Indole-Containing Natural Products 2019–2022: Isolations, Reappraisals, Syntheses, and Biological Activities

Indole alkaloids represent a large subset of natural products, with more than 4100 known compounds. The majority of these alkaloids are biologically active, with some exhibiting excellent antitumor, antibacterial, antiviral, antifungal, and antiplasmodial activities. Consequently, the natural products of this class have attracted considerable attention as potential leads for novel therapeutics and are routinely isolated, characterized, and profiled to gauge their biological potential. However, data on indole alkaloids, their various structures, and bioactivities are complex due to their diverse sources, such as plants, fungi, bacteria, sponges, tunicates, and bryozoans; thus, isolation methods produce an incredible trove of information. The situation is exacerbated when synthetic derivatives, as well as their structures, bioactivities, and synthetic schemes, are considered. Thus, to make such data comprehensive and inform researchers about the current field’s state, this review summarizes recent reports on novel indole alkaloids. It deals with the isolation and characterization of 250 novel indole alkaloids, a reappraisal of previously reported compounds, and total syntheses of indole alkaloids. In addition, several syntheses and semi-syntheses of indole-containing derivatives and their bioactivities are reported between January 2019 and July 2022.


Introduction
Despite the decreasing interest of modern pharmaceuticals in pursuing natural products as leads for new medicine [1], natural products and their derivatives still represent a significant fraction of approved drugs, with more than 22 compounds in the WHO list of essential medicine being sourced exclusively from flowering plants [2]. Furthermore, approximately 40% of all available medicine is either a natural product or a semi-synthetic derivative [3,4]. This ubiquity of natural products in medicine can be explained by their inherent nature as secondary metabolites. These compounds have been fine-tuned over millennia, acquiring biological functions to boost their host's survivability [5]. Thus, these compounds can regulate endogenous defense mechanisms and interactions with other organisms, explaining their potency as antiviral, antibacterial, and antitumor agents [6]. Natural products also represent desired targets in synthesis due to their structural complexity or potent bioactivity.

Isolation Methods
The isolation methods generally included using organic solvents (dichloromethane, ethanol, ethyl acetate, or methanol) to extract a crude mixture of alkaloids directly from the source (bacteria, fungi, dried plant material, etc.). This mixture was then concentrated and resuspended in acidic media (acidified using HCl, H 2 SO 4 , or tartaric acid). Next, the organic layer containing fats and other dissolved material was removed, while the aqueous layer was isolated and basified using NH 3 , NH 4 OH, or NaOH. Subsequently, the organic layer was concentrated and purified by column chromatography, affording pure alkaloids. Extracted with CH 3 OH, and acidified using saturated tartaric acid (pH = 2-3); extracted with EtOAc; aq. phase basified using saturated NaOH (pH = 9-10) and extracted with DCM [55] 196 to 200 Fusarium Extracted with EtOAc [56] 201 to 204 Beauveria Extracted with EtOAc [57] 205 to 209 Gelsemium elegans Extracted with 95% EtOH; suspended in water, acidified using 0.3 M HCl (pH = 3), and partitioned with CHCl 3 ; aq. phase basified using NH3 soln.

Structure Reappraisals
Due to the stereochemical richness of natural products and structural complexity, extensive spectroscopic analyses are required to elucidate their complete structures. Occasionally, some fragments within the compounds are misidentified, possibly due to a lack of spectroscopic data resulting from insufficient quantities of the compound to analyze. Later, due to contradictory results (often emerging from the synthesis of homologs or the analysis of their decomposition products), the postulated structures of compounds are reevaluated, and the errors in the initially proposed structures are rectified.
Cottoquinazolines E-G (251 to 253) were isolated from the fungus Neosartorya fischeri, found in the medicinal arthropod Cryptotympana atrata [73]. The fumiquinazoline-type alkaloids were reinvestigated due to unresolved absolute configuration of compounds 251 and 252. Extensive spectroscopic analysis, electronic circular dichroism data, and X-ray crystallography confirmed the absolute configurations of all three indole alkaloids ( Figure 12). Cottoquinazoline E had two posited steric conformations: cottoquinazoline E (a) with C-3R, C-14R, and C-16S configurations or cottoquinazoline E (b) with C-3S, C-14S, and C-16R configurations. In addition, spectroscopic studies confirmed the structure of 251 and its configurations (C-3S, C-14S, and C-16S). Similarly, the absolute configurations of 252 were changed from C-3R and C-14R to C-3S and C-14S, and 253 was modified from C-16R to C-16S [74].
Cottoquinazolines E-G (251 to 253) were isolated from the fungus Neosartorya fischeri found in the medicinal arthropod Cryptotympana atrata [73]. The fumiquinazoline-type al kaloids were reinvestigated due to unresolved absolute configuration of compounds 251 and 252. Extensive spectroscopic analysis, electronic circular dichroism data, and X-ray crystallography confirmed the absolute configurations of all three indole alkaloids ( Figure  12). Cottoquinazoline E had two posited steric conformations: cottoquinazoline E (a) with C-3R, C-14R, and C-16S configurations or cottoquinazoline E (b) with C-3S, C-14S, and C 16R configurations. In addition, spectroscopic studies confirmed the structure of 251 and its configurations (C-3S, C-14S, and C-16S). Similarly, the absolute configurations of 252 were changed from C-3R and C-14R to C-3S and C-14S, and 253 was modified from C-16R to C-16S [74].  Echinosulfone A (254) and echinosulfonic acids A-C (255-257) were initially isolated from Echinodictyum, a marine sponge [75]. Similarly, echinosulfonic acid D (258) was isolated from a Psammoclema sponge, and its structure was assigned on the basis of the previous report [76]. However, discrepancies arose when decomposition products of 254 were analyzed using X-ray diffraction. Thus, the original alkaloids were reisolated from sponges in the Crella genus. Their structures were re-evaluated using 1D and 2D NMR data in conjunction with MS fragmentation, X-ray crystallography, and DFT predictions of 13 C-NMR shifts. For compound 254, the original sulfonyl bridge between the two indole monomers was modified to a carbonyl bridge, while the N-1-bonded carboxylic acid was replaced with an N-1-bonded sulfonic acid ( Figure 11). Similar changes were made in compounds 255 to 258, where the C1 -bonded sulfonic acid was changed with the C1bonded methoxycarbonyl group. The N-1-bonded methoxycarbonyl was replaced with an N-1-bonded sulfonic acid moiety ( Figure 13) [77]. data in conjunction with MS fragmentation, X-ray crystallography, and DFT predictions of 13 C-NMR shifts. For compound 254, the original sulfonyl bridge between the two indole monomers was modified to a carbonyl bridge, while the N-1-bonded carboxylic acid was replaced with an N-1-bonded sulfonic acid ( Figure 11). Similar changes were made in compounds 255 to 258, where the C1ʹʹ-bonded sulfonic acid was changed with the C1ʹʹbonded methoxycarbonyl group. The N-1-bonded methoxycarbonyl was replaced with an N-1-bonded sulfonic acid moiety ( Figure 13) [77]. Protoaculeine B (259) is the N-terminal residue of aculeine B, a peptide toxin that is a potent membrane disruptor [78]. Aculeines are found in the aqueous extracts of Axinyssa aculeata, a marine sponge. Aculeine B consists of a 44 amino-acid long sequence (containing three disulfide bonds), connected to a long-chain polyamine (LCPA; consisting of a 1,3-propanediamine oligomer) by a tryptophan-derived amino acid (259). A homolog of 259 was synthesized, but its spectroscopic and chemical properties differed significantly from its natural counterpart. Thus, the structure of compound 259 was re-evaluated, and its C-3-bonded amino-acid chain was modified into a fused pyridine-2-carboxylic acid ring bonded to the LCPA via a propane group ( Figure 14) [79]. Lyaline (260) was isolated from Pauridiantha paucinervis, and was reported as an unusual harman-1,4-dihydropyridine derivative [80]. However, efforts to synthesize the monoterpene indole alkaloid resulted in discrepancies (the synthetic product was extremely unstable; nevertheless, the natural analog did not show any degradation) [81]. Thus, the absolute configuration of C-15 was elucidated as S, and a single bond between C-21 and N-1 was added. As Δ20 from the original structure was reduced, it was revised Protoaculeine B (259) is the N-terminal residue of aculeine B, a peptide toxin that is a potent membrane disruptor [78]. Aculeines are found in the aqueous extracts of Axinyssa aculeata, a marine sponge. Aculeine B consists of a 44 amino-acid long sequence (containing three disulfide bonds), connected to a long-chain polyamine (LCPA; consisting of a 1,3propanediamine oligomer) by a tryptophan-derived amino acid (259). A homolog of 259 was synthesized, but its spectroscopic and chemical properties differed significantly from its natural counterpart. Thus, the structure of compound 259 was re-evaluated, and its C-3-bonded amino-acid chain was modified into a fused pyridine-2-carboxylic acid ring bonded to the LCPA via a propane group ( Figure 14) [79].
compounds 255 to 258, where the C1ʹʹ-bonded sulfonic acid was changed with the C1ʹʹ bonded methoxycarbonyl group. The N-1-bonded methoxycarbonyl was replaced with an N-1-bonded sulfonic acid moiety ( Figure 13) [77]. Protoaculeine B (259) is the N-terminal residue of aculeine B, a peptide toxin that is a potent membrane disruptor [78]. Aculeines are found in the aqueous extracts of Axinyssa aculeata, a marine sponge. Aculeine B consists of a 44 amino-acid long sequence (contain ing three disulfide bonds), connected to a long-chain polyamine (LCPA; consisting of a 1,3-propanediamine oligomer) by a tryptophan-derived amino acid (259). A homolog o 259 was synthesized, but its spectroscopic and chemical properties differed significantly from its natural counterpart. Thus, the structure of compound 259 was re-evaluated, and its C-3-bonded amino-acid chain was modified into a fused pyridine-2-carboxylic acid ring bonded to the LCPA via a propane group ( Figure 14) [79]. Lyaline (260) was isolated from Pauridiantha paucinervis, and was reported as an un usual harman-1,4-dihydropyridine derivative [80]. However, efforts to synthesize the monoterpene indole alkaloid resulted in discrepancies (the synthetic product was ex tremely unstable; nevertheless, the natural analog did not show any degradation) [81] Thus, the absolute configuration of C-15 was elucidated as S, and a single bond between C-21 and N-1 was added. As Δ20 from the original structure was reduced, it was revised Lyaline (260) was isolated from Pauridiantha paucinervis, and was reported as an unusual harman-1,4-dihydropyridine derivative [80]. However, efforts to synthesize the monoterpene indole alkaloid resulted in discrepancies (the synthetic product was extremely unstable; nevertheless, the natural analog did not show any degradation) [81]. Thus, the absolute configuration of C-15 was elucidated as S, and a single bond between C-21 and N-1 was added. As ∆20 from the original structure was reduced, it was revised as S ( Figure 15). Lastly, considering the revised structure of 260, it was established as the first naturally occurring nacycline analog [82]. as S ( Figure 15). Lastly, considering the revised structure of 260, it was established as the first naturally occurring nacycline analog [82].

Total Synthesis of Indole Alkaloids
(±)-Conolidine (269), a potent nonopioid analgesic, was synthesized in six steps without any nonstrategic redox manipulations. It was isolated from the stem bark of Tabernae-

Total Synthesis of Indole Alkaloids
(±)-Conolidine (269), a potent nonopioid analgesic, was synthesized in six steps without any nonstrategic redox manipulations. It was isolated from the stem bark of Tabernaemontana divaricate in 2004 [83], and its first total synthesis was carried out in 2011 with a nine-step synthetic route in an 18% overall yield [84]. The latest total synthesis attempts primarily utilized gold(I)-catalyzed Conia-ene and Pictet-Spengler reactions, producing 269 in six steps in overall 19% yield ( Figure 16). They also used DFT calculations to develop a successful scheme [85].

Total Synthesis of Indole Alkaloids
(±)-Conolidine (269), a potent nonopioid analgesic, was synthesized in six steps without any nonstrategic redox manipulations. It was isolated from the stem bark of Tabernaemontana divaricate in 2004 [83], and its first total synthesis was carried out in 2011 with a nine-step synthetic route in an 18% overall yield [84]. The latest total synthesis attempts primarily utilized gold(I)-catalyzed Conia-ene and Pictet-Spengler reactions, producing 269 in six steps in overall 19% yield ( Figure 16). They also used DFT calculations to develop a successful scheme [85]. For the total synthesis of compound 269, compound 261 was treated with n-butyllithium and dropwise addition to N-tosylpyrrolidone affording 262 that underwent a nucleophilic reaction with 1-bromo-2-butyne to give compound 263. Next, compound 263 was treated with TBAF to remove the N-1-tosyl group producing compound 264. Afterward, 264 was added to TiPSOTf in 2,6-lutidine, affording a pair of stereoisomers of 265 (E:Z = 8:92). Its gold-catalyzed reaction using [JohnPhosAu(CH3CN)]SbF6 produced For the total synthesis of compound 269, compound 261 was treated with n-butyllithium and dropwise addition to N-tosylpyrrolidone affording 262 that underwent a nucleophilic reaction with 1-bromo-2-butyne to give compound 263. Next, compound 263 was treated with TBAF to remove the N-1-tosyl group producing compound 264. Afterward, 264 was added to TiPSOTf in 2,6-lutidine, affording a pair of stereoisomers of 265 (E:Z = 8:92). Its gold-catalyzed reaction using [JohnPhosAu(CH 3 CN)]SbF6 produced compounds 266 and 267. The latter was exposed to sodium naphthalenide to deprotect its N-2 tosyl group affording 268. Lastly, a Pictet-Spengler reaction using TFA and paraformaldehyde in acetonitrile was used to convert 268 into 269 [85].
Psammocindoles A and B (212 and 213) significantly increased adiponectin production. Thus, to further investigate their pharmacology and assign the absolute configuration of compound 212 at C-2 , the two alkaloids and their congener, psammocindole C (214), were synthesized. In addition, enantiomers of psammocindole D (286), and four N-lactam analogs, isopsammocindoles A-D (287 to 290), were produced. The synthetic scheme was four steps long, and the overall yields were 23%, 21%, and 21% for compounds 212, 213, and 214, respectively ( Figure 17). As a result, the R-configuration of compound 212 at C-2 was assigned [61].
To confirm the structure of amakusamine (233), it was synthesized by the same research group that first isolated it. Upon initial failure of a direct debromination of 5,6methylenedioxyindole, they used an alternative reaction scheme containing three steps and obtained an overall yield of 56% ( Figure 18). The synthetic method also enabled the synthesis of 15 synthetic derivatives of compound 233 (294 to 308) for an SAR analysis ( Figure 19) [66]. The synthesis involved the reaction of 6-nitropiperonal (291) with NBS in concentrated H2SO4, producing 292. A Henry reaction (using Al2O3 as a base and nitromethane) followed by dehydration using acetic anhydride transformed 292 into 293. However, since the latter was crystalline and hard to dissolve in many solvents, its crude mixture was reduced directly by an excess of iron in acetic acid, affording compound 233 [66]. (R)-2-Methylbutan-1-amine was prepared in an 88% enantiomeric excess using a five-step synthetic scheme, while the other amines were obtained commercially. The amines (270 to 273) were reacted with ethyl bromoacetate, affording compounds 274 to 277. Subsequently, further condensation with propionyl chloride produced compounds 278 to 281, which were used to prepare the N-alkyl-α,β-unsaturated γ-lactams (282 to 285) by an intramolecular Claisen condensation. Lastly, condensation between the lactams (at C-3) and the indole's nitrogen afforded compounds 212 to 214 and 286 to 290, with approximately 30% yields for all analogs [61].
To confirm the structure of amakusamine (233), it was synthesized by the same research group that first isolated it. Upon initial failure of a direct debromination of 5,6methylenedioxyindole, they used an alternative reaction scheme containing three steps and obtained an overall yield of 56% ( Figure 18). The synthetic method also enabled the synthesis of 15 synthetic derivatives of compound 233 (294 to 308) for an SAR analysis ( Figure 19) [66]. The synthesis involved the reaction of 6-nitropiperonal (291) with NBS in concentrated H 2 SO 4 , producing 292. A Henry reaction (using Al 2 O 3 as a base and nitromethane) followed by dehydration using acetic anhydride transformed 292 into 293. However, since the latter was crystalline and hard to dissolve in many solvents, its crude mixture was reduced directly by an excess of iron in acetic acid, affording compound 233 [66].   The first total synthesis of griseofamine B (314) and its three stereoisomers, 16-epigriseofamine B, ent-griseofamine B, and 11-epi-griseofamine B (317, 321, and 323), was carried out in 2022. Compound 314 is an indole-tetramic acid alkaloid isolated from the Penicillium griseofulvum fungus in 2018 [86]. In the total synthesis, 4-bromo tryptophan methyl ester hydrochloride was used as the starting reagent, with its l-enantiomer being used to synthesize compounds 314 and 317. d-Enantiomer 4-bromo tryptophan methyl ester hydrochloride was used to synthesize compounds 321 and 323. All four compounds were obtained in five steps with yields of 18%, 5%, 19%, and 5%, respectively ( Figure 20) [87].

Synthesis of Indole Derivatives
As β-carboline derivatives generally display only moderate cytotoxicity, new synthetic derivatives were designed to contain a hydroxycinnamic acid moiety inferring HDACinhibitory properties that provide synergy and improve their antiproliferative effects [88]. These derivatives (347 to 352) differed only in the number of carbons connecting the βcarboline with the hydroxycinnamic acid motif and in the substituents on the phenyl ring in the hydroxycinnamic acid moiety ( Figure 21).
Neopeltolide, a marine natural product, was isolated from a sponge in the neopeltidae family. It is a highly potent antitumor agent (IC 50 < 1 nm) and strongly inhibits cytochrome bc1, an essential component of the mitochondrial respiratory chain [89]. Compounds (404 to 424 and 438 to 443) were synthesized, replacing its macrolactone ring with an indole having potential as a fungicide. The derivatives were synthesized in two series. The first series (404 to 424) contained an ester linkage between the oxazole and indole heterocycles ( Figure 22). The second series (313 to 318), guided through the bioactivities of the compounds in the first series (Table 2) and the docking studies of leads, replaced the linkage with an amide group and contained only fluorine or methoxy substituents ( Figure 22) [90]. The reaction of prop-2-yn-1-amine (353) with NaHCO 3 in 1,4-dioxane affords 354. Carboxylation of 354 using n-butyllithium in THF and a CO 2 atmosphere produced 355 that was reduced to 356 using Lindlar's catalyst. Compound 356 reacted with l-serine methyl ester hydrochloride, N-methylmorpholine, and isobutyl chloroformate to produce 357. Compound 357 was reacted with DAST, DBU, and BrCCl 3 at a low temperature to yield 358, and LiOH hydrolyzed it to give 359. Separately, methyl indole-4carboxylate (360) was treated with N-chlorosuccinimide in acidic conditions, producing 361. It was then reacted with substituted benzyl chlorides and NaH in THF, yielding 362 to 382, which were reduced by DIBAL-H, giving 383 to 403. Lastly, 359 was reacted with each of the 21 intermediates to give derivatives 404 to 424. The second derivatives were synthesized by reacting 4-nitroindole (425) with substituted benzyl bromides and NaH in anhydrous DMF, yielding 426 to 431. They were reduced using iron and NH 4 Cl in EtOH and water, affording 432 to 437. Another time, 359 reacted with each of the six derivatives, producing derivatives of 438 to 443 [90].

Synthesis of Indole Derivatives
As β-carboline derivatives generally display only moderate cytotoxicity, new synthetic derivatives were designed to contain a hydroxycinnamic acid moiety inferring HDAC-inhibitory properties that provide synergy and improve their antiproliferative effects [88]. These derivatives (347 to 352) differed only in the number of carbons connecting (E)-Ferulic acid (324) and p-coumaric acid (325) were esterified using SOCl2 in CH3OH, yielding compounds 326 and 327. These compounds were then treated with ωdibromoalkanes (1,2-dibromoethane, 1,2-dibromopropane, and 1,2-dibromobutane) in the presence of K2CO3 to produce compounds 328 to 333. Using a Pictet-Spengler reaction, l-tryptophan (334) was transformed into 335 with 4-methoxybenzaldehyde. Compound 335 was then esterified using SOCl2 in CH3OH, giving compound 336, which was oxidized using KMnO4 in DMF, affording compound 337. It was then reacted with hydrazine monohydrate to give compound 338 and was converted into 339 using NaNO2. Next, compound 339 underwent a Curtis rearrangement to produce compound 340. Lastly, compounds 328 to 333 were reacted with 340, yielding residues 341 to 346, which were then treated with NH2OK to produce derivatives 347 to 352.
Neopeltolide, a marine natural product, was isolated from a sponge in the neopeltidae family. It is a highly potent antitumor agent (IC50 < 1 nm) and strongly inhibits cytochrome bc1, an essential component of the mitochondrial respiratory chain [89]. Compounds (404 to 424 and 438 to 443) were synthesized, replacing its macrolactone ring with an indole having potential as a fungicide. The derivatives were synthesized in two series. The first series (404 to 424) contained an ester linkage between the oxazole and indole heterocycles ( Figure 22). The second series (313 to 318), guided through the bioactivities of the compounds in the first series (Table 2) and the docking studies of leads, replaced the linkage with an amide group and contained only fluorine or methoxy substituents ( Figure 22) [90]. The reaction of prop-2-yn-1-amine (353) with NaHCO3 in 1,4-dioxane affords 354. Carboxylation of 354 using n-butyllithium in THF and a CO2 atmosphere produced 355 that was reduced to 356 using Lindlar's catalyst. Compound 356 reacted with l-serine methyl ester hydrochloride, N-methylmorpholine, and isobutyl chloroformate to produce 357. Compound 357 was reacted with DAST, DBU, and BrCCl3 at a low temperature to yield 358, and LiOH hydrolyzed it to give 359. Separately, methyl indole-4-carboxylate (360) was treated with N-chlorosuccinimide in acidic conditions, producing 361.  Celastrol (444) is a friedelane-type triterpenoid isolated from Tripterygium wilfordii and possesses immunosuppressive properties [91]. However, it has toxic properties. Therefore, its synthetic derivatives were prepared to obtain lower cytotoxicity. Ten celastrol derivatives (456 to 465) with indole substituents were synthesized that differed only in the substituents attached to the indole group ( Figure 23) [92]. In the synthesis, compound 444 was converted into 445 via a nucleophilic reaction using propargyl bromide and NaHCO3 in DMF at room temperature. Compound 445 was transformed using a Celastrol (444) is a friedelane-type triterpenoid isolated from Tripterygium wilfordii and possesses immunosuppressive properties [91]. However, it has toxic properties. Therefore, its synthetic derivatives were prepared to obtain lower cytotoxicity. Ten celastrol derivatives (456 to 465) with indole substituents were synthesized that differed only in the substituents attached to the indole group ( Figure 23) [92]. In the synthesis, compound 444 was converted into 445 via a nucleophilic reaction using propargyl bromide and NaHCO 3 in DMF at room temperature. Compound 445 was transformed using a Friedel-Crafts reaction using substituted indoles and FeCl 3 ·6H 2 O in DCM, giving compounds 446 to 455. These compounds were acetylated using DMAP and Ac 2 O immediately without purification, yielding derivatives 456 to 465. Phidianidine A is a marine natural product isolated from Phidiana militaris, a mollusk. The natural product and its synthetic analogs possess both cytotoxic and immunosuppressive activities. However, the antifouling properties of its derivatives were not previously explored despite its resemblance with other potent antifouling MNPs. Therefore, 10 synthetic derivatives (479 to 488) having primary amines, guanidines, and a quaternary ammonium compound were produced ( Figure 24) [93]. Initially, the diamines 466 to 469 (propan-1,3-diamine, butan-1,4-diamine, and pentan-1,5-diamine) and 6-aminohexanol (474) were protected using Boc2O. Then, compound 474 was converted into compound 475 using methane sulfonyl chloride and methylamine. Separately, 6-bromoindole (476) was converted into derivative 477 using oxalyl chloride. It was then reduced to compound 478 using hydrazine and sodium methoxide. The derivative 478 was then reacted with the diamines 470 to 473 and 475 and deprotected using TFA, affording the primary amines 479 to 483. Additionally, compounds 479 to 481 and 483 were reacted with DIPEA and 489 to yield guanidine derivatives 484 to 487. Furthermore, compound 479 was reacted with NaBH3CN in formaldehyde and acetic acid, producing quaternary ammonium derivative 488 [93]. Phidianidine A is a marine natural product isolated from Phidiana militaris, a mollusk. The natural product and its synthetic analogs possess both cytotoxic and immunosuppressive activities. However, the antifouling properties of its derivatives were not previously explored despite its resemblance with other potent antifouling MNPs. Therefore, 10 synthetic derivatives (479 to 488) having primary amines, guanidines, and a quaternary ammonium compound were produced ( Figure 24) [93]. Initially, the diamines 466 to 469 (propan-1,3-diamine, butan-1,4-diamine, and pentan-1,5-diamine) and 6-aminohexanol (474) were protected using Boc 2 O. Then, compound 474 was converted into compound 475 using methane sulfonyl chloride and methylamine. Separately, 6-bromoindole (476) was converted into derivative 477 using oxalyl chloride. It was then reduced to compound 478 using hydrazine and sodium methoxide. The derivative 478 was then reacted with the diamines 470 to 473 and 475 and deprotected using TFA, affording the primary amines 479 to 483. Additionally, compounds 479 to 481 and 483 were reacted with DIPEA and 489 to yield guanidine derivatives 484 to 487. Furthermore, compound 479 was reacted with NaBH 3 CN in formaldehyde and acetic acid, producing quaternary ammonium derivative 488 [93].

Semi-Synthesis of Indole Alkaloids
Fradcarbazole A, a novel staurosporine-type indole alkaloid containing a thiazole group, was isolated from a mutant strain of Streptomyces fradiae and semi-synthesized from staurosporine by the same group [94,95]. To enhance its efficacy as an antitumor agent, 14 derivatives (535 to 548) of fradcarbazole A were synthesized by variation only in the substituents on two indole units ( Figure 25) [96]. In the synthesis, 5fluoro/chloro/bromo/methoxy-tryptamines (489 to 493) were protected using Boc 2 O to give derivatives 494 to 498 and oxidized using DDQ, producing compounds 499 to 503. TFA-mediated Boc deprotection resulted in molecules 504 to 508. Staurosporine (509) was protected using Boc 2 O, giving 510, which was halogenated using NCS and NBS (compounds 511 and 512), and deprotected by TFA (compounds 513 and 514). These derivatives and compound 509 were converted to compounds 515 to 517 using TCDI. These com-pounds were alkylated by CH 3 I in CH 3 CN, affording compounds 518 to 520. They were then converted into compounds 521 to 534 via a nucleophilic reaction with 504 to 508. Lastly, intramolecular cyclization reactions of compounds 521 to 534 resulted in 535 to 548 [96].
(propan-1,3-diamine, butan-1,4-diamine, and pentan-1,5-diamine) and 6-aminohexanol (474) were protected using Boc2O. Then, compound 474 was converted into compound 475 using methane sulfonyl chloride and methylamine. Separately, 6-bromoindole (476) was converted into derivative 477 using oxalyl chloride. It was then reduced to compound 478 using hydrazine and sodium methoxide. The derivative 478 was then reacted with the diamines 470 to 473 and 475 and deprotected using TFA, affording the primary amines 479 to 483. Additionally, compounds 479 to 481 and 483 were reacted with DIPEA and 489 to yield guanidine derivatives 484 to 487. Furthermore, compound 479 was reacted with NaBH3CN in formaldehyde and acetic acid, producing quaternary ammonium derivative 488 [93]. (−)-Melodinine K (556), a complex bisindole alkaloid, was semi-synthesized, using (−)-tabersonine as the starting reagent. The aspidosperma-aspidosperma-type alkaloid was isolated from the Melodinus tenuicaudatus plant in 2010. Its cytotoxicity was tested by the same group and was reported to be more potent than cisplatin and vinorelbine in four of the five cancer cell lines [97]. The synthetic route adopted had six steps in its most extended linear sequence (eight steps overall) and afforded compound 556 with a 4% yield ( Figure 26) [98]. While planning its synthesis, 556 was divided into two fragments, both of which could be synthesized from (−)-tabersonine (549). The total synthesis of 549 was accomplished by the same group in 2013 [99]. However, this precursor was mainly isolated from the seeds of Voacanga africana (a small tree). It was bio-transformed into compound 550 using T16H yeast and allylated by allyl bromide and K 2 CO 3 in DMF, resulting in 551, the northern fragment of compound 556. Separately, compound 549 was protected using TrocCl, affording 552, which was further converted to 553 by TFA and m-CPBA. Compound 553 was reacted with m-CPBA in DCM, producing its N-oxide analog 554.
On the basis of the amount of 561 isolated, the overall yield was 28% (Figure 27) [100]. 5-Methyl-1H-indol-4-ol (557) was acetylated with acetic anhydride and NaHCO 3 in toluene into compound 558. It was then treated with oxalyl chloride, followed by dimethylamine in THF, to result in 559, which, on reduction by LiAlH 4 , resulted in 560. Compound 560 was incubated with PsiK and ATP for 16 h; the analysis of the mixture by HPLC indicated that 90% of it was successfully converted into 5-methylpsilocybin (561) with an overall isolated yield of only 40% [100]. from staurosporine by the same group [94,95]. To enhance its efficacy as an antitumor agent, 14 derivatives (535 to 548) of fradcarbazole A were synthesized by variation only in the substituents on two indole units ( Figure 25) [96]. In the synthesis, 5fluoro/chloro/bromo/methoxy-tryptamines (489 to 493) were protected using Boc2O to give derivatives 494 to 498 and oxidized using DDQ, producing compounds 499 to 503. TFA-mediated Boc deprotection resulted in molecules 504 to 508. Staurosporine (509) was protected using Boc2O, giving 510, which was halogenated using NCS and NBS (compounds 511 and 512), and deprotected by TFA (compounds 513 and 514). These derivatives and compound 509 were converted to compounds 515 to 517 using TCDI. These compounds were alkylated by CH3I in CH3CN, affording compounds 518 to 520. They were then converted into compounds 521 to 534 via a nucleophilic reaction with 504 to 508. Lastly, intramolecular cyclization reactions of compounds 521 to 534 resulted in 535 to 548 [96]. pound 550 using T16H yeast and allylated by allyl bromide and K2CO3 in DMF, resulting in 551, the northern fragment of compound 556. Separately, compound 549 was protected using TrocCl, affording 552, which was further converted to 553 by TFA and m-CPBA. Compound 553 was reacted with m-CPBA in DCM, producing its N-oxide analog 554. Compound 554 was treated with TFAA in DCM and coupled with 551 in a Polonovski-Potier reaction, which resulted in 555. Compound 555 was deprotected by treating with Pd(PPh3)4, affording 556 [98].  5-Methylpsilocybin (561), a novel analog of the psychedelic psilocybin, was produced by enzymatically phosphorylating its synthetic precursor, 5-methylpsilocin (560). The 4-hydroxytryptamine kinase (PsiK) enzyme was purified from the Psilocybe cubensis fungus. On the basis of the amount of 561 isolated, the overall yield was 28% (Figure 27) [100]. 5-Methyl-1H-indol-4-ol (557) was acetylated with acetic anhydride and NaHCO3 in toluene into compound 558. It was then treated with oxalyl chloride, followed by dimethylamine in THF, to result in 559, which, on reduction by LiAlH4, resulted in 560. Compound 560 was incubated with PsiK and ATP for 16 h; the analysis of the mixture by HPLC indicated that 90% of it was successfully converted into 5-methylpsilocybin (561) with an overall isolated yield of only 40% [100].         Table 2 only includes the bioactivities of novel indole alkaloids that were isolated, synthesized, and tested from 2019-2022 to enable an extensive structure-activity relationship analysis; the activities of previously reported natural products (which were tested alongside the novel indole alkaloids) are also included. These compounds are referred to by their trivial names. Furthermore, since most articles presented a range of biological activities, we only summarize these activities in this section.
Compounds 30 to 31 showed no antifungal, antibacterial, and cytotoxicity activities. A congener, baraphenazine E, exhibited some cytotoxicity and antibacterial activity, indicating that its C-11 -amide may be responsible for its moderately potent activities, as the other alkaloids contained a carboxylic acid group instead. All compounds showed no activity or toxicity in an axolotl embryo tail regeneration assay [25]. The chlorinated cyclic hexapeptides, 32-36, exhibited no cytotoxicity (tested on HeLa cells). However, compounds 32 and 33 displayed moderate and weak antibacterial activities, respectively, as replacing the phenylalanine residue (32) with a leucine residue (33) could decrease antibacterial activity. Similarly, replacing the isoleucine residue (32 vs. 33) with a valine residue (35 vs. 36) completely suppressed activity. Compounds 38 and 39 showed no cytotoxicity, while compound 37 demonstrated moderate cytotoxicity on HT-29 cells [26]. Compounds 41 and 43 showed no antibacterial activity, while compounds 40, 42, 44, and 45 displayed moderate to weak inhibitory activity against Xanthomonas oryzaepv. Oryzae (rice bacterial leaf blight). Treatment with compound 40 at 100 µg/mL offered 61% protection, while treatment with 200 µg/mL offered 82% protection from the same pathogen. Furthermore, chaetogline A displayed antifungal properties against S. sclerotiorum (EC 50 = 10.3 µg/mL), while 40 to 45 were inactive. The results indicated that the C-19 carboxylic acid in compound 44 is necessary for its antibacterial activity, while the C-19 ester in chaetogline A is essential for its antifungal activity. Similarly, a second N-methylated indole increased antibacterial activity (41 vs. 42) [28].
A mixture of compounds 51-54 was evaluated. It was found to be extremely potent and selective inhibitors of the U87 and SKOV3 cell lines with an EC 50 of 0.41 nM and 0.75 nM, respectively (SI > 7000; cytotoxicity was also tested against MDA-MB-221 and HCT116 cell lines). The mechanism of action is not proven similar to that of plinabulin (a similar diketopiperazine) due to the nonexistence of vascular disruption and tubulin binding studies [30]. Among compounds 55-62, only 55 and 56 displayed moderate cytotoxicity, while all other tested indole alkaloids were inactive. Comparing compound 60 with asperversiamide H (both inactive) demonstrated that replacing the C-11 proton with a methoxy group did not significantly affect activity [31]. Compound 63 demonstrated potent antiplasmodial activity, inhibiting more than 90% of liver-stage growth of P. falciparum at 6.1 µM. It showed similar antimalarial activity to primaquine [32]. Compounds 64-66 exhibited weak antibacterial activity against the ATCC-4 strain of H. influenzae, and compounds 64 and 66 showed slightly more activity than compound 65. The results indicated that replacing the C-19α proton with a hydroxy group did not alter the bioactivities of (3β,6β,23-trihydroxyolean-12-en-28-oic acid vs. 3β,6β,19α,23-tetrahydroxyolean-12en-28-oic acid; MIC = 18.8 µg/mL for both), suggesting that it is not a determinant for activities. Additionally, naucleidinal most potently inhibited H. influenzae strain ATCC-4 (MIC = 3.1 µg/mL) [33].
Compounds 80, 81, 82, 85, and 88 displayed no activities against the 5-HT1A receptor, a therapeutic target in CNS diseases. Of the remaining four compounds, 83 and 87 displayed weak to moderate activity, while compounds 84 and 86 showed potent agonism. The results demonstrate the importance of stereochemistry in biological systems; 84 and 86 contain a C-16S, whereas their diastereomers 85 and 87 were inactive, having a C-16R configuration. Additionally, a vinyl group at C-20 instead of an ethyl group (86 vs. 84) increased bioactivity. Furthermore, docking studies showed that 84 interacted with the 5-HT1A receptor primarily through its two hydrogen bonds between the indole-nitrogen (NH-1) and Asp116, Tyr390. Alternatively, the interaction of compound 86 comprised only one hydrogen bond between the indole-nitrogen (NH-1) and Asn386 [37].
Compounds 89-98 were assessed for cytotoxicity and acetylcholinesterase (AChE) inhibition; none of the alkaloids showed cytotoxicity. Compound 98 moderately inhibited AChE, and docking studies were used to investigate its interactions with the enzyme's active site. The C-22 carbonyl, N-1, N-4, and the hydroxy group at C-5 and C-6 formed hydrogen bonds with Tyr124, Tyr341, Arg296, and His287, respectively, indicating that both the carbonyl group at C-22 and the indole ring's N-1 were important for its activity [38]. Compounds 99-105 were tested for quorum-sensing inhibition, cytotoxicity, and antibacterial activity. The only bioactive alkaloids were 104 and 105, which displayed only weak quorum-sensing inhibition in C. violaceum strain CV026. Despite stereochemistry being essential in determining bioactivities, enantiomers 103 and 104 displayed the same activities. Additionally, scequinadoline G inhibited quorum-sensing most potently (MIC = 16 µg/well) [39].
Compounds 106-108 showed no cytotoxicity and antibacterial activity, possibly due to their self-fluorescence. Slight modifications such as reducing ∆2 (solitumine B vs. solitumine A; both inactive) or replacing the C-19-bonded NH 2 with an -OH (106 vs. 107) showed no detectable differences, suggesting that they are not significant determinants of bioactivity [40]. Compound 109 displayed weak antiviral activity against the herpes simplex virus 2 (HSV-2). At the same time, other amphiepicoccins, such as amphiepicoccin B, C, D, and H, were also tested for their antiviral and antibacterial (against B. subtilis) activities. Amphiepicoccin C and H displayed weak antiviral activities (IC 50 = 64 and 29 µM), while amphiepicoccin D and H showed moderate to weak antibacterial activities (MIC = 13 and 25 µM). The rest were inactive. The carbonyl at C-5 is a critical determinant of bioactivity if replacing it with a hydroxy group significantly decreased activity (amphiepicoccin C vs. B). Interestingly, reducing the number of sulfur atoms in the C-2 to C-7 sulfide bridge from two to one (amphiepicoccin D vs. H) meaningfully increased antiviral activity but moderately decreased antibacterial activity [41].
Compounds 110-114 displayed moderate to strong antiparasitic activities against the P. falciparum strain FcB1. No cytotoxicity was detected against MRC-5 cells. The results showed that the reduction of ∆19 slightly boosted activity (111 vs. 110), but replacing C-22 with a proton significantly suppressed bioactivity (112 vs. 113) [42]. Compounds 115-123 were evaluated for cytotoxicity, but only 123 showed potent activity, while 117-119 showed only weak cytotoxicity. Considering compound 123 had a sulfide bridge, compounds 118 and 119 contained an SCH 3 -group at C-6, and compounds 120 to 122 were sulfur-free analogs, especially the sulfide bridge greatly influenced the activity of diketopiperazine [43].
Compounds 124-126 were tested for their ability to promote triglyceride accumulation in 3T3-L1 cells. While compounds 124 and 125 showed no significant activity, compound 126 was a moderate promoter. Additionally, no compound exhibited toxicity against 3T3-L1 cells at 50 µM. Analyzing the structure-activity relationships yielded two important moieties responsible for the bioactivities of compounds: the pyrazino [2,1-b]quinazoline-3,6-dione core and the unsaturated isopropyl group at C-3. Both these motifs are present in scequinadoline D, scequinadoline E (most potent among the compounds tested; EC 50 = 0.27 and 0.36 µM), while compound 126 only contains the tricyclic core. Suppose the core was replaced with another tricyclic system (quinazoline-pyridine-tetrahydrofuran) or a quinazolinone ring suppressed activity (124, scedapin C, and scedapin A; all inactive). Replacing the unsaturated isopropyl group with its saturated analog also reduced activity (125, fiscalin C, epi-fiscalin C, scequinadoline A, scequinadoline B, and fiscalin B; all inactive). Furthermore, substituents at C-22 also modulated activity, with ethyl and methyl groups resulting in the most potent analogs (scequinadoline D, scequinadoline E) [44].
Among compounds 127-136, only compounds 127-129 and 132 were tested for their activities based on chemometric results. Among those three, only compound 127 was active, potently inhibiting MAO-A, while weakly inhibiting MAO-B and BChE. Cimitrypazepine and fargesine were also weak to moderate inhibitors of BChE (18% and 41% inhibition at 10 µM) and potent inhibitors of MAO-A (IC 50 = 1.4 µM for both). Interestingly, replacing the tertiary amine in cimitrypazepine with an N-oxide (as in fargesine) significantly increased BChE inhibition while not affecting MAO-A inhibition. Furthermore, replacing the tertiary amine with a quaternary ammonium ion, as in compound 127, slightly boosted both BChE and MAO-A inhibition. Conversely, the glucosylated alkaloids 129 and 132 were inactive due to the bulkiness of the glucose group, which hampered the interaction of the alkaloids with the enzyme's active site [45].
Compound 137 displayed no antibacterial or cytotoxic activities but moderately and selectively inhibited the Hepatitis C virus (SI > 35) [46]. Compounds 138-141 were tested for antibacterial, antifungal activities, and α-glucosidase inhibition but were inactive. Compound 141 was weak against mushroom tyrosinases inhibitor, and it is the first example of inhibiting tyrosinases by a thiodiketopiperazine. Changing the steric configuration of C-2 from R to S increased bioactivity (139 vs. 141). Additionally, the presence of an OCH 3 /SCH 3 group at C-2 and an N-4 -CH 3 group is necessary for the bioactivities of compounds. Furthermore, a second indole group significantly boosted bioactivity (Leptosin D, IC 50 = 28.4 µM) [47].
Among compounds 142-150, only 145 showed moderate cytotoxicity against SW480 while being inactive against other cell lines. The cytotoxicity of tabernaricatine C was also tested, and it potently inhibited HL-60 and SW480 cells (EC 50 = 3.2 and 3.5 µM). Previously, other alkaloids such as taburnaemine D, tabernaemine I, tabercorine B, tabernaelegantine B, conodurine, and tabernaricatine C (among others), along with compounds 142-150, were tested for their inhibitory activity against autophagic flux. However, only bisindoles were active, with the most potent inhibitor being taburnaemine D (EC 50 = 12.9 µM). The linkage pattern of the two indole units might be a crucial factor in determining the activities of the compound [48].
Compounds 160-172 displayed weak to moderate cytotoxicities when tested against cell lines U251 and U87MG, and none of them exhibited antimicrobial activities. Nonetheless, results indicated that the methyl substituent on N-1 (the nitrogen of the indole ring) was an important determinant of activity as replacing it with a proton drastically lowered activity (160, 162, or 170 vs. 161, 163, or 171, respectively). Similarly, an OH group at C-13 completely suppressed the cytotoxicity (164 vs. 165), and an α-OH group at C-16 deactivated 169 compared to 168. Furthermore, replacing the C-9 carbonyl with an acetyl group (resulting in the oxidation of the C-9-N-2 bond) meaningfully boosted the activity, which is evident by comparing compound 171 with marine carboline C (EC 50 = 12, 13 µM against U251, U87MG cells) and to a lower extent 170 with 168. Furthermore, the steric configurations of the substituents also modulated the activity by replacing the β-OH group on C-16 with an α-OH group, causing a noteworthy increase in cytotoxicity (166 vs. 167). Interestingly, changing the geometric configuration of ∆15 from Eto Z-configuration slightly boosted the potency of methylated-indole analogs (160 vs. 162). Nonetheless, non-methylated indole analogs (161 vs. 163) were inactive [51].
Compounds 173 and 174 are inactive in vitro antibacterial assays. Nevertheless, they enhanced the activity if co-administered with imipenem (imipenem; MIC = 16-32 µg/mL). Considering that krisynomycin (MIC = 0.25-0.5 µg/mL with 4 µg/mL of imipenem) was more chlorinated than 174, which was more chlorinated than 173, the extent of chlorination was thought to be a significant cause of the imipenem-potentiating effects of the compound. Furthermore, chlorination in the alkaloid resulted in increased potentiating effects of imipenem at lower doses [52].
Among compounds 175-185, only 177 and 185 inhibited the DC secretion of IL-12p40, while 176 increased the secretion of DC cytokine IL-10. Compounds 177 and 185 did not affect the viability of DCs, demonstrating that the decrease in secretion was not due to cell damage. The results indicated that the nature of the C-14 substituent did not affect bioactivity noticeably. Replacing a proton with a hydroxy (180 or 182 vs. 181 or 183) or methoxy (182 vs. 184) did not meaningfully affect activity. Furthermore, pyrrolo [2,3b]indole (180 to 184) and oxindole (178 and 179) alkaloids were found to be inactive. On the other hand, 6-bromoindole alkaloids (177 and 185) were active, suggesting that modified indoles decreased the bioactivities of compounds [53]. Compound 186 demonstrated no antiplasmodial activity against the ring stage of the P. falciparum strain 3D7 nor the P. falciparum strain Dd2. It was non-cytotoxic against HEK293 cells, too [54].
The cytotoxicities of compounds 187-195 against various cell lines were determined .  Molecules 187, 191, 192, and 193 exhibited moderate cytotoxicities. The only difference between 190 and 191 was the oxidation of C-18, suggesting that the absence of the carbonyl group significantly increases the compound's activity. Furthermore, replacing a proton with a hydroxy group at C-2 causes a significantly suppressed activity (192 vs. 191). Stereochemistry (192 vs. 194 and 193 vs. 195) was also a foremost determinant of the activities of alkaloids; switching C-14 from an S to R configuration significantly reduced their activities [55].
Compounds 196-200 were not cytotoxic against the A549 cell line. Similarly, only 196, 197, and 198 inhibited the Zika virus. Previously reported activities of natural products suggested that the F-ring was a significant factor of antiviral activity; an open F-ring afforded an inactive compound (emindole SB), while a furan F-ring yielded the most potent compound in the series, JBIR-03 (EC 50 = 4.2 µM). However, compared to JBIR-03, the weaker activities of fusaindoterpene A (EC 50 = 12 µM) and asporyzin A (EC 50 = 18 µM) indicated that B-and C-rings also modulate bioactivity. Furthermore, adding an oxygen atom into the eight-membered ring of asporyzin A (as in fusaindoterpene A) slightly increased activity [56].
Compounds 201-204 were tested against the HEI-OC1 cell line to determine their protecting ability. Compound 201 displayed potent protecting effects (optical density = 0.7, compared to the blank control's 0.5), while the other compounds were inactive. The alkaloids were also tested to determine if they stimulated glucose uptake in cultured rat L6 myoblasts. Only compounds 203 and 204 promoted glucose uptake in the myoblasts. Among the non-novel natural products tested, beauverolide Ka displayed both protective ability (optical density = 0.69) and promotion of glucose uptake (9 mmol/L). The results indicated that a hydroxy substituent on C-13 (para-substituent of the phenyl ring) instead of a proton decreased protective ability significantly (beauverolide Ka vs. 203 and 201 vs. 204). Interestingly, replacing the indole ring, the C-2 substituents, or the C-13 proton in beauverolide Ka drastically decreased its glucose uptake promotion activity, evident when comparing it to beauveamide E (inactive), 201, and 203 [57].
The analgesic effects of compounds 205-209 were studied, with compound 207 exhibiting more potent analgesic activities than morphine at 0.04 and 0.2 mg/kg doses. Since 205 was more active than humantenine N 4 -oxide and humantenine, its oxazolidinone ring may enhance bioactivity. Conversely, compound 207 and N-desmethoxyhumantenine were more potent than alkaloids with an N-1-OCH 3 motif (206, humantenine N 4 -oxide, 11-hydroxyhumantenine, humantenine, and humantenirine), indicating that the group might cause a decline in activity. Moreover, compound 208 displayed lower activity than epi-koumidine and koumidine, inferring that the N 4 -oxide (not possessed by the latter two) decreased the activity of sarpagine-type alkaloids. Furthermore, compound 206, 11hydroxyhumantenine, and humantenirine contained a hydroxy or methoxy substituent at C-11and showed less potent activities, suggesting that these groups decreased the activities of humantenine-type alkaloids [58]. Compound 210 demonstrated no cytotoxicity against the MRC-5 cell line; however, it potently inhibited P. falciparum strain FcB1. Comparing its antiplasmodial activity with vobasine (IC 50 = 22.5 µM) revealed that the C-3 bonded -SH group is necessary for the activity of vobasane-type alkaloids; nevertheless, vobasine contains a carbonyl. Additionally, the discovery of compound 210 is of particular biosynthetic interest, as it was proposed to be a step in forming the ion brunonines A and B [59].
Compound 211 displayed moderate to robust cytotoxicities against various cell lines and selectivity (SI > 5) when tested against HT-29 and CCD-18Co cells. It induced mitochondrial apoptosis, and in vitro tubulin polymerization assays confirmed it as a microtubulestabilizing agent. It was also predicted to bind at the taxol-binding site in β-tubulin. The docking models showed that compound 211 had several nonpolar interactions and hydrogen bonds with the amino-acid residues in the binding pocket, resulting in its calculated interaction strength being more significant than paclitaxel, which was consistent with the observed microtubule-stabilizing activity. Compound 211 also satisfied the criterion of drug-likeness, with a score of 3.28 (vincristine 4.03, Taxol 0.82), well within the proposed acceptable range of 2-7 [60,101].
The adiponectin secretion-promoting activities of compounds 212-214 and their synthetic isomers, 286-290, were investigated in hBM-MSCs. However, the EC 50 of compounds 214 and 286-290 was greater than 10 µM. Compounds 212 and 213 were more potent than bezafibrate and increased lipid accumulation in differentiated adipocytes. The results suggested that they improved insulin sensitivity (like pioglitazone). Comparing the activities of the alkaloids demonstrated the importance of handedness in biological systems; enantiomers of the compound 212 and 286 displayed significantly less potency. A bulky phenyl group at C-2 may suppress bioactivity by hindering the interactions of compounds with the target's active sites (213 vs. 214) [61].
Docking studies showed the indole acetonitrile in compounds 215 and 216, and other compounds such as 3-indole acetonitrile and 4-methoxy-3-indole acetonitrile, which strongly bind with the HA and form hydrogen bonds with the THR59F and GLU90D residues. This hydrogen bonding possibly modulated the function of HA and produced the supposedly antiviral activities of compounds. Nevertheless, despite reporting compound 215 as an inhibitor of HA, no quantitative information is available [62].
Compounds 217-227 displayed weak to moderate cytotoxicities against various cell lines, while compound 228 was inactive. Western blot analysis showed that compound 218 induced apoptosis of MCF-7 cells via the Bcl-2/caspase-3/PARP pathway. Results revealed that reducing ∆19 caused a decrease in cytotoxicity (218 vs. 219 and 223 vs. 220), which infers that structural rigidity is required to exhibit cytotoxicity. Interestingly, the geometric conformation of ∆19 did not significantly influence activity, as evident when comparing the activities of the Z-isomer (271) and the E-isomer (272) [63]. No cytotoxicity or antibacterial activity was detected when compounds 229 and 230 were tested. Compound 229 displayed weak antifungal activity against F. oxysporum [64]. Compounds 231 and 232 showed no cytotoxicity, and only compound 231 showed weak antimycobacterial activity. Replacing the C-12 proton in compound 231 with a methoxy group as in 232 completely lost the activity [65].
Compound 233 and its novel synthetic derivatives (294-308) were tested for their inhibition of RANKL-induced multinuclear osteoclasts in RAW264 cells. The weaker activity of 295 compared to compound 233 demonstrated the importance of ∆2 for bioactivity. If the methylenedioxy ring was replaced with the two methoxy groups, as in 296, slightly enhanced activity was observed. The results also indicated that the presence and positions of bromine atoms are a significant determinant of bioactivity as 5,6-methylenedioxyindole. Shifting the position of bromines as in 294 or replacing them with chlorines as in 299 significantly suppressed the activities. While the bromine at C-4 was believed essential for activity, the bromine at C-7 slightly enhanced activity when C-4 was unsubstituted (298 compared to 5,6-methylenedioxyindole). Moreover, the presence of chlorines slightly improved potency when bromines were absent (299 versus 5,6-methylenedioxyindole). Similarly, N-acyl derivatives were used to study the effects of varying chain lengths on activity. Compounds 300-302 with C2-C8-alkyl chains showed activity, but compounds 303 (C14-alkyl chain) and 304 (benzoyl derivative) showed no activity, demonstrating that long or bulky groups cause a decline in activity. Furthermore, the effects of substituents at C-3 were studied by comparing the activities of compounds 305, 306, and 307, which contained methyl, hydroxymethyl, and formyl substituents, respectively. Although compounds 305 and 306 had similar activities as 233, compound 307 was less potent. Compound 308, a tryptamine derivative, was more potent but was found cytotoxic at 25 µM, while none of the other derivatives showed toxicity even at 50 µM [66].
While compounds 235 and 236 were not tested, other compounds-(1H-indol-3-yl)oxoacetamide, and indole-3-aldehyde-were also produced by E. coli harboring the EDB cluster and were examined for their ability to repel C. elegans. The two molecules had a chemotaxis index of −0.18 and −0.08 at 0.2 mM, implying moderate repulsion and confirming that P. fluorescens NZI7 s EDB cluster enables it to convert external tryptophan into indole-containing molecules that repel their predators, C. elegans [68]. Compound 237 (and, to a much lesser extent, 238) inhibited LPS-induced B-cell proliferation, with the former displaying high selectivity (SI > 548). The results indicated that the C-6 methoxy group of compound 237 is essential for activity. Replacing it with a proton, as in 238, led to an increase in toxicity and a sharp decline in inhibitory activity. Compound 237 was also inactive against the four tyrosine kinases ibrutinib (the positive control) inhibited, suggesting that compound 237 exhibited its activity via a new mechanism. However, neither compound inhibited concanavalin-A-induced T-cell proliferation [69].
Among compounds, 239-244, 239 and 242 exhibited cytotoxic effects, indicating that the N-OH group was a key determinant of activity (239 compared to 240). Similarly, compound 242 had more potent activity than compound 241, suggesting that the more rigid cyclized form of compound 242 improved the cytotoxicity. Molecule 239 also caused mitochondrial dysfunction in HeLa cells, and compound 239-induced cell apoptosis was related to generating reactive oxygen species in HeLa cells. Furthermore, compound 239 increased the phosphorylation levels of JNK, ERK, and p38 in HeLa cells, suggesting that compound 239 activated the MAPK signaling pathway, which helps mediate cell proliferation and apoptosis [70,102].
The hepatoprotective activities of compounds 245-248 were assayed in transgenic fluorescent zebrafish with liver injury. Quantitative analyses showed that 20 µM of compounds 245, 246, and 248 exhibited moderate hepatoprotective activities equal to those of the positive control (50 µM S-adenosyl-l-methionine). In addition, C-3 substituents were also a significant determinant of bioactivity as replacing the 2-aminoethanal group in 248 with a methyl formate group (as in 247) or with a 2-aminopropanoic acid group (as in C-mannosyl tryptophan) resulted in inactivity [71].
Although compounds 249 and 250 displayed moderate to strong antiviral activities, they were toxic against MDCK and Hep-2 cells with CC 50 values ranging from 13 to 31 µM, drastically lowering their therapeutic ratios. Interestingly, the hemiketal form (250) of compound 249 displayed more potent antiviral activities while also being more toxic [72].
The antibacterial activities of compounds 314, 317, 321, and 323 were evaluated but were considerably less potent than their congener, griseofamine A's activity (MIC = 8-16 µg/mL). Since all compounds tested contained the 3-acyltetramic acid moiety, the putative notion that the motif was a major determinant of antibacterial activity was not supported [87]. Compounds 347-352 displayed moderate to strong cytotoxicities against various cell lines. The results indicated that increasing the length from four to five (counting the heteroatoms) of the linker chain increased cytotoxicity (347 vs. 348 and 350 vs. 351). However, a further increase (from five to six) suppressed bioactivity (348 vs. 349 and 351 vs. 352). Replacing the methoxy group in the trisubstituted phenyl ring with a proton weakened the activities of derivatives (347, 348, and 349 vs. 350, 351, and 352) [88]. Moreover, compound 348 displayed the most robust cytotoxicity in the series while exhibiting minimal toxicity on normal hepatic LO2 cells (showing high selectivity). It also inhibited HDAC1 and HDAC6 while enhancing the acetylation levels of histone H3 and α-tubulin. In addition, it induced more significant cancer cell apoptosis than SAHA by regulating the expression of apoptotic proteins Bax, Bcl-2, and caspase 3. Compound 348 also induced important autophagic flux activity in Bel7402 cells by increasing the expression of Beclin-1 and LC3-II proteins and suppressing LC3-I and p62 production. Furthermore, compound 348 also significantly inhibited PI3K/Akt/mTOR signaling, an essential cell-growth-promoting pathway aberrantly activated in many cancers [88,103].
Compounds 404-424 and 438-443 were evaluated for their inhibitory activity against porcine SCR (mixture of bc1 and complex II). The first series (404-424) showed weak activities, except 411, only inhibiting 10-49% of SCR at 10 µM. The binding of the lead compound 411 of the first series was analyzed with the complex. The carboxyl oxygen in carbamate formed an H-bond with Glu271. However, the indole and phenyl rings were almost perpendicular with Phe274, resulting in weak interactions with the residue. Previous studies by the same group showed that the π-π interactions between neopeltolide and Phe274 were essential for activity [104]. Furthermore, the carboxyl oxygen in the ester did not form an H-bond with His161, also an essential factor for bioactivity [104]. The second series (438-443) was synthesized with an amide linker to improve the interactions between the derivatives and key residues. Similarly, it contained only fluorine-and methylsubstituted benzyls as substituents on the indole ring (first series derivatives with such substituents were generally more potent). These modifications considerably increased the potency of the second series derivatives. Again, the binding mode of the most potent derivative 442 was analyzed. It formed π-π interactions with Phe274 and an H-bond with Glu271 and His161. Interestingly, the two positional isomers of compound 442 (which differed in the positions of substituents on the benzyl group), 441 (o-methyl), and 443 (pmethyl) were considerably less potent, demonstrating that m-methyl was the most bioactive configuration. In contrast, for the fluorine substituents, o-fluorine was the most bioactive configuration (438 vs. 439 vs. 440) [90].
None of the celastrol-indole derivatives (456-465) inhibited concanavalin A-induced Tcell proliferation or displayed cytotoxicities. [92] Phidianidine A and its synthetic structure variants (479-488) were screened against the settlement and metamorphosis of Amphibalanus improvisus cyprids, with compounds 486 and 487 exhibiting the most potent activities. It demonstrated that only the guanidine derivatives showed significant activity, while primary amine (479-483) or quaternary ammonium 488 derivatives were relatively inactive. Additionally, increasing chain length increased bioactivity among the guanidine derivatives (484-487). Furthermore, since both compounds 486 and 487 were more potent than phidianidine A (EC 50 = 4.0 µg/mL), the original scaffold's transformation into a simpler, more bioactive structure that did not contain the constrained 1,2,4-oxadiazole ring was deemed successful [93].
Compounds 535-548 displayed potent cytotoxicity against the MV4-11 cell line. In addition, compound 540 was the most powerful derivative in the series and induced apoptosis of MV4-11 cells. It also arrested cells in the G0/G1 phase and reduced the expression of FLT-3, CDK2, and c-kit. The preliminary structure-activity relationship study yielded interesting results: if the substituent on C-3 was a proton (535-538), the most activity-boosting substituent on C-5 was a methoxy group (538). Additionally, if C-3 was bonded to chlorine (539-543), then fluorine on C-5 produced 540 (the most potent derivative overall). Lastly, for bromine on C-3 (544-548), a proton on C-5 afforded the most potent bromine-containing derivative 544 [96]. However, compound 561 was tested for psychedelic-like activity using the mouse head-twitch response assay, which showed that it was more potent than dimethyltryptamine but less potent than psilocybin [100].

Conclusions
The 354 novel indole alkaloids discussed-consisting of 250 isolated and 87 synthesizedrepresent only a tiny fraction of the known indole-alkaloids, according to both the number and the structural diversity of predicted indole-containing compounds yet to be discovered. This massive gap between known and predicted indole alkaloids and the group's potent, selective, and variegated bioactivities represents a worthwhile opportunity for chemists, who can leverage modern spectroscopic and biological tools to determine the alkaloids' structures and mechanisms of action efficiently. This article described recent advances in the field to draw further attention to unique alkaloids that either possess unprecedented motifs or display substantial activities against their targets and to provide a convenient, easily accessible list of information regarding the isolations and bioactivities of alkaloids. Similarly, reappraisals were included to demonstrate the efficacy of modern-day spectroscopic techniques. At the same time, syntheses (both total and semi) were discussed to highlight the second aspect of drug development: optimizing the lead.

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