Chemistry and bioactivities of natural steroidal alkaloids

Mei-Ling Xiang, Bin-Yuan Hu, Zi-Heng Qi, Xiao-Na Wang, Tian-Zhen Xie, Zhao-Jie Wang, Dan-Yu Ma,Qi Zeng and Xiao-Dong Luo,2*

Abstract Steroidal alkaloids possess the basic steroidal skeleton with a nitrogen atom in rings or side chains incorporated as an integral part of the molecule.They have demonstrated a wide range of biological activities, and some of them have even been developed as therapeutic drugs, such as abiraterone acetate (Zytiga?), a blockbuster drug, which has been used for the treatment of prostate cancer.Structurally diverse natural steroidal alkaloids present a wide spectrum of biological activities, which are attractive for natural product chemistry and medicinal chemistry communities.This review comprehensively covers the structural classification, isolation and various biological activities of 697 natural steroidal alkaloids discovered from 1926 to October 2021, with 363 references being cited.

Keywords: Steroidal alkaloids, Chemistry, Bioactivities, Solanaceae, Liliaceae, Apocynaceae, Buxaceae

1 Introduction

Steroidal alkaloids are nitr ogenous derivatives of natural steroids.They are an important class of alkaloids and conventional secondary metabolites that occur in plants including Solanaceae, Liliaceae, Apocynaceae, Buxaceae,amphibians and marine organisms.Previous research results exhibited that steroidal alkaloids possess potential anticancer, anticholinergic, antimicrobial, anti-inflammatory and analgesic, anti-myocardial ischemia, anti-giogenesis effects and other activities.

Steroidal alkaloids are already launched as drugs, such as abiraterone acetate, marketed as Zytiga?by Janssen Biotech (a subsidiary of Johnson & Johnson), is a steroidal antiandrogen medication approved by the Food and Drug Administration (FDA) for the treatment of metastatic castration resistant prostate cancer (mCRPC)in 2011 and metastatic high-risk castration-sensitive prostate cancer (mCSPC) in 2018 [1].Zytiga?is a blockbuster drug on the prostate cancer market, and in 2020,it generated almost $2.4 billion in sales from the Johnson& Johnson annual report, with ongoing research into its application for additional indications.Natural steroidal alkaloid cyclovirobuxine D (203) is the main active component of oral drug “huangyangning” tablets listed in the Chinese pharmacopeia 2015.This drug, discovered from a folk prescription in the treatment of rheumatic disease,was approved by the China Food and Drug Administration (CFDA) in 2009 to treat cardiovascular and cerebrovascular diseases, such as coronary heart disease, angina pectoris, arrhythmia, heart failure, hypertension and cardiac neurosis [2].Several steroidal glycoalkaloids from the local plantSolanum linnaeanumpossess activity of slowing skin cancer growth in horses and cattle, in whichα-solamargine (500) andα-solasonine (501) were identified.These two active compounds were subsequently developed into a topical treatment for keratoses, basal cell carcinomas, and squamous cell carcinomas, which were marketed in Australia signed Curaderm [3].

Sheep ranchers experienced outbreaks of cyclopic lambs, leading to the discovery of cyclopamine (432)as a plant derived teratogen [4].Cyclopamine was the first compound found to antagonize the Hedgehog (Hh)signaling pathway, the constitutive activation of which is intimately implicated in many human malignancies [5].Vismodegib and sonidegib, cyclopamine derivatives and Hh pathway inhibitors, were FDA-approved for the treatment of basal cell carcinoma and acute myeloid leukemia,respectively [6].In 2016 cyclopamine was identified as a potent inhibitor of human respiratory syncytial virus(hRSV) replication [7].

Phyllobates terribilisfrogs, made into poison darts by Central American indigenous people, advertise their lethal armament with their gaudy colors.Batrachotoxin (615) is a potent neurotoxin in the skin secretions of these frogs and as a tool to study voltage-sensitive sodium channels of excitable membranes [8, 9].Toxicity is widespread among living organisms and how these frogs avoid poisoning themselves remains a mystery.A study addressed that a single rat muscle Na+channel mutation confers batrachotoxin autoresistance [10].

Some reviews related to steroidal alkaloids have been presented since 1953.For example, the chemistry of these alkaloids from the Liliaceae and Solanaceae [11–15], the Apocynaceaethe [16], the Buxaceae [17, 18], the marine organisms [19], synthesis of cephalostatins and ritterazines [20], biosynthesis of Buxaceae alkaloids [21], and biological activities [22, 23].A comprehensive review was published in 1998 concerning the developments in the field of steroidal alkaloids [24].In consideration of these reviews providing little information about recent research, we provide an updated review in a concise form, covering comprehensive structure classification,resources, biosynthesis and bioactivities of natural steroid alkaloids reported from 1926 to October 2021.

This review will help the scientific community understand natural steroidal alkaloids overall and compactly.We comprehensively summarize 16 structural subtypes of steroidal alkaloids along with their bioactivities and toxicity.In addition, steroidal alkaloids (362–365, 381)whose names were not proposed by authors were presented only with numbers in the tables.

2 Basic skeletal classification

Steroidal alkaloids possess the basic steroidal skeleton with a nitrogen atom in rings or side chains incorporated as an integral part of the molecule [24].In general, steroidal alkaloids can be classified into monomeric and dimeric on the basis of the carbon framework.Monomeric steroidal alkaloids, possessing a pregnane (C21),cyclopregnane (C24), cholestane (C27) and other carbon heterocyclic skeletons, were isolated from plants,amphibians and some marine sponges.Dimeric steroidal alkaloids, a class of bis-steroidal pyrazine alkaloids, were only found in marine organisms.Figure 1 lists the different types of natural steroidal alkaloids.

2.1 Monomeric steroidal alkaloids

2.1.1 Pregnane alkaloids

The occurrence of 177 pregnane alkaloids (1–177),however, is not restricted to the Apocynaceae family,which is also found in Buxaceae, such asSarcococcaandPachysandra.

2.1.1.1 Conanine typeConanine type alkaloids are characteristic of an 18,20-epimino five-membered E ring, and most of them contain an amino or oxygen at C-3 (Fig.2).Alkaloids (1–36) were isolated from various plants of the family Apocynaceae, such asHolarrhena,Funtumia,Malouetia, andWrightia(Table 1).

Fig.1 Classification of steroidal alkaloids

Fig.2 Structures of conanine type steroidal alkaloids 1–36

For alkaloids with nitrogen substituents at C-3, only 53–62 and 172–176 bear 3αsubstituents, whereas most compounds possess the 3βconfiguration.All of them except 143–154 contain nitrogen substituents at C-20, which is a common feature of pregnane type alkaloids.Salignarine A (167) has a novel structure with an epoxide functionality at C-5–C-6 [44].Two compounds, pachysanone (148) and pachysanonin (149)

Table 1 Structures and sources of conanine type steroidal alkaloids 1–36

Conessine (1) was the first and most common conanine type alkaloid isolated from the seeds ofHolarrhena antidysenterica[25].Rings A and B of the pregnane moiety were dehydrogenated to form a conjugated system comprising two double bonds in regholarrhenine C (28), funtudienine (29), mokluangin D (35).Compounds 14–16 and 20–27 have secondary and tertiary amino group of the nitrogen in the heterocyclic ring, respectively, but both of them lack the C-3 amino function and possess a 1,4-dien-3-one system in ring A.Mokluangin B (32) contains a novel structure with the amide carbonyl group instead of the methyl group at C-20, whose structure was elucidated by analysis of NMR and MS spectroscopic data [26].

2.1.1.2 Paravallarine typeParavallarine type alkaloids bear a pregnane-(18 → 20)-lactone skeleton (Fig.3) [42].Currently, eight compounds (37–44) of this type have been found only in Apocynaceae family, includingKibataliaandParavallaris(Table 2).

Fig.3 Structures of paravallarine type steroidal alkaloids 37–44

Table 2 Structures and sources of paravallarine type steroidal alkaloids 37–44

The structure of 20-epi-kibataline (37) contains a rare configuration 20R, while the configuration 20Sis proposed for all remaining compounds [43].Compounds 38–40 differ from others possessing an opposite orientation at C-3.The structure of kibalaurifoline (44) was carefully established from 2D NMR analyses, in which the conjugated system of the two double bonds Δ4(5)and Δ6(7)was determined from the HMBC spectrum [42].

2.1.1.3 Pregnane typeNearly all the reported pregnane type alkaloids, share the 5α-pregnane steroidal skeleton with varying functionalities such as an amino function at C-3 and C-20 that may be modified by methyl, benzoyl and aliphatic groups (Fig.4).A total of 133 new alkaloids(45–177) were isolated fromSarcococcaandPachysandraof the Buxaceae family andHolarrhenaof the Apocynaceae family (Table 3).bear a 3,4-dimethylpent-3-enoyloxy substituent at C-11,a rare functional group in natural products [45].Compounds 155–164, bear a (3′sopropyl)-β-lactam ring at the C-3 position, whereas compound 165 bears a phthalimido moiety at the same position [46].Spiropachysine(166) possesses a five membered-ring spiro-lactam and a disubstituted benzene ring at C-3 [46].Compounds 168–171 display a structural modification that has not been reported from this genus, viz.the epoxy ring at C-16/C-17.N-methylfuntumafrine (172) shows a novel structure with an acetyl group at C-17 [47].

Fig.4 Structures of pregnane type steroidal alkaloids 45–177

Table 3 Structures and sources of pregnane type steroidal alkaloids 45–177

Table 3 (continued)

Table 3 (continued)

2.1.2 Cyclopregnane alkaloids

TheBuxusgenus of the Buxaceae family is a rich source of cyclopregnane alkaloids, and 116 cyclopregnane alkaloids (178–293) have been reported fromB.sempervirens, B.longifolia, B.hildebrandtii, B.bodinieri, B.hyrcana, B.microphylla, B.papillosa, B.wallichiana,B.rugulosa, B.natalensis,andB.macowanii.

Cyclopregnane alkaloids, also known as triterpenoid alkaloids, possess a unique pregnane type structure with C-4 methyl groups, a 9β,10β-cycloartenol system,and a degraded C-20 side chain.All alkaloids possess a nitrogen function at C-3 and/or C-20, which may be unmethylated, partially methylated, or fully methylated.Structurally, the majority of these alkaloids contain either a 9β,19-cyclo-14α-methylpregnane type or a 9(10 → 19)abeo-14α-methylpregnane type, having a characteristic substituent pattern at C-4.

2.1.2.1 9β,19-Cyclo-14α-methylpregnane typeOut of the 116 cyclopregnane alkaloids, 50 (178–227) belong to this type, which are characteristic of the genusBuxus(Table 4).This type of compound is characterized by a pentacyclic 4,4,14-trimethyl-9,19-cyclopregnane skeleton(Fig.5).

Table 4 Structures and sources of 9β,19-Cyclo-14α-methylpregnane type steroidal alkaloids 178–227

Table 4 (continued)

Fig.5 Structures of 9β,19-cyclo-14α-methylpregnane type steroidal alkaloids 178–227

Cyclobuxine D (178) was the first steroidal alkaloid fromBuxusbearing a C-4 methylene substituent [85].Later, cyclobuxamidine (179) and trans-cyclosuffrobuxinine (213) of this type were isolated fromB.longifolia[86].Buxbodine A (181) has a unique structure due to the lack of a keto or an amino functionality at the C-3 position [87].Typically, all alkaloids of this type have a C-3 amino or carbonyl group, except for buxmicrophylline K (186), which has a hydroxyl group substituted at C-3[88].In compoundstrans-cyclosuffrobuxinine (213) and sempervirone (215), the methylamino group at C-20 is eliminated and the secondary alcohol at C-16 is oxidized[86, 89].Buxozine C (214) with a tetrahydro-oxazine ring joining the C-16αand the C-20 nitrogen has been isolated fromB.papillosa.The mass spectra of compound 214 exhibit the ions at m/z 127 and 113 due to cleavage of ring D, and these ions serve as diagnostic features to determine the presence of a tetrahydro-oxazine ring in ring D [90].

2.1.2.2 9(10→19)Abeo-14α-methylpregnanealkaloidsThis type contains a tetracyclic system in which 9,19 bond fission has occurred to give seven-membered ring B (Fig.6).Sixty-six alkaloids (228–293) were isolated from the genusBuxus(Table 5).

Fig.6 Structures of 9(10 → 19)abeo-14α-methylpregnane type steroidal alkaloids 228–293

Table 5 Structures and sources of 9(10 → 19)abeo-14α-methylpregnane type steroidal alkaloids 228–293

Table 5 (continued)

Alkaloids 248–254 and 290–291 are members of the class having a tetrahydro-oxazine moiety incorporated in ring A, while in compound 288 an oxazine ring is attached to ring D [89].The presence of this ring can be easily recognized by the1H NMR spectrum exhibiting the presence of two pairs of AB doublets at δ 3.20–4.50[91].Compounds 255–258 belong to a unique class having a conjugated triene system at △1,2, △10,19and △9,11.Compounds 259–264, 282–284 and 292 belong to the rarely occurring class having an additional tetrahydrofuran ring incorporated in their structures through the ether linkage between C-10, C-2, or C-10 and C-23.Buxalongifolamidine (268) and 270–272 containing a hydroxyl group at C-10 may support the plausible biosynthesis of the ether linkage in these alkaloids [107].Compounds 285–287 and 292 are rare cyclopregnane alkaloids featuring an epoxy motif [108].Sempervirooxazolidine (289) also represents a novel structure having an oxazolidine moiety incorporated in its structure at C-2 and C-3 [96].The compound 17-Oxo-3-benzoylbuxadine(293) having a carbonyl group at C-17 has been isolated fromB.hyrcana[94].

2.1.3 Cholestane alkaloids

Based on the carbon framework, C27alkaloids can be divided into two types: C-nor-D-homosteroidal alkaloids and cholestane alkaloids.The former, usually referred to asVeratrumsteroidal alkaloids, characterised by a fivemembered C-ring and six-membered D-ring system, can be further divided into cevanine, veratramine, and jervine types.The latter, usually namedSolanumsteroidal alkaloids, containing the common ABCD steroid skeleton, generally occurring as glycosides, can be grouped into spirosolane, solanidine and verazine types.

A total of 310 new members (294–603) were derived mainly from the genusSolanumin the Solanaceae family, and the generaVeratrumandFritillariain Liliaceae family.

2.1.3.1 Cevanine typeMembers of the cevanine type are characterized by the hexacyclic benzo [7, 8]fluoreno[2,1-β]quinolizine nucleus (Fig.7) [15].This type is the largest representative group of C-nor-Dhomosteroidal alkaloids, and currently comprises 91 new members (294–384) fromVeratrumandFritillariagenera in the Liliaceae family (Table 6).

Fig.7 Structures of cevanine type steroidal alkaloids 294–384

Table 6 Structures and sources of cevanine type steroidal alkaloids 294–384

Table 6 (continued)

Veratrenone (294), the first alkaloid with a cevanine skeleton fromV.album, was investigated in 1974 [118].The structure of isobaimonidine (301), the C-6 epimer of baimonidine (299), was deduced by chemical transformation [119].Eleven glycoalkaloids (302, 317, 326,329–331, 333, 337, 341, 343 and 384) have been isolated from variousFritillariaspecies in cevanine-type alkaloids.Alkaloid pingbeinone (372) has a unique structure with a lack of a C-18 methylene unit, and its structure could be unequivocally established by X-ray diffraction of its corresponding hydroiodide salt [120].Alkaloids 349–368 belong to the rarely occurring class of cholestane alkaloids having a tetrahydrofuran ring incorporated in their structures.Heilonine (371),the first example with an aromatic D-ring in the group of cevanine alkaloids, was isolated fromFritillaria ussuriensisin the group of Kaneko [121].Compounds 373–376 are four unique steroidal alkaloids with a seven-membered G-ring formed by a connection between C-18 and C-27.Taipaienine (377) [122]and yibeisine (378) [123], which are unique in bearing a C-25 hydroxyl moiety as of a cevanine system, have been isolated fromFritillaria taipaiensisandFritillaria pallidiflora, respectively.Compounds 318–321 and frititorine B (384) [124] are steroidal alkaloidN-oxide derivatives.Neoverataline A (379) and neoverataline B (380), having a novel 3,4-secocevane-4,9-olid-3-oic acid skeleton, were obtained from the genusVeratrum[125].Compound 381 possesses a rare 9-hydroxy moiety within cevanine-type alkaloids [126].

2.1.3.2 Veratramine typeThe veratramine type of steroidal alkaloids, in which ring E of cevane has been opened at C-18 (Fig.8).Compounds 385–411, a total of 27 veratramine alkaloids, have been found inVeratrumandFritillaria(Table 7).

Fig.8 Structures of veratramine type steroidal alkaloids 385–411

Table 7 Structures and sources of veratramine type steroidal alkaloids 385–411

Thirteen alkaloids, 387, 390–394, 402–408 containing an aromatic D-ring are unusual in C27steroidal alkaloids,and concurrently 387 is a steroidal alkaloidN-oxide derivative.A chemical investigation of the hypogeal parts ofFritillaria imperialisfurnished two unique bases,impranine (398) and dihydroimpranine (399), which have a methyl group at C-12.This is the first time the novel “impranane” class derived from the veratramine skeleton has been found in the genusFritillaria[179].Veratravine A (405) and zhebeisine (411) contain a new oxazinane ring F forming a rare 6/6/5/6/6/6 fused-ring system.

2.1.3.3 Jervine typeThe steroidal alkaloids of the jervine subgroup are hexacyclic compounds that have the tetrahydrofuran E ring fused onto a methylpiperidine F ring system forming an ether bridge between carbon atoms C17and C23(Fig.9) [15].The jervine type currently consists of 29 new members (412?440) fromVeratrumandFritillaria(Table 8).

Fig.9 Structures of jervine type steroidal alkaloids 412–440

Table 8 Structures and sources of jervine type steroidal alkaloids 412–440

Verdine (412) was first separated from the bulbs ofVeratrum dahuricumin 1980 [191], and its structure was finally elucidated in 1984 by X-ray diffraction [192].Two new steroidal alkaloids, kuroyurinidine(417) [193] and 23-isokuroyurinidine (418) [194],bearing C-2β, C-3α, and C-6βhydroxyl groups, were found in the genusFritillaria.Whole plants ofVeratrum taliensehave yielded a novel steroidal alkaloid,6,7-epoxyverdine (437), whose structure with an epoxide functionality at C-5/C-6 was determined by 2D NMR spectroscopic analysis [195].Yibeinone A (439)features a jervine skeleton with a rare 12α,13α-epoxy ring [156].

2.1.3.4 Spirosolane typeSpirosolane alkaloids (441–526) have a unique 1-oxa-6-azaspiro[4.5] decane ring system in ring E, which can form a spirosolane 22-αN type and 22-βN type (Fig.10) [205].They were reported fromSolanumandLycopersiconin the Solanaceae family,Fritillaria meleagrisandLilium longiflorumin the Liliaceae family (Table 9).

Fig.10 Structures of spirosolane type steroidal alkaloids 441–526

Table 9 Structures and sources of spirosolane type steroidal alkaloids 441–526

Table 9 (continued)

Table 9 (continued)

Spirosolane alkaloids generally occur as glycosides.Compounds 441– 451 have no double bond between C-5 and C-6, and the nitrogen atom in the F ring is always in theα-orientation (22-αN).Ring F can contain other moieties, such as hydroxyl or acetyl groups.Most glycosidic units are attached to the aglycone at the hydroxyl group at C-3, but some of them may be attached at other locations, such as C-6, C-7, C-23, C-25, and C-27.For example, esculeoside A (448) and lycotetraose G (449)have one glucose linked to ring-F at C-25 and C-23,respectively [206].Compounds 474–515 are the largest members in spirosolane alkaloids, with 22-βN and double bonds between C-5 and C-6.Almost all spirosolane alkaloids at C-16 are in theβ-orientation, however, 504 is an exception since it possesses a 16β-H in its E ring[207].There are many substitutions and changes in these compounds, such as 475, 478 and 498 have a hydroxy group on C-27 in the F ring, and 486, 497, 499 and 507 have a hydroxy group on C-12 in the C ring.Five rare C-3 amino spirosolane alkaloids, 517–521 [208, 209] were isolated from aerial parts of the genusSolanum.

2.1.3.5 Solanidine typeIn the solanidine type, the side-chain of a C27steroid has been converted into an indolizidine ring (Fig.11).Solanidine alkaloids (527–555)currently include 29 novel members fromVeratrum,FritillariaandSolanum(Table 10).

Fig.11 Structures of solanidine type steroidal alkaloids 527–555

Table 10 Structures and sources of solanidine type steroidal alkaloids 527–555

Table 10 (continued)

α-Solanine (528) was found mainly in the tuber of potato (Solanum tuberosumL.) and in the whole plant of the nightshade (Solanum nigrumLinn.) of the Solanaceae family [255].The bulbs ofF.delavayiyielded(22R,25S)-solanid-5-enine-3β,5α,6β-triol (550) [133], the first example with a glycol moiety at the A- and B-rings in the group of solanidine alkaloids.An investigation ofV.dahuricumfurnished unusual glycoalkaloid 552 [194],which represents the first member of a new class with a 15,16-secosolanida-5,14-diene skeleton.The two novel compounds, 553 [256] and 554 [257], bearing a methylene substituent at C-20 and C-7, respectively, were structurally elucidated by extensive 2D NMR analysis.

2.1.3.6 Verazine typeMembers of the verazine type,having a 22/23,26-epiminocholestane skeleton, are characterized by the absence of ring E and the presence of a piperidine ring D and consist of 46 new members (556–601) (Fig.12).They were obtained fromVeratrum,Fritillaria,Allium victorialisandZygadenus sibiricusin the Liliaceae family, and only oneSolanumspecies,Solanum Hypomalacophyllum(Table 11).

Fig.12 Structures of verazine type steroidal alkaloids 556–601

Table 11 Structures and sources of verazine type steroidal alkaloids 556–601

Table 11 (continued)

The alkaloidal fraction ofVeratrum stenophyllumgave a new 3β,20β-dihydroxy-△5–22,26-epiminocholestane alkaloid, Stenophylline B (564).Its structure was established on the basis of spectroscopic comparisons with known verazine-type alkaloids [268].Rhamnoveracintine (576), having a five-membered heterocyclic ring and L-rhamnose as structural features, is the first example of a C26steroidal alkaloid from the aerial parts of aVeratrumspecies [269].Ebeietinone (586), the first example of a verazine type alkaloid with a 5β-hydroxyl group, was structurally assigned based on MS and NMR and confirmed by X-ray crystallography [270].Verdinine (587)[271], fetisinine (588) [179] and isoecliptalbine (597)[203], exhibiting a pyridine ring as a structural feature,were pyridyl-pregnane-type steroidal alkaloids, and their structural assignment was performed by extensive spectroscopic techniques and some chemical transformations.

2.1.3.7 OthersTwo distinctive alkaloids, veragranine A (602) and veragranine B (603), featuring a 6/6/6/5/6/6 polycyclic structure (Fig.13), in which a previously unidentified linkage of C-12/23 generates a rigid skeleton,resulting in a new subtype of cholestane steroidal alkaloid,were isolated fromVeratrum grandiflorum[290].

Fig.13 Structures of others cholestane steroidal alkaloids 602–603

2.1.4 Miscellaneous monomeric steroidal alkaloids

2.1.4.1 SamandarinesApproximately 11 samandarines(604–614) are a unique class of steroidal alkaloids isolated and characterized from terrestrial salamanders of the genusSalamandra(Table 12).They differ from other types since they are built by a seven-membered A-ring with nitrogen at position 3.Therefore, they belong to the uncommon group of 3-aza-A-homo-5α,10α-androstans,an androstane with an N-enlarged A-ring (Fig.14) [291].

Table 12 Structures and sources of samandarines 604–614

Fig.14 Structures of samandarines 604–614

Samandarines can be further grouped according to their constitution.The first group consists of molecules with an oxazolidine system present, including 604–607,608 and 610–612.Members of the second group, e.g.cycloneosamandion (609), lack an intact oxazolidine system, whereas they share a carbinolamine function [292].A third group consists of samandarines in which both the oxazolidine system and the carbinolamine group are missing, and only samanine (613) and samanone (614)were described from this group.

2.1.4.2 BatrachotoxinsOnly 7 batrachotoxins (615–621) were isolated in minute quantities from the skins of poison arrow frogs (Phyllobates aurotaenia) as well as from the skins and feathers of New Guinea birds (genusPitohuiandIflita) (Table 13).They exhibit novel structural features, including a steroid-based pentacyclic core skeleton, an intramolecular 3-hemiketal, and a sevenmembered oxazapane ring (Fig.15) [301].

Table 13 Structures and sources of batrachotoxins 615–621

Fig.15 Structures of batrachotoxins 615–621

The structure of pseudobatrachotoxin (616) is the 20α-p-bromobenzoate of batrachotoxinin A (615)[302].Batrachotoxin (617) is the 20αester of 615 with 2,4-dimethylpyrrole-3-carboxylic acid [302], while homobatrachotoxin (618) is the 20αester of 615 with2-ethyl-4-methylpyrrole-3-carboxylic acid [303].These structures were confirmed by partial synthesis of batrachotoxin selective acylation.

2.1.4.3 PlakinaminesConsiderable research effort has been focused on the discovery of new bioactive natural products from marine animals.A number of new steroidal alkaloids have been isolated in the process,mostly from marine invertebrates.A marine sponge of the genusPlakinaandCorticiumsp.yielded nineteen new steroidal alkaloids (622–640), namely, plakinamine(Table 14).Plakinamines have modified ergostane-type steroidal cores, as they possess nitrogen substitution at C-3 in the A ring and linear or cyclized nitrogenous side chains (Fig.16) [305].

Fig.16 Structures of plakinamines 622–640

Table 14 Structures and sources of plakinamines 622–640

Plakinamine G (630) bearing a rare side chain with anα,β-unsaturatedγ-lactam ring was structurally assigned by 2D NMR spectroscopy and accurate mass measurements (HR-EIMS) [306].Most plakinamines contain a substituted pyrrolidine ring in the steroidal side chain, only in plakinamine I (633) the pyrrolidine nitrogen forms an additional fused piperidine ring system [307].The first natural representative of steroidal alkaloids with a double bond at C-2 and an amine substituent at C-4 was plakinamine J (634) [307].Three new steroidal alkaloids, plakinamine L, M and P (638–640),have unprecedented acyclic side chains, while other compounds contain cyclized nitrogenous side chains.

2.1.4.4 CortistatinsKobayashi et al.isolated a new family of abeo-9(10-19)-androstane-type steroidal alkaloids with oxabicyclo[3.2.1]octane called cortistatins, from the Indonesian marine spongeCorticium simplex(Fig.17,Table 15) [314].Up to now, this family has 11 members(641–651), with the B and C rings connected through an interesting and characteristic oxo-bridge.

Fig.17 Structures of cortistatins 641–651

Table 15 Structures and sources of cortistatins 641–651

Cortistatins A–D (641–644) and J–L (651, 648–649)have a 5-membered E-ring decorated with a unique isoquinoline moiety at C-17.

2.2 Dimeric steroidal alkaloids

2.2.1 Cephalostatins

The 20 cephalostatins (652–671) have been isolated only in one marine organism:Cephalodiscus gilchristi, a tiny marine worm predominantly found in shallow and temperate waters (Table 16).The structure of cephalostatins,characterized by an adissymmetric bis-steroidal pyrazine framework, consisting of 13 rings is quite unusual(Fig.18) [20].

Table 16 Structures and sources of cephalostatins 652–671

Fig.18 Structures of cephalostatins 652–671

The atypical C-22′ spiroketals involving C-18′ in most cephalostatins 1–4 (652?654, 659), 9–11 (664,655?656), 14–19 (668?671, 657?658) and C-12′ in cephalostatins 5 (660) and cephalostatins 6 (661) are also noteworthy.Cephalostatins 10 (655), 11 (656) [318], and 13 (667) [319] with an oxygen substituent (OMe or OH)at the 1α- or 1′α-positions, close to the central pyrazine ring, are rare in this type alkaloids.Both cephalostatins 5 (660) and 6 (661) contain an aromatic C′ ring that is rather unusual in naturally occurring steroids [320].The only symmetric cephalostatin 12 (666) containing two identical steroid units is unique [319].Cephalostatin 16(670), the only compound contains the [4.5] spiroketal system with the unusual 22Sconfiguration (not yet confirmed by synthesis) in the right side steroid unit, whereas other cephalostatins have a common [4.4] spiroketal system in the right steroidal unit [321].

2.2.2 Ritterazines

The ritterazine class of steroidal alkaloids comprises 26 compounds (672–697), all of which were found in the lipophilic extract of the tunicateRitterella tokiokacollected off the Izu Peninsula by Fusetani and colleagues(Table 17).They are spiroketal-containing steroidal heterodimers (Fig.19).Ritterazines and cephalostatins share common structural features, in which two highly oxygenated hexacyclic steroidal units are fused via a pyrazine ring at C-2 and C-3 and both side chains of the steroidal units form either [4.4] or [4.5] spiroketals [20].

Fig.19 Structures of ritterazines 672–697

Table 17 Structures and sources of ritterazines 672–697

The hydroxyl groups of cephalostatins at C-12, C-17,C-23, and C-26 are more oxygenated in the right side hemispheres than in ritterazines, which is hydroxylated only at C-12, while the left side hemisphere ritterazines are more oxygenated, with C-7′, C-12′, C-17′ and C-25′ being hydroxylated.All cephalostatins containβ-hydroxyl oxygen substituents at the C-12 position,while some ritterazines bear carbonyl groups at this position.In the original paper, the configuration of ritterazine B (674) at the spiro carbon atom was mistaken for the same as in cephalostatin 1 in 1995 [328].However,this has been recently revised by Phillips and Shair, who synthesized the right half of ritterazine B in 2007 [329].Ritterazines J–M (683–686), exhibited the presence of the [4.5] spiroketal system on both sides of the alkaloid molecule, but only one of them, ritterazine K (684), was symmetrical [330].In the original paper ritterazine M(686) was erroneously assigned as theSconfiguration at C-22, along with an incorrect configuration at C-12[330].A chemical synthesis of this compound by Fuchset al.allowed correcting the structure [331].Ritterazines A (672), T (673), D (676), E (677), N (687), O (688), U-X(693–696), and Z (697) have a unique five-membered C ring on their right side, which is a rearranged steroid nucleus, the same asVeratrumalkaloids.Structural abbreviations used in this review are illustrated in Fig.20.

Fig.20 Structural abbreviations used in this review

3 Biological activities

3.1 Anticancer effects

Most steroidal alkaloids showed anticancer activity as cytotoxicity with the IC50values listed in Table 18.Among the seven human cancer cell lines SMMC-772,A-549, SK-BR-3, PANC-1, K562, SGC7901 and HL-60,the most sensitive cell line according to sarcovagine D(116) was SK-BR-3, which had an IC50value of 2.25 μM.[79].Holamine (145) and funtumine (154) exhibited anticancer activity against human colon adenocarcinoma(HT-29) with IC50values of 31.06 and 22.36 μM, respectively.The study demonstrated that 145 and 154 induced cytotoxicity through the induction of apoptosis in HeLa,MCF-7, and HT-29 cancer cells [83].Then, they induced apoptosis through the elevation of reactive oxygen species (ROS), mitochondrial function modulation, the perturbation of F-actin polymerization, and caspase-3 induction, which were all more prominent in HeLa cells[334].

Table 18 Cytotoxic activity of steroidal alkaloids against tumor cell lines

Table 18 (continued)

Table 18 (continued)

Cyclopamine (432), a Hedgehog (Hh) signaling pathway antagonist, was first identified as a potent teratogen in animals.Among the nine human pancreatic cell lines examined, the IC50values of cyclopamine ranged from8.79 to more than 30 μM [335].In addition, 432 also showed prominent anticancer effects, including smallcell lung cancer (SCLC) [336], oral squamous cell carcinoma (OSCC) [337], breast cancer [338], pancreatic cancer [339], hepatocellular carcinoma (HCC) [340] and human erythroleukemia cells [341].Furthermore, 432 induced apoptosis in HCC cells through inhibition of the Sonic Hh signaling pathway by downregulating Bcl-2[340].In addition, 432 could induce apoptosis and upregulate cyclooxygenase-2 (COX-2) expression which plays a crucial role in the proliferation and differentiation of leukemia cells [341].

Tomatidine (458) and solasodine (513), important alkaloids found in a large number ofSolanumspecies,exerted cytotoxic activity against HBL-100 cells [342].They had a weak inhibitory effect on MCF-7, HT-29 and HeLa cells by blocking the cell cycle in the G0/G1phase [343].α-Solamargine (500) andα-solasonine(501), the two glycosides of 513, differed only in their carbohydrate moieties, which are used in the treatment of keratoses, basal cell carcinomas, and squamous cell carcinomas [344].Moreover, 500 was significantlycytotoxic to the human tumor cell lines H441, H520,H661, H69, HeLa, A549, MCF-7, K562, HCT116, U87 and HepG2 with IC50values from 2.1 to 8.0 μM [345,346].The cellular and molecular mechanism of 500 anti-human breast cancer cells HBL-100, ZR-75-1 and SK-BR-3 were investigated, and it was concluded that this compound could activate apoptotic proteins and inhibite anti-apoptotic, so it has great potential as an anti-human breast cancer candidate drug [347].The target ofα-solanine (528) inducing apoptosis in HepG2cells seemed to be mediated by the inhibition of the expression of Bcl-2 protein [255].

Cephalostatin 1–20 (652–671) were significantly cytotoxic to the human tumor cell lines BXPC-3, MCF-7,SF-268, NCI-H460, KM20L2 and DU-145.Of these compounds, cephalostatin 2 (653) was the most active compound, with GI50(growth inhibition of 50%) values in the range of 0.0056–0.11 nM.Importantly, compared with the cephalostatins 9 (664) and 20 (665), the inhibitory effects of 653 and cephalostatin 1 (652) were significantly increased by 100–1000 times.From this evidence, it was clear that the spirostanol structure must be intact and was the critical center for antineoplastic activities.The opening of the left-side spiro-ring significantly reducedthe inhibition of these carcinoma cells.A significant contribution of the presence of a hydroxy group at C-8′ to antineoplastic potency was evident by comparing the activity of cephalostatins 2 (653) and 1 (652), which was further supported by the cancer growth inhibitory activity of cephalostatins 20 (665) and 9 (664).Compounds 653 and 665, in which the hydroxy substitution at C-8′,

had considerably increased activity compared with compounds 652 and 664, respectively [326].

Ritterazines A–Z (672–697) were all significantly cytotoxic to the human tumor cell lines of P388 murine leukemia cells.Of these compounds, ritterazine B (674)was the most active with an IC50value of 0.00015 μg/mL.The presence of both the terminal 5/6 spiroketal and the hydroxyl groups was found to be especially important for pronounced inhibition of P388 cells.Ritterazines B (674)and F (678), which have terminal 5/6 spiroketal, showed high cytotoxicity against P388 cells, whereas ritterazine C, possessing 5/5 spiroketal structure, showed a lower significant level of cytotoxicity [333].

3.2 Anticholinergic effects

Some pregnane and cyclopregnane type alkaloids are distributed in many genera of Apocynaceae and display significant anticholinergic activity.Cholinesterase (ChE),divided into two enzymes acetylcholinesterase (AChE)and butyrylcholinestarase (BChE), have been identified as potential targets in the treatment of AD, myasthenia gravis and glaucoma.The IC50values of AChE and BchE inhibited by most steroidal alkaloids are listed in Table 19.

Table 19 Cholinesterase-inhibiting activities of steroidal alkaloids

Table 19 (continued)

Phulchowkiamide A (121), containing a carbonyl group at C-4 along with the tigloylamino moiety at position C-3, was found to be the most potent inhibitor of AChE and BChE among these alkaloids with IC50values of 0.5 and 0.4 μM, respectively [71].Similarly, compounds such as sarsalignone (64) [68], sarsaligenone (65) [70],sarcovagine D (116), sarcovagenine C (117) [71], and hookerianamide F (122) [73], which have in common with 121, displayed higher inhibitory activity than other compounds.In general, theα,β-unsaturated carbonyl group and tigloylamino moiety might be considered to be important factors to increase the activity.

From the list, we found that some alkaloids, including axillarine C (96), hookerianamide B (102), hookerianamide C (103), saligenamide D (140), cyclovirobuxeine A(202), hyrcanone (273), impericine (312), delavine (325),and persicanidine A (345), appeared to be more selective inhibitors of BChE.The presence of a C-2βhydroxy group, as in 2-hydroxysalignamine (49), saligenamide C(93), axillarine C (96), axillarine F (97), salonine A (98),and hookerianamide A (137) caused a negative effect on the inhibitory activity towards both AChE and BChE.In general, pregnane alkaloids were more selective than cyclopregnane alkaloids towards AChE and BChE.This might be due to the effect of the C-4 methyl groups and the cyclopropane ring in cyclopregnane alkaloids that decreased the activity.

3.3 Antimicrobial effects

Steroidal alkaloids are considered a part of plant chemical defenses against various pathogens, namely, fungi, bacteria, and viruses.Epipachysamine-E-5-ene-4-one (66)and iso-N-formylchonemorphine (90) showed strong antibacterial activity against a wide range of pathogenic bacteria (Bacillus cereus,Klebsiella pneumoniae,Staphylococcus aureusandPseudom aeruginosa) with minimum inhibitory concentrations (MICs) of 0.0312–0.2500(mg/mL), compared with the widely used antibiotics amoxicillin and ampicillin (0.0625–0.2500 mg/mL) [59].The five pregnane alkaloids sarcovagine C (80), hookerianamide I (107), chonemorphine (108),N-methypachysamine A (109) and hookerianamide H (123), were all active in antibacterial properties againstBacillus subtiliswith MIC values of lower than 20 μg/mL, and most of them displayed moderate to good antibacterial activities againstMicrococcus luteus,Streptococcus faecalis, andPseudomonas pallida[349].As saligcinnamide (85),Namethyl epipachysamine D (86) and epipachysamine D(87) had the same skeleton, and possessed potent antibacterial activity against seven human pathogenic bacteria with inhibition zones ranging from 6 to 12 mm [67].

(+)-16α,31-Diacetylbuxadine (278) exhibited significant antibacterial activity with zones of inhibition (ZI) of 14–19 mm againstK.pneumoniaeandSalmonella typhiand moderate to weak activity (ZI = 4–12 mm) against other seven human pathogenic bacterias [92].Neoverataline A (379), neoverataline B (380), stenophylline B(564), veramiline-3-O-β-D-glucopyranoside (566) and jervine (427) were tested for antifungal properties against the phytopathogensPhytophthora capisisandRhizoctonia cerealis,among which 380, 564 and 566 displayed strong activity againstP.capisiswith MICs at 120, 80 and 80 μg/mL, respectively.The MIC of triadimefon, a positive control, againstP.capisiswas 80 μg/mL [125].

Tomatidine (458) potentiated the action of several aminoglycoside antibiotics (gentamicin, kanamycin,tobramycin, amikacin and streptomycin) againstS.aureus, and the synergy between 458 and aminoglycosides could help reduce the incidence of resistance.Furthermore, 458 affected the haemolytic ability ofS.aureusand repressed several agr-regulated virulence factors [350].α-Chaconine (527),α-solanine (528),α-solamargine (500),α-solasonine (501), andα-tomatine(459) showed antimalarial activity, among which the most active compound 527 had no additive effect with 528.When orally administered at 7.5 mg/kg/day for 4 days, 527 suppressed the parasitemia level by 71.38%[351].Among the four mycobacterial species,Mycobacterium tuberculosis,Mycobacterium avium,Mycobacterium intracellulareandMycobacterium simiae,plakinamine P (640) exhibited the strongest antibacterial effect againstM.tuberculosis, giving a MIC of 1.8 μg/mL[313].

3.4 Anti-inflammatory and analgesic effects

Solasodine (513) significantly reduced the inflammatory reaction to carrageenan-induced rat paw oedema from 19.5 to 56.4% [352].In addition, the antinociceptive activity of 513 was evaluated by a hot plate, formalin,and writhing tests.513 caused a significant decrease in nociception at a dose of 8 mg/kg in acetic acid-induced mice abdominal constrictions, with a maximum inhibition of 61%, compared to indomethacin (74%).It could also significantly reduce the painful sensation caused by formalin and produce a significant increase in the pain threshold in the hot plate test.Overall, the results suggested that 513 may possess analgesic activity through both central and peripheral mechanisms [353].

The data provided by Chiu et al.suggested that tomatidine (458) inhibited NF-κB nuclear translocation and c-Jun N-terminal kinase activation, thereby decreasing the expression of COX-2 and inducible cytotoxic nitric oxide (NO) synthase, which might be beneficial for antiinflammatory therapy.They also found 458 had a better anti-inflammatory effect than solasodine (513) in Lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages [354].

α-Chaconine (527) and solanidine (535) were responsible for the anti-inflammatory effect, which was dependent on reducing the production of interleukin-2 and interleukin-8 induced by canidin A in Jurkat cells, and the induced NO production by LPS stimulated macrophages[355].

3.5 Anti-myocardial ischemia effects

Buxus microphyllais often used to treat cardiovascular and cerebrovascular diseases asafolk medicine in China.Cyclovirobuxine D (203) was the most potent component contributing to the anti-myocardial ischemia effects of the “huangyangning” tablet.This Chinese drug is used to treat cardiovascular and cerebrovascular diseases and has been developed successfully for more than 10 years in China.In the myocardial ischemia model induced by isoprenaline or pituitrin, 1.1 and 2.2 mg/kg cyclovirobuxine D could improve model rat plasma superoxide dismutase(SOD) activation, and reduce the plasma MDA, LDH,and phosphocreatine kinase (CPK) contents of model rats [356].The main mechanism of 203 in treating acute myocardial ischemia may be attributed to inhibiting blood stasis and thrombosis, enhancing NO release, and opening KATPchannels [357].In addition, data from a study of rats with congestive heart failure showed significant benefits after oral administration of 203, indicating that it may be a promising and useful drug in the treatment of cardiac dysfunction [358].

3.6 Anti-giogenesis effects

Cortistatins, novel steroidal alkaloids extracted fromCorticium sponge, showed highly selective anti-proliferative activity against human umbilical vein endothelial cells (HUVECs), which inhibited the formation of original capillaries, a process known as angiogenesis[314].Among the eleven cortistatins A–J (641?651),cortistatins A (641) and J (651) showed the most strongest anti-proliferative action against HUVECs with IC50values of 1.8 and 8 nM, which were 3000 and 300–1100 times more selective than normal human dermalfibroblast (NHDF) and tumor cell murine neuroblastoma cells(Neuro2A), respectively.[315].

3.7 Others

Among the four conanine-type alkaloids, conessine (1), conimin (9), mokluangin A (10), and irehline(36), 36 showed the most effective antimalarial activity(IC50= 1.2 μM) againstPlasmodium falciparum,comparable to that of the positive control dihydroartemisinine with an IC50of 3.7 nM [41].

The anti-tussive activity of three steroidal alkaloids was also investigated.yibeinone C (347), imperialine (335),yibeinone B (402), and showed an apparent concentration-dependent relaxation of isolated tracheal preparation, amongst 347 and 335 showed significant effects with pA2values of 6.19 and 8.41, and EC50values of 0.65 μmol/L and 4.40 nmol/L, respectively [156].

The five steroidal alkaloids puqienine A (400), puqienine B (401), puqietinone (577),N-demethylpuqietinone(579), and puqietinonoside (580) could significantly prolong the latent period and reduce the number of coughs in ammonia-induced mouse cough models at doses of 5 and 10 mg/kg, confirming their antitussive activity compared to the positive control codeine.The presence of these compounds may be responsible for the traditional use ofFritillaria puqiensisin cough remedies [188].

Plakinamines J (634), N (636), and O (637), containing a substituted pyrrolidine ring, showed potent antiproliferative activity against seven human colon carcinoma cell lines with mean GI50values of 11.5, 2.4 and 1.4 μM,respectively, whereas plakinamine I (633) with the pyrrolidine nitrogen formed an additional fused piperidine ring system that exhibited relatively weak activity [311].

4 Toxicity

Jervine (427) and cyclopamine (432), veratrum alkaloids isolated fromVeratrum californicum, had prominent teratogenic activity to produce synophthalmia and related cephalic malformations in sheep, cattle, goats and rabbits [359, 360].In addition, the presence of C-5,C-6 olefinic linkages in the framework of jervanes was found to be a critical structural factor to enhance teratogenicity induction [361].

In both pregnant and nonpregnant mice, tomatidine(458), solasodine (513), and solanidine (535) induced an increase in liver weight after being fed a diet containing 2.4 mmol/kg of these aglycones for 14 days[362].

In terms of the LC50and EC50after 96 h of exposure,α-chaconine (527) was teratogenic and more embryotoxic thanα-solanine (528) in frogs.The carbohydrate side chain attached to the 3-OH group of solanidine(535), the only difference between these two compounds, appeared to be an important factor in governing teratogenicity [363].

A pathophysiological study showed that isorubijervine (543) and rubijervine (544) were highly toxic compounds with LD50values of 1.14 and 1.77 mg/kg in mice, respectively.They also exerted the strongest ability to inhibit the sodium channel NaV1.5, which plays an essential role in cardiac physiological function [263].

5 Summary

Natural steroidal alkaloids with diverse bioactivities and high toxicity keep them one of the highlighted types of natural products.In this review, the structural diversity and biological activities of 697 natural steroidal alkaloids have been summarized and it is likely that many more steroidal alkaloids with novel structures will be discovered, especially rings E and F.Additionally, the high medicinal potential of cyclovirobuxine D, cyclopamine,α-solamargine,α-solasonine, cephalostatin 1 and many other members of this intriguing family of natural products is far from being exploited.Therefore, future research in this field will further contribute to understanding their full potential in drug development.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China(32170405) and Yunnan Science and Technology Project (202105AE160006,2019FY003004) for partial financial support.

Author contributions

M-LX searched the literature, collected the data, and drafted the manuscript;B-YH, Z-HQ, T-ZX and Z-JW provided corrective works of phytochemistry and biological activities; X-NW, D-YM and QZ revised the chemical structures.X-DL conceived the projects, revised manuscript and provided financial support.All authors read and approved the final manuscript.

Declarations

Competing interests

The authors declare that there are no conflicts of interest associated with this work.

Author details

1Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan Provincial Center for Research & Development of Natural Products, School of Chemical Science and Technology, Yunnan University,Kunming 650091, People’s Republic of China.2State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,Chinese Academy of Sciences, Kunming 650201, People’s Republic of China.

Received: 6 April 2022 Accepted: 12 April 2022