Synthesis of tris-indolylmethanealkaloids by harnessing the nucleophilic reactivity of Indole-BX and studying their interactions with hemoglobin

Abstract

In the current investigation, we have synthesized tris-indolylmethanealkaloid (TIM) derivatives by exploration of the nucleophilicity of cyclic hypervalent iodine reagent Indole-BX and also described the nature of the interaction between hemoglobin (Hb) with tris-indolylmethanealkaloids (TIM) 10 and 5 using different multi-spectroscopic studies. Fluorescence spectroscopy investigations have revealed a stronger interaction of TIM 10 with Hb than that of TIM 5, which is almost structurally similar. Circular dichroism (CD) analysis indicates that the native Hb conformation unfolds in the presence of both compounds. According to the thermal melting investigation, both TIM 10 and 5 have a small effect on Hb stability. This study demonstrates that subtle structural variations in TIM 10 and 5 can drastically alter interaction properties, potentially aiding drug discovery.

---

Introduction

Indole is an essential heterocyclic molecule used in many fields of science and industry.^1–11 Its flexibility in chemical synthesis and its presence in natural products continue to motivate research and development across a variety of sectors, including materials science and medicine.^12–15 Bis- and tris-indole derivatives are important compounds and have been found to have many potential bioactivities.^16–20 Isolation as well as synthesis of bis-indolylmethanes (BIMs) (1, 2) (arundine, vibrindole),^21,22 bis-indolylarsinolmethane BIAMs (3, 4) (arsinol line A),^23–30 tris-indolylmethane (TIM) (5) have received considerable attention from the scientific community (Fig. 1).

Fig. 1 Privileged structures of bis- and tris-indolylmethanealkaloids.

Hemoglobin (Hb) is one of the most important proteins in our body. It is a tetramer protein, consisting of a pair of α chains and a pair of β chains. Each α chain consists of 141 amino acids, and each β chain consists of 146 amino acids.³¹ Heme, an iron-containing porphyrin ring, is present in every chain.³² Hb is generally contained within erythrocytes, with only a small amount in plasma. Hb is mainly associated with the gaseous transport in our body. It generally collects oxygen from the lungs, releases it in tissues, collects carbon dioxide from tissues, and delivers it to the lungs.³³ Additionally, it also interacts with different endogenous and exogenous compounds reversibly, such as 2,3-bisphosphoglycerate,³⁴ alkaloids,³⁵ flavonoids,³⁶ and food colorants, etc.³⁷ Among different compounds, alkaloids are an important group due to their various medicinal properties, such as, antioxidant,³⁸ antitumor,³⁹ neuroprotective,⁴⁰ anti-inflammatory,⁴¹ and analgesic⁴² activities. Hence, the interaction between Hb and different alkaloids has been well studied.⁴³ Among the various interactions of this class of molecules, those involving tris-indole derivatives with Hb remain unexplored, aside from a few reports on their antitumor⁴⁴ and cytotoxic activities.⁴⁵

As a continuation of our work^46–48 in the field of hypervalent iodine chemistry, we wish to explore Waser's Indole-BX⁴⁹ for the synthesis of different indole derivatives. In general so far this Indole-BX were used as an electrophilic indole source from hypervalent iodine surrogates by suppressing the innate nucleophilicity of Indole nucleus. However, recently, Yoshikai reported⁵⁰ that many hypervalent iodine reagents also act as nucleophilic group-transfer agents. Motivated by this finding, we plan to investigate the possibility that Indole-BX may function as a nucleophile.

In this study, we first synthesized bis- and tris-indole derivatives from Indole-BX. Then, we examined how structural changes in the ligands affect their interactions with hemoglobin (Hb) using various analytical methodologies. This understanding will be especially important when dealing with novel agents, as it will help predict interactions and their consequences. Besides that, the study also provides useful information to understand how interactions between Hb and ligands affect the normal structure and function of Hb.

Experimental sections

Materials

Lyophilized human Hb and methanol were obtained from Sigma-Aldrich. The Hb concentration was calculated by spectrophotometric absorbance considering the molar extinction coefficient of Hb at 276 nm (120 808 M⁻¹ cm⁻¹).³⁵ All experiments were executed using a buffer containing 50 mM KCl, 10 mM KH₂PO₄, and 1 mM K₂EDTA (pH 7.4) at room temperature (25 °C). In each experiment, a minimal methanol concentration (less than 1% by volume) was used. TIMs were dissolved in methanol.

Absorption spectroscopy

The UV-Vis absorption spectra of Hb alone and in the presence of TIMs were recorded on a Hitachi UH5300 spectrophotometer. In this experiment, the concentration of Hb (2.5 µM) was kept constant, and the concentration of TIMs was gradually increased from 0 to 9.5 µM. The experiment was executed using a cuvette with a 1 cm path length. The spectra were collected with a data interval of 1 nm and a scanning speed of 400 nm min⁻¹.

The binding constant (K_b) between Hb and TIMs was calculated considering the absorbance of Hb at 405 nm using the modified Benesi–Hildebrand equation.⁵¹

where A₀, A, and A₁ are the absorbance intensities of Hb in the absence of ligands, at an intermediate ligand concentration, and at an infinite ligand concentration (representing saturation level of interaction, respectively). K_b is the binding constant. C was the concentration of ligands. The plot of 1/ΔA vs. 1/C yields a straight line, and the corresponding binding constant (K_b) was determined from the intercept to slope ratio of the aforesaid plot.

Fluorescence spectroscopy

The fluorescence properties of Hb were investigated using fluorescence spectroscopy. Fluorescence spectroscopy was performed on a Fluoromax-4C spectrofluorometer (Horiba Scientific) using a cuvette with a 1 cm path length at 298, 303, and 310 K. In this experiment, the concentration of Hb (2.5 µM) was kept constant, and the concentration of TIMs was gradually increased from 0 to 9.5 µM. In this study, the Hb was excited at 280 nm.

Stoichiometry

The stoichiometry between Hb and TIMs was performed using a UV-Vis spectrophotometer. In this experiment, the overall concentration (C_Hb + C_Ligand) was held constant, while the molar fractions of Hb and TIMs were continuously changed. The difference in absorbance was calculated (ΔA = A_Hb − A_Hb+Ligand) and plotted against the molar fraction of the ligand. The Job plot was carried out, considering the variation in absorbance at the Soret band.

Circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy of Hb in the absence and presence of TIMs was taken using a Jasco J-1500 CD spectrometer. In this study, the concentration of Hb was kept at 3 µM, and the concentration of TIMs was increased to obtain Hb: TIMs ratios of 1 : 1 to 1 : 4. The study was performed in a 1 mm path-length cuvette. The scanning speed of the CD spectrometer was 100 nm min⁻¹. Each spectrum was the average of three individual scans. CD results were converted to mean residue ellipticity (MRE) in deg cm² dmol⁻¹ using the following formula:
where C is the molar protein concentration, n is the number of amino acid residues, and l is the path length of the cuvette in centimeters.

The quantity of α-helix in different Hb samples can be estimated from the MRE value at 222 nm using the equation of Chen et al.⁵²

UV-Melting study

Thermal denaturation studies of Hb in the absence and presence of TIMs were performed on a Hitachi UH5300 absorption spectrophotometer equipped with a Peltier temperature controller. In this study, the concentration of Hb was kept at 5 µM, and TIMs were used throughout the course at a 1 : 1 ratio. The experiment was performed in a cuvette with a 1 cm path length. The samples were heated at the rate of 1 °C min⁻¹, and the change in absorbance was monitored at 280 nm.

Results and discussion

Synthesis of TIMs from Indole-BX

To establish the optimal reaction conditions for the desired transformation, a series of experiments was conducted using benzaldehyde (6a) and N-methylated Indole-BX (7a) as the model substrates (Table 1). Inspired by earlier reports on bis-indole formation from aldehydes and simple indoles,^53–56 we initially screened those reported conditions using Indole-BX in place of a regular indole. However, attempts with various additives in the absence of a transition-metal catalyst were unsuccessful. When NBS (10 mol%) or NH₄Cl (1 equiv.) was used either neat or in MeCN, no product formation was observed, even after prolonged heating at 120 °C (entries 1–3).

Table 1 Optimization of the reaction conditions

S. no.	Indole-BX (equiv.)	Catalyst (4 mol%)	Additive	Solvent	T (°C)	Time (h)	Yield (%)
Optimized reaction conditions: 6a (1 equiv.), 7a (2 equiv.), [RhCp*Cl2]2 (4 mol%), AgSbF6 (20 mol%), gl. AcOH (5 mol%), HFIP (1.5 mL), 110 °C for 6 h; ND = not detected.
1.	2	—	NBS (10 mol%)	—	rt-120	24	ND
2.	2	—	NH4Cl (1 equiv.)	—	120	24	ND
3.	2	—	NH4Cl (1 equiv.)	MeCN	120	24	ND
4.	2	—	I2 (20 mol%)	MeCN	rt-120	24	ND
5.	2	—	I2 (20 mol%)	HFIP	rt-120	24	ND
6.	2	—	gl. AcOH (10 mol%)	—	rt-120	24	ND
7.	2	—	gl. AcOH (10 mol%)	HFIP	rt-120	24	ND
8.	2	—	gl. AcOH (10 mol%)	DCE	rt-120	24	ND
9.	2	[RhCp*Cl2]2	gl. AcOH (5 mol%)	DCE	rt-120	8	46
			AgSbF6 (20 mol%)
10.	2	[RhCp*Cl2]2	gl. AcOH (5 mol%)	HFIP	110	6	62
			AgSbF 6 (20 mol%)
11.	2	—	gl. AcOH (5 mol%)	HFIP	110	6	ND
			AgSbF6 (20 mol%)
12.	2	[RhCp*Cl2]2	AgSbF6 (20 mol%)	HFIP	110	8	7
13.	2	[RhCp*Cl2]2	gl. AcOH (5 mol%)	HFIP	110	12	11
14.	2	FeCl3	gl. AcOH (5 mol%)	HFIP	rt-120	24	ND
			AgSbF6 (20 mol%)
15.	2	Ru(PPh3)2Cl2	gl. AcOH (5 mol%)	HFIP	rt-120	24	ND
			AgSbF6 (20 mol%)

---

Similarly, the use of I₂ (20 mol%) in MeCN or HFIP, as well as glacial AcOH (10 mol%) in different solvents, such as HFIP or DCE, failed to yield the desired product 8a (entries 4–8). Encouraged by previous reports on C–H activation using Rh(III) complexes, we turned our attention to [RhCp*Cl₂]₂ in combination with AgSbF₆ as a halide abstractor. Gratifyingly, when [RhCp*Cl₂]₂ (4 mol%) was used along with gl. AcOH (5 mol%) and AgSbF₆ (20 mol%) in DCE, the desired product 8a was obtained in 46% yield after 8 h (entry 9). A further enhancement in yield was observed when HFIP was employed as the solvent at a slightly elevated temperature (110 °C), affording the product in 62% yield within 6 h (entry 10). Control experiments confirmed the essential roles of both the Rh(III) catalyst and additives. In the absence of the rhodium catalyst (entry 11), or when either gl. AcOH (entry 12) or AgSbF₆ (entry 13) was omitted; a significant drop in yield was noted, underscoring the synergistic effect of all three components. Attempts to replace Rh(III) with other metal catalysts such as FeCl₃ and Ru-based complexes proved ineffective under similar conditions (entries 14 and 15), with no detectable product formation. These results clearly indicate that the combination of [RhCp*Cl₂]₂ (4 mol%), glacial AcOH (5 mol%), and AgSbF₆ (20 mol%) in HFIP at 110 °C provides the most efficient and reproducible conditions for the desired transformation.

With this result, we can say that our hypothesis regarding the innate nucleophilicity of indole also holds for Indole-BX reagents. Encouraged by the optimized conditions, we next investigated the substrate scope to evaluate the versatility of our methodology (Scheme 1). Although numerous protocols for synthesizing bis- and tris-indolyl frameworks were reported in the literature, both metal-free and metal-catalyzed,^53–78 often providing high yields under simple and economic conditions, our focus here is on showcasing the unique reactivity of Indole-BX reagents. While our isolated yields are modest in comparison, the method's adaptability and potential for biological application, particularly in haemoglobin interaction studies, prompted us to pursue both the developed protocol and literature-reported approaches for scale-up. Upon introducing electron-donating substituents such as methyl groups at the ortho- and para-positions of the aromatic aldehyde (6) (Scheme 1(A)), under the optimized conditions with N-methylated Indole-BX (7a), we observed a slight increase in yields for the corresponding bis-indole adducts (8b and 8c: 65% and 70%, respectively). Halogen-substituted substrates (para-F) also performed well, affording product 8d in shorter reaction times (2 h) with a moderate 60% yield, underscoring the robustness of the reaction conditions. The electron-donating group (para-NO₂) also gives comparable yields (8e: 58%). For the tris-indole series (Scheme 1(B)), employing N-methylated derivatives of both indole-3-aldehyde and Indole-BX reagent, afforded the desired product (10) in 56% yield. In contrast, use of NH-indole-3-aldehyde led to a reduced yield (5, 44%), possibly due to subtle electronic differences or variations in solubility and hydrogen-bonding behaviour under the reaction conditions. Moreover, the reaction with other heteroaromatic aldehydes, such as pyrrole-2-carboxaldehyde, resulted in the formation of the expected product in very low yield (11:18%), whereas thiophene-2-carboxaldehyde failed to yield the corresponding product (12); instead, a complex reaction mixture was observed. We tested aromatic and heteroaromatic aldehydes as well as an aliphatic aldehyde (such as pentanal) under the optimized conditions, but no product formation was observed. Inspired by the well-documented biological relevance of TIMs in medicinal chemistry,^16–20 we extended our methodology toward their synthesis with an eye on probing their interactions with haemoglobin in future studies.

Scheme 1 Substrate scope: (A) variation with aryl aldehyde; (B) variation with heteroaryl aldehyde.

A plausible mechanism for this transformation is illustrated in Scheme 2. In the presence of AgSbF₆, a partially naked cationic Rh(III) species (A) is generated in situ, which undergoes oxidative addition with Indole-BX (7) to form intermediate B. A coordination followed by nucleophilic addition of the aldehyde (6/9) to B affords intermediate C through attack of the indole moiety at the formyl carbon, followed by coordination of the resulting alkoxide oxygen to the rhodium centre.^79,80 A subsequent hydride migration to rhodium furnishes intermediate D along with the formation of ketone 13. The rhodium catalyst is then regenerated through the elimination of 2-iodobenzoic acid (14), which can re-enter the catalytic cycle via a new oxidative addition with another molecule of Indole-BX (7) to regenerate B. Resembles to the earlier step [B-C], the newly formed ketone 13 undergoes nucleophilic addition by indole and coordinate to the Rh(III) centre, generating intermediate E. Reductive elimination from E regenerates the active catalyst. It may produce a peroxy-type intermediate F. Subsequent nucleophilic attack of water at the carbonyl carbon, followed by cleavage through intermediate G, delivers the desired products (8/5/10/11) along with molecular oxygen and 2-iodobenzoic acid (14).

Scheme 2 Proposed reaction mechanism.

UV-Vis absorption spectroscopy

UV-Vis absorption spectra of Hb possess two major absorption peaks: one at 269 nm corresponding to the phenyl groups of tryptophan and tyrosine residues in the globin portion, and another at 405 nm, called the Soret band, which arises from the heme group. The first peak arises from the π → π* transition of the phenyl amino acids, and the second peak appears due to the π → π* transition in the porphyrin ring of the heme group. Gradual addition of these two TIMs induces Hb spectral changes. These two compounds have no absorption, so the spectral change in Hb is solely due to their interaction with Hb. Following these two TIMs, the intensity of the Soret band decreased, while the maximum absorption wavelengths remained constant. These results indicate that the heme remains unexposed from the crevices at the exterior of the subunit, and these compounds are readily bound to the hydrophobic pocket of Hb.³⁵ The percentage of hypo-chromicity is high in the case of TIM 10 (26.47%) compared to TIM 5 (22.87%) (Fig. 2). Besides that, the binding constant between Hb and TIM 10 was 1.51 × 10⁵ M⁻¹, and Hb and TIM 5 was 5.67 × 10⁴ M⁻¹ (Fig. S1). This result indicates that the interaction between Hb and TIM 10 is stronger than that between Hb and TIM 5.

Fig. 2 UV-Vis spectroscopic study of the interaction between (A) Hb and TIM 10, and (B) Hb and TIM 5.

Fluorescence spectroscopy

The tryptophan residues of Hb are mainly responsible for its fluorescence. In fluorescence spectrophotometry, efficient energy transfer from tryptophan residues to heme significantly quenches the protein's inherent fluorescence. Hence, in buffer, Hb exhibits very low fluorescence. In this study, TIM 10 induced quenching of the fluorescence property of Hb, indicating interaction between Hb and TIM10. However, TIM 5 entered the hydrophobic cavity of Hb and induced the exposure of heme. As a result, the TIM 5 induced a small increase in the fluorescence intensity of Hb, and the fluorescence quenching of tryptophan by heme was suppressed at different temperatures (Fig. 3 and Fig. S5).⁸¹ To gain a comprehensive understanding of the quenching mechanism of the interaction between Hb and TIM 10, the fluorescence spectroscopy results were evaluated using the Stern–Volmer equation.⁸²

F₀/F = 1 + K_q × τ₀[L] = 1 + K_sv

Where F₀ and F are the fluorescence intensity of Hb in the absence and presence of TIM 10, respectively. Additionally, K_q, τ₀, [L], and K_sv are the quenching rate constant, the average lifetime of Hb, the concentration of the TIM 10, and the Stern–Volmer association constant, respectively. In this study, the K_sv values of the interaction between Hb and TIM 10 at different temperatures were reduced with an increase in temperature of the reaction condition (Fig. S2–S4 and Table S1). The result indicated that TIM 10 induced quenching of the fluorescence property of Hb was static in nature.⁸³ To understand the nature of the interaction between Hb and TIM 10, the fluorescence spectroscopic study was performed at three different temperatures, and the results were interpreted using the modified Stern–Volmer equation.⁸⁴

F₀/(F₀ − F) = 1/f_a + 1/K_af_a[L]

where, f_a, and K_a are the fraction of accessible fluorescence and the modified Stern–Volmer association constant, respectively. The linear nature of the curve of F₀/(F₀ − F) vs. 1/[L] indicated static quenching between Hb and TIM 10 (Fig. S2–S4).⁸⁵ Four types of noncovalent bonds play a major role in the interaction between a protein and a ligand, including hydrogen bonds, van der Waals forces, electrostatic forces, and hydrophobic interactions. To obtain information about the nature of interaction between Hb and TIM 10, thermodynamic parameters such as enthalpy change (ΔH°) and entropy change (ΔS°) of the binding interaction were required to calculate from van’t Hoff's equation

ln K_a = −ΔH°/RT + ΔS°/R

where R is the gas constant and T is the temperature. The values of ΔH° and ΔS° were obtained from the linear van’t Hoff plot. The value of ΔG° was calculated from the following equation

ΔG° = ΔH° − TΔS° = −RT ln K_a.

Fig. 3 Fluorescence spectra of Hb in the absence and presence of TIM 10 (A), and TIM 5 (B).

It was found that both calculated ΔH° and ΔG° of the interaction between Hb and TIM 10 were negative in nature, which implied that the acting bonds were mainly hydrogen bonds and van der Waals forces. The negative value of ΔG° indicated that the interaction was spontaneous (Table S1).^86,87

Stoichiometry

The stoichiometry of Hb and TIMs was determined by the continuous variation method. This method allows the protein–ligand complex's characteristics to be recognized. In this experiment, the total concentration of (C_Hb + C_Ligand) was kept constant, while the molar fraction of Hb and TIMs was varied. Then, changes in the absorbance of the Soret band were recorded and plotted against the molar fraction of the ligand. It has been found that Hb interacts with TIMs in a 1 : 1 ratio (Fig. 4).

Fig. 4 Study of stoichiometry between Hb and TIM 10 (A), and Hb and TIM 5 (B).

Circular dichroism (CD) spectroscopy

To look into the potential impact of these two TIMs binding on the secondary structure of Hb, CD spectroscopy was performed. In buffer media, two negative ellipticity peaks from Hb generally appear, one at 208 nm and another at 222 nm. The peak at 208 appears due to the π → π* transition of the α-helix, while the peak at 222 nm corresponds to π → π* transition of both the α-helix and β-sheets. The percentage of α-helix in native Hb was 49.72%, whereas upon interaction with TIM 10 and TIM 5, the percentage of α-helix decreased to 39.22% and 39.53%, respectively (Fig. 5). Besides that, no alterations in the peaks of maxima were found. Therefore, it can be concluded that the interaction of these two compounds with Hb induced the unfolding of the native conformation of Hb and the π–π stacking interaction force between Hb and TIMs.

Fig. 5 Circular dichroism spectra of Hb binding with TIM 10 (A), and TIM 5 (B). Black line = Hb, red line = Hb + TIM (1 : 1 ratio), blue line = Hb + TIM (1 : 2 ratio), green line = line = Hb + TIM (1 : 4 ratio).

UV melting study

The effects of these two TIMs on Hb stability were examined using UV melting. The melting temperature of control Hb was 62.5 °C. In the presence of these two compounds, the melting temperature of Hb was altered to 62.39 °C by TIM 10 and 62.66 °C by TIM 5 (Fig. 6). Hence, this study indicates that these two compounds have the least effect on Hb stability.

Fig. 6 Melting study of Hb in the absence and presence of different TIMs in a 1 : 1 ratio; black line = Hb, red line = Hb + TIM 10, and blue line = Hb + TIM 5.

Conclusion

In conclusion, we synthesised a series of bis-indole derivatives using cyclic Indole-BX, in which indole is transferred to the carbonyl carbon as an unusual nucleophilic source in hypervalent iodine chemistry. We also synthesised tris-indole derivatives, TIM5 and TIM10, using our protocol. Several recent studies have shown molecular interactions between proteins and ligands using multi-spectroscopic techniques. The interaction between these two almost similar structures with Hb will depict a picture for drug designing. The spectrophotometric titration study of the interaction between these two ligands and Hb exhibited hypochromic changes in the UV-Vis spectra of Hb, denoting the formation of complexes. TIM 10 induced much greater hypochromicity in Hb compared to TIM 5, indicating a stronger interaction between Hb and TIM 10 than between Hb and TIM 5. The strong binding affinity between TIM 10 and Hb is also supported by the calculated binding constants. TIM 10 induced quenching of the fluorescence property of Hb and exhibited a large binding constant between Hb and TIM 10. These results indicate stronger complex formation between Hb and TIM 10 than between Hb and TIM 5.⁸⁸ Stoichiometry studies revealed that TIM 10 and 5 interact with Hb in a 1 : 1 ratio.

Furthermore, CD findings suggested that the presence of TIM 10 and TIM 5 caused unfolding of the native Hb configuration. Melting experiments revealed that both compounds slightly affect Hb stability. This study helps understand how a small structural alteration (the presence of a Met group) can drastically alter the interaction process, which may be important for pharmaceutical manufacturing. This research may be crucial for pharmaceutical and medicinal chemists to establish and comprehend how these alkaloids interact with Hb in blood plasma. The finding may shed light on the varied behaviors of these alkaloids in biological systems and aid understanding of drug transport across membranes. This approach can also be readily applied to drugs of a similar nature and helps in understanding the pharmacokinetics and biological activities of these alkaloids. As a result, this work will aid in understanding the structural basis for screening and the design of appropriate synthetic compounds, which will be critical to advancing clinical and pharmaceutical research.

Author contributions

S. D.: writing – original draft, visualization, methodology, investigation, formal analysis, data curation. M. D. B.: writing – original draft, visualization, methodology, investigation, formal analysis, data curation. S. B.: writing – review & editing, investigation, visualization, formal analysis. R. S.: investigation, formal analysis, data curation. B. R.: investigation, formal analysis, data curation. S. B.: writing – review & editing, supervision, project administration, formal analysis, funding acquisition, conceptualization. R. K. N.: writing – review & editing, supervision, project administration, formal analysis, funding acquisition, conceptualization.

Conflicts of interest

There are no conflicts to declare

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information (SI). Supplementary information is available with experimental procedure, charecterization data and biological studies results. See DOI: https://doi.org/10.1039/d5nj03073k.

Acknowledgements

SD is thankful to DST-INSPIRE, Govt. of India, for her PhD fellowship (SRF) (IF 190973). MDB thanks the University of Calcutta for providing a fellowship (URF, Fellow ID F-2495). Sagar Bag thanks UGC, Government of India, for providing a fellowship and research grant (UGC-Senior Research Fellowship, NTA reference number: 201610001623). SB thanks “Intramural Seed Money Research Committee, SBV” for “SBV-Seed money” (SBV/IRC/SEED MONEY/167/2023). RKN & BR are thankful to SERB (ANRF)-Govt. of India for financial assistance (project No. EEQ/2022/000930 and EEQ/2021/000635, respectively). RKN is also thankful to JU for financial assistance with seed money (Ref. No. S-3/178/23). We also thank DST for the instrumental facility at Jadavpur University under the FIST scheme SR/FST/CS-II-032/2014(G).

References

1. W. J. Houlihan, Chemistry of Heterocyclic Compounds: Indoles, Part Two, Wiley Interscience, New York, 1972.
2. R. J. Sundberg, Indoles, Elsevier, 1996.
3. J. A. Joule and K. Mills, Heterocyclic Chemistry, Blackwell Science, Oxford, UK, 4th edn, 2000.
4. A. Takeda, S. Kamijo and Y. Yamamoto, Indole synthesis via palladium-catalyzed intramolecular cyclization of alkynes and imines, J. Am. Chem. Soc., 2000, 122, 5662–5663.
5. K. R. Roesch and R. C. Larock, Synthesis of isoindolo [2,1-a] indoles by the palladium-catalyzed annulation of internal acetylenes, J. Org. Chem., 2001, 66, 412–420.
6. A. Lerchner and E. M. Carreira, First total synthesis of (±)-strychnofoline via a highly selective ring-expansion reaction, J. Am. Chem. Soc., 2002, 124, 14826–14827.
7. H. Kusama, J. Takaya and N. Iwasawa, A facile method for the synthesis of polycyclic indole derivatives: The generation and reaction of tungsten-containing azomethine ylides, J. Am. Chem. Soc., 2002, 124, 11592–11593.
8. L. Ackermann, General and efficient indole syntheses based on catalytic amination reactions, Org. Lett., 2005, 7, 439–442.
9. N. L. Segraves and P. Crews, Investigation of brominated tryptophan alkaloids from two thorectidae sponges: Thorectandra and Smenospongia, J. Nat. Prod., 2005, 68, 1484–1488.
10. S. A. Patil, R. Patil and D. D. Miller, Indole molecules as inhibitors of tubulin polymerization: potential new anticancer agents, Future Med. Chem., 2012, 4, 2085–2115.
11. T. V. Sravanthi and S. L. Manju, Indoles—A promising scaffold for drug development, Eur. J. Pharm. Sci., 2016, 91, 1–10.
12. B. Biersack and R. Schobert, Indole compounds against breast cancer: recent developments, Curr. Drug Targets, 2012, 13, 1705–1719.
13. A. Ahmad, B. Biersack, Y. Li, D. Kong, B. Bao, R. Schobert, S. B. Padhye and F. H. Sarkar, Targeted regulation of PI3K/Akt/mTOR/NF-κB signaling by indole compounds and their derivatives: mechanistic details and biological implications for cancer therapy, Anti-Cancer Agents Med. Chem., 2013, 13, 1002–1013.
14. S. Olgen, Recent development of new substituted indole and azaindole derivatives as anti-HIV agents, Mini-Rev. Med. Chem., 2013, 13, 1700–1708.
15. N. K. Kaushik, N. Kaushik, P. Attari, N. Kumar, C. H. Kim, A. K. Verma and E. H. Choi, Biomedical importance of indoles, Molecules, 2013, 18, 6620–6662.
16. F. A. Wati, M. Santoso, Z. Moussa, S. Fatmawati, A. Fadlan and Z. M. A. Judeh, Chemistry of trisindolines: natural occurrence, synthesis and bioactivity, RSC Adv., 2021, 11, 25381–25421.
17. C. Bonnesen, I. M. Eggleston and J. D. Hayes, Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines, Cancer Res., 2001, 61, 6120–6130.
18. C. Hong, G. L. Firestone and L. F. Bjeldanes, Bcl-2 family-mediated apoptotic effects of 3,3′-diindolylmethane (DIM) in human breast cancer cells, Biochem. Pharmacol., 2002, 63, 1085–1097.
19. S. Safe, S. Papineni and S. Chintharlapalli, Cancer chemotherapy with indole-3-carbinol, bis (3′-indolyl) methane and synthetic analogs, Cancer Lett., 2008, 269, 326–338.
20. M. Rahimi, K.-L. Huang and C. K. Tang, 3,3′-Diindolylmethane (DIM) inhibits the growth and invasion of drug-resistant human cancer cells expressing EGFR mutants, Cancer Lett., 2010, 295, 59–68.
21. R. Bell, S. Carmeli and N. Sar, Vibrindole A, a metabolite of the marine bacterium, Vibrio parahaemolyticus, isolated from the toxic mucus of the boxfish Ostracion cubicus, J. Nat. Prod., 1994, 57, 1587–1590.
22. M. Shiri, M. A. Zolfigol, H. G. Kruger and Z. Tanbakouchian, Bis-and trisindolylmethanes (BIMs and TIMs), Chem. Rev., 2010, 110, 2250–2293.
23. G. de la Herran, A. Segura and A. G. Csaky, Benzylic substitution of gramines with boronic acids and rhodium or iridium catalysts, Org. Lett., 2007, 9, 961–964.
24. X. Guo, S. Pan, J. Liu and Z. Li, One-pot synthesis of symmetric and unsymmetric 1,1-Bis-indolylmethanes via tandem iron-catalyzed C–H bond oxidation and C–O bond cleavage, J. Org. Chem., 2009, 74, 8848–8851.
25. C. Praveen, Y. W. Sagayaraj and P. T. Perumal, Gold(I)-catalyzed sequential cycloisomerization/bis-addition of o-ethynylanilines: An efficient access to bis (indolyl) methanes and di (indolyl) indolin-2-ones, Tetrahedron Lett., 2009, 50, 644–647.
26. D. Xia, Y. Wang, Z. Du, Q.-Y. Zheng and C. Wang, Rhenium-catalyzed regiodivergent addition of indoles to terminal alkynes, Org. Lett., 2012, 14, 588–591.
27. L. Zhang, C. Peng, D. Zhao, Y. Wang, H.-J. Fu, Q. Shen and J.-X. Li, Cu(II)-catalyzed C–H (SP³) oxidation and C–N cleavage: base-switched methylenation and formylation using tetramethylethylenediamine as a carbon source, Chem. Commun., 2012, 48, 5928–5930.
28. M. P. Muñoz, M. C. de la Torre and M. A. Sierra, Platinum-catalysed bisindolylation of allenes: a complementary alternative to gold catalysis, Chem. – Eur. J., 2012, 18, 4499–4504.
29. J. Beltrá, M. C. Gimeno and R. P. Herrera, A new approach for the synthesis of bisindoles through AgOTf as catalyst, Beilstein J. Org. Chem., 2014, 10, 2206–2214.
30. S. Karmakar, P. Das, D. Ray, S. Ghosh and S. K. Chattopadhyay, Ag(I)-Catalyzed domino cyclization–addition sequence with simultaneous carbonyl and alkyne activation as a route to 2,2′-disubstituted bisindolylarylmethanes, Org. Lett., 2016, 18, 5200–5203.
31. M. Yuan, Q. Nong, H. Guo, Y. Li, H. Tian, J. Zhang, L. Liu, B. He, L. Hu and G. Jiang, pH-dependent cadmium binding to hemoglobin: Implications for human excretion, Sci. Total Environ., 2025, 958, 177700.
32. Y. Yuan, V. Simplaceanu, J. A. Lukin and C. Ho, NMR investigation of the dynamics of tryptophan side-chains in hemoglobins, J. Mol. Biol., 2002, 321, 863–878.
33. S. Singh, P. Gopi, P. Sharma, M. S. S. Rani, P. Pandya and M. S. Ali, Hemoglobin targeting potential of aminocarb pesticide: Investigation into dynamics, conformational stability, and energetics in solvent environment, Biochem. Biophys. Res. Commun., 2024, 736, 150896.
34. G. M. Levitzky, Pulmonary Physiology, McGraw Hill, 2007.
35. M. D. Burman, S. Bag, S. Ghosal, S. Karmakar, G. Pramanik, R. K. Chinnadurai and S. Bhowmik, Exploring the structural importance of the C3=C4 double bond in plant alkaloids harmine and harmaline on their binding interactions with hemoglobin, ACS Omega, 2023, 8, 37054.
36. J.-L. Yuan, H. Liu, X. Kang, Z. Lv and G.-L. Zou, Characteristics of the isomeric flavonoids apigenin and genistein binding to hemoglobin by spectroscopic methods, J. Mol. Struct., 2008, 891, 333.
37. A. Basu and G. S. Kumar, Interaction of the dietary pigment curcumin with hemoglobin: energetics of the complexation, Food Funct., 2014, 5, 1949.
38. C.-Y. Chen and Y. Zhang, Berberine: An isoquinoline alkaloid targeting the oxidative stress and gut-brain axis in the models of depression, Eur. J. Med. Chem., 2025, 290, 117475.
39. Q. Cao, Q. Wang, X. Wu, Q. Zhang, J. Huang, Y. Chen, Y. You, Y. Qiang, X. Huang, R. Qin and G. Cao, A literature review: mechanisms of antitumor pharmacological action of leonurine alkaloid, Front. Pharmacol., 2023, 14, 1272546.
40. K. Bhardwaj, R. Bhargav, J. Patocka, R. Sharma, Z. Navratilova, P. Oleksak and K. Kuca, Dendrobine: a neuroprotective sesquiterpenic alkaloid for the prevention and treatment of diseases: a review, Mini Rev. Med. Chem., 2024, 24, 1395–1408.
41. R. G. Geetha and S. Ramachandran, Recent advances in the anti-inflammatory activity of plant-derived alkaloid rhynchophylline in neurological and cardiovascular diseases, Pharmaceutics, 2021, 13, 1170.
42. K. Ya, W. Tangamornsuksan, C. N. Scholfield, J. Methaneethorn and M. Lohitnavy, Pharmacokinetics of mitragynine, a major analgesic alkaloid in kratom (Mitragyna speciosa): A systematic review, Asian J. Psychiatr., 2019, 43, 73–82.
43. M. Gaurav, A. Natesh, A. Arundhati and D. Mariam, Biochemical aspects of hemoglobin-xenobiotic interactions and their implications in drug discovery, Biochimie, 2021, 191, 154–163.
44. E. V. Stepanova, A. A. Shtil’, S. N. Lavrenov, V. M. Bukhman, A. N. Inshakov, E. P. Mirchink, A. S. Trenin, O. A. Galatenko, E. B. Isakova, V. A. Glazunova, L. G. Dezhenkova, E. S. Solomko, E. E. Bykov and M. N. Preobrazhenskaya, Tris (1-alkylindol-3-yl) methylium salts as a novel class of antitumor agents, Russ. Chem. Bull., 2010, 59, 2259–2267.
45. S. N. Lavrenov, Y. N. Luzikov, E. E. Bykov, M. I. Reznikova, E. V. Stepanova, V. A. Glazunova, Y. L. Volodina, V. V. Tatarsky Jr, A. A. Shtil and M. N. Preobrazhenskaya, Synthesis and cytotoxic potency of novel tris (1-alkylindol-3-yl) methylium salts: Role of N-alkyl substituents, Bioorg. Med. Chem., 2010, 18, 6905–6913.
46. S. Das, T. Datta, M. A. Sk, B. Roy and R. K. Nandi, Isoxazole group directed Rh(III)-catalyzed alkynylation using TIPS-EBX, Org. Biomol. Chem., 2024, 22, 6922.
47. P. Pal, J. Waser and R. K. Nandi, Umpolung of Electron-Rich Heteroarenes with Hypervalent Iodine Reagents, Heterocycles, 2021, 103, 555.
48. (a) S. Das, S. Bag, M. A. Sk, P. Majumdar, U. K. Das, S. Bhowmik and R. K. Nandi, Uracil-BX: A New Class of Cyclic Hypervalent Iodine Reagents for Unraveling Approach Towards Umpolung Functionalization and VEGF G-Quadruplex DNA Identification. ChemRxiv, 2025, preprint DOI:10.26434/chemrxiv-2025-tn425; (b) S. Das, M. A. Sk, P. Majumdar, U. K. Das and R. K. Nandi, Uracil-BX: Bench-Stable Cyclic Hypervalent Iodine Reagents for Umpolung Functionalization, Org. Lett., 2025, 27, 8158–8163.
49. P. Caramenti, S. Nicolai and J. Waser, Indole-and Pyrrole-BX: Bench-Stable Hypervalent Iodine Reagents for Heterocycle Umpolung, Chem. – Eur. J., 2017, 23, 14702.
50. C. Arakawa, K. Kanemoto, K. Nakai, C. Wang, S. Morohashi, E. Kwon, S. Ito and N. Yoshikai, Carboiodanation of arynes: Organoiodine(III) compounds as nucleophilic organometalloids, J. Am. Chem. Soc., 2024, 146, 3910.
51. S.-Q. Liu, J.-J. Xu and H.-Y. Chen, A reversible adsorption–desorption interface of DNA based on nano-sized zirconia and its application, Colloids Surf., B, 2004, 36, 155.
52. Y.-H. Chen and J. T. Yang, A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism, Biochem. Biophys. Res. Commun., 1971, 44, 1285.
53. H. Koshima and W. Matsusaka, N-Bromosuccinimide catalyzed condensations of indoles with carbonyl compounds under solvent-free conditions, J. Heterocycl. Chem., 2002, 39, 1089.
54. S. Naskar, A. Hazra, P. Paira, K. B. Sahu, S. Banerjee and N. B. Mondal, NH₄Cl-promoted synthesis of symmetrical and unsymmetrical triindolylmethanes under solvent-free conditions, J. Chem. Res., 2008, 2008, 568.
55. A. Hazra, P. Paira, K. B. Sahu, S. Banerjee and N. B. Mondal, Molecular iodine: an efficient catalyst for the synthesis of both symmetrical and unsymmetrical triindolylmethanes (TRIM), Catal. Commun., 2008, 9, 1681.
56. M. T. El Sayed, K. M. Ahmed, K. Mahmoud and A. Hilgeroth, Synthesis, cytostatic evaluation and structure activity relationships of novel bis-indolylmethanes and their corresponding tetrahydroindolocarbazoles, Eur. J. Med. Chem., 2015, 90, 845.
57. X. Qi, H.-J. Ai, N. Zhang, J.-B. Peng, J. Ying and X.-F. Wu, Palladium-catalyzed carbonylative bis (indolyl) methanes synthesis with TFBen as the CO source, J. Catal., 2018, 362, 74.
58. W. Xiu, W. Zhenhua, G. Zhang, W. Zhang, Y. Wu and Z. Gao, Tunable Titanocene Lewis Acid Catalysts for Selective Friedel–Crafts Reaction of Indoles and N-Sulfonylaldimines, Eur. J. Org. Chem., 2016, 502.
59. J. Yang, Y. Zhang, H.-C. F. Wong, J. Huang, Y.-L. S. Tse and Y.-Y. Yeung, Design and Applications of Cyclopropenium Chalcogen Dihalides in Catalysis via C (sp³)–H··· X Interactions, ACS Catal., 2024, 14, 3018.
60. S. Khaksar, S. M. Vahdat, M. Gholizadeh and S. M. Talesh, An expeditious and efficient synthesis of symmetrical tris (indolyl) methanes under catalyst-free conditions in fluorinated alcohols, J. Fluorine Chem., 2012, 136, 8.
61. K. Singh, S. Sharma and A. Sharma, Unique versatility of Amberlyst 15. An acid and solvent-free paradigm towards synthesis of bis (heterocyclyl) methane derivatives, J. Mol. Catal. A: Chem., 2011, 347, 34.
62. S. N. Lavrenov, Y. N. Luzikov, E. E. Bykov, M. I. Reznikova, E. V. Stepanova, V. A. Glazunova, Y. L. Volodina, V. V. Tatarsky Jr, A. A. Shtil and M. N. Preobrazhenskaya, Synthesis and cytotoxic potency of novel tris (1-alkylindol-3-yl) methylium salts: Role of N-alkyl substituents, Bioorg. Med. Chem., 2010, 18, 6905.
63. Z. H. Zhang and J. Lin, Efficient and convenient method for the synthesis of symmetrical triindolylmethanes catalyzed by iodine, Synth. Commun., 2007, 37, 209.
64. M. Chakrabarty, S. Sarkar, A. Linden and B. K. Stein, First general solvent-free synthesis of symmetrical triindolylmethanes using acid-washed montmorillonite clay, Synth. Commun., 2004, 34, 1801.
65. X. L. Luo, W. J. Ji, Z. L. Yu, M. M. Wang, C. B. Pang, X. Q. Chu and D. Ge, Heteroarylation of Organophosphonium Salts with Indoles in Water: Synthesis of Symmetrical and Unsymmetrical 3,3′-Bisindolylmethane Derivatives, Eur. J. Org. Chem., 2024, e202400964.
66. P. Nath, M. L. Deb and P. K. Baruah, Synthesis of tris (indolyl) methanes by using dimethyl carbonate as a single carbon source via CH functionalization approach, Chem. Pap., 2024, 78, 1117.
67. M. L. Deb, P. J. Borpatra, C. D. Pegu, R. Thakuria, P. J. Saikia and P. K. Baruah, Iodine/tert-Butyl Hydroperoxide-Mediated Reaction of Indoles with Dimethylformamide/Dimethylacetamide to Synthesize Bis-and Tris (indolyl) methanes, ChemistrySelect, 2017, 2, 140.
68. M. Barbero, S. Cadamuro, S. Dughera, C. Magistris and P. Venturello, A new practical synthesis of triaryl and trisindolylmethanes under solvent-free reaction conditions, Org. Biomol. Chem., 2011, 9, 8393.
69. M. Kargar, R. Hekmatshoar and A. Mostashari, Synthesis of novel methylene bridge functionalized bis (indolyl) methanes thorough a double Michael addition, Heterocycles, 2011, 83, 2535.
70. D. G. Gu and S. J. Ji, Various tri (indolyl) methanes were synthesized by condensation of indole and its derivatives with indole-3-carboxaldehyde using acidic ionic liquids [hmim] HSO₄/EtOH as an efficient and green catalyst system, Chin. J. Chem., 2008, 26, 578.
71. X. F. Zeng, S. J. Ji and X. M. Su, Facile Synthesis of Symmetrical Triindolylmethanes Catalyzed by Iodine under Solvent-free Condition, Chin. J. Chem., 2008, 26, 413.
72. N. G. Singh, C. Kathing, J. W. S. Rani and R. L. Nongkhlaw, Synthesis of pharmacologically active bis (indolyl) and tris (indolyl) derivatives using chlorotrimethylsilane, J. Chin. Chem. Soc., 2014, 61, 442.
73. E. Akgün, M. Tunali and U. Pindur, Proton acid-catalysed acylation of indoles by 2-alkoxy-1, 3-dioxolanes, Arch. Pharm., 1987, 320, 397.
74. S. Tuengpanya, C. Chantana, U. Sirion, W. Siritanyong, K. Srisook and J. Jaratjaroonphong, One-pot solvent-free synthesis of triaryl-and triheteroarylmethanes by Bi (OTf) 3-catalyzed Friedel-Crafts reaction of arenes/heteroarenes with trialkyl orthoformates, Tetrahedron, 2018, 74, 4373.
75. M. Chakrabarty, N. Ghosh, R. Basak and Y. Harigaya, Dry reaction of indoles with carbonyl compounds on montmorillonite K10 clay: a mild, expedient synthesis of diindolylalkanes and vibrindole A, Tetrahedron Lett., 2002, 43, 4075.
76. H. Veisi, M. Ataee, P. Darabi-Tabar, E. Amiri and A. R. Faraji, One-pot conversion of aromatic compounds to the corresponding bis (indolyl) methanes by the Vilsmeier–Haack reaction, C. R. Chim, 2014, 17, 305.
77. V. Nair, K. G. Abhilash and N. Vidya, Practical synthesis of triaryl-and triheteroarylmethanes by reaction of aldehydes and activated arenes promoted by gold(III) chloride, Org. Lett., 2005, 7, 5857.
78. V. D. Kadu, A. A. Patil and P. R. Shendage, Oxone-promoted synthesis of bis (indolyl) methanes from arylmethylamines and indoles, J. Mol. Struct., 2022, 1267, 133502.
79. Y. Lian, R. G. Bergman, L. D. Lavis and J. A. Ellman, Rhodium (III)-catalyzed indazole synthesis by C–H bond functionalization and cyclative capture, J. Am. Chem. Soc., 2013, 135, 7122–7125.
80. B. Jia, Y. Yang, X. Jin, G. Mao and C. Wang, Rhenium-Catalyzed Phthalide Synthesis from Benzamides and Aldehydes via C–H Bond Activation, Org. Lett., 2019, 21, 6259–6263.
81. W. Liu, X. Guo and R. Guo, The interaction between hemoglobin and two surfactants with different charges, Int. J. Biol. Macromol., 2007, 41, 548.
82. J. R. Lakowicz, Principles of fluorescence spectroscopy, Springer, 3rd edn, 2006.
83. W. He, H. Dou, Z. Li, X. Wang, L. Wang, R. Wang and J. Chang, Investigation of the interaction between five alkaloids and human hemoglobin by fluorescence spectroscopy and molecular modeling, Spectrochim. Acta, Part A, 2014, 123, 176–186.
84. S. Lehrer, Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion, Biochemistry, 1971, 10, 3254–3263.
85. W. R. Ware, Oxygen quenching of fluorescence in solution: an experimental study of the diffusion process, J. Phys. Chem., 1962, 66, 455–458.
86. A. Sharifi-Rad, J. Mehrzad, M. Darroudi, M. R. Saberi and J. Chamani, Oil-in-water nanoemulsions comprising Berberine in olive oil: biological activities, binding mechanisms to human serum albumin or holo-transferrin and QMMD simulations, J. Biomol. Struct. Dyn., 2021, 39, 1029–1043.
87. M. Kaffash, S. Tolou-Shikhzadeh-Yazdi, S. Soleimani, S. Hoseinpoor, M. R. Saberi and J. Chamani, Spectroscopy and molecular simulation on the interaction of Nano-Kaempferol prepared by oil-in-water with two carrier proteins: an investigation of protein–protein interaction, Spectrochim. Acta, Part A, 2024, 309, 123815.
88. S. Rezaei, H.-S. Meftah, Y. Ebtehajpour, H. R. Rahimi and J. Chamani, Investigation on the effect of fluorescence quenching of calf thymus DNA by piperine: caspase activation in the human breast cancer cell line studies, DNA Cell Biol., 2024, 43, 26–38.

---