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Fischer Indole Synthesis

What is fischer indole synthesis.

Indole synthesis has provided an ample playground for both organic chemists and medicinal chemists. Each year, thousands of indole derivatives are made in pursuit of life-saving medicines.

The Fischer indole synthesis is one of the oldest and most effective methods of indole development and was first invented by Fischer in 1883. A variety of indole may be prepared from aryl hydrazines and substituted ketones or aldehydes in the presence of Brønsted or Lewis acids.

The most useful route to indoles is the Fischer indole synthesis in which an aromatic phenylhydrazone is heated in acid. Phenylhydrazone is the condensation product from a phenylhydrazine and an aldehyde or ketone. Ring closure involves a cyclic rearrangement process.

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Fischer indole synthesis reaction, fischer indole synthesis reaction mechanism, drawbacks of fischer indole synthesis, features of fischer indole synthesis, recommended videos, frequently asked questions on fischer indole synthesis.

The Fischer indole synthesis has become the most popular method to prepare indole rings since its discovery in 1883 by Emil Fischer. In essence, the Fischer indole synthesis can be regarded as the cyclization of an arylhydrazone, prepared from aryl hydrazine and aldehyde or a ketone by treatment with an acid catalyst or effected thermally to form the indole nucleus. The mechanism has been the subject of intensive investigations for over a century, and many intermediates have been isolated and characterized.

The Fischer indole synthesis reaction is given below.

There has probably been more work carried out on the synthesis of indoles than on any other single heterocyclic system, and consequently, many routes are available; ring syntheses of benzofurans and benzo thiophenes have been much less studied. The Fischer indole synthesis now more than 100 years old, is still widely used – an aryl hydrazone is heated with an acid, a multi-step sequence ensues, ammonia is lost, and indole is formed.

The synthesis is often carried out by subjecting an equimolar mixture of the aryl hydrazine and aldehyde or ketone directly to the indolization conditions without isolation of the hydrazone. Similarly, aryl hydrazones, prepared by reduction of the corresponding aryldiazonium salt or N-nitroso arylalkylamine or by a palladium mediated coupling reaction , can be subjected to the indolization conditions directly in the presence of the carbonyl moiety without isolation of the aryl hydrazone. Such methods are useful when the aryl hydrazone intermediates are unstable or toxic.

The arylhydrazone prepared from condensation of an aryl hydrazine and a carbonyl compound, undergoes protonation and isomerization to the enamine tautomer. The protonated enamine tautomer then undergoes an irreversible electrocyclic rearrangement that is [3,3] – sigmatropic rearrangement where the N-N bond is broken.

The resulting double imine then re-aromatizes the benzene ring to provide an anilino imine, whose nucleophilic amine group attacks the imine intramolecularly to afford the amino indoline. Loss of a molecule of ammonia and aromatization then deliver the indole.

The hydrazine behaves as an amine towards a carbonyl compound and forms the imine like the product, a hydrazone. The cycle rearrangement involves the enamine tautomer of this hydrazone and proceeds because the cyclic flow of electrons forms a strong C-C bond whilst cleaving a weak N-N bond. This produces what appears to be a di-imine.

One of these is involved in rearomatization and creates nan aromatic amine. This then attacks the other imine function, and we get the nitrogen equivalent of a hemiketal. Finally acid catalysed elimination of ammonia gives the aromatic indole system.

Unfortunately, the reaction fails with acetaldehyde and cannot, therefore be used to synthesize indole itself. It is possible to use the keto acid pyruvic acid instead and decarboxylate the product to yield indole.

The key characteristics of the Fischer Indole Synthesis are:

• The indole formation can be done in a single pot because it is not required to separate the intermediate aryl hydrazones.
• Unsymmetric ketones give two region-isomeric 2,3-displaced indols with a region-selectivity depending on medium acidity, hydrazine substitution and steric effects.
• 1,2-diketones can give both mono-indoles and bis-indoles, which are usually formed by strong acid catalysts in refluxing alcohols.

Fischer- Indole Synthesis

How is indole produced?

Indole is produced by reductive deamination of tryptophan via the intermediate molecule indole pyruvic acid. Tryptophanase catalyzes the deamination process from which the tryptophan molecule group amine (-NH 2 ) is extracted. The final reaction products are indole, pyruvate, ammonium ( NH 4 + ) and water.

What is an indole group?

Indole is an organic heterocyclic aromatic compound with formula C8H7N. It has a bicyclic composition, consisting of a six-piece benzene ring fused to a five-piece pyrrole ring. Indole is commonly spread in the natural world and can be produced by a wide range of bacteria.

Where is indole found?

An indole is a solid man at room temperature. It occurs naturally in human faeces and has a strong faecal odour. At very low concentrations, however, it has a floral scent and is a component of many floral scents (such as orange blossoms) and perfumes. This exists in coal tar as well.

What does indole smell like?

Indoles are chemical compounds that can look like jasmine (Jasmine is naturally indolent) or faeces. They can also be made synthetically, and perfumers use them to enhance the floral scent. For some people, the indols smell like a sour odour. Others have an animal piss or fur scent.

What does indole positive mean?

The indole test is a biochemical study conducted on bacterial organisms to assess the ability of the organism to transform tryptophan into indole. This separation is done by a series of various intracellular enzymes, a mechanism commonly referred to as “tryptophanase.”

How is indole detected?

The indole test is used to assess the ability of the body to break down the amino acid tryptophan to form the indole compound. Indole production is observed by Kovac or Ehrlich’s reagent containing 4 (p)-dimethylamino benzaldehyde, which interacts with indole to create a red coloured compound.

Is indole acidic or basic?

Indoles, consisting of a pyrrole ring fused to benzene to form 2, 3-benzopyrrole, are compounds forming an indole moiety. Indole is a solid, partially soluble compound (in water) and an extremely weak acidic compound (essentially neutral).

What is the starting material used in Fischer indole synthesis?

The synthesis of Fischer indole is an organic reaction used by an acid catalyst to convert phenyl hydrazine and an aldehyde or ketone to an indole. By the acid catalysed reaction of the hydrazine with the carbonyl, the cycle starts with the formation of a phenylhydrazone.

Why do bacteria produce indole?

In the inquiry of bacterial contact, several chemical compounds have been found and each has a biological tale to tell. Indole is a direct consequence of amino acid catabolism, signals in multidrug exportation, cell division inhibition, stress tolerance, and biofilm-forming.

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Fischer Indole Synthesis

Definition: what is fischer indole synthesis, examples of fischer indole synthesis, mechanism of fischer indole synthesis.

The Fischer indole synthesis is an organic reaction used to convert a phenylhydrazine and an aldehyde or ketone to an indole using an acid catalyst, like Brønsted or Lewis acids. An application of this reaction is the synthesis of antimigraine drugs belonging to the triptan class [1-8] .

The history of this reaction goes back to 1883 when the reaction was first discovered by a German chemist Emil Fischer.

The Fisher indole synthesis is used to synthesize 2-phenylindole and tetrahydrocarbazole [2,3,9] .

Phenylhydrazone is formed from a condensation reaction of phenylhydrazine and an aldehyde or ketone. The phenylhydrazone converts into an indole in the presence of an acid catalyst [2-8,10] .

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Related Reactions Gewald Reaction Synthesis of indoles

Fischer Indole Synthesis

The conversion of aryl hydrazones to indoles; requires elevated temperatures and the addition of Brønsted or Lewis acids. Some interesting enhancements have been published recently; for example a milder conversion when N -trifluoroacetyl enehydrazines are used as substrates. ( abstract ).

Mechanism of the Fischer Indole Synthesis

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• Fischer Indole Synthesis

What is meant by Fischer Indole Synthesis?

One of the oldest and most powerful methods of indole development is the Fischer Indole Synthesis. It was first patented in 1883 by Fischer. A variety of indole can be prepared from aryl hydrazine and substituted ketones or aldehydes in the presence of Brønsted or Lewis acids. It is a chemical reaction that generates the aromatic heterocyclic indole from a (substituted) phenylhydrazine and an aldehyde or ketone, under acidic conditions.

Fischer indole synthesis (FIS) is said to have a wide range of applications. This includes the synthesis of indole rings that are often present in the overall synthesis of natural products as a framework. Those are particularly found in the alkaloid domain, which comprises a ring system known as an indole alkaloid.

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The ability to use a wide range of alcohols instead of their oxidized equivalents is an obvious advantage of this process. The sequence can be performed in one pot, tolerates substitution on both hydrazine and alcohol, and gives moderate to excellent yields to the indoles. Synthesis is often done by subjecting the equimolar mixture of aryl hydrazine and aldehyde or ketone directly to the conditions of idolization without hydrazone isolation. Similarly, aryl hydrazone, prepared by a reduction of the connected aryl diazonium salt or N-nitroso arylalkylamine salt, can be directly subjected to idolization conditions in the presence of carbonyl without aryl hydrazone isolation.

The Fischer Indole Synthesis’s Main Features include:

The indole formation can be carried out in one pot, as the intermediate aryl hydrazones need not be isolated.

Two region-isomeric 2,3-disubstituted indoles are provided by unsymmetrical ketones with region-selectivity depending on medium acidity, hydrazine substitution, and steric effects.

1,2-diketones can provide both mono and bis-indoles, usually forming mono-indoles in refluxing alcohols with strong acid catalysts.

Fischer Indole Synthesis Mechanism

Fischer indole synthesis converts aldehyde or ketone aryl hydrazones into aryl hydrazones in the presence of an acid catalyst, Indoles.

The arylhydrazone, prepared from the condensation of the aryl hydrazine and carbonyl compound, is protonated and isomerized to the receiver of the enamine. An irreversible electrocyclic rearrangement is then subjected to the protonated enamine tautomer, which is [3,3]-sigmatropic rearrangement, where the N-N bond is broken.

A cyclic amino acetal (or aminal) forms the resulting imine, which removes NH 3 under acid catalysis, resulting in an energetically favorable aromatic indole. The Fischer indole synthesis in which an aromatic phenylhydrazone is heated in acid is the most useful route to the indoles. The condensation product of phenylhydrazine and an aldehyde or a ketone is phenylhydrazone. A cyclic rearrangement mechanism is involved in ring closure.

Fischer Indolization

A convergent approach known as Fischer Indole Synthesis has been developed to access the fused indoline ring system found in a multitude of bioactive molecules. The approach involves the condensation of hydrazines with latent aldehydes via an interrupted Fischer indolization sequence. This eventually delivers indoline-containing products. The method is convergent, mild, easy to operate, broad in reach, and can be used for accessing enantioenriched goods.

The first catalytic Fischer asymmetric Indolization has been made. 4-substituted cyclohexanone-derived phenylhydrazones undergo strongly enantioselective indolization in the presence of a 5 mol percent loading of a novel spirocyclic chiral phosphoric acid. The addition of a weakly acidic cation exchange resin, which removes the ammonia produced, has achieved efficient catalyst turnover. The reaction can be performed under mild conditions and gives different genera of 3-substituted tetrahydro carbazoles. 

An effective way to create fused indoline ring systems present in a variety of natural alkaloids is the interrupted Fischer indolization. In alkaloids and perophoramidine, a succinct approach to the complete synthesis of communes is given. The technique is based on the use of the disrupted Fischer indolization to create the natural products’ tetracyclic indoline nucleus. Studies will be presented to test the reach and limitations of this strategy.

The disrupted Fischer indolization reaction could also be used to complete the formal overall synthesis of the natural product pyrrolidinoindoline debromoflustramine B (5) Pyrrolidinone was generated using a standard two-step sequence to hemiaminal 40.

Fischer Indole Synthesis Procedure

A heterocycle of considerable significance to biological systems is Indole. In proteins, one of the main charge carriers involved in electron transfer is the redox-active indole side-chain of tryptophan 1,2. The optical properties of indole make tryptophan one of the principal intrinsic fluorophores in the study of protein fluorescence. Indole is generated via the intermediate molecule indole pyruvic acid by reductive deamination of tryptophan. Tryptophanase catalyzes the deamination mechanism from which the amine (-NH 2 ) group of the tryptophan molecule is removed. Indole, pyruvate, ammonium and water are the final reaction components.

The Fischer indole synthesis in which an aromatic phenylhydrazone is heated in acid is the most useful route to the indoles. An achiral molecular unit is the parent indole; the creation of chiral products using a FIS is by applying alpha-branched carbonyl molecules.

FAQs on Fischer Indole Synthesis

Q1. What is the Initial Material which is used in Fischer Indole Synthesis? 

Ans: An effective catalyst for aerobic dehydrogenation of the 3° indolines to the corresponding indoles is a transition-metal/quinone complex. In the synthesis of main intermediates into pharmaceutically essential molecules, the usefulness of the process is demonstrated.

Tetramethylammonium fluoride (TMAF) allows various amides, indoles, pyrroles, imidazoles, alcohols, and thiols to be directly and selectively methylated. Operational simplicity, broad reach, and ease of purification define the process.

Q2. Why do Bacteria Produce Indole?

Ans: Indole is a direct result of amino acid catabolism, multidrug export signals, inhibition of cell division, tolerance to stresses, and formation of biofilm. Escherichia coli tryptophanase produces indole from tryptophan and exports it via the AcrEF pump. To guide transcription of tnaAB, astD, and gab in E, Indole acts as an autoinducer. Indole signaling increases the development of indole itself as that encodes tryptophanase.

During biofilm formation, the molecule indole was shown to activate the signaling cascade. Bacteria could also be further processed to create different derivatives that could be involved in biofilm formations.

Indole-3-acetonitrile (IAN) has been found mainly in plants of the family Brassicaceae, including Arabidopsis thaliana and Brassica Chinensis,86 which in adequate amounts, produce a wide range of indole compounds.

Fischer Indole Synthesis in the Gas Phase, the Solution Phase, and at the Electrospray Droplet Interface

• Focus: Bio-Ion Chemistry: Interactions of Biological Ions with Ions, Molecules, Surfaces, Electrons, and Light : Research Article
• Published: 13 February 2017
• Volume 28 , pages 1359–1364, ( 2017 )

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• Ryan M. Bain 1 ,
• Stephen T. Ayrton 1 &
• R. Graham Cooks 1  

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Previous reports have shown that reactions occurring in the microdroplets formed during electrospray ionization can, under the right conditions, exhibit significantly greater rates than the corresponding bulk solution-phase reactions. The observed acceleration under electrospray ionization could result from a solution-phase, a gas-phase, or an interfacial reaction. This study shows that a gas-phase ion/molecule (or ion/ion) reaction is not responsible for the observed rate enhancement in the particular case of the Fischer indole synthesis. The results show that the accelerated reaction proceeds in the microdroplets, and evidence is provided that an interfacial process is involved.

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Introduction

In the course of spray-based ionization, such as desorption electrospray ionization (DESI) [ 1 ] or paper spray ionization [ 2 ], derivatization reactions can be performed in order to improve the limits of detection for compounds that are difficult to ionize [ 3 – 5 ]. This is done during DESI by adding a reagent to the primary spray solvent, which desorbs the analyte from the surface and into the secondary droplets, where the reaction occurs during passage to the mass spectrometer (MS) [ 6 ]. In paper spray ionization, reactions are often performed by adding reagents to the paper substrate prior to analysis, and the reaction either occurs in the thin film on the paper or in the droplets after they are emitted from the paper tip [ 7 – 10 ]. Derivatizing reagents for ambient ionization mass spectrometry, organized by specific functional group, have been cataloged in a review article [ 3 ].

Electrospray ionization mass spectrometry (ESI-MS) is gaining recognition as a robust tool for reaction monitoring [ 11 – 15 ] and, alternatively, as a method for rapid screening of reactions to establish whether products are generated [ 16 , 17 ]. In the first application, reaction products are simply sampled from the reaction mixture, whereas in the latter, products can be generated during the ESI event. Different conditions (initial droplet size, solvent, concentrations of reagents, droplet flight time, etc.) control these two particular outcomes. Numerous reactions (a score or so) have been found to have significantly faster rates in ESI droplets when compared with the corresponding bulk-phase reactions, and a number of these reactions have been cataloged in recent review articles [ 12 , 18 ]. Amongst these accelerated reactions are quinolone and isoquinolone synthesis [ 19 ], Hantzsch synthesis of 1,4-dihydropyridines [ 20 ], hydrazone formation [ 6 , 21 ], and Claisen-Schmidt condensation [ 22 , 23 ]. The studies of the effects of pH [ 24 ], concentration [ 7 ] and surface activity [ 25 ] (a description of the likelihood of a given species being at the surface of a droplet) in small droplet systems suggested that the underlying cause of reaction rate acceleration lies in surface effects. Studies using acoustically levitated droplets [ 26 ], Leidenfrost levitated droplets [ 27 ], microfluidics [ 28 ], thin films [ 29 ], and “on water” chemistry [ 30 , 31 ] have helped characterize acceleration phenomena in confined volumes.

It is known that the pH at the surface of electrosprayed droplets decreases [ 32 – 34 ] during the electrospray ionization (ESI) process. Droplets likely undergo desolvation and fission during their flight time [ 35 – 38 ]. Correlations between surface activity and reaction acceleration in ESI support the hypothesis that reaction acceleration is connected with surface activity [ 39 , 40 ]. The main models of ESI for small molecules (the charge residue and ion evaporation models [ 36 , 39 – 41 ]) suggest that at some point in the process of ionization, surface active molecules exist in a partially solvated form. In the charge residue model, solvent molecules continuously evaporate to yield dry ions, so there is presumably a point in the process when ions are partially solvated. In the alternative ion evaporation model, ions exist at the surface of charged droplets, where they are thought to be partially solvated at the air–droplet interface; dry ions are subsequently ejected by Coulombic forces [ 40 ]. In some cases, it has been shown that the acceleration of reactions in electrospray droplets requires that the distance between the nESI emitter and the ion transfer capillary be increased well beyond normal operating distances [ 20 , 21 , 24 ]; therefore, observed acceleration factors in chemical reactions under these conditions are thought to be due to reactions of partially solvated ions (increased distance between the nESI emitter and ion transfer capillary corresponds to more time for droplet evaporation as well as time for reaction). However, a gas-phase reaction mechanism is also possible since gas-phase ion/molecule reactions are much faster than solution phase reactions [ 42 ].

This study explores the possible contributions of a gas-phase mechanism by examining a reaction that yields different products in the gas phase from the solution phase. The Fischer indole synthesis was selected for its well documented and distinctive gas-phase ion chemistry [ 43 , 44 ]. In the gas phase, phenylhydrazine ( 1 ) and acetone ( 2 ), when combined, immediately form the protonated acetone phenylhydrazone ( 3a ) . This species has been reported to instantly cyclize to ( 3b ) based on evidence from mass spectrometry [ 43 , 44 ]. This cyclization leaves the isomeric species 3a and 3b indistinguishable. Evidence for this proposal comes from gas-phase collision-induced dissociation (CID) of the ion 3b , which gives a fragment ion shown to have the same structure as protonated methylindole ( 4 ). Interestingly, under the conditions used here, the solution-phase reaction does not proceed to 4 , even upon overnight reflux; instead, it forms a mixture of isomeric enamine and imine products ( 5 & 6 ) (Scheme  1 ). The solution-phase formation of 5 and 6 is presumably due to excess acetone in solution.

Two routes by which phenylhydrazine ( 1 ) and acetone ( 2 ) react in two phases

Experimental

An LTQ and LTQ-XL-Orbitrap (Thermo Scientific, San Jose, CA, USA) fitted with nanoelectrospray ionization (nESI) [ 45 ] sources were used for these experiments. The nESI emitter was a borosilicate glass capillary (1.5 mm o.d., 0.86 mm i.d.; Sutter Instruments, Novato, CA, USA) pulled to a tip of approximately 5 μm. A potential of 2.0 kV was applied to the inserted platinum wire to produce an electrospray. Experiments in which the emitter to MS entrance distance was varied (Figure  1 ) were performed using a modified moving stage with a 3D printed electrode holder to maintain alignment (See Figure  S1 in Supplementary Information).

Effect of distance between the sprayer and ion transfer capillary on the progress of the Fischer indole synthesis reaction. At greater distance, the products are more abundant than at shorter distances, indicating that the electrospray process is accelerating the reaction

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Reactions were prepared by combining 10 μL phenylhydrazine (Eastman, Kingsport, TN, USA), 200 μL acetone, and 50 μL 1.0 M HCl in methanol (prepared by diluting 37% HCl (Mallinckrodt, St. Louis, MO, USA) in methanol to 1.0 M). An excess of acetone was used unlike a standard Fischer indole synthesis. The excess acetone allows the second (and, upon additional reflux of 48 h, the third) acetone addition(s), which compete with and prevent the formation of the standard methyl indole product ( 4 ).

Results and Discussion

Collision-induced dissociation of 3b.

Analysis by nESI of the freshly prepared reaction mixture yielded a signal corresponding to 3b at m/z 149, which dominates the full-scan mass spectrum (Figure  2a ). This reaction product was isolated and fragmented using CID, and it lost ammonia to produce the previously reported [ 43 , 44 ] methyl indole product ion, 4 (Figure  2b ). The ion at m/z 134 is thought to be an impurity arising from the reaction of residual aniline in the stock phenylhydrazine with acetone to form the corresponding imine. It is also possible—and more likely—that this signal arises from in situ formation of aniline from phenylhydrazine followed by aniline’s subsequent reaction with acetone. The molecular formula of 4 was verified using an LTQ-XL-Orbitrap, which confirmed the assignment C 9 H 12 N + with an error of 0.119 ppm. The MS/MS scan provided a spectrum identical to authentic material prepared from aniline and acetone (Supplementary Figure  S2 ).

(a) Full scan mass spectrum of freshly prepared reaction mixture with base peak corresponding to 3 and with 5 and/or 6 as well as m/z 134 resulting from a side reaction between acetone and aniline. (b) Product ion scan of isolated m/z 149 with a 1.0 Thomson isolation width and a normalized collision energy of 25 [arb units] produces primarily compound 4

The role of a possible gas-phase reaction in the open air (prior to entering the ion transfer capillary) was then explored. This was done by allowing acetone and phenylhydrazine vapor to mix from two separate, open vials. A corona discharge was struck between a platinum electrode and the ion transfer capillary to facilitate atmospheric pressure chemical ionization (APCI). The full scan mass spectrum showed 3 as the dominant species, indicating that the gas-phase reaction produces only this product. The absence of ions 5 and 6 even in the more selective MS/MS mode is noted. A product ion scan from isolated 3b produces methylindole ( 4 ) by elimination of ammonia as expected.

Solution Phase Imine/Enamine Formation

Reactions performed in the bulk solution phase were analyzed after 2 d of standing at room temperature (Figure  3a ). Analysis of the solution by MS showed that the reaction provides 5 and/or 6 . This demonstrates that the reaction in solution does not proceed to methylindole over time but instead favors enamine formation ( 5 and/or 6 ), represented in the mass spectrum as m/z 189.

(a) Full scan MS of the reaction mixture allowed to stand for 48 h provides the ions 5 and/or 6 exclusively. (b) Product ion scan of isolated m/z 189 shows the loss of ammonia ( 7 ) and the loss of 56 ( 8 ) as explained in Scheme  2

When the reaction mixture was heated to reflux (24 h), it yielded mainly the ion at m/z 189, as well as the product of an additional acetone addition at m/z 229 (see Supplemental Information ). Further reflux (48 h) and addition of acetone promotes the conversion of m/z 189 to 229. The product ion scan of the isolated ion at m/z 189 yielded fragment ions due to the loss of neutral species with masses of 17 and 56 Da. The loss of 17 corresponds to the loss of ammonia, yielding the acetone imine of methylindole ( 8 ). The loss of 56 is not a subsequent fragmentation of the enamine of methylindole (confirmed by MS 3 ); rather, it corresponds to protonated 2-aminoindole ( 7) . The suggested structure of compound 7 arises from a postulated fragmentation mechanism, whereby a rearrangement is followed by elimination of butene as shown in Scheme  2 .

Postulated fragmentation mechanism to form 2-amino indole (7) by rearrangement followed by loss of neutral butene. The charge site is not known

Compound 7 has previously been reported as a product of thermal activation of an intermediate species in the reaction of phenylhydrazine and acetone [ 44 ] when nebulized by electrospray; however, the phase in which it forms (solution/gas phase) had not been delineated. This work shows that a solution-phase mechanism was likely to be responsible for the formation of the precursor ion that fragments to produce 7 in the previous literature.

Reaction Acceleration in Charged Droplets

A significant excess of acetone was required to observe accelerated formation of the methyl amino-indole ( 3 ) with distance. This is most likely due to the volatility of acetone compared with the phenylhydrazine and methanol. At lower concentrations, the acceleration with distance decreases and eventually ceases. This suggests that, as expected, nESI can be used safely to monitor reaction kinetics provided the limitations imposed by possible reaction acceleration are recognized and avoided. The acceleration effect can be seen in Figure  4 , and the acceleration factor is 10 if the m/z 189 (corresponding to 5 / 6 ) is added back. That is, the acceleration factors was estimated as the ratio of product ( 5 + 6 + 7 )/starting material ( 3 ) in the droplet phase as opposed to bulk solution. This acceleration shows that these accelerated reactions favor a solution-phase droplet mechanism over an alternative gas-phase mechanism. This is evident from the fact that the two reagents in the gas phase produced 3 and not 5 / 6 ; the ESI droplet experiment (using extended distance between spray emitter and ion transfer capillary) produced accelerated formation of 5 and/or 6 with respect to 3 .

NanoESI of fresh reaction mixture at a distance of 0.5 cm (a) and 7 cm (b) between the ion transfer capillary and the tip of the nESI source shows an increase in the abundance of 5 and/or 6 with respect to 3 (an impurity reacting with acetone at m/z 134 and the fragment of 5 ( 7 ) are also present in the full scan spectrum). The spectra are each an average of 10 scans at unit resolution

In an effort to probe reaction at the surface of the droplet, two different surfactants (triton x-100 and pentadecanoic acid) were doped into the reaction mixture. Fresh reaction mixtures were prepared and surfactant was added to the mixture at 1% v/v before analysis. The spectra acquired in the presence of surfactant were of lower quality and contained many interfering peaks; however, the peaks of interest were significant and reproducible in the MS full scan. Surfactants reduced the surface tension of the droplet, creating smaller droplets sooner [ 46 ]. This is because the Rayleigh limit (the point at which droplets undergo fission) is reached faster as the surface tension is reduced. Surfactants decrease surface tension and the distance required to achieve equal acceleration effects without surfactants falls from 7 to 3 cm. Interestingly, as the distance was increased further (to the 7 cm distance, which yielded acceleration without surfactant, the spectrum recorded using surfactant was again dominated by 3 . This is interpreted as indicating a contribution of a gas-phase reaction to the observed mass spectrum, as the droplets were allowed to completely desolvate, yielding dry reagent ions, which produced the gas phase product, 6 . This result was confirmed to be an artifact of the spray process and not due to bulk-phase reaction by returning the sprayer to the 3 mm distance and repeating the distance experiment and acquiring the same result as previously discussed. This observation is significant as it bolsters the hypothesis that the observed acceleration does indeed proceed via a solution-phase mechanism.

The formation of reaction products 5 and 6 can be accelerated by increasing the distance between the nESI emitter and the ion transfer capillary. As this variant of the Fischer indole synthesis forms a different final product in solution as opposed to gas phase, it has been shown that the reaction accelerated in ESI favors the solution-phase product over the ion-molecule gas-phase products. When surfactants are added, acceleration can be achieved at a smaller distance between the ion transfer capillary and the nESI emitter compared with the reactions accelerated without surfactants, pointing to the importance of surface reactions.

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Bain, R.M., Ayrton, S.T. & Cooks, R.G. Fischer Indole Synthesis in the Gas Phase, the Solution Phase, and at the Electrospray Droplet Interface. J. Am. Soc. Mass Spectrom. 28 , 1359–1364 (2017). https://doi.org/10.1007/s13361-017-1597-z

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Received : 28 November 2016

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DOI : https://doi.org/10.1007/s13361-017-1597-z

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Exploring the World of Indole: Synthesis, Chemistry and Biofunctions

Indole, with the chemical formula C 8 H 7 N, is a remarkable aromatic and heterocyclic organic compound. Its unique structure features a fused combination of a six-membered benzene ring and a five-membered pyrrole ring. The advancement of indole chemistry can be attributed to the widespread use of the renowned indigo dye. In 1886, Baeyer successfully derived indole from indigo by employing isatin and oxindole, and he further elucidated its structural composition. Indole is naturally present in various sources, including the high-boiling fraction of coal tar, jasmine oil, diverse floral species, pitch, and even in fecal matter, typically in concentrations of up to 2.5%.

This comprehensive article provides a detailed overview of indole, covering its general information, physico-chemical properties, an exploration of its biofunctions, tests for indole detection, chemical properties, synthesis methods, a conclusive summary, and a bibliography.

General Information About Indole [1-6]

Other synonyms names of Indole are: 2,3-Benzopyrrole; 1-Benzazole; Ketole; 1-Azaindene; Benzopyrrole; 2,3-Benzopyrole

IUPAC Name of Indole: 1H-indole

CAS number is 120-72-9

Physico-Chemical Properties of Indole [1-6]

• Molecular Formula C 8 H 7 N
• Molar Weight 117.15 g/mol
• Boiling point 254-254 °C
• Melting Point 52-52.5 °C
• Solubility: hot water and 0.36 g/100 g (at 25 °C); hot alcohols (35.9 g/100 g ethanol at 20 °C); ether; benzene; toluene; naphtha; Soluble in fixed oils; propylene glycol; insoluble in mineral oil and glycerol.
• Color/Form: Colorless to yellowish scales, turning red on exposure to light and air.
• Odor: Unpleasant odor at high concentration, odor becomes floral at higher dilutions

Structural formula present on Figure 1 .

Crystalline solid of the Indole can be seen in the picture provided in Figure 2 .

General Chemical Information of Indole [7-9]

This compound falls under the category of organic substances known as indoles. Indoles are compounds that contain an indole component, which is composed of a pyrrole ring fused to a benzene ring, resulting in the formation of 2,3-benzopyrrole. Indole, with the chemical formula C 8 H 7 N, is an aromatic and heterocyclic organic compound. It possesses a bicyclic structure, comprising a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environment and can be synthesized by various bacteria. As an intercellular signaling molecule, indole regulates numerous aspects of bacterial physiology, including spore formation, plasmid stability, drug resistance, biofilm formation, and virulence. Tryptophan, an amino acid, is an indole derivative and serves as the precursor for the neurotransmitter serotonin.

Indole exists as a solid at room temperature. It occurs naturally in human feces, emitting a distinct fecal odor. However, at very low concentrations, it exhibits a floral fragrance and is utilized as a constituent in many perfumes. It is also found in coal tar.

The corresponding substituent for indole is referred to as indolyl. Indole undergoes electrophilic substitution, primarily at position 3. Substituted indoles serve as structural components of tryptophan-derived tryptamine alkaloids, including serotonin (a neurotransmitter), melatonin (a hormone), dimethyltryptamine, and psilocybin (naturally occurring psychedelic drugs). Other indolic compounds include the plant hormone auxin (indolyl-3-acetic acid, IAA), tryptophol, the anti-inflammatory drug indomethacin, and the beta-blocker pindolol.

The name “indole” is a combination of the terms “indigo” and “oleum” since indole was first isolated by treating indigo dye with oleum.

The study of indole chemistry originated from the investigation of the dye indigo. Indigo can be transformed into isatin and subsequently into oxindole. In 1866, Adolf von Baeyer reduced oxindole to indole using zinc dust. In 1869, he proposed a formula for indole.

Certain derivatives of indole played a crucial role as dyes until the late 19th century. In the 1930s, interest in indole grew when it was discovered that the indole substituent is present in many significant alkaloids, known as indole alkaloids (such as tryptophan and auxins). Today, indole chemistry remains an active area of research.

Biofunction of Indole

Indole is synthesized through the shikimate pathway using anthranilate. It serves as an intermediate in the biosynthesis of tryptophan, where it remains within the tryptophan synthase molecule after the removal of 3-phospho-glyceraldehyde and before the condensation with serine. When the cell requires indole, it is typically produced from tryptophan through the action of tryptophanase.

As an intercellular signaling molecule, indole plays a regulatory role in various aspects of bacterial physiology, encompassing spore formation, plasmid stability, drug resistance, biofilm formation, and virulence. Numerous derivatives of indole hold significant cellular functions, including functioning as neurotransmitters like serotonin.

Tests for Indole [9]

Ehrlich’s Test Figure 4

This test illustrates the capability of certain bacteria to break down the amino acid tryptophane into indole, which accumulates in the medium. The production of indole serves as a significant criterion in the identification of Enterobacteria. Most strains of E. coli, P. vulgaris, P. rettgeri, M. morgani, and Providencia species possess the enzymatic machinery to degrade tryptophane, resulting in the release of indole. This process involves a cascade of intracellular enzymes collectively known as “tryptophanase.” The test is commonly employed as part of the IMViC procedures, designed to differentiate members of the Enterobacteriaceae family.

In the case of non-fermenters and anaerobes, a modified version of the test utilizing Ehrlich’s reagent (employing ethyl alcohol instead of isoamyl alcohol) is employed.

Principle of Ehrlich’s Indole Test: Tryptophane, an amino acid, can undergo deamination and hydrolysis by bacteria that express the enzyme tryptophanase. The enzymatic action leads to the generation of indole through reductive deamination, with indolepyruvic acid serving as an intermediate molecule. Tryptophanase catalyzes the deamination process, where the amine (-NH 2 ) group of the tryptophane molecule is removed. The end products of this reaction include indole, pyruvic acid, ammonium (NH 4 + ), and energy. The presence of pyridoxal phosphate as a coenzyme is necessary for the enzymatic activity.

When indole is mixed with Kovac’s Reagent, consisting of hydrochloric acid, p-dimethylaminobenzaldehyde, and amyl alcohol, the solution undergoes a color change from yellow to cherry red. Since amyl alcohol is not soluble in water, the red coloration forms as an oily layer on top of the broth. In the spot test, indole reacts with p-Dimethylaminocinnamaldehyde (DMACA) in the filter paper matrix under acidic conditions, resulting in the formation of a blue to blue-green compound. This Indole Spot Reagent has been proven effective in detecting indole production among members of the Enterobacteriaceae family and certain anaerobic species.

Chemical Properties of Indole

In contrast to most amines, indole does not exhibit basic properties. The aromatic nature of the ring, similar to pyrrole, prevents the lone pair of electrons on the nitrogen atom from being available for protonation. However, strong acids like hydrochloric acid can protonate indole. Interestingly, indole is primarily protonated at the C3 position rather than N1 due to the enamine-like reactivity of the portion of the molecule located outside the benzene ring. The protonated form of indole has a pKa of -3.6. The sensitivity of many indolic compounds, including tryptamines, to acidic conditions is attributed to this protonation phenomenon.

In terms of reactivity, indole shares similarities with benzene but is generally more reactive. The lone pair of electrons on the nitrogen atom in indole contributes to an aromatic sextet, which hinders easy protonation and imparts a lack of basic properties. However, in the presence of strong bases, indole exhibits weak NH-acidic properties.

As a weak acid, indole can form N-sodium indole in a solution containing sodium in liquid ammonia (NH 3 ), and N-potassium indole with the addition of potassium hydroxide (KOH) at a temperature of 130 °C. ( Figure 6 )

Acetylation of indole takes place at position 3 when using acetic acid, while in the presence of sodium acetate (CH 3 COONa), acetylation occurs at position 1. Acetic anhydride leads to the formation of 1,3-diacetylindole. Indole readily undergoes attachment to unsaturated ketones and nitriles through the alpha, beta double bond. It’s worth noting that indole exhibits acidophobic properties, meaning it has a tendency to avoid or resist interactions with acids. Figure 7.

Indole exhibits aromatic characteristics due to its ring structure. Electrophilic substitution reactions primarily take place at the carbon atom located at position 3. Nitration of indole is commonly achieved using benzoyl nitrate as the reagent of choice. Figure 8 .

The bromination of indole using dioxane dibromide. Figure 9

The chlorination of indole utilizing SO 2 Cl 2 . Figure 10

The alkylation of indole employing highly reactive alkyl halides. Figure 11

The aminomethylation of indole through the Mannich reaction. Figure 12

The Vilsmeier reaction involving indole with DMF (dimethylformamide) and POCl 3 (phosphoryl chloride) is a synthetic method used to introduce a formyl group (CHO) onto the indole ring. In this reaction, DMF acts as a reactant and a solvent, while POCl 3 serves as a reagent for the formylation process. The Vilsmeier reaction with indole, DMF, and POCl 3 results in the formation of N-formylindole. This reaction is commonly employed in organic synthesis to modify the indole structure and create functionalized derivatives. Figure 13

In the process of hydrogenation, when indole is subjected to mild conditions, the reduction selectively occurs on the pyrrole ring. However, under more severe conditions, both the pyrrole and benzene rings undergo reduction. Figure 14

The dimerization of indole refers to the chemical reaction in which two indole molecules combine to form a dimeric compound. This reaction can occur through various methods, such as oxidative coupling or acid-catalyzed condensation. The resulting dimeric product can exhibit unique chemical properties and potentially serve as a building block for the synthesis of more complex indole derivatives. The dimerization of indole represents an important avenue in indole chemistry, providing opportunities for the creation of novel compounds with diverse applications in pharmaceuticals, materials science, and other fields. Figure 15

The azo coupling of indole involves the reaction of an indole compound with an aromatic diazonium salt, resulting in the formation of an azo compound. This reaction typically occurs under mild conditions and is facilitated by a suitable coupling agent or catalyst. The azo coupling of indole allows for the introduction of azo functional groups into the indole structure, leading to the formation of novel compounds with diverse properties. Figure 16

The oxidation of indoles utilizing potent oxidizing agents results in the cleavage of the pyrrole ring. This reaction leads to the fragmentation of the indole structure, resulting in the formation of distinct chemical species. The use of strong oxidizing agents facilitates the breaking of chemical bonds within the pyrrole ring, leading to the generation of diverse products with altered properties. Figure 17 .

The sulfonation of indole using pyridine sulfotrioxide is a chemical reaction that introduces a sulfonic acid group (-SO 3 H) onto the indole molecule. Pyridine sulfotrioxide acts as a sulfonating agent, facilitating the addition of the sulfonic acid group to the indole ring. Figure 18

Synthesis of Indole [10-16]

Indole, along with its various derivatives, can be synthesized through a diverse array of methods. The primary pathways employed in industrial settings involve commencing with aniline and subjecting it to vapor-phase reactions with ethylene glycol, facilitated by catalysts. Typically, these reactions are carried out within the temperature range of 200 to 500 °C. Remarkably, the yields achieved can reach an impressive 60%. Additionally, other compounds such as formyltoluidine, 2-ethylaniline, and 2-(2-nitrophenyl)ethanol can serve as precursor materials for indole synthesis, undergoing cyclization reactions to yield the desired product. Figure 19

The Leimgruber–Batcho indole synthesis represents an efficient and reliable approach to the synthesis of indole and its substituted derivatives. Originally introduced in a patent publication back in 1976, this method stands out for its high-yielding nature and ability to generate substituted indole compounds. It holds particular significance within the pharmaceutical industry, as numerous pharmaceutical drugs incorporate specifically substituted indoles in their molecular structures. The Leimgruber–Batcho indole synthesis involves a series of organic reactions that transform o-nitrotoluenes into indole compounds. The initial step entails the formation of an enamine through the utilization of N,N-dimethylformamide dimethyl acetal and pyrrolidine. Subsequently, in the second step, the desired indole is produced through reductive cyclization. The reductive cyclization step can be achieved using various agents, including Raney nickel and hydrazine as illustrated in the provided scheme. Alternatively, palladium-on-carbon with hydrogen, stannous chloride, sodium hydrosulfite, or iron in acetic acid can also serve as effective reducing agents in this process. Figure 20

The Fischer indole synthesis is a notable chemical transformation that enables the production of the aromatic heterocycle known as indole. This reaction involves the interaction of a (substituted) phenylhydrazine with an aldehyde or ketone under acidic conditions. The discovery of this reaction is credited to Emil Fischer in 1883. Presently, the synthesis of antimigraine drugs belonging to the triptan class often employs the Fischer indole synthesis as a key step. Various catalysts can facilitate this reaction, including Brønsted acids such as HCl, H 2 SO 4 , polyphosphoric acid, and p-toluenesulfonic acid, as well as Lewis acids like boron trifluoride, zinc chloride, iron chloride, and aluminum chloride. These catalysts play a crucial role in promoting the conversion of the reactants into the desired indole compound. Figure 21

Indole is a versatile and significant heterocyclic compound that plays a crucial role in various fields, including pharmaceuticals and organic synthesis. Its synthesis can be achieved through several efficient methods, such as the Leimgruber-Batcho indole synthesis and the Fischer indole synthesis. These methods enable the production of indole and its substituted derivatives with high yields. The pharmaceutical industry particularly benefits from indole synthesis, as many drugs incorporate specifically substituted indole moieties for their desired biological activity. The reductive cyclization and enamine formation steps are key aspects of indole synthesis, and various catalysts, both Brønsted acids and Lewis acids, can be employed to facilitate the reactions. Overall, indole and its derivatives continue to be of significant interest in scientific research and drug development, showcasing their importance in the field of organic chemistry.

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In 1883, E. Fischer and F. Jourdan treated pyruvic acid 1-methylphenylhydrazone with alcoholic hydrogen chloride and generated 1-methylindole-2-carboxylic acid. Preparing indoles by heating the arylhydrazones of either aldehydes or ketones in the presence of a protic or Lewis acid is now known as the Fischer indole synthesis. Since its discovery, it has remained the most important method of preparing substituted indoles.

Main features of the Fischer indole synthesis:

• The indole formation can be carried out as a one-pot synthesis, as it is not necessary to isolate the intermediate arylhydrazones
• Unsymmetrical ketones give two region-isomeric 2,3-disubstituted indoles, with the region-selectivity dependent on acidity of the medium, substitution of the hydrazine, and steric effects
• 1,2-diketones can give both mono and bis-indoles, the mono-indoles usually forming with strong acid catalysts in refluxing alcohols

Mechanism of the Fischer indole synthesis

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A three-component Fischer indole synthesis

Vol: 3 (2008) > Issue: 8 (August)

Protocol | DOI: 10.1038/nprot.2008.94

• Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, New York, USA
• Present address: Novomer Inc., 950 Danby Road Suite 198, Ithaca, New York 14850, USA.

This protocol describes a three-component approach to multiply-substituted indoles from nitriles, organometallic reagents and arylhydrazine hydrochloride salts. The condensation of organolithium or Grignard reagents with nitriles produces

This protocol describes a three-component approach to multiply-substituted indoles from nitriles, organometallic reagents and arylhydrazine hydrochloride salts. The condensation of organolithium or Grignard reagents with nitriles produces metalloimines, which under acidic conditions and in the presence of arylhydrazines lead to arylhydrazones, the starting materials for the Fischer indole reaction. Combining this approach with the Fischer indole reaction produces indoles in an efficient, one-pot process. The procedure takes ∼20 h to complete: 3 h for metalloimine formation, 15 h for the Fischer indole reaction and 2 h for isolation and purification.

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Citations (9)

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Fischer indole synthesis applied to the total synthesis of natural products

First published on 15th November 2017

One of the oldest and most useful reactions in organic chemistry is the Fischer indole synthesis (FIS). It is known to have a wide variety of applications including the synthesis of indole rings, often present as the framework in the total synthesis of natural products, particularly those found in the realm of alkaloids, which comprise a ring system known as an indole alkaloid. In this review, we are trying to emphasize the applications of FIS as an old reaction, which is currently applied to the total synthesis of biologically active natural products and some other complex targets.

1. Introduction

Pd-promoted reaction is also a perfect approach for the synthesis of indoles. In this approach o -haloaniline is usually used as starting material, which upon treatment with appropriate unsaturated units, generates new carbon–carbon and carbon–nitrogen bonds for the construction of the indole core. 8–12

Buchwald et al. presented an approach for the syntheses of FIS precursors. They developed a Pd-catalyzed cross coupling reaction for the synthesis of N -aryl benzophenone hydrazones, which are used as common precursors in the typical FIS. 13 The same group also reported a facile, efficient and general Pd-catalyzed approach for the synthesis of a wide range of arylhydrazines or arylhydrazones, which are typical precursors for the FIS. 14

FIS which is rarely called Fischer indolization, has been accomplished first by Emil Fischer and Friedrich Jourdan in 1883. 15 FIS is significant among the well-established classical methods which efficiently results in the synthesis of the bioactive indole scaffold that is usually found in alkaloids and in different valuable medicament. 16,17 The FIS usually gives a facile, effective protocol for the conversion of enolizable N -arylhydrazones into indoles using an acid as catalyst. 18 In FIS, the selection of acid catalyst is very decisive. Brønsted acids such as HCl, H 2 SO 4 and PTS were frequently employed effectively in this reaction. 19,20 Lewis acids such BF 3 /etherate, ZnCl 2 , FeCl 3 , and AlCl 3 are also beneficial catalysts for this reaction. 21,22 Several reviews have been reported the selected examples of FIS acid catalysis. 23

A few arylhydrazines are commercially available; they are generally synthesized by reduction of aryl diazonium salts, which in turn can be provided from the appropriate aniline derivatives. Alternatively, aryl diazonium salts can directly be transformed to hydrazones via the Japp–Klingemann reaction. 24 The Japp–Klingemann reaction involves the reaction of the aryl diazonium salt with an active methylenyl or methinyl compound in the presence of an appropriate either acid or base to afford an azo compound, which under either basic or acidic, or even thermal conditions can be transformed into the corresponding hydrazone. 24

Indole is the most powerful pharmacodynamic core known in several naturally occurring compounds. 12–25 Indoles are known as privileged structures due to their unique roles in different biochemical procedures. 26,27

In the group of molecules having biological properties that were known for a long period of time, melatonin 3 as a common cycle of circadian, tryptophan as a vital amino acid utilized in sleep disorders and depressive states treatment, tryptamine 1 like serotonin 2 realized as growth factors in plants, are important neurotransmitter. Furthermore, strychnine 5 is a potent stimulant of central nervous system (CNS), LSD 6 is a powerful hallucinogen, and also reserpine was applied as antihypertensive ( Fig. 1 ). 28

Esrine appears in the Physostigma venenosums seeds has been suggested for the curing of Alzheimer's disease. Serotonin, the vasoconstrictor hormone, serves as a neurotransmitter in animals. Dimeric vinca alkaloids including vincristine and vinblastine extracted from Catharanthus roseus , which are utilized in cancer and Hodgkin's diseases treatment. 28

Due to the importance of indole derivatives, 29 it has been published different reviews in the synthesis of indoles and their applications in the total synthesis of natural product. 25,30–33 Because of the large number of biologically fascinating natural products containing poly-substituted indole moieties, FIS has attained significant synthetic attention. 34

In continuation of our interest in applications of name reactions in the total synthesis of natural products 35–47 and in the synthesis of heterocyclic systems, 48–52 in this review, we try to highlight the applications of FIS in total synthesis of biologically active natural products.

2. Applications of Fischer indole synthesis in the total synthesis of natural products using

2.1. aryl hydrazines.

The starting materials for the total synthesis of strychnine 5 was the 2-veratrylindole 10 that was synthesized by FIS from phenylhydrazine 8 and acetoveratrone 9 . The first steps in this methodology involves the introduction of the 2-aminoethyl chain to the β-position of 2-veratrylindole 10 . After several steps, compound 10 was transformed to strychnine 5 ( Scheme 1 ). 57,58

Aspidospermidine is a member of aspidosperma alkaloids including a pentacyclic ring system, which was separated from Aspidosperma (a genus of flowering plant in the family Apocynaceae) quebracho blanco and other aspidosperma species. 59,60 The total synthesis of alkaloids aspidospermine 11 was first achieved and reported in 1963 by Stork and Dolfini. 61 It was used in the treatment of erectile impotence, and decreasing the benign prostatic hyperplasia (BPH) symptoms, in guinea pig and also in rabbit corpus spongiosum and cavernosum due to its inhibition of smooth muscle contractions.

The parent indole is an achiral molecular unit; the formation of chiral products by using a FIS is by no means unusual. The application of α-branched carbonyl molecules for example can result in the synthesis of indolenine derivatives having a quaternary stereocenter in the 3-position. For the total synthesis of (±)-aspidospermine 11 , Stork and co-workers acquired merit of this reactivity. This method was started from butyraldehyde 12 , which transformed into complex cyclohexanone 13 upon several steps. The FIS of the cyclohexanone 13 and hydrazine 14 afforded the indolenine 15 that has been transformed into the desired 11 via imine reduction and N -acetylation ( Scheme 2 ). 61

Remarkably, in chemistry of alkaloid, attention in carbazole alkaloids has raised significantly throughout the past years because of the desired ability of novel kinds of pharmacologically active compounds. Therefore, for example various carbazoles, oxotetrahydrocarbazoles, tetrahydrocarbazoles, mukonine and glycozoline isomers, prenylcarbazoles, carbazomycins, amino-and nitrocarbazoles as well as pyrido[ b ]carbazole derivatives (such as ellipticines 16 and analogues) contain anti-convulsant, anti-tumour, anti-inflammatory, anti-histamine, psychotropic, antibiotic and fungistatic activities. 62

The tetracyclic natural product ellipticine (5,11-dimethyl-6 H -pyrido[4,3- b ]carbazole) 16 has been extracted in 1959 from the Ochrosia elliptica Labill plant material. 63 This small tropical evergreen tree goes to the Apocynaceae family and included various other alkaloids, involving 9-methoxyellipticine. As ellipticine 16 was extracted from various other Apocynaceae plants class ( Ochrosia acuminate , Ochrosia moorei and Ochrosia vieillardii ) and from strychnos dinkagei of the Loganiaceae class. The ellipticine class of complexes use their biological property through various styles of action, the most well-developed of that are insertion with topoisomerase II inhibition and DNA. Recently, other types of action were demonstrated, involving kinase inhibition, communication with bio-oxidation, p53 transcription factor and adduct formation. 64

As depicted in Scheme 3 , the total synthesis of ellipticine 16 initiated from the allylic alcohol 17 . A [3,3]-sigmatropic rearrangement with propionic anhydride 18 gave the carboxylic acid 19 . Next, the carboxylic acid 19 treated with phenylhydrazine 8 to provide phenylhydrazone 20 continued by the FIS to provide indole 21 . The latter afforded the corresponding natural product ellipticine 16 in several reaction steps. 65

A series of 11-alkylbenzo[ a ]carbazole derivatives 22 and their dihydro analogues have been prepared and examined for their binding attraction for the estrogen receptor and their antiestrogenic and estrogenic effects in the immature mouse. They also showed mammary tumor inhibiting property. Furthermore, benzo[ a ]-carbazole derivatives are flat polycycles, which contain the intercalating potential into the DNA. In 1986 von Angerer and Prekajac described the total synthesis of the 11-alkyl-11 H -benzo[ a ]carbazoles 22 . In this approach, the FIS of the arylhydrazine hydrochlorides 23 and the tetralones 24 gave the 5,6-dihydro-11 H -benzo[ a ]carbazoles 25 . Next, the latter has been transformed into the corresponding natural product 22b through subjection to different chemical reactions ( Scheme 4 ). 66

The Strychnos alkaloids are obtained from preakuammicine and appearently generated from secologanin and tryptophan through geissoschizine and strictosidine which is actually a monoterpenoid indole alkaloids biosynthetic pathway. Several mechanisms have been presupposed to interconnect those of the Strychnos type with the Corynanthe alkaloid geissoschizine, but the main features of the rearrangement to preakuammicine and dehydropreakuammicine stay still unknown. The Strychnos alkaloids have obtained less attention from synthetic standpoint than other kinds of indole alkaloids such as Yohimbe, Iboga , Aspidosperma and Corynanthe . 67

The indole alkaloids bearing a nonrearranged secologanin scaffold contain different structural varieties. Among these, the alkaloids of the uleine family (dasycarpidan stereoparent) and the Strychnos alkaloids along with the Aspidospermatan biogenetic subtype (condyfolan stereoparent) are identified by the presence of a 1,5-methanoazocino[4,3- b ] indole moiety having two-carbon chain, typically an ethyl group, at the bridge carbon.

An asymmetric total synthesis of the alkaloids of the uleine family, nordasycarpidone 26 , dasycarpidol 27 , dasycarpidone 28 and tubotaiwin 29 were achieved via formation of the tetracyclic intermediate 34 , that were synthesized through FI reaction. This approach was initiated from 4-piperidineacetates cis - 31 and trans - 31 , which formed from l-benzyl-3-ethyl-4-piperidone 30 and their transformation to the desired 4-acetonylpiperidines 32 . Then, the conversion of piperidine cis - 32 into the bridged 2-azabicyclo [3.3.1]-nonane 33 and the FIS of the latter afforded the desired compound 34 . The FIS of ketone 33 has been examined by applying different acid catalysts. The best consequence has been provided once the phenylhydrazone from 33 has been refluxed in acetic acid. Based on these reaction conditions the desired tetracycle 34 has been produced as the only isolable product, but in satisfactory yield. The methanoazocinoindole 34 as a usual main intermediate. This tetracyclic compound includes a C-20 ethyl group equatorial with respect to the piperidine ring, namely, with the identical relative stereochemistry as uleine, dasycarpidone, and the Aspidospermatan alkaloids. Therefore, after several steps, tetracycle 34 afforded the alkaloids nordasycarpidone 26 and dasycarpidone 27 in 73% and 76% yields, respectively. Lastly, NaBH 4 reduction of dasycarpidone 27 resulted in the alkaloid dasycarpidol 28 . On the other hand, methanoazocinoindole 34 , afforded (±)-tubotaiwin 29 upon several steps ( Scheme 5 ). 68,69

Archer and co-workers employed an identical approach for the synthesis of 5,11-demethylellipticines (9-hydroxy-6 H -pyrido[4,3- b ] carbazole). 68 In the current method, initially, the reaction of enamine 36 and methyl vinyl ketone 37 afforded a mixture of trans - and cis -ketones 38 that have individually transformed into indole 40 through FI reaction. Some of the nonlinear pyrido[3,4- c ]carbazole (17%) have been synthesized from the cis -ketone. Next, dehydrogenation and demethylation gave the corresponding natural product 9-hydroxy-6 H -pyrido[4,3- b ]carbazole 35 ( Scheme 6 ). 70

Indolocarbazole alkaloids exhibit an increasing figure of natural products extracted from slime molds, marine sources and soil organisms. Several of them exhibited significant biological property. For example, staurosporine and rebeccamycin are known as antitumor, protein kinase C and topoisomerase I inhibitors, respectively. Arcyriaflavin A 41 is proven to be an inhibitor of human cytomegalovirus replication. Particularly, a synthetic derivative, NB-506, is now under clinical trials as antitumor agents. 71

Similarly, a concise and extremely significant synthesis of indolo[2,3- a ]pyrrolo[3,4- c ]carbazole derivatives like arcyriaflavin A 41 via a double FIS has been effectively demonstrated by Bergman and Pelcman in 1989. 70,71 This approach is based on a double FIS of the bis(phenylhydrazone) 45 . The latter was synthesized through Diels–Alder reaction of market purchasable 2,3-bis(trimethylsilyloxy) butadiene 42 with the dienophiles 43 , affording the cycloadducts 44 . This cycloadduct was treated with phenylhydrazine 8 in acetic acid and MeOH to afford 45 . The double FIS of 45 using polyphosphoric acid trimethylsilyl ester (PPSE) as the cyclization agent, led to arcyriaflavin A 41 in 68% yield ( Scheme 7 ). The novel method gave a concise and very significant synthesis of indolo[2,3- a ]pyrrolo[3,4- c ]carbazole derivatives and appropriate in the synthesis of a large range of functionalized products and does not need expensive or in available starting compounds. 72

Physostigmine is an alkaloid which is extracted from the seeds of Physostigma venenosum (Calabal beans) and it is clinically effective as a anticholinergic drug. In addition, its enantiomer protects against organophosphate poisoning. Physostigmine's analogous have indicated a therapeutic power in Alzheimer's disease, 2 and recently is in phase II efficacy trials. Additionally, physostigmine has an important role in neuroscience such as a powerful potent morphine-like narcotic agonist activity of (−)-eserine in vivo . 74

On the other hand, in another route, the lactol 61 upon several steps provided (−)-esermethole 51 . Since 51 has formerly been converted into natural (−)-physostigmine 53 in two steps through (−)-eseroline 52 , these reactions are considered as steps of a formal synthesis of the natural product ( Scheme 9 ). 75

Bioactivity-directed isolation of the obtain of the cyanophyte Tolypothrix tjipanasensis has resulted in the isolation of novel N -glycosides of indolo[2,3- a ]carbazole derivatives planned tjipanazole derivatives Al, A2, B, Cl, C2, C3, C4, D, E, Fl, F2, Gl, G2, I and J. Tjipanazoles are known as novel antifungal agents extracted from the blue-green alga Tolypofhrix tjipanasensis . They showed moderate fungicidal property (strain DB-l-l) against Aspergillus flavus , Trichophyton mentagrophytes and Candida albicans . 76

The total synthesis of tjipanazole D 64 and E 65 has been achieved by Bonjouklian and co-workers in 1991, 75 which relied on FIS reaction. Initially, under air, two equivalents of p -chlorophenylhydrazine hydrochloride 66 and 1,2-cyclohexanedione 67 provided 6-chloro-1-(2-(4-chlorophenyl)hydrazono)-2,3,4,9-tetrahydro-1 H -carbazole 68 . The latter based on a FIS gave tjipanazole D 64 in 54% yield. Subsequently, tjipanazole E 65 has been extracted as a minor component through coupling reaction of 64 and 1-bromo-α- D -glucopyranosyl-2,3,4,6-tetraacetate 69 after elimination of the masking substituents ( Scheme 10 ). 76

A novel and efficient synthesis of the pyrrolo[4,3,2- d , e ] quinoline system was accomplished. It is a typical class of marine alkaloids contains the discorhabdins, prianosins and other antineoplastic. These include the discorhabdins, prianosins, damirones, iso-batzellines and batzellines which are extracted from wakayin and sponges, isolated from the Fijian ascidian Clauelina sp. Makaluvamine is a member of the pyrroloiminoquinone family and synthesis of makaluvamine D 70 , a mammalian topoisomerase II inhibitor is discovered by the sponge Zyzzya cf. marsailis . They also showed power in vitro cytotoxicity in the direction of the human colon tumor cell line HCT 116 and lead into cancer chemotherapy. 77

The ibophyllidine alkaloids compose a small group of indole alkaloids of the ibogan type which is explored during the last twenty years and identified by the presence of the structurally unusual pyrrolizino [l,7- c , d ] carbazole ring system. It actually containes a pyrrolidine D-nor ring instead of the piperidine ring that usually existed in the monoterpenoid indole alkaloids. Much research has been typifieded the synthesis of alkaloids with the former feature, by the Strychnos alkaloids and Aspidosperma although less attention has been performed to ibophyllidine alkaloids 5 but deethylibophyllidine is chosen as the synthetic target. 79

The total synthesis of (±)-deethylibophyllidine 78 was accomplished in eight steps starting from O -methyltyramine 79 in 5.5% overall yield in that regioselective FIS to a tetracyclic ring system as a main step. The synthesis was initiated by market purchasable O -methyltyramine (4-methoxyphenethylamine) 79 that has transformed into cis -octahydroindolone 80 by an enantioselective method in 54% overall yield in several steps. The FIS of the phenylhydrazone and ketone 80 occurred regioselectively in acetic acid as an acid catalyst and solvent to give the tetracyclic 81 in 60% yield. It is remarkable that the sulfoxide substituent did not endure Pummerer rearrangement in the presence of the acidic conditions needed for both the hydrolytic elimination of the enol ether and the FIS. β-Amino sulfoxide scaffold is more reactive than those sulfoxides bearing an electron-withdrawing group at the α-position. Thus, β-amino sulfoxide is useful from the synthetic point of view, thus employed in initial step of the synthesis with the requisite oxidation level at the methylene carbon linked to the nitrogen atom. In the following, after several steps the tetracyclic 81 was transformed into (±)-deethylibophyllidine 78 in 50% yield ( Scheme 12 ). 80,81

Murrayafoline A 82 , extracted from the root of various species of the genus Glycosmis , Murraya and Clausena (Rutaceae), displays potent fungicidal property against Cladosporium cucumerinum and growth inhibitory property on cell cycle M-phase inhibitory, human fibrosarcoma HT-1080 cells and apoptosis inducing properties on mouse tsFT210 cells. 82

In 1998, Murakami and co-workers described the six-step total synthesis of murrayafoline A 82 with 40% yield. 83 The current method initiated by the treatment of aminophenol 83 through the 2-hydrazino-5-methylphenol 84 to give the O -methanesulfonyl (mesyl) derivative 86 . Then, the provided O -mesylphenylhydrazone 86 has been exposed to FIS to give the tetrahydrocarbazole derivatives 87 and 88 in 63% and 3% yield, respectively. Lastly, after multi reactions the mesyloxy compound 87 gave murrayafoline A 82 ( Scheme 13 ). 83

A catalytic enantioselective synthesis of 20-deethyltubifolidine 89 by using the heterobimetallic enantioselective catalyst (ALB-KO- t -Bu-MS 4A) has been achieved in 1998. 84 Initially, the catalytic enantioselective Michael addition of cyclohexenone 90 and dimethyl malonate 91 , by utilizing AlLibis(binaphthoxide) complex (ALB) as catalyst, provided optically pure 92 in 99% ee and 94% yield even at ambient temperature. Then, optically pure compound 92 transformed into the indole derivative 93 in 92% yield, via an extremely regioselective FI approach continued through decarbalkoxylation (the ee of 93 has been shown to be 99%). Then, the latter has been transformed to 20-deethyltubifolidine 89 , through a multi-step reaction with an overall yield of 27% ( Scheme 14 ). 84

A catalytic enantioselective synthesis of the strychnos alkaloid tubifolidine 94 , was extracted from the leaves of pleiocarpa tubicina , has been accomplished in an extremely stereocontrolled method. 84 This total synthesis based on the chiral indoles 93 as a main intermediate to put the remaining enantioselective centers by using substrate control. This compound has been provided through an extremely regioselective FIS between ketone 92 , which has been provided through enantioselective Michael reaction, and phenylhydrazine hydrochloride in AcOH at 80 °C. The latter has lastly converted to the tubifolidine 94 via a multi-step synthesis in 24% overal yield ( Scheme 15 ). 84

Several indole alkaloids contain the basic tetracyclic framework with eventually groups for example a methyl substituent on nitrogen 5 or 12 and/or hydroxy or methoxy substituents on carbon 1, 2, 3 or 4. A 9-azabicyclo [3.3.1] nonanone was applied during a tried synthesis of ajmaline via a FI reaction. ( endo , endo )-9-benzyl-9-azabicyclo [3.3.1] nonane-2,6-diol 96 that already gave a simple admittance to enantioselective synthesis of indolizidine and quinolizidine alkaloids, can also be applied in principle for the formation of macroline/sarpagine type alkaloids. Ketone 97 has been produced in 37% yield from readily accessible ( endo , endo )-9-benzyl-9-azabicyclo [3.3.1] nonane-2,6-diol 96 after several steps.

Next, ketone 97 was treated with functionalized phenylhydrazines 98 under reflux in hydrochloric acid-saturated MeOH. The desired derivatives 99 were readily provided via an abnormal FI. Then two intermediates 95a and 95b were directly produced through Swern oxidation of 99a and 99b , respectively. These two intermediates can be utilized for the formation of macroline/sarpagine kind alkaloids ( Scheme 16 ). 85

The Aspidosperma group demonstrates one of the widest class of indole alkaloids, having more than 250 products extracted from different biological sources. An important member of this group is tabersonine 103 that exhibits a main role in the synthetic chemistry of Aspidosperma alkaloids and biosynthesis. Initially, tabersonine has been extracted from Amsonia tabernaemontana in 1954 by Le Men and co-workers. 86 Soon upon the first report, the alkaloid has been extracted from various other natural sources, demonstrating its relative biological abundance.

In 2001, Rawal and his group described the twelve-step enantioselective total synthesis of (±)-tabersonine 103 with overall yield of 70–80%. 87 The enantioselective total synthesis of racemic tabersonine was initiated from market purchasable monoacetal 104 . Upon several steps, carbamate 105 has been provided in 88% yield. Subsequent, reaction of the silyl enol ether 105 with dilute HCl provided a clean hydrolysis to bicyclic ketone 106 , with the cis stereochemistry intact. This transformation, sacrificed the double bond position, which has been accomplished via the first cycloaddition. In providing for FIS, ketone 106 has been transformed into the phenylhydrazone via heating it with phenylhydrazine hydrochloride 23a by using Na 2 CO 3 . In the following, the crude hydrazone has been refluxed at 95 °C in glacial AcOH. A weakly acidic medium found to favor idolization toward the more functionalized carbon. Based on these reaction conditions, the indolization provided efficiently and in satisfactory yield (93% overall) but gave both possible indole isomers in roughly equal quantities. The two isomers have been easily isolated via column chromatography. Hence, by using FI reaction, this reaction didn't have stereocontrol. Next, the tetracyclic indole 107 , upon several steps, afforded (±)-tabersonine ( rac - 103 ), in 70–80% yield ( Scheme 18 ). 87

Peduncularine 109 is a member of the indole alkaloids class with a monoterpene unit like the aliphatic portion, which firstly was extracted by Bick and co-workers in 1971, from the Tasmanian shrub Aristotelia peduncularis . 88 Alkaloids have been derived from some of other elaeocarpaceous plants from A. serrata (New Zealand), A. chilensis (Chile) and also mostly from New Guinea. Its configuration associated alkaloids aristoteline, peduncularine, tasmanine, aristoserratine, sorelline and hobartine. That is also re-divided biogenetically pattern with a rearranged geranyl and tryptamine subunit. These natural productions such as peduncularine have indicated cytotoxic activity against cell lines of breast cancer and other biological activities.

In 2002, Roberson and co-workers achieved a relatively short total synthesis of (±)-peduncularine 109 in sixteen steps from market purchasable 1,4-cyclohexadiene 110 . 89 This compound could provide acetate 111 , after several steps. The latter has been exposed to FIS to supply alcohol 112 with concomitant deprotection of the C-8 alcohol. The latter upon several steps produced (±)-peduncularine 109 with appropriate yield. The main step of in the current total synthesis is the [3 + 2] annulation reaction of an allylic silane by using chlorosulfonyl isocyanate, that transported the desired bicyclic nucleus of the naturally occurring compounds ( Scheme 19 ). 89

A novel group of 5-heteroaryl-functionalized 1-(4-fluorophenyl)-3-(4-piperidinyl)-1 H -indoles as enormously selective and potentially CNS-active α 1 -adrenoceptor antagonists was shown by Eilbracht and co-workers. 90 The corresponding products were provided from the antipsychotic sertindole 113 . The structure–affinity relationships of the 5-heteroaryl substituents and the groups on the piperidine nitrogen atom were optimized with respect to affinity for α 1 adrenoceptors and selectivity in respect to dopamine (D 1–4 ) and serotonin (5-HT 1A–1B and 5-HT 2A,2C ) receptors. 91

A usual aspect of unusual antipsychotics for example clozapine, olanzapine, sertindole 113 and seroquel is nanomolar attraction for α 1 adrenoceptors additionally to their attractions for serotonin 5-HT 2A and dopamine D 2 and receptors. The real balanced attractions for these receptors might underlie the enhanced form of these drugs (enhanced rate between doses containing antipsychotic property and extrapyramidal side effects) as contrasted to classical antipsychotic drugs, for example haloperidol. The phenylindole framework of sertindole 113 is a promising pattern for the growth of main acting α 1 antagonists. Replacing of the 5-chloro atom in sertindole 113 with polar substituents including functionalized aminomethyl and carbamoyl groups provided a novel group of particular α 1 adrenoceptor antagonists. This research demonstrated that 5-substituents mixing hydrogen bond acceptor possessions with steric bulk in the plane of the indole core are necessary to provide high affinity for adrenergic α 1 receptors mixed with satisfactory selectivity in respect to dopamine D 2 and serotonin 5-HT 2A and 5-HT 2C receptors.

Tryptamine imitative is specifically included in many biological processes, such as melatonin in serotonin in neurological processes or in the circadian rhythm control. Therefore, tryptamine containing the nucleus indole and its imitative were employed for the reaction of various diseases such as migraine (for example Sumatriptan), schizophrenia ( e.g. Sertindole) and depression ( e.g. D -tryptophan). In this approach, firstly, tandem hydroformylation-FIS gave appropriate admittance to indole 116 initiating from the readily accessible olefin 114 through transformation with market purchasable 4-bromophenylhydrazine 115 and afforded indole 116 in 39% yield. The latter has been then transformed into the corresponding sertindole 113 , upon several reactions ( Scheme 20 ). 90,91

Meridianins are brominated 3-(2-aminopyrimidine)-indoles, which are purified from Aplidium meridianum , an Ascidian from the South Atlantic (South Georgia Islands). Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed. 92

The compounds, except meridianin G and the related iso-meridianins C, were found to inhibit CDKs, GSK-3, PKA and other protein kinases in the low micromolar range. Meridianins B and meridianin E were the most potent inhibitors while meridianin G, isomeridianin C and G were essentially inactive. Meridianins B and E were selected for further studies on selectivity and cellular effects. 93

Franco and co-workers described synthesis of iso-meridianin derivatives 117 via microwave irradiation (MWI). 93 The synthetic method contains six steps, in which FIS is considered as the main reaction. This group selected isocytosine 118 as a suitable starting compound, since the hydroxyl group at position 4 permits an appropriate functionalization and introduction of the requisite C-2 carbonyl group. In this procedure, isocytosine 118 has been transformed into the corresponding methyl ketone 119 upon different steps. The latter based on normal conditions afforded the desired phenylhydrazone derivatives 120a and 120b in 90% yield. The desired products 120a and 120b have been employed without more purification. The FIS was the main step of this method, so many efforts by utilizing various catalysts, solvents and heating conditions were attempted. As a result, the usage of zinc chloride and MWI provided a quick, clean and quantitative elimination of the Boc group. Although, addition of a small amount of dimethylformamide prior to MWI with zinc chloride into 120a and 120b provided the desired iso-meridianin G 117a and iso-meridianin C 117b in satisfactory yields, respectively ( Scheme 21 ). 93

The total synthesis of 8-desbromohinckdentine A 121 has been effectively achieved and described by Liu and co-workers in 2003. 94 In this route, 2-(2-bromophenyl)-indole 123 , has been formed through the FIS from phenylhydrazine hydrochloride 23a and 2-bromoacetophenone 122 . Upon several steps, indole 123 has been converted into the corresponding single dibrominated natural product 121 ( Scheme 22 ). 94

(±)-Aspidospermidine 124 is one of the aspidosperma alkaloids family. Aspidosperma alkaloids structurally have pentacyclic [6.5.6.6.5] ABCDE ring system skeleton with a common structural feature which is cis -stereocenters at C-7, C-21, and C-20 (all carbon quaternary). Some parts of this kind of alkaloids like vinblastine and vincristine have been used as cancer chemotherapy medications. Additionally tabersonine (possess inhibitory effect against SK-BR-3 human cancer cell lines which is better than cisplatin), jerantinine-E (more potent in vitro cytotoxicity against human KB cells, IC50 <1 μg mL −1 ), and vincadifformine (cytotoxic), are pharmacologically important alkaloids. 95

Total synthesis of (+)-aspidospermidine 124 has been described by Aube and co-workers in 2005. 96 The main reactions used in the total synthesis of this pentacyclic Aspidosperma alkaloid contain a deracemizing imine alkylation/Robinson annulation sequence, a selective “redox ketalization”, and also an intramolecular Schmidt reaction. A FIS happened on a tricyclic ketone similar to the sequence described in their aspidospermine synthesis. The synthetic method utilized 2-ethylcyclopentanone 125 as the precursor. The latter provided tricyclic lactam 126 as a single diastereomer in 82% chemical yield and 84% enantioselectivity. Compound 126 did not show amenable to a direct FIS reaction and so has been reduced in a three-step procedure to the desired ketoamine 13 by using of selective protection of the ketone carbonyl continued through a reduction/deprotection reaction. The reaction of ketoamine 13 with phenylhydrazine 8 , acid and lastly lithium aluminium hydride gave the target (+)-aspidospermidine ( 124 , 51% yield from 13 ) along with 13% of a by-product 127 . This by-product probably arose through the FI of the less-functionalized enamine isomer ( Scheme 23 ). 96

Another approach has been described by Canesi and co-workers for the synthesis of (±)-aspidospermidine 124 in 12 steps. 97 This method, relied on ‘‘aromatic ring umpolung’’ was initiated from a polyfunctionalized phenol 128 . Upon diverse steps, this compound can be transformed into main intermediate 13 . In the following, FIS of tricycle 13 and phenylhydrazine 8 under reflux condition resulted in the hydrazone 129 that is transformed to imine 130 in AcOH. The latter is reduced in the same pot by using lithium aluminium hydride to provide (±)-aspidospermidine 124 , in 43% yield ( Scheme 24 ). 97

Also, in another method, for the total synthesis of (+)-aspidospermidine 124 , 1-( p -methoxy benzyl)piperidin-2-one 131 upon several steps provided tricyclic core 13 . The latter is a privileged tricyclic nucleus having the crucial C-20 all-carbon quaternary stereocenter would be a significant issue in providing enantioselective synthesis of 124 and structurally related bisindole alkaloids. Also, FI cyclization reaction of 13 produced dehydroaspidospermidine that on reduction by using lithium aluminium hydride afforded corresponding naturally occurring compound (+)-aspidospermidine 124 in 50% yield ( Scheme 25 ). 95

Cryptosanguinolentine 132 or called isocryptolepine is the most important member of indoloquinoline alkaloids, which was separated from the roots of the West African plant Cryptolepis sanguinolenta . In traditional folk medicine was used for treatment of fevers such as fever of malaria. Various types of hetero-aromatic alkaloids have potential applications in the medicinal field. Many such alkaloids intercalate in the DNA double helix resulting in changes in DNA conformation so they can inhibit DNA transcription and replication. X-ray analysis and spectroscopy can investigate the mode and strength of binding of these alkaloids to DNA. In addition, some N -methyl derivatives of these ring systems exhibit important cytotoxic and antimicrobial activities. 98

Haplophytine 135 (C 27 H 31 O 5 N 3 ), the important indole alkaloid, and cimicidine (C 23 H 28 O 5 N 2 ) were isolated from the Mexican plant Haplophyton cimicidum 's dried leaves, first time in 1952 by Snyder and co-workers. 100 Huplophyton cimicidum produces the aspidospermine and the biogenetically related eburnamine kinds of alkaloids. Haplophytine 135 is constituted of two parts that are joined by the forming of a quaternary carbon center. The right-half part is a hexacyclic aspidosperma class of alkaloid, known as aspidophytine, which is acquired by the acidic degradation of (+)-haplophytine. The left-half part has an unique structure, that includes a bicyclo[3.3.1] framework which possesses bridged aminal and ketone activities. Both alkaloids are toxic to insects, but most of the toxicity is owed by the haplophytine conLent (I) also they are toxic to German roaches on contact, injection and ingestion. The LD/50 dosage of cimicidine is about 60 γ/g (contact, 48 h) and for haplophytine is 18 γ/g. Haplophytine caused extended paralysis at dosage levels under the LD/50 value. The total crude alkaloid has been discovered to be toxic to an extensive range of insects containing Mexican bean beetle larvae, European corn borers, egg-plant lace bugs, Colorado potato beetle larvae and adults, codling moths and grasshoppers. 101

The first total synthesis of (+)-haplophytine 135 has been reported by using several key steps including intramolecular Mannich reaction, oxidative rearrangement, the FIS and Friedel–Crafts alkylation reaction. 102 Ueda and co-workers demonstrated the first total synthesis of (+)-haplophytine 135 in 2009 via an effective method. 102 Haplophytine is contained of two segments that are linked by the construction of a quaternary carbon center. Total synthesis of (+)-haplophytine 135 was initiated from market purchasable 7-benzyloxyindole 136 and upon various steps afforded 137 as the main precursor of the FI reaction. The reaction of hydrazine 137 with tricyclic ketone 138 , synthesized by d’Angelo and co-workers by using 50% H 2 SO 4 provided the desired hydrazone. 103 Upon widespread optimization, this group ultimately known that cautious control of the reaction temperature and the suitable selection of solvent and acid were necessary to favorably attain the corresponding indolenine 139 over the indole 140 in satisfactory yield. Therefore, the reaction with p -toluenesulfonic acid in tert -butanol at 80 °C provided indolenine 139 in 47% yield accompanied with indole 140 in 29% yield. Lastly, imine 139 after different steps provided (+)-haplophytine 218 ( Scheme 27 ). 102

Minfiensine is an indole alkaloid which is extracted from the African plant named Strychnos minfiensis by Massiot and co-workers in 1989. 104 Minfiensine has significant biological functions containing anticancer activities. Additionally, various types of the Strychnos indole alkaloids have interesting anticancer functions too. 104

In 2011, a short total synthesis of minfiensine 141 was accomplished in ten steps. 105 The assembly of minfiensine 141 started with the FIS. Therefore, the reaction of cheap and market purchasable phenylhydrazine 8 and 1,4-cyclohexanedione monoethylene acetal 142 at ambient temperature continued by heating at 190 °C provided the corresponding indole product 143 in 89% yield. Next, the corresponding indole after several steps afforded (±)-minfiensine 141 in 95% yield ( Scheme 28 ). 105

(+)-Aspidoalbidine (or (+)-fendleridine) is an Aspidosperma alkaloid separated from the seeds of Aspidosperma fendleri . The name fendleridine was related to the name of the tree, by Burnell and co-workers in 1966. 106 Brown found (+)- N -acetylaspidoalbidine in 1963, 107 they gave the name “aspidoalbidine” to the postulated simplest member by analogy to aspidospermidine, discovered by Biemann two years before. Acetylaspidoalbidine is a member of Aspidosperma family, which is isolated from the Venezuelan tree species and Aspidosperma rhombeosignatum markgraf and Aspidosperma fendleri woodson. The Aspidosperma family is one of the indole alkaloid families also many of them have significant biological activities. 108

In 2004, Kam and co-workers isolated Mersicarpine 148 from the stem-bark of the Kopsia arborea and Kopsia fruticosa . 110 The tetracyclic dihydroindole mersicarpine was exhibited in another kind of Kopsia , viz. , K. singapurensis too, which has a typical seven-membered cyclic imine joined with indoline and δ-lactam. The Kopsia has intriguing biological activities. Additionally other alkaloids were obtained from kopsia such as mersicarpine, pericidine, arboricinine, valparicine, arboflorine, arboricine and arboloscine. 111

In 2013, Iwama and co-workers accomplished an extremely significant enantioselective total synthesis of (±)-mersicarpine 148 by using an 8-pot/11-step sequence in 21% chemical yield that initiated from market purchasable 2-ethylcyclohexanone. 112 The features of this procedure were FIS, simple admittance to the azepinoindole framework, the short-step and significant synthesis. This method was started with the FIS by utilizing optically active ketoester 149 , which synthesized in 99% enantioselectivity based on the procedure by d’Angelo and Desmaële. 113 The latter has been reacted with phenylhydrazine 8 in AcOH at 120 °C to provide the desired tetracyclic tetrahydrocarbazole 150 in excellent yield without isolation of the transient tricyclic tetrahydrocarbazole 151 . After several steps by using various routes, the corresponding compounds 150 and 151 produced benzyloxycarbonyl (Cbz) carbamate 152 , that is as a main intermediate to form (±)-mersicarpine 148 . This group tried to examine the reaction conditions of FIS reaction that afforded tricyclic tetrahydrocarbazole 150 without the production of the lactam ring. Relied on the previous observation, using phenylhydrazine hydrochloride in MeOH and in the presence of acetic acid afforded the corresponding tricyclic tetrahydrocarbazole 150 , selectively. The effect of acid on the ratio of 150 to 151 was examined. As a result, methanesulfonic acid was found as a suitable acid for the improvement in selectivity of the FIS. More outstandingly, it was found that the product ratio was extremely sensitive to the quantity of acid employed. Therefore, the reaction in the presence of phenylhydrazine 8 and methanesulfonic acid afforded 151 in excellent yield and selectivity. Finally, the (−)-mersicarpine 148 was synthesized in 21% yield ( Scheme 30 ). 112

The Aspidosperma alkaloids own a main place in natural chemistry inventions due to their different biological activities and extensive range of compound structural variations. Acid cleavage of haplophytine (a dimeric indole alkaloid was found in the Haplophyton cimicidum 's leaves) guided to aspidophytine, a lactonic aspidospermine member of alkaloid that has been recommended to be used in its synthesis and be a biosynthetic pioneer of haplophytine. 114

In 2013 Satoh and co-workers described a total synthesis of (−)-aspidophytine 153 and also the initial total synthesis of its congeners, (+)-cimicidine 154 and (+)-cimicine 155 , have been achieved in a divergent method. Preparation of the aspidosperma scaffold has been performed via FIS. 115 The regiochemistry of the FIS was powerfully reliant on the type of acid, and a weak acid, including AcOH gave the corresponding indolenine isomer in satisfactory selectivity. In this method, total synthesis of (−)-aspidophytine 153 and (+)-cimicidine 154 was initiated from market purchasable ketoester 156 , which has been provided from optically active tricyclic aminoketone 138 upon several steps. 102 The latter compound 138 treated with functionalized phenylhydrazine 72 via FIS under reflux in benzene to afford hydrazone 157 which has been exposed to different acidic conditions. Finally, this group provided the corresponding indolenine 158 in 48% accompained with 5% of indole 159 once reaction has been completed in AcOH at 100 °C. Pentacyclic indolenine 158 provided (−)-aspidophytine 153 after several steps. Subsequent, they demonstrated the total synthesis of (+)-cimicidine 154 by using the usual intermediate 158 through diverse reactions ( Scheme 31 ). 115

The efficacy of the convergent synthetic approach has been shown by achievement of the first total synthesis of (+)-cimicine 155 . Therefore, FIS through 2-methoxyphenylhydrazine 14 and aminoketone 138 provided hydrazine 160 which provided 41% of the corresponding indolenine compound 161 in satisfactory regioselectivity and 6% of compound 162 Next, upon different steps, imine 161 gave (+)-cimicine 155 ( Scheme 32 ). 115

The efficiency of this synthetic approach for manufacturing extremely substituted aspidosperma alkaloids has been completely exhibited via the divergent synthesis of these three aspidosperma alkaloids from the usual tricyclic aminoketone intermediate. Furthermore, in this research, the regiochemistry of the FIS has been powerfully affected by acidity of acids, which produced the corresponding indolenine isomer in satisfactory selectivity ( Scheme 32 ). 115

Akuammilines have an indolenine or indoline core, which is combined to a polycyclic framework. The complicated structures of these molecules have biosynthetic source and biological efficacy that can show activities for combating plasmodial, viral, and cancerous diseases. The alkaloids fraction of alstonia scholarisleaf, vallesamine, picrinine and scholaricine, may make the analgesic and anti-inflammatory effects based on in vivo and in vitro screening. Picrinine is a component of the akuammiline family of alkaloids, which has six stereogenic centers, five of them are contiguous, and includes two N , O -acetal linkages within its polycyclic skeleton. Picrinine 163 reveals anti-inflammatory activity via inhibition of the 5-lipoxygenase enzyme. 17 Picrinine was firstly extracted from the leaves of Alstonia scholaris in 1965. 116 The plant Alstonia scholaris , also called the Dita Bark tree, has been a rich origin of alkaloids. They can be extracted from its flowers, bark, seeds, leaves, fruitpods and roots. These alkoldes have been used to treat chronic respiratory diseases, for treatment of malaria and dysentery in Southeast Asia, and have been used as traditional medicines to treat various ailments in livestock and humans for the centuries. Even in china it is used for their antitussive and antiasthmatic properties also to release tracheitis and cold symptom. In addition to picrinine and scholaricine, three new indole alkaloids, 5- epi -nareline ethyl ether, nareline ethyl ether and scholarine-V(4)-oxide were separated from the leaf extract of Alstonia scholaris . 117

Smith and co-workers described the first total synthesis of the akuammiline alkaloid picrinine 163 in which a main step of their total synthesis was FIS. 118 Synthesis of this natural product has been initiated from sulfonamide 164 , which is available from market purchasable or can be easily synthesized. Then, sulfonamide 164 upon various steps produced carbonate 165 that was an appropriate initiating compound for FIS. In the critical FI, trifluoroacetic acid stimulated reaction of carbonate 165 and phenylhydrazine 8 gave the hexacyclic indolenine 166 with complete diastereoselectivity. This conversion is identified as one of the most complex cases of the FIS. It should be mentioned that indolenine 166 is in equilibrium with its hydrate, hence, purification and 2D NMR analysis were essential to assist structure clarification. However, upon numerous steps picrinine 163 has been provided. This synthetic method shows short assembly of a main FIS to forge the natural product's carbon scaffold, and a number of delicate late-stage conversions to furnish the total synthesis ( Scheme 33 ). 118

Family of the Vinca class of alkaloid was the subject of synthetic studies with attendant and important biological properties. For example the pentacyclic vindoline 167 , which is the main alkaloid, isolated from the plant Catharanthus roseus . Due to its antimitotic properties compound 167 has been recently being used in the clinical treatment of different human cancers. 119

Members of the Vinca group of alkaloid have been the topic of extensive synthetic studies. The total synthesis of 167 was initiated from phenylhydrazine 8 and cyclohexane-1,4-dione monoethylene ketal 142 as precursor for the first FIS to provide product 143 (94%). Next, the latter has been transformed into the pentacyclic compound 167 by using different steps in chiral, non-racemic form and in ca. 33% yield ( Scheme 34 ). 120

The spiroindimicins are a member of structurally alkaloids extracted from the deep-sea-derived marine actinomycete Streptomyces sp. SCSIO 03032. Marine actinomycete Streptomyces SCSIO 03032 can produce polyketide macrolactam heronamides, α-pyridone antibiotic piericidins, lynamicins and polyketide macrolactam heronamides. Deep-sea organisms have survived under their hard environment by adjusting an extensive range of their metabolic pathways and biochemical processes. The deep-sea-derived marine actinomycete Streptomyces sp. SCSIO 03032, that causes a range of structurally unprecedented natural products such as spiroindimicins A–D, dichlorinated bisindole alkaloids possessing unique heteroaromatic frameworks featuring or spiro-rings. Some analogues of bisindole alkaloids are DNA-topoisomerase I inhibitors and protein kinase inhibitors which have been used in cancer clinical trials. 121

Sperry and co-workers in 2016 exhibited effective usage of the FIS to generate a pentacyclic spirobisindole. 122 In this synthetic method, firstly, requisite spiroindolinyl pentanone 172 has been produced from the treatment of iodoaniline 170 and bromide 171 in high yield. Then, with the spiroindolinyl pentanone 172 , the stage was set for the critical FI reaction. After heating a solution of 172 in hand, and 4-chlorophenylhydrazine in AcOH under reflux, the spirobisindole 174 has been provided in high yield, representing the timeless efficacy of this classic reaction in complex natural product synthesis. Subsequent, the latter transformed into the natural products (±)-spiroindimicin C 168 , that after reductive amination produced (±)-spiroindimicin B 169 ( Scheme 35 ). 122

Montamine isolated from the seeds of Centaurea montana. The genus Centaurea have been applied in folk medicine for the treatment of different diseases. Centaurea montana , known as the mountain cornflower, is a native plant in Australia and Europe. Montamine has a unique dimeric N , N ′-diacyl hydrazide structure and shows cytotoxicity against CaCo-2 colon cancer cells (IC 50 = 43.9l M). 123

In 2015, Xu and co-workers described the total synthesis of alkaloid natural product montamine analogue 175 in 55% yield. 124 As depicted in Scheme 36 , this group provided 3-(2-hydroxyethyl)-5-methoxyindole (usually found as 5-methoxytryptophol, 176 ) through the FIS with p -methoxyphenylhydrazine hydrochloride 39 and 2,3-dihydrofuran 73 . Whereas the yield of this treatment was modest, it nevertheless gave a reasonable, one-step method to an indole suitably substituted having an oxygen group at the 5-position and ethyl alcohol scaffold at the 3-position. Alcohol 176 gave the advanced montaine analogue 175 in several steps ( Scheme 36 ). 124

The monoterpene indole alkaloids are an extremely different group of naturally occurring compounds, which were provided in a widespread series of medicinal plants. They have natural structural complication and a number of significant biological properties, which qualifies a figure of them to be principle candidates for anti-arrhythmic, anti-malarial and anti-cancer agents. 125 Dixon and co-workers, in 2016, achieved a novel method for the difference total synthesis of (±)-vincadifformine, (±)-vincaminorine, (±)-quebrachamine, (±)- N -methylquebrachamine and (±)-minovine and, each in slighter than 10 linear steps in perfect diastereoselectivities. 126

Initially, for the synthesis of (±)-minovine 177 , this route was started with the formation of aldehyde 183 from 3-ethyl-2-piperidone 182 upon two steps. The production of the indole functionality continued effectively by Stork's modification of the FIS of aldehyde 183 with phenylhydrazine hydrochloride 23a which produced indole 184 in 62% yield. The latter after different steps produced the desired alkaloid, (±)-minovine 177 in 52% yield. Another skeletally distinct alkaloid, (±)-vincaminorine 178 has been provided as a single diastereomer in the reaction vessel in 31% yield, which could be more transformed into N -methylquebrachamine 179 in 61% yield. Moreover, in another route, indole 184 can produce (±)-vincadifformine 180 and (±)-quebrachamine 181 with 84% and 71% yields, respectively. 126 This approach that gives a concise and different synthetic method to various vincadifformine-type, quebrachamine-type and iboga-type alkaloids. Strategically, the novel method shows a key late-stage formation of reactive enamine functionality from stable indole-linked δ-lactam through an extremely chemoselective Ir( I ) mediated reduction ( Scheme 37 ). 126

Ergot alkaloids were in the group of the first fungal-derived naturally occurring compounds recognized, inspiring pharmaceutical requests in infective diseases, CNS disorders, cancer and migraine. Aurantioclavine 185 has been first extracted from the fungus Penicillium aurantiovirens in 1981. It has become an striking target for whole synthesis campaigns because of the interesting synthetic challenge presented by the fused azepinoindole nucleus and its jub as a biosynthetic pioneer to the communesin alkaloids, that show cytotoxicity against leukemia cell rules. 127

In 2016, total synthesis of (−)-aurantioclavine has been accomplished by Cho and co-workers. 128 However, the significant step of this total synthesis was the production of 3,4-fused tricyclic indole derivatives via intramolecular FIS of aryl hydrazides, that contain a carbonyl group including a side chain connected to the meta -position of the aromatic ring. The intramolecular FIS approach does not need cumbersome prefunctionalization, and therefore, it may act to simplify the formation of polycyclic indole alkaloids. The novel approach initiated with market purchasable ( S )-β-amino-3-iodo-benzene ethanol 186 . The latter after several steps gave aryl hydrazide 187 in 70% yield that exposed to the standard intramolecular FIS conditions and treated to generate the corresponding indole derivatives 188 in 72% yield. The latter underwent several chemical synthetic conversions to make the corresponding (−)-aurantioclavine 185 ( Scheme 38 ). 128

2.2. Japp–Klingemann reaction

The first naturally occurring carbazole alkaloid extracted from Murraya koenigii Spreng (Rutaceae) was Murrayanine 194 . However, later from other species of the genus Clausena and Murraya and displays important biological property. For example, it has exhibited to powerfully prevent the aggregation of platelet, serve as an antifungal and antibacterial agent, and contain cytotoxic property. 130

Chakraborty and co-workers reported total synthesis of murrayanine 194 in 1968. 131 In this approach, initial Japp–Klingemann reaction between phenyldiazonium chloride 195 and 2-hydroxymethylene-5-methylcyclohexanone 196 led to hydrazone 197 . Next, FIS of hydrazone 197 in the presence of acetic acid/hydrochloric acid under reflux afforded 1-oxo-3-methyl-1,2,3,4-tetrahydrocarbazole 198 . Finally, the latter was then transformed into the corresponding natural product murrayanine 194 upon different chemical reactions ( Scheme 40 ). 131

Murrayacine 199 , that is an alkaloid extracted from Murraya koenigii stem bark and Clausena heptaphylla , is known in spices, herbs and (curryleaf tree). 132 The total synthesis of murrayacine 199 , has been demonstrated by Chakraborty and co-workers in 1973. 133 7-Hydroxy-6-(hydroxymethyl)-2,3,4,9-tetrahydro-1 H -carbazol-1-one 202 . Lastly, the latter has been transformed to the corresponding murrayacine 199 ( Scheme 41 ). 133

Carbazole derivatives contain a significant group of heterocycles that are found for their powerful antibacterial, antitumor, anti-inflammatory, antihistamine and psychotropic, activities. Natural 1-oxygenated carbazole alkaloids are principally isolated from the genera Clausena and Murraya species, and in the case of 2- and 3-functionalized classes, their biogenesis were developed. 134

The total synthesis of natural products mukolidine 203 and mukoline 204 has been achieved by Chakraborty and co-workers in 1982. 135 As depicted in Scheme 42 , this total synthesis initiated through a Japp–Klingemann reaction of toluenediazonium chloride 205 and 2-hydroxymethylenecyclohexanone 191 to give hydrazone 206 . Next, FIS of the latter afforded the 1-oxotetrahydrocarbazole 207 . Subsequent, the desired 1-oxotetrahydrocarbazole 207 has been transformed into mukolidine 203 that reduced with NaBH 4 , to provide mukoline 204 . 135

The total synthesis of heptazolidine 208 , a carbazole alkaloid extracted from Clausena heptaphylla , has been achieved by Chakraborty and co-workers in 1985. 136 In this approach, the main intermediate is 2-hydroxy-3-methoxy-6-methyltetrahydrocarbazole 215b . Initially, diazoaryl derivative 209 reacted with 2-hydroxymethylene-5-methylcyclohexanone 196 via Japp–Klingemann reaction to give the corresponding phenylhydrazone 210 . The latter was then subjected into FIS to afford drocarbazole 211 , which upon Wolff–Kishner reduction provided the tetrahydrocarbazole 215b . The tetrahydrocarbazole 215b has been produced via reaction between 4-methylcyclohexanone 213 and 3-acetoxy-4-methoxyphenylhydrazine hydrochloride 212 to the phenylhydrazone 214 , FI reaction, and ester removal. The latter has been transformed into the corresponding natural product heptazolidine 208 through subjection to various chemical reactions ( Scheme 43 ). 136

The carbazole alkaloids exhibit a big and constructional rich group of naturally occurring compounds, which are provided by a range of terrestrial plants. Especially, plants inside the Rutaceae group are remarkable producers of these products, with the genus Murraya affording the maximum figure of distinctive structures. Usually known in the external Himalayas and on the Indian peninsula, the leaves of M. koenigii (L.) Spreng are with other things, broadly used as a spice flavoring and giving it the name “curry-leaf tree” by the people of these areas. Extracts obtained of the plant are employed in local medicine due to their antimicrobial property. 137

The first total synthesis of a natural dimeric carbazole alkaloid, (±)-bismurrayaquinone-A 216 was demonstrated by Bringmann and co-workers in 1995. 138 In this synthetic method, firstly, the desired hydrazone 197 has been produced from phenyldiazonium chloride 195 and 2-hydroxymethylene-5-methylcyclohexanone 196 through Japp–Klingemann reaction. Then, the desired hydrazone 197 has been exposed to FIS by using acetic acid/hydrochloric acid to give 1-oxo-3-methyl-1,2,3,4-tetrahydrocarbazole 198 . Subsequent, the latter has been transformed into the desired 216 in 73% yield, after several steps. Through chromatography on a chiral phase, the two enantiomers of (±)-bismurrayaquinone-A 216 have been separated ( Scheme 44 ). 138

Indole alkaloids are significant natural products due to their structural association to the important amino acid, tryptophan and the important metabolites of tryptophan, for instance the neurotransmitter serotonin. One class of indole alkaloids was extracted from Alstonia species. 139 Tryprostatin A 217 was extracted as secondary metabolites of a marine fungal strain BM939 and depicted to entirely prevent respectively cell cycle progression of tsFT210 cells in the G2/M phase at a final concentration of 50 μg mL −1 of 217 . Tryprostatins A 217 include a 2-isoprenyltryptophan scaffold and a proline residue that include the diketopiperazine unit. 140

Chowdhury and co-workers extracted two novel carbazole alkaloids planned as koenigine-quinone A and koenigine-quinone B from the alcoholic extract of the stem bark of Murraya koenigii Spreng. 142 In 1998, they have demonstrated the structures given for koeniginequinone A 220a and B 220b using a FI of the desired phenylhydrazones 222a,b as the main step. The phenylhydrazone derivatives 222 have been provided via a Japp–Klingemann reaction between 2-hydroxymethylene 5-methylcyclohexanone 196 and the aryldiazonium chloride derivatives 221a and 221b , respectively and transform into the 1-oxotetrahydrocarbazole 223a and 223b in the presence of acid. Next, koeniginequinone A 220a A and 220b B have been produced in 65.5% and 72% yield, respectively, through a multi-step reaction ( Scheme 46 ). 142

Melatonin ( N -acetyl-5-methoxy tryptamine) 3 (darkness hormone) is an indolamine hormone which is made by the photoreceptor cells of the retina and the pineal gland in vertebrates, additionally it was first isolated in 1958 by Lerner and Case at Yale University. 143 They found the light-related features within the skin cells of amphibians. The molecule produced the collection of the pigment melanin inside the melanocytes which is causing the skin to lighten. The hormone is increased at night and has been conducting as a time signal for an organism's annual (circannual) and daily (circadian) biological rhythms. Melatonin regulates sleep/wake patterns and also synchronizes the release of other hormone. Furthermore, melatonin has been displayed medicinally functions as inhibitor of the onset of Alzheimer's disease, treatment of sleep disorders and in protection against oxudative stress. 143

In recent years, much was appealed about the therapeutic possessions of the hormone melatonin. This interest has resulted in the of publication of two general scientific books preserving that the hormone can cure the symptoms of some kinds of cancer, acting as hypertensive in case of high blood pressure, treating Alzheimer's disease, AIDS, and coronary heart disease as well as being used as sleep aid, sexual vivacity, and durability. Therefore, making it a phenomenon drug of the 1990s. Melatonin is mostly formed in the pineal gland, a peasized organ placed in the center of the brain, and to a minor extent in the retina. Melatonin is found valuable in some problems for example coronary heart disease and aging. 144

The total synthesis of melatonin 3 needed phenylhydrazone 226 , which is synthesized through coupling diazotized 4-methoxyaniline 224 and 2-oxopiperidine-3-carboxylic acid 225 . This has been continued via FI cyclization reaction to give 6-methoxy-1-oxotetrahydro-β-carboline 227 . The latter afforded the corresponding melatonin 3 in several reaction steps in 41% yield ( Scheme 47 ). 145

The first asymmetric total synthesis of (−)-gilbertine 228 , a member of the uleine type indole alkaloids, has been demonstrated in 2004 by Blechert and Jiricek in a seventeen-steps reaction with a 5.5% yield. 146 The uleine alkaloid (−)-gilbertine 228 has been extracted by Miranda and Blechert in 1982 from the Brazilian tree Aspidosperma gilbertii (A. P. Duarte). 147 This method was initiated from 2-allylcyclohexenone 229 , that could be readily synthesized from o -anisic acid through Birch conditions on a large scale, and dimethylmalonate as a market purchasable starting compound. Upon a multi-step reaction, 2-allylcyclohexenone 229 has been converted into compound 230 , which was an appropriate precursor for FIS. That products have been isolated from the FIS depended powerfully on both the solvent and the p K a of the acid promoter. Pivalic acid afforded no transformation, and formic acid reaction led to deprotection of the alcohol and production of the formylester 231b , while trifluoracetic acid afforded the deprotected hydroxyindole 231a . Also, p -TsOH in tetrahydrofuran afforded only decomposition; although, in toluene the corresponding indole 231c could be provided in 72% yield. The Japp–Klingemann FI procedure has been utilized effectively as a convergent synthetic method for the formation of the corresponding tetrahydrocarbazole 231c . Then, the latter has been transformed into the corresponding natural product (−)-gilbertine 228 ( Scheme 48 ). 147

3. Miscellaneous

Alkaloids, for example manzamines and lamellarins, having diversely structures and significant biological properties provided by marine plants, microbes and invertebrates importantly encouraged interdisciplinary studies by biologists and chemists worldwide. Among the marine alkaloids, the structurally and discorhabdins associated alkaloids are a special group of nitrogenous pigments belonging to the pyrroloiminoquinone-kind alkaloids family. This class of naturally occurring compounds contains a typical nucleus pyrrolo[2,3,4- d , e ]quinoline tetracyclic framework linked to a spiro-substituent at the C-6 location. 148

The pyrroloquinoline alkaloids, found as the discorhabdins, are known in the sponges of the genus Latrunculia du Bocage along the New Zealand coast. These quinonimine alkaloids are responsible for the pigmentation possessed by the sponges, and several of the compounds in this group, together with the structurally related makaluvamines, prianosins, and epinardins, show antitumor activity. 149

In 1997, Heathcock and co-workers revealed the approach relied on the FIS to make 7-hydroxy-indole derivatives in satisfactory yield. 150 The unusual FIS is a common problem once an ortho group is current on the phenylhydrazone. Mixtures of indole derivatives are constantly provided, and yields of the corresponding indole are commonly low. They could evade this problem by using constrain the hydrazine that it could merely endure electrocyclization in the expected way. Such a limitation would result if the oxygen and nitrogen atoms could be connected by a short tether. Finally, this group examined the formation and usage of an appropriately-substituted benzoxazine. The requisite phenylhydrazine 242 has been formed in four steps from 2-amino-4-nitrophenol 241 in 60% yield. Next, the reaction with 4-benzyloxybutanal 243 gave hydrazone 244 in 93% yield. There are several formerly found instances of employing hydrazones of 4-aminobenzoxazines identical to hydrazone 244 in the FI reaction. In each of these cases, two carbon tether was an essential structural part in the desired compound. In this approach, they explored to employ the tether to suppress the unusual FI reaction, then eliminated it when the indole had been synthesized. The similar approach has been employed to form other indole derivatives containing different groups on the benzene ring. In each situation, no proof of contaminating indole products has been realized. Since the dimethylene chain is easily provided and then cleaved, this “tether” approach may show to be an important approach for overpowering the unusual FI reaction. Next, after several steps, indole 245 afforded the pyrrolo[2,3,4- d , e ]quinoline core 246 , that is a structural part known in a range of naturally occurring compounds involving the discorhabdin alkaloids, the isobatzellines, and wakayin, batzellines, the makaluvamines, damir ones A and B, and terrestrially-obtained haematopodin. 150 The latter after several steps afforded discorhabdins C 237 , E 238 , D 239 and the dethia analogue 240 ( Scheme 50 ). 149

Eudistomidin-A is an alkaloid separated from Okinawan tunicate, Eudistoma glaucus , which has a calmodulin antagonistic effect. Recently, calmodulin antagonists have been effective as device for investigating physiological activities of calmodulin, a ubiquitous Ca 2+ -binding protein that acts as a main mediator regulating cellular function and a difference of cellular enzyme system. 151

In 1998, the initial total synthesis of the marine alkaloid eudistomidin-A 247 in 72% yield presented a FIS as a key step has been described by Murakami and co-workers. 152,153 This synthetic method was started from 2-amino-5-bromophenol 248 that has been transformed into ethyl pyruvate 2-(4-bromo-2-tosyloxyphenyl)hydrazone 249 . FIS of the hydrazone 249 has effectively occurred with polyphosphoric acid (PPA) to give the desired ethyl 5-bromo-7-tosyloxyindole-2-carboxylate 250 as the only separable product (41% yield). The latter afforded eudistomidin-A 247 after several steps ( Scheme 51 ). 152,153

seco -Duocarmycins show a great guarantee as ultrapotent cytotoxins but due to poverty of therapeutic index, they are unsuccessful in advance clinical. seco -Duocarmycins include spirocyclization of a deep-embedded chloromethylindoline fragment to prompt production of an N3-adenine covalent. This spiracyclization can be stopped by blocking the seco -duocarmycins OH group. 154 The natural antibiotic (+)-duocarmycin SA 251 is a powerful cytostatic agent ( Scheme 52 ). (+)-Duocarmycin SA with an IC 50 of 10 pM (cancer line L1210) shows high cytotoxicity power, which is candidate for cancer treatment. The cytotoxicity of 251 , the biologically and structurally of related (+)-CC-1065 are created by an alkylation of N-3 of adenine in AT-rich parts of the minor groove of the DNA by reaction with the spiro -[cyclopropane-cyclohexadienone] moiety in 251 and (+)-CC-1065. Duocarmycin SA 251 looks better than CC-1065 because it hasn't deadly hepatotoxic side effect which is appeared with (+)-CC-1065. Besides (+)-duocarmycin SA 251 is a strongest one in cytotoxic potency and solvolytic stability and the most stable part is this class agents additionally glycosylated seco analogues of duocarmycin and CC-1065 are extremely encouraging for the critical cancer's treatment in an antibody-directed enzyme prodrug therapy. 155

A concise and significant synthesis of seco -duocarmycin SA 251 , an extremely strong cytostatic agent and direct precursor of the natural product duocarmycin SA 251 , was accomplished in 2003 by Tietze and co-workers. 155 The synthetic procedure includes a FIS to show the heterocyclic moiety as a main reaction. The total synthesis of seco -duocarmycin SA 251 was initiated by diazotation of market purchasable 2-methoxy-4-nitroaniline 251 , which upon two steps produced the hydrazone 253 extremely easily with an overall yield of 69%. Then, FI reaction happened by heating at 120 °C by using of polyphosphoric acid and xylene as co-solvent to provide methyl 7-methoxy-5-nitro-1 H -indole-2-carboxylate 254 in 64% yield. In the following, upon several steps seco -duocarymcin SA 251 has been synthesized in 91% yield ( Scheme 52 ). 155

Such as Evodia officinalis and Evodia rutaecarpa, rutaecarpine 255 is the major of indoloquinazoline alkaloid, which is extracted from Rutaceous plants. Traditional medicinal has long used this plant for treatment of inflammation-related symptoms. Studies currently shows this anti-inflammatory function is related to its component rutaecarpine, exhibiting a selective and powerful COX-2 inhibited activity. Furthermore, rutaecarpine has other functions such as the analgesic, antianoxic, vasorelaxing, cytotoxic and antiplatelet. 156

The total synthesis of nosiheptide 260 was achieved by Bentley and co-workers and reported in 2004. This approach has been accomplished through a FIS as a main step. 160 The starting hydrazine was synthesized from the market purchasable 3-amino-4-chlorobenzoic acid 261 via diazotisation and reduction with SnCl 2 , and has been instantaneously reacted with methyl 2-oxobutanoate to provide the hydrazone 262 . In the following, FI cyclisation reaction of hydrazone 262 by using PPA in AcOH produced the indole 263 in 87% yield. The corresponding indole 263 has been transformed into the nosiheptide 260 via several chemical transformations ( Scheme 54 ). 160

Sempervirine 264 , an alkaloid isolated from the roots of Gelsemium sempervirens , in 1916 is known as an antiproliferative agent both in vitro and in vivo . 161 Earlier in a high throughput screening (HTS) campaign of natural products, sempervirine was discovered as a MDM2 E3 ubiquitin ligase inhibitor. Sempervirine is known to stabilize p53 tumor suppressor protein levels by blocking its proteasomal degradation via an ubiquitin-dependent pathway. It inhibits both murine double minutes-2 (MDM2) dependent p53 ubiquitinylation and MDM2 auto-ubiquitinylation. Thus, cancer cells carrying wild-type p53 when treated with this compound induce stabilization of p53 leading to apoptosis. Sempervirine is also known to intercalate DNA, and inhibits DNA topoisomerase I; therefore, it is considered as a potential lead in anticancer therapeutics. Some of the known members of this family such as flavopereirine, serpentine and alstonine exhibit a variety of biological activities, for example anti-HIV, antipsychotic, sedative and immunostimulant activities together with notable cytostatic effects. 162

In 2006, Lipinska and co-workers reported a unified synthetic approach for the total synthesis of zwitterionic indolo[2,3- a ]quinolizine alkaloid in five steps via FIS as one of the main steps. 1 This total synthesis was initiated from the accessible 5-acetyl-3-methylthio-1,2,4-triazine 268 that has been synthesized in 40% yield in two-step reaction from 3-methylthio-1,2,4-triazine 267 which can be provided on a large laboratory-scale by using the glioxal 265 and S -methylthiosemicarbazide hydroiodide 266 . In the following, the corresponding compound 268 , was transformed into 3-acetyl-1-methylthiocycloalka[ c ]pyridine 270 via various stages. The acetyl substituent stays in compound 270 that affords admittance to the synthesis of the indole scaffold through the FIS. The FIS of the phenylhydrazone 270 into 271 requisite MWI of the reaction mixture (substrate with zinc chloride (ZnCl 2 ) solution in triethylene glycol (TEG)). The latter can then be converted into the sempervirine 264 ( Scheme 55 ). 1

4. Conclusion

Conflicts of interest.

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• Published: 06 September 2024

Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution

• Santosh R. Sukka   ORCID: orcid.org/0000-0003-2334-4685 1 ,
• Patrick B. Ampomah   ORCID: orcid.org/0000-0003-0561-1145 1 ,
• Lancia N. F. Darville 2 ,
• David Ngai 1 ,
• Xiaobo Wang   ORCID: orcid.org/0000-0001-6044-914X 1 ,
• George Kuriakose 1 ,
• Yuling Xiao 3 ,
• Jinjun Shi   ORCID: orcid.org/0000-0001-9200-5068 3 ,
• John M. Koomen   ORCID: orcid.org/0000-0002-3818-1762 2 ,
• Robert H. McCusker   ORCID: orcid.org/0000-0001-8006-3228 4 &
• Ira Tabas   ORCID: orcid.org/0000-0003-3429-1515 1 , 5  

Nature Metabolism ( 2024 ) Cite this article

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• Cell signalling
• Molecular medicine
• Monocytes and macrophages

Macrophage efferocytosis prevents apoptotic cell (AC) accumulation and triggers inflammation-resolution pathways. The mechanisms linking efferocytosis to resolution often involve changes in macrophage metabolism, but many gaps remain in our understanding of these processes. We now report that efferocytosis triggers an indoleamine 2,3-dioxygenase-1 (IDO1)-dependent tryptophan (Trp) metabolism pathway that promotes several key resolution processes, including the induction of pro-resolving proteins, such interleukin-10, and further enhancement of efferocytosis. The process begins with upregulation of Trp transport and metabolism, and it involves subsequent activation of the aryl hydrocarbon receptor (AhR) by the Trp metabolite kynurenine (Kyn). Through these mechanisms, macrophage IDO1 and AhR contribute to a proper resolution response in several different mouse models of efferocytosis-dependent tissue repair, notably during atherosclerosis regression induced by plasma low-density lipoprotein (LDL) lowering. These findings reveal an integrated metabolism programme in macrophages that links efferocytosis to resolution, with possible therapeutic implications for non-resolving chronic inflammatory diseases, notably atherosclerosis.

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Efferocytosis in atherosclerosis

Macrophages use apoptotic cell-derived methionine and DNMT3A during efferocytosis to promote tissue resolution

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All data supporting the study are available in the manuscript and supplementary information. Source data are provided with this paper.

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Acknowledgements

We thank R. Ramakrishnan (Columbia University) for his guidance on statistical analysis of the data in this study. We thank B. Gerlach for his invaluable initial discussions, which greatly contributed to the development of this research. This work was supported by NIH/NHLBI grant nos. R35-HL145228 and P01-HL087123 (to I.T.) and R01-HL159012 (to J.S. and I.T.). D.N. was supported by American Heart Association postdoctoral award no. 24POST1192241. Immunofluorescence imaging experiments were conducted in the Columbia Center for Translational Immunology Core Facility, funded by NIH grant nos. P30CA013696, S10RR027050 and S10OD020056. Flow cytometry experiments were conducted using the Herbert Irving Comprehensive Cancer Center Flow Cytometry Shared Resources, funded in part through NIH grant no. P30CA013696. Samples for histological analysis were prepared in the Molecular Pathology Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant no. P30CA013696. The confocal microscopy work in this study was conducted in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grants nos. P30CA013696 and S10RR025686. This work was supported in part by the Proteomics & Metabolomics Core at Moffitt Cancer Center and funded as part of an NCI-designated Comprehensive Cancer Center (P30 CA076292).

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Department of Medicine, Columbia University Irving Medical Center, New York, NY, USA

Santosh R. Sukka, Patrick B. Ampomah, David Ngai, Xiaobo Wang, George Kuriakose & Ira Tabas

Proteomics and Metabolomics Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

Lancia N. F. Darville & John M. Koomen

Center for Nanomedicine and Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

Yuling Xiao & Jinjun Shi

Department of Animal Sciences, Integrative Immunology and Behavior Program and Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, IL, USA

Robert H. McCusker

Departments of Physiology and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA

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Contributions

S.R.S. and I.T. conceived the project. P.B.A., D.N., X.W., J.S. and R.H.M. provided additional intellectual input in the development of the project. L.N.F.D. conducted the LC–MS analyses under the guidance of J.M.K. G.K. and Y.X. helped with the mouse atherosclerosis experiments. S.R.S. and I.T. wrote the manuscript and the other co-authors provided comments and revisions.

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Correspondence to Santosh R. Sukka or Ira Tabas .

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Nature Metabolism thanks Derek W. Gilroy, Laurent Yvan-Charvet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina in collaboration with the Nature Metabolism team.

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Extended data

Extended data fig. 1 related to fig. 1 : macrophage efferocytosis drives trp metabolism..

a , Tryptophan and kynurenine content expressed as pmol/μg cell protein in BMDMs incubated ± ACs from the experiment in Fig. 1a ( n  = 6 biological replicates/group). b,c , Additional metabolite values from BMDMs incubated ± ACs from the experiment in Fig. 1a ( n  =  6 biological replicates/group). Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test.

Extended Data Fig. 2 Related to Fig. 1 : Examples of extracted ion chromatograms.

a , Reversed phase separation and mass spectrometry detection of 12 neat tryptophan metabolite standards (10 ng) individually analyzed. Each trace shows ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from the Atlantis T3 reversed phase column. b , Reversed phase separation on an Atlantis T3 reversed phase column and mass spectrometry detection of tryptophan metabolites from an AC- wild type sample. Each trace shows ion signal at a given mass-to-charge ratio (m/z) with a mass tolerance of 5 ppm. For both sets of chromatograms, data are shown for: ( i ) 2 − picolinic acid), ( ii ) nicotinic acid, ( iii ) nicotinamide, ( iv ) quinolinic acid, ( v ) nicotinamide adenine dinucleotide (NAD)+, ( vi ) serotonin, ( vii ) DL-kynurenine, ( viii ) N-formylkynurenine, ( ix ) L-tryptophan, ( x ) tryptamine, ( xi ) kynurenic acid, and ( xii ) anthranilic acid. The normalization level (NL) indicates the intensity of the base peak for each spectrum. In b, although more background was observed in the extracted ion chromatogram for anthranilic acid, the anthranilic acid signal is easily differentiated from the background.

Extended Data Fig. 3 Related to Fig. 1 : Absolute quantitation of kynurenine and tryptophan.

a-d , Quantitation using a known amount of isotope-labeled standards. a,b , Examples of extracted ion chromatograms showing reversed phase separation and mass spectrometry detection for kynurenine (10 ng) and D 4 -kynurenine (50 ng); and tryptophan (10 ng) and 13 C 11 -tryptophan (50 ng). Each trace shows the ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from an Atlantis T3 reversed phase column. c,d , Positive ion mass spectra of kynurenine and D 4 -kynurenine; and tryptophan and 13 C 11 -tryptophan. e-h , Quantitation of kynurenine and tryptophan in an AC − wild type sample. e,f , Examples of extracted ion chromatograms showing reversed phase separation and mass spectrometry detection for endogenous kynurenine and spiked D 4 -kynurenine (50 ng); and endogenous tryptophan and spiked 13 C 11 -tryptophan (50 ng). Each trace shows the ion signal at a given mass-to-charge ratio (m/z) as the compounds elute from an Atlantis T3 reversed phase column. g,h , Positive ion mass spectra of endogenous kynurenine and D 4 -kynurenine; and endogenous tryptophan and 13 C 11 -tryptophan. The normalization level (NL) indicates the intensity of the base peak for each spectrum.

Extended Data Fig. 4 Related to Fig. 1 : The role of SLC36A4 in Trp metabolism in efferocytosing macrophages.

a , Immunofluorescence microscopy of SLC36A4 (green) and LAMP-1 (red) in Scr- or siSlc36a4-transfected BMDMs incubated 45 mins with PKH26-labelled ACs (pseudocolored white); DAPI (blue) nuclear stain. Scale bar, 50 μm. Another set of cells was assayed for Slc36a4 mRNA and immunoblotted for SLC36A4 ( n  =  3 biological replicates/group). b , Immunofluorescence microscopy of SLC36A4 (green) in BMDMs incubated with PKH26-labelled ACs (red) for 45 min. Scale bar, 50 μm. White arrows , engulfed ACs; blue arrows , unengulfed ACs. c , Representative image of Scr-transfected macrophages not incubated with ACs and then stained for SLC36A4 (green) and DAPI (blue); note low expression of SLC36A4 compared with AC + macrophages in Fig. 1b . Image is representative of 3 biological triplicates. Scale bar, 50 μm. d , BMDMs were incubated ± apoptotic Jurkat cells (apJCs) or apoptotic macrophages (apMϕs), chased for 3 h, and assayed for Slc36a4 ( n  =  6 biological replicates/group). e , BMDMs were incubated ± ACs or PS-beads for 1 h, chased for 3 h, and immunoblotted for SLC36A4. f , BMDMs pre-treated with ± 20 µM MG132 were incubated ± ACs for 1 h, chased for 3 h, and immunoblotted for SLC36A4. g , Scr- or siSlc36a4-transfected BMDMs were incubated with PKH26-labelled ACs (red) and quantified for the percentage of PKH26 + macrophages ( arrows ) of total macrophages. Scale bar, 50 μm ( n  =  5 biological replicates/group). h , Tryptophan and kynurenine in Scr- or siSlc36a4-transfected BMDMs incubated with ACs (see Fig. 1d ; n  =  6 biological replicates/group). i , BMDMs were incubated ± apoptotic macrophages for 1 h and assayed for Ido1 ( n  =  3 biological replicates/group). j , Scr- or siSLC36A4-transfected HMDMs were for SLC36A4 ( n  =  3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Source data

Extended data fig. 5 related to figs. 2 and 3 : additional data on the roles of slc36a4 and ido1 in resolution..

a , Control or IDO1-KO BMDMs incubated with PKH26-labelled ACs were quantified for percent PKH26 + macrophages ( arrows ). Scale bar, 50 μm ( n  =  6 biological replicates/group). b , Scr- or siIdo1-transfected BMDMs incubated with pHrodo-Red-labelled ACs were quantified for percent pHrodo-Red + macrophages of total macrophages by flow cytometry ( n  =  3 biological replicates/group); immunoblotted for IDO1 ( n  =  3 samples/group); and assayed for Ido1 ( n  =  6 biological replicates/group). c , Flow cytometry contour plots for the experiment in Fig. 2b . d , BMDMs treated with ACs ± epacadostat were assayed for Arg1 and Mcf2 after a 6-h or 2-h chase, respectively ( n  = 3 biological replicates/group). e,f , Contour plots for the experiments in Fig. 2g, h . g , Scr- or siIDO1-transfected HMDMs were assayed for IDO1 ( n  =  3 biological replicates/group). h , Scr- or siSlc36a4-transfected BMDMs were incubated ± apoptotic macrophages, chased for 6 h, and assayed for Tgfb1 and Il10 ( n  =  3 biological replicates/group). i , Scr- or siSlc36a4-transfected BMDMs were pre-treated ± kynurenine and then incubated ± ACs for 1 h, chased for 6 h in Trp-deficient medium, and assayed for Tgfb1 and Il10 mRNA. Right, normal and Trp-depleted media were assayed for Trp by LC − MS/MS ( right ) ( n  =  3 biological replicates/group). j , BMDMs pre-treated for 1 h with vehicle or 50 µM Trp were incubated ± ACs, chased for 6 h, and assayed for Tgfb1 and Il10 ( n  =  3 biological replicates/group). k , Scr- or siSlc36a4-transfected BMDMs were pre-treated ± 50 μM Trp for 1 h, incubated ± ACs. and assayed for Tgfb1 and Il10 ( n  =  3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Extended Data Fig. 6 Related to Figs. 4 and 5 : Additional in-vivo and in-vitro data on the IDO1-Kyn-AhR pathway.

a-c , The thymi of the mice from Fig. 4g–e were immunostained for IDO1 (red) and Mac2 (green) and quantified for IDO1 MFI in Mac2 + areas ( arrows ). Also shown are thymus weight, thymus cellularity, and F4/80 + macrophages/thymus ( n  =  8 mice/group). d , Control and Ido1 −/− BMDMs ( top 2 graphs ), or Scr- and siSlc364-transfected BMDMs ( bottom graph ), were pre-treated for 1 h ± Kyn and incubated ± ACs. After a 6-h chase, the cells were assayed for Cyp1a1 and Cyp1b1 ( n  =  3 biological replicates/group). e , BMDMs were incubated ± apoptotic macrophages and assayed for Cyp1a1 . f , Scr- or siSLC36A4-transfected HMDMs were incubated ± ACs, chased for 3 h, and assayed for CYP1A1 and CYP1B1 ( n  =  3 biological replicates/group). g , Scr- or siAhr-transfected BMDMs were incubated with DiD-labelled ACs and quantified for percent DiD + macrophages ( arrows ). Scale bar, 50 μm ( n  =  4 biological replicates/group). h , BMDMs pre-treated ± CH223191 were assayed for continuing efferocytosis as in Fig. 2a . Arrows , PKH26 + PKH67 + macrophages. Scale bar, 50 μm ( n  =  3 biological replicates/group). i , As in panel h, but one of the cohorts was also treated with cytochalasin D before the second round of efferocytosis. Arrows , PKH26 + PKH67 + macrophages. Scale bar, 50 μm ( n  =  3 biological replicates/group). j , Scr- or siAhr-transfected BMDMs were assayed for Ahr ( n  =  3 biological replicates/group) and immunoblotted for AhR protein ( n  =  3 samples/group). k , Scr- or siAHR-transfected HMDMs were assayed for AHR ( n  =  3 biological replicates/group). l , Scr- or siArnt-transfected BMDMs were assayed for Arnt ( n  =  3 biological replicates/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Extended Data Fig. 7 Related to Figs. 6 and 7 : Additional in-vivo and in-vitro data on the role of AhR in efferocytosis-induced resolution.

a , BMDMs were incubated for 1 h with 100 μM Kyn alone or with control (non-PS) or PS beads ± Kyn and then assayed for Cyp1a1 and Ido1 after a 3-h chase ( n  =  4 samples/group). b , BMDMs incubated with ACs, PS-beads (PS), Kyn, or PS-beads + Kyn were assayed for Tgfb1 after a 6-h chase ( n  =  4 samples/group). c , BMDMs incubated with 50 μM Trp, PS-beads (PS), Trp and PS-beads, or 100 μM Kyn and PS-beads for 1 h were assayed for Cyp1a1 and Il10 after a 3-h or 6-h chase, respectively ( n  = 3 biological replicates/group). d , BMDMs pre-treated ± U0126 were incubated with ACs for 1 h and then immunoblotted for AhR and ARNT after a 3-h chase ( n  =  3 samples/group). e , BMDMs treated with PS-beads (PS) and Kyn ± U0126 for 1 h were assayed for Tgfb1 or Ido1 after a 3-h chase or 6-h chase, respectively ( n  = 3 biological replicates/group). f , BMDMs incubated ± ACs ± CH223191 for 1 h were assayed Hsp90 or Xap2 after a 3-h chase ( n  =  3 biological replicates/group). g , Scr-, siHsp90-, or siXap2-transfected BMDMs were incubated with PKH26-labelled ACs and quantified for percent PKH26 + macrophages ( arrows ). Scale bar, 50 μm ( n  =  3 biological replicates/group). h , Proposed pathway (created using BioRender.com): Trp form an efferocytosed AC (AC1) is transported into the macrophage by SLC36A4 and then converted to Kyn by IDO1. Kyn and activated ERK induce Hsp90 and Xap2, leading to AhR-ARNT-mediated transcription of Tgfb1 , Il10 , and Ido1 and Rac1-mediated AC2 internalization (continuing efferocytosis). i-k , The thymi of the mice from Fig. 7 were immunostained for AhR (red) and Mac2 (green) and quantified for AhR MFI in Mac2 + areas ( arrows ). Also shown are thymus weight and F4/80 + macrophages/thymus ( n  =  8 mice/group). All mRNA data are expressed relative to the indicated control groups. Data are mean ± SEM, and significance was determined by two-tailed Student’s t-test or one-way ANOVA with Fisher’s LSD post-hoc analysis.

Extended Data Fig. 8 Related to Fig. 8 : Systemic and lesional parameters in control and Mϕ-IDO1-iKO BMT Ldlr −/− mice.

Ldlr −/− mice were transplanted with BM from Ido1 fl/fl (Control) or Ido1 fl/fl Cx3cr1cre ERT2+/− (Mϕ-IDO1-iKO) mice and then fed the Western diet for 16 weeks. One cohort from each group was harvested (Baseline), and the rest of the mice were switched to chow diet, injected with HDAd-LDLR virus, and given tamoxifen. After 7 weeks, the mice were harvested (Regression). a-k , Body weight, total plasma cholesterol, fasting blood glucose, complete blood count ( n  = 9–10 mice/group). WBC, white blood cell; NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils, RBC, red blood cells; PLT, platelets. l , Immunostaining of IDO1 (red) and Mac2 (green) in regressing aortic root lesions, with quantification of IDO1 MFI in Mac2 + and Mac2 − areas. Arrows , examples of IDO1-Mac2 co-localization. DAPI was used for nuclear staining. Scale bar, 25 μm ( n  = 10 mice/group). m , Quantification of lesion area, based on H&E staining of the aortic root lesions ( n  = 10 mice/group). n , The regressing aortic root lesions of Control and Mϕ-IDO1-iKO groups were immunostained for Mac2 (macrophages; green) and TGF-β1 or IL-10. (red) Arrows, examples of colocalization of Mac2 and TGF-β1 (top) and Mac2 and IL-10 (bottom). DAPI (blue) was used for nuclear staining. Scale bar, 50 μm. o , The total number of Mac2 + cells per lesion section was quantified in regressing aortic root lesions ( n  = 10 mice/group). The data are expressed as mean ± SEM, and significance was determined by one-way ANOVA with Fisher’s LSD post-hoc analysis for panels a-k and m, and by Student’s t-test for panels l and o.

Supplementary information

Supplementary tables 1–5.

Supplementary Table 1: Macrophage efferocytosis drives tryptophan metabolism. Supplementary Table 2: The average and s.d. of retention time and m/z of tryptophan metabolites using LC–MS. Supplementary Table 3: Macrophage efferocytosis drives tryptophan metabolism in an SLC36A4-dependent manner. Supplementary Table 4: List of antibodies. Supplementary Table 5: Primer sequences and RNAi list.

Reporting Summary

Source data figs. 1, 5, and 6.

Unprocessed western blots for Figs. 1, 5, and 6.

Source Data Extended Data Fig. 4–7

Unprocessed western blots for Extended Data Figs. 4–7.

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Sukka, S.R., Ampomah, P.B., Darville, L.N.F. et al. Efferocytosis drives a tryptophan metabolism pathway in macrophages to promote tissue resolution. Nat Metab (2024). https://doi.org/10.1038/s42255-024-01115-7

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DOI : https://doi.org/10.1038/s42255-024-01115-7

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Towards new delivery agents for boron neutron capture therapy: synthesis and in vitro evaluation of a set of fluorinated carbohydrate derivatives.

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1. Introduction

2. results and discussion, 2.1. chemical synthesis and structural characterization, 2.2. early-stage in vitro assessment, 3. conclusions, 4. materials and methods, 4.1. synthetic protocols, 4.2. substrate-specific analytical data, 4.3. cytotoxicity studies, 4.4. affinity studies, 4.5. boron uptake studies, 4.6. plasma stability studies, 4.7. plasma protein binding studies, supplementary materials, author contributions, data availability statement, acknowledgments, conflicts of interest.

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Matović, J.; Järvinen, J.; Sokka, I.K.; Imlimthan, S.; Aitio, O.; Sarparanta, M.; Rautio, J.; Ekholm, F.S. Towards New Delivery Agents for Boron Neutron Capture Therapy: Synthesis and In Vitro Evaluation of a Set of Fluorinated Carbohydrate Derivatives. Molecules 2024 , 29 , 4263. https://doi.org/10.3390/molecules29174263

Matović J, Järvinen J, Sokka IK, Imlimthan S, Aitio O, Sarparanta M, Rautio J, Ekholm FS. Towards New Delivery Agents for Boron Neutron Capture Therapy: Synthesis and In Vitro Evaluation of a Set of Fluorinated Carbohydrate Derivatives. Molecules . 2024; 29(17):4263. https://doi.org/10.3390/molecules29174263

Matović, Jelena, Juulia Järvinen, Iris K. Sokka, Surachet Imlimthan, Olli Aitio, Mirkka Sarparanta, Jarkko Rautio, and Filip S. Ekholm. 2024. "Towards New Delivery Agents for Boron Neutron Capture Therapy: Synthesis and In Vitro Evaluation of a Set of Fluorinated Carbohydrate Derivatives" Molecules 29, no. 17: 4263. https://doi.org/10.3390/molecules29174263

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