Indole Nitrogen Research Articles

Activity and Biocatalytic Potential of an Indolylamide Generating Thioesterase.

The chemical synthesis of N-acyl indoles is hindered by the poor nucleophilicity of indolic nitrogen, necessitating the use of strongly basic reaction conditions that encumber elaboration of highly functionalized scaffolds. Herein, we describe the total chemoenzymatic synthesis of the bulbiferamide natural products by the biochemical activity reconstitution of a nonribosomal peptide synthetase assembly line-derived (NRPS-derived) thioesterase that neatly installs the macrocyclizing indolylamide. The enzyme represents a starting point for biocatalytic access to macrocyclic indolylamide peptides and natural products.

Tryptophanol-derived oxazoloisoindolinone fluorescent probes for cellular localization studies of p53 activators

The protein p53 is a transcription factor with several key roles in cells, including acting as a tumour suppressor. In most human cancers its tumour suppressor function is inactivated, either through inhibition by negative regulators or by mutation in the TP53 gene. Thus, there is a high interest in developing molecules able to activate p53 tumour suppressor activity. Tryptophanol-derived isoindolinones are known to act as wild-type and mutant p53 activators. Specifically, SLMP53-1 is a non-fluorescent wild-type and mutant p53 R280K reactivator, with potent in vivo anti-tumour activity in HCT116 and MDA-MB-231 mice xenograft models. With the aim of studying tryptophanol-derived isoindolinones intracellular localization by fluorescence microscopy, three SLMP53-1 based fluorescent probes were prepared. Here we report the design, synthesis, photophysical characterization, antiproliferative activity and cell localization studies of these fluorescent probes.The previously described structure–activity relationships of the SLMP53-1 scaffold set the basis for the design the fluorescent probes. The probes were prepared by connecting a small fluorophore (dansyl or 7-nitrobenzofurazan) to the indole nitrogen of the tryptophanol-derived oxazoloisoindolinone SLMP53-1 through two different linkers. The antiproliferative activity and cell localization studies of the three fluorescent probes were performed in HCT116 cells. The three probes showed enhanced internalization when compared with their fluorophore-linker intermediates, good photo-stability and high affinity for the endoplasmic reticulum, indicating the potential involvement of endoplasmic reticulum in the mechanism of action of tryptophanol-derived oxazoloisoindolinones.

Rotameric heterogeneity of conserved tryptophan is responsible for reduced photochemical quantum yield in cyanobacteriochrome slr1393g3.

The red/green cyanobacteriochrome (CBCR) slr1393g3 exhibits a quantum yield of only 8% for its forward photoconversion significantly lower than other species from the same CBCR subfamily. The cause for this reduced photoconversion is not yet clear, although in the related NpR6012g4 dark-state structural heterogeneity of a paramount Trp residue has been proposed to cause the formation of nonproductive subpopulation. However, there is no such information on the equivalent residue in slr1393g3, W496. Here we use solid-state NMR to explore all possible sidechain rotamers of this Trp residue and their local interactions at the atomic level. The indole nitrogen (Nε1) is used as an NMR probe, achieved by site-specific 15N-indole labeling of a quadruply Trp-deleted variant and trehalose vitrification technique. The data reveal a set of seven indole rotamers of W496 with four distinct environments for the Nε1-H group. Only a minority population of 20% is found to retain the π-stacking and hydrogen-bonding interactions with the chromophore in the dark state that has been assigned to account for complete forward photoconversion. Our results demonstrate the direct role of W496 in modulating the forward quantum yield of slr1393g3 via rearrangement of its sidechain rotameric conformations.

A novel exopolysaccharide-producing bacterium, Pseudescherichia liriopis sp. nov. isolated from Liriope platyphylla, enhances the growth of Daucus carota subsp. sativus under drought and salinity stress.

Biological and abiotic stresses in plant growth are associated with reduced crop yields. Therefore, improving plant stress resistance can be a crucial strategy to improve crop production. To overcome these problems, plant growth-promoting bacteria are emphasized as one of the alternative tools for sustainable agriculture. This study found a novel strain (L3T) of a plant growth-promoting bacterium in fermented Liriope platyphylla fruit. Strain L3T showed the ability to promote plant growth. The L3T strain promoted plant growth of D. carota subsp. sativus, increasing the length (increase rate compared to the control group, 36.98%), diameter (47.06%), and weight of carrots (81.5%), ultimately increasing the edible area. In addition, we confirmed that plant growth was improved even in situations that inhibited plant growth, such as salinity and drought stress. Strain L3T performed indole production, siderophore production, phosphate solubilization, and nitrogen fixation, all characteristics of a strain that promotes plant growth. Genome analysis revealed genes involved in the growth promotion effects of strain L3T. Additionally, the properties of exopolysaccharides were identified and characterized using FTIR, TGA, and UHPLC. Our results demonstrated that L3 isolated from fermented L. platyphylla fruit can be used to simultaneously alleviate drought and NaCl stress.

Analytical and behavioral characterization of 1-hexanoyl-LSD (1H-LSD).

The development of lysergic acid diethylamide (LSD) derivatives and analogs continues to inform the design of novel receptor probes and potentially new medicines. On the other hand, a number of newly developed LSD derivatives have also emerged as recreational drugs, leading to reports of their detection in some countries. One position in the ergoline scaffold of LSD that is frequently targeted is the N1-position; numerous N1-alkylcarbonyl LSD derivatives have been reported where the acyl chain is attached to the indole nitrogen, for example, in the form of linear n-alkane substituents, which represent higher homologs of the prototypical 1-acetyl-N,N-diethyllysergamide (1A-LSD, ALD-52). In this study, 1-hexanoyl-LSD (1H-LSD, SYN-L-027), a novel N1-acyl LSD derivative, was characterized analytically using standard techniques, followed by evaluation of its in vivo behavioral effects using the mouse head-twitch response (HTR) assay in C57BL/6J mice. 1H-LSD induced the HTR, with a median effective dose (ED50) of 192.4 μg/kg (equivalent to 387 nmol/kg), making it roughly equipotent to ALD-52 when tested previously under similar conditions. Similar to other N1-acylated analogs, 1H-LSD is anticipated to by hydrolyzed to LSD in vivo and acts as a prodrug. It is currently unknown whether 1H-LSD has appeared as on the research chemical market or is being used recreationally.

Phosphate-solubilizing rhizobacteria and their effects on the growth and phosphorus uptake by wheat plants

Wheat represents a fundamental component of the diet that contributes to ensuring global food security. However, its cultivation faces various abiotic and biotic constraints that result in fluctuating yields. The use of plant growth promoting rhizobacteria (PGPR) with the ability to solubilize phosphate presents itself as an environmentally friendly solution aimed to at enhancing sustainable agriculture practices. The objective of the present study is to assess the potential of PGPR in augmenting both yield and phosphorus (P) levels in wheat plants (Triticum durum L). under semi-controlled environmental conditions. Initially, the strains under investigation were screened for their capacity to dissolve different forms of mineral complex P, along with other plant growth promoting attributes such as iron chelation, Indole acetic acid (IAA) production, nitrogen fixation, and production of exopolysaccharides. Plant response to inoculation with PGPR was studied through an experiment conducted in a greenhouse involving four distinct treatments: (1) Control plants without any inoculation, (2) Inoculation with the PGP27 strain (Rahnella aquatilis), (3) Inoculation with the PGP25 strain (Erwinia persicina), and (4) Inoculation with the PGP374 strain (Raoultella terrigena). Results demonstrated that the studied strains exhibited significant promoting activities, including the solubilization of complex phosphate, the production of exopolysaccharides, IAA, siderophores, and atmospheric nitrogen fixation. Furthermore, the inoculation of plants with PGPR led to an increase in both biomass and plant phosphate concentrations, surpassing those observed in the control group. In conclusion, the findings propose that species like Raoultella and Erwinia have the potential to offer agronomic advantages as rhizobacteria that promote plant growth.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Novel 6-alkyl-bridged 4-arylalkylpiperazin-1-yl derivatives of azepino[4,3-b]indol-1(2H)-one as potent BChE-selective inhibitors showing protective effects against neurodegenerative insults

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-β (Aβ) peptide at 100 μM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 μM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.

Panchagavya-Derived Brevibacillus brevis S1-3: Insights from the Draft Genome on its Antimicrobial and Plant Growth-Promoting Ability

Panchagavya has traditionally been used in Indian Ayurvedic practices because of its pro-agricultural and medicinal properties. This study presents the draft genome of a new Brevibacillus brevis S1-3 strain isolated from the fermented product Panchagavya. Through whole-genome sequencing, we determined that the genome of B. brevis S1-3 was 6,348,716 base pairs with a GC content of 54.3%. Genome assembly revealed the presence of 6107 protein-coding genes, 186 tRNA genes, and 13 rRNA genes. Genome annotation and analysis identified the genes involved in metabolism and other cellular processes. We also predicted the presence of several gene clusters associated with plant growth promotion, including indole acetic acid (IAA), gibberellic acid, ammonia, and nitrogen. Our study also revealed the genes responsible for the production of secondary metabolites that displayed a significant correlation with antimicrobial activity. Our results provide new insights into the genomic basis of the plant growth-promoting abilities of B. brevis and pave the way for further research in this field.

Generation of streptocarbazoles with cytotoxicities by pathway engineering and insights into their biosynthesis.

Indolocarbazoles (ICZs) are a group of antitumor agents, with several analogs used in clinical trials. Therefore, the identification of novel ICZ compounds is important for drug discovery. Streptocarbazoles harbor unique N-glycosidic linkages (N13-C1' and N12-C3'), distinguishing them from the representative ICZ compound staurosporine; however, their biosynthesis remains unclear. In this study, two new streptocarbazoles (1 and 3) with cytotoxic activities were obtained by manipulating the staurosporine biosynthetic gene cluster spc followed by heterologous expression. The biosynthetic pathway of streptocarbazoles was proposed, and their productions were improved through the overexpression of the key enzymes involved. This study enriches the structural diversity of ICZ compounds and would facilitate the discovery of new streptocarbazoles via synthetic biological strategies.

Transition-Metal-Free Synthesis of 2-Substituted Benzo[cd]Indoles via the Reaction of 1-Halo-8-lithionaphthalenes with Nitriles.

A simple and effective organolithium approach to the synthesis of 2-substituted benzo[cd]indoles from peri-dihalonaphthalenes and nitriles has been developed. The reaction proceeds via a surprisingly easy intramolecular aromatic nucleophilic substitution facilitated by the "clothespin effect". The discovered transformation provides good isolated yields, allows usage of an extensive range of nitriles, and demonstrates a good substituents tolerance. UV-absorption and NMR spectra of the obtained benzo[cd]indoles and their protonated forms demonstrated exclusive protonation to the indole nitrogen atom even in the presence of two NMe2 groups in positions 5 and 6 (i. e. "proton sponge" moiety).

CoO/Co-graphene quantum dots as an oxidative mimetic nanozyme for the colorimetric detection of L-cysteine.

The preparation of cobalt-based nanozymes with high oxidase-like activity still needs more efforts. In this paper, we report the synthesis of a CoO/Co-tryptophan-functional graphene quantum dot hybrid (CoO/Co-Try-GQD). Firstly, cobalt ions coordinate with the indole nitrogen on Try-GQD to form a complex, followed by thermal reduction and oxidation. The resulting hybrid presents a three-dimensional network structure, and CoO/Co nanoparticles are uniformly dispersed on the graphene sheet with an average size of 10 ± 0.24 nm. This unique structure improved the oxidase-like activity of the hybrid, enabling it to catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to rapidly produce deep blue ox-TMB with a strong absorbance at 652 nm (A652). A colorimetric method was developed for the highly sensitive determination of L-cysteine (L-cys) based on the inhibition of the hybrid's oxidase-like activity and low A652 caused by the binding of L-cys with Co atoms on CoO/Co via the Co-S bond. The A652 linearly decreased with increasing L-cys concentration in the range of 0.05-2 μM, and the detection limit was 0.032 μM. Further, the established method has been successfully applied to the determination of L-cys in milk.

Heavy Metal Co-Resistance with Antibiotics amongst Plant Growth Promoting Bacteria Isolates from Rhizosphere of Nypa Fruticans

Heavy metal co-resistance with antibiotics has the potential to amplify antimicrobial resistance genes in the environment, and this could have an impact on clinical settings. This study aimed to investigate the presence of heavy metal co-resistance with antibiotics among plant growth promoting bacterial isolates from the rhizosphere of Nypa fruticans. The heavy metals content of the water habouring the Nypa fruticans including copper(0.07mg/L), chromium (2.3 mg/L), lead (4.22 mg/L), iron (12.4 mg/L), cadmium (0.45 mg/L), nickel (3.12 mg/L), arsenic (0.47 mg/L) and zinc (5.37 mg/L) were above acceptable limits of WHO. A total of 10 bacteria including Bacillus subtilis, Bacillus cereus, Pseudomonas fluorescens, Klebsiella oxytoca, Serratia marcescens, Achromobacter denitrificans, Bulkholderia vietnamiensis and Azospirrilum brasilense, Azobacter vinelandii and Enterobacter aerogenes were isolated and identified to specie level using 16s RNA gene sequences, which showed above 95 % similarity with reference sequences from GenBank. The isolates showed high resistance to lead, chromium and zinc. Most of the isolates were resistant to streptomycin, ciprofloxacin and augmentin. Also, most of the isolates demonstrated positive growth promoting activities of indole acetic acid production, nitrogen fixation, siderophore production and phosphate solubilization. Pot trials on the effect of heavy metal and antibiotic tolerant bacteria on growth of beans (Phaseolus vulgaris l.) in heavy metal polluted soil revealed that beans treated with consortiumof all the isolates recorded high shoot and root length. Hence these isolates can be used for bioremediation of metal polluted site and as well act as plant growth promoting bacterial in agricultural system.

Asymmetric N‐Alkylation of 1H‐Indoles via Carbene Insertion Reaction

AbstractAn intermolecular enantioselective N‐alkylation reaction of 1H‐indoles has been developed by cooperative rhodium and chiral phosphoric acid catalyzed N−H bond insertion reaction. N‐Alkyl indoles with newly formed stereocenter adjacent to the indole nitrogen atom are produced in good yields (up to 95 %) with excellent enantioselectivities (up to >99 % ee). Importantly, both α‐aryl and α‐alkyl diazoacetates are tolerated, which is extremely rare in asymmetric X−H (X=N, O, S et al.) and C−H insertion reactions. With this method, only 0.1 mol % of rhodium catalyst and 2.5 mol % of chiral phosphoric acid are required to complete the conversion as well as achieve the high enantioselectivity. Computational studies reveal the cooperative relay of rhodium and chiral phosphoric acid, and the origin of the chemo and stereoselectivity.

Asymmetric N-Alkylation of 1H-Indoles via Carbene Insertion Reaction.

An intermolecular enantioselective N-alkylation reaction of 1H-indoles has been developed by cooperative rhodium and chiral phosphoric acid catalyzed N-H bond insertion reaction. N-Alkyl indoles with newly formed stereocenter adjacent to the indole nitrogen atom are produced in good yields (up to 95 %) with excellent enantioselectivities (up to >99 % ee). Importantly, both α-aryl and α-alkyl diazoacetates are tolerated, which is extremely rare in asymmetric X-H (X=N, O, S et al.) and C-H insertion reactions. With this method, only 0.1 mol % of rhodium catalyst and 2.5 mol % of chiral phosphoric acid are required to complete the conversion as well as achieve the high enantioselectivity. Computational studies reveal the cooperative relay of rhodium and chiral phosphoric acid, and the origin of the chemo and stereoselectivity.

Draft genome sequence of a halotolerant plant growth-promoting bacterium Pseudarthrobacter oxydans NCCP-2145 isolated from rhizospheric soil of mangrove plant Avicennia marina

BackgroundLimited knowledge exists regarding the diversity of mangrove plant-associated Pseudarthrobacter species. This study presents the first draft genome and phylogenetic analysis of a Pseudarthrobacter oxydans NCCP-2145, isolated from the rhizospheric soil of Aviciana marina, a native mangrove plant found in Miani Hor, Lasbela-Baluchistan, Pakistan. ResultsThe genome of P. oxydans NCCP-2145 comprises 4,495,869 base pairs, with a G+C content of 65.9% and 4,207 coding sequences. Genome annotation revealed the presence of multiple biosynthesis pathways. The analysis also identified genes responsible for plant growth-promoting traits, such as the synthesis of indole acetic acid, nitrogen fixation, and phosphorus solubilization. Experimental evaluations confirmed strain NCCP-2145 positive reactions for phosphorus solubilization, indole-3-acetic acid production, and ammonia production. Furthermore, strain NCCP-2145 exhibited tolerance to heavy metals (nickel, copper, and cadmium) and salinity levels up to 10% NaCl. Antibiotic susceptibility testing indicated resistance only to ceftazidime and the combination of amoxicillin/clavulanic acid. For phylogenomic analysis, strain NCCP-2145 was analyzed and compared to the closely related validly published type species P. oxydans DSM 20119T, revealing a similarity score of 98.64% based on 16S rRNA gene sequences and 89.1% based on DNA-DNA hybridization, confirming its classification as a member of species, P. oxydans. ConclusionsThis study significantly contributes to our understanding of the genomic characteristics, functional capabilities, and potential plant growth-promoting attributes of P. oxydans NCCP-2145. Future research should focus on unraveling the precise mechanisms underlying its plant growth-promoting abilities and exploring practical applications in sustainable agriculture and environmental restoration.How to cite: Bushra R, Uzair B, Ali A, et al. Draft genome sequence of a halotolerant plant growth-promoting bacterium Pseudarthrobacter oxydans NCCP-2145 isolated from rhizospheric soil of mangrove plant Avicennia marina. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.08.003.

Synthesis of β-Carbolines with Electrocyclic Cyclization of 3-Nitrovinylindoles.

The β-carboline motif is common in drug discovery and among numerous biologically active natural products. However, its synthetic preparation relies on multistep sequences and heavily depends on the type of substitution required in the core of the desired β-carboline target. Herein, we demonstrate that this structural motif can be accessed with the microwave-assisted electrocyclic cyclization of heterotrienic aci (alkylideneazinic acid) forms of 3-nitrovinylindoles. The reaction can start with 3-nitrovinylindoles themselves under two sets of conditions. The first one involves microwave irradiation of butanolic solutions of 3-nitrovinylindoles, whereas the second one consists of prior Boc protection of indolic nitrogen, where the protecting group cleanly comes off during the course of the reaction. Alternatively, the reaction can start with 3-nitrovinylindoles prepared in situ using various processes. Finally, the reaction may utilize indoles with β-nitrostyrenes, likely involving the intermediacy of spirocyclic oxazolines, which rearrange to similar heterotrienic systems undergoing cyclization to β-carbolines. As part of this study, several natural products, namely, alkaloids norharmane, harmane, and eudistomin N, were synthesized.

Green Halogenation of Indoles with Oxone-Halide.

Oxidative functionalization of indoles is one of the most widely used approaches to exploit the synthetic utility of indoles. In continuation of our research interest in the green oxidation of indoles, we further explore the oxidation of indoles with oxone-halide and discover that the protecting group on the nitrogen of indoles plays a decisive role in controlling the pathways of indole oxidation with oxone-halide. An electron-withdrawing group on the nitrogen of indoles (N-EWG) enables C2 halogenation with stoichiometric halide, while C3 halogenation could be selectively achieved by using stoichiometric halide without dependence on the electronic property of the protecting group on the indole nitrogen. Different from our previous results obtained by using catalytic halide, these findings lead to the development of an environmentally friendly, efficient, and mild protocol for access to 2- or 3-haloindoles (chloro and bromo). As compared to the previous synthetic methods for 2-/3-haloindoles, our method exploits the in situ-generated reactive halogenating species from oxone-halide for halogenation of indoles and thus eliminates the use of stoichiometric halogenating agents and the production of toxic and hazardous organic byproducts derived from oxidants.

Paenibacillus sp. S1S22 suşu ile Ulva lactuca Ekstresinin Kombine Kullanımının Domates Bitkisinin Tohum Çimlenmesi ve Büyüme Üzerine Etkilerinin Belirlenmesi

In recent years, plant growth promoting bacteria (PGPB) have been used as biofertilizers that increase agricultural productivity and plant resistance to changing environmental conditions such as drought, salinity and heat stress. One of the proposed new approaches to increase plant productivity, especially under stress conditions, is using algae extracts, which usually consist of naturally occurring bioactive compounds. Paenibacillus sp. S1S22 isolated from sediment was tested for its performance in indole acetic acid production (IAA), nitrogen (N) fixation, phosphate solubilization, proteolytic activity, and cellulose activity. Its antimicrobial activity was also determined against different pathogens of Paenibacillus sp. S1S22. Tomato seeds were incubated with the S1S22 strain, and the S1S22 strain supplemented with U. lactuca. The seeds incubated with deionized water were used as control. As a result, we demonstrated that the addition of U. lactuca extract to tomato seedlings incubated with Paenibacillus sp. S1S22 strain increased the root and stem length compared to the control. The results indicate that macroalgae in combination with PGPR may be a much more effective way of promoting plant growth.

Base-Controlled Regiospecific Mono-Benzylation/Allylation and Diallylation of 4-Aryl-5-indolyl-1,2,4-triazole-3-thione: Thio-Aza Allyl Rearrangement

The regiospecific S-benzylation/allylation of two 4-aryl-5-indolyl-1,2,4-triazole-3-thione precursors was carried out using Et3N as a base. Allyl group migration from exocyclic sulfur to the triazole nitrogen (N3) was successfully achieved in a short time via thermal fusion without the need for any catalyst. The allylation of indole nitrogen, along with exocyclic sulfur or triazole nitrogen (N3), was carried out using K2CO3 as stronger base. S,N-Diallylated products were converted to N,N-diallylated analogues using a simple fusion approach. Structural analyses of the two newly synthesized hybrids 2b and 5b investigated via the X-ray diffraction of a single crystal combined with Hirshfeld calculations. The compound 5b was crystallized in a monoclinic crystal system and the P21/c space group, whereas in compound 2b, the crystal system comprises the less symmetric triclinic and P − 1 space group. The asymmetric unit contains one and two molecules of 5b and 2b, respectively, while the unit cell contains four molecules in both cases. Hirshfeld analysis was performed in both systems to analyze the non-covalent interactions that control molecular packing. For 5b, C…H, N…H, S…H, Cl…N and H…H interactions are the most significant. Their percentages are 23.7, 8.8, 4.5, 1.2 and 48.2, respectively. In the case of 2b, the Cl…C, S…N, C…H, H…H and N…H interactions have the upper hand in molecular packing. In one unit, the percentages of these contacts are 2.3, 0.9, 26.8, 38.7 and 9.3%, while in the other unit, the corresponding values are 4.4, 1.3, 22.1, 43.6 and 9.0%, respectively.