Facile Multicomponent Synthesis, Computational, and Docking Studies of Spiroindoloquinazoline Compounds

Abstract

Synthesis of spiroindoloquinazolines via one-pot three-component condensation reactions of tryptanthrin, malononitrile or ethylcyanoacetate, and nucleophiles was carried out in MeOH using triethylamine as the base catalyst under reflux conditions. This method has the advantages of short reaction time, excellent yields, and an easy work-up procedure. The structural properties of the compound 4c {2-amino-6-methyl-spiro-[12H]Indolo(2,1-b)quinazoline-12-one]12′,4-pyrano(2,3-c)pyrazole]-3-carbonitrile} and the obtained crystal were analyzed by the DFT theoretical calculation method, using the B3LYP 6-311G(d,p) basis set and were found to be in agreement with the experimental results. The importance of biological application and drug design of the same compound was verified by molecular docking studies.

Introduction

Synthesis of complex organic molecules by conventional methods involves a large number of synthetic operations, extraction, and purification processes. A conventional method leads not only to inefficient synthesis but also to the production of a large amount of waste. Multicomponent reactions (MCRs) play an essential task in organic synthesis, since they normally take place in one pot, afford high atom economy and selectivity, and offer the possibility of diversifying products by varying the different reactants.¹ MCRs are performed without any isolation of the intermediates, thus saving energy and raw materials and reducing the duration of the reaction.² Hence, the proposal of new MCRs has received great attention from research groups working in the fields of medicinal chemistry, drug discovery, and materials science. In addition, improvements in the already known MCRs are also of substantial interest in current organic synthesis. Quinoxalines are an important class of benzoheterocyclic derivatives that find pharmacological applications as an antidepressant, antifungal, antibacterial, and antitumor agents.³⁻⁶ Tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) and its derivatives are indoloquinazoline alkaloids found in many kinds of plants and have various biological activities such as antiinflammatory, immunomodulatory, and antitumor activities.^7,8 In addition, these compounds inhibit the activity of microorganisms and parasites.^9,10 In recent years, tryptanthrin has received much attention as an anticancer agent.¹¹ Tryptanthrin was recognized with outstanding cytotoxicity against human breast carcinoma (MCF-7), lung carcinoma (NCI-H460), and central nervous system carcinoma (SF-268) cell lines. On the other hand, pyrans are an important building block unit and are present in many plants.¹² Pyrans and their derivatives have received a considerable interest because of biological properties such as anticancer activity and diuretic, spasmolytic, and hypertensive agents.^13,14 Interestingly, when the quinazoline ring of the spiroquinazoline system with pyrans is joined to a heterocyclic structure with a spiro carbon at the C-12 site, the resulting compounds show a wide spectrum of pharmacological activities. Also, the oxadiazoline structure has many pharmacological activities. The oxadiazoline moiety at C-12 of the indolo[2,1-b]quinazoline systems leads to more significant biological activity.¹⁵

A few reports are available on the synthesis of γ-spirolactones, γ-spiroiminolactones,^16,17 and spiroquinazoline derivatives,^18,19 using tryptanthyrin as one of the starting materials and spiroindoloquinazoline using ammonium acetate and DABCO.^20,21 We are reporting herewith a facile and efficient one-pot synthesis of functionalized spiroindoloquinazoline compounds involving a three-component condensation reaction catalyzed by triethylamine using the substrates tryptanthrin, an active methylene compound, and a nucleophile carried out in MeOH using triethylamine as the base catalyst under reflux conditions (Scheme 1).

Scheme 1. Synthesis of Spiroindoloquinazoline Compounds.

Quantum chemical calculations such as DFT are of great interest in studying the properties of organic molecules and predicting the properties of the molecule with reasonable computational accuracy.²² This could also provide extensive help in comparing the data with experimental results. Based on the literature survey, the crystal structure and theoretical calculations of this compound 4c have not been reported so far. Therefore, in this view of advantages, we have made an attempt to study the molecule properties like geometrical parameters, chemical reactivity, dipole moment, vibrational energies, molecular energies, and so forth. By the DFT method using the B3LYP/6-311G(d,p) basis set and using the optimized structure, we have calculated all the properties of the optimized molecule. Apart from this, we have also tried to find out the electronic properties of the molecule using the TD-DFT method.²² Pharmacological significance of these compounds encourages us to perform the small molecule docking analysis of this class of compounds. It is a computational technique and helps in predicting the interactions between compounds and the protein receptors. We have performed the docking analysis of compound 4c using Autodock vina.²³

Materials and Methods

Experimental Section

All the chemicals were used as received from Sigma-Aldrich (USA), Spectrochem (India), and SRL (India). Melting points were recorded in open capillaries and are uncorrected. Fourier transform infrared spectra (FT-IR) were recorded in the wave number range of 4000–400 cm^–1 and are recorded on a Thermo Mattson Satellite FT-IR spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker UltraShield spectrometer (400 and 100 MHz) or Bruker UltraShield spectrometer (300 and 75 MHz) in a DMSO-d₆ solvent using TMS as an internal standard. Mass spectra were recorded on a Waters^(R)Micromass^(R)Q-TOF Micro mass spectrometer. Solvents used were of commercial grade and purified before use by distillation.

General Procedure for the Synthesis of Compound 4

A mixture of tryptanthrins (1a–c) (1 mmol), malononitrile or ethylcyanoacetates (2a,b) (1 mmol), and nucleophiles (3a–e) (1 mmol) and triethylamine (1.2 mmol) in MeOH (5 mL) was stirred under reflux conditions for the time indicated in Table 2. After completion of the reaction, the solvent was evaporated under a vacuum. The residue was extracted with ethyl acetate, washed with 10% HCl, and then neutralized with 5% Na₂CO₃ and dried over anhydrous Na₂SO₄. The solvent was removed to dryness which was purified by flash column chromatography (silica gel, mixtures of petroleum ether/ethyl acetate) to afford desired pure products 4.

Table 2. Synthesis of Spiroindoloquinazoline Compounds.

entry	tryptanthrin 1	active methylene compound 2	nucleophile 3	time (min)	product/[yield %]	observed m. pt.	reported m. pt. (references)
1	1a	2a	3a	60	4a [85]	296–298	>27020
2	1a	2a	3b	60	4b [86]	266–268	>27020
3	1a	2a	3d	60	4c [86]	296–298	this work
4	1a	2a	3e	90	4d [88]	305–310	this work
5	1a	2b	3a	90	4e [82]	262–265	this work
6	1a	2b	3b	90	4f [84]	212–214	this work
7	1a	2b	3c	90	4g [88]	258–260	this work
8	1a	2b	3d	60	4h [82]	226–230	this work
9	1b	2a	3a	90	4i [86]	305–310	>27020
10	1b	2a	3b	90	4j [85]	264–268	>27020
11	1b	2a	3c	90	4k [81]	242–246	>27020
12	1b	2a	3d	90	4l [86]	212–214	this work
13	1c	2a	3a	120	4m [87]	278–280	this work
14	1c	2a	3b	90	4n [88]	254–256	this work
15	1c	2a	3d	120	4o [85]	212–214	this work

Results and Discussion

As the first step, the reaction between tryptanthrin 1a, malononitrile 2a, and dimedone 3a, as a model reaction, was investigated under solvent- and catalyst-free conditions. Only dicyanomethylene adduct 5a was obtained in 92% yield, and expected product 4a was not observed (Table 1, entry 1). Even when the reaction was carried out in MeOH under catalyst-free conditions, only dicyanomethylene derivative 5a was obtained in 93% yield (Table 1, entry 2). When the above-mentioned reaction was carried out with various catalysts such as montmorillonite-K10 clay, I₂, and Amberlite IRA-400 Cl in MeOH under reflux conditions too, only 5a was obtained (Table 1, entries 3–5). When the reaction of tryptanthrin 1a, malononitrile 2a, and dimedone 3a in MeOH and the presence of the Et₃N catalyst under reflux conditions was carried out, 4a was obtained as the major product and 5a as a minor product (Table 1, entry 6). When this reaction was also carried out at room temperature, the yield of 4a was decreased and that of 5a was increased (Table 1, entry 7). When the catalyst load was doubled, there was no significant change in yield, and a lower catalyst load led to a lower yield of 4a (Table 1, entry 8, 9). The reaction was also carried out in various solvents such as DMSO, MeCN, EtOH, and H₂O. Product yields in DMSO were similar to those in MeOH, and the remaining solvents were found to be not suitable (Table 1, entry 10–13). Other amines such as l-proline, pyridine, and piperidine failed to catalyze the reaction (Table 1, entry 14–16). Therefore, the optimum conditions for this reaction are refluxing in solvents such as MeOH and DMSO and using Et₃N as a catalyst.

Table 1. Optimization of Reaction for the Synthesis of 4a.

					product yield (%)
entry	catalyst (equiv)	solvent	temp (°C)	time (min)	4a	5a
1			reflux	120		92
2		MeOH	reflux	120		93
3	K10-Clay(1 equiv)	MeOH	reflux	120		94
4	Iodine (1 equiv)	MeOH	reflux	120		93
5	IRA-400 Cl (1 equiv)	MeOH	reflux	120		92
6	Et3N (1 equiv)	MeOH	reflux	60	85	8
7	Et3N (1 equiv)	MeOH	RT	60	40	17
8	Et3N (2 equiv)	MeOH	reflux	60	85	10
9	Et3N (0.5 equiv)	MeOH	reflux	60	63	19
10	Et3N (1 equiv)	DMSO	reflux	60	80	10
11	Et3N (1 equiv)	MeCN	reflux	60	Trace	52
12	Et3N (1 equiv)	EtOH	reflux	60
13	Et3N (1 equiv)	H2O	reflux	60
14	l-proline (1 equiv)	MeOH	reflux	60	Trace
15	pyridine (1 equiv)	MeOH	reflux	60	Trace
16	piperidine (1 equiv)	MeOH	reflux	60	Trace

The condensation reaction of an equal ratio of tryptanthrin (1a), malononitrile (2a), and dimedone (3a) in the presence of 1 equiv of triethylamine in MeOH under reflux conditions furnished 2-amino-7,7-dimethyl-5,12′-dioxo-5,6,7,8-tetrahydro-12′H spiro[chromene-4,6′-indolo(2,1-b)quinazoline]-3-carbonitrile 4a with 84% yield within 60 min. Encouraged by this result, we extended the reaction of tryptanthrins (1a–c) and cyanoacetic acid derivatives 2a,b (cyanoacetic ester, malononitrile) with other nucleophiles 3a–e (1,3-dicarbonyl compounds, 3-methyl-1H-pyrazol-5-one, and 1-naphthol) under similar conditions (MeOH/triethylamine) which gave corresponding compounds of spiroindoloquinazoline compounds, 4a–o, in moderate to high yields (84–87%), and the results are summarized in Table 2. However, when the reaction was carried out with ethyl cyanoacetate 2b at room temperature, it showed a combination of starting materials, and product 4 was obtained only in trace amounts. When the reaction was carried out in EtOH under reflux conditions, only the starting compounds (3a–e) were obtained with trace amounts of product 4. Interestingly, we found that MeOH under reflux conditions proceeded and gave unexpected products 4e–h (Table 2), and it was observed that compound 4e by the ¹HNMR spectrum showed a single peak at δ 3.15 ppm confirming the presence of the methoxy group. The ¹³C NMR spectrum also demonstrates that the resonance peak at 49.8 corresponds to the methoxy group. Moreover, the presence of the molecular ion peak at 470.1724 (M)⁺ in the mass spectrum was due to the formation of compound 4e. All these results suggest that the ethyl group of the ester is replaced by the methyl group originating from the solvent, MeOH.

It was observed that electron-withdrawing groups such as bromo and nitro substituted on tryptanthrins did not alter the yield of the products. Among the various nucleophiles, dimedone 3a, 1,3-cyclohexanedione 3b, 4-hydroxycoumarin 3c, 3-methyl-1H-pyrazol-5-one 3d, and 1-naphthol 3e gave the best yield. Good yield was also observed for 1,3-cyclohexanedione 3b and dimedone 3a (86, 85%, respectively) as a nucleophile. Significantly, the reaction with 3-methyl-1H-pyrazol-5-one 3d also afforded a good yield (Table 2, entry 3). When the reaction was carried out with 3-methyl-1H-pyrazol-5-one 3d, trypthanthrins (1a–c), and active methylene compounds (2a,b) in MeOH, better yields (82—86%) were observed under reflux conditions compared to the same reaction carried out at room temperature (40%).

The reaction between tryptanthrin 1a, malononitrile 2a, and dimedone 3a was carried out to produce 2-amino-7,7-dimethyl-5,12′-dioxo-5,6,7,8-tetrahydro-12′H spiro[chromene-4,6′-indolo(2,1-b)quinazoline]-3-carbonitrile 4a (shown in Scheme 2). In the first step of the reaction, possibly the Knoevenagel reaction between tryptanthrin 1a and malononitrile 2a leads to the formation of dicyanomethylene derivative 5a. The subsequent hydroxide-promoted Michael addition of cyclic 1,3-diketone 3a to 5a results in electron-deficient 2-amino-7,7-dimethyl-5,12′-dioxo-5,6,7,8-tetrahydro-12′H spiro[chromene-4,6′-indolo(2,1-b)quinazoline]-3-carbonitrile 4a.

Scheme 2. Plausible Mechanism for the Formation of Compound 4.

All the synthesized compounds were characterized and confirmed using spectroscopic techniques (IR, ¹HNMR, ¹³CNMR, and HRMS). Final structure proof of compound 4c was obtained from single-crystal X-ray studies (Figure 1).²⁵

Figure 1.

Spiroindoloquinazoline compounds from 4a–o.

Computational Details

Computational studies of synthesized compound 4c was carried out in the DFT method using Gaussian 09 software²⁴ and the B3LYP/6-311 G(d,p) basis set, and the optimized molecular structure was predicted. The geometrical parameters like bond length and bond angle of the compound were analyzed using Chemcraft software. Theoretical vibrational frequency data are compared with experimental results, and they are in good agreement with each other. The TD-DFT method was employed to study the electronic properties of the compound in DMSO as the solvent. Also, the frontier molecular orbital energies (FMO) were calculated as the energy gap between HOMO and LUMO helps us to predict the reactivity of the compound. The molecular electrostatic potential (MEP) is viewed using GaussView software. To learn about the nonlinear optical properties of the molecule, dipole moment and polarizability were theoretically calculated. The molecular docking studies were carried out using Autodock vina software.

Molecular Geometry

Single-crystal XRD of compound 4c was done using a crystal of dimension 0.35 × 0.30 × 0.25 mm³ with graphite monochromatic Mo Kα radiation (0.71073). The studied compound has a space group of P2₁/c, and the unit cell parameters are a = 11.3245(4) Å, b = 12.1767(5) Å, C = 17.3982(8) Å, α, γ = 90°, β = 104.506°,V = 2322.64(17) ų, and Z = 4. The crystallographic details are given in Table 3.

Table 3. Crystallographic Data of Compound 4c.

identification code	945,362
empirical formula	C24H20N6O3S
formula weight	472.52
temperature/K	293(2)
space group	P21/c
a/Å	11.3245(4)
b/Å	12.1767(5)
c/Å	17.3982(8)
α/°	90.00
β/°	104.506(2)
γ/°	90.00
volume/Å3	2322.64(17)
Z	4
ρcalcg/cm3	1.351
μ/mm–1	0.178
F(000)	984.0
crystal size/mm3	0.35 × 0.30 × 0.25
Radiation	Mo Kα (λ = 0.71073)
2Θ range for data collection/°	3.72 to 55
index ranges	–9 ≤ h ≤ 14, –13 ≤ k ≤ 15, –22 ≤ l ≤ 22
reflections collected	24,269
independent reflections	5337 [Rint = 0.0344, Rsigma =]
data/restraints/parameters	5337/7/337
goodness-of-fit on F2	1.027
final R indexes [I ≥ 2σ (I)]	R1 = 0.0579, wR2 = 0.1537
final R indexes [all data]	R1 = 0.0862, wR2 = 0.1751
largest diff. peak/hole/e Å–3	0.77/–0.68

The crystallographic structure obtained by solving the structure using SHELX was used to optimize the structure, and the ORTEP diagram of the compound is given in Figure 2.²⁵

Figure 2.

(a) ORTEP diagram and (b) optimized structure.

From the optimized structure, the geometrical parameters (bond length and bond angle) were calculated theoretically. The calculated parameters are given in Table S1. A plot of the calculated and experimental bond length and bond angle is done to check the accuracy of calculation and is shown in Figure 3.

Figure 3.

Plot of calculated versus experimental bond lengths and bond angles.

From the graph, a linear relationship was observed between experimental and theoretical values of the bond length and bond angle. The correlation coefficient (R²) calculated was unity and 0.9938 for the bond length and bond angle, respectively, which shows good agreement between experimental and theoretical results.

Furthermore, the compound is analyzed with weak hydrogen bonding between N–H···O, and this plays an important role in the stability of the molecule.

Frontier Molecular Orbital Analysis

FMO analysis explains the chemical reactivity of a compound which includes two molecular orbitals such as HOMO or highest occupied molecular orbital and LUMO or lowest unoccupied molecular orbital. HOMO is the outermost orbital with electrons, and it acts as a donor, whereas LUMO is the outermost orbital without electrons, and it acts as an acceptor. The HOMO–LUMO plot is given in Figure 4. The HOMO energy is calculated as −6.0208 eV, and LUMO energy is −1.3285, and the energy gap which is the difference in energies of HOMO and LUMO predicts that the reactivity of the molecule is 4.6924 eV.

Figure 4.

Frontier molecular orbital of compound 4c.

In addition to this, it also helps us to calculate various other factors like chemical hardness (η), electron affinity (EA), ionization potential (IP), electrophilicity (ω), and electronegativity (χ) using the equations derived from the Koopmans theorem²⁶ and are given in Tables 4 and 5.

1

2

3

4

5

6

Table 4. Hydrogen Bond Measurements for Compound 4c.

D	H	A	d(D–H)/Å	d(H–A)/Å	d(D–A)/Å	D–H–A/°
N4	H4A	O11	0.900(10)	2.028(12)	2.898(3)	162(2)
N4	H4B	N52	0.897(10)	2.073(10)	2.968(3)	176(2)
N6	H6A	O33	0.900(10)	1.893(11)	2.790(5)	174(3)

Table 5. Global Reactivity Parameters of Compound 4c.

orbital energy	energy (eV)
EHOMO	–6.02084
ELUMO	–1.3285
ΔE	4.6924
chemical potential (μ)	–6.6857
chemical hardness (η)	2.34615
softness (S)	0.2131
electronegativity(χ)	6.6857
Global electrophilicity index (ω)	9.5259

Molecular Electrostatic Potential

The MEP is a three-dimensional plot that helps to predict the interaction of the molecule with its surrounding molecules.²⁷ The MEP predicts the site of electrophilic and nucleophilic attack. The different color regions in the MEP plot represent different electrostatic potentials. The red region represents the most negative electrostatic potential, and blue represents the most positive electrostatic potential, and green represents zero electrostatic potential. The MEP plot of compound 4c is given in Figure 5.

Figure 5.

Molecular electrostatic potential diagram of compound 4c.

The color code of this electron density diagram lies in the range of −8.234 and 8.234 a.u. The negative region is around oxygen and nitrogen atoms. The red color around the O atom indicates the possibility of hydrogen bonding. The positive region is around the N atom.

Electronic Properties

The electronic spectrum of the molecule provides a deep insight into the charge transfer in the excited and ground state of the molecule. Thus, to record UV visible absorption spectra of the compound, the TD-DFT method was employed using the B3LYP basis set and Gaussian 09 package. The absorption spectra of compound 4c are obtained as shown in Figure 6.

Figure 6.

Absorption spectra of compound 4c.

In an organic molecule, absorption spectra may be observed due to transitions from π–π* and/or n–π* in the range of 200–700 nm. In compound 4c, the maximum absorption peak (λ_max) is observed at 284.67 nm with an oscillator strength of 0.2816.

Non-linear Optical Studies

Organic NLO properties have been at the forefront in the recent past, as they have gained their importance in telecommunications, signal processing, and optical interconnections.²⁸ Therefore, we computed the total dipole moment (μ), polarizability (α), and first-order hyper polarizability (β) of compound 4c as shown in Table 6.

7

8

9

Table 6. Polarizability Characteristics of Compound 4c.

components of μ	values	components of α	values	components of β	Values
μx	–1.8586	αxx	–157.766	βxxx	53.9989
μy	–1.5608	αyy	–165.2621	βyyy	12.727
μz	–4.1896	αzz	–163.978	βzzz	–125.4794
μ(D)	4.841822	αxy	–13.4191	βxyy	–17.2272
		αxz	–4.2283	βxxy	–47.194
		αyz	–12.6253	βxxz	30.3625
		α (a.u)	–162.3353667	βxzz	–66.7209
		α (e.s.u)	–2.40581Ε–23	βyzz	–61.1666
		Δα (a.u)	273.3469351	βyyz	31.2512
		Δα (e.s.u)	4.051 × 10–23	βxyz	38.4196
				βtot (a.u)	118.8342
				βtot e.s.u	1.026 × 10–30

The complete equation for calculating the magnitude of the first-order hyper polarizability is as follows

10

From the calculations, it is observed that the dipole moment of the compound is 4.8418 Debye units, and the polarizability (α_total) is 4.05 × 10^–23, and the first-order polarizability is 1.0266 × 10^–30. NLO properties of compound 4c are greater than those of urea which is considered to be the prototypical compound. Theoretically, the NLO property of the compound is greater than that of urea, which makes it a good NLO component.

Vibrational Analysis

The theoretical FT-IR spectrum of the compound was deduced by using the B3LYP/6-311G (d,p) basis set and compared with the experimental FT-IR data. Both the theoretical and experimental responses are illustrated in Figures 7 and 8.

Figure 7.

Theoretical FT-IR spectrum.

Figure 8.

Experimental FT-IR spectrum 4c.

There is a slight difference in the position of peaks. The −CN peak is observed at 2190 cm^–1 in experimental observation. However, when calculated theoretically, there is a slight difference, and it appears at 2500 cm^–1. The slight deviations in theoretical intensities from the experimental values may be attributed to the fact that the theoretical wave numbers are obtained from the isolated molecule in the gaseous phase, and the experimental wave numbers are obtained from the isolated molecule in the solid state.

Molecular Docking

Molecular docking is an important technique in drug discovery. Molecular docking provides computer-assisted drug design. It is a convenient tool that helps to study the mechanism of protein–ligand interactions and helps in analyzing the protein ligand-binding site.

The phospholipase inhibitory effect of these molecules are already explored using molecular docking studies.²⁰ Thus, we decided to carry out the molecular docking studies of compound 4c, to check other significant biological activity of the compound. The smile format of the compound was used in PASS online (prediction of activity of spectra for substances). This provides an insight into the various biological activities of the compound, like Hepatic disorder treatment, liver fibrosis treatment, antiallergic, antimitotic, and antiasthmatic treatments. It can also act as a fibroblast growth factor agonist with a high P_a (probability to be active) value of 0.671. Two proteins were chosen out of the various choices according to their characteristics that meet docking conditions. Two target proteins 4POK and 4IZE of resolution 1.70 and 2.00 A⁰, respectively, were chosen for docking.

Both ligand and target proteins were prepared for docking analysis in Pymol software. Dock suite 4.2.6 software was used for studying the interaction between the protein and ligand and their binding affinity. The Lamarckian Genetic Algorithm (LGA) feature in Auto Dock software was utilized for the docking process. Discovery Studio Visualizer 4.0 software was used to visualize the interaction of the ligand with the target protein. The molecular interaction between the ligand and protein along with their binding site is illustrated in Figure 9.

Figure 9.

Diagrammatic representation of ligand interactions with proteins (a) 4POK and (b) 4IZE.

The various parameters of docking are listed in Table 7. Table 7 gives information about the target protein bond distance, inhibition constant, and RMSD values. The inhibition constant (ki) value provides information regarding the medication required to inhibit the activity of the protein. The obtained inhibition constant for the ligand with two given target proteins 4POK and 4IZE is 29.05 and 19.02 μM, respectively. The binding energy exhibited by these proteins 4POK and 4IZE is −6.91 and −7.72 kcal/mol, respectively. Lower binding energy is shown by the 4IZE protein which suggests that this protein can bind easily with a ligand molecule. Also, a lower inhibition constant for the protein 4IZE suggests that only less quantity of medication is required in the case of this protein.

Table 7. Docking Parameters of Ligand (Compound) 4c.

protein (PDB)	bond distance (Å)	estimated inhibition constant (μM)	binding energy (kcal/mol)	intermolecular energy (kcal/mol)	RMSD
4POK	5.75, 5.32, 5.04, 4.91, 4.58, 2.62, 1.94, 1.98	8.64	- 6.91	–7.21	29.48
4IZE	5.36, 5.03, 4.82, 4.78, 2.20, 2.14, 1.93, 1.91, 1.86	2.19	- 7.72	–8.02	20.01

The binding of various enzymes on the ligand and the type of bond formed by them are shown in Figure 10; the PHE A:151 forms hydrogen bonding with the nitrogen atom of −NH₂ and the pyrazole ring of the ligand. The amino acid TYR A 150 forms a carbon–hydrogen bond with the ligand, and the amino acid VAL A44 forms a π-alkyl bond with the benzene ring. PHE A:9 forms a π–π stacked and π–π T-shaped bond with the benzene ring.

Figure 10.

Binding activity of various enzymes of proteins on the ligand: (a) 4POK and (b) 4IZE.

Figure 10 explains that the amino acid ILE A:149, LEU A:97, and PRO A:95 forms hydrogen bonding with the ligand molecule, whereas amino acids ILE A:150 and lEU A:151 form a π-alkyl bond with the ligand. These computational explanations suggest that this compound can act against 4POK and 4IZE as fibroblast growth factor agonists.

Conclusion

In summary, we have developed an easy and clean one-pot, three-component reaction for the synthesis of spiroindoloquinazoline compounds in methanol in the presence of triethylamine as a catalyst. This method is simple, efficient, and comes with easy work-up procedures. Theoretical DFT calculations were performed for compound 4c, and the properties were in good agreement with the experimental values. Molecular docking was also carried out for selective compound 4c as the ligand and using two proteins 4POK and 4IZE, and the results suggest that the ligand binds actively with the target protein and indicate that compound 4c can act as a fibroblast growth factor agonist.

Spectroscopic Data for Selected Compounds

(4a): White solid, mp 296 °C; Rf (40% EtOAc–petroleum ether) 0.69, FT-IR ν_max (KBr): 3408, 3276, 2198, 1660, 1600, 1464, 1358, 1322, 1266 cm^–1; ¹H NMR (300 MHz, DMSO-d₆): δ = 8.45 (d, 1H, J = 8.1 Hz, ArH), 8.33 (d, 1H, J = 7.8 Hz, ArH), 7.88–7.76 (m, 2H, ArH), 7.66–7.63 (m, 1H, ArH), 7.55 (s, 2H, NH₂), 7.52–7.35 (m, 3H, ArH), 2.70–2.67 (m, 2H, CH₂), 2.11–2.10 (m, 2H, CH₂), 1.05 (s, 3H, CH₃), 1.01 (s, 3H, CH₃); ¹³C NMR (75 MHz, DMSO-d₆): δ = 195.4, 164.4, 162.6, 158.9, 158.3, 147.1, 138.6, 135.6, 134.9, 129.1, 127.3, 127.1, 126.4, 123.8, 120.6, 117.3, 115.8, 111.2, 57.9, 49.8, 47.8, 40.3, 32.1, 27.4, 27.0; HRMS m/z calcd for C₂₆H₂₁N₄O₃, (M + H)⁺ 437.1623; found, 437.1614.

(4e): White solid, mp 262 °C; Rf (40% EtOAc–petroleum ether) 0.66, FT-IR ν_max (KBr): 3346, 3077, 2954, 1684, 1603, 1463, 1353, 1313, 1224 cm^–1; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.62 (d, 1H, J = 7.5 Hz, ArH), 8.47 (d, 1H, J = 7.0 Hz, ArH), 7.72–7.71 (m, 2H, ArH), 7.51–6.79 (m, 6H, ArH, NH₂), 3.15 (s, 3H, OCH₃), 2.67–2.47 (m, 2H, CH₂), 2.18–2.03 (m, 2H, CH₂), 1.11 (s, 3H, CH₃), 1.03 (s, 3H, CH₃); ¹³C NMR (125 MHz, DMSO-d₆): δ = 195.4, 164.9, 162.6, 158.9, 158.3, 147.1, 138.6, 135.6, 134.9, 129.1, 127.3, 127.1, 126.4, 123.8, 120.6, 117.2, 115.8, 111.2, 57.9, 49.8, 47.8, 40.3, 32.1, 27.4, 27.0; HRMS m/z calcd for C₂₇H₂₄N₃O₅, (M + H)⁺ 470.1724; found, 470.1716.

(4i): White solid, mp 305 °C; Rf (40% EtOAc–petroleum ether) 0.67, FT-IR ν_max (KBr): 3406, 3270, 2960, 2196, 1659, 1598, 1465, 1354, 1320, 1221, 560 cm^–1; 7.49–6.47 (m, 8H, ArH, NH₂), 6.70 (s, 1H, ArH), 2.63 (s, 2H, CH₂), 2.22–2.17 (m, 2H, CH₂), 1.02–1.08 (s, 6H, CH₃); ¹³CNMR (100 MHz, DMSO-d₆): δ = 195.9, 164.9, 163.2, 159.4, 158.8, 147.6, 139.0, 136.1, 135.4, 129.6, 127.9, 127.8, 127.6, 126.9, 124.3, 121.1, 117.8, 116.3, 111.7, 58.4, 50.3, 48.4, 32.6, 27.9, 27.5; HRMS m/z calcd for C₂₆H₁₉N₄O₃BrNa, (M + Na)⁺ 537.0545; found, 537.0538.

(4m): White solid, mp 278 °C; Rf (40% EtOAc–petroleum ether) 0.71, FT-IR (KBr): ν_max = 3425, 2200, 1692, 1596, 1516, 1465, 1342, 1267, 1204; ¹HNMR (500 MHz, DMSO-d₆& CDCl₃): δ = 8.46 (d, J = 10.0 Hz, 1H, ArH), 8.32–8.18 (m, 1H, ArH), 7.82 (t, J = 9.5 Hz, 1H, ArH), 7.00–7.56 (m, 1H, ArH), 7.47–7.34 (m, 3H, NH₂, ArH), 7.14–7.11 (m, 1H, ArH), 2.71–2.59 (m, 2H, CH₂), 2.14–2.04 (m, 2H, CH₂), 1.05 (s, 3H, CH₃), 1.02 (s, 3H, CH₃); ¹³C NMR (125 MHz, DMSO-d₆): δ = 195.2, 164.5, 159.4, 159.2, 147.6, 139.1, 134.8, 129.4, 128.5, 127.6, 127.5, 126.8, 123.9, 123.3, 122.1, 121.2, 117.7, 116.3, 109.8, 78.9, 57.9, 50.4, 47.3, 39.3, 32.3, 27.7, 27.6; ESI m/z calcd for C₂₆H₁₉N₅O₅, 481.4596; found, 481.4595.

Data and Software Availability

In our study, computational studies were carried out using the Gaussian 09 package which is available at www.gaussian.com.PDB structures are available from RCSBPDB (https://www.rcsb.org). Molecular docking studies were performed using software AutoDock Tools. (http://autodock.scripps.edu/resources/adt).The ligand and protein molecule preparation was done using PYMOL which is available at http://pymol.sourceforge.net.The docked structures were viewed in Discovery Studio which can be obtained from https://discover.3ds.com/discovery-studio-visualizer.The FT-IR spectra were drawn using Origin Lab which is available at https://www.originlab.com. Produced and analyzed data are available from the authors upon request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c06781.

• Experimental procedures and compound characterization data (PDF)

The authors declare no competing financial interest.