 218.7 µg/mL) than the
wild-type RAD+ strain (IC12 1542.7 µg/mL) [73]. Since the mutant strain is characterized by DNA repair-deficiency, lycorine activity
(4) is a good indicator of its topoisomerase activity [73]. This can be extrapolated to reflect cancer cells, where there are significant topoisomerase
activity levels and extraordinarily high levels of DNA activity [71].
It was earlier seen that out of the 2 minor alkaloid constituents of P. maritimum, bicolorine (9) was a better antiproliferative agent than norismine (21) against the effects of PC-3 prostate cancer cells [16]. The adjoining component of this study was the test via the wound healing assay (WHA) to ascertain what effects these compounds had on PC-3
cell migration [16]. Here too bicolorine (9) exhibited better activity than norismine (21), allowing only 38.1% migration of PC-3 cells compared to 100% for the latter [16]. At the equimolar concentration (50 µM) of the test substrates, the standard antimigratory
compound Z-4-ethylmercaptophenylmethylene hydantoin permitted 23.8% migration [16]. Apart from this, it has prior been seen that some alkaloids of the Amaryllidaceae
possessed encouraging anti-invasive
activities [74]. Although the inhibition of metastasis and tumor invasion are attractive possibilities
in cancer chemotherapy, a drug for either of these processes is still to see commercial
fruition [75]. While the most promising anticancer drug targets of the Amaryllidaceae (such as
narciclasine 2) are viewed primarily as selective apoptosis inducers, the fact that they can mediate
in tumor invasion is also encouraging [74]. For example, 2,3,4,7-O-tetraacetylnarciclasine (80) (at 0.1 µM) allowed only 2% invasion of collagen type I fibers by HeLa tumor cells
[74].
Several studies outlined above have been used to examine the neuroprotective effects
of Amaryllidaceae alkaloids [19], [37], [38], [39], [45]. All of these had the SH-SY5Y neuroblastoma cell line in common as the target to
probe the cytotoxicity-related effects of Amaryllidaceae alkaloids [19], [37], [38], [39], [45]. SH-SY5Y cells were initially cloned from the bone marrow of a neuroblastoma sufferer.
Since they are phenotypically adrenergic but also express some dopaminergic markers,
they are particularly useful in the study of neurodegenerative diseases [76]. The suggestion made in consideration of the effects of galanthamine (1), norgalanthamine
(30), N-chloromethylgalanthamine (43), and N-chloromethyllycoramine (44) against CoCl2- and H2O2-induced injuries in SH-SY5Y cells was that they could be of neuroprotective benefit
in instances involving hypoxic injury or oxidative damage, such as in neurodegeneration
but also brain cancers [37]. The same suggestion has been made concerning the neuroprotective effects of several
alkaloids from L. longituba against not only CoCl2- and H2O2-induced injuries but also those caused by the amyloid β-protein Aβ
25 – 35
[19]. Apart from this, it was shown that while galanthamine (1), as desired, did not affect SH-SY5Y cell proliferation, it did protect cells from
the detrimental effects of the Aβ
1 – 42 protein, notably those arising as a consequence of induced genomic
instability [38]. The micronucleus assay showed that Aβ
1 – 42 exposure, in addition to causing considerable DNA damage, had a significant impact
on nuclear division as gauged by the nuclear division index (NDI) [38]. Treatment with Aβ
1 – 42 significantly decreased the NDI but correspondingly increased some key markers linked
to genomic instability such as micronuclei, nucleoplasmic bridges, and nuclear bud
numbers compared to untreated controls [38]. By contrast, exposure to galanthamine caused a significant reduction in these genomic
instability events [38]. These results suggested that galanthamine may be exerting its effect by regulating
cell loss and prompting an antigenotoxic response [38]. It was also suggested that its antigenotoxic effect could be related to galanthamineʼs
capacity to
activate the requisite enzymes that stimulate both detoxification and DNA repair
[38]. Galanthamine was also able to shield SH-SY5Y cells from the detrimental effects
of both rotenone/oligomycin A and okadaic acid, as observed above [39]. From this, it was suggested that galanthamine might be acting through multitarget
synergistic mechanisms to reduce the neuroinflammation and oxidative stress generated
by these substances [39].
Inflammatory cells are prominent in the tumor micro-environment, which has manifold
functions in the neoplastic process, particularly in cell migration, cell proliferation,
and cell survival [77]. The anomalous cell proliferation levels in cancer are reciprocated by a highly
aberrant and sustained immune response, as seen through cancer patientsʼ pain and
discomfort [77]. One of the key steps in the inflammatory response is the activation of phospholipase
A2, which stimulates fatty acid activity, notably that of arachidonic acid from various
membrane lipids [77]. Once released, arachidonic acid is oxidized by either constitutive cyclooxygenase-1
(COX-1) or inducible cyclooxygenase-2 (COX-2) into prostanoids such as thromboxane
and prostaglandins [77]. As pro-inflammatory agents, prostaglandins have key functions in inflammation-related
events
such as pain, fever, swelling, and tenderness [77]. Cyclooxygenase inhibition is thus a viable means of intervening in such processes
[77]. Given the significant potential of the Amaryllidaceae in cancer chemotherapy, not
surprisingly, some studies have elected to examine the COX-inhibitory effects of its
alkaloid principles [24], [55], [78]. The diamine norbelladine (17) inhibited COX-1 activity by 51% and COX-2 by 25% at a relatively low concentration
of 0.25 µM [78]. The corresponding standards aspirin and NS-398 at equimolar concentrations reflected
inhibition values of 35%, respectively [78]. Also, norbelladine displayed dose-dependent DPPH-radical scavenging activity and
inhibited ROS production in a cell-free xanthine/xanthine-oxidase system [78]. Since ROS is intrinsically involved with NF-κB activation, which in turn is closely associated with COX-2 expression, norbelladine
(17) was also probed for effects on the nuclear factor NF-κB [78]. To this extent, NF-κB activity was dose-dependently inhibited by norbelladine in THP-1 leukemia cells
that were stimulated by LPS treatment [78]. Overall, this suggested that norbelladine may manifest its effects via antioxidant and anti-inflammatory pathways as indicated by radical scavenging activity
and COX and NF-κB inhibition [78]. Eight alkaloid entities were identified in the Chinese plant L. aurea, all of which, as indicated above, were subjected to a 7 cell line anticancer screen
[24]. A scan for cyclooxygenase inhibitory activity was also carried out using the COX-1
and COX-2
enzyme substrates [24]. In the event, the bicolorine analog 1,2-dihydroxy-5,6-dihydrobicolorine (26) was over 2 times more active against COX-2 (27.6% inhibition) than COX-1 (12.9%
inhibition) [24]. These measurements were made against the corresponding reference standards NS-398
and SC-560, which reflected substantial inhibitory values of 97.1% and 63.2%, respectively
[24]. Identifying several unusual cripowellin compounds (16, 73, 74, 75, 76) formed the basis for studying the Chinese medicinal plant C. latifolium described above [55]. The studyʼs biological component involved a screen for cytotoxic effects against
7 lung cancer cell lines and a probe for antioxidant and anti-inflammatory effects
[55]. In the event, potent activities were detected for all 5 compounds against COX-2,
where inhibitory values
were > 90% relative to NS-398 (97.0% inhibition) [55]. Although the COX-1 activities were comparatively moderate, all of the isolated
compounds were more active than the reference standard SC-560 (63.5% inhibition) [55]. In the antioxidant evaluation, all 5 compounds (16, 73, 74, 75, 76) were shown to be less effective than the standard Trolox (IC50 43.2 µM) at radical scavenging in the DPPH assay [55]. However, compounds 74, 75, and 76 proved effective at scavenging the ABTS radical, with IC50 values in each case seen to be better than that observed for Trolox (IC50 80.9 µM) [55].
Apoptosis (or programmed cell death) is recognized as the chief pathway by which the
cytotoxic effects of Amaryllidaceae alkaloids are manifested in cancer cells [79]. This phenomenon has been intensively examined for key representatives in the phenanthridone,
lycorane, and crinane alkaloid groups of the Amaryllidaceae [79]. Although there is a paucity of information on the apoptosis-inducing effects of
the familyʼs minor alkaloid groups, some studies have addressed various aspects of
this topic in recent years [14], [20], [21], [42], [80]. The first of these was based on the B-seco alkaloid ismine (7) ([Fig. 2]), as well as 4-ethyl-5,6-dihydrobicolorine (81), a synthetic analog of 5,6-dihydrobicolorine (24), which
were probed for effects about Wnt/β-catenin signaling [80]. To this extent, it was demonstrated that 4-ethyl-5,6-dihydrobicolorine (81) activated Wnt signaling in a dose- and Wnt ligand-dependent manner, none of which
by comparison were observed for ismine (7) [80]. It also acted synergistically with the Wnt3a ligand to upregulate some Wnt target
genes, such as Axin2, DKK1, and NKD1 [80]. A synergistic effect was also seen for 4-ethyl-5,6-dihydrobicolorine (81) together with Wnt3a to stabilize cytosolic and nuclear β-catenin [80]. These results indicated that 4-ethyl-5,6-dihydrobicolorine (81) is an activator in the Wnt/β-catenin signaling pathway, acting upstream of β-catenin and functioning only in the presence of the Wnt3a ligand [80]. The Wnt/β-catenin pathway
performs several key functions in embryonic development, such as cell differentiation,
cell proliferation, and cell survival [81]. It has also been linked to several human diseases, including bone disorders, Alzheimerʼs,
and cancer [81]. It is believed that Wnt signaling could be involved in apoptosis through a variety
of avenues, including SFRP2 gene expression, WntBMP signaling, GSK, 3-β-NF-κB, β-catenin activation, and JNK signaling [81]. The bicolorine-related structure zephycandidine A (77) was described following a further study of the Chinese plant Z. candida
[57], which had prior yielded 15 alkaloids from its whole-plant material [17]. As noted above, it proved to be more active than cis-platin in each of the 5 cancer cell lines screened [57]. This study
also involved a probe of the apoptotic effects manifested by zephycandidine A
in HL-60 leukemia cells, where it exhibited its best activity (IC50 1.98 µM) relative to cis-platin (IC50 3.12 µM) [57]. Under inverted phase-contrast microscopy, zephycandidine A (at 3.0 µM) caused significant
cell shrinkage and other abnormal morphological changes [57]. Annexin V and PI staining subsequently indicated an apoptotic incidence of 80.4%
for zephycandidine A relative to etoposide phosphate (VP16) (83.8%) [57]. Furthermore, zephycandidine A produced considerable changes in the expression of
key apoptosis-related proteins, including Bax, Bcl-2, cleaved caspase-3, and PARP
as determined by Western blotting using β-actin as an internal standard [57]. Overall, the evidence suggested that zephycandidine A-induced apoptosis in leukemia
cells is mediated by the activation of caspase-3, upregulation of Bax, downregulation
of Bcl-2, and degradation of PARP [57]. Caspase-3 is the chief executioner protein responsible for the cleavage of several
proteins, including the nuclear enzyme PARP, which, when achieved, sets into motion
a cascade of events culminating with apoptotic cell death [82]. Trisphaeridine (8) and sanguinine (38) were both shown to be inactive against HepG2 and MCF7 cells, as referenced above
[21]. Despite this, both compounds were also probed for caspase-3 activation via in silico docking simulations [21]. Each showed favorable binding affinity (− 6.0 and − 6.1 kcal/mol, respectively)
at the active site relative to a standard tetrapeptide ligand (− 6.0 kcal/mol), a
fragment of caspase-3 used to standardize protein-ligand binding [21]. Despite the overwhelming evidence supporting the apoptosis-inducing effects of
Amaryllidaceae alkaloids [83], the fact that they are also lethal to apoptosis-resistant cells suggests that they
can manifest their cytotoxicities via other pathways of cell death.
A further area explored to unravel the molecular basis of Amaryllidaceae alkaloidsʼ
cytotoxicity is their interaction with the kinase class of proteins [13], [14], [47]. Kinases are enzymes responsible for the phosphorylation of other protein molecules,
an essential reaction in cellular metabolism using ATP as the phosphate donor [84]. It has been estimated that as much as 30% of all human proteins may be modified
by kinase activity [84]. As such, they are known to mediate in most cellular pathways, particularly those
involved in signal transduction such as transcription, cell cycle progression, cell
differentiation, cell movement, apoptosis, cytoskeletal rearrangement, and metabolism
[84]. With this as a backdrop, a library of 28 Amaryllidaceae alkaloids incorporating
most structural groups of the
family was examined for effects on the serine/threonine protein kinase, GSK-3β (glycogen synthase kinase-3β) [13], [47]. Although GSK-3β is most closely associated with glycogen metabolism, it has also been shown to be
a promising inhibitor target in Alzheimerʼs disease, bipolar disorder, and stroke
drug development [13], [47]. GSK-3β has also garnered interest in the cancer arena, where it is believed to have a modulatory
function in neoplastic transformation via Wnt/catenin and PI3K/Akt signaling [85]. All 28 Amaryllidaceae alkaloids tested were shown to be active, with inhibitory
activities ranging from 10.7% to 66.0% [13], [47]. The minor alkaloid group representatives belladine (6) and tetrahydromasonine (82) exhibited
moderate to mild inhibitory activities (34.4% and 22.4%, respectively) [13], [47].
Other targets probed with links to cancer include Aurora kinase A, histone deacetylase
2 (HDAC2), and vascular endothelial growth factor R2 (VEGF-R2) [14]. Although these were approached from an in silico modeling perspective, they nevertheless provided useful insight into the interaction
of some Amaryllidaceae alkaloids with these interesting protein targets [14]. The study involved a structure-based virtual screening of 313 Amaryllidaceae alkaloids
using the extra precision (XP) method associated with the docking program Glide to
assess their suitability as ligands for the 3 targets [14]. The minor alkaloid group representative N-formylismine (83) was rated amongst the top 10 candidates in terms of its binding affinity (− 8.41 kcal/mol)
for Aurora kinase A [14]. Six of the top 10 ligands predicted to have HDAC2 affinity were
belladine-related compounds including, norbelladine (17) (− 9.65 kcal/mol), 4-O-methylnorbelladine (18) (− 9.54 kcal/mol), 4-O,N-dimethylnorbelladine (19) (− 10.36 kcal/mol), craugsodine (84) (− 9.55 kcal/mol), isocraugsodine (85) (− 9.5 kcal/mol,) and latisodine (86) (− 9.34 kcal/mol) [14]. Ismine (7) (− 9.33 kcal/mol), trisphaeridine (8) (− 10.35 kcal/mol), 4-O-methylnorbelladine (18) (− 10.81 kcal/mol), 4-O,N-dimethylnorbelladine (19) (− 10.16 kcal/mol), 3,4-dihydro-3-methoxytrisphaeridine (23) (− 10.58 kcal/mol), isocraugsodine (85) (− 9.24 kcal/mol), and 3-hydroxytrisphaeridine (87) (− 10.71 kcal/mol) were identified among the best candidates as ligands for VEGF-R2
[14]. By controlling chromatid segregation, Aurora kinases play a crucial role in cellular
division [86]. Furthermore, any defects in chromatid segregation can produce genetic instability,
a condition commonly associated with tumorigenesis [86]. Histone deacetylases are responsible for removing the acetyl group from an ε-N-acetyl lysine residue on a histone molecule, allowing the histone to wrap more tightly
around DNA [87]. This is significant since DNA expression is regulated by both acetylation and de-acetylation
reactions [87]. Although HDAC inhibitors such as valproic acid have long been associated with neurological
therapeutics, the approval of vorinostat and istodax for cutaneous T-cell lymphoma
has reignited interest in such molecules in the cancer arena [87]. As far as the Amaryllidaceae alkaloids are concerned, it has been shown that lycorine-mediated
inhibition of HDAC is partly responsible for G0/G1
cell cycle arrest and apoptosis induction in K562 cells [88]. Vascular endothelial growth factor (VEGF) is a signal protein that stimulates the
formation of blood vessels during both vasculogenesis and angiogenesis [89]. Since new blood vessel formation (angiogenesis) is necessary for tumor growth,
invasion, and metastatic dissemination, VEGF inhibitors such as bevacizumab have come
to play a pivotal role in cancer chemotherapy [89].
Further areas explored to delineate the cytotoxicity of Amaryllidaceae alkaloids relate
to tyrosinase and AKR1C3 inhibition as well as effects pertaining to serotonin re-uptake
[12], [20], [46]. Trisphaeridine (8), 3-methoxy-3,4-dihydrotrisphaeridine (23), and 5,6-dihydrobicolorine (24) were among the 28 compounds identified in Narcissus tazetta subsp. chinensis, a study referred to earlier in the text [20]. Tested for their abilities to inhibit theophylline-induced melanogenesis in B16
4A5 melanoma cells, all of these compounds were shown to be active, with inhibitions
ranging from 14.6% to 61.7% [20]. Given the observed antimelanogenesis effects, these alkaloids were also probed
for inhibitory activity towards tyrosinase, a key enzyme in melanin biosynthesis [20].
However, none of them exhibited tyrosinase-inhibitory activity using either L-tyrosine
or L-DOPA as substrates, suggesting that tyrosinase inhibition is not involved in
the antimelanogenesis effects of these compounds [20]. The aldo-keto reductase AKR1C3 (also referred to as 17β-hydroxysteroid dehydrogenase type 5) is linked to various hormone-dependent and hormone-independent
cancers [12]. There is a notable elevation of AKR1C3 activity in castration-resistant prostate
cancers, while modulation of its expression is known to reduce testosterone, 5α-dihydroxytestosterone, and androgen-dependent gene expression levels [90]. Although the number of studies subjecting Amaryllidaceae alkaloids to prostate
cancers is limited [79], the one above notably identified bicolorine (9) (IC50 15 µM) as more active than
Z-4-ethylmercaptophenylmethylene hydantoin (IC50 37.4 µM) against PC-3 cells [16]. Bicolorine (9) also fully inhibited (100%) the migration of PC-3 cells in a wound healing evaluation
compared to norismine (21) (38.1%), highlighting its significance in the tumor invasion and metastasis aspects
of cancer biology [16]. However, none of the minor alkaloids subsequently examined for AKR1C3 activity,
including galanthamine (1), belladine (6), and tetrahydromasonine (82), were able to register inhibitory activities of > 10% relative to indomethacin (98%
inhibition) [12]. Montanine (13) ([Fig. 3]) proved to be more active than coccinine (68) in each of the 6 cell lines screened above for cytotoxic effects, including MCF7,
Hs578T, MDA-MB-231, HCT-15, A549, and SKMEL-28 cells [46]. Interestingly, montanine (IC50 121.3 µM) had prior also proved to be better than coccinine (IC50 196.3 µM) at binding the serotonin transporter protein SERT, although these effects
were weak relative to citalopram (IC50 7.7 nM) and paroxetine (IC50 0.8 nM) [68]. By inhibiting serotonin re-uptake, SERT is associated primarily with antidepressant
effects [91]. However, there is mounting evidence suggesting that serotonin can act as a growth
factor, affecting both cancer cell growth and survival [92]. Furthermore, significant levels of serotonin and 5-hydroxytryptamine (5-HT), as
well as their respective synthesizing enzymes tryptophan hydroxylase (TPH) 1 and 2,
are observable in tissue or cancer cells extracted from cancer patients [93]. From this, it was suggested that the observed
antiproliferative effects could be explained by the binding of montanine (13), and to a lesser extent, of coccinine (68), to SERT, thereby dysregulating the 5-HT system and inhibiting cancer cell growth
[46].
