N-Heterocyclic Carbene-Catalyzed Facile Synthesis of Phthalidyl Sulfonohydrazones: Density Functional Theory Mechanistic Insights and Docking Interactions

Abstract

N-heterocyclic carbene catalysis reaction protocol is disclosed for the synthesis of phthalidyl sulfonohydrazones. A broad range of N-tosyl hydrazones react effectively with phthalaldehyde derivatives under open-air conditions, enabling the formation of a new C–N bond via an oxidative path. The reaction proceeds under mild reaction conditions with broad substrate scopes, wide functional group tolerance, and good to excellent yields. The mechanistic pathway is studied successfully using control experiments, competitive reactions, ESI-MS spectral analyses of the reaction mixture, and computational study by density functional theory. The potential use of one of the phthalidyl sulfonohydrazone derivatives as the inhibitor of β-ketoacyl acyl carrier protein synthase I of Escherichia coli is investigated using molecular docking.

Introduction

Chemical modification on medicinally important natural products or drug molecules is an established strategy to develop chemical entities and prodrugs with enhanced performance or introducing different clinical applications. Sulfonamide-based organic molecules find profound applications in the field of medicine and pharmaceutical areas.¹ Hydrazones are a significant class of organic molecules for drug design and synthesis. Of late, these functional compounds have been developed with numerous biological activities. Some hydrazone derivatives have proven to possess antimicrobial,² antimalarial,³ antiviral,⁴ vasodilator,^5a anti-inflammatory,^5b anticancer,^5c and antituberculosis⁶ activities (A–C, Figure 1). Tosyl hydrazone compounds are found as antibacterial, antifungal, and anticancer agents (D–G, Figure 1).⁷ Moreover, tosyl hydrazones are treated as versatile and useful partners in organic synthesis.⁸ In particular, under basic conditions, the tosyl hydrazone moieties are easily converted into diazo compounds,⁹ which can undergo insertion reactions, leading to the construction of various chemical bonds like C–C, C–N, C–Si, etc. In addition, hemiaminal esters constitute crucial building blocks for several biological systems. In general, these compounds are obtained via a dynamic kinetic resolution process using various substrates.¹⁰ Among the various types of sulfonamides, phthalidyl sulfonamide promoters were found with impressive success. Phthalidyl sulfonamides derivatives are an important class of heterocycles having potent biological and photophysical properties. For instance, hydrochlorothiazide is a diuretic medication for the treatments of high blood pressure, congestive heart failure, diabetes insipidus, and renal tubular acidosis.¹¹ Saccharin is an artificial sweetener and food additive. Sulfamethoxazoles are known as antibiotics used for bacterial infections such as urinary tract infections, bronchitis, and prostatitis.¹²

Figure 1.

Few drugs and bioactive molecules (A–G) containing hydrazone and tosyl hydrazone units.

Sustainable N-heterocyclic carbene (NHC) organocatalysis is established as a powerful tool in modern organic synthesis to access functional molecules.¹³ Chi and co-workers reported an enantioselective method for the synthesis of chiral phthalidyl ester via NHC-catalyzed acetalization of carboxylic acids using a stoichiometric amount of oxidant (eq i, Scheme 1).^14a Very recently, the same group has disclosed the reaction of N-aryl sulfonamides with phthalaldehydes producing optically enriched phthalidyl sulfonamides under NHC organocatalysis and subsequent oxidation (eq ii, Scheme 1). However, the reaction did not proceed without the use of 3,3′,5,5′-tetra-tert-butyldiphenoquinone as an externally supplied oxidant (1 equiv).^14b Aerial oxidation in NHC catalysis is well documented in the literature to access aromatic esters or carboxylic acids from aromatic aldehydes with alcohols or nonactivated aldehydes, respectively.¹⁵ Thus, we examined the NHC-catalyzed C–O/C–N coupled reaction using molecular oxygen from air (eq iii, Scheme 1).

Scheme 1. Synthesis of Phthalidyl Esters (eq i), Sulfonamides (eq ii), and Our Approach (eq iii) to Phthalidyl Sulfonohydrazones.

Inspired by the very interesting results, we assume that the sulfonohydrazide moiety-containing phthalidyl scaffold could be an appropriate modification of existing phthalidyl sulfonamide derivatives, which were found having diverse bioactivity. To the best of our knowledge, until date, there is no report regarding the bioactivity assay of phthalidyl sulfonohydrazone derivatives, as their synthesis is unknown in the literature. Our aim was to synthesize organic molecules, which in turn looked like as sulfonamides, hemiaminal ester, and phthalidyl sulfonohydrazone. In this context, we disclosed NHC access to bioactive phthalidyl sulfonohydrazone from phthalaldehyde and N-tosyl hydrazones in good to excellent yield under aerobic oxidation (eq iii, Scheme 1). The mechanistic pathways are explored in detail using the density functional theory (DFT) computational study. Further, molecular docking is performed to see the potential bioactivity of one of the reported compounds.

Results and Discussion

At the outset of our studies, we have investigated the reaction employing N-tosyl hydrazone derivative (2b) and phthalaldehyde (1a) as the model substrates (Table 1) with the variation of catalytic amounts of various NHCs in open air. The oxidative NHC-catalyzed reaction was initially attempted using imidazolium salt 3a or thiazolium salt 3b along with various bases and solvents (entries 1–9, Table 1). These reactions were either unsuccessful or only a trace of the desired product (4b) was obtained in these cases. Gratifyingly, the desired product was obtained in 55% yield on the treatment of the substrate with thiazolium salt 3c and base DBU in dichloromethane solvent for 12 h at ambient temperature (entry 10). To our delight, the yield (86%) and reaction rate (the reaction time decreased to 2 h) were drastically improved on the use of Cs₂CO₃ as a base in acetonitrile (entry 11). The yield of the 4b was not improved at all on changing the reaction medium and base (entries 12–15). On the enhancement of catalyst loading (12 mol %) and reaction time (6 h), the yield was not significantly improved (88%, entry 16). A decrease of catalyst loading (8 and 6%, respectively) led to the lowering of the reaction rate and yield (entries 17 and 18). The reaction did not proceed in the absence of NHC (entry 19). Thus, we found the optimized conditions for this reaction using Cs₂CO₃ as the base in acetonitrile solvent with NHC 3c as the efficient catalyst in air to produce the desired phthalidyl sulfonohydrazone derivative in 2 h with 86% yield (entry 11). Also, we have performed the reaction under inert atmosphere conditions using “kahrasch oxidant” (3,3′,5,5′-tetra-tert-butyldiphenoquinone, DPQ) and observed a moderate yield.^14b

Table 1. Screening for Optimized Reaction Conditions.

entrya	catalyst	base	solventb	time (h)	yield (%)c
1	3a	DBU	DCM	12	ndd
2	3a	DBU	DMF	12	nd
3	3a	Cs2CO3	DCM	12	nd
4	3a	DBU	THF	12	nd
5	3a	NaOAc	THF	12	nd
6	3b	Cs2CO3	THF:EtOH(1:1)	12	10
7	3b	Cs2CO3	DCM	12	<5
8	3b	DBU	THF	12	trace
9	3b	NaOAc	THF:EtOH(1:1)	12	trace
10	3c	DBU	DCM	12	55
11	3c	Cs2CO3	CH3CN	2	86
12	3c	Cs2CO3	DMF	12	42
13	3c	Cs2CO3	THF	12	65
14	3c	Cs2CO3	THF:EtOH(1:1)	12	15
15	3c	NaOAc	EtOH	12	10
16d	3c	Cs2CO3	CH3CN	6	86
17e	3c	Cs2CO3	CH3CN	8	70
18f	3c	Cs2CO3	CH3CN	12	40
19g		Cs2CO3	CH3CN	12	nd
20h	3c	Cs2CO3	CH3CN	8	84

^aReaction conditions: phthalaldehyde (1, 1.0 mmol), N-tosyl hydrazone (2, 1.0 mmol), solvent (5 mL), NHC precursor 3a–c (10 mol %), and base (20 mol %) were stirred at ambient temperature.

^bMolecular sieves (4 Å) and air used.

^cYield of the product obtained after purification by silica gel column chromatography.

^d3c: 12 mol %.

^e3c: 8 mol %.

^f3c: 6 mol %.

^gWithout NHC, nd: 4 not detected.

^hGram-scale synthesis.

General applicability of the developed reaction conditions (entry 11, Table 1) using various substituted N-tosyl hydrazones (2) and phthalaldehyde (1) to obtain functionalized phthalidyl sulfonohydrazide moieties (4a–s) was framed in Scheme 2. N-tosyl hydrazones were derived from aryl aldehydes in this case. Electron-donating groups in the aromatic ring of N-tosyl hydrazones performed well under these optimized reaction conditions to yield the product (4b–h) in 75–86%. Moderate to good yields (53–73%) were observed for N-tosyl hydrazones having an electron-withdrawing halogen or nitro group (4i–m). The reaction also went well when sterically hindered aldehyde precursors from naphthalene, biphenyl, and pyrene were tested under the reaction conditions to furnish desired products (4n–p) in 65, 63, and 60% yields, respectively. The reaction is also in consistent with heterocyclic N-tosyl hydrazones (4q, r). N-tosyl hydrazine from cinnamaldehyde also tolerated in this reaction protocol to furnish corresponding phthalidyl sulfonohydrazide 4s in 68% yield. In general, all of these allowed installation of a great diversity of substituents in the sulfonohydrazone template.

Scheme 2. Synthesized Phthalidyl Sulfonohydrazones (4) from N-Tosyl Hydrazones.

We further studied the N-tosyl hydrazones derived from aryl ketones as substrates to react with phthalaldehydes under the optimized reaction conditions (Scheme 3). N-tosyl hydrazones were well tolerated in the reaction protocol irrespective of the major electronic effects of the substituents in the aryl part of the hydrazones and furnished the desired products 4t–v in good yields (60–70%). N-tosyl hydrazones with a heteroaryl moiety in the structure reacted well and furnished the product (4w) in 64% yield. Regioselectivity studies of unsymmetrical phthalaldehyde were investigated, which afford 4x as a single isomer with good yield (60%). A gram-scale synthesis of 4b under the optimized reaction conditions was also verified to afford the desired product in 84% yield (entry 20). The structure of the all unknown compounds (4a–x) was determined unambiguously by recording NMR, FT-IR, and HRMS spectra and single-crystal XRD data analyses of compound 4m (Supporting Information).

Scheme 3. Synthesized Phthalidyl Sulfonohydrazones (4) from Ketohydrazones.

To elucidate the reaction pathway, ESI-MS and DFT calculations were performed. We assume that a nucleophilic NHC-aldehyde adduct (Breslow intermediate) (I) is formed initially, which upon aerial oxidation generates the azolium hydroperoxy intermediate II(15d,15e) (Scheme 4). Intermediate II converts into the acyl azolium intermediate III, with liberation of the hydroperoxy anion (oxidative path). The intermediate III further undergoes nucleophilic attack by deprotonated N-tosyl hydrazone, followed by O–C coupling to give intermediate IV through intramolecular annulation.¹⁴ The intermediate IV upon fragmentation may yield a phthalidyl sulfonohydrazone as the final product along with the regeneration of the active catalyst. In the following sections, we will discuss the detailed reaction mechanisms, including the formation of the Breslow intermediate, aerial oxidation of the Breslow intermediate, nucleophilic attack of deprotonated N-tosyl hydrazone, O–C coupling, and dissociation of the NHC catalyst.

Scheme 4. Plausible Mechanistic Pathway.

DFT Study: Formation of the Breslow Intermediate

The suggested mechanism involves the nucleophilic attack of NHC to the electrophilic aldehyde group of phthaladehyde, giving rise to the Breslow intermediate (Figure 2). At first, the reaction mixture leads to the formation of free NHC as a result of proton transfer from the azolium cation to the base, CS₂CO₃ (Figures S30 and S31, Supporting Information). The free energy profile shows that the formation of NHC occurs with a minimal activation barrier of 2.07 kcal/mol. Then, NHC generates a vdW complex (C1-s) by the nucleophilic attack to aldehyde. It is observed that the zwitterionic intermediate (Int1-s) occurs through the transition state (TS1-s) with a free energy barrier of 10.38 kcal/mol from separated reactants. The generated intermediate exhibits a decrease in a free energy of −3.74 kcal/mol compared to separated reactants, which suggests an exergonic reaction process. The solvent-phase geometries corresponding to the Breslow intermediate formation are given in Figure S32 (Supporting Information). At TS1-s, the distance between NHC carbene C and carbonyl C shortens from 3.333 Å (C1-s) to 2.282 Å (TS1-s), demonstrating the gradual formation of the C–C bond. Finally, the zwitterionic intermediate (Int1-s) is generated with a C–C bond distance of 1.523 Å. The Breslow intermediate is formed by 1,2-proton transfer in the zwitterionic intermediate. In our recent study, we have shown that direct proton transfer has a very high free activation energy as a consequence of strained TS and the proton transfer process proceeds through the acid/base pair catalyzed energetically favorable pathway.¹⁶ Here, Cs₂CO₃ helps the 1,2-proton transfer path and the reaction proceeds through the stepwise reaction. In this pathway, the protonation of the O atom occurs before the C–H cleavage takes place. On the other hand, the hydrogen abstraction has an activation free energy of 12.40 kcal/mol from the separated reactants.

Figure 2.

Free energy diagram for forming the Breslow intermediate (I) from NHC with phthalaldehyde.

Aerial Oxidation of the Breslow Intermediate

The Breslow intermediate (I) reacts with molecular oxygen, leading to the triplet intermediate, Int3-t (Figure 3). At a distance of 2.790 Å, O₂ engages in a moderate O–H···O hydrogen bonding with the Breslow intermediate (Figure S33, Supporting Information). Then, the triplet O₂ takes the proton from the OH group of the Breslow intermediate in Int4-t to generate the HOO^– ion. A strong H-bonding is formed when the HOO^– ion interacts with carbonyl group (Figure S33, Supporting Information). The hydroperoxide intermediates (II-s and II-t) are formed when the HOO– reacts at the electrophilic carbon center through a barrierless process. Since the II-s is energetically more stable than the corresponding triplet state (II-t), an intersystem crossing process containing a transition from triplet to singlet occurs. During the reaction, there is an elimination of a HOO^– ion from II and III being formed. However, the dissociation of zwitterionic prerequisites a significant electrostatic effort. Moreover, the HOO^– ion is a very strong base and nucleophile. Thus, there is a requirement of receiver species that can capture this leaving anion to assist the process. The second aldehyde or solvent molecule could act as a receiver.^15d Finally, the oxidative product (III) is formed and is exergonic by 2.30 kcal/mol.

Figure 3.

Free energy diagram of the aerial oxidation pathway to generate the acyl azolium intermediate (III). Orange color, triplet pathway; violet color, singlet pathway.

Intramolecular Annulation

As proposed, the nucleophilic addition of N-tosyl hydrazone to the oxidized Breslow intermediate (III) starts with a vdW complex, Int 5-s (Figure 4). Before the nucleophilic addition of N-tosyl hydrazine, it should be deprotonated at the N center. In this study, we have considered Cs₂CO₃ to remove the hydrogen in N-tosyl hydrazone. The production of cesium salt (Int 5-s) is energetically favored. Once it is deprotonated, the nucleophilic attack of N-tosyl hydrazine occurs at the carbonyl group in III. Two stereoisomeric channels associated with the nucleophilic attack of the re or siface of the carbonyl carbon are feasible. The lengths of the C–N bonds are 1.866 Å (Re-TS3) and 1.981 Å (Si-TS3) (Figure S34, Supporting Information). Thus, the transition state TS3 is the stereoselectivity determining step responsible for the R or S configuration of III. Thus, the reaction proceeds through O–C coupling to generate intermediate IV. Finally, there is extrusion of the NHC catalyst from intermediate IV and it produces the final product (4). The final step in the reaction follows a pathway through transition state TS4, where the activation free energy barrier is 10.86 kcal/mol higher than that of the intermediate IV. A free energy difference of 3.83 kcal/mol between (S)-TS4 and (R)-TS4 can be attributed to the fact that (R)-TS4 is sterically less crowded compared to (S)-TS4. At (R)-TS4, the distance between NHC carbene C and ester C is measured to be 2.084 Å, whereas at (S)-TS4, this distance increases to 2.129 Å (Figure S34, Supporting Information). From the separated reactants, the formation of the final product is strongly exergonic by −23.63 kcal/mol.

Figure 4.

Free energy diagram for forming the final product hemiaminal ester along with the regeneration of the active catalyst. Red color, proceeds through the Re face; violet color, proceeds through the Si face.

In this section, we will investigate the potential of one of the phthalidyl sulfonohydrazone derivatives (4f) as the inhibitor of β-ketoacyl acyl carrier protein synthase I (KAS I) of Escherichia coli. Escherichia coli, commonly known as E. coli, is a prevalent bacterium responsible for various bacterial infections in humans (Figure 5). These infections encompass a wide range of conditions, including cholecystitis, bacteremia, cholangitis, urinary tract infections, and traveler’s diarrhea, as well as clinical infections like neonatal meningitis and pneumonia. Certainly, three types of β-ketoacyl acyl carrier protein synthase (KAS) enzymes play a crucial role in addressing bacterial resistance issues. Thus, targeting these enzymes can be an effective strategy in tackling antibiotic resistance. Disruption of KAS I can impede the synthesis of essential fatty acids, crucial for bacterial membrane formation and cell growth. Thiolactomycin (TLM), a unique thiolactone molecule comprising natural products, inhibits bacterial cell growth by impeding the β-ketoacyl-ACP synthase activity. Thus, it becomes possible to compare the binding activity of TLM with the final product 4f (R/S). The docking studies are performed with the protein (1FJ4)¹⁷ with a resolution factor of 2.35 Å. The active site of KAS I procedures a catalytic triad hole consisting of His–His–Cys.^18,19 The docking study shows that TLM binds at the active site and forms a hydrogen bond with His333, contributing to the stabilization of the protein–inhibitor complex. The noncovalent interactions of TLM with the amino acids at the active site are elucidated in Figure S35 (Supporting Information). The calculated binding energy of reference TLM is −6.15 kcal/mol, whereas (R)-4 shows a favorable binding energy of −6.87 kcal/mol. (S)-4 has a slightly weak binding interaction (−5.02 kcal/mol) at the active site compared to reference TLM. Thr-300 exhibits strong hydrogen bonding interactions with both compounds (Figure 5 and Figure S36, Supporting Information). Based on the phthalidyl sulfonohydrazone–receptor interactions, it is suggested that these leads have the potential to be developed into effective antimicrobial drugs targeting Gram-negative E. coli.

Figure 5.

Glide molecular docking interactions of the receptor (PDB ID: 1FJ4) with (R)-4f. (a) Protein–ligand schematic interaction diagram of the protein and (R)-4f complex. (b) Binding pose of (R)-4f in the active site of the receptor.

Conclusions

In conclusion, this study offers an excellent methodology for the synthesis of new phthalidyl sulfonohydrazone compounds using NHC-catalyzed reaction conditions under open air. The reaction mechanism proceeds through the formation of the Breslow intermediate, followed by aerial oxidation. Ultimately, phthalidyl sulfonohydrazones are formed via intramolecular annulation. The full mechanism is supported by both experimental and computational methods. Finally, the phthalidyl sulfonohydrazide-β-ketoacyl acyl carrier protein synthase I interactions are studied successfully using molecular docking, which suggests the potential of phthalidyl sulfonohydrazones to be effective as an antimicrobial drug targeting Gram-negative E. coli.

Experimental Section

All reagents were purchased from commercial suppliers and used without further purification, unless otherwise specified. Commercially supplied ethyl acetate and petroleum ether were distilled before use. All solvents were dried through usual methods. The petroleum ether used in our experiments has a boiling range of 60–80 °C. Analytical thin-layer chromatography was performed on 0.25 mm extra-hard silica gel plates with a UV254 fluorescent indicator. The reported melting points are uncorrected. The ¹H NMR and ¹³C NMR spectra were recorded at ambient temperature using both 300 MHz spectrometers (300 MHz for ¹H and 75 MHz for ¹³C). Chemical shifts are reported in ppm with respect to tetramethylsilane internal reference, and coupling constants are reported in Hz. Proton multiplicities are represented as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), and m (multiplet). The infrared spectra were recorded on an FT-IR spectrometer in thin films. HRMS data were recorded on a Q-tof-micro quadruple mass spectrophotometer.

General Procedure for the Synthesis of Phthalidyl Sulfonohydrazide Derivatives (4, GP-1)

In a 25 mL round-bottom flask, phthalaldehyde (1, 1.0 mmol) and N-tosyl hydrazones¹ (2, 1.0 mmol) were added in CH₃CN (5 mL) in the presence of thiazolium bromide 3c (10 mol %) in open-air conditions, and Cs₂CO₃ (20 mol %) was added to this reaction mixture and stirred at room temperature for 2–5 h. Upon completion (monitored through TLC), the reaction mixture was filtered through the Celite bed and evaporated in a rotary evaporator under reduced pressure and then extracted with CH₂Cl₂ (2 × 15 mL). The combined organic layer was washed with water (3 × 10 mL) and dried over anhydrous Na₂SO₄, filtered, and evaporated in a rotary evaporator under reduced pressure at room temperature. The residue was chromatographed on a silica gel column (60–120 mesh) using ethyl acetate–petroleum ether (9 to 20%, v/v) as an eluent, which afforded the corresponding hemiaminal phthalidyl ester derivatives (4).

(E)-N′-Benzylidene-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4a)

Compound 4a was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-benzylidene-4-methylbenzenesulfonohydrazide (2a, 1 mmol) as the starting material and afforded the title compound 4a (using 10% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 73% (296.7 mg); M.P.: 155–157 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.59 (s, 1H), 7.90–7.85 (m, 3H), 7.70–7.65 (m, 1H), 7.57–7.52 (m, 2H), 7.48 (s, 1H), 7.39–7.24 (m, 7H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 160.3, 145.3, 144.9, 134.6, 133.8, 133.0, 131.5, 130.4, 129.8, 129.1, 128.6, 128.1, 127.2, 125.5, 123.2, 88.3, 21.7; FT-IR (KBr, cm^–1): 1766, 1596, 1427, 1358, 1044, 956; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₉N₂O₄S [M + H]⁺: 407.1066, found 407.1070.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(2,4,6-trimethylbenzylidene)benzenesulfonohydrazide (4b)

Compound 4b was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(2,4,6-trimethylbenzylidene)benzenesulfonohydrazide (2b, 1 mmol) as the starting material and afforded the title compound 4b (using 9% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as brown solid, Yield: 86% (385.7 mg); M.P.: 135–137 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.94 (s, 1H), 7.90–7.85 (m, 3H), 7.70–7.66 (m, 1H), 7.58–7.56 (m, 2H), 7.55–7.51 (m, 1H), 7.35 (d, J = 8.1 Hz, 2H), 6.74 (m, 2H), 2.45 (s, 3H), 2.22 (s, 3H), 1.94 (s, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 162.1, 145.2, 145.1, 140.5, 138.8, 134.5, 134.0, 130.4, 129.8, 129.7, 129.1, 127.5, 127.0, 125.5, 123.4, 88.3, 21.7, 21.1, 21.0; FT-IR (KBr, cm^–1): 1772, 1600, 1435, 1400, 1170, 1048, 960; HRMS (ESI-TOF) m/z Calcd for C₂₅H₂₅N₂O₄S [M + H]⁺: 449.1535, found 449.1539.

(E)-N′-(2,5-Dimethylbenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4c)

Compound 4c was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(2,5-dimethylbenzylidene)-4-methylbenzenesulfonohydrazide (2c, 1 mmol) as the starting material and afforded the title compound 4c (using 9% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 84% (365.0 mg); M.P.: 138–140 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.76 (s, 1H), 7.89–7.86 (m, 3H), 7.71–7.66 (m, 1H), 7.57–7.52 (m, 2H), 7.49 (s, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.05–7.03 (m, 2H), 6.97–6.94 (m, 1H), 2.48 (s, 3H), 2.19 (s, 3H), 2.07 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 160.8, 145.3, 144.9, 135.6, 135.3, 134.5, 133.9, 131.9, 131.0, 130.8, 130.4, 129.8, 129.1, 129.0, 127.3, 125.4, 123.3, 88.3, 21.7, 20.7, 20.7, 19.6; FT-IR (KBr, cm^–1): 1770, 1598, 1440, 1397, 1167, 1049, 962; HRMS (ESI-TOF) m/z Calcd for C₂₄H₂₃N₂O₄S [M + H]⁺: 435.1379, found 435.1381.

(E)-N′-(4-Methoxybenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4d)

Compound 4d was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(4-methoxybenzylidene)-4-methylbenzenesulfonohydrazide (2d, 1 mmol) as the starting material and afforded the title compound 4d (using 15% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as colorless liquid, Yield: 80% (349.2 mg); ¹H NMR (300 MHz, CDCl₃): δ 8.54 (s, 1H), 7.85–7.80 (m, 3H), 7.67–7.62 (m, 1H), 7.53–7.48 (m, 2H), 7.42 (s, 1H), 7.33 (d, J = 8.7 Hz, 4H), 6.87 (d, J = 9.0 Hz, 2H), 3.77 (s, 3H), 2.43 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 162.9, 162.4, 145.0, 144.7, 134.3, 133.8, 130.2, 130.0, 129.5, 129.0, 127.1, 125.4, 125.3, 114.0, 88.2, 55.3, 21.6; FT-IR (neat, cm^–1): 1768, 1609, 1522, 1424, 1167, 1039, 950; HRMS (ESI-TOF) m/z Calcd for C₂₃H₂₁N₂O₅S [M + H]⁺: 437.1171, found 437.1175.

(E)-N′-(2-Methoxybenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4e)

Compound 4e was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(2-methoxybenzylidene)-4-methylbenzenesulfonohydrazide (2e, 1 mmol) as the starting material and afforded the title compound 4e (using 14% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 78% (340.4 mg); M.P.: 125–127 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.96 (s, 1H), 7.88–7.86 (m, 3H), 7.70–7.65 (m, 1H), 7.57–7.54 (m, 2H), 7.48 (s, 1H), 7.36–7.24 (m, 4H), 6.84–6.73 (m, 2H), 3.83 (s, 3H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.8, 158.7, 156.9, 145.1, 144.9, 134.4, 132.9, 130.3, 129.7, 129.2, 126.7, 125.4, 123.3, 121.5, 120.6, 111.0, 88.3, 55.6, 21.7; FT-IR (KBr, cm^–1): 1764, 1612, 1521, 1420, 1163, 1037, 947; HRMS (ESI-TOF) m/z Calcd for C₂₃H₂₁N₂O₅S [M + H]⁺: 437.1171, found 437.1176.

(E)-4-Methyl-N′-(4-methylbenzylidene)-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4f)

Compound 4f was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(4-methylbenzylidene)benzenesulfonohydrazide (2f, 1 mmol) as the starting material and afforded the title compound 4f (using 10% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 77% (323.8 mg); M.P.: 120–122 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.55 (s, 1H), 7.86–7.80 (m, 3H), 7.67–7.62 (m, 1H), 7.53–7.48 (m, 2H), 7.43 (s, 1H), 7.32 (d, J = 8.1 Hz, 2H), 7.26–7.23 (m, 2H), 7.06 (d, J = 7.8 Hz, 2H), 2.43 (s, 3H), 2.30 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.8, 161.9, 145.2, 144.8, 142.3, 134.5, 133.9, 130.33, 130.25, 129.7, 129.4, 128.3, 127.2, 125.4, 123.2, 88.3, 21.8, 21.6; FT-IR (KBr, cm^–1): 1767, 1598, 1432, 1350, 1162, 1069, 940; HRMS (ESI-TOF) m/z Calcd for C₂₃H₂₁N₂O₄S [M + H]⁺: 421.1222, found 421.1220.

(E)-N′-(4-Isopropylbenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4g)

Compound 4g was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(4-isopropylbenzylidene)-4-methylbenzenesulfonohydrazide (2g, 1 mmol) as the starting material and afforded the title compound 4g (using 9% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as brown solid, Yield: 75% (336.0 mg); M.P.: 170–172 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.59 (s, 1H), 7.88–7.84 (m, 3H), 7.69–7.63 (m, 1H), 7.55–7.50 (m, 2H), 7.46 (s, 1H), 7.36–7.28 (m, 4H), 7.14 (d, J = 8.1 Hz 2H), 2.92–2.83 (m, 1H), 2.44 (s, 3H), 1.22–1.19 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 161.6, 153.0, 145.1, 144.8, 134.4, 133.8, 130.6, 130.2, 129.7, 129.0, 128.3, 127.1, 126.7, 125.3, 123.1, 88.2, 34.0, 23.58, 23.56, 21.6; FT-IR (KBr, cm^–1): 1771, 1608, 1440, 1387, 1172, 1041, 942; HRMS (ESI-TOF) m/z Calcd for C₂₅H₂₅N₂O₄S [M + H]⁺: 449.1535, found 449.1540.

(E)-N′-(4-(Dimethylamino)benzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4h)

Compound 4h was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(4-(dimethylamino)benzylidene)-4-methylbenzenesulfonohydrazide (2h, 1 mmol) as the starting material and afforded the title compound 4h (using 20% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as red solid, Yield: 75% (337.1 mg); M.P.: 160–163 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.48 (s, 1H), 7.80 (d, J = 8.1 Hz, 3H), 7.62–7.59 (m, 1H), 7.52–7.44 (m, 2H), 7.38 (s, 1H), 7.33–7.30 (m, 4H), 6.53 (d, J = 8.7 Hz, 2H), 2.96 (s, 6H), 2.44 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.8, 166.6, 152.6, 144.7, 144.6, 134.19, 134.16, 130.2, 130.1, 129.4, 129.1, 127.1, 125.2, 123.1, 120.0, 111.1, 88.4, 39.9, 21.6; FT-IR (KBr, cm^–1): 1769, 1594, 1444, 1379, 1174, 1042, 945; HRMS (ESI-TOF) m/z Calcd for C₂₄H₂₄N₃O₄S [M + H]⁺: 450.1488, found 450.1485.

(E)-N′-(4-Chlorobenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4i)

Compound 4i was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(4-chlorobenzylidene)-4-methylbenzenesulfonohydrazide (2i, 1 mmol) as the starting material and afforded the title compound 4i (using 10% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 73% (321.8 mg); M.P.: 190–192 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.54 (s, 1H), 7.91–7.84 (m, 3H), 7.71–7.66 (m, 1H), 7.58–7.54 (m, 2H), 7.49 (s, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.24 (m, 4H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 158.0, 145.5, 144.8, 137.5, 134.6, 133.6, 131.5, 130.4, 129.9, 129.2, 129.0, 128.9, 127.2, 125.5, 123.3, 88.2, 21.8; FT-IR (KBr, cm^–1): 1780, 1595, 1462, 1333, 1168, 1087, 814; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₈ClN₂O₄S [M + H]⁺: 441.0676, found 441.0680 (one of the major peaks).

(E)-N′-(4-Bromobenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4j)

Compound 4j was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(4-bromobenzylidene)-4-methylbenzenesulfonohydrazide (2j, 1 mmol) as the starting material and afforded the title compound 4j (using 10% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 70% (339.7 mg); M.P.: 196–198 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.53 (s, 1H), 7.91–7.84 (m, 3H), 7.71–7.66 (m, 1H), 7.58–7.49 (m, 2H), 7.49 (s, 1H), 7.41–7.15 (m, 2H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 157.8, 145.5, 144.8, 134.6, 133.6, 132.0, 130.5, 129.9, 129.3, 129.0, 127.2, 126.0, 125.5, 123.2, 88.2, 21.8; FT-IR (KBr, cm^–1): 1781, 1592, 1471, 1331, 1170, 1070, 812; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₈BrN₂O₄S [M + H]⁺: 485.0171, found 485.0176 (one of the major peaks).

(E)-N′-(3-Fluorobenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4k)

Compound 4k was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(3-fluorobenzylidene)-4-methylbenzenesulfonohydrazide (2k, 1 mmol) as the starting material and afforded the title compound 4k (using 11% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 65% (275.9 mg); M.P.: 186–188 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.53 (s, 1H), 7.89 (t, J = 8.7 Hz, 3H), 7.72–7.67 (m, 1H), 7.59–7.51 (m, 3H), 7.36 (d, J = 8.1 Hz, 2H), 7.27–7.20 (m, 1H), 7.09–6.94 (m, 3H), 2.44 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 164.3 (C–F, ¹J_C–F = 245.8 Hz), 161.0 (C–F, ¹J_C–F = 245.8 Hz), 156.5 (C–F, ⁴J_C–F = 2.2 Hz), 156.4 (C–F, ⁴J_C–F = 2.2 Hz), 145.6, 144.9, 135.3 (C–F, ³J_C–F = 7.7 Hz), 135.2 (C–F, ³J_C–F = 7.7 Hz), 134.6, 133.6, 130.5, 130.3 (C–F, ³J_C–F = 8.0 Hz), 130.2 (C–F, ³J_C–F = 8.0 Hz), 129.9, 129.0, 127.2, 125.5, 124.23 (C–F, ⁴J_C–F = 2.8 Hz), 124.19 (C–F, ⁴J_C–F = 2.8 Hz), 123.2, 118.4 (C–F, ²J_C–F = 21.4 Hz), 118.2 (C–F, ²J_C–F = 21.4 Hz), 113.9 (C–F, ²J_C–F = 22.5 Hz), 113.6 (C–F, ²J_C–F = 22.5 Hz), 88.2, 21.7; FT-IR (KBr, cm^–1): 1778, 1602, 1513, 1362, 1322, 1160, 1054, 932; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₈FN₂O₄S [M + H]⁺: 425.0971, found 425.0975.

(E)-N′-(3,4-Dichlorobenzylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4l)

Compound 4l was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(3,4-dichlorobenzylidene)-4-methylbenzenesulfonohydrazide (2l, 1 mmol) as the starting material and afforded the title compound 4l (using 9% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as yellow solid, Yield: 55% (261.4 mg); M.P.: 196–198 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.46 (s, 1H), 7.93–7.85 (m, 3H), 7.70 (t, J = 7.2 Hz, 1H), 7.61–7.54 (m, 2H), 7.51 (s, 1H), 7.38–7.30 (m, 4H), 7.11–7.08 (m, 1H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 154.6, 145.7, 144.7, 135.4, 134.7, 133.4, 133.09, 133.07, 130.7, 130.6, 130.0, 129.5, 129.0, 127.1, 126.6, 125.5, 123.2, 88.1, 21.8; FT-IR (KBr, cm^–1): 1779, 1593, 1475, 1337, 1165, 1057, 931; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₇Cl₂N₂O₄S [M + H]⁺: 475.0286, found 475.0281 (one of the major peaks).

(E)-4-Methyl-N′-(4-nitrobenzylidene)-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4m)

Compound 4m was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(4-nitrobenzylidene)benzenesulfonohydrazide (2m, 1 mmol) as the starting material and afforded the title compound 4m (using 18% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as reddish brown solid, Yield: 53% (239.3 mg); M.P.: 160–162 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.55 (s, 1H), 8.10–8.07 (m, 2H), 7.96–7.89 (m, 3H), 7.72 (t, J = 7.5 Hz, 1H), 7.64–7.57 (m, 3H), 7.40–7.28 (m, 4H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.4, 151.2, 149.0, 145.9, 144.7, 139.0, 134.7, 133.3, 130.6, 130.1, 128.9, 128.1, 127.2, 125.6, 123.8, 123.2, 88.1, 21.7; FT-IR (KBr, cm^–1): 1767, 1597, 1480, 1340, 1170, 1060, 935; HRMS (ESI-TOF) m/z Calcd for C₂₂H₁₈N₃O₆S [M + H]⁺: 452.0916, found 452.0921.

(E)-4-Methyl-N′-(naphthalen-1-ylmethylene)-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4n)

Compound 4n was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(naphthalen-1-ylmethylene)benzenesulfonohydrazide (2n, 1 mmol) as the starting material and afforded the title compound 4n (using 12% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as brown solid, Yield: 65% (296.7 mg); M.P.: 154–156 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.21 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.95–7.91 (m, 3H), 7.86–7.78 (m, 2H), 7.69- 7.66 (m, 1H), 7.63–7.60 (m, 1H), 7.57–7.55 (m, 2H), 7.48–7.43 (m, 2H), 7.40–7.32 (m, 4H), 2.41 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.7, 161.0, 145.4, 145.0, 134.6, 133.8, 133.6, 132.2, 130.5, 130.4, 130.0, 129.9, 129.2, 128.6, 128.5, 127.5, 127.4, 126.3, 125.5, 125.0, 124.3, 123.4, 88.5, 21.7; FT-IR (KBr, cm^–1): 1765, 1590, 1509, 1320, 1172, 1065, 944; HRMS (ESI-TOF) m/z Calcd for C₂₆H₂₁N₂O₄S [M + H]⁺: 457.1222, found 457.1221.

(E)-N′-([1,1′-Biphenyl]-2-ylmethylene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4o)

Compound 4o was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-([1,1′-biphenyl]-2-ylmethylene)-4-methylbenzenesulfonohydrazide (2o, 1 mmol) as the starting material and afforded the title compound 4o (using 10–20% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as orange solid, Yield: 63% (304.0 mg); M.P.: 170–172 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.45 (s, 1H), 7.87 (d, J = 7.5 Hz, 1H), 7.74–7.69 (m, 3H), 7.56–7.49 (m, 6H), 7.45–7.39 (m, 2H), 7.32–7.25 m, 3H), 7.19–7.15 (m, 3H), 2.44 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 159.1, 145.2, 144.9, 143.6, 138.8, 134.5, 134.0, 131.0, 130.5, 130.4, 130.2, 129.8, 129.0, 128.5, 127.9, 127.6, 126.3, 125.6, 123.2, 88.1, 21.8; FT-IR (KBr, cm^–1): 1770, 1594, 1490, 1318, 1175, 1062, 947; HRMS (ESI-TOF) m/z Calcd for C₂₈H₂₃N₂O₄S [M + H]⁺: 483.1379, found 483.1377.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(pyren-2-ylmethylene)benzenesulfonohydrazide (4p)

Compound 4p was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(pyren-1-ylmethylene)benzenesulfonohydrazide (2p, 1 mmol) as the starting material and afforded the title compound 4p (using 13% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as brown oily liquid, Yield: 60% (318.3 mg); ¹H NMR (300 MHz, CDCl₃): δ 9.55 (s, 1H), 8.25–8.21 (m, 2H), 8.13–8.10 (m, 2H), 7.98 (t, J = 4.2 Hz, 6H), 7.91–7.84 (m, 3H), 7.67–7.63 (m, 2H), 7.59 (s, 1H), 7.56–7.51 (m, 1H), 7.33 (d, J = 8.1 Hz, 2H), 2.38 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.8, 160.3, 145.4, 145.0, 134.6, 133.9, 130.9, 130.5, 129.9, 129.8, 129.2, 129.1, 127.4, 127.1, 126.3, 126.2, 126.0, 125.5, 125.1, 124.6, 123.4, 122.3, 88.6, 21.7; FT-IR (neat, cm^–1): 1771, 1593, 1489, 1317, 1171, 1063, 948; HRMS (ESI-TOF) m/z Calcd for C₃₂H₂₃N₂O₄S [M + H]⁺: 531.1379, found 531.1384.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(thiophen-2-ylmethylene)benzenesulfonohydrazide (4q)

Compound 4q was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(thiophen-2-ylmethylene)benzenesulfonohydrazide (2q, 1 mmol) as the starting material and afforded the title compound 4q (using 15% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 75% (309.4 mg); M.P.: 164–166 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.71 (s, 1H), 7.86–7.81 (m, 3H), 7.68–7.63 (m, 1H), 7.54–7.50 (m, 2H), 7.40 (s, 1H), 7.35–7.25 (m, 4H), 6.97 (t, J = 4.2 Hz, 1H), 2.42 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 155.9, 145.3, 144.7, 137.7, 134.5, 133.8, 132.7, 130.8, 130.4, 129.8, 129.1, 127.6, 127.2, 125.5, 123.2, 88.2, 21.8; FT-IR (KBr, cm^–1): 1791, 1588, 1432, 1342, 1167, 1062, 946; HRMS (ESI-TOF) m/z Calcd for C₂₀H₁₇N₂O₄S₂ [M + H]⁺: 413.0630, found 413.0633.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(pyridin-3-ylmethylene)benzenesulfonohydrazide (4r)

Compound 4r was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(pyridin-3-ylmethylene)benzenesulfonohydrazide (2r, 1 mmol) as the starting material and afforded the title compound 4r (using 17% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 70% (285.2 mg); M.P.: 170–172 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.56–8.47 (m, 3H), 7.92–7.87 (m, 3H), 7.73–7.67 (m, 1H), 7.60–7.50 (m, 4H), 7.36 (d, J = 8.1 Hz, 2H), 7.18–7.14 (m, 1H), 2.44 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.6, 153.5, 151.8, 149.6, 145.7, 144.8, 134.9, 134.7, 133.9, 133.4, 130.5, 130.0, 129.2, 129.0, 127.2, 125.5, 124.0, 123.7, 123.2, 88.1, 21.7; FT-IR (KBr, cm^–1): 1783, 1607, 1436, 1355, 1168, 1048, 942; HRMS (ESI-TOF) m/z Calcd for C₂₁H₁₈N₃O₄S [M + H]⁺: 408.1018, found 408.1015.

4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-((1E,2E)-3-phenylallylidene)benzenesulfonohydrazide (4s)

Compound 4s was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and 4-methyl-N′-((1E,2E)-3-phenylallylidene)benzenesulfonohydrazide (2s, 1 mmol) as the starting material and afforded the title compound 4s (using 13% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as orange solid, Yield: 68% (294.1 mg); M.P.: 160–162 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.42 (d, J = 9.3 Hz, 1H), 7.88–7.85 (m, 3H), 7.69 (t, J = 7.2 Hz, 1H), 7.58–7.52 (m, 2H), 7.43 (s, 1H), 7.39–7.31 (m, 7H), 6.95 (d, J = 16.2 Hz, 1H), 6.57–6.49 (m, 1H), 2.45 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.5, 164.5, 145.1, 144.6, 144.2, 134.9, 134.4, 133.7, 130.3, 129.8, 129.7, 129.0, 128.7, 127.4, 127.2, 125.4, 125.4, 124.1, 123.1, 88.0, 21.6; FT-IR (KBr, cm^–1): 1767, 1650, 1600, 1435, 1352, 1171, 1069, 923; HRMS (ESI-TOF) m/z Calcd for C₂₄H₂₁N₂O₄S [M + H]⁺: 433.1222, found 433.1224.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(1-phenylethylidene)benzenesulfonohydrazide (4t)

Compound 4t was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(1-phenylethylidene)benzenesulfonohydrazide (2t, 1 mmol) as the starting material and afforded the title compound 4t (using 11% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 65% (273.3 mg); M.P.: 164–166 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.81–7.76 (m, 3H), 7.61 (t, J = 7.5 Hz, 1H), 7.52–7.46 (m, 4H), 7.41–7.29 (m, 6H), 2.55 (s, 3H), 2.49 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 180.5, 168.5, 144.8, 144.2, 136.2, 134.2, 134.1, 131.4, 130.5, 129.4, 129.3, 128.4, 127.2, 127.1, 125.6, 122.7, 88.7, 21.8, 18.4; FT-IR (KBr, cm^–1): 1782, 1603, 1470, 1365, 1170, 1049, 942; HRMS (ESI-TOF) m/z Calcd for C₂₃H₂₁N₂O₄S [M + H]⁺: 421.1222, found 421.1219.

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(1-(p-tolyl)ethylidene)benzenesulfonohydrazide (4u)

Compound 4u was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(1-(p-tolyl)ethylidene)benzenesulfonohydrazide (2u, 1 mmol) as the starting material and afforded the title compound 4u (using 10% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as colorless oily liquid, Yield: 70% (304.1 mg); ¹H NMR (300 MHz, CDCl₃): δ 7.81–7.75 (m, 3H), 7.62–7.57 (m, 1H), 7.48–7.41 (m, 4H), 7.36–7.28 (m, 3H), 7.09 (d, J = 8.1 Hz, 2H), 2.52–2.49 (m, 6H), 2.34 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 180.1, 168.5, 144.8, 144.2, 142.0, 134.2, 134.0, 133.5, 130.5, 129.4, 129.3, 129.0, 127.3, 127.1, 125.5, 122.7, 88.8, 21.8, 21.4, 18.2; FT-IR (neat, cm^–1): 1772, 1605, 1436, 1355, 1164, 1073, 942; HRMS (ESI-TOF) m/z Calcd for C₂₄H₂₃N₂O₄S [M + H]⁺: 435.1379, found 435.1382.

(E)-N′-(1-(4-Bromophenyl)ethylidene)-4-methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)benzenesulfonohydrazide (4v)

Compound 4v was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-N′-(1-(4-bromophenyl)ethylidene)-4-methylbenzenesulfonohydrazide (2v, 1 mmol) as the starting material and afforded the title compound 4v (using 9% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 60% (299.6 mg); M.P.: 170–172 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.77 (d, J = 7.8 Hz, 3H), 7.61 (d, J = 7.8 Hz, 1H), 7.50–7.33 (m, 9H), 2.52–2.49 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 179.5, 169.2, 145.0, 144.1, 134.9, 134.1, 131.6, 130.6, 129.5, 129.3, 128.7, 126.8, 126.4, 125.6, 124.0, 122.6, 88.7, 21.8, 18.2; FT-IR (KBr, cm^–1): 1785, 1593, 1433, 1345, 1168, 1072, 949; HRMS (ESI-TOF) m/z Calcd for C₂₃H₂₀BrN₂O₄S [M + H]⁺: 499.0327, found 499.0329 (one of the major peaks).

(E)-4-Methyl-N-(3-oxo-1,3-dihydroisobenzofuran-1-yl)-N′-(1-(thiophen-2-yl)ethylidene)benzenesulfonohydrazide (4w)

Compound 4w was prepared following the GP-1 using phthalaldehyde (1a, 1 mmol) and (E)-4-methyl-N′-(1-(thiophen-2-yl)ethylidene)benzenesulfonohydrazide (2w, 1 mmol) as the starting material and afforded the title compound 4w (using 15% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 64% (273.0 mg); M.P.: 160–164 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.80–7.75 (m, 3H), 7.64–7.59 (m, 2H), 7.49–7.44 (m, 2H), 7.37–7.28 (m, 3H), 7.22–7.16 (m, 2H), 2.51–2.50 (m, 6H); ¹³C NMR (75 MHz, CDCl₃): δ 175.6, 168.5, 144.8, 144.2, 139.5, 134.1, 134.0, 130.5, 129.4, 129.3, 128.6, 127.1, 126.1, 126.0, 125.5, 122.7, 88.7, 21.8, 18.5; FT-IR (KBr, cm^–1): 1792, 1590, 1434, 1340, 1166, 1067, 945; HRMS (ESI-TOF) m/z Calcd for C₂₁H₁₉N₂O₄S₂ [M + H]⁺: 427.0786, found 427.0781.

(E)-N-(5-Methoxy-3-oxo-1,3-dihydroisobenzofuran-1-yl)-4-methyl-N′-(4-methylbenzylidene)benzenesulfonohydrazide (4x)

Compound 4x was prepared following the GP-1 using 4-methoxyphthalaldehyde (1b, 1 mmol) and (E)-4-methyl-N′-(4-methylbenzylidene)benzenesulfonohydrazide (2f, 1 mmol) as the starting material and afforded the title compound 4x (using 15% ethyl acetate–petroleum ether (v/v) as an eluent for purification) as white solid, Yield: 60% (270.3 mg); M.P.: 148–150 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.56 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.42–7.28 (m, 7H), 7.21–7.17 (m, 1H), 7.11 (d, J = 7.8 Hz, 2H), 3.84 (s, 3H), 2.45 (s, 3H), 2.34 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 168.8, 162.6, 161.5, 145.1, 142.3, 136.9, 134.0, 130.3, 129.7, 129.4, 129.1, 128.8, 128.4, 124.1, 123.0, 107.6, 88.2, 55.8, 21.7, 21.6; FT-IR (KBr, cm^–1): 1765, 1597, 1430, 1355, 1165, 1068, 937; HRMS (ESI-TOF) m/z Calcd for C₂₄H₂₃N₂O₅S [M + H]⁺: 451.1328, found 451.1331.

Computational Methods

All the geometries considered in this study have been fully optimized using the dispersion-corrected PBE0-D3^20,21 functional with the def2-TZVPP basis set.²² The solvent effects were considered via the COSMO solvation model²³ with acetonitrile solvent medium. The vibrational frequencies of each stationary point were carried out at the same level of theory to classify the stationary points either as real minima (with no imaginary frequencies) or as transition state with only one imaginary frequency. All the calculations were performed using Gaussian 16.²⁴

Molecular docking was performed using the Schrodinger Suite molecular modeling package (version 2021-3) using the default parameters. Co-crystal structures of thiolactomycin with β-ketoacyl-[acyl carrier protein] 2 synthase (PDB: 1FJ4),¹⁷ with a resolution of 2.35 Å, were used as templates and were prepared using the Protein Preparation Wizard. In this step, force field atom types and bond orders were assigned, missing atoms were added, tautomer/ionization states were assigned, and the tautomers of ionizable residues (Asn, Gln, and His residues) were adjusted to optimize the hydrogen bond network. Hydrogen-constrained energy minimization was then performed. Glide SP docking was used to grant full flexibility of ligands into the active site.^25,26 A postdocking minimization, in which only the ligands were flexible, was performed on the output complexes. The binding energies were calculated for each ligand.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c08529.

• ESI-MS spectra; spectroscopic and XRD data; NMR spectra; computational details; and additional figures (PDF)

Accession Codes

CCDC 2080548 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Author Contributions

^# T.G. and D.B. contributed equally.

The authors declare no competing financial interest.