Bisindole Alkaloids from Alstonia Species: Comparison
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Bisindoles are structurally complex dimers and are intriguing targets for partial and total synthesis. They exhibit stronger biological activity than their corresponding monomeric units. Bisindole alkaloids are naturally occurring alkaloids containing two indole nuclei and are the products of late-stage biosynthetic processes in higher plants by combining two monomeric units. Depending on the monomeric units involved, bisindoles can be a homo- or heterodimer. As a result, bisindole alkaloids comprise much higher structural complexity than both of the monomeric units that comprise them.

Alstonia

, a major genus in the Apocynaceae family of plants, has more than 150 species and is found all over the world. Robert Brown named it in 1811 in honor of Charles Alston (1685–1760), an eminent botanist at the University of Edinburgh. The

Alstonia

genus’ trees and shrubs are prevalent in the tropical and subtropical parts of Africa, Asia, and Australia. They contribute significant pharmacological activity, including anticancer, antileishmanial, antimalarial, antitussive, antiviral, antiarthritic, and antibacterial activities. 

  • Apocynaceae
  • Alstonia species
  • Sarpagine-macroline-ajmaline type indole alkaloids
  • Pleiocarpamine
  • Bisindole synthesis
  • Isolation of bisindoles
  • Bioactivity of bisindoles

1. Bisindole Alkaloids in drug discovery

 

Nature has been a substantial and sustainable pool of biologically active compounds. Since ancient times natural product extracts (in crude form) have been used in traditional and folk medicines in many countries. In modern times pure (isolated) natural products and their derivatives play an important role in drug discovery, as indicated by their prevalence in approved drugs for clinical use. Out of the 1881 newly FDA-approved drugs over the last four decades (1 January 1981 to 30 September 2019), a significant portion comprising 506 (26.9%) were either natural products or derived from or inspired by natural products [1]. It is expected that the advent of modern and innovative technologies such as computational software, cheminformatics, artificial intelligence, automation, and quantum computing will further boost natural product-based drug discovery. A synergy among these technological milestones would accelerate hit to lead to clinic pathways of drug discovery, and natural products are expected to remain an important source [2]. Moreover, pharmacophores and their unique stereochemical interactions with natural products may stimulate more demanding targets such as protein–protein interactions in the near future and open up a new avenue in modern drug discovery [3]. The majority of biologically active natural products are produced in plants, known traditionally as medicinal plants. Alkaloids, the most important class of natural products with structural diversity and significant pharmacological effects, are mainly found in higher plants such as the Apocynaceae, Ranunculaceae, Papaveraceae, and Leguminosae families [4][11]. These natural products, along with flavonoids, fatty acids, etc., are the major classes of secondary metabolites that are believed to be parts of the plants’ defense mechanism. To date, many monoterpenoid indole and bisindole alkaloids have been found in the Alstonia genus [5][4]. Modern clinical application of many of these alkaloids are similar to their traditional or folklore applications; for example, cocaine and morphine were used as anesthetics while caffeine and nicotine were used as stimulants [6][12]. Recently, Fielding et al. illustrated that several anti-coronavirus alkaloids showed potential therapeutic value against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in their in silico studies [7][9].

The indole motif is present in many naturally occurring and biologically active compounds. Some of them have been used in the clinic. In addition, there are numerous examples of synthetic compounds with useful medicinal properties that bear the indole moiety. Consequently, the indole scaffold is one of the few “privileged structures” in modern medicinal chemistry and drug discovery [8][9]. The prevalence of bioactivity of indole-containing molecules may be attributed to their similarity to the essential amino acid tryptophan, as well as important biomolecules such as tryptamine and serotonin. Many plant extracts, which likely include alkaloids, have been used from time immemorial in folk medicines for fever, general weakness, dysentery, pain, liver diseases, gastrointestinal diseases, and cancer [10]. Currently, there are many indole alkaloid-based marketed drugs such as sumatriptan for the treatment of migraine; vincristine and vinblastine for the treatment of various cancers, including leukemia and lung cancer; as well as reserpine for the treatment of hypertension and to decrease severe agitation in patients with mental disorders [11].
The indole motif is present in many naturally occurring and biologically active compounds. Some of them have been used in the clinic. In addition, there are numerous examples of synthetic compounds with useful medicinal properties that bear the indole moiety. Consequently, the indole scaffold is one of the few “privileged structures” in modern medicinal chemistry and drug discovery [13,14]. The prevalence of bioactivity of indole-containing molecules may be attributed to their similarity to the essential amino acid tryptophan, as well as important biomolecules such as tryptamine and serotonin. Many plant extracts, which likely include alkaloids, have been used from time immemorial in folk medicines for fever, general weakness, dysentery, pain, liver diseases, gastrointestinal diseases, and cancer [15]. Currently, there are many indole alkaloid-based marketed drugs such as sumatriptan for the treatment of migraine; vincristine and vinblastine for the treatment of various cancers, including leukemia and lung cancer; as well as reserpine for the treatment of hypertension and to decrease severe agitation in patients with mental disorders [16].
More structural diversity can be achieved via bisindole-based drug discovery by changing both monomeric units to furnish unnatural and
pseudo
-natural alkaloids. As such, bisindole alkaloids offer a large pool of natural, semi-natural, and chimeric drug candidates that have greater drug-like characteristics. As a result, due to their important biological applications and complex structural features, bisindole alkaloids have engendered the profound interest of synthetic organic and medicinal chemists, computational chemists, and chemical biologists.

2. Isolation and Plant' Morphology of Bisindoles  from Alstonia Species

 Among various species of the

 

 Among various species of the

Alstonia

 genus, 

A. macrophylla

 and 

A. angustifolia

 are the two major sources of bisindole alkaloids discussed herein (

vide infra

, 

Table 1

). (+)-Alstomacroline 

1

, a bisindole alkaloid consisting of a macroline and an ajmaline unit (monomeric units not shown here: please visit

https://doi.org/10.3390/molecules26113459

for details), was isolated from the bark

of A. macrophylla [12] and the leaves, stem-bark, and root-bark extracts of 

 [18] and the leaves, stem-bark, and root-bark extracts of 

A. scholaris, A. glaucescens, and A. macrophylla[13][14]. (+)-Alstomacrophylline 

[19,20]. (+)-Alstomacrophylline 

2

 (macroline–macroline-type) was isolated from the bark 

of A. macrophylla [12] and the leaves, stem-bark, and root-bark extracts of 

 [18] and the leaves, stem-bark, and root-bark extracts of 

A. scholaris, A. glaucescens, and A. macrophylla [14]. (-) Alstonisidine 

 [20]. (-) Alstonisidine 

3

, which contains a quebrachidine  and a macroline  unit, was isolated from the bark of

A. muelleriana [15][16]. The structure of (-)-alstonisidine 

 [21,22]. The structure of (-)-alstonisidine 

3 was confirmed by X-ray crystallographic data [17]. Yeap et al. recently isolated seven novel bisindoles from the methanol extract of the stem-bark of Malayan 

 was confirmed by X-ray crystallographic data [23]. Yeap et al. recently isolated seven novel bisindoles from the methanol extract of the stem-bark of Malayan 

A. penangiana [18]. This includes (-)-angustilongine E 

 [5]. This includes (-)-angustilongine E 

6

, (-)-angustilongine F 

7

, (+)-angustilongine G 

8

, (+)-angustilongine H 

9

, (+)-angustilongine J 

10

, (+)-angustilongine K 

11

, and (-)-angustilongine L 

12

 (macroline–pleiocarpamine type). (+)-Angustilongine K 

11

 was converted into (+)-

di

-

O

-acetylangustilongine K 

13

 by stirring it with 10 equivalents of pyridine and 15 equivalents of acetic anhydride for 6 h at room temperature in 

95% yield [18]. Among those, angustilongine G 

 yield [5]. Among those, angustilongine G 

8

 and angustilongine H 

9 are C-19 methyl substituted [19] bisindoles. The structures of the angustilongines were confirmed by various spectroscopic data, including 

 are C-19 methyl substituted [24] bisindoles. The structures of the angustilongines were confirmed by various spectroscopic data, including 

1

H NMR, 

13C NMR, 2D NMR, IR, and HRMS by Yeap et al. [18]. Angustilongine E 

C NMR, 2D NMR, IR, and HRMS by Yeap et al. [5]. Angustilongine E 

6

, angustilongine F 

7

, angustilongine G 

8

, angustilongine H 

9

, angustilongine J 

10

, and angustilongine K 

11

 are macroline–sarpagine coupled bisindoles. Angustilongine G 

8

 and angustilongine H 

9 differ in stereochemistry only at the C-20 position.

 differ in stereochemistry only at the C-20 position.
 
 

Table 1.

Structures of bisindole alkaloids from

Alstonia

species including semi-synthetic derivatives.
 
Macroline-macroline type
Molecules 26 03459 i012 Molecules 26 03459 i013 Molecules 26 03459 i014
Molecules 26 03459 i015 Molecules 26 03459 i016 Molecules 26 03459 i017
Molecules 26 03459 i018 Molecules 26 03459 i019 Molecules 26 03459 i020
Molecules 26 03459 i021 Molecules 26 03459 i022 Molecules 26 03459 i023

[14]. (-)-Anhydromacralstonine 

 extracts [20]. (-)-Anhydromacralstonine 

27

 was isolated from the stem-bark of 

A. angustiloba [23] and contains (-)-alstophylline  and (+)-macroline  as monomeric units. Another (-)-alstophylline 

 [28] and contains (-)-alstophylline  and (+)-macroline  as monomeric units. Another (-)-alstophylline 

28

 and (+)-macroline  monomeric bisindole, (+)-

Des-N’a

-methylanhydromacralstonine 

30,

 was isolated from the bark of 

A. muelleriana [14][30], the stem-bark of 

 [20,35], the stem-bark of 

A. angustifolia [28], and 

 [33], and 

A. glabriflora [26]. (+)-Macrocarpamine 

 [31]. (+)-Macrocarpamine 

31

, a hetero-dimeric bisindole containing a (+)-pleiocarpamine  and (-)-anhydromacrosalhinemethine monomeric unit was isolated from the leaves, stem-bark, and root-bark of

 A. scholaris, A. glaucescens, and A. macrophylla [14], as well as the stem-bark of 

 [20], as well as the stem-bark of 

A. angustifolia [23]. 10-Methoxymacrocarpamine 

 [28]. 10-Methoxymacrocarpamine 

34

 and 10-methoxymacrocarpamine 

N4′

-oxide 

35

 are structurally related bisindoles. These bisindoles were isolated from 

A. angustifolia leaves [31].

 leaves [36].

Lim et al. [28] reported the isolation of new macroline–macroline-type bisindoles, (-)-perhentidine A 

Lim et al. [33] reported the isolation of new macroline–macroline-type bisindoles, (-)-perhentidine A 

36

, (-)-perhentidine B 

37

, and (-)-perhentidine C 

38

 from the ethanolic extract of the stem-bark of Malayan 

A. macrophylla

 and 

A. angustifolia.

 The structurally related bisindole, (-)-perhentinine 

39

 (macroline–macroline-type), was isolated from the stem-bark and leaves of 

A. macrophylla

 and the leaves of 

A. angustifolia [12]. The exact structure of (-)-perhentinine 

 [18]. The exact structure of (-)-perhentinine 

39

 was confirmed by X-ray crystallography, converting it into the dimethyl diiodide salt by treating it with an excess of iodomethane. The X-ray crystallographic data of (-)-perhentinine 

39

 also helped in the structural characterizations of perhentidines A–C (

36–38) [28]. Tan et al. isolated three macroline–sarpagine-type bisindoles: (+)-perhentisine A 

) [33]. Tan et al. isolated three macroline–sarpagine-type bisindoles: (+)-perhentisine A 

40

, (-)-perhentisine B 

41

, and (+)-perhentisine C 

42

 from the stem-bark of 

A. angustifolia as a light yellow-colored oil together with other bisindoles [23]. The structures of these bisindoles were also elucidated using various NMR and MS techniques [23]. (+)-Villastonine 

 as a light yellow-colored oil together with other bisindoles [28]. The structures of these bisindoles were also elucidated using various NMR and MS techniques [28]. (+)-Villastonine 

43

, a macroline–pleiocarpamine-type bisindole, was isolated from the stem-bark, root-bark, and leaves of various 

Alstonia 

species, including 

A. spectabilis [24] and 

 [29] and 

A. muelleriana [32] by LeQuesne et al.,

 [37] by LeQuesne et al.,

A. macrophylla [25][27][33], and 

 [30,32,38], and 

A. angustifolia [31][34]. Schmid et al. elucidated the structure of (+)-villalstonine 

 [36,39]. Schmid et al. elucidated the structure of (+)-villalstonine 

43 by spectroscopic means, accompanied by degradation, and Nordman et al. confirmed the structure by X-ray crystallography [33][35].

 by spectroscopic means, accompanied by degradation, and Nordman et al. confirmed the structure by X-ray crystallography [38,40].

Table 2.

  Isolation of bisindoles from various part' of

Alstonia

 species.
Bisindoles Alstonia

Species		 Morphology and References
(+)-Alstomacroline 1 A. scholaris, A. glaucescens, and A. macrophylla extracts Leaves, stem-bark, and root-bark [13][14][19,20]
A. macrophylla Bark [12][18]
(+)-Alstomacrophylline 2 A. macrophylla Bark [13][19]
  A. scholaris, A. glaucescens, and A. macrophylla extracts Leaves, stem-bark, and root-bark [14][20]
Molecules 26 03459 i024 Molecules 26 03459 i025 Molecules 26 03459 i026
Macroline-sarpagine type
Molecules 26 03459 i027 Molecules 26 03459 i028 Molecules 26 03459 i029
Molecules 26 03459 i030 Molecules 26 03459 i031 Molecules 26 03459 i032
Molecules 26 03459 i033 Molecules 26 03459 i034 Molecules 26 03459 i035
Molecules 26 03459 i036 Molecules 26 03459 i037
Macroline-ajmaline type
Molecules 26 03459 i038 Molecules 26 03459 i039
Macroline-pleiocarpamine type
Molecules 26 03459 i040 Molecules 26 03459 i041 Molecules 26 03459 i042
Molecules 26 03459 i043 Molecules 26 03459 i044 Molecules 26 03459 i045
Molecules 26 03459 i046
Two macroline units are contained in (-)-lumusidine A 

14

, (-)-lumusidine B 

15

, (-)-lumusidine C 

16

, and (-)-lumusidine D 

17

 bisindoles. They were isolated from the stem-bark of 

A. macrophylla and the structures were confirmed via NMR spectroscopy, mass spectrometry, UV spectroscopy, and X-ray crystallography [12]. After isolation, the group of Kam et al. converted oily (-)-lumusidine A 

 and the structures were confirmed via NMR spectroscopy, mass spectrometry, UV spectroscopy, and X-ray crystallography [18]. After isolation, the group of Kam et al. converted oily (-)-lumusidine A 

14

, (-)-lumusidine B 

15

, and (-)-lumusidine D 

17 into the corresponding crystalline dimethyl diiodide salts (structures not shown) by treatment with an excess of iodomethane for 24 h. The crystalline salts were employed to obtain X-ray crystallographic data to elucidate the exact stereochemical confirmation [12]. (-)-Lumusidine D 

 into the corresponding crystalline dimethyl diiodide salts (structures not shown) by treatment with an excess of iodomethane for 24 h. The crystalline salts were employed to obtain X-ray crystallographic data to elucidate the exact stereochemical confirmation [18]. (-)-Lumusidine D 

17 is also known as thungfaine [20]. (+)-Lumutinine A 

 is also known as thungfaine [25]. (+)-Lumutinine A 

18

, (-)-lumutinine B 

19

, (+)-lumutinine C 

20

, and (+)-lumutinine D 

21

 are linearly fused bisindoles isolated from the stem-bark of 

A. macrophylla as a light yellowish oil [21]. (+)-Lumutinine A 

 as a light yellowish oil [26]. (+)-Lumutinine A 

18

 and (-)-lumutinine B 

19

 are macroline–macroline-type bisindoles, while (+)-lumutinine C 

20

, (+)-lumutinine D 

21

, and (+)-lumutinine E 

22 are macroline–sarpagine-type bisindoles. The structures of the lumutinines were elucidated using spectroscopic means including 1D and 2D NMR, IR, as well as mass spectrometric analysis [21]. The structure of (+)-lumutinine D 

 are macroline–sarpagine-type bisindoles. The structures of the lumutinines were elucidated using spectroscopic means including 1D and 2D NMR, IR, as well as mass spectrometric analysis [26]. The structure of (+)-lumutinine D 

21 was confirmed by X-ray crystallographic data [22]. (+)-Lumutinine E 

 was confirmed by X-ray crystallographic data [27]. (+)-Lumutinine E 

22

, a macroline–sarpagine-type bisindole, was isolated from the stem-bark of 

A. angustifolia [23].

 [28].
(+)-Macralstonidine 

23

 (macroline–sarpagine-type) was isolated from the bark of 

A. macrophylla [24][25], as well as from 

 [29,30], as well as from 

A. somersentenis [24] and 

 [29] and 

A. spectabilis [26]. (+)-Macralstonine 

 [31]. (+)-Macralstonine 

24

 was isolated from the leaves, stem-bark, and root-bark extracts of 

A. scholaris, A. glaucescens, and A. macrophylla extracts [14], 

 extracts [20], 

A. macrophylla [24][25][27][28][29], 

 [29,30,32,33,34], 

A. muelleriana [30], 

 [35], 

A. angustifolia [31], as well as from

 [36], as well as from

 A. glabriflora [26]. The structure of (+)-macralstonine 

 [31]. The structure of (+)-macralstonine 

24 was confirmed by various NMR spectroscopy, mass spectrometry, and X-ray crystallography [28]. The (+)-macralstonine 

 was confirmed by various NMR spectroscopy, mass spectrometry, and X-ray crystallography [33]. The (+)-macralstonine 

24

-related bisindole, (+)-

O

-acetylmacralstonine 

25,

 was isolated from the leaves, stem-bark, and root-bark extracts of 

A. scholaris, A. glaucescens, and A. macrophylla [14]. Also, (+)-

 [20]. Also, (+)-

O-

methylmacralstonine 

26

 was isolated from the leaves, stem-bark, and root-bark of 

A. scholaris, A. glaucescens, and A. macrophylla extracts

(−) Alstonisidine 
3
A. muelleriana Bark [15][16][21,22]
(−)-Angustilongine E 6 A. penangiana Stem-bark [18][5]
(−)-Angustilongine F 7 A. penangiana Stem-bark [18][5]
(+)-Angustilongine G 8 A. penangiana Stem-bark [18][5]
(+)-Angustilongine H 9 A. penangiana Stem-bark [18][5]
(+)-Angustilongine J 10 A. penangiana Stem-bark [18][5]
(+)-Angustilongine K 11 A. penangiana Stem-bark [18][5]
(−)-Angustilongine L 12 A. penangiana Stem-bark [18][5]
(−)-Anhydromacralstonine 27 A. angustifolia Stem-bark [23][28]
(+)-Des-N’a-Methylanhydromacralstonine 30 A. muelleriana Bark [14][20]
A. angustifolia Stem-bark [28][33]
A. muelleriana Leaves, stem-bark and root-bark [14][30][20,35]
(−)-Lumusidine A 14 A. macrophylla Stem-bark [12][18]
(−)-Lumusidine B 15 A. macrophylla Stem-bark [12][18]
(−)-Lumusidine C 16 A. macrophylla Stem-bark [12][18]
(−)-Lumusidine D 17 A. macrophylla Stem-bark [12][18]
(+)-Lumutinine A 18 A. macrophylla Stem-bark [21][26]
(−)-Lumutinine B 19 A. macrophylla Stem-bark [21][26]
(+)-Lumutinine C 20 A. macrophylla Stem-bark [21][26]
(+)-Lumutinine D 19 A. macrophylla Stem-bark [21][26]
(+)-Lumutinine E 21 A. angustifolia Stem-bark [23][28]
(+)-Macralstonidine 23 A. macrophylla Bark [24][25][29,30]
A. somersentenis Bark [24][29]
N
-oxide 
46
A. angustifolia
Leaves
[
36
]
[
41
]
Villalstonine 

N

(4)-oxide 

44

 was isolated from the stem-bark of 

A. angustifolia [23]. It was also isolated from the leaves, stem-bark, and root-bark of 

 [28]. It was also isolated from the leaves, stem-bark, and root-bark of 

A. scholaris, A. glaucescens, and A. macrophylla extracts [14]. Moreover, two villalstonine 

 extracts [20]. Moreover, two villalstonine 

44

-related bisindoles, (+)-10-methoxy villalstonine 

45

 and 10-methoxy villalstonine 

N

(4)-oxide 

46

, were isolated from the leaves of 

A. angustifolia [36]. Villalstonidine A 

 [41]. Villalstonidine A 

47

, villalstonidine B 

48

, villalstonidine C 

49

, villalstonidine D 

50

, villalstonidine E 

51

, (+)-villalstonidine F 

52

, and villalstonine 

N

(4)-oxide 

44

 are macroline–pleiocarpamine-type bisindoles and are close in structure to villalstonine 

43

. Villalstonidines A–D (

47–50)

 were isolated from the stem-bark of 

A. angustifolia [23]. Additionally, (+)-villalstonidine F 

 [28]. Additionally, (+)-villalstonidine F 

52

 {

N

(1)-demethylderivative of villalstonine 

43

} and (+)-villalstonidine B 

48

 were isolated from the stem-bark of 

A. macrophylla [22].

 [27].

3. Bioactivity of Bisindoles from Alstonia Species

Studies from various groups have shown that bisindoles have anticancer activity in different cell lines, including  vincristine-resistant KB/VJ300 cells. Bisindoles are also reported to have other biological activities, including antiprotozoal activity against

 

Studies from various groups have shown that bisindoles have anticancer activity in different cell lines, including  vincristine-resistant KB/VJ300 cells. Bisindoles are also reported to have other biological activities, including antiprotozoal activity against

Plasmodium falciparum

 and antileishmanial activity against promastigotes of 

Entamoeba histolytica

. The reported biological activity of bisindoles from various 

Alstonia

 species including the semi-synthetic derivatives reviewed herein are listed in 

Table 3

. (+)-Alstomacroline 

1

 and (+)-alstomacrophylline 

2

 bisindoles were active against the K1 (multi-drug resistant) strain of 

P. falciparum

 with an IC

50

 1.12 ± 0.35 μM and IC

50 1.10 ± 0.30 μM, respectively [37]. Newly isolated macroline-sarpagine-type bisindoles, (−)-angustilongines E 

 1.10 ± 0.30 μM, respectively [42]. Newly isolated macroline-sarpagine-type bisindoles, (−)-angustilongines E 

6

, (-)-angustilongine F 

7

, (+)-angustilongine G 

8

, (+)-angustilongine H 

9

, (+)-angustilongine J 

10

, and (+)-angustilongine K 

11

 showed

in-vitro

growth inhibitory activity against human cancer cell lines, inclusive of KB, vincristine-resistant strains of KB, HCCT 116, PC-3, MDA-MB-231, LNCaP, MCF7, HT-29, and A549 cells with IC

50 values ranging from 0.02 to 9.0 μM in the study from the group of Kam [18]. Kam et al. [12] also reported the anticancer activity of (−)-lumusidine A 

 values ranging from 0.02 to 9.0 μM in the study from the group of Kam [5]. Kam et al. [18] also reported the anticancer activity of (−)-lumusidine A 

14

, (−)-lumusidine B 

15

, (−)-lumusidine C 

16

, and (−)-lumusidine D 

17

. These bisindoles were cytotoxic against KB/VJ300 cells ranging from IC

50 0.16 to 5.03 μg/mL (μM) values with 0.12 μM vincristine added [12]. Lumutinine A 

 0.16 to 5.03 μg/mL (μM) values with 0.12 μM vincristine added [18]. Lumutinine A 

18

, lumutinine B 

19

, lumutinine C 

20

, lumutinine D 

21

, and lumutinine E 

22

 exhibited moderate anticancer activity against KB/VJ300 cells ranging in IC

50 value from 0.10 to 4.61 μg/mL (μM) again with 0.12 μM vincristine added in the studies from the same group (Kam) [12].

 value from 0.10 to 4.61 μg/mL (μM) again with 0.12 μM vincristine added in the studies from the same group (Kam) [18].

Table 3.

Bioactivity of bisindoles (including semisynthetic derivatives) from

Alstonia

 species.

Bisindoles

Bioactivity

 References
(+)-Alstomacroline 1 Antimalarial, with IC50 values of 1.12 ± 0.35 and 10.0 ± 0.4 μM against the K1 strain and T9-96 strain of P. falciparum, respectively. [14][20]
(+)-Alstomacrophylline 2 Antimalarial, with an IC50 value of 1.10 ± 0.30 μM against the K1 strain of P. falciparum. [14][20]
Angustilongines E, F, G, H, J, and K (6–11) Anticancer, cytotoxic against various human cancer cell lines including KB, vincristine-resistant KB, HCCT 116, PC-3, MDA-MB-231, LNCaP, MCF7, HT-29, and A549 cells with IC50 values ranging from 0.02 to 9.0 μM. [18][5]
(−)-Lumusidine A, B, and C (14–16) Anticancer, moderately cytotoxic in KB/VJ300 cells with IC50 values of 0.16, 0.70, and 1.19 μg/mL (μM), respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(−)-Lumusidine D 17 Anticancer, cytotoxic in KB/VJ300 cells with an

IC50 value of 5.03 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth.		 [12][18]
Lumutinine A, B, C, D, and E

(18–22)		 Anticancer, moderately cytotoxic in KB/VJ300 cells with IC50 0.21, 0.10, 4.61, 3.93, and 2.74 μg/mL (μM) values, respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(+)-Macralstonine 24 Anticancer, strongly cytotoxic in KB/VJ300 cells with an IC50 1.71 μg/mL (μM) value. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
Antimalarial, active against the K1 strain of P. falciparum with an IC50 8.92 ± 2.95 μM value. [14][20]
(−)-Anhydromacralstonine 27 Anticancer, moderately cytotoxic in KB/VJ300 cells with

an IC50 value of 0.44 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth.		 [12][18]
(+)-O-Acetyl macralstonine 25 Antimalarial, with IC50 values 0.53 ± 0.09 and 12.4 ± 1.6 (μM) against the K1 strain and T9-96 strain of P. falciparum, respectively. [14][20]
(+)-O-Methyl macralstonine 26 Antimalarial, active against the K1 strain of P. falciparum with an IC50 0.85 ± 0.20 μM value. [14][20]
O-Acetyl-E-seco-macralstonine 53 Anticancer, strongly cytotoxic in KB/VJ300 cells with an IC50 value of 0.27 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(−)-Perhentidine A 36 and (-)-perhentidine B 37 Anticancer, strongly cytotoxic in KB/VJ300 cells with IC50 values of 2.29 and 0.84 μg/mL (μM), respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(−)-O-Acetylperhentidine A 54 and (-)-O-Acetylperhentidine B 55 Anticancer, strongly cytotoxic in KB/VJ300 cells with IC50 0.36 and 0.28 μg/mL (μM) values, respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(−)-Perhentinine 39 and O-Acetylperhentinine 56 Anticancer, cytotoxic in KB/VJ300 cells with

IC50 values of 0.52 and 0.30 μg/mL (μM), respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth.		 [12][18]
(+)-Macralstonidine 23 Anticancer, moderately cytotoxic in KB/VJ300 cells with an IC50 value of 0.13 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(+)-Macrocarpamine 21 Anticancer, strongly cytotoxic in KB/VJ300 cells with an IC50 value of 0.53 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
Villalstonine 
N
(4)-oxide 
44
Antileishmanial activity against promastigotes of L. mexicana with an IC50 value of 80.3 μM (amphotericin B was used as the positive control). [34][39]
Antimalarial, active against the K1 strain of P. falciparum with an IC50 10.7 ± 1.9 (μM) value. [14][20]
(+)-Villalstonidine B 48 and

(+)-villalstonidine F 52		 Anticancer, strongly cytotoxic in KB/VJ300 cells with IC50 values of 0.35 and 5.64 μg/mL (μM), respectively. The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
(+)-Villalstodinine D 50 Antileishmanial, active against promastigotes of L. mexicana with an IC50 value of 120.4 μM (amphotericin B was used as the positive control). [34][
A. spectabilis Bark [26][31]
(+)-Macralstonine 24 A. scholaris, A. glaucescens, and A. macrophylla extracts
Strong antimalarial activity against the K1 strain of P. falciparum with an IC50 value of 0.36 ± 0.06 μM.

Active against the T9-96 strain of P. falciparum with an IC50 >39 μM value.		 [14][20] Leaves, stem-bark and root-bark [13][19]
(+)-O-Acetylmacralstonine 25                                      A. macrophylla [24
  Strong antiprotozoal activity in vitro against E. histolytica and P. falciparum with ED50 8.12 (95% C.I.) μM and ED50 9.36 (95% C.I.) μM values, respectively. [38][43]
(+)-Villalstonine 43 Anticancer, cytotoxic in KB/VJ300 cells with an IC50 value of 0.42 μg/mL (μM). The assay with 0.12 μM added vincristine did not influence KB/VJ300 cell growth. [12][18]
Anticancer, cytotoxic against the HT-29 cell line with an ED50 8.0 μM value (paclitaxel was used as the positive control). [34][39]
Antimalarial, with IC50 values of 0.27 ± 0.06 and 0.94 ± 0.07 μM against the K1 strain and T9-96 strain of P. falciparum, respectively. [14][20]
Antiamoebic activity against E. histolytica with an ED50 of 2.04 μM. [38][43]
39]
(+)-Villalstonidine E 51 Anticancer, cytotoxic against HT-29 cell lines with an ED50 6.5 μM value (paclitaxel was used as the positive control). [34][39]
][25][27]
Antileishmanial against promastigotes of L. mexicana with an IC50
[28][29][29,30,32,33,34]
 78 μM value (amphotericin B was used as the positive control). [34][39]
A. angustifolia [31][36]
A. muelleriana [30][35]
A. glabriflora Mgf. [26][31]
  A. scholaris, A. glaucescens, and A. macrophylla extracts [14][20]
(+)-O-Methylmacralstonine 26 A. scholaris, A. glaucescens, and A. macrophylla extracts [14][20]
(+)-Macrocarpamine 31 A. scholaris, A. glaucescens, and A. macrophylla extracts Leaves, stem-bark, and root-bark [14][20]
  A. angustifolia Stem-bark [23][28]
10-Methoxy macrocarpamine 34 A. angustifolia Leaves [36][41]
10-Methoxy macrocarpamine 4′-N-oxide 35 A. angustifolia Leaves [36][41]
(−)-Perhentidine A 36 A. macrophylla and A. angustifolia Stem-bark [23][28][28,33]
(−)-Perhentidine B 37 A. macrophylla and A. angustifolia Stem-bark [28][33]
(−)-Perhentidine C 38 A. macrophylla and A. angustifolia Stem-bark [23][28][28,33]
(−)-Perhentinine 39 A. macrophylla Stem-bark [12][18]
A. angustifolia Leaves [12][18]
A. macrophylla Leaves [12][18]
(+)-Perhentisine A 40 A. angustifolia Stem-bark [23][28]
(−)-Perhentisine B 41 A. angustifolia Stem-bark [23][28]
(+)-Perhentisine C 42 A. angustifolia Stem-bark [23][28]
  A. muelleriana Leaves and stem-bark [32][37]
(+)-Villalstonidine A 47 A. angustifolia Stem-bark [23][28]
(+)-Villalstonidine B 48 A. angustifolia Stem-bark [23][28]
A. macrophylla Stem-bark [22][27]
(+)-Villalstonidine C 49 A. angustifolia Stem-bark [23][28]
(+)-Villalstonidine D 50 A. angustifolia Stem-bark [23][28]
(+)-Villalstonidine E 51 A. angustifolia Stem-bark [23][28]
(+)-Villalstonidine F 52 A. macrophylla Stem-bark [22][27]
(+)-Villalstonine 43 A. angustifolia Leaves and stem-bark [23][31][34][28[,3636,39],41]
A. macrophylla [36][41]
A. villosa [36][41]
A. verticillosa [36][41]
A. somersentensis [36][41]
Villalstonine N(4)-oxide 44 A. angustifolia Stem-bark [23][28]
A. scholaris, A. glaucescens, and A. macrophylla extracts Leaves, stem-bark, and root-bark [14][25][27][33][20,30,32,38]
(+)-10-Methoxy villalstonine 45 A. angustifolia Leaves [36][41]
10-Methoxy villalstonine 4′-
(+)-Macralstonine 

24

 was active against the K1 strain of 

P. falciparum

 (IC

50

 8.92 ± 2.95 μM). Notably, the derivatives of (+)-macralstonine 

24

 were more active than (+)-macralstonine 

24

 itself. (+)-

O

-methyl macralstonine 

26

 and (+)-

O-

acetyl macralstonine 

25

 demonstrated more potent activity against the K1 strain of 

P. falciparum

 with IC

50

 values of 0.85 ± 0.20 μM and IC

50 0.53 ± 0.09 μM, respectively [14]. Likely, the functionalization facilitated the transportation of these bisindoles through the cell membranes of parasites and red blood cells, which would have enhanced the activity as lipophilicity rose [14]. (+)-

 0.53 ± 0.09 μM, respectively [20]. Likely, the functionalization facilitated the transportation of these bisindoles through the cell membranes of parasites and red blood cells, which would have enhanced the activity as lipophilicity rose [20]. (+)-

O

-acetyl macralstonine 

25

 and (+)-alstomacroline 

1

 were also somewhat active against the T9-96 strain of 

P. falciparum

 with IC

50 values of 12.4 and 10.2 μM, respectively [14]. Heterodimeric alkaloid (−)-anhydromacralstonine 

 values of 12.4 and 10.2 μM, respectively [20]. Heterodimeric alkaloid (−)-anhydromacralstonine 

27

 showed moderate cytotoxicity (IC

50 value of 0.44 μg/mL (μM) in KB/VJ300 cells with 0.12 μM of vincristine added [12]). Another semisynthetic analog of macralstonine 

 value of 0.44 μg/mL (μM) in KB/VJ300 cells with 0.12 μM of vincristine added [18]). Another semisynthetic analog of macralstonine 

24

, the related 

O

-acetyl-

E-seco

-macralstonine 

53

, showed strong anticancer activity. It was prepared by the reaction of macralstonine 

24 with acetic anhydride/pyridine in DCM [12]. It demonstrated potent activity against vincristine-resistant KB/VJ300 cells with an IC

 with acetic anhydride/pyridine in DCM [18]. It demonstrated potent activity against vincristine-resistant KB/VJ300 cells with an IC

50 value of 0.27 μg/mL (μM), with 0.12 μM of vincristine added to the assay [12]. (+)-Macralstonidine 

 value of 0.27 μg/mL (μM), with 0.12 μM of vincristine added to the assay [18]. (+)-Macralstonidine 

23

 was found to exhibit moderately active anticancer activity. It was active against KB/VJ300 cells with an IC

50 value of 0.13 μg/mL (μM) [12].

 value of 0.13 μg/mL (μM) [18].
(−)-Macrocarpamine 

31

 exhibited antiprotozoal and anticancer activity in various studies. It showed significant antiprotozoal activity in vitro in studies from Wright et al. against 

E. histolytica 

and

 P. falciparum

 with ED

50 values of 8.12 (95% C.I.) μM and 9.36 (95% C.I.) μM, respectively [38]. Keawpradub et al. reported significant activity of (−)-macrocarpamine 

 values of 8.12 (95% C.I.) μM and 9.36 (95% C.I.) μM, respectively [43]. Keawpradub et al. reported significant activity of (−)-macrocarpamine 

31

 against the K1 strain of 

P. falciparum

 with an IC

50 value of 0.36 μM [14]. Furthermore, (−)-macrocarpamine 

 value of 0.36 μM [20]. Furthermore, (−)-macrocarpamine 

31

 showed antimalarial activity against the T9-96 strain of 

P. falciparum

 with an IC

50

 > 39 μM. The ancient folklore use of the extracts from 

A. angustifolia in Malaya for treatment of malaria and dysentery is supported by these in vitro studies [39]. (−)-Macrocarpamine 

 in Malaya for treatment of malaria and dysentery is supported by these in vitro studies [44]. (−)-Macrocarpamine 

31

 was strongly cytotoxic in KB/VJ300 cells with an IC

50 value of 0.53 μg/mL (μM) [12]. (−)-Perhentidine A 

 value of 0.53 μg/mL (μM) [18]. (−)-Perhentidine A 

36

 and (−)-perhentidine B 

37

 showed strong cytotoxicity against KB/VJ300 cells with IC

50 values of 2.29 and 0.84 μg/mL (μM), respectively [12]. 

 values of 2.29 and 0.84 μg/mL (μM), respectively [18]. 

O

-Acetylperhentidine A 

54

 and 

O

-acetylperhentidine B 

55

 also exhibited strong cytotoxicity against KB/VJ300 cells with IC

50 values of 0.36 and 0.28 μg/mL (μM), respectively [12]. (−)-Perhentidine A 

 values of 0.36 and 0.28 μg/mL (μM), respectively [18]. (−)-Perhentidine A 

36

 and (−)-perhentidine B 

37

 were treated individually by dropwise addition of acetic anhydride in a py/DCM solution, followed by stirring at room temperature for 2 h to furnish the semisynthetic (−)-

O

-acetylperhentidine A 

54

, and (−)-

O

-acetylperhentidine B 

55, respectively [12]. (−)-Perhentinine 

, respectively [18]. (−)-Perhentinine 

39

 and 

O

-acetylperhentinine 

56

 were cytotoxic against KB/VJ300 cells with IC

50 values of 0.52 and 0.30 μg/mL (μM), respectively, in the studies by Kam et al. [12].

 values of 0.52 and 0.30 μg/mL (μM), respectively, in the studies by Kam et al. [18].
(+)-Villalstonine 

43

 has demonstrated various biological activities including anticancer, antimalarial, and antiamoebic activity. (+)-Villastonine 

43 was 1/15th as potent as chloroquine (antimalarial drug) against malaria [40]. It exhibited potent antiplasmodial activity against the multidrug-resistant K1 strain of 

 was 1/15th as potent as chloroquine (antimalarial drug) against malaria [45]. It exhibited potent antiplasmodial activity against the multidrug-resistant K1 strain of 

P. falciparum

 with an IC

50 value of 0.27 μM [14]. Wright et al. tested this compound for antiamoebic activity against 

 value of 0.27 μM [20]. Wright et al. tested this compound for antiamoebic activity against 

E. histolytica [38]. (+)-Villalstonine 

 [43]. (+)-Villalstonine 

43

 showed six times less activity (ED

50

 11.8 μM) than the antiamoebic drug emetine (ED

50

 2.04 μM). These results also concur with the use of various parts of the 

A. angustifolia plant from ancient times to treat malaria and amoebic dysentery [38]. Moreover, (+)-villalstonine 

 plant from ancient times to treat malaria and amoebic dysentery [43]. Moreover, (+)-villalstonine 

43

 was cytotoxic against KB cells with an ED

50 value of 11.6 μM [38]. (+)-Villalstonine 

 value of 11.6 μM [43]. (+)-Villalstonine 

43

 was also potent against the T9-96 strain of 

P. falciparum

 with an IC

50

 value of 0.94 μM. It also showed anticancer activity against the HT-29 cell line with an ED

50

 value of 8.0 μM (paclitaxel was the positive control). Also, it was cytotoxic against vincristine-resistant KB/VJ300 cells with an IC

50 value of 0.42 μg/mL (μM) [12]. On the other hand, the derivative of (+)-villalstonine 

 value of 0.42 μg/mL (μM) [18]. On the other hand, the derivative of (+)-villalstonine 

43

, villalstonine 

N(4)

-oxide 

44

 was less potent (IC

50

 10.7 ± 1.9) than (+)-villalstonine 

43

 itself. The increase in the ionic charge (decrease in lipophilicity) might have reduced the ability of villalstonine 

N(4)

-oxide 

44 to cross through the cell membranes of red blood cells or the parasites, which if correct, would explain the weaker activity [14]. Villalstonine 

 to cross through the cell membranes of red blood cells or the parasites, which if correct, would explain the weaker activity [20]. Villalstonine 

N

(4)-oxide 

44

 is also an antileishmanial bisindole. It was active against promastigotes of 

Leishmania mexicana

 with an IC

50 value of 80.3 μM [34]. (+)-Villalstonine 

 value of 80.3 μM [39]. (+)-Villalstonine 

43

-related alkaloids (+)-villalstonidine B 

48

 and (+)-villalstonidine F 

52

 were found to be strongly cytotoxic against KB/VJ300 cells with IC

50 values of 0.35 and 5.64 μg/mL (μM), respectively [12]. (+)-Villalstonidine D 

 values of 0.35 and 5.64 μg/mL (μM), respectively [18]. (+)-Villalstonidine D 

50

 exhibited antileishmanial activity. It was active against promastigotes of 

L. mexicana

 with an IC

50 value of 120.4 μM in a study from Pan et al. [34]. Another (+)-villalstonine 

 value of 120.4 μM in a study from Pan et al. [39]. Another (+)-villalstonine 

43

-related alkaloid, (+)-villalstonidine E 

51,

 demonstrated anticancer and antimalarial activity. It was cytotoxic against the HT-29 cell lines with an ED

50

 value of 6.5 μM and was active against promastigotes of 

L. mexicana

 with an IC

50 value of 78 μM in the study from the same group [34]. Many bisindoles are yet to be screened for their activity because of the paucity of material; however, their significant role in future drug discovery should be considered.

 value of 78 μM in the study from the same group [39]. Many bisindoles are yet to be screened for their activity because of the paucity of material; however, their significant role in future drug discovery should be considered.
 

45. Conclusions

 

Many

Alstonia

species are rich in indole alkaloids including bisindoles. Bisindole alkaloids including semisynthetic derivatives have been found to possess significant bioactivity, including anticancer, antileishmanial, and antimalarial properties, and thus are promising leads for nature-inspired drug discovery and development. Unnatural medicinal compounds formed by combining bioactive mismatched monomeric units can furnish novel medicinal compounds. Incorporating unnatural enantiomers of monomeric alkaloids into bisindoles can provide access to novel and unnatural bioactive compounds that may have better activity and in vivo stability depending on their metabolism. Several  bisindoles along with their corresponding monomeric units  has been successfully synthesized (please visit

https://doi.org/10.3390/molecules26113459  for details) however; many bisindoles still await their total synthesis as well as biological screening.

  for details) however; many bisindoles still await their total synthesis as well as biological screening.
 
 
 
 
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