6-((2-Oxoindolin-3-ylidene)hydrazineylidene)indolo[2,1-b]qui-nazolin-12(6H)-one

Abstract

A novel unsymmetrical azine, 6-((2-oxoindolin-3-ylidene)hydrazineylidene)indolo[2,1-b]quinazolin-12(6H)-one, was synthesized through a condensation reaction between tryptanthrin-6-hydrazone and isatin in chloroform under reflux conditions. Structural characterization revealed the compound exists as a mixture of geometric isomers with one predominant form. Density functional theory (DFT) calculations identified the E,E configuration as the most stable isomer. The isomerization barriers for both C=N bonds were calculated at approximately 18.5 kcal/mol via nitrogen inversion. Given the established biological activities of tryptanthrin and isatin derivatives, this hybrid azine represents a promising lead compound for developing bifunctional drug candidates.

1. Introduction

Imine-functionalized heterocycles constitute a valuable class of compounds characterized by versatile reactivity and pharmacological relevance. Azines, which follow the general formula RR′C=N–N=CR″R″′ and are considered derivatives of hydrazine, serve as key representatives. Interest in these compounds is driven particularly by their therapeutic activities [1,2]. The synthesis of azines can be performed via the condensation of hydrazine with two equivalents of carbonyl compound (aldehyde or ketone) under reflux [3,4]. A complementary preparative method employs the reaction of a nucleophilic carbene with a diazoalkane [5].

The influence of substituents on conjugation within the azine core remains underexplored. A detailed investigation into their isomerism, physicochemical properties, and electronic structures is therefore essential, especially for azines incorporating pharmacologically relevant heterocycles. Tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione, 1), isatin (indoline-2,3-dione, 2), and their derivatives are highly promising compounds due to their well-established biological profiles [6,7,8,9]. In this work, we have synthesized the first representative of an unsymmetrical azine linking tryptanthrin and isatin, namely 6-((2-oxoindolin-3-ylidene)hydrazineylidene)indolo[2,1-b]quinazolin-12(6H)-one, which is of interest as a potential biologically active compound that could find application in medicinal chemistry, particularly for anticancer drug development.

2. Results and Discussion

2.1. Synthesis

Tryptanthrin-6-hydrazone (3) was prepared beforehand via the selective nucleophilic addition of hydrazine hydrate to the more electrophilic C-6 carbonyl of tryptanthrin (1) (Scheme 1). This transformation typically involves heating of tryptanthrin with excess hydrazine hydrate in an organic solvent [10].

The target unsymmetrical azine 6-((2-oxoindolin-3-ylidene)hydrazineylidene)indolo[2,1-b]quinazolin-12(6H)-one (4) was prepared for the first time by condensing tryptanthrin-6-hydrazone (3) with isatin (2) under reflux in chloroform for 5 h, without added acid or base catalysts (Scheme 2). Complete conversion was monitored by TLC (hexane/EtOAc, 2:1), affording the crude brick-orange precipitate upon filtration. Final purification by silica gel column chromatography (CHCl₃ eluent) provided the analytically pure product in 69% yield.

According to the NMR data (DMSO-d₆), the purified compound 4 was obtained as a mixture of geometric isomers, with one being predominant. The ¹H NMR spectrum revealed a minor isomer, as evidenced by an approximately 1:6 ratio of integral intensities for the low-field doublets near 8.3 and 8.5 ppm corresponding to protons in the tryptanthrin fragment. Other signals also exhibited minor counterparts (Figure S1), which could not be attributed to protons in the starting compounds. Separation of the isomers proved impossible, likely due to a relatively low energy barrier for isomerization of the carbon-nitrogen double bond [4,11] (see also the DFT results described in Section 2.2). In this context, the absence of separate ¹³C NMR signals for the Z/E isomers stems from rapid isomerization across a low barrier, which averages resonances on the ¹³C NMR timescale [12].

The main characteristics of the title compound 4 are as follows: brick-orange powder, m.p. 277–280 °C, low soluble in chloroform and DMSO.

2.2. DFT Investigation of Geometric Isomerism

Due to the presence of two imine bonds, azines may display geometric isomerism [13]. To assess the energetic preferences among the four possible isomers of compound 4, we employed density functional theory (DFT) computations. Initial conformational exploration for each configuration (E,E; Z,Z; Z,E; E,Z) was carried out based on molecular mechanics (see Materials and Methods). Subsequent geometry optimizations were performed using the PBEh-3c method [14] followed by M06-2X/def2-TZVP refinements [15,16]. Computations revealed the E,E form as the energetically favored isomer. The relative Gibbs free energies of the Z,Z, Z,E, and E,Z structures are 1.1–2.3 kcal/mol above the energy minimum of the E,E isomer conformation (Figure 1). Notably, all the optimized structures exhibited deviations from planarity, characterized by C=N-N=C dihedral angles of 132° (E,E), 103° (Z,Z), 109° (Z,E), and 107° (E,Z).

Energy barriers for isomer interconversion were estimated using the nudged elastic band (NEB) approach [17,18] with refinement at the M06-2X/def2-TZVP level. The Gibbs free energy barriers for the E,E → Z,E and E,E → E,Z processes are nearly 18.5 kcal/mol (Figure 1). The mechanism of isomerization involves nitrogen atom in-plane inversion, evidenced by N-N=C bond angles of approximately 169° (tryptanthrin-adjacent) and 170° (isatin-adjacent) in the transition state structures. The calculated energy barriers are sufficiently elevated to impede rapid interconversion between the geometric isomers at ambient temperatures, thereby enabling the resolution of distinct signals for a minor isomer in the ¹H NMR spectrum. Nevertheless, these barriers are of a close resemblance to the rotational energy barrier associated with the partial double-bond character of the C-N bond in acetamide [19], which allows for conformational equilibration on a longer timescale, thus rendering the isolation of pure isomers infeasible under standard laboratory conditions.

3. Materials and Methods

3.1. General

All reagents were purchased from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on Merck plates (Merck KGaA, Darmstadt, Germany), silica gel 60, F254 with visualization under UV light (254 nm). Column chromatography was carried out using silica gel 60 (0.063–0.200 mm). LC/MS analysis was performed on an Agilent Infinity chromatograph with an AccurateMass QTOF 6530 mass detector (Santa Clara, CA, USA). Chromatographic conditions: column Zorbax EclipsePlusC18 1.8 μm, 2.1 × 50 mm; eluent H₂O: ACN (85%); flow 0.2 mL/min. Ionization source: ESI in positive mode. ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer (Bruker corporation, Billerica, CA, USA) at 400 MHz and 100 MHz, respectively, in DMSO-d₆ with TMS as the internal standard. The melting point of the synthesized compound was measured using a Melting Point Apparatus SMP30 (Cole-Parmer Instrument Company, Vernon Hills, IL, USA), heating rate 3.0 °C/min. Elemental analysis was made using a Carlo Erba analyzer (Thermo Fisher Scientific, Waltham, MA, USA). IR spectrum was recorded on a FT-IR spectrometer Agilent Cary 630 (Agilent Technologies, Santa Clara, CA, USA).

3.2. Synthesis and Characterization

A mixture of tryptanthrin-6-hydrazone (0.1311 g, 0.5 mmol) and isatin (0.074 g, 0.5 mmol) in chloroform (50 mL) was refluxed for 5 h, with reaction progress monitored by TLC (hexane:ethyl acetate, 2:1 v/v). Upon completion, the brick-orange precipitate was filtered off, yielding the crude product (0.171 g). Purification by column chromatography (silica gel, chloroform) afforded 0.135 g (69%) of the title compound as an orange solid (m.p. 277–280 °C). ¹H NMR (400 MHz, DMSO-d₆): δ 6.89–7.03 (m, 2H, H-5′, H-6′), 7.34–7.40 (m, 0.25H, H-8, H-7′ of minor isomer), 7.41–7.51 (m, 1.7H, H-8, H-7′), 7.68–7.76 (m, 3H, H-2, H-9, H-4′), 7.94–8.00 (m, 3H, H-3, H-7, H-10), 8.11 (d, 0.15H, J 8 Hz, H-7 of minor isomer), 8.28 (d, 0.15H, J 8 Hz, H-4 of minor isomer), 8.35 (d, 0.85H, J 8 Hz, H-4), 8.50 (d, 0.14H, J 8 Hz, H-1 of minor isomer), 8.54 (d, 0.85H, J 8 Hz, H-1), 11.05 (s, 1H, NH) (atom numbering is shown in Scheme 3). ¹³C NMR (100 MHz, DMSO-d₆): δ 111.1, 115.9, 116.7, 119.2, 122.2, 122.6, 126.8, 126.9, 128.5, 128.6, 128.8, 129.0, 134.2, 134.6, 135.0, 141.6, 145.4, 145.5, 147.0, 148.0, 158.4, 163.7. Elemental analysis: found, %: C, 70.32; H, 3.46; N, 17.74, C₂₃H₁₃N₅O₂, calculated, %: C, 70.58; H, 3.35; N, 17.89. IR (cm^–1): 3532 ν(N-H), 1729, 1674 ν(C=O), 1636, 1606 ν(C=N), 1595 (arom.), 1157 ν(N-N). LC/MS (ESI+)—m/z: 392.1153 experimental ([C₂₃H₁₃N₅O₂ + H]⁺ = 392.1148 theor.).

The ¹H NMR and ¹³C NMR spectra are shown in Figures S1 and S2. The IR spectrum is presented in Figure S3.

3.3. Computational Details

Conformational searches for compound 4 were conducted using the GFN-FF force field [20] with the GOAT algorithm [21] in ORCA software [22] (version 6.1.0). Additional searches were performed with VConf 2.0 program (VeraChem, LLC, Germantown, MD, USA). Both methods overcame stereochemical constraints and generated conformational ensembles for all the four geometric isomers. Up to 25 low-energy conformers from the combined ensembles were optimized with the PBEh-3c composite DFT method [14]. The lowest-energy conformer for each isomer was subsequently refined at the M06-2X/def2-TZVP level in the gas phase. Transition states for Z,E isomerization were located using the climbing image nudged elastic band (CI-NEB-zoom) technique with HF-3c energy evaluations [23] in ORCA 6.1.0 and refined similarly to the geometric isomers. The nature of all stationary points was confirmed by vibrational frequency analysis.

4. Conclusions

In summary, we have synthesized the novel unsymmetrical azine 6-((2-oxoindolin-3-ylidene)hydrazineylidene)indolo[2,1-b]quinazolin-12(6H)-one through a straightforward condensation reaction between tryptanthrin-6-hydrazone and isatin, achieving high yield and purity. This compound represents the first reported hybrid linking the tryptanthrin and isatin scaffolds via an azine bridge, expanding the chemical diversity of such pharmacologically relevant heterocycles [8]. Structural characterization confirmed its identity as a mixture of geometric isomers with one predominant form, while DFT calculations elucidated the geometric isomerism, identifying the E,E configuration as the most stable with noticeable energy differences (1.1–2.3 kcal/mol) among alternatives. The calculated isomerization barriers (~18.5 kcal/mol) via nitrogen inversion highlight challenges in the isomer separation, consistent with behaviors observed in related imine systems [13]. Given the established pharmacological potentials of tryptanthrin and isatin derivatives, this azine is promising as a lead for developing bifunctional agents targeting cancer pathways.