Chemical Diversity and Bioactivities of Monoterpene Indole Alkaloids (MIAs) from Six Apocynaceae Genera

By the end of the twentieth century, the interest in natural compounds as probable sources of drugs has declined and was replaced by other strategies such as molecular target-based drug discovery. However, in the recent times, natural compounds regained their position as extremely important source drug leads. Indole-containing compounds are under clinical use which includes vinblastine and vincristine (anticancer), atevirdine (anti-HIV), yohimbine (erectile dysfunction), reserpine (antihypertension), ajmalicine (vascular disorders), ajmaline (anti-arrhythmic), vincamine (vasodilator), etc. Monoterpene Indole Alkaloids (MIAs) deserve the curiosity and attention of researchers due to their chemical diversity and biological activities. These compounds were considered as an impending source of drug-lead. In this review 444 compounds, were identified from six genera belonging to the family Apocynaceae, will be discussed. These genera (Alstonia, Rauvolfia, Kopsia, Ervatamia, and Tabernaemontana, and Rhazya) consist of 400 members and represent 20% of Apocynaceae species. Only 30 (7.5%) species were investigated, whereas the rest are promising to be investigated. Eleven bioactivities, including antibacterial, antifungal, anti-inflammatory and immunosuppressant activities, were reported. Whereas cytotoxic effect represents 47% of the reported activities. Convincingly, the genera selected in this review are a wealthy source for future anticancer drug lead.


Introduction
Alkaloids are basic nitrogenous natural metabolites with structural diversity and molecular conformity. They displayed interesting bioactivities and are known to perform an important role in plant protection. The majority of them were discovered from plants and recently recorded Ca 21,000 [1,2]. The alkaloids are generally derived from amino acids that are containing one or more nitrogen atoms. These precursors are playing a rule in their classification. Also, the biosynthetic pathway of alkaloids can be named according the amino acid source [3]. Thus, they can be categorized into several groups based on associated moieties, including piperidine, pyrrolidine, pyrrole, pyridine, quinolone, isoquinoline, indole, quinolizidine, pyrrolizidine, tropane, benzylisoquinoline, purine, β-carboline, indolinics and quinolizidine.
Terpenoids are considered to be interesting natural products that have chemical diversity and different bioactivities. Common terpenoids have been reported from marine sources [4]. Whereas, the plants were listed as an important source of such metabolites. Terpenoids include several subclasses according to the number of carbo-skeleton; monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesterterpenes (C 25 ), triterpenes (C 30 ), and tetraterpenes (C 40 ).
Monoterpene indole alkaloids (MIAs) are metabolites containing a bicyclic structure of a benzene ring fused to a five-membered pyrrole ring. It is a noteworthy that the occurrence of multipart alkaloids is largely restricted to limited number of plant families. (e.g., Apocynaceae, Loganiaceae, and Rubiaceae) [5][6][7][8]. These families are closely taxonomically related. Also, on the chemical aspect, they are recognized to have apparent uniformity in the building blocks of these alkaloids. MIAs have been proposed to be sourced from strictosidine, which originates from the condensation of tryptophan with secologanin (C 10 or C 9 part), which can be divided into linear six carbon (6 C), one carbon (1 C) and three carbon (3 C) units ( Figure 1). The connection between them requires proving. The nine-carbons fragment may be formed by the loss at certain stage of one of the carbons from the 3 C unit, and there are also a few indole bases which appear to have ended up without the 3 C or the 1 C units. Three hypothetical building blocks, Types I, II and III. It is nevertheless a useful way of dividing indole alkaloids into groups based on their sub architecture. Since Type I alkaloids are by far the most numerous, they may be the source of Type II and III. It was suggested by LeMen and Tylor that the convention be extended to cover Type II and III alkaloids as illustrated in Figure 1. On these hypothetical bases, the MIAs categorized according to their biogenic pathway in three main groups, corynanthe, aspidosperma and iboga [9]. Recently, strictosidine has been considered as the building block of MIAs biosynthesis [10]. MIAs have been proposed to arise from strictosidine, which itself originates from the condensation of tryptophan with secologanin in a 1:1 ratio. Strictosidine has been elaborated to give an impressive array of structural variants. This type of alkaloids possess 18 (or 19) carbon atoms on its skeleton. Additionally, the MIAs could be produced from tryptophan and secologanin in 1:2 or 2:1 ratio. According to this arrangement, three types (classes) of monoterpenes were constructed, including, corynanthe (e.g., ajmalicine), aspidosperma (e.g., tabersonine) and iboga (e.g., catharanthine) [11][12][13].
Apocynaceae contains about 250 genera and 2000 species [14]. Five sub-families are classified under Apocynaceae, including, Apocynoideae, Asclepiadoideae, Periplocoideae, Rauvolfioideae, and Secamonoideae. Apocynaceae species ranged from shrubs to trees. The characteristic features of these plants include colorful flowers and opposite leaves. Traditionally, species of this family have been used for the treatment of fever, malaria, gastrointestinal ailments, diabetes, and pain [15]. Additionally, some species have shown antiplasmodial and anticancer activities [14]. Several Apocynaceae MIAs have been used as anticancer, analgesic, anti-inflammatory and anti-spasmodic agents. For example, vinblastine, vinorelbine, vincristine, and vindesine were utilized as anticancer agents, whereas ajmalicine and ajmaline were used in the treatment of cardiovascular disorders ( Figure 2) [2]. Catharanthus roseus and Rauvolfia serpentine are members of Apocynaceae and are known as sources of bioactive indole alkaloids [16]. Reserpine has been used as a tranquillizer, whereas vinblastine and vincristine have been used as anti-leukemic agents [17]. Vincristine and vinblastine were among the earliest anti-tumor agents, and since 1965 have been used as tubulin polymerization inhibitors. They have been used in combination for the treatment of acute lymphoblastic leukemia and also against both Hodgkin's and non-Hodgkin lymphoma. Additionally, strychnine is potent muscle contracting agent whereas, yohimbine has been used for the treatment of sexual dysfunction and investigated as a remedy for type-2 diabetes in animal and human models. There are several publications interested in the terpene indole alkaloids of individual species of the family Apocynaceae. The current review organizes the reported MIAs considering the historical aspect in each selected genus. Moreover, these MIAs were biosynthetically classified according to the tepenoidal fragment, i.e., corynanthe, aspidosperma, or iboga. Also, it focuses on the origin, structural diversity and biological activities exerted by 444 (Table 1) monoterpene indole alkaloids which have been reported from selected six genera of the family Apocynaceae (Alstonia, Kopsia, Ervatamia, Rauvolfia, Tabernaemontana and Rhazya), in the period between 2010 and December 2020. The listed metabolites are categorized under 26 subclasses, ajmaline, akuamiline, akuammidine, akuammicine, apparicine, aspidofractinine, aspidospermatan, eburnane, flabelliformide, kopsine, macroline, macroline oxindole, macroline-akuammiline, methyl chanofruticosinate, nareline, paucidactine, picrinine, pleiocarpamine, sarpagine, scholaricine, secodine, strictosidine, strychnos, vincamine, vincorine and vobasine (Figures 3 and 4).  Additionally, the future prospective and emphasizing the research gaps and highlighting the roadmap to discover the potent bioactive monoterpenoid alkaloids, which could be a drug lead from the six genera. Also, this review will discuss the reported structural activity relationships.

Alstonia
Plants of the genus Alstonia are grown in Africa and Asia. It includes 60 species, which were recognized as rich source of heterocyclic monoterpene indole alkaloids. It has different names according to the geographical sources, including Devil tree, Australian fever bush, dita bark, Australian quinine, fever bark and palimara. Alstonia bark shows potent therapeutic effects including anti-inflammatory, antirheumatic, analgesic, antidiabetic, antimalarial, antipyretic, antihelminthic, antibiotic, antimicrobial, anticancer, antibacterial and antitussive effects [18][19][20].
Alsmaphorazines A (23) and B (24) ( Figure 6) were identified from the leaves of malaysian A. pneumatophore. The chemical structures were determined on the basis of 2D NMR and MS spectral analysis. These compounds had an unprecedented skeleton containing an 1,2-oxazine (six-member ring) and an isoxazolidine (five-member ring) [36]. The absolute configuration of alsmaphorazine B was determined using CD spectral analysis. The absolute configuration of alsmaphorazine B (24) was studied by comparing its experimental CD spectrum with the calculated CD spectrum, with the CD calculations performed by Turbomole 6.1using the Time-Dependent Density Functional Theory (TD-DFT-B3LYP/TZVPP) level of theory on RI-DFTBP386LYP/TZVPP optimized geometries. Compound 23 inhibited the production of nitric oxide (NO) in an LPS-stimulated J774.1 cell with an IC 50 value = 49.2 µM, without affecting the cell viability, whereas compound 24 showed no inhibitory effect at 50.0 µM. Compound 23 was more potent as an anti-inflammatory agent due to the presence of a hydroxyl group at C-12 [36]. Alstrostines A (25) and B (26) were determined as derived from the condensation of tryptophan and secologanin in a ratio of 1:2. They were isolated from Alstonia rostrata [37]. The structures were established by measuring 1 H, 13  Alstrostines C-F (27-30) ( Figure 6) were isolated from the leaves and twigs of Chinese A. rostrata [38]. Compounds 27-30 showed a characteristic UV absorption at 326, 275 and 214 nm, which indicated the presence of an indole alkaloid with a β-anilineacrylate system. The chemical structure elucidation was confirmed by 1D and 2D NMR. Compounds 27-30 showed weak cytotoxicity against five human cancer cells, breast (SK-BR-3), human myeloid leukemia (HL-60), pancreatic (PANC-1), hepatocellular carcinoma (SMMC-7721) and lung (A-549) cells, with IC 50 values > 40 µM [38].
Alstolactines A (52), B (53), and C (54) (Figure 9) were isolated from the leaves of chines A. scholaris [43]. The structures were identified by extensive spectroscopic data analyses and X-ray diffraction analyses. The absolute stereochemistry was deduced from crystal X-ray diffraction. These compounds are biosynthetically originated from picrinine, which is the main metabolite in A. scholaris. Compounds 52-54 exhibited no effects against four bacterial strains: Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus [43]. Moreover, Alistonitrine A (55) (Figure 9) had an unprecedented caged carbon skeleton with a unique 6/5/6/5/5/6 ring system and also contained three nitrogen atoms. It was isolated from the same species [12]. Its structure and absolute configuration were established by extensive spectroscopic analyses and electron circular dichroism calculations. Compound 55 exhibited no activity as an anti-inflammatory in both NF-κB and HIF-α models [12].
A review entitled "Alstonia scholaris and Alstonia macrophylla: A comparative review on traditional uses, phytochemistry and pharmacology" was published in 2014 and mentioned the compounds obtained from A. scholaris from 1976 to 2009, and from A. macrophylla from 1987 to 2013 [82]. A review published in 2018 entitled "The alstoscholarisine compounds: isolation, structure determination, biogenesis, biological evaluation and synthesis" studied the alstoscholarisine compounds obtained from A. scholaris [83]. Furthermore, a review published in 2016 called "An overview phytochemistry and chromatographic analysis of Alstonia scholaris used as a traditional medicine" discussed A. scholaris compounds which were reported between 1965 and 2009 [84].

Kopsia
Kopsia (Family Apocynaceae) contained 30 species with a distribution in China, India, Southeast Asia, and Australia. Sixteen species were grown in Malaysia [85], and five species were grown in Thailand [86]. These plants are considered as rich sources of indolecontaining compounds. Traditionally, some of the species have been used for the treatment of tonsillitis, dropsy and rheumatoid arthritis. Several species have been reported to have antitumor, antimanic, antitussive and antileishmanial effects [87][88][89]. A review published in 2017 was interested in reporting indole alkaloids from genus kopsia plants regarding reversing multidrug resistance in vincristine-resistant KB cells for example, kopsirensine B, arboloscine A [90], grandilodines A and C, and lapidilectine B [91,92].

Rauvolfia
Rauvolfia (family Apocynaceae) contains 60 species. It contains trees or shrubs that are distributed in Africa, America, and Asia [131]. Rauvolfia serpentine is one of the most important medicinal plant that has been considered as a drug lead for a long time [132]. Rauvolfia has been used traditionally for the treatment of several diseases, such as high blood pressure (hypertensive), fever (malaria), arrhythmia, cancer, oxidative stress, microbial problems, intestinal spleen ailments, and various mental disorders [133]. Therapeutically, it is a source of monoterpenoid indoles, including ajmaline (antiarrhythmic), ajmalicine, yohimbine, reserpine (antihypertensive), and serpentine [133].

Ervatamia
The genus Ervatamia contains 120 species. It is distributed in Asia and Australian. Of which, fifteen species and five varieties are grown in south China. Ervatamia is a rich source of iboga-type MIAs, which is characterized by structural novelty and biological diversity including neuroprotective, anti-tumor, and anti-addiction activities [151][152][153].
Compound 306 exhibited significant protective effects toward MPP + (1-methyl-4phenylpyridinium) and induced damage in primary cortical neurons with an IC 50 = 12.5 µM. Parkinson's disease (PD) is caused by MPP + a toxic agent that interferes with the function of mitochondria, thus causing neuronal damage and death. Brain-derived neurotrophic factor (BDNF) was used as a positive control and showed an inhibitory effect, with an IC 50 value = 200 ng/mL [49].
The Ervatamia genus is known to produce iboga-type indole derivatives, which contain two subclasses, flabelliformide-type (364, 365) and apparicine-type (368) (Figure 28), with compounds belongonging to the main class corynathe. The iboga-type showed an interesting bioactivity in the nervous system.

Tabernaemontana
The Genus Tabernaemontana (subfamily Rauvolfioideae) contains 110 species, which are distributed throughout tropical and subtropical regions. Thirty species are grown in Brazil, whereas, 44 species were grown in America and the rest in different places around the world. Traditionally, the plants of this genus have been used for the treatment of hypertension, sore throat, and abdominal pain [6,192]. A review article entitled "Brazilian Tabernaemontana genus: indole compounds and phytochemical activities" activities was published in 2016 [6]. It concerned in the monomeric and dimeric MIAs reported from the genus. A review article entitled: A review on tabernaemontana spp.: Multipotential medicinal plant, shows the MIAs reported from this genus until 2015 [6].
The Tabernaemontana genus produced iboga type indoles, which contained four subclasses, such as vincamine-type, apparicine-type and akuammidine, these compounds which belongs to the main class aspidosperma and corynanthe, respectively.

Biosynthesis of Monoterpenoid Indole Alkaloids
Monoterpenoidal indoles are obtained from the reaction of tryptamine with secologanin terpenoid. Condensation of tryptamine with Secologanin produces strictosidine by the Mannich-link reaction. The deglycosylation of strictosidine converts it to a hemiacetal. Opening the hemiacetal led to forming an aldehyde group, which then reacts with the (N-4) secondary amine of strictosidine to form 4,21-dehydrocorynanthenine. Allylic isomerization moves the double bond of vinyl to a conjugation with iminium nitrogen that generates dehydrogeissoschizine, which is then cyclized to form cathenamine. The reduction of cathenamine in the presence of NADPH forms ajmalicine (corynanthe-type) [229].
The formation of Preakuammicine occurs from dehydrogeissoschizine. Preakuammicine intermediate (strychnos-type) is the common precursor of the strychnos, aspidosperma and iboga indole alkaloids. Preakuammicine reduced to form stemmadenine, then rearranged to form the acrylic ester dehydrosecodine, which is a common intermediate for iboga and aspidosperma skeletons. Tabersonine (aspidosperma type) and catharanthine (iboga type) are formed the Diels-Alder reaction (Scheme 1) [229]. Polyneuridine aldehyde (sarpagan type) is an intermediate compound of the ajmaline pathway. The possibility of a mechanism where the sarpagan bridge enzyme converts an isomer of 4,21-dehydrogeissoschizine to polyneuridine aldehyde is shown (Scheme 2). Polyneuridine aldehyde methyl ester is hydrolyzed by polyneuridine aldehyde esterase, generating an acid which decarboxylates, to yield epi-vellosamine. Epi-vellosamine transforms to the ajmaline alkaloid vinorine. The hydroxylation of vinorine to vomilene is caused by the vinorine hydroxylase enzyme. After formation of vomilene, two step reduction occurs. First, the indolenine bond is reduced by an NADPH enzyme to yield 1,2-dihydrovomilenene. The second step, reducing the 1,2-dihydrovomilenene to acetylnorajmaline by a 1,2-dihydrovomilenene reductase enzyme. The acetyl linkage of acetylnorajmaline is hydrolyzed by acetylesterase to yield norajmaline. Finally, the production of ajmaline by N-methyl transferase of a methyl group at the indole nitrogen of norajmaline occurs (Scheme 2) [229,230].
It is noteworthy to mention that, sarpagine, ajmaline, and macroline alkaloids are biosynthetically similar or all derived from the same origin. Whereas, sarpagine can be converted into macroline by means of Michael addition [231], on the other hand macroline can be converted into sarpagine by through a retro-Michael reaction [231][232][233]. Similarly, some sarpagine-containing alkaloids can be converted into ajmalines under strong acidic conditions, which refers to the great similarity between them [233].

Conclusions and Future Prospectives
Natural products have an unprecedented molecular conformity with a diversity of functionalities. These characteristics enable them to produce biological effects, which validates the initial step for a drug lead. In recent years, the majority of new drugs reported have been natural or originated from natural sources. Alkaloids are an important source of drugs. It is noteworthy that, many alkaloids displaying fascinating molecular structures with diverse physiological and pharmacological effects have been isolated from plant families. The Apocynaceae family has been noted as a unique producer of biologically active natural metabolites such as vincristine, vinblastine, reserpine and yohimbine. This review is interested in discussing the metabolites produced from six genera belong to the family Apocynaceae. These six genera contain 400 species, which represent 20% of the Apocynaceae family. Only 30 species, which represent 7.5% of the total species of the six genera were studied. Chemical investigation of these genera led to the reporting of 444 MIAs, in the period between 2010 until December 2020, which were discussed in this review. Figure 34 illustrates the number of compounds isolated from the six species; there are 157 (35.4%), 126 (28.4%), 66 (14.9%), 48 (10.8%), 27 (6.1%), and 20 (4.4 %), from Alstonia, Kopsia, Ervatamia, Tabernaemontana, Rhazya and Rauvolfia, respectively. We believe that the six genera are interesting candidate for further investigation. This record coincided with the data illustrated in Figure 35. For example, Alstonia scholaris is a species that belongs to the genus Alstonia that has produced the highest number of MIAs (71 compounds) and represents 45.2 % of the MITs identified from the same genus between 2010 and 2020. The second and third most interesting species are Kopsia officinalis and Kopsia pauciflora which produced 45 and 27 compounds, respectively. These two species represent 35.7% and 21.4% of the total compounds produced from the genus Kopsia. The fourth most interesting species belong to the genus Alstonia (Alstonia mairei), which produced 26 compounds and represents 16.5 % of the MITs identified from the genus Alstonia.  It is interesting that the majority of compounds were isolated from twigs and leaves as illustrated in Figure 36. Additionally, the majority of the examined species belonging to the selected six genera were Chinese species and led to the identification of 360 compounds.  Figure 37 presents the biological activities of the compounds. The prominent activity was cytotoxicity followed by anti-inflammatory and antimicrobial activities. Thus, these compounds could be a source of anticancer drugs. The family of terpene indole alkaloids has been discovered for over a century. There are numbers of total syntheses studies of these intricate scaffolds have been achieved. Additionally, several reviews and book chapters, as well as the references therein, are interested in the synthetic efforts have been reported.

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

A431
Dermatoma cancer A-549 Lung cancer AChE Acetylcholinesterase B16F10 Melanogenesis activity BEN-MEN-1 Meningioma BGC-823 Human gastric carcinoma CAL-27 Head and neck squamous cell carcinomas CCF-STTG1 Astrocytoma CHG-5 Glioma CI Confidence intervals Detroit-562 Head and neck squamous cell carcinomas ED 50 Median effective dose F.sp. Forma specialis, abbreviated f. sp., is an informal taxonomic grouping allowed by the International Code of Nomenclature for algae, fungi, and plants HCT 116 Human colorectal carcinoma HeLa Human Gastric cancer Hep-2 Head and neck squamous cell carcinomas HepG2 Human hepatocellular HIF-α Hypoxia-inducible factor HL-60 Human myeloid leukemia HS-1 Dermatona cancer HS-4 Dermatona cancer HT-29 Human colorectal carcinoma IC 50 Half maximal inhibitory concentration ID 50 Median infective dose IL-1β Interleukin 1