Design, Synthesis, Antimicrobial Evaluation, and Molecular Modeling Studies of Novel Indolinedione-Coumarin Molecular Hybrids.

Keeping in view various pharmacological attributes of indole and coumarin derivatives, a new series of indolindione-coumarin molecular hybrids was rationally designed and synthesized. All synthesized hybrid molecules were evaluated for their antimicrobial potential against Gram-negative bacterial strains (Escherichia coli and Salmonella enterica), Gram-positive bacterial strains (Staphylococcus aureus and Mycobacterium smegmatis), and four fungal strains (Candida albicans, Alternaria mali, Penicillium sp., and Fusarium oxysporum) by using the agar gel diffusion method. Among all synthetics, compounds K-1 and K-2 were found to be the best antimicrobial agents with the minimum inhibitory concentration values of 30 and 312 μg/mL, against Penicillium sp. and S. aureus, respectively. The biological data revealed some interesting facts about the structure-activity relationship which state that the electronic environment on the indolinedione moiety and carbon chain length between indolinedione and triazole moieties considerably affect the antimicrobial potential of the synthesized hybrids. Various types of binding interactions of K-2 within the active site of S. aureus dihydrofolate reductase were also streamlined by molecular modeling studies, which revealed the possible mechanism for potent antibacterial activity of the compound.

Introduction

Nowadays, microbial resistance has become a serious threat to n class="Species">human health, which is a major problematic and most challenging issue to worldwide researchers. According to the World Health Organization, 50 000 people including male, female, and children are dying everyday from microbial infections around globe.[1] A report revealed by WHO stated that approximately 16 million people died in 1990 which was further decreased by 1% in 2010 (15 million), and it is estimated to reach 13 million by 2050. No doubt the number of deaths by microbial infection is falling but with a very slow rate, may be due to burst increase in the world population and microbial resistance.[2] The recent focus on the development of antimicrobial agents has failed to reach the expectations because of high risk of toxicity and insufficient antimicrobial activity as well as microbial resistance, which led to a search for novel antimicrobial agents.[3] In the recent past, numerous research strategies have been successfully utilized to fight against multidrug-resistant microbial infection. It has been reported that solo targeting agents are more prone to drug resistance, which fails the expected successful drug candidate.[4] A general belief is that drugs affecting more than a single target or multiple sites of the single target showed higher potency and lower resistance as compared to solo targeting agents.[5−9] This can only be achieved by the molecular hybridization technique.

Molecular hybridization is an approach n class="Chemical">for drug development in which two different active pharmacophores are clubbed together with or without the help of a linker. In these days, this approach is the most popular one in the development of novel drug entities to target multiple sites.[10] Therefore, hybrid molecules can attenuate the risk of multiple drug resistance as well as a drug–drug interaction which could be useful for humanity to resist microbial resistance.

Coumarin is a plant secondary metabolite, that exhibits a wide variety on class="Chemical">f biological activities such as antimicrobial and its derivatives have reported for anticoagulant, anti-inflammatory, antimicrobial, anti-HIV, antioxidant, antiallergic, anticancer, antiproliferative, and antiviral activities.[11−15] There are numerous evidence on coumarin-based hybrid molecules with promising antimicrobial potential which includes coumarin–theophylline hybrids (1), coumarin–carbonodithioate hybrids (2), coumarin–benzimidazole hybrids (3), coumarin–imidazole hybrids (4), coumarin–thiazolyl hybrids (5), coumarin–sulfonamide hybrids (6), 2-aminothiazole hybrids (7), and coumarin–dihydropyrimidine hybrids (8)[16−23] (Figure )

Figure 1

Recently reported hybrid molecules as antimicrobial agents.

2,3-Indolinedione, an n class="Chemical">indole derivative, is considered as a pharmacological active nucleus with its vast biological applications. It is generally found in many natural products such as fungal metabolites, indole alkaloids, and marine natural products. There is a number of research articles describing the antimicrobial potential of indolinedione-based hybrids such as propylene-tethered ciprofloxacin–isatin hybrids (9) and indole isatin hybrids (10)[24,25] (Figure ).

These significant reports provide a strong rationale n class="Chemical">for the inclusion of these two active moieties into a single molecular architecture, which could exhibit low toxicity with significant antimicrobial activity.

Triazole is also an important heterocyclic moiety which plays an imperative role in medicinal chemistry and can be used n class="Chemical">for the synthesis of numerous biologically active molecules such as antimicrobial, anticancer, antioxidant, anti-inflammatory, antiviral, and anticonvulsant.[26−32] This elite class of moiety can then be considered to incorporate into the molecules to exhibit potential results. There are a number of molecules exhibiting excellent antimicrobial activity such as fluorinated chalcone-1,2,3-triazole conjugates (11), coumarin–1,2,3-triazole hybrids (12), dehydroacetic acid-chalcone-1,2,3-triazole hybrids (13), 1,2,3-triazole-linked chalcone and flavone hybrids (14), theophylline containing 1,2,3-triazole nucleoside derivatives (15), ciprofloxacin-triazole conjugates (16), and 4-substituted 1,2,3-triazole coumarin hybrids (17).[33−38] This active moiety can then be selected to incorporate into target molecules as a linker between two active pharmacophoric moieties, that is, coumarin and indolinedione (Figure ).

Figure 2

Variously reported triazole-linked hybrid molecules as antimicrobial agents.

Variously reported triazole-linked hyn class="Chemical">brid molecules as antimicrobial agents.

Keeping in view the problem of antimicrobial resistance as a major limitation on class="Chemical">f the currently available antimicrobial drugs, the present study targets at the synthesis of triazole-tethered indolinedione–coumarin hybrids by using a click chemistry approach (Figure ) and evaluation against various bacterial and fungal strains. The zone of inhibition was determined for all synthetics, and the potent compounds were selected to calculate their minimum inhibitory concentration (MIC) values. Furthermore, various binding interactions of the most potent compound were also explored by using molecular modeling studies.

Figure 3

Design strategy of indolinedione–coumarin hybrid molecules.

Design strategy of n class="Chemical">indolinedione–coumarin hybrid molecules.

Results and Discussion

Indolinedione–n class="Chemical">coumarin hybrids were synthesized by following the synthetic Scheme . First, substituted indolinedione was stirred with 1,2-dibromoalkanes (1 equiv) at room temperature by using dimethylformamide (DMF) as the solvent and K2CO3 (1.5 equiv) as the base. The resulting product was then dissolved in DMF and then NaN3 (1 equiv) was added. The reaction mixture was stirred at room temperature to form 1-(4-azidoalkyl)indoline-2,3-diones.

Scheme 1

Synthesis of Indolinedione–Coumarin Hybrids

Reagents and conditions: (a) K2CO3, DMF, 2 h, stir, rt; (b) NaN3, DMF, 1 h, stir, rt; (c) propargyl bromide, K2CO3, DMF, 2 h, stir, rt; and (d) sodium ascorbate, CuSO4, DMF, 15 min, rt.

Reagents and conditions: (a) K2CO3, n class="Chemical">DMF, 2 h, stir, rt; (b) NaN3, DMF, 1 h, stir, rt; (c) propargyl bromide, K2CO3, DMF, 2 h, stir, rt; and (d) sodium ascorbate, CuSO4, DMF, 15 min, rt.

Separately, 4(prop-2-ynyloxy)-2H-chromen-2-one (n class="Chemical">PHC) was obtained by stirring the reaction mixture of 4-hydroxycoumarin and propargyl bromide (1.2 equiv to 4-hydroxycoumarin) in DMF under basic conditions (K2CO3; 1.5 equiv).

This 4-(prop-2-ynyloxy)-2H-chromen-2-one (n class="Chemical">PHC) was further reacted with various 1-(4-azidoalkyl)indoline-2,3-dione analogues in the presence of pentahydrate CuSO4 (catalytic amount) and sodium ascorbate (as a reducing agent of CuSO4), in DMF at room temperature to obtain the desired hybrid compounds. 1H NMR and 13C NMR spectroscopic techniques were used to characterize the synthesized compounds, and all spectral data were found in accordance with assumed structures.

In Vitro Evaluation

Antibacterial Activity

All synthesized hybrids were tested against two Gram-negative bacterial strains (n class="Species">Escherichia coli and Salmonella enterica) and two Gram-positive strains (Staphylococcus aureus and Mycobacterium smegmatis). The results showed that almost all of the compounds are active against the tested micro-organisms. Among these strains, S. aureus was the most sensitive one, and E. coli was the most resistant strain to the hybrid molecules. Compound K-2 was found to be the most potent which exhibited 2.5 and 1.3 cm inhibition zone against S. aureus and S. enterica, respectively (Figure A). An interesting structure–activity relationship has also been established from Table which revealed that electron density on the fifth position of the indolinedione moiety greatly influences the antibacterial potential. Activity increased with increase in electronegativity on the fifth position of indolinedione. It has also been cleared from the data that the chain length of two carbon atoms which joins the triazole moiety to indolinedione, was a most tolerable linker. Thus, the overall preference order for antibacterial potential for indolinedione is F > Cl > Br > I > NO2 > OCH3 > H and for linker spacer length: n = 1 > 2 > 3 (Figure ). For determination of MIC of the most potent hybrid (K-2) against S. aureus, different concentrations were prepared and tested. Results showed that the compound at a concentration of 5 mg/mL was quite effective against the tested organism and exhibited a maximum zone of inhibition of 2.5 cm (Figure a), whereas negative control colistin and dimethylsulphoxide (DMSO) showed no zone of inhibition (Figure b). MIC of K-2 was found to be 312 μg/mL where the minimum zone of inhibition of 0.1 cm was observed, which was found comparable to that of norfloxacin against S. aureus (MIC = 128 μg/mL).[39]

Figure 4

(a) Zone of inhibition exhibited by compound K-2 against S. aureus; (b) negative control colistin & DMSO; (c) maximum zone of inhibition at a concentration of 30 μg/mL with negative control fluconazole and DMSO; (d) effect of different concentrations 15, 7.5, 3.75, and 1.87 μg/mL of compound K-1 against Penicillium sp.

Table 1

Results of the Antimicrobial Activitya,b

	zone of inhibition (cm)
s.no	sample	S. enterica	E. coli	S. aureus	M. smegmatis	C. albicans	A. mali	Penicillium sp.	F. oxysporum
1	K-1	1.3 ± 0.2		0.5 ± 0.2				2.5 ± 0.2
2	K-2	1.3 ± 0.2		2.5 ± 0.5			0.4 ± 0.1	1.8 ± 0.5
3	K-3	1.1 ± 0.1		2.1 ± 0.4			0.5 ± 0.1	1.0 ± 0.2
4	K-4	0.9 ± 0.1		1.7 ± 0.6	0.5 ± 0.1			1.6 ± 0.6	0.3 ± 0.2
5	K-5		1.1 ± 0.2	1.2 ± 0.6		0.3 ± 0.2		1.8 ± 0.4	0.6 ± 0.1
6	K-6	1.1 ± 0.2		0.9 ± 0.4	0.3 ± 0.2			1.4 ± 0.3
7	K-7	0.8 ± 0.1		0.6 ± 0.2				1.2 ± 0.6	1.0 ± 0.1
8	L-1			1.2 ± 0.1				1.7 ± 0.4	0.7 ± 0.2
9	L-2	1.2 ± 0.2		0.8 ± 0.4	0.4 ± 0.2		0.6 ± 0.2	1.3 ± 0.6
10	L-3	1.2 ± 0.1		0.7 ± 0.3			0.9 ± 0.1	1.5 ± 0.4
11	L-4			0.4 ± 0.1	0.7 ± 0.1			1.0 ± 0.4	0.5 ± 0.2
12	L-5	1.1 ± 0.3		0.5 ± 0.1
13	L-6	0.7 ± 0.1		0.2 ± 0.1		0.6 ± 0.1		0.2 ± 0.03
14	L-7		0.5 ± 0.1	0.3 ± 0.1				0.1 ± 0.04
15	M-1	1.1 ± 0.2			0.5 ± 0.1		0.2 ± 0.1
16	M-2			0.6 ± 0.1	0.4 ± 0.2		0.3 ± 0.2
17	M-3			0.5 ± 0.2	0.3 ± 0.2		0.5 ± 0.1
18	M-4	1.1 ± 0.1	0.7 ± 0.1						0.6 ± 0.1
19	M-5								0.7 ± 0.2
20	M-6		0.4 ± 0.1		0.6 ± 0.1
21	M-7		0.5 ± 0.1	0.4 ± 0.1

Zones of inhibitions (cm) were measured by using agar gel diffusion assay. The results are the mean ± SD of three replicate experiments.

No zone of inhibition observed.

Figure 5

Structure–activity relationship of hybrid molecules.

(a) Zone of inhibition exhibited by compound K-2 against n class="Species">S. aureus; (b) negative control colistin & DMSO; (c) maximum zone of inhibition at a concentration of 30 μg/mL with negative control fluconazole and DMSO; (d) effect of different concentrations 15, 7.5, 3.75, and 1.87 μg/mL of compound K-1 against Penicillium sp.

Structure–activity relationship of hyn class="Chemical">brid molecules.

Zones of inhibitions (cm) were measured by using n class="Chemical">agar gel diffusion assay. The results are the mean ± SD of three replicate experiments.

No zone on class="Chemical">f inhibition observed.

Antifungal Activity

Synthesized compounds were also tested against four n class="Chemical">fungal strains, namely, Candida albicans, Alternaria mali, Penicillium sp., and Fusarium oxysporum by using the agar gel diffusion method. Most of the compounds were active against Penicillium sp. amongst which compound K-1 was endowed with the most prominent zone of inhibition, that is, 2.5 cm. K-2 was found to be the second most potent compound against Penicillium sp. with 1.3 cm inhibition zone. The compounds were inactive against the C. albicans fungal strain which indicates that indolinedione–coumarin hybrids could be used for the treatment of infectious disease, associated with Penicillium sp. The structure–activity relationship revealed that the unsubstituted indolinedione moiety is the most crucial one for antifungal activity. Further activity falls in the order of the electronegative effect with exception of fluorine and chlorine atoms. The preference order for antifungal activity thus became: R = H > Cl > F > Br > I > NO2 > OCH3. The effect of chain length was found similar to that of antibacterial in the order of n = 1 > 2 > 3 (Figure ). To determine MIC of the most potent compound K-1, different concentrations were tested against Penicillium sp. Results in (Figure c) showed that the compound at a concentration of 30 μg/mL was quite effective against the tested organism and exhibited a maximum zone of inhibition of 2.5 cm, whereas DMSO showed no zone of inhibition. The results were found comparable to that of clinically used drug fluconazole against Penicillium sp. which was found to exhibit an MIC value of 4.072 μg/mL.[40] It was clearly observed that with a decrease in concentration from 30 to 7.5 μg/mL, there was a decrease in the zone of inhibition and for last two concentrations (3.75 & 1.875 μg/mL), no zone of inhibition was observed (Figure d). Although synthesized compound K-1 at a concentration of 15 and 7.5 μg/mL affects the growth of the tested organism, it also showed more effect on sporulation/pigmentation. Spores were not pigmented in the presence of the compound.

The most potent hybrid compounds n class="Gene">K-1 and K-2 were also tested for their toxicity to the microbial strains. Both the compounds at their MIC values did not kill the microbial strains and they only found to inhibit the growth of the microbes. This confirmed that the observed potential of the compounds is solely due to their inherent antimicrobial activity.

Overall biological data suggested that the most potent compounds (K-1 and K-2) could act as hit lead molecules n class="Chemical">for further development of potent antimicrobial agents. As hybrid molecules, these agents perform better results as compared to standard drugs, colistin and fluconazole (marketed drugs). In contrast to this, the use of bifunctionalities in a single molecular architecture could also mitigate the drug–drug interactions as well as the chance of microbial resistance. Most interestingly, compound K-1 significantly inhibits the growth of S. aureus. S. aureus is one of the most common causes of infective endocarditis, bacteremia, and various drastic skin and soft tissue infections. It can lay dormant for years undetected in the human body and may cause dreadful conditions if not treated. In this regard, the study suggests that K-1 could be used as a future drug candidate to treat various infections attributed to S. aureus.

Docking Studies

For deep and clear perception to analyze the molecular mechanism on class="Chemical">f the most potent antibacterial compound K-2, molecular modeling studies were performed on S. aureus dihydrofolate reductase (DHFR). DHFR is a key enzyme in folate metabolism and a valuable target for antibacterial drugs. The docking protocol was validated by reproducing the X-ray derived conformation of 7-aryl-2,4-diaminoquinazoline at the DHFR binding site (PDB entry: 3SRQ). The three-dimensional (3D) coordinates of 7-aryl-2,4-diaminoquinazoline were extracted and docked at the active site using GoldScore, ChemScore, ASP, and ChemPLP scoring functions. GoldScore has reproduced the best fit conformation of 7-aryl-2,4-diaminoquinazoline with a root-mean-square deviation of 0.104 which ensures the reliability of the selected docking protocol. Therefore, GoldScore was selected as a fitness function for the docking study. Ten conformations of K-2 were generated and ranked according to their GoldScore. The conformation with the highest GoldScore (61.63) was selected for discussion.

The binding site residues and overall binding mode of K-2 suggest that it n class="Disease">fits well in the binding cavity and gets stabilized by various electrostatic interactions (Figure ). The major interactions between K-2 and DHFR include van der Waal’s, π–π stacking, and H-bond interactions. The 5-fluoro-indoline-2,3-dione moiety gets positioned in a cavity formed by Val7, Ala8, Ile15, Leu21 (hydrophobic residues) and Trp23 (aromatic residue). Here, the backbone −NH of Ala8 has shown an H-bond interaction with carbonyl oxygen present at the benzene ring of the indolinedione moiety (H-bond acceptor; d = 1.382 Å). The triazole ring is involved in a face-to-face π–π stacking interaction with Phe93 (d = 4.035 Å) (Figure a). This arrangement of energetically favorable π–π stacking interactions has also seen in the cocrystal structure of DHFR with 7-aryl-2,4-diaminoquinazoline (d = 4.603 Å)[41] (Figure a). The coumarin moiety is surrounded by hydrophobic residues which include Leu29, Val32, Lys33, Ile51, and Leu55 (Figure b). It is perfectly sandwiched between the aliphatic side chain of Leu29 and Leu55 and stabilized by van der Waals and/or dispersion interactions. The study suggests that K-2 may block the activity of DHFR sufficiently enough to prevent the substrate from binding to its active site.

Figure 6

(a) Docking conformation of K-2 at the active site of DHFR (hydrogens which are involved in H-bond interactions are shown); (b) two-dimensional depiction of various residues involved in D–R interactions.

(a) Docking conformation on class="Chemical">f K-2 at the active site of DHFR (hydrogens which are involved in H-bond interactions are shown); (b) two-dimensional depiction of various residues involved in D–R interactions.

In Silico Studies

Compounds which exhibited above 0.5 cm zone of inhibition in sensitive microbial strains, namely, n class="Species">S. aureus and Penicillium sp. (K-1 to K-7 and L-1 to L-3) were subjected to determine their physicochemical properties by using web-based software, MarvinSketch (http://www.chemaxon.com/) and PreADMET (http://preadmet.bmdrc.org/) (Table ). The results of the in silico study revealed that all compounds are less likely to cause neurotoxicity and could not hinder the normal action of neuronal cells, confirmed by the predicted in silico values. Table summarized the drug likeliness properties of the synthesized compounds which were determined by the online softwares, ChemAxon and MarvinSketch. Table indicated that all synthesized compounds fit well with the Lipinski rule of five. Tabular values indicated that (a) all synthetics have molecular weights within the limits of 180–500, except K-5, (b) synthesized compounds have no H-bond donating site at all, (c) compounds have hydrogen bond accepting ability but complies with H-bond acceptor criteria (<10), (d) the molar refractivity was found in the range of 121.16–134.53 cm3 mol–1, which also obeys the accepting limit of 40–130, and (e) the log P of all compounds indicate that the compounds are not very lipophilic (<5.6). From these results, it can be stated that the compounds could be pharmacologically efficient for clinical use in the future as they follow the Lipinski rule of five except K-5.

Table 2

In Silico ADME Properties of Active Hybrid Molecules

	absorption				distribution
compound	human intestinal absorption (HIA) %	in vitro Caco-2 cell permeability (nm/s)	in vitro MDCK cell permeability (nm/s)	in vitro skin permeability (log Kp) cm/h	in vitro plasma protein binding (%)	in vivo blood brain barrier penetration (C.brain/C.blood)
K-1	99.64	20.78	8.06	–4.27	91.10	0.06
K-2	99.65	21.29	3.01	–4.46	91.83	0.07
K-3	99.57	21.23	0.92	–4.31	96.85	0.08
K-4	98.10	21.45	0.04	–3.87	97.40	0.03
K-5	97.95	27.26	0.45	–4.26	98.67	0.07
K-6	92.61	10.54	2.15	–4.15	99.18	0.05
K-7	99.47	22.52	2.52	–4.38	90.96	0.06
L-1	99.71	23.58	5.33	–4.23	92.21	0.09
L-2	99.72	22.17	3.51	–4.44	92.68	0.10
L-3	99.44	21.31	0.87	–4.27	97.85	0.13

Table 3

Physicochemical Parameters of Active Hybrid Molecules

compound	molecular weight	no. of H-bond donors	no. of H-bond acceptors	molar refractivity	log P	no. of Lipinski violation
K-1	416	0	6	121.16	1.64	0
K-2	434	0	6	121.38	1.78	0
K-3	450	0	6	125.97	2.24	0
K-4	495	0	6	128.79	2.41	0
K-5	542	0	6	134.53	2.57	2
K-6	461	0	8	128.49	1.58	0
K-7	446	0	7	127.63	1.48	0
L-1	430	0	6	126.03	1.70	0
L-2	448	0	6	126.25	1.84	0
L-3	464	0	6	130.83	2.30	0

Conclusions

In the present study, indolinedione–n class="Chemical">coumarin molecular hybrids were designed, synthesized, and characterized by using 1H NMR and 13C NMR. All synthetics were assessed for the antimicrobial activity against a panel of Gram-positive and Gram-negative bacterial and fungal strains and their zone of inhibition was calculated. Among all synthetics, compounds K-1 and K-2 were found to exhibit a maximum zone of inhibition (2.5 cm) against Penicillium sp. and S. aureus, respectively. The most potent antibacterial compound K-2 displayed an MIC of 312 μg/mL, while the most potent antifungal compound K-1 exhibited an MIC value of 30 μg/mL. The structure–activity relationship established from biological results states that the electronic environment on the indolinedione moiety greatly affected the antimicrobial activity of the hybrid molecules. The activity decreased with the increase in chain length between the triazole and indolinedione moieties. Moreover, various binding interactions of the most potent compound K-2, within the catalytic active site of S. aureus DHFR, were also streamlined by using docking studies which revealed the probable mechanism for its potent antibacterial potential. Thus, the overall study concluded that indolinedione–coumarin hybrids could act as a hit lead for further developments of antimicrobial agents.

Experimental Section

Materials and Measurements

The chemical reagents were procured from CDH, Sigma-Aldrich, and Loba, India. All yields ren class="Chemical">fer to isolated products after purification. Products were characterized by comparing with authentic samples and by spectroscopic techniques, that is, 1H and 13C NMR, elemental analysis. AVANCE III HD 500 MHz Bruker Biospin and JEOL AL 300 MHz machines were used to record the NMR spectra. The spectra were recorded by dissolving in CDCl3 and DMSO-d6 relative to tetramethylsilane (TMS) (0.00 ppm). In 1H NMR, chemical shifts were reported in δ values using the internal standard (TMS) with a number of protons, multiplicities (s-singlet, d-doublet, t-triplet, q-quartet, and m-multiplet), and coupling constants (J) in hertz (Hz). Melting points were determined in open capillaries and were uncorrected.

General Procedure for the Synthesis of 4-(Prop-2-ynyloxy)-2H-chromen-2-one (PGC)

In 50 mL of n class="Chemical">DMF, 4-hydroxy coumarin (20 g) was dissolved with the addition of propargyl bromide (1.2 equiv) and K2CO3 (1.5 equiv). The reaction mixture was stirred at room temperature, and the reaction was continuously monitored by thin-layer chromatography (TLC). After the completion of reaction, the reaction mixture was poured on crushed ice. The solid product of propargylated coumarin thus obtained was filtered, washed with cold water, and air dried. The physical data of propargylated coumarin are given below:

PGC

Yield 80%; mp 103–107 °C. 1H n class="Chemical">NMR (CDCl3, 500 MHz, δ, TMS = 0): 3.22–3.24 (m, 1H, −CH–propargylic), 4.92–4.94 (m, 2H, −CH2−), 5.84 (d, 1H, J = 12 Hz, −CH−), 7.25–7.27 (m, 2H, ArH), 7.56 (s, 1H, ArH), 7.77 (d, 1H, J = 8 Hz, ArH). 13C NMR (CDCl3, 125 MHz, δ, TMS = 0): 57.38, 76.50, 79.32, 91.71, 115.39, 116.60, 123.16, 124.28, 132.83, 153.17, 162.02, 164.24.

General Procedure for the Synthesis of 1-(2-Bromoethyl)indoline-2,3-dione

Indoline-2,3-dione (1 equiv) was dissolved in n class="Chemical">DMF (in minimum amount), dibromoethane (1 equiv) and K2CO3 (1.5 equiv) were added. The reaction mixture was stirred at room temperature. After the completion of reaction as confirmed by TLC, the reaction mixture was poured on crushed ice. The impure product so obtained was filtered, air dried, and subjected to column chromatography (hexane/ethylacetate as eluent) to gain the desired product, that is, 1-(2-bromoethyl)indoline-2,3-dione. All other 1-bromoalkylindoline-2,3-diones were synthesized via the same procedure as mentioned above using various dibromoalkanes.

General Procedure for the Synthesis of 1-(4-Azidobutyl)indoline-2,3-dione (IBA)

In minimum amount of n class="Chemical">DMF, 1-(4-bromobutyl)indoline-2,3-dione (1 equiv) was dissolved, sodium azide (1 equiv) was added, and the mixture was stirred at room temperature. After the completion of reaction, it was poured on crushed ice, precipitated (1-(4-azidobutyl)indoline-2,3-dione), and thus were collected by simple filtration. The physical data of 1-(4-azidobutyl)indoline-2,3-dione are given below:

IBA

Yield 78%; mp 50–55 °C. 1H n class="Chemical">NMR (CDCl3, 500 MHz, δ, TMS = 0): 1.67–1.70 (m, 2H, −CH2−), 1.79–1.84 (m, 2H, −CH2−), 3.38 (t, 2H, J = 6.5 Hz, −CH2−), 3.77 (t, 2H, J = 7.5 Hz, −CH2−), 6.92 (d, 1H, J = 7.5 Hz, 1H, ArH), 7.12–7.15 (m, 1H, ArH), 7.60–7.63 (m, 2H, ArH). 13C NMR (CDCl3, 125 MHz, δ, TMS = 0): 24.54, 26.21, 39.58, 50.81, 110.02, 117.64, 123.79, 125.55, 138.37, 150.69, 158.21, 183.27.

All other 1-azidoalkylindoline-2,3-dione analogues were synthesized by n class="Chemical">following the abovementioned procedure.

General Procedure for the Synthesis of Triazole-Linked Indoline-2,3-dione–Coumarin Hybrids

In DMF, n class="Chemical">1-azidoalkylindoline-2,3-dione(1 equiv) and 4-(prop-2-ynyloxy)-2H-chromen-2-one (PHC) (1 equiv) were dissolved. The catalytic amount of pentahydrate copper sulfate (CuSO4·5H2O) and its reducing agent, sodium ascorbate was added in it. The reaction mixture was kept aside for some time, at room temperature. After the completion of reaction as confirmed by TLC, the reaction mixture was filtered directly on crushed ice to remove the excess of CuSO4 and sodium ascorbate. The solidified final product thus obtained was filtered and air dried. The physical data of all synthesized bifunctional hybrids are given below:

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)indoline-2,3-dione (K-1)

Yield 78%, mp 104–108 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 4.21 (s, 2H, −CH2−), 4.77 (s, 2H, −CH2−), 5.38 (s, 2H, −CH2−), 6.11 (s, 1H, −CH−), 6.91 (d, 1H, J = 7.8 Hz, ArH), 7.08 (t, 1H, ArH), 7.41–7.46 (m, 2H, ArH), 7.54–7.56 (m, 2H, ArH), 7.68–7.71 (m, 2H, ArH), 8.49 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.64, 62.93, 91.50, 110.52, 115.43, 116.84, 117.64, 123.29, 123.79, 124.64, 125.00, 133.33, 138.59, 150.54, 153.11, 158.55, 162.14, 164.73, 183.32. Anal. Calcd for C22H16N4O5: C, 63.46; H, 3.87; N, 13.46. Found: C, 63.26; H, 3.98; N, 13.15.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-fluoroindoline-2,3-dione (K-2)

Yield 79%, mp 74–78 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 4.22 (s, 2H, −CH2−), 4.76 (s, 2H, −CH2−), 5.39 (s, 2H, −CH2−), 6.14 (s, 1H, −CH−), 6.94 (s, 1H, ArH), 7.40–7.47 (m, 4H, ArH), 7.69–7.71 (m, 2H, ArH), 8.51 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.61, 63.06, 79.06, 79.32, 79.58, 91.60, 111.80, 112.00, 112.07, 115.45, 116.87, 118.58, 118.64, 123.22, 124.60, 133.28, 146.86, 153.16, 158.59, 162.03, 164.72, 182.73. Anal. Calcd for C22H15FN4O5: C, 60.83; H, 3.48; F, 4.37; N, 12.90. Found: C, 60.52; H, 3.50; F, 4.21; N, 12.96.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-chloroindoline-2,3-dione (K-3)

Yield 79%, mp 160–164 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 4.16 (s, 2H, −CH2−), 4.70 (s, 2H, −CH2−), 5.34 (s, 2H, −CH2−), 6.09 (s, 1H, −CH−), 6.88 (d, 1H, J = 8.5 Hz, ArH), 7.35–7.40 (m, 2H, ArH), 7.56–7.57 (m, 2H, ArH), 7.64–7.67 (m, 2H, ArH), 8.45 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.59, 63.15, 91.68, 112.35, 115.49, 116.90, 119.13, 123.23, 124.48, 124.66, 126.67, 128.02, 133.26, 137.35, 149.19, 153.20, 158.34, 161.99, 164.74, 182.25. Anal. Calcd for C22H15ClN4O5: C, 58.61; H, 3.35; Cl, 7.86; N, 12.43. Found: C, 58.33; H, 3.54; Cl, 7.77; N, 12.55.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-bromoindoline-2,3-dione (K-4)

Yield 83%, mp 74–78 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 4.15 (s, 2H, −CH2−), 4.70 (s, 2H, −CH2−), 5.34 (s, 2H, −CH2−), 6.10 (s, 1H, −CH−), 6.84 (d, 1H, J = 8 Hz, ArH), 7.37–7.41 (m, 2H, ArH), 7.65–7.71 (m, 4H, ArH), 8.44 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.55, 63.15, 91.69, 112.80, 115.52, 116.92, 119.52, 123.26, 124.71, 126.56, 127.24, 133.29, 140.17, 149.57, 153.21, 155.86, 162.02, 164.77, 182.12. Anal. Calcd for C22H15BrN4O5: C, 53.35; H, 3.05; Br, 16.13; N, 11.31. Found: C, 53.44; H, 3.01; Br, 16.33; N, 11.12.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-iodoindoline-2,3-dione (K-5)

Yield 81%, mp 88–91 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 4.17 (s, 2H, −CH2−), 4.68 (s, 2H, −CH2−), 5.37 (s, 2H, −CH2−), 6.08 (s, 1H, −CH−), 6.82 (d, 1H, J = 8 Hz, ArH), 7.35–7.39 (m, 2H, ArH), 7.63–7.65 (m, 2H, ArH), 7.84–7.87 (m, 2H, ArH), 8.61 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.59, 63.23, 91.74, 112.86, 115.57, 116.91, 119.59, 123.24, 124.70, 126.53, 127.30, 133.34, 140.19, 149.52, 153.27, 155.90, 161.97, 164.73, 182.53. Anal. Calcd for C22H15IN4O5: C, 48.73; H, 2.79; I, 23.40; N, 10.33. Found: C, 48.93; H, 2.68; I, 23.44; N, 10.22.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-nitroindoline-2,3-dione (K-6)

Yield 83%, mp 104–108 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 4.14 (s, 2H, −CH2−), 4.65 (s, 2H, −CH2−), 5.37 (s, 2H, −CH2−), 6.11 (s, 1H, −CH−), 6.83 (s, 1H, ArH), 7.41–7.44 (m, 2H, ArH), 7.61–7.64 (m, 2H, ArH), 7.96–7.99 (m, 2H, ArH), 8.76 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.71, 63.32, 91.65, 112.82, 115.55, 116.96, 119.62, 123.43, 124.76, 126.52, 127.13, 135.33, 146.56, 152.43, 153.86, 155.95, 162.45, 164.78, 182.72. Anal. Calcd for C22H15N5O7: C, 57.27; H, 3.28; N, 15.18. Found: C, 57.42; H, 3.13; N, 15.45.

1-(2-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-methoxyindoline-2,3-dione (K-7)

Yield 75%, mp 70–74 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 3.68 (s, 3H, −OCH3), 4.12 (s, 2H, −CH2−), 4.69 (s, 2H, −CH2−), 5.32 (s, 2H, −CH2−), 6.06 (s, 1H, −CH−), 6.80 (d, 1H, J = 8.0 Hz, ArH), 7.07–7.09 (m, 2H, ArH), 7.33–7.39 (m, 2H, ArH), 7.63–7.66 (m, 2H, ArH), 8.42 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.61, 56.24, 63.06, 91.57, 109.50, 111.73, 112.08, 115.45, 116.87, 118.19, 123.26, 124.44, 124.64, 126.59, 133.30, 144.45, 153.67, 156.15, 158.61, 162.08, 164.77, 183.56. Anal. Calcd for C23H18N4O6: C, 61.88; H, 4.06; N, 12.55. Found: C, 60.99; H, 4.23; N, 12.32.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)indoline-2,3-dione (L-1)

Yield 76%, mp 122–125 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 2.23 (s, 2H, −CH2−), 3.75 (s, 2H, −CH2−), 4.52 (s, 2H, −CH2−), 5.41 (s, 2H, −CH2−), 6.15 (s, 1H, −CH−), 7.11–7.18 (m, 2H, ArH), 7.32–7.41 (m, 2H, ArH), 7.54–7.75 (m, 4H, ArH), 8.37 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.00, 37.36, 47.71, 63.29, 91.78, 110.97, 115.54, 116.94, 118.19, 123.25, 123.61, 124.70, 124.89, 125.81, 133.31, 138.45, 150.84, 153.23, 158.82, 162.03, 164.82, 183.74. Anal. Calcd for C23H18N4O5: C, 64.18; H, 4.22; N, 13.02. Found: C, 64.33; H, 4.11; N, 13.41.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-fluoroindoline-2,3-dione (L-2)

Yield 79%, mp 94–97 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 2.18 (s, 2H, −CH2−), 4.19 (s, 2H, −CH2−), 4.77 (s, 2H, −CH2−), 5.37 (s, 2H, −CH2−), 6.15 (s, 1H, −CH−), 6.93 (s, 1H, ArH), 7.43–7.48 (m, 4H, ArH), 7.70–7.74 (m, 2H, ArH), 8.50 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.87, 47.56, 63.21, 79.32, 79.31, 79.54, 91.66, 111.83, 112.12, 112.21, 115.43, 116.82, 118.52, 118.59, 123.32, 124.64, 133.19, 146.82, 153.14, 158.73, 162.23, 164.69, 183.74. Anal. Calcd for C23H17FN4O5: C, 61.61; H, 3.82; F, 4.24; N, 12.49. Found: C, 61.45; H, 3.95; F, 4.06; N, 12.53.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-chloroindoline-2,3-dione (L-3)

Yield 71%, mp 166–169 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 2.20 (s, 2H, −CH2−), 3.74 (s, 2H, −CH2−), 4.51 (s, 2H, −CH2−), 5.41 (s, 2H, −CH2−), 6.15 (s, 1H, −CH−), 7.03–7.04 (m, 1H, ArH), 7.34–7.41 (m, 2H), 7.66–7.96 (m, 4H, ArH), 8.35 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 27.87, 37.40, 47.65, 63.30, 86.47, 91.78, 113.48, 115.54, 116.95, 123.35, 124.70, 125.76, 132.53, 133.32, 145.86, 150.17, 153.23, 158.17, 162.03, 164.83, 182.41. Anal. Calcd for C23H17ClN4O5: C, 59.43; H, 3.69; Cl, 7.63; N, 12.05. Found: C, 59.60; H, 3.55; Cl, 7.75; N, 12.01.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-bromoindoline-2,3-dione (L-4)

Yield 80%, mp 74–78 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 2.16 (s, 2H, −CH2−), 4.17 (s, 2H, −CH2−), 4.72 (s, 2H, −CH2−), 5.35 (s, 2H, −CH2−), 6.08 (s, 1H, −CH−), 6.86 (d, 1H, J = 8 Hz, ArH), 7.38–7.42 (m, 2H, ArH), 7.64–7.70 (m, 4H, ArH), 8.46 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.66, 47.59, 63.12, 91.71, 112.89, 115.56, 116.97, 119.57, 123.29, 124.76, 126.58, 127.28, 133.32, 140.19, 149.59, 153.28, 155.89, 162.06, 164.79, 182.56. Anal. Calcd for C23H17BrN4O5: C, 54.24; H, 3.36; Br, 15.69; N, 11.00. Found: C, 54.28; H, 3.15; Br, 15.78; N, 11.03.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-iodoindoline-2,3-dione (L-5)

Yield 83%, mp 98–102 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 2.13 (s, 2H, −CH2−), 4.14 (s, 2H, −CH2−), 4.70 (s, 2H, −CH2−), 5.35 (s, 2H, −CH2−), 6.11 (s, 1H, −CH−), 6.81 (s, 1H, ArH), 7.36–7.39 (m, 2H, ArH), 7.65–7.68 (m, 2H, ArH), 7.80–7.84 (m, 2H, ArH), 8.59 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.69, 47.65, 63.28, 91.72, 112.81, 115.54, 116.98, 119.63, 123.23, 124.72, 126.57, 127.33, 133.37, 140.14, 149.56, 153.24, 155.94, 161.94, 164.76, 182.63. Anal. Calcd for C23H17IN4O5: C, 49.66; H, 3.08; I, 22.81; N, 10.07. Found: C, 49.55; H, 2.88; I, 22.84; N, 10.01.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-nitroindoline-2,3-dione (L-6)

Yield 79%, mp 106–109 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 2.14 (s, 2H, −CH2−), 4.15 (s, 2H, −CH2−), 4.65 (s, 2H, −CH2−), 5.34 (s, 2H, −CH2−), 6.10 (s, 1H, −CH−), 6.83 (s, 1H, ArH), 7.42–7.45 (m, 2H, ArH), 7.61–7.67 (m, 3H, ArH), 7.97–7.99 (m, 1H, ArH), 8.76 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 28.56, 47.73, 63.31, 91.64, 112.83, 115.56, 116.92, 119.65, 123.43, 124.77, 126.52, 127.16, 135.34, 146.53, 152.46, 153.82, 155.94, 162.55, 164.74, 182.72. Anal. Calcd for C23H17N5O7: C, 58.11; H, 3.60; N, 14.73. Found: C, 58.19; H, 3.51; N, 14.99.

1-(3-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)-5-methoxyindoline-2,3-dione (L-7)

Yield 76%, mp 87–90 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 2.19–2.24 (m, 2H, −CH2−), 3.71–3.76 (m, 3H, −OCH3), 4.50–4.53 (m, 2H, −CH2−), 5.41 (s, 2H, −CH2−), 6.15 (s, 1H, −CH−), 7.10–7.14 (m, 2H, ArH), 7.21–7.23 (m, 1H, ArH), 7.32–7.41 (m, 2H, ArH), 7.64–07.75 (m, 2H, ArH), 8.37 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 27.99, 36.26, 37.34, 47.71, 56.36, 63.30, 91.77, 109.76, 112.04, 115.54, 116.93, 118.68, 123.35, 124.09, 124.69, 133.31, 144.59, 153.22, 156.16, 158.82, 162.03, 164.83, 183.98. Anal. Calcd for C24H20N4O6: C, 62.60; H, 4.38; N, 12.17. Found: C, 62.66; H, 4.13; N, 12.34.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)indoline-2,3-dione(M-1)

Yield 74%, mp 146–150 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 1.58–1.61 (m, 2H, −CH2−), 1.92–1.95 (m, 2H, −CH2−), 3.69–3.72 (m, 2H, −CH2−), 4.44–4.47 (m, 2H, −CH2−), 5.40 (s, 2H, −CH2−), 6.14 (s, 1H, −CH−), 7.11–7.18 (m, 2H, ArH), 7.34–7.41 (m, 2H, ArH), 7.53 (d, 1H, J = 7 Hz, ArH), 7.63–7.66 (m, 2H, ArH), 7.73 (d, 1H, J = 7.5 Hz, ArH), 8.38 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.25, 27.42, 49.53, 63.33, 91.77, 111.06, 115.54, 116.91, 118.01, 123.33, 123.58, 124.66, 124.90, 133.26, 138.51, 151.03, 153.22, 158.67, 161.98, 164.81, 183.86. Anal. Calcd for C24H20N4O5: C, 64.86; H, 4.54; N, 12.61. Found: C, 64.97; H, 4.32; N, 12.85.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-fluoroindoline-2,3-dione (M-2)

Yield 77%, mp 110–1114 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 1.59 (s, 2H, −CH2−), 1.87 (s, 2H, −CH2−), 3.70 (s, 2H, −CH2−), 4.44 (s, 2H, −CH2−), 5.34 (s, 2H, −CH2−), 6.13 (s, 1H, −CH2−), 6.91 (s, 1H, −CH−), 7.44–7.49 (m, 4H, ArH), 7.72–7.74 (m, 2H, ArH), 8.52 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.44, 27.34, 47.52, 63.25, 79.32, 79.38, 79.53, 91.65, 111.83, 112.11, 112.29, 115.42, 116.81, 118.55, 118.68, 123.32, 124.61, 133.23, 146.81, 153.15, 158.77, 162.19, 164.71, 183.72. Anal. Calcd for C24H19FN4O5: C, 62.34; H, 4.14; F, 4.11; N, 12.12. Found: C, 62.62; H, 4.25; F, 3.99; N, 12.19.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-chloroindoline-2,3-dione (M-3)

Yield 70%, mp 170–172 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 1.56 (s, 2H, −CH2−), 1.92 (s, 2H, −CH2−), 3.68 (s, 2H, −CH2−), 4.44 (s, 2H, −CH2−), 5.39 (s, 2H, −CH2−), 6.13 (s, 1H, −CH−), 7.20–7.40 (m, 3H, ArH), 7.56–7.73 (m, 4H, ArH), 8.39 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.14, 27.30, 49.56, 63.37, 91.75, 112.75, 115.52, 116.89, 119.43, 123.33, 124.37, 124.67, 127.79, 133.27, 137.29, 149.53, 153.20, 158.46, 162.01, 164.82, 182.75. Anal. Calcd for C24H19ClN4O5: C, 60.19; H, 4.00; Cl, 7.40; N, 11.70. Found: C, 60.30; H, 3.88; Cl, 7.47; N, 11.64.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-bromoindoline-2,3-dione (M-4)

Yield 76%, mp 75–80 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 1.57 (s, 2H, −CH2−), 1.92 (s, 2H, −CH2−), 3.69 (s, 2H, −CH2−), 4.44 (s, 2H, −CH2−), 5.40 (s, 2H, −CH2−), 6.14 (s, 1H, −CH−), 7.16 (s, 1H, ArH), 7.34–7.41 (m, 2H, ArH), 7.66–7.80 (m, 4H, ArH), 8.38 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.13, 27.31, 49.55, 63.36, 88.90, 91.77, 113.20, 115.27, 115.53, 116.91, 119.83, 123.34, 124.46, 127.10, 133.27, 140.09, 147.92, 153.21, 158.30, 161.99, 164.81, 182.61. Anal. Calcd for C24H19BrN4O5: C, 55.08; H, 3.66; Br, 15.27; N, 10.71. Found: C, 55.25; H, 3.44; Br, 15.45; N, 10.55.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-iodoindoline-2,3-dione (M-5)

Yield 83%, mp 97–102 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 1.58 (s, 2H, −CH2−), 1.91 (s, 2H, −CH2−), 3.71 (s, 2H, −CH2−), 4.44 (s, 2H, −CH2−), 5.38 (s, 2H, −CH2−), 6.08 (s, 1H, −CH−), 6.84 (s, 1H, ArH), 7.34–7.37 (m, 2H, ArH), 7.67–7.69 (m, 2H, ArH), 7.76–7.79 (m, 2H, ArH), 8.61 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.54, 27.43, 48.99, 63.56, 91.76, 112.82, 115.58, 117.12, 119.67, 123.25, 124.77, 126.53, 127.33, 133.32, 140.11, 149.45, 153.00, 155.95, 161.94, 164.71, 182.73. Anal. Calcd for C24H19IN4O5: C, 50.54; H, 3.36; I, 22.25; N, 9.82. Found: C, 50.51; H, 3.45; I, 22.12; N, 10.00.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-nitroindoline-2,3-dione (M-6)

Yield 80%, mp 110–112 °C. 1H n class="Chemical">NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 1.57 (s, 2H, −CH2−), 1.92 (s, 2H, −CH2−), 3.66 (s, 2H, −CH2−), 4.43 (s, 2H, −CH2−), 5.37 (s, 2H, −CH2−), 6.10 (s, 1H, −CH−), 6.84 (s, 1H, ArH), 7.44–7.47 (m, 2H, ArH), 7.62–7.67 (m, 2H, ArH), 7.94–7.96 (m, 2H, ArH), 8.72 (s, 1H, ArH). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 24.34, 27.65, 48.56, 63.65, 91.34, 112.87, 115.52, 116.97, 119.63, 123.46, 124.73, 126.55, 127.12, 135.35, 146.56, 152.43, 153.84, 155.96, 162.53, 164.77, 182.69. Anal. Calcd for C24H19N5O7: C, 58.90; H, 3.91; N, 14.31. Found: C, 58.99; H, 3.76; N, 14.42.

1-(4-(4-((2-Oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5-methoxyindoline-2,3-dione (M-7)

Yield 73%, mp 97–100 °C. 1H n class="Chemical">NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 1.57 (s, 2H, −CH2−), 1.92 (s, 2H, −CH2−), 3.67 (s, 2H, −CH2−), 3.75 (s, 3H, −OCH3), 4.44 (s, 2H, −CH2−), 5.39 (s, 2H, −CH2−), 6.13 (s, 1H, −CH−), 7.09–7.11 (m, 2H, ArH), 7.20–7.22 (m, 1H, ArH), 7.33–7.35 (m, 1H, ArH), 7.39–7.40 (m, 1H, ArH), 7.65–7.67 (m, 1H, ArH), 7.72–7.73 (m, 1H, ArH), 8.37 (s, 1H, ArH). 13C NMR (d-DMSO, 125 MHz, δ, TMS = 0): 24.23, 27.39, 49.53, 56.35, 63.32, 91.74, 109.78, 112.13, 115.52, 116.90, 118.18, 123.33, 124.23, 124.68, 125.58, 133.28, 144.78, 153.20, 156.14, 158.67, 162.02, 164.83, 184.12. Anal. Calcd for C25H22N4O6: C, 63.29; H, 4.67; N, 11.81. Found: C, 63.45; H, 4.47; N, 11.92.

In Vitro Antimicrobial Activity

Agar plate din class="Chemical">ffusion assay was used to evaluate the antimicrobial activity. Twenty different compounds were screened for antimicrobial activity against test organisms, namely, E. coli, S. enterica, S. aureus, M. smegmatis, C. albicans, A. mali, Penicillium sp., and F. oxysporum. A quantity of 50 μL of each synthesized compound was added in the wells of nutrient agar plates (for bacteria) and potato dextrose agar plates (for fungi), seeded with 100 μL of activated test cultures (E. coli, S. enterica, S. aureus, M. smegmatis, C. albicans, A. mali, Penicillium sp., and F. oxysporum). The plates were incubated at 37 °C (bacteria) and 30 °C (fungi) under aerobic conditions for 24–48 h, and the clear zones of inhibition around the compounds were measured and compared with the negative control.[42]

MIC Determination

To determine MIC, different concentrations of the active compound (K-2: 5, 2.5, 1.25, 0.625, 0.312, and 0.156 mg/mL) were prepared in DMSO, and 50 μL of each concentration was added in the well of the nutrient agar plate, seeded with S. aureus. The plates were incubated at 37 °C for 24 h, and the clear zones of inhibition around the different concentrations of the compound were measured and compared with the negative control.

Similarly, for determining MIC on class="Chemical">f K-1, different concentrations (30, 15, 7.5, 3.75, and 1.875 μg/mL) were prepared in DMSO, and 50 μL of each concentration was added in the well of the potato dextrose agar plate, seeded with Penicillium sp. The plates were incubated at 30 °C for 24–48 h, and the clear zones of inhibition around the different concentrations of the compound were measured and compared with the negative control.

Docking Study

The X-ray-derived 3D co-ordinates of n class="Species">S. aureus DHFR bound with 7-aryl-2,4-diaminoquinazoline were retrieved from the protein data bank (PDB entry: 3SRQ; resolution 1.69 Å).[41] GOLD software version 5.5 was used to perform the docking study.[42] Gold executes genetic algorithm-based ligand docking to optimize the conformation of the ligand at the receptor binding site. GoldScore comprises four components: protein–ligand hydrogen bond energy, protein–ligand van der Waals (vdW) energy, ligand internal vdW energy, and ligand torsional strain energy. The structure of K-2 was drawn in ChemDraw Ultra (2006) and subjected to energy minimization using the MM2 force field as implemented in Chem 3D Ultra software.[43,44] The compounds were docked ten times, and each pose was ranked according to its GoldScore fitness function. The conformation with the highest score was selected for discussion.