Accessing Promising Passerini Adducts in Anticancer Drug Design

Abstract

The 3-component Passerini reaction (3CPR), discovered little more than 100 years ago, has been demonstrated in the last few decades to be a valuable tool for accessing structural diversity and complexity, essential topics to consider in drug discovery programs. Focusing on accessing a fine-tuned family of α-acyloxyamide–oxindole hybrids, we underline herein our latest insights regarding the use of this mild reaction approach to obtain promising anticancer agents. Cheap and commercially available isatin was used as starting material. The library of α-acyloxyamide–oxindole hybrids was tested against six human solid-tumor cell lines; among them, non-small cell lung carcinoma, cervical and colon adenocarcinoma, and breast and pancreas cancer. The most potent compound displayed GI₅₀ values in the range of 1.3–21 µM.

1. Introduction

Multicomponent reactions (MCRs) are a valuable tool in drug design and development programs, providing easy access to great structural diversity in mild reaction conditions with a considerable reduction in chemical waste [1,2,3]. This was demonstrated by its application in the pharmaceutical industry in the development of many commercially available drugs [4]. Compared to multistep reaction protocols, the use of MCR offers immediate advantages from a synthetic, economic, and environment point of view. Extraordinary synthetic efficiency and atom- and step economy, together with a considerable reduction in waste generation, makes these particular protocols very attractive for accessing libraries of complex molecules of biological interest (and others). In the last decade, our group has been active in the synthesis of bioactive oxindole-type hybrids, manipulating the isatin core (positions N1, C3 and C5 of the aromatic ring) with resourceful chemical transformations [5,6,7,8,9,10,11]. Taking advantage of the transition-metal-catalyzed arylation reactions in racemic and asymmetric fashion, libraries of 3-hydroxy-3-aryl-oxindole [5,6,9] and 3-amino-3-aryl-oxindole [8] derivatives were obtained in good to excellent yields and enantioselectivities, proving to be promising hit scaffolds as cholinesterase inhibitors [5,6,8] (important targets for neurodegenerative diseases like Alzheimer’s) and anticancer agents [9] (Figure 1). Multistep protocols were also developed in order to obtain families of 3-protected-N,C5-substituted-oxindole derivatives, with potent activity against butyrylcholinesterase, a well-known target involved in the study of Alzheimer’s disease [7]. The use of isatin as a component in MCR is well established in the literature [12,13], demonstrating easy access to structural diversity in the quest for new bioactive and druglike molecules.

Very recently, we reported our latest findings regarding the use of the Ugi MCR approach to obtain large libraries of isatin-based α-acetamide–carboxamide–oxindole hybrids (Figure 1), promising anticancer agents, in a mild and fast sustainable reaction process [10,11]. Focused on increasing the cytotoxicity of the drug candidates in several cancer cell lines (breast, lung, colon, etc.) and within the course of our research work–plan regarding the manipulation of the isatin core, we decided to investigate another MCR, the 3-component Passerini reaction (3CPR), to access 3,3-amido-esther-oxindole hybrids. The secular Passerini reaction was the first isocyanide-based 3-component MCR, discovered by Mario Torquato Passerini in 1921 [14], and engages an aldehyde, a carboxylic acid, and an isocyanide (Scheme 1) [15]. Overall, the most accepted mechanism starts with the activation of the aldehyde by the carboxylic acid, forming a hydrogen-bonded intermediate which undergoes the addition of an isocyanide, forming a nitrilium intermediate (α-addition of the electrophilic carbonyl component and the nucleophilic carboxylate to the chameleonic isocyanide). Mumm-type rearrangement gave access to the α-acyloxyamide product (Scheme 1). Despite being much less explored than the related Ugi 4-component reaction [16], the literature findings have demonstrated that 3CPR has emerged in the last decade as a powerful tool to access libraries of bioactive complex molecules [17].

However, and as far as we know, isatin-based 3CPR was still poorly explored in the literature. Eleven years ago, Esmaeili and co-workers reported the first version of the 3CPR, using isatin as ketone component. Using CH₃CN as the solvent, at 80 °C, they could access a library of 15 isatin–Passerini adducts in moderate to high yield. [18] In the same year, the Biju group reported a similar synthetic outcome, using a solvent-free approach, at 100 °C, accessing a library of 26 isatin–Passerini adducts in low to high yield. [19] In 2016, Shaabani and co-workers explored the use of DES (Deep Eutectic Solvents) as environmental benign media to perform the 3CPR. Only two examples were reported regarding the use of isatin as a ketone component [20]. Investigating Ugi MCRs on isatin scaffolds, our group very recently reported an interesting formation of the isatin–Passerini adducts as secondary products using optimized conditions for the Ugi protocol. The cheap and commercially available InCl₃ was used as a catalyst in mild reaction conditions [21]. Quite recently, Salami, Krause, and co-workers investigated the use of mechanochemical activation in the 3CPR. They found out that the methodology works well when using isatin as a starting material and water as a solvent. A library of 30 isatin–Passerini adducts was obtained in moderate to high yields [22,23].

Taking advantages of the MCR tool kit to obtain libraries of oxindole-type hybrids in drug design, and focused on the modification of the isatin core to increase the cytotoxicity of the scaffolds, we decided to explore the 3CPR in detail, since 3-substituted oxindole derivatives have proved their value as powerful anticancer agents [24,25].

2. Results and Discussion

2.1. The Isatin-Based 3-Component Passerini Reaction

Searching for new and powerful anticancer agents is still a very challenging and entirely relevant area of study, considering that cancer is still an incurable, complex, and fatal disease.

The development of imatinib (Gleevec^®) [26], the tyrosine kinase inhibitor synthetized 30 years ago and still prescribed to treat several types of cancer, marked a new era in drug discovery, setting a precedent for designing highly selective and potent drugs that achieve target-specific binding across diverse therapeutic areas. Inspired by this, we decided to explore the 3CPR’s ability to access α-acyloxyamide-oxindole hybrids 4, using isatin derivatives 1 as starting material. Taking advantage of the scarce literature findings, we decided to start our reaction scope by using a microwave reactor, which has proven to be efficient in this kind of MCR [27]; however, as far as we know, it has not been tested with ketones. The results can be seen in

Table 1. Survey of the 3CPR using microwave reaction conditions.

Entry a	1	2	Equiv.	3	Equiv.	Solvent	Temp./°C	T/h	4	Yield c/%
1	1a	2b	1.0	3a	2.0	Toluene	60	0.25	4aba	0
2	1a	2b	1.5	3a	2.0	Toluene	120	0.5	4aba	60
3	1a	2b	2.0	3a	1.5	Toluene	120	3	4aba	91
4	1a	2b	3.0	3a	2.4	Toluene	120	3	4aba	59
5	1a	2b	2.0	3a	1.5	1,4-Dioxane	120	3	4aba	93
6	1a	2a	2.0	3a	1.5	Toluene	120	3	4aaa	85
7	1a	2a	3.0	3a	2.4	Toluene	120	3	4aaa	72
8	1a	2b	2.0	3b	1.5	Toluene	120	3	4abb	76
9	1a	2b	2.0	3b	1.5	Toluene	120	1	4abb	57
10	1a	2b	1.5	3b	1.2	Toluene	120	1	4abb	49
11	1a	2b	2.0	3b	1.5	DMF	120	1	4abb	11
12	1a	2b	2.0	3b	1.5	1,4-Dioxane	120	1	4abb	50
13	1a	2c	2.0	3b	1.5	Toluene	120	3	4acb	86
14	1b	2c	2.0	3b	1.5	Toluene	120	3	4bcb	89
15 b	1b	2c	1.3	3b	1.0	CH3CN	80	2	4bcb	35
16 b	1b	2c	2.5	3b	2.0	CH3CN	80	2	4bcb	50
17 b	1b	2c	4.0	3b	2.0	CH3CN	80	2	4bcb	33

^a Reaction conditions: 1a (0.4 mmol) or 1b (0.6 mmol), 2, 3, and the solvent (3 mL) were added to a microwave vial and stirred at the indicated temperature for a certain time. ^b 4Å MS (100 mg) were added to the reaction vial. ^c Isolated yield.

.

In our first attempt, N-benzyl isatin 1a, together with 2-iodobenzoic acid 2b, tert-butyl isocyanide 3a, and toluene, were put on a microwave vial at 60 °C with a short reaction time (15 min). The reaction failed to obtain the desired Passerini-adduct product 4aba (Table 1, entry 1). Increasing the temperature, the reaction time (120 °C in 30 min), and the equivalents of 2b (1.5 equivalents), 4aba could be obtained in 60% yield (Table 1, entry 2). Within 3 h, 4aba could be obtained in 91% yield, using a slight excess of 2b (2 equivalents) and decreasing 3a slightly (1.5 equivalents) (Table 1, entry 3). We found out that the yield of 4aba decreased significantly when the stoichiometry of 2b (3 equivalents) and 3a (2.4 equivalents) were increased considerably (see Table 1; compare entries 3 and 4). Like toluene, 1,4-dioxane was also a good solvent for acquiring the desired 4aba in very good yield (see Table 1; compare entries 3 and 5). Using the same reaction conditions to obtain 91% of 4aba (Table 1, entry 3), we decided to evaluate the reaction scope, testing the behavior of other components (Table 1, entries 6, 8, 13 and 14). No significant differences were noted in the yield of 4aaa using the corresponding 2-bromobenzoic acid 2a (Table 1; compare entries 3 and 6). Using the bulky cyclohexyl isocyanide 3b, the corresponding 4abb was obtained at 76% (Table 1; compare entries 3 and 8). Using the unsubstituted benzoic acid 2c, the corresponding 4acb was obtained in 86% yield, as expected (Table 1; compare entries 8 and 13). N-Methyl isatin 1b was also tested in this microwave reaction scope, and no variance in the yield was noted comparatively to the corresponding benzyl adduct 4acb (Table 1; compare entries 13 and 14). Other variables were tested in this microwave reaction outcome. For instance, regarding the 3CPR to access 4aaa, when an excess of 2a (3 equivalents) and 3a (2.4 equivalents) were used, the yield of 4aaa slightly decreased (Table 1, entries 6 and 7). An attempt to reduce the time of the reaction was also evaluated when accessing 4abb. The yield decreased when the reaction was performed in just 1 h (Table 1; compare entries 8 and 9). Setting the reaction time to 1 h, when the amounts of 2b and 3b were reduced to 1.5 and 1.2 equivalents, respectively, the yield of 4abb was 49% (Table 1; compare entries 9 and 10). Other solvents were tested to access 4abb, like DMF and 1,4-dioxane. Similar behavior to that reported previously for 4aba (Table 1, entries 3 and 5) was found for 4abb when 1,4-dioxane was used as a solvent (Table 1; compare entries 9 and 12). The use of DMF as a solvent afforded 4abb in only 11% yield (Table 1, entry 11). In an attempt to decrease the reaction temperature, CH₃CN was used as solvent in the synthesis of 4bcb (Table 1, entries 15–17). Using 80 °C and 2 h of reaction time, the stoichiometric amounts of the components 2c and 3b were evaluated. The use of molecular sieves (MS) as an additive (water scavenger) [18] was also tested in the reaction optimization. We found similar behavior to that reported previously in the yield of 4bcb when low and high amounts of 2c and 3b were used (Table 1; compare entries 15 and 17, respectively).

Our next step was to test the reaction outcome in batch, using the Radley’s^® 12-position carousel system. After recognizing that the reaction does not work at room temperature (even after 7 days stirring, probably due to the less reactive and bulky ketone [15] 1), and based on the results obtained previously using the microwave (Table 1), we start by using N-benzyl isatin 1a, benzoic acid 2c, and cyclohexylisocyanide 3b as components, with aprotic CH₃CN as a solvent [28] to obtain 4acb in several reaction conditions. The results can be seen in

Table 2. Survey of the 3CPR using batch reaction conditions with a Radley’s^® 12-position carousel system.

Entry a	(2a) Equiv.	(3b) Equiv.	Solvent	Additive	Temp./°C	t/h	Yield d/%
1	2.0	1.5	CH3CN	4Å MS b	80	24	34
2	1.3	1.0	CH3CN	4Å MS b	80	16	33
3	2.0	2.0	CH3CN	4Å MS b	80	87	73
4	2.0	2.0	CH3CN	4Å MS b,c	80	15	30
5	2.0	2.0	CH3CN	4Å MS b	100	24	86
6	2.0	2.0	CH3CN	-	100	24	69
7	2.0	2.0	CH3CN	5Å MS	100	24	35
8	2.0	2.0	CH3CN	4Å MS	100	24	83
9	2.0	2.0	MeOH	4Å MS b	100	24	21
10	2.0	2.0	EtOH	4Å MS b	100	24	21
11	2.0	2.0	2-MeTHF	4Å MS b	100	24	74
12	2.0	2.0	TFE	4Å MS b	100	24	70
13	2.0	2.0	CH2Cl2	4Å MS b	60	24	54
14	2.5	2.5	CH3CN	4Å MS b	100	24	55

^a Reaction conditions: 1a (1.0 mmol), 2a, 3b, MS (100 mg) and solvent (3 mL) were added to a Radley’s^® 12-position carousel reactor tube and stirred at the indicated temperature for a certain time. ^b Powder. ^c ZnF₂ (10 mol%) was added to the reaction media. ^d Isolated yield. TFE: 2,2,2-trifluoroethanol.

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The use of 2.0 equivalents of 2 and 3 was established as the optimal stoichiometric amounts for accessing high yields of 4acb (Table 2; compare entries 1, 2 and 3). When a slight excess was used, the yield of 4acb decreased significantly (Table 2; compare entries 5 and 14). Regarding reaction temperature and time, the reaction works better when using 100 °C and 24 h (Table 2, entry 5). No significant differences were noted by using powered or granulated 4Å MS (Table 2; compare entries 5 and 8), unlike the use of different sizes of MS (Table 2, entry 7) as water scavengers. ZnF₂ has proven to be highly efficient as a catalyst in the 4C-Ugi reaction reported by our group. [10,11] We decided to test it in this 3CPR approach. A poor yield of 30% was obtained for 4acb, demonstrating that the reaction works better without a catalyst (Table 2, entry 4). As demonstrated in Table 2 (entries 9 to 13), the solvent had a clear influence on reaction yield. Polar protic solvents like alcohols were not a good choice to access the Passerini adducts, as demonstrated by the low yields of 4acb (Table 2, entries 9 and 10). In our case, the use of high polar protic 2,2,2-trifluoroethanol (TFE) afforded the desired 4acb in 70% yield (Table 2, entry 12). According to the literature [15], typical low polarity aprotic solvents such as CH₂Cl₂, 2-MeTHF, or CH₃CN are usually the ones that provide the best yields of the Passerini adducts, which was demonstrated in this study, since CH₃CN provided the best yield of 4acb (Table 2, entries 5 and 8).

Considering the use of 2.0 equivalents of the component’s acid 2 and isocyanide 3, and CH₃CN (a key solvent in drug development [29]), as the optimized reaction conditions (Table 2, entry 5), we proceed with the study of the substrate scope in order to obtain a structurally versatile library of Passerini adducts 4. A broad selection of in-house synthesized isatin derivatives 1, commercially available carboxylic acids 2, and isocyanides 3 were tested to achieve such a goal (Figure 2 and Scheme 2).

Using benzoic acid 2c and cyclohexyl isocyanide 3b, several isatin derivatives 1 were evaluated in this reaction approach. The best yield of 4 was obtained when N-benzyl isatin 1a was used (Scheme 2, 4acb, 86% yield). Usually, when N1-unsubstituted or C5-substituted 1 was used, very low yields of the corresponding 4 adducts were obtained (see, for instance, Scheme 2; compounds 4ccb, 4dcb, and 4ecb, with 16%, <5%, and <10%, respectively). Compounds 4icb, 4jcb, and 4kcb from N1-aliphatic derivatives of 1 were obtained in modest yields (26%, 50%, and 35%, respectively; Scheme 2). The less bulky 1b (Figure 2) was used for isocyanide and carboxylic acid screenings. A comparison between compound 4bcb and the compounds 4bca, 4bcc, and 4bcd demonstrated the efficiency of the tert-butyl 3a and benzyl 3c isocyanides above the bulky 3b and the electron-withdrawing 3d (Scheme 2; a yield range from 23% to 66%). Regarding carboxylic acid 2 screening, we found out that the 3CPR has a great scope. No significant differences were found regarding ortho-substituted derivatives in benzoic acid derivatives 2 (Figure 2), since the yields of 4 were moderate, ranging from 32% (4bda) to 56% (4bba) (Scheme 2). The best yields were obtained when meta-substituted derivatives were used (4bpa with 83% yield and 4boa with 88% yield) and when the benzoic acid 2 has two substituents in the aromatic ring (4bna with 89% yield, 4bfa with 88% yield, and 4bqa with 83% yield) (Scheme 2). The reaction was not efficient when 2-phenylacetic acid 2h and formic acid 2i were used, obtaining 4bha and 4bia adducts in less than 30% yield. On the contrary, using myristic acid 2j, the corresponding 4bja was obtained in 82% yield (Scheme 2), indicating a good tolerance for aliphatic long-chain carboxylic acids. Also, a good tolerance was established for the use of heterocyclic carboxylic acid components 2k, 2l, 2m and 2u (Figure 2), since the corresponding Passerini adducts 4bka, 4bla, 4bma, and 4bua were obtained in 32%, 70%, 58%, and 83%, respectively (Scheme 2). Interestingly, slight differences were noted in the yield of 4 depending on the position of the substituent in the aromatic ring of 2. The formation of 4 was favored when electron-donating groups like NH₂ or OH were present on the meta-position of the benzoic acid derivative 2 (see, for instance, compounds 4bsa, 4bqa, and 4bpa with 44%, 83%, and 83% of yield, respectively (Scheme 2)), demonstrating electronic effects.

Overall, the versatility of this MCR was demonstrated by the high reagent scope, providing easy access to libraries of α-acyloxyamide-oxindole hybrids 4 in moderate to good yields (Scheme 2). To check the synthetic value of the method, the synthesis of 4bba and 4fba were performed at gram-scale, successfully. The yields obtained for the Passerini adducts were higher for the case of 4bba and consistent for 4fba (Scheme 3).

2.2. Post-Passerini Reaction Approaches

One of the main advantages of using MCR in drug-discovery programs is the high level of scaffold diversity and complexity, in an atom- and step-economy approach. We decided to explore the usefulness of the Passerini adducts 4 in post-Passerini reactions of interest, making them a more druglike species. Taking advantage of the bromoacetic moiety on Passerini-adduct 4avc, we decided to use N,N’-diisopropylethylamine (DIPEA) as the non-nucleophilic base to access spiro-oxindole derivatives, privileged scaffolds for anticancer agents [25,30]. Despite not being optimized, the highly valuable spiro-oxindole 5 was obtained in 10% yield (Scheme 4A). Arylamides are also important building blocks in several biological active molecules. Cross-coupling reactions are indispensable tools and robust transformations for the formation of new C-N bonds. [31] The amination reactions of aryl halides have been impressively explored on the last decades, with the Buchwald–Hartwig [32,33] and the Ullman [34,35] catalyzed coupling reactions being remarkable examples. After several failed attempts using the Pd-catalyzed Buchwald–Hartwig system on the 4bba Passerini adduct, we decided to use the copper-amino acid catalyzed C-N coupling methodology [36] to access the 7-member spiro-oxindole derivative (Scheme 4B). Despite the reaction having failed to reach the 7-member spiro-oxindole, a 5-member spiro-oxindole derivative 6 was obtained instead, in low yield (Scheme 4B). The mechanism is unclear, but we believe that some rearrangement might be involved in the formation of 6.

In an attempt to obtain primary aromatic amines from aromatic halides, using sodium azide, we applied our previous established protocol [7], using the Passerini-adduct 4fba (Scheme 4C). Despite the successful introduction of the amine unit in position C5 of the aromatic ring of 4fba, the harsh reaction conditions led to the cleavage of the ester group of the scaffold [37], leading to the 3-hydroxy-derivative 7, in 13% yield (Scheme 4C).

2.3. Antiproliferative Activity

Next, we ran a preliminary screening to check the biological activity of the Passerini adducts. A panel of six representative human solid-tumor cell lines served as a model to test the antiproliferative activity of the compounds. The panel comprised lung (A549 and SW1573), cervix (HeLa), pancreas (MIA PaCa-2), breast (T-47D), and colon (WiDr) cancer cell lines. The test followed our implementation of the NCI protocol [38]. Thirty-eight samples underwent screening, whilst compounds 4bha, 4bna, 4bpa and 4bta were discarded due to poor solubility under the experimental conditions. The results (Figure 3) revealed that the majority of the compounds were inactive (GI₅₀ > 100 μM against all cell lines).

Table 3. Antiproliferative activity (GI₅₀, μM) of Passerini adducts 4 active against human solid-tumor cell lines ^a.

Compound	A549	HeLa	MIA PaCa-2	SW1573	T-47D	WiDr
4avc	21 ± 2.9	4.3 ± 1.6	2.0 ± 0.4	1.3 ± 0.2	6.3 ± 1.6	20 ± 5.9
4bac	>100	87 ± 19	>100	>100	>100	53 ± 23
4bfa	44 ± 17	36 ± 9.3	58 ± 12	31 ± 7.0	26 ± 5.0	43 ± 16
4bga	70 ± 28	41 ± 7.9	45 ± 8.9	59 ± 16	44 ± 13	41 ± 14
4dcb	59 ± 13	36 ± 4.5	36 ± 4.4	49 ± 12	40 ± 3.2	42 ± 1.4
4faa	78 ± 7.4	31 ± 8.2	84 ± 23	75 ± 26	22 ± 3.8	31 ± 6.8
4fba	48 ± 4.0	40 ± 2.7	37 ± 13	43 ± 18	13 ± 0.4	24 ± 9.7
4fcb	>100	43 ± 8.4	78 ± 38	38 ± 8.8	41 ± 16	60 ± 20
4hcb	32 ± 1.0	24 ± 6.8	27 ± 7.3	31 ± 4.9	25 ± 10	29 ± 6.6
4kcb	79 ± 36	37 ± 0.9	>100	>100	>100	35 ± 6.7
4lcb	15 ± 1.6	26 ± 8.5	6.3 ± 2.1	7.1 ± 2.4	94 ± 11	70 ± 27

^a Values represent mean ± standard deviation of three independent experiments.

shows the antiproliferative effects (50% growth inhibition, GI₅₀) of active compounds. According to the data, compound 4avc was the most potent compound of the series, with GI₅₀ values in the range 1.3–21 μM. The α-bromo ester fragment is essential for the activity, since its modification led to an inactive derivative 5. This result is consistent with a previous report on the antiproliferative effects of Ugi adducts of isatin [21]. When considering selectivity, compound 4avc showed preferential activity against HeLa, MIA PaCa-2, SW1573, and T-47D cells. Interestingly, SW1573 and MIA PaCa-2 cells were more sensitive to 4avc than A549 cells. A difference between these cell lines is the mutation in KRAS protein. The formers are KRAS G12C, whilst the latter is KRAS G12S. This difference was crucial for the different bioactivity profile of the Ugi adducts of isatin [39]. Interestingly, adduct 4lcb also exhibited a preferential activity in SW1573 and MIA PaCa-2 cells. The Ugi adducts exhibited significantly greater differences in GI₅₀ values across KRAS cell lines compared to the Passerini adducts. For example, the GI₅₀ values for Ugi adducts varied from 1.3 μM in A549 cells to 2.3 nM in SW1573 cells and 3.8 nM in MIA PaCa-2 cells. In contrast, the Passerini adducts showed more moderate variation, with GI₅₀ values of 21 μM in A549 cells, 1.3 μM in SW1573 cells, and 2.0 μM in MIA PaCa-2 cells (Table 3). Our results demonstrate that the presence of the α-halo ester on the isatin scaffold is key for the selectivity.

3. Materials and Methods

3.1. General Information

All reagents were obtained from Sigma–Aldrich, Acros, BLDPharm, and TCI, and were used as received. Solvents were used as received. Reactions were conducted in microwave vials, round-bottom flasks, or in a Radley’s^® 12-position carousel reactor. Microwave reactions were conducted with a Biotage Initator+ Microwave System with an automated position system. Column chromatography was carried out on silica gel (Carlo Erba, 40–63 μm, 60Å). Thin-layer chromatography (TLC) was carried out on aluminum-backed Kieselgel 60 F254 plates (Merck and Macherey–Nagel). Plates were visualized either by UV light or with phosphomolybdic acid in ethanol. Melting points (m.p.) were determined with a Barnstead Electothermal 9100 apparatus and are uncorrected. NMR spectra were recorded with a Bruker Avance III instrument (400 MHz). Chemical shifts (δ) are given in parts per million (ppm) with respect to the solvent (CDCl₃, 1H: δ = 7.26 ppm, 13C: δ = 77.2 ppm; DMSO-d6, 1H: δ = 2.50 ppm, 13C: δ = 39.5 ppm). Coupling constants (J) are reported in Hz and refer to apparent peak multiplicities. Splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), br (broad), dd (double of doublets), dt (double of triplets). Mass spectra (MS) were recorded with a quadrupole mass spectrometer Waters ZQ4000 (Chemistry department, University of Salamanca). The ionization was performed by ESI and the samples were infused in methanol.

Isatin derivatives 1c and 1d were commercially available and used as received. The other isatin derivatives were synthesized using our previously reported procedures [40,41,42]. The characterization of compounds 4ccb, 4bcb, 4fcb, 4bca, 4bcd, 4baa, 4bda, 4bea, 4bka, and 4bla can be found in the literature [18,19,20].

3.2. The General Procedure for the Synthesis of α-Acyloxyamide-Oxindole Hybrids 4 Using the 3CPR

Microwave Reactor: In a microwave vial with a magnetic stirrer, the corresponding isatin derivative 1a–l (0.4 mmol or 0.6 mmol), the carboxylic acid 2a–v, the isocyanide 3a–d, and the solvent (3 mL) were added. The vial was closed with a proper cap and the reaction mixture was left stirred at the indicated temperature for a certain time. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified in a short chromatographic glass column with SiO₂ flash using hexane and AcOEt mixtures as the eluents.

Radley’s^® 12-position Carousel: In a glass reactor tube with a magnetic stirrer, the corresponding isatin derivative 1a–l (1.0 mmol), the carboxylic acid 2a–v (2.0 mmol), the isocyanide 3a–d (2.0 mmol), powder 4Å MS (100 mg), and CH₃CN (3 mL) were added. The vial was closed with a proper cap and the reaction mixture was left stirred at 100 °C for 24 h. The solvent was evaporated under reduced pressure, and the crude reaction mixture was purified in a short chromatographic glass column with SiO₂ flash using hexane and AcOEt mixtures as the eluents.

3-(Cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4ccb) [20]: 1c (147.1 mg, 1.0 mmol), 2c (159 mg, 2.0 mmol, 2 equivalents), 3b (124 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4ccb as a white solid (69.4 mg, 16% yield), m.p. = 205.3–298.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 0.83–0.90 (m, 1H, CH₂), 1.28–1.42 (m, 4H, CH₂), 1.60–1.64 (m, 1H, CH₂), 1.71–1.76 (m, 2H, CH₂), 1.95–2.03 (m, 2H, CH₂), 3.81–3.89 (m, 1H, CH), 6.77 (d, 1H, NH), 6.87 (d, J = 8 Hz, 1H, Ar), 7.01 (t, J = 8 Hz, 1H, Ar), 7.24–7.32 (m, 2H, Ar), 7.47 (t, J = 8 Hz, 2H, Ar), 7.61 (t, J = 8 Hz, 1H, Ar), 7.99–8.02 (m, 2H, Ar), 8.48 (s br, 1H, NH). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.75, 24.79, 25.55, 32.65, 32.88, 49.06, 81.27, 110.93, 123.15, 124.39, 125.47, 128.54, 128.80, 130.08, 130.83, 134.09, 142.39, 163.37, 163.90, 172.80. HRMS (ESI) m/z: calculated for C₂₂H₂₂O₄N₂Na [M]⁺ 401.1472, found 401.1465.

3-(Cyclohexylcarbamoyl)-5-methyl-2-oxoindolin-3-yl benzoate (4dcb): 1d (100 mg, 0.62 mmol), 2c (152 mg, 2.0 mmol, 2 equivalents), 3b (149 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4dcb as a white solid (6 mg, <5% yield), m.p. = 139.5–142.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.24–1.36 (m, 5H, CH₂), 1.60–1.65 (m, 1H, CH₂), 1.69–1.78 (m, 2H, CH₂), 1.96–2.05 (m, 2H, CH₂), 2.26 (s, 3H, CH₃), 3.82–3.87 (m, 1H, CH), 6.74–6.80 (m, 2H, NH + Ar), 7.08 (d, J = 8 Hz, 1H, Ar), 7.13 (s, 1H, Ar), 7.46–7.49 (m, 2H, Ar), 7.62 (t, J = 8 Hz, 1H, Ar), 8.01 (d, J = 8 Hz, 2H, Ar), 8.09–8.12 (s br, 1H, NH). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 21.22, 24.76, 24.81, 25.57, 32.68, 32.91, 49.05, 81.33, 110.51, 125.16, 125.41, 128.61, 128.81, 130.10, 131.26, 132.82, 134.08, 139.74, 163.42, 163.89, 172.75. HRMS (ESI) m/z: calculated for C₂₃H₂₄O₄N₂Na [M]⁺ 415.1628, found 415.1621.

5-Bromo-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4hcb): 1h (100 mg, 0.44 mmol), 2c (108 mg, 2.0 mmol, 2 equivalents), 3b (110 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4hcb as a white solid (46 mg, 33% yield), m.p. = 147.3–149.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.32–1.46 (m, 5H, CH₂), 1.62–1.65 (m, 1H, CH₂), 1.72–1.78 (m, 2H, CH₂), 1.95–2.00 (m, 2H, CH₂), 3.80–3.87 (m, 1H, CH), 6.74 (d, J = 8 Hz, 1H, Ar), 6.80 (d, 1H, NH), 7.35–7.40 (m, 2H, Ar), 7.48 (t, J = 8 Hz, 2H, Ar), 7.63 (t, J = 8 Hz, 2H, Ar), 7.98–8.00 (m, 2H, Ar), 8.73 (s, 1H, NH). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.66, 24.71, 25.41, 32.53, 32.77, 49.18, 80.85, 112.42, 115.53, 127.22, 127.32, 128.81, 130.02, 133.56, 134.26, 141.47, 162.90, 163.75, 172.23. HRMS (ESI) m/z: calculated for C₂₂H₂₁O₄N₂BrNa [M]⁺ 479.0577, found 479.0568.

3-(Cyclohexylcarbamoyl)-1-methyl-2-oxoindolin-3-yl benzoate (4bcb) [19]: 1b (100 mg, 0.62 mmol), 2c (152 mg, 2.0 mmol, 2 equivalents), 3b (149 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bcb as a yellow solid (96 mg, 40% yield), m.p. = 145.7–149.2 °C.

3-(Cyclohexylcarbamoyl)-1,5-dimethyl-2-oxoindolin-3-yl benzoate (4ecb): 1e (100 mg, 0.57 mmol), 2c (139 mg, 2.0 mmol, 2 equivalents), 3b (136 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4ecb as a white solid (12 mg, <10% yield), m.p. = 178.7–181.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.22–1.42 (m, 5H, CH₂), 1.60–1.65 (m, 1H, CH₂), 1.71–1.78 (m, 2H, CH₂), 1.96–2.04 (m, 2H, CH₂), 2.29 (s, 3H, CH₃), 3.30 (s, 3H, CH₃), 3.79–3.86 (m, 1H, CH), 6.78–6.82 (m, 2H, NH + Ar), 7.16–7.18 (m, 2H, Ar), 7.47 (t, J = 8 Hz, 2H, Ar), 7.61 (t, J = 8 Hz, 1H, Ar), 7.99 (d, J = 8 Hz, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 21.21, 24.75, 24.81, 25.59, 27.09, 32.68, 32.92, 49.03, 81.15, 108.66, 124.93, 125.12, 128.69, 128.78, 130.05, 131.18, 132.91, 134.01, 142.81, 163.45, 163.81, 171.48. HRMS (ESI) m/z: calculated for C₂₄H₂₆O₄N₂Na [M]⁺ 429.1785, found 429.1775.

5-Bromo-3-(cyclohexylcarbamoyl)-1-methyl-2-oxoindolin-3-yl benzoate (4fcb) [18]: 1f (100 mg, 0.42 mmol), 2c (102 mg, 2.0 mmol, 2 equivalents), 3b (103 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4fcb as a pale yellow solid (52 mg, 22% yield), m.p. = 190.1–192.3 °C. HRMS (ESI) m/z: calculated for C₂₃H₂₃O₄N₂BrNa [M]⁺ 493.07334, found 493.0725.

1-Benzyl-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4acb): 1a (243 mg, 1.0 mmol), 2c (244 mg, 2.0 mmol, 2 equivalents), 3b (250 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4acb as a pale yellow solid (384 mg, 86% yield), m.p. = 140.0–142.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.28–1.40 (m, 5H, CH₂), 1.62–1.66 (m, 1H, CH₂), 1.74–1.79 (m, 2H, CH₂), 2.02–2.04 (m, 2H, CH₂), 3.84–3.93 (m, 1H, CH), 4.95 (d, J = 16 Hz, 1H, CH₂), 5.14 (d, J = 16 Hz, 1H, CH₂), 6.69 (d, J = 8 Hz, 1H, Ar), 6.79–6.81 (s br, 1H, NH), 7.01 (t, J = 8 Hz, 1H, Ar), 7.22 (t, J = 8 Hz, 1H, Ar), 7.26–7.38 (m, 4H, Ar), 7.44–7.51 (m, 4H, Ar), 7.63 (t, J = 8 Hz, 1H, Ar), 8.02 (d, J = 8 Hz, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.76, 24.78, 25.57, 32.76, 32.98, 44.47, 48.93, 81.25, 110.10, 123.24, 123.76, 127.15, 127.67, 128.81, 128.96, 130.06, 130.70, 134.07, 135.25, 144.26, 163.54, 163.64, 171.63. HRMS (ESI) m/z: calculated for C₂₉H₂₈O₄N₂Na [M]⁺ 491.1941, found 491.1931.

5-Bromo-3-(cyclohexylcarbamoyl)-1-(3-methoxybenzyl)-2-oxoindolin-3-yl benzoate (4gcb): 1g (100 mg, 0.28 mmol), 2c (70 mg, 2.0 mmol, 2 equivalents), 3b (72 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4gcb as a white solid (67 mg, 41% yield), m.p. = 154.6–157.3 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.24–1.45 (m, 5H, CH₂), 1.62–1.66 (m, 1H, CH₂), 1.75–1.78 (m, 2H, CH₂), 2.00–2.05 (m, 2H, CH₂), 3.74 (s, 3H, OCH₃), 3.83–3.91 (m, 1H, CH), 5.09 (s, 2H, CH₂), 6.68–6.73 (m, 2H, Ar), 6.81 (d, 1H, NH), 7.05 (t, J = 8 Hz, 1H, Ar), 7.14 (d, J = 4 Hz, 1H, Ar), 7.27 (t, J = 8 Hz, 1H, Ar), 7.32 (d, J = 8 Hz, 1H, Ar), 7.44–7.51 (m, 3H, Ar), 7.64 (t, J = 8 Hz, 1H, Ar), 7.98–8.01 (m, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.71, 24.75, 25.52, 32.70, 32.92, 44.59, 48.93, 55.76, 81.40, 109.93, 112.51, 112.58, 116.39, 123.43, 125.26, 128.43, 128.86, 129.91, 130.77, 133.29, 134.17, 135.03, 143.89, 159.72, 163.48, 163.58, 171.51. HRMS (ESI) m/z: calculated for C₃₀H₂₉O₅N₂BrNa [M]⁺ 599.1152, found 599.1143.

1-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4lcb): 1l (100 mg, 0.31 mmol), 2c (76 mg, 2.0 mmol, 2 equivalents), 3b (78 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4lcb as a pale yellow solid (90 mg, 52% yield), m.p. = 168.1–173.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.27–1.42 (m, 5H, CH₂), 1.61–1.65 (m, 1H, CH₂), 1.73–1.76 (m, 2H, CH₂), 1.95–2.01 (m, 2H, CH₂), 3.79–3.86 (m, 1H, CH), 5.17 (s br, 2H, CH₂), 5.45–5.53 (m, 2H, CH₂), 6.75 (d, 1H, NH), 6.92–6.93 (m, 1H, Ar), 7.02 (t, J = 8 Hz, 1H, Ar), 7.21–7.33 (m, 8H, CH + Ar), 7.48 (t, J = 8 Hz, 2H, Ar), 7.63 (t, J = 8 Hz, 1H, Ar), 7.91–7.94 (m, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.76, 24.78, 25.54, 32.69, 32.91, 49.00, 81.17, 110.13, 123.44, 123.58, 125.14, 128.16, 128.44, 128.71, 128.82, 129.11, 129.99, 130.85, 134.18, 134.58, 143.56, 163.42, 163.65, 171.11. HRMS (ESI) m/z: calculated for C₃₂H₃₁O₄N₅Na [M]⁺ 572.2268, found 572.2256.

1-Allyl-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4icb): 1i (100 mg, 0.53 mmol), 2c (130 mg, 2.0 mmol, 2 equivalents), 3b (132 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4icb as a white solid (57 mg, 26% yield), m.p. = 136.5–138.9 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.21–1.43 (m, 5H, CH₂), 1.60–1.64 (m, 1H, CH₂), 1.72–1.77 (m, 2H, CH₂), 1.96–2.04 (m, 2H, CH₂), 3.80–3.89 (m, 1H, CH), 4.38–4.49 (m, 2H, CH₂), 5.28 (d, J = 12 Hz, 1H, CH₂), 5.46 (d, J = 20 Hz, 1H, CH₂), 5.87–5.96 (m, 1H, CH), 6.77 (d, 1H, NH), 6.89 (d, J = 8 Hz, 1H, Ar), 7.04 (t, J = 8 Hz, 1H, Ar), 7.31–7.35 (m, 2H, Ar), 7.47 (t, J = 8 Hz, 2H, Ar), 7.61 (t, J = 8 Hz, 1H, Ar), 7.98–8.01 (m, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.72, 24.75, 25.53, 32.68, 32.91, 42.89, 48.92, 81.10, 109.84, 117.87, 123.14, 123.84, 125.22, 128.57, 128.76, 129.99, 130.66, 134.01, 144.34, 163.44, 163.60, 171.23. HRMS (ESI) m/z: calculated for C₂₅H₂₆O₄N₂Na [M]⁺ 441.1785, found 441.1776.

(E)-1-(But-2-en-1-yl)-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl benzoate (4jcb): 1j (100 mg, 0.49 mmol), 2c (121 mg, 2.0 mmol, 2 equivalents), 3b (123 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4jcb as a white solid (105 mg, 50% yield), m.p. = 130.1–133.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.24–1.43 (m, 5H, CH₂), 1.60–1.64 (m, 1H, CH₂), 1.71–1.77 (m, 5H, CH₃ + CH₂), 1.95–2.02 (m, 2H, CH₂), 3.80–3.87 (m, 1H, CH), 4.31–4.43 (m, 2H, CH₂), 5.50–5.57 (m, 1H, CH), 5.82–5.88 (m, 1H, CH), 6.76 (d, 1H, NH), 6.91 (d, J = 8 Hz, 1H, Ar), 7.04 (t, J = 8 Hz, 1H, Ar), 7.31–7.35 (m, 2H, Ar), 7.45–7.48 (m, 2H, Ar), 7.61 (t, J = 8 Hz, 1H, Ar), 7.99 (d, J = 8 Hz, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 17.91, 24.73, 24.76, 25.57, 32.69, 32.92, 42.46, 48.92, 81.07, 109.93, 123.06, 123.72, 124.03, 125.20, 128.66, 128.76, 129.34, 130.03, 130.66, 133.98, 144.52, 163.42, 163.67, 171.17. HRMS (ESI) m/z: calculated for C₂₆H₂₈O₄N₂Na [M]⁺ 455.1941, found 455.1934.

3-(Cyclohexylcarbamoyl)-2-oxo-1-(prop-2-yn-1-yl)indolin-3-yl benzoate (4kcb): 1k (100 mg, 0.54 mmol), 2c (132 mg, 2.0 mmol, 2 equivalents), 3b (134 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4kcb as a white solid (81 mg, 35% yield), m.p. = 145.7–147.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.23–1.40 (m, 5H, CH₂), 1.59–1.63 (m, 1H, CH₂), 1.70–1.74 (m, 2H, CH₂), 1.94–2.01 (m, 2H, CH₂), 2.33–2.34 (m, 1H, CH), 3.80–3.87 (m, 1H, CH), 4.53–4.62 (m, 2H, CH₂), 6.75 (d, 1H, NH), 7.08 (t, J = 8 Hz, 1H, Ar), 7.13 (d, J = 8 Hz, 1H, Ar), 7.35–7.47 (m, 4H, Ar), 7.57–7.61 (m, 1H, Ar), 7.99 (d, J = 8 Hz, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.59, 24.62, 25.39, 29.92, 32.49, 32.71, 48.87, 72.93, 76.11, 80.77, 109.94, 123.47, 124.03, 124.91, 128.26, 128.65, 129.91, 130.70, 133.97, 143.23, 162.95, 163.62, 170.54. HRMS (ESI) m/z: calculated for C₂₅H₂₄O₄N₂Na [M]⁺ 439.1628, found 439.1622.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl benzoate (4bca) [19]: 1b (100 mg, 0.62 mmol), 2c (152 mg, 1.2 mmol, 2 equivalents), 3a (140 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bca as a pale yellow solid (142 mg, 63% yield), m.p. = 144.3–146.7 °C.

3-(Benzylcarbamoyl)-1-methyl-2-oxoindolin-3-yl benzoate (4bcc): 1b (108.8 mg, 0.67 mmol), 2c (152 mg, 1.2 mmol, 2 equivalents), 3c (151 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bcc as a white solid (177 mg, 66% yield), m.p. = 180.3–182.9 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 3.33 (s, 3H, CH₃), 4.52 (dd, J = 6 Hz, 2H, Ar), 4.64 (dd, J = 6 Hz, 2H, Ar), 6.94 (d, J = 8 Hz, 1H, Ar), 7.06–7.10 (m, 1H, Ar), 7.31–7.45 (m, 10H, Ar + NH), 7.57–7.61 (m, 1H, Ar), 7.95–7.98 (m, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.06, 43.93, 81.12, 109.03, 123.36, 124.32, 124.88, 127.66, 127.79, 128.39, 128.74, 128.93, 130.03, 131.00, 134.06, 137.57, 145.24, 163.79, 164.46, 171.36. HRMS (ESI) m/z: calculated for C₂₄H₂₀O₄N₂Na [M]⁺ 423.1315, found 423.1306.

3-((2-Ethoxy-2-oxoethyl)carbamoyl)-1-methyl-2-oxoindolin-3-yl benzoate (4bcd) [19]: 1b (100 mg, 0.62 mmol), 2c (152 mg, 1.2 mmol, 2 equivalents), 3d (135 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bcd as a white solid (57 mg, 23% yield), m.p. = 132.5–135.9 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-bromobenzoate (4baa) [19]: 1b (172 mg, 1.1 mmol), 2a (402 mg, 2.0 mmol, 2 equivalents), 3a (200 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4baa as a white solid (163 mg, 35% yield), m.p. = 158.9–160.1 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-iodobenzoate (4bba): 1b (170 mg, 1.1 mmol), 2b (496 mg, 2.0 mmol, 2 equivalents), 3a (200 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bba as a white solid (209 mg, 56% yield), m.p. = 192.4–196.3 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.41 (s, 9H, 3H), 3.31 (s, 3H, CH₃), 6.91 (d, J = 8 Hz, 1H, Ar), 7.01 (s br, 1H, NH), 7.09 (t, J = 8 Hz, 1H, Ar), 7.20 (t, J = 8 Hz, 1H, Ar), 7.35–7.44 (m, 3H, Ar), 7.74 (d, J = 8 Hz, 1H, Ar), 7.98 (d, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.02, 28.76, 52.43, 82.12, 93.24, 109.01, 123.19, 124.01, 125.18, 128.38, 130.84, 132.39, 132.57, 133.44, 141.35, 145.55, 163.26, 163.73, 171.12. HRMS (ESI) m/z: calculated for C₂₁H₂₁O₄N₂INa [M]⁺ 515.0438, found 515.0433.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-chlorobenzoate (4bda) [19]: 1b (100 mg, 0.62 mmol), 2d (307 mg, 1.2 mmol, 2 equivalents), 3a (140 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bda as a white solid (80 mg, 32% yield), m.p. = 149.1–152.4 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-fluorobenzoate (4bea) [19]: 1b (100 mg, 0.62 mmol), 2e (174 mg, 1.2 mmol, 2 equivalents), 3a (140 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bea as a white solid (92 mg, 39% yield), m.p. = 161.5–163.4 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 3-hydroxybenzoate (4bpa): 1b (100 mg, 0.62 mmol), 2p (171 mg, 1.2 mmol, 2 equivalents), 3a (140 μL, 1.2 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bpa as a white solid (196 mg, 83% yield), m.p. = 245.7–247.2 °C. ¹H NMR (DMSO-d6, 400 MHz) δ: 1.34 (s, 9H, CH₃), 3.18 (s, 3H, CH₃), 7.02–7.12 (m, 3H, Ar), 7.36–7.40 (m, 3H, Ar), 7.46–7.52 (m, 2H, Ar), 7.62 (s br, 1H, NH), 10.03 (s br, 1H, OH). ¹³C APT NMR (DMSO-d6, 100 MHz) δ: 26.53, 28.28, 51.55, 81.25, 109.06, 116.25, 120.66, 121.39, 122.62, 122.92, 125.89, 129.18, 130.12, 130.44, 145.21, 157.61, 163.26, 164.08, 170.98. HRMS (ESI) m/z: calculated for C₂₁H₂₂O₅N₂Na [M]⁺ 405.1421, found 405.1413.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 3-bromobenzoate (4boa): 1b (100 mg, 0.62 mmol), 2o (250 mg, 1.24 mmol, 2 equivalents), 3a 140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4boa as a white solid (234 mg, 88% yield), m.p. = 201.7–204.7 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.42 (s, 9H, CH₃), 3.31 (s, 3H, CH₃), 6.75 (s br, 1H, NH), 6.91 (d, J = 8 Hz, 1H, Ar), 7.08 (t, J = 8 Hz, 1H, Ar), 7.36–7.40 (m, 2H, Ar), 7.59–7.61 (m, 2H, Ar), 7.82–7.84 (m, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.05, 28.66, 52.28, 81.25, 108.95, 123.39, 124.49, 124.77, 127.49, 129.32, 130.93, 131.40, 132.17, 145.14, 162.87, 163.18, 171.58. HRMS (ESI) m/z: calculated for C₂₁H₂₁O₄N₂BrNa [M]⁺ 467.0577, found 467.0571.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-aminobenzoate (4bta): 1b (100 mg, 0.62 mmol), 2t (170 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bta as a pale yellow solid (78 mg, 33% yield), m.p. = 264.8–267.1 °C. ¹H NMR (DMSO-d6, 400 MHz) δ: 1.32 (s, 9H, CH₃), 3.18 (s, 3H, CH₃), 6.53 (s br, 2H, NH₂), 6.62 (t, J = 8 Hz, 1H, Ar), 6.76 (d, J = 8 Hz, 1H, Ar), 7.02–7.08 (m, 2H, Ar), 7.28–7.33 (m, 1H, Ar), 7.37 (t, J = 8 Hz, 1H, Ar), 7.49 (d, J = 8 Hz, 1H, Ar), 7.53 (s br, 1H, NH). ¹³C APT NMR (DMSO-d6, 100 MHz) δ: 26.52, 28.26, 51.49, 80.79, 106.76, 108.99, 114.94, 116.69, 122.52, 122.78, 126.27, 130.23, 131.37, 135.08, 145.10, 152.07, 164.30, 164.56, 171.29. HRMS (ESI) m/z: calculated for C₂₁H₂₃O₄N₃Na [M]⁺ 404.1581, found 404.1573.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 3-aminobenzoate (4bsa): 1b (100 mg, 0.62 mmol), 2s (170 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bsa as a pale yellow solid (105 mg, 44% yield), m.p. = 174.7–176.9 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.42 (s, 9H, CH₃), 3.30 (s, 3H, CH₃), 6.78 (s br, 1H, NH), 6.87–6.91 (m, 2H, Ar), 7.05 (t, J = 8 Hz, 1H, Ar), 7.20–7.24 (m, 2H, Ar), 7.32–7.38 (m, 3H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.03, 28.71, 52.22, 81.17, 108.88, 116.04, 119.79, 120.38, 123.27, 124.13, 125.22, 129.52, 129.65, 130.78, 145.28, 146.84, 163.39, 163.93, 171.73. HRMS (ESI) m/z: calculated for C₂₁H₂₃O₄N₃Na [M]⁺ 404.1581, found 404.1575.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2,4-dibromobenzoate (4bna): 1b (100 mg, 0.62 mmol), 2n (347 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bna as a white solid (290 mg, 89% yield), m.p. = 193.2–195.7 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.43 (s, 9H, CH₃), 3.31 (s, 3H, CH₃), 6.76 (s br, 1H, NH), 6.92 (d, J = 8 Hz, 1H, Ar), 7.10 (t, J = 8 Hz, 1H, Ar), 7.38–7.41 (m, 1H, Ar), 7.87–7.88 (m, 1H, Ar), 8–02 (s, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.11, 28.65, 29.83, 52.44, 81.53, 109.06, 123.38, 123.60, 124.35, 124.88, 131.14, 131.72, 131.74, 131.86, 138.85, 139.25, 145.12, 161.56, 162.49, 171.36. HRMS (ESI) m/z: calculated for C₂₁H₂₀O₄N₂Br₂Na [M]⁺ 544.9682, found 544.9672.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 4-aminobenzoate (4bra): 1b (100 mg, 0.62 mmol), 2r (170 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bra as a white solid (48 mg, 20% yield), m.p. = 169.3–170.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.42 (s, 9H, CH₃), 3.29 (s, 3H, CH₃), 4.27 (s br, 2H, NH₂), 6.58 (d, J = 8 Hz, 2H, Ar), 6.78 (s br, 1H, NH), 6.88 (d, J = 8 Hz, 1H, Ar), 7.04 (t, J = 8 Hz, 1H, Ar), 7.29 (d, J = 8 Hz, 1H, Ar), 7.35 (t, J = 8 Hz, 1H, Ar), 7.72 (d, J = 8 Hz, 2H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 26.97, 28.69, 52.11, 80.90, 108.83, 113.84, 117.16, 123.12, 123.73, 125.77, 130.58, 132.12, 145.24, 152.11, 163.62, 163.84, 172.11. HRMS (ESI) m/z: calculated for C₂₁H₂₃O₄N₃Na [M]⁺ 404.1581, found 404.1574.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 4-fluoro-2-iodobenzoate (4bfa): 1b (100 mg, 0.62 mmol), 2f (330 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bfa as a pale yellow solid (278 mg, 88% yield), m.p. = 166.1–169.5 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.41 (s, 9H, CH₃), 3.32 (s, 3H, CH₃), 6.91 (d, J = 8 Hz, 1H, Ar), 6.97 (s br, 1H, NH), 7.07–7.14 (m, 2H, Ar), 7.36–7.43 (m, 2H, Ar), 7.71 (dd, J = 8 Hz, 1H, Ar), 7.78–7.82 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.08, 28.78, 52.51, 82.15, 93.85, 93.94, 109.09, 115.69, 115.90, 123.33, 124.24, 125.01, 128.65, 128.89, 130.94, 134.19, 134.28, 145.48, 162.66, 162.86, 163.06, 165.24, 171.22. HRMS (ESI) m/z: calculated for C₂₁H₂₀O₄N₂FINa [M]⁺ 533.0344, found 533.0334.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-bromo-4-(trifluoromethyl)benzoate (4bga): 1b (100 mg, 0.62 mmol), 2g (334 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bga as a pale yellow solid (197 mg, 61% yield), m.p. = 149.8–152.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.40 (s, 9H, CH₃), 3.32 (s, 3H, CH₃), 6.93 (d, J = 8 Hz, 1H, Ar), 6.97 (s br, 1H, NH), 7.10 (t, J = 8 Hz, 1H, Ar), 7.35–7.40 (m, 2H, Ar), 7.65 (d, J = 8 Hz, 1H, Ar), 7.89 (d, J = 8 Hz, 1H, Ar), 7.94 (s, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 26.99, 28.60, 29.72, 52.40, 82.28, 109.08, 121.46, 123.33, 123.76, 124.56, 124.69, 131.04, 131.38, 132.17, 133.07, 145.46, 162.26, 162.89, 170.78. HRMS (ESI) m/z: calculated for C₂₂H₂₀O₄N₂BrF₃Na [M]⁺ 535.0451, found 535.0443.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 3-hydroxy-4-methoxybenzoate (4bqa): 1b (100 mg, 0.62 mmol), 2q (208 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bqa as a pale yellow solid (196 mg, 83% yield), m.p. = 148.8–151.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.44 (s, 9H, CH₃), 3.30 (s, 3H, CH₃), 3.92 (s, 3H, OCH₃), 5.99 (s br, 1H, OH), 6.82–6.84 (m, 2H, NH + Ar), 6.89 (d, J = 8 Hz, 1H, Ar), 7.05 (t, J = 8 Hz, 1H, Ar), 7.31 (d, J = 8 Hz, 1H, Ar), 7.35 (t, J = 8 Hz, 1H, Ar), 7.46 (d, J = 4 Hz, 1H, Ar), 7.49–7.52 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.03, 28.70, 52.22, 56.19, 81.21, 108.87, 110.16, 115.78, 121.45, 123.25, 123.51, 123.98, 125.42, 130.70, 145.25, 145.56, 151.36, 163.35, 163.49, 171.90. HRMS (ESI) m/z: calculated for C₂₂H₂₄O₆N₂Na [M]⁺ 435.1527, found 435.1519.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-phenylacetate (4bha): 1b (100 mg, 0.62 mmol), 2h (168 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bha as a white solid (36 mg, <5% yield), m.p. = 210.8–203.6 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.27 (s, 9H, CH₃), 3.23 (s, 3H, CH₃), 3.73 (d, J = 4 Hz, 2H, CH₂), 6.29 (s br, 1H, NH), 6.84 (d, J = 8 Hz, 1H, Ar), 7.02 (d, J = 8 Hz, 1H, Ar), 7.07–7.09 (m, 1H, Ar), 7.25–7.27 (m, 2H, Ar), 7.28–7.39 (m, 4H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 26.91, 28.55, 41.10, 51.98, 81.10, 108.92, 123.06, 123.11, 125.28, 127.62, 128.99, 129.35, 130.76, 133.13, 145.39, 163.34, 168.01, 171.25. HRMS (ESI) m/z: calculated for C₂₂H₂₄O₄N₂Na [M]⁺ 403.1628, found 403.1619.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl formate (4bia): 1b (100 mg, 0.62 mmol), 2i (46.8 µL, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bia as a white solid (53 mg, 29% yield), m.p. = 163.4–165.7 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.38 (s, 9H, CH₃), 3.25 (s, 3H, CH₃), 6.67 (s br, 1H, NH), 6.87 (d, J = 8 Hz, 1H, Ar), 7.07 (t, J = 8 Hz, 1H, Ar), 7.25–7.27 (m, 1H, Ar), 7.34–7.39 (m, 1H, Ar), 7.97 (s, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 26.97, 28.60, 52.30, 80.77, 109.05, 123.34, 123.61, 124.78, 131.01, 145.22, 157.20, 162.92, 170.79. HRMS (ESI) m/z: calculated for C₁₅H₁₈O₄N₂Na [M]⁺ 313.1159, found 313.1153.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl tetradecanoate (4bja): 1b (100 mg, 0.62 mmol), 2j (283 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bja as a white solid (233 mg, 82% yield), m.p. = 136.3–138.8 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 0.86–0.89 (m, 3H, CH₃), 1.24–1.25 (m, 20H, CH₂), 1.38 (s, 9H, CH₃), 2.34–2.48 (m, 2H, CH₂), 3.25 (s, 3H, CH₃), 6.64 (s br, 1H, NH), 6.85 (d, J = 8 Hz, 1H, Ar), 7–05 (t, J = 8 Hz, 1H, Ar), 7.23 (d, J = 8 Hz, 1H, Ar), 7.34 (t, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 14.25, 22.80, 24.72, 26.93, 28.63, 28.95, 29.32, 29.46, 29.50, 29.68, 29.75, 29.77, 32.03, 33.93, 52.10, 80.88, 108.89, 123.14, 123.38, 125.54, 130.65, 145.30, 163.42, 170.69, 171.71. HRMS (ESI) m/z: calculated for C₂₈H₄₄O₄N₂Na [M]⁺ 495.3193, found 495.3186.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl picolinate (4bka) [19]: 1b (100 mg, 0.62 mmol), 2k (153 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bka as a white solid (83 mg, 32% yield), m.p. = 161.5–163.0 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl furan-2-carboxylate (4bla) [19]: 1b (100 mg, 0.62 mmol), 2l (139 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bla as a pale yellow solid (154 mg, 70% yield), m.p. = 193.0–195.6 °C.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 5-nitrofuran-2-carboxylate (4bma): 1b (100 mg, 0.62 mmol), 2m (190 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bma as a pale yellow solid (133 mg, 58% yield), m.p. = 210.1–212.8 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.44 (s, 9H, CH₃), 3.29 (s, 3H, CH₃), 6.86 (s br, 1H, NH), 6.92 (d, J = 8 Hz, 1H, Ar), 7.08 (t, J = 8 Hz, 1H, Ar), 7.29–7.34 (m, 3H, Ar), 7.40 (t, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.05, 28.59, 52.42, 81.66, 109.18, 111.66, 120.65, 123.50, 123.83, 124.27, 131.39, 143.01, 145.50, 153.56, 162.58, 170.32. HRMS (ESI) m/z: calculated for C₁₉H₁₉O₇N₃Na [M]⁺ 424.1115, found 424.1104.

1-Benzyl-3-(benzylcarbamoyl)-5-bromo-2-oxoindolin-3-yl 2-bromobenzoate (4mac): 1m (171 mg, 0.5 mmol), 2a (201 mg, 1.0 mmol, 2 equivalents), 3c (123 μL, 1.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4mac as a pale yellow solid (229 mg, 67% yield), m.p. = 133.8–135.0 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 4.52–4.64 (m, 2H, CH₂), 4.96–5.12 (m, 2H, CH₂), 6.57 (d, J = 8 Hz, 1H, Ar), 7.29–7.40 (m, 10H, Ar), 7.44–7.45 (m, 3H, Ar), 7.51–7.53 (m, 1H, Ar), 7.64–7.66 (m, 1H, Ar), 7.83–7.85 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 44.18, 44.66, 81.82, 111.77, 115.89, 121.58, 126.77, 127.06, 127.20, 127.81, 127.88, 127.97, 128.03, 129.05, 130.11, 133.23, 133.68, 134.04, 134.69, 134.72, 137.13, 143.70, 163.05, 163.99, 170.44. HRMS (ESI) m/z: calculated for C₃₀H₂₂O₄N₂Br₂Na [M]⁺ 654.9839, found 654.9830.

3-(tert-Butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 1H-pyrrole-2-carboxylate (4bua): 1b (100 mg, 0.62 mmol), 2u (138 mg, 1.24 mmol, 2 equivalents), 3a (140 μL, 1.24 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4bua as a white solid (196 mg, 83% yield), m.p. = 196.1–199.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.41 (s, 9H, CH₃), 3.25 (s, 3H, CH₃), 6.24–6.26 (m, 1H, Ar), 6.77 (s br, 1H, NH), 6.85–6.87 (m, 2H, Ar), 6.95–6.96 (m, 1H, Ar), 7.02–7.06 (m, 1H, Ar), 7.28–7.37 (m, 2H, Ar), 9.49 (s br, 1H, NH). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 26.94, 28.64, 52,15, 80.88, 108.81, 110.81, 114.89, 116.71, 120.67, 123.18, 123.59, 123.79, 125.23, 125.57, 130.70, 145.74, 158.24, 163.47, 171.74. HRMS (ESI) m/z: calculated for C₁₉H₂₁O₄N₃Na [M]⁺ 378.1424, found 378.1417.

1-Benzyl-3-(tert-butylcarbamoyl)-2-oxoindolin-3-yl 2-iodobenzoate (4aba): 1a (102 mg, 0.4 mmol), 2b (209 mg, 0.8 mmol, 2 equivalents), 3a (95 μL, 0.8 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4aba as a pale yellow solid (74 mg, 30% yield), m.p. = 144.0–145.8 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.43 (s, 9H, CH₃), 5.05 (q, J = 16 Hz, 2H, CH₂), 6.68 (d, J = 8 Hz, 1H, Ar), 7.02–7.06 (m, 2H, NH + Ar), 7.19–7.24 (m, 2H, Ar), 7.28 (d, J= 8 Hz, 1H, Ar), 7.33–7.37 (m, 2H, Ar), 7.42–7.47 (m, 4H, Ar), 7.80 (dd, J = 8 Hz, 1H, Ar), 8.00 (dd, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 28.85, 44.51, 52.49, 82.35, 93.37, 110.21, 123.22, 123.89, 125.31, 127.27, 127.64, 128.43, 128.92, 130.69, 132.49, 133.62, 134.45, 135.27, 141.43, 144.61, 163.33, 163.73, 171.28. HRMS (ESI) m/z: calculated for C₂₇H₂₅O₄N₂INa [M]⁺ 591.0751, found 591.0743.

1-Benzyl-3-(cyclohexylcarbamoyl)-2-oxoindolin-3-yl 2-iodobenzoate (4abb): 1a (113 mg, 0.47 mmol), 2b (209 mg, 0.8 mmol, 2 equivalents), 3b (104 μL, 0.8 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4abb as a pale yellow solid (91 mg, 31% yield), m.p. = 155.2–157.4 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.22–1.39 (m, 5H, CH₂), 1.61–1.64 (m, 1H, CH₂), 1.73–1.76 (m, 2H, CH₂), 1.98–2.00 (m, 2H, CH₂), 3.82–3.89 (m, 1H, CH), 4.96 (d, J = 16 Hz, 1H, CH₂), 5.14 (d, J = 16 Hz, 1H, CH₂), 6.67 (d, J = 8 Hz, 1H, Ar), 7.01–7.08 (m, 2H, NH + Ar), 7.20–7.28 (m, 3H, Ar), 7.33–7.37 (m, 2H, Ar), 7.40–7.48 (m, 4H, Ar), 7.80 (dd, J = 8 Hz, 1H, Ar), 8.01 (dd, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.89, 24.91, 25.56, 32.94, 33.20, 44.50, 49.14, 82.25, 93.34, 110.22, 123.18, 123.76, 125.30, 127.20, 127.64, 128.45, 128.94, 130.75, 132.61, 133.66, 134.47, 135.25, 141.43, 144.61, 163.50, 163.72, 171.10. HRMS (ESI) m/z: calculated for C₂₉H₂₇O₄N₂INa [M]⁺ 617.0908, found 617.0895.

1-Benzyl-3-(tert-butylcarbamoyl)-2-oxoindolin-3-yl 2-bromobenzoate (4aaa): 1a (101 mg, 0.4 mmol), 2a (169 mg, 0.8 mmol, 2 equivalents), 3a (95 μL, 0.8 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4aaa as a pale yellow solid (115 mg, 52% yield), m.p. = 175.8–176.0 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.43 (s, 9H, CH₃), 5.05 (s, 2H, CH₂), 6.67 (d, J = 8 Hz, 1H, Ar), 7.03 (t, J = 8 Hz, 1H, Ar), 7.13 (s br, 1H, NH), 7.21 (dt, J = 8 Hz, 1H, Ar), 7.28 (d, J = 8 Hz, 1H, Ar), 7.33–7.37 (m, 3H, Ar), 7.40–7.42 (m, 2H, Ar), 7.45–7.47 (m, 2H, Ar), 7.69–7.71 (m, 1H, Ar), 7.83–7.86 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 28.82, 44.52, 52.40, 82.41, 110.23, 121.30, 123.23, 123.39, 125.45, 127.24, 127.63, 127.83, 128.93, 130.71, 130.78, 133.27, 133.83, 134.60, 135.29, 144.70, 163.16, 163.53, 171.21. HRMS (ESI) m/z: calculated for C₂₇H₂₅O₄N₂BrNa [M]⁺ 543.0889, found 543.0883.

1-Benzyl-3-(benzylcarbamoyl)-2-oxoindolin-3-yl 2-iodobenzoate (4abc): 1a (420 mg, 1.8 mmol), 2b (892 mg, 3.6 mmol, 2 equivalents), 3c (438 μL, 3.6 mmol, 2 equivalents), powder 4Å MS (200 mg), and CH₃CN (6 mL) were used to obtain the corresponding 4abc as a white solid (152 mg, 14% yield), m.p. = 142.8–145.3 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 4.49–4.54 (m, 1H, CH₂), 4.62–4.67 (m, 1H, CH₂), 5.06 (q, J = 16 Hz, 2H, CH₂), 6.71 (d, J = 8 Hz, 1H, Ar), 7.05 (t, J = 8 Hz, 1H, Ar), 7.18 (t, J = 8 Hz, 1H, Ar), 7.24–7.43 (m, 11H, Ar), 7.48 (d, J = 8 Hz, 2H, Ar), 7.77–7.80 (m, 1H, Ar), 7.95 (d, J = 8 Hz, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 44.06, 44.57, 93.46, 110.30, 123.29, 123.96, 125.03, 127.25, 127.69, 127.92, 128.06, 128.36, 128.96, 130.90, 132.47, 133.63, 134.18, 135.17, 137.37, 141.44, 144.61, 163.64, 164.45, 170.97. HRMS (ESI) m/z: calculated for C₃₀H₂₃O₄N₂INa [M]⁺ 625.0595, found 625.0584.

1-Benzyl-3-(benzylcarbamoyl)-2-oxoindolin-3-yl 2-bromoacetate (4avc): 1a (565 mg, 2.4 mmol), 2v (667 mg, 4.8 mmol, 2 equivalents), 3c (585 μL, 4.8 mmol, 2 equivalents), powder 4Å MS (200 mg), and CH₃CN (6 mL) were used to obtain the corresponding 4avc as a white solid (167 mg, 14% yield), m.p. = 196.7–197.2 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 3.89 (q, J = 16 Hz, 2H, CH₂), 4.47–4.62 (m, 2H, CH₂), 5.00 (q, J = 16 Hz, 2H, CH₂), 6.68 (d, J = 8 Hz, 1H, Ar), 7.05 (t, J = 8 Hz, 1H, Ar), 7.16 (s br, 1H, NH), 7.22–7.41 (m, 12H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 24.84, 43.94, 44.55, 81.98, 110.38, 123.49, 123.53, 124.25, 127.16, 127.78, 127.93, 128.97, 129.00, 131.20, 134.93, 137.33, 144.48, 163.85, 164.08, 170.60. HRMS (ESI) m/z: calculated for C₂₅H₂₁O₄N₂BrNa [M]⁺ 515.0577, found 515.0571.

3-(Benzylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-bromobenzoate (4bac): 1b (300 mg, 2.0 mmol), 2a (804 mg, 4.0 mmol, 2 equivalents), 3c (487 μL, 4.0 mmol, 2 equivalents), powder 4Å MS (200 mg), and CH₃CN (6 mL) were used to obtain the corresponding 4bac as a pale yellow solid (314 mg, 33% yield), m.p. = 142.8–145.3 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 3.33 (s, 3H, CH₃), 4.46–4.51 (m, 1H, CH₂), 4.57–4.63 (m, 1H, CH₂), 6.93 (d, J = 8 Hz, 1H, Ar), 7.09 (t, J = 8 Hz, 1H, Ar), 7.30–7.42 (m, 9H, Ar), 7.49 (s br, 1H, NH), 7.61–7.63 (m, 1H, Ar), 7.77–7.79 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.09, 44.02, 82.12, 109.14, 121.42, 123.30, 123.79, 124.97, 127.70, 127.86, 127.92, 128.93, 130.47, 131.07, 132.94, 133.77, 134.57, 137.34, 145.61, 163.09, 164.44, 170.80. HRMS (ESI) m/z: calculated for C₂₄H₁₉O₄N₂BrNa [M]⁺ 501.0420, found 501.0410.

5-Bromo-3-(tert-butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-bromobenzoate (4faa): 1f (259 mg, 1.1 mmol), 2a (402 mg, 2.0 mmol, 2 equivalents), 3a (200 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4faa as a white solid (328 mg, 58% yield), m.p. = 195.9–198.1 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.41 (s, 9H, CH₃), 3.29 (s, 3H, CH₃), 6.79 (d, J = 8 Hz, 1H, Ar), 7.09 (s br, 1H, NH), 7.39–7.43 (m, 3H, Ar), 7.50 (dd, J = 8 Hz, 1H, Ar), 7.69–7.71 (m, 1H, Ar), 7.79–7.82 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.14, 28.72, 52.55, 81.62, 110.46, 115.74, 121.44, 126.69, 127.22, 127.86, 130.22, 133.23, 133.65, 134.04, 134.72, 144.72, 162.83, 163.11, 170.62. HRMS (ESI) m/z: calculated for C₂₁H₂₀O₄N₂Br₂Na [M]⁺ 544.9682, found 544.9675.

5-Bromo-3-(tert-butylcarbamoyl)-1-methyl-2-oxoindolin-3-yl 2-iodobenzoate (4fba): 1f (268 mg, 1.1 mmol), 2b (496 mg, 2.0 mmol, 2 equivalents), 3a (200 μL, 2.0 mmol, 2 equivalents), powder 4Å MS (100 mg), and CH₃CN (3 mL) were used to obtain the corresponding 4fba as a white solid (311 mg, 48% yield), m.p. = 196.7–202.4 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 1.41 (s, 9H, CH₃), 3.29 (s, 3H, CH₃), 6.79 (d, J = 8 Hz, 1H, Ar), 6.99 (s br, 1H, NH), 7.21 (dt, J = 8 Hz, 1H, Ar), 7.43 (t, J = 8 Hz, 1H, Ar), 7.48–7.52 (m, 2H, Ar), 7.74 (dd, J = 8 Hz, 1H, Ar), 7.98–8.01 (m, 1H, Ar). ¹³C APT NMR (CDCl₃, 100 MHz) δ: 27.14, 28.75, 52.61, 81.54, 93.41, 110.45, 115.72, 127.06, 127.18, 128.44, 132.44, 133.60, 133.77, 133.99, 141.48, 144.63, 162.62, 163.69, 170.67. HRMS (ESI) m/z: calculated for C₂₁H₂₀O₄N₂BrINa [M]⁺ 592.9543, found 592.9536.

3.3. The Post-Passerini Reaction Transformations

1,4′-Dibenzylspiro[indoline-3,2′-morpholine]-2,3′,6′-trione (5): The compound 4avc (150 mg, 0.3 mmol) and CH₂Cl₂ (5 mL) were added to a round-bottom flask with a magnetic stirrer. DIPEA (58 µL, 0.34 mmol, 1.1 equivalents) was added, and the reaction was left stirring at room temperature for 24 h. The reaction mixture was quenched with H₂O (10 mL) and extracted with CH₂Cl₂ (3 × 10 mL). After being dried with Na₂SO₄ and filtered, the solvent was evaporated under reduced pressure. The crude product was purified in a short chromatographic glass column with SiO₂ flash and Hex:AcOEt (5:1) and (2:1) as eluents. The corresponding compound 5 was obtained as a white solid (8.3 mg, 7% yield), m.p. = 105.0–106.7 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 4.65 (d, J = 16 Hz, 1H, CH₂), 4.83–4.96 (m, 2H, CH₂), 5.08 (s, 2H, CH₂), 5.38 (d, J = 16 Hz, 1H, CH₂), 6.72 (d, J = 8 Hz, 1H, Ar), 7.07 (t, J = 8 Hz, 1H, Ar), 7.19 (d, J = 8 Hz, 1H, Ar), 7.27–7.39 (m, 11H, Ar). ¹³C NMR (CDCl₃, 100 MHz) δ: 42.95, 44.02, 64.11, 79.66, 110.45, 123.97, 124.88, 125.86, 127.10, 127.95, 128.05, 128.65, 128.78, 129.13, 131.57, 134.57, 136.06, 144.01, 167.34, 168.56, 170.89. HRMS (ESI) m/z: calculated for C₂₅H₂₀O₄N₂Na [M]⁺ 435.1315, found 435.1309.

1-Methyl-3′H-spiro[indoline-3,1′-isobenzofuran]-2,3′-dione (6): In a Radley’s^® 12-position carousel reactor tube, CuI (3.6 mg, 0.019 mmol, 5 mol%), DMAP (9.3 mg, 0.076 mmol, 20 mol%), and DMSO (2 mL) were added, and the mixture was left stirring for 30 min at 30 °C. Then, compound 4bba (184 mg, 0.38 mmol) and KOH (53 mg, 0.95 mmol, 2.5 equivalents) were added in the reaction tube and the reaction was left stirring at 120 °C for 5 h. The reaction mixture was quenched with H₂O (10 mL) and extracted with AcOEt (3 × 10 mL). After being dried with Na₂SO₄ and filtered, the solvent was evaporated under reduced pressure. The crude product was purified in a short chromatographic glass column with SiO₂ flash and Hex:AcOEt (5:1) and (2:1) as eluents. The corresponding compound 6 was obtained as a white solid (9.3 mg, 9% yield), m.p. = 174.7–176.9 °C. ¹H NMR (CDCl₃, 400 MHz) δ: 3.30 (s, 3H, CH₃), 6.98–7.03 (m, 2H, Ar), 7.06–7.11 (m, 2H, Ar), 7.45 (t, J = 8 Hz, 1H, Ar), 7.58–7.64 (m, 2H, Ar), 8.00–8.03 (m, 1H, Ar). ¹³C NMR (CDCl₃, 100 MHz) δ: 27.15, 84.57, 109.27, 121.97, 123.95, 124.95, 125.35, 126.08, 126.26, 130.48, 131.79, 134.91, 144.76, 146.95, 169.70, 171.32. HRMS (ESI) m/z: calculated for C₁₆H₁₁O₃NNa [M]⁺ 288.0631, found 288.0626.

5-Amino-N-(tert-butyl)-3-hydroxy-1-methyl-2-oxoindoline-3-carboxamide (7): In a Radley’s^® 12-position carousel reactor tube under nitrogen atmosphere, compound 4fba (158 mg, 0.28 mmol), CuI (58 mg, 0.28 mmol, 1 equivalent), L-Proline (46 mg, 0.4 mmol, 1.3 equivalents), NaN₃ (40 mg, 0.6 mmol, 2 equivalents), and DMSO (1 mL) were added, and the mixture was left stirring at 100 °C for 24 h. The reaction mixture was quenched with brine (10 mL) and extracted with AcOEt (3 × 10 mL). After being dried with Na₂SO₄ and filtered, the solvent was evaporated under reduced pressure. The crude product was purified in a short chromatographic glass column with SiO₂ flash and Hex:AcOEt (1:1) and AcOEt as eluents. The corresponding compound 7 was obtained as a white solid (10 mg, 13% yield), m.p. = 220.0–222.4 °C. ¹H NMR (DMSO-d6, 400 MHz) δ: 1.29 (s, 9H, CH₃), 3.01 (s, 3H, CH₃), 4.88 (s br, 2H, NH₂), 6.51 (d, J = 8 Hz, 1H, Ar), 6.55 (s, 1H, Ar), 6.67 (d, J = 8 Hz, 1H, Ar), 7.02 (s, 1H, OH), 7.20 (s br, 1H, NH). ¹³C APT NMR (DMSO-d6, 100 MHz) δ: 26.01, 28.40, 50.38, 78.26, 108.94, 110.35, 113.74, 130.86, 134.32, 144.71, 168.10, 173.73. HRMS (ESI) m/z: calculated for C₁₄H₁₉O₃N₃Na [M]⁺ 300.1319, found 300.1314.

3.4. Antiproliferative Bioassays

The antiproliferative tests were conducted using our implementation of the NCI protocol [38]. The cell-seeding densities were 2500 (A549, HeLa, MIA PaCa-2, and SW1573) or 5000 (T-47D and WiDr) cells/well. Stock solutions of isatin-based Passerini adducts were prepared in DMSO at 40 mM. The maximum test concentration was 100 μM. The exposure time was set at 48 h. At the end of the exposure, the SRB protocol was applied. The results were expressed as 50% growth inhibition (GI₅₀).

4. Conclusions

A library of 44 α-acyloxyamide–oxindole hybrids was obtained efficiently using the valuable 3CPR approach. The reaction was optimized in terms of temperature, time, solvent, and the presence of additives, using a microwave or Radley’s carousel reactors. The component scope was also evaluated, proving that the 3CPR has a great tolerance towards isatin, carboxylic acid, and isocyanide derivatives. In general, moderate yields of the desired α-acyloxyamide–oxindole hybrids were obtained in 24 h, using CH₃CN as the solvent. The success of the gram-scale reaction was a clear demonstration of the competence of the synthetic method. Post-Passerini reactions also supported the versatility of the α-acyloxyamide–oxindole hybrids’ scaffold in accessing other interesting derivatives. Most of the library of the α-acyloxyamide–oxindole hybrids was evaluated regarding their antiproliferative activity in six human solid-tumor cell lines, with 4avc being the most potent compound of the series, with GI₅₀ values in the range 1.3–21 μM. Further studies on the mode of action and lead discovery are in progress and will be reported shortly.