Synthesis of N-isoindolinonyl peptides via Pd-catalyzed C(sp²)–H olefination–activation and their conformational studies

Abstract

Isoindolinone is a constituent of several natural products that show a wide range of bioactivity, such as anticancer, antimicrobial, antiviral and anti-inflammatory properties. It would be interesting to explore the carbonyl group (H-bond acceptor) of isoindolinone and its structural and conformational changes. However, the synthesis of isoindolinone-comprising peptides in short steps is challenging. Herein, we have developed a synthetic methodology for introducing the isoindolinone residue to peptides via Pd-catalyzed C(sp²)–H activation/olefination, and demonstrated the conformational changes owing to the isoindolinone scaffold. Hence, isoindolinonyl peptides provide an avenue for the synthesis of novel foldamers and therapeutic agents.

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Introduction

Isoindolinone is heterocyclic compound with a benzo fused γ-lactam scaffold that is a constituent of various natural products and synthetic bioactive molecules such as pagoclone, indoprofen, erinacerins, lennoxamine, lenalidomide, isohericenone, and pazinaclone (Fig. 1a).^1–3 These molecules show antimicrobial, antioxidant, antifungal, antiviral, anxiolytic, anti-Parkinson's, anti-inflammatory, antihypertension, and anticancer activities.⁴ In the repertoire of isoindoline scaffolding into peptides, the incorporation of isoindolinone moiety into naturally occurring bioactive cyclic peptides enhances their bioactivity. Fenestin A and zygosporamide are marine-derived natural cyclic peptides possessing a wide range of bioactivity, including anticancer. Jin has synthesized Fenestin analogues containing isoindolinone derivatives that improved their apoptosis of tumor cells and led to cycle arrest in the G2/M phase.⁵ Zygosporamide cyclic depsipeptides isolated from marine-derived fungus and their analogues are also synthesized by incorporating the isoindolinone moiety, which significantly improved their bioactivity (Fig. 1b).⁶ Nevertheless, Chen has synthesized various stapled peptides by incorporating isoindolinone derivatives (Fig. 1c).⁷ Thus, the demand for isoindolinone-containing peptides has suddenly increased and become a lucrative research area for both chemists and biologists. In the literature, various heterocyclic scaffolds have been used for the structural and conformational changes of native peptides.^8–10 The isoindolinone moiety of isoindolinonyl peptides can also participate in intramolecular hydrogen bonding with the other amide N–H of peptides and play a significant role in the conformational changes of peptides.

Fig. 1 Isoindolinone derivative: (a,b) Isoindolinone related natural peptides; (c) stapled peptides.

The classical synthesis of isoindolinone compounds is achieved through multiple challenging steps.^11–13 In recent times, various heterocycle compounds are synthesized in one step from an economical reactant precursor through metal-catalyzed C–H activation, which is a modern synthetic methodology. However, the incorporation of isoindolinone residue in the native peptides is challenging through chemical synthesis. Recently, we reported the synthesis of isoindolinone-containing amino acid ester derivatives through Pd-catalyzed C–H activation/olefination from N-benzoyl amino acid esters and olefins without using an auxiliary directing group, which is a modern synthetic methodology by C–H activation.¹⁴ Transition metal-catalysed C–H activation and C–H olefination are emerging synthetic methodologies for the synthesis of various natural products and their precursors, including isoindolinone derivatives.^15–18 The Pd-catalysed C(sp²)–H olefination methodologies have reached a new milestone owing to their easy handling and cost effectiveness.^18–29 The Co-/Rh-catalyzed substrate-specific C(sp²)–H olefination is also known.^30–32 The regioselective metal-catalyzed C–H activation also requires a directing group and ligand for metal complexation before inert C–H activation and functionalization. The directing groups could be auxiliary, transient or intrinsic. The directing group containing arylamides has also been used for synthesizing isoindolinone derivatives from non-activated olefins through C–(sp²)–H olefination.³³ For example, the quinoline directing group containing arylamides gives N-quinolinyl substituted isoindolinones from acrylate in the presence of Pd(II)-catalyst. However, N-tosylated benzamide requires a ligand to prepare isoindolinone derivatives from olefin through Pd-catalyzed olefination.^34,35 Amino acids and peptides are natural ligands of various transition metals and are being used as the directing group/ligand in the different transition metal-catalyzed C–H activation/C-olefination reactions.³⁶ Yu has explored amino acids/peptides as the ligand or directing group in the metal-catalyzed C(sp³)–H functionalization of di-/tri/tetra-peptides at the N-terminus/site-specific C–H alkynylation/β-C–H arylation.^37–39 Albericio has shown the synthesis of stapled peptides by the late-stage C(sp³)–H activation of peptides.^40,41 Daugulis has used 2-thiomethylaniline as the directing group for C(sp³)–H functionalization of N-protected amino acids at the β-position with different aryl halides.⁴² The metal-catalyzed C–H olefinations of amino acid/peptide-related compounds have generated opportunities to prepare various synthetic and natural peptide analogues.^43,44 Metal-catalyzed olefination of ligand enabled an aryl amide with olefin, which is an emerging synthetic methodology attractive for the synthesis of substituted aryl amide derivatives.^24,45–51 Recently, Maiti has explored Pd-catalyzed 8-AQ-directed and template-based regioselective C–H olefinations with non-active aliphatic olefins.^47–53 Wang has demonstrated a Pd-catalyzed ortho-olefination reaction and cyclization reaction at benzylsufonamide to yield benzosultam peptidomimetics.⁵⁴ The synthesis of isoindolinone derivatives without comprising amino acids was accomplished from activated arylamides and non-reactive olefins through transition-metal-catalyzed C–H activation using metal ions Sc, Yb, Au, Rh, Ir, Ru, Co, Cu, Ni, Zn, In, and Pd (Fig. 2a and b).⁵⁵ Herein, we have rationally designed peptides containing an isoindolinone olefin ester at the N-terminal to explore the role of isoindolinone's lactam carbonyl in the folding of peptide structure through noncovalent interactions (Fig. 2c). This report describes the post-synthesis isoindolinonyl peptides from N-arylamide peptides and olefins through Pd-catalysed C(sp²)–H olefination. Their conformational analysis was also studied by NMR and computational technique. To examine the biocompatibility and therapeutic value, cell cytotoxicity studies were also performed before exploring their therapeutic value.

Fig. 2 (a) The previously reported C–H hydroarylation, (b) C–H functionalization and (c) (this report) post-synthesis of isoindolinyl peptides.

Results and discussion

The conjugation of isoindolinone scaffold with peptides could be achieved by two ways: (a) coupling of previously synthesized isoindolinonyl amino acid carboxylates at the free NH group of the designed peptides; (b) C(sp²)–H olefination/activation reaction at the N-benzamide of target peptides. However, we were unable to achieve the mono carboxylate derivative of isoindolinoyl amino acid ester through the base-catalyzed hydrolysis under mild condition (Scheme 1a). Thus, we attempted C(sp²)–H olefination/activation at the N-benzamide of peptides. We synthesized dipeptides (1–4), containing glycine, valine, phenyl alanine and phenyl glycine ester, and their N-benzamide peptides (6a–6d) with benzoic acid under peptide coupling conditions. These peptides were subjected to Pd-catalyzed C–H activation/olefination reactions with different olefins by using our previously reported method.⁵⁶ Unfortunately, we could not obtain any olefinated/activated product. We noticed the formation of isoindolinonyl peptides (8a) from peptide 6a and olefin methyl acrylate (7a) under Cu(OAc)₂/NaOAc at 100 °C in the presence of Pd catalyst.⁵⁴ Importantly, additive Cu(OAc)₂/NaOAc was found essential for mono C–H olefination/activation reaction in N-benzamide peptides (Scheme 1b). Similarly, we synthesized other isoindolinonyl peptides 8b–8d from peptide 6a and different olefins: ethyl acrylate (7b), n-butyl acrylate (7c), and t-butyl acrylate (7d). Next, peptide 6b was treated with various olefins (7a–7d) under similar optimized reaction conditions of mono C(sp²)–H olefination/activations that produced isoindolinonyl peptides 9a–9d (Scheme 1b). We performed similar reaction with N-benzamide of additional phenyl ring – containing dipeptides (6c–6d) and different olefins (7a–7d). Pleasantly, we isolated isoindolinonyl peptides 10a–10d/11a–11d from the respective peptides and olefins through chemoselective/regioselective C–H olefination/activation at only the ring. Herein, we could not get C(sp²)–H olefination reaction at the phenyl glycine residue of peptide 6c and phenyl alanine residue of peptide 6d though there is presence of a probable intrinsic amide-directing group. Thus, only benzamide ring is susceptible to the C–H olefination which led to the formation of isoindolinonyl peptides.

Scheme 1 Synthesis of isoindolinonyl peptide derivatives with different peptides and olefins.

To explore the diversity of arylamide substrates for Pd-catalyzed isoindolinonyl peptide synthesis, we synthesized arylamide dipeptides 13a/13b from 2-napthoic acid and dipeptides 4b/4c (Scheme 2). Similarly, we synthesized arylamide peptide substrates 16a/16b from 4-biphenyl carboxylic acid and peptides 4b/4c. These peptides were subjected to Pd-catalyzed olefination reaction under optimized conditions. Importantly, these peptides also produced chemoselective naphthyl isoindolinonyl peptides 14a–14d from the respective 2-napthoyl peptides 13a/13b and acrylate 7a/7b, as well as biphenyl isoindolinonyl peptides 17a–17d from the respective 4-biphenyl-containing peptides 16a/16b and acrylate 7a/7b. Here too, we could not achieve C(sp²)–H olefination reaction at the phenyl glycine residue of peptide 13b/16b though there is presence of probable intrinsic amide-directing group.

Scheme 2 Synthesis of isoindolinonyl peptides with different aromatic acids.

Further, we examined the C–H olefination/isoindolinonylation reaction with different N-benzamide dipeptides and olefines under Pd-catalysed optimized reaction conditions. We synthesized arylamide peptides 19a–19c containing different types of benzoyl residues (18), such as p-methyl benzoyl in 19a, p-tert-butylbenzoyl in peptide 19b, and m-nitrobenzoyl in peptide 19c (Scheme 3). These peptides were subjected to olefination with different acrylates (7) under Pd-catalyzed olefination reaction condition. We isolated isoindolinone derivatives 20a–20b from p-methylbenzoyl peptide and 21a–21d from p-tert-butyl benzoyl peptides though their yields are low. However, nitrobenzoyl peptide (19c) could not give any C–H olefinated/activated product. It seems an electron-withdrawing group possibly perturbs the metal (Pd-II) complexation of the phenyl ring of nitrobenzoyl peptide. Thus, unsubstituted aryl amides are more suitable substrates for Pd- catalysed C(sp²)–H olefination/activation reactions under the optimized conditions.

Scheme 3 Synthesis of aryl-substituted isoindolinonyl peptide derivatives.

Further, we attempted to apply this methodology for the synthesis of isoindolinone-containing tri-peptides. Thus, we synthesized the benzamide of tri-peptides (22) such as BzNHGly-Ala-Phe (22a) and BzNHVal-Leu-Phe-OMe (22b). These peptides were subjected to Pd-catalyzed olefination/activation reaction with olefin (7a) under the above optimized reaction conditions (Scheme 4). Importantly, benzamide peptides 22a–22b also produced the respective isoindolinone derivative peptides 23/24 though their yields are quite low (∼25–30%) as compared to dipeptides.

Scheme 4 Synthesis of substituted isoindolinonyl tri-peptide.

Herein, we propose the reaction mechanism of isoindolinone-peptide synthesis through Pd-catalyzed C(sp²)–H olefination/activation (Fig. 3). Additive Cu(OAc)₂ facilitates the formation of a stable palladacycle with amide carbonyl through N,O-type chelation by preventing N,N covalent chelation. In the literature, the coordination of amide carbonyl with catalyst Pd(OAc)₂ is known as well.⁵⁷ The benzoyl amino acid residue of the peptide (reactant6a) acts as the intrinsic directing group for Pd complexation (5-membered palladacycle) and proceeds to C(sp²)–H activation at the ortho position of its phenyl ring (I-1). Olefin forms Pd complex I-2 by displacement of ligand OAc, and then proceeds to I-3 through 1,2-migration as the intermediate I-3. This intermediate (I-3) leads to olefinated product (6′) via β-hydride elimination reaction. Again, palladacycle forms with the benzoyl amino acid residue of olefinated peptide (6′) and proceeds to C(sp²)–H activation at olefin as the intermediate I-4, which leads to stable isoindolinonyl peptides (8a) via reductive elimination. The regeneration of catalyst occurs in situ through reoxidation.

Fig. 3 The proposed mechanism for isoindolinone peptide synthesis.

After the successful synthesis of isoindolinonyl-conjugated peptides through C–H activation, we attempted to find the conformation of isoindolinyl tripeptides (23) in solution by NMR technique. We recorded their NMR spectra (¹H/¹H–¹HCOSY/¹H–¹H NOESY) only in DMSO-d₆ solvent because of poor solubility (or insolubility) in other solvents. Their spectra are provided in the ESI (Fig. S88†). The chemical shift of all protons were assigned from the COSY spectra, then the NOE cross peaks were used to find the interaction of non-vicinal protons. Our analyses helped to find the 1H–1H interactions which are in close proximity. In Fig. 4, the phenyl ring proton (d) of the isoindolinone residue interacts with the methyl protons of acrylate residue. Olefin protons of acrylate residue exhibit two cross peaks with glycinate protons (a) and alaninyl methyl protons. Thus, the orientation of isoindolinyl residue is in close proximity with alanine methyl (i + 2) to form a stable conformation in solution.

Fig. 4 NOESY spectra of isoindolinone derivative 23.

The DMSO-d₆ titration NMR (¹H-NMR) experiment is a well-established technique to find the intramolecular hydrogen bonding N–H in CDCl₃.⁵⁸ Herein, we performed the DMSO-d₆ titration ¹H-NMR experiment with representative isoindolinonyl peptides derived from methyl acrylates (8a–11a/14a/14c/17a/17c) and the control peptide BzNHGly-ValOMe (6b). Their titration profiles are depicted in Fig. 5, while their spectra are provided in ESI.† However, peptides 23 and 24 are insoluble in CDCl₃ (soluble only in DMSO). In Fig. 5B, we have also extracted a bar diagram for peptides vs. Δδ(ppm), where Δδ(ppm) = (δ_final − δ_initial). The control peptide (6b) has two amide N–H groups, whose titration profile shows that one N–H is unaffected while the other has a small downfield shift with DMSO-d₆ titration owing to the strong and moderate intramolecular hydrogen bonding, respectively. The isoindolinonyl peptides (8a–11a/14a/14c/17a/17c) have one amide N–H that exhibits a small downfield shift (∼0.4–1.3 ppm) with DMSO-d₆ titration, possibly due to intramolecular hydrogen bonding with amide N–H (Fig. 5A and B). The amide N–H groups of peptide 11a and 9a have small shifts as compared to other isoindolinonyl peptides (Fig. 5B). Thus, sequence-specific isoindolinone carbonyl (C=O) has significant roles in the intramolecular hydrogen bonding with amide N–H, which could be useful for tuning the peptide folding.

Fig. 5 DMSO-d₆ titration NMR profile of isoindonyl derivatives (A) and Bar diagram of isoindolinyl peptides vs. Δδ(ppm), where Δδ(ppm) = (δ_final − δ_initial) (B).

Next, we performed computational studies with representative isoindolinonyl peptides derived from methyl acrylates (8a–11a/14a/14c/17a/17c/23/24) and the control peptide BzNHGly-ValOMe (6b) using MMFF94 force field and GMMX. We visualized their conformations on PyMol (Fig. 6). The control peptide (6b), without containing isoindolinone scaffold, exhibits hydrogen bonding between the benzoyl carbonyl and the second amino acid residue valine N–H (i + 3) with bond distance of 1.9 Å (N–H⋯O=C) and bond angle ∠N–H–O ∼ 148° that nearly matched with the γ-turn. Isoindolinone-containing dipeptides (8a–11a/14a/14c/17a/17c) exhibit intramolecular hydrogen bonding between the isoindolinone carbonyl (C=O) and N–H of the second amino acid residue, with bond distance (N–H⋯O) ∼2.0 and bond angle ∠N–H–O ∼150° with the respective substituents of the amino acid and nature of the aryl group of the isoindolinone residue. In the case of isoindolinonyl tri-peptides (23/24), they exhibit two intramolecular hydrogen bonds between isoindolinonyl carbonyl and the i + 3 amide (N–H⋯O=C). The bond length and angle of the hydrogen bonds are almost same, ∼2.0 Å/∼150° for peptide 23, and possibly lead to the formation of a helix. In contrast, for 24, the bond length and angle of hydrogen bonds are different, at ∼2.0 Å/∼150° (strong) and ∼2.8 Å/136° (weak), presumably due to the leu residue at the 2^nd position, and may lead to only γ-turn structure (Fig. 6). These data strongly support that isoindolinone-containing peptides are potential building blocks for designing unique foldamers.

Fig. 6 Conformation of isoindolinone peptides (computationally optimized structure of isoindolinone peptides MMFF94 and GMMX).

Finally, we examined the cell cytotoxicity of isoindolinonyl peptides derived from methyl acrylates (8a–11a/14a/14c/17a/17c/23/24) with Hek293T cells using MTT assay. Their cytotoxicity data are provided in ESI (Fig. S89†), which indicate that isoindolinonyl peptides have negligible cytotoxicity as compared to the control (DMSO).

Conclusions

Isoindolinonyl-incorporated cyclopeptides are emerging therapeutic target drug molecules, and their synthesis involves multiple challenging steps. Herein, we have developed a novel methodology for the synthesis of isoindolinone scaffold comprising di/tri-peptides through Pd-catalyzed C(sp²)–H olefination/activation from various arylamide peptides and acrylate olefins. We have also studied the conformational changes of those peptides owing to the isoindolinone residue. Our conformational analyses strongly support the formation of γ-turn in dipeptide and helical formation in less stearic tripeptides. Finally, these peptides are biocompatible owing to their negligible cytotoxicity effect on Hek293T cells. Hence, isoindolinone-containing peptides are potential building blocks for novel foldamers with therapeutic value.

Experimental details

Material and instrumentation

All required materials were obtained from commercial suppliers and used without purification. Dry DMF was freshly prepared by distilling over calcium hydride. Reactions were monitored by thin-layer chromatography, visualized by UV and ninhydrin. Column chromatography was performed in 100–200 mesh silica. Mass spectra were obtained from Bruker Micro TOF-Q II Spectrometer. NMR spectra were recorded on Bruker AV-400 for ¹H (400 MHz) and ¹³C (100.6 MHz). ¹H and ¹³C NMR chemical shifts were recorded in ppm downfield from tetramethylsilane; splitting patterns are abbreviated as s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; dq, doublet of the quartet; m, multiplet.

General procedure for benzamide derivative peptide synthesis

Benzoic acid or its derivative was dissolved in DMF before triethylamine (TEA, 3.0 eq.), EDC.HCl (1.3 eq.) and HOAT (1.3 eq.) were added, followed by free N-terminal peptide methyl ester (1.2 eq.). Further, this mixture was heated to 60 °C for 8–12 h. The reaction was monitored by TLC. The crude reaction mixture was concentrated under reduced pressure before water was added. The aqueous layer was extracted with EtOAc. The organic layer was combined, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified with column chromatography by EtOAc/hexane solvent system to yield the corresponding substrates.

General procedure for Pd-catalysed reactions

Typically, amide substrates were placed in a 15 ml sealed reaction tube under the indicated reaction conditions. The mixture was stirred at 100 °C for 12–24 h, cooled to room temperature, and then diluted with EtOAc. The resulting solution was filtered through a Celite pad, concentrated under reduced pressure, and the product was further purified by column chromatography by the EtOAc/hexane solvent system and was typically obtained as a white solid.

Methyl benzoylglycylglycinate (6a). ¹H NMR (400 MHz, CDCl₃) δ 7.85 (t, J = 8.0 Hz, 2H), 7.62 (d, J = 4.0 Hz, 1H), 7.50 (t, J = 8.0 Hz, 1H), 7.45 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 4.21 (d, J = 4.0 Hz, 2H), 4.05 (d, J = 4.0 Hz, 2H), 3.72 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.28 (s), 169.88 (s), 168.04 (s), 133.35 (s), 131.91 (s), 128.59 (s), 127.23 (s), 52.41 (s), 43.57 (s), 41.20 (s). White solid (68% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₂H₁₄N₂O₄ 251.1032, found 251.1040.

Methyl (Z)-2-(2-(2-((2-methoxy-2-oxoethyl)amino)-2-oxoethyl)-3-oxoisoindolin-1-ylidene)acetate (8a). ¹H NMR (400 MHz, CDCl₃) δ 9.07 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.79 (s, 1H), 4.52 (s, 2H), 4.04 (d, J = 4.0 Hz, 2H), 3.81 (s, 3H), 3.72 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 169.77 (s), 167.26 (s), 166.82 (s), 166.08 (s), 147.65 (s), 133.88 (s), 133.79 (s), 131.55 (s), 129.32 (s), 128.27 (s), 123.56 (s), 99.68 (s), 52.49 (s), 51.82 (s), 43.39 (s), 41.23 (s). White solid (81% yield). ESI-HRMS m/z [M + Na]⁺ calcd for C₁₂H₁₄N₂O₄ 355.0906, found 355.1041.

Ethyl (Z)-2-(2-(2-((2-methoxy-2-oxoethyl)amino)-2-oxoethyl)-3-oxoisoindolin-1-ylidene)acetate (8b). ¹H NMR (400 MHz, CDCl₃) δ 9.09 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 7.4 Hz, 1H), 7.71 (t, J = 8.3 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 6.49 (d, J = 4.0 Hz, 1H), 5.80 (s, 1H), 4.54 (s, 2H), 4.28 (q, J = 8.0 Hz, 2H), 4.06 (d, J = 5.4 Hz, 2H), 3.74 (s, 3H), 1.36 (t, J = 8.0 Hz, 3H). ¹³C NMR (176 MHz, CDCl₃) δ 169.70 (s), 167.27 (s), 166.85 (s), 165.65 (s), 147.37 (s), 133.87 (s), 133.76 (s), 131.31 (s), 129.32 (s), 128.32 (s), 123.81 (s), 100.29 (s), 60.77 (s), 52.42 (s), 43.47 (s), 41.24 (s), 14.24 (s). White solid (71% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₇H₁₈N₂O₆ 347.1243, found 347.1243.

Butyl (Z)-2-(2-(2-((2-methoxy-2-oxoethyl)amino)-2-oxoethyl)-3-oxoisoindolin-1-ylidene)acetate (8c). ¹H NMR (400 MHz, CDCl₃) δ 9.10 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 6.47 (s, 1H), 5.81 (s, 1H), 4.54 (s, 2H), 4.23 (t, J = 8.0 Hz, 2H), 4.06 (d, J = 8.0 Hz, 2H), 3.74 (s, 3H), 1.72–1.67 (m, 2H), 1.49–1.41 (m, 2H), 0.98 (t, J = 8.0 Hz, 3H). ¹³C NMR (176 MHz, CDCl₃) δ 169.68 (s), 167.26 (s), 166.87 (s), 165.77 (s), 147.35 (s), 133.89 (s), 133.75 (s), 131.30 (s), 129.31 (s), 128.33 (s), 123.80 (s), 100.30 (s), 64.70 (s), 52.40 (s), 43.48 (s), 41.24 (s), 30.68 (s), 19.17 (s), 13.73 (s). White solid (61% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₉H₂₂N₂O₆ 375.1558, found 375.1553.

tert-Butyl (Z)-2-(2-(2-((2-methoxy-2-oxoethyl)amino)-2-oxoethyl)-3-oxoisoindolin-1-ylidene)acetate (8d). ¹H NMR (400 MHz, CDCl₃) δ 9.02 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 6.57 (s, 1H), 5.74 (s, 1H), 4.50 (s, 2H), 4.04 (d, J = 4.0 Hz, 2H), 3.71 (s, 3H), 1.55 (s, J = 8.0 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 169.71 (s), 167.28 (s), 167.09 (s), 165.03 (s), 146.31 (s), 133.97 (s), 133.61 (s), 131.21 (s), 129.33 (s), 128.21 (s), 123.41 (s), 102.55 (s), 81.19 (s), 52.41 (s), 43.44 (s), 41.22 (s), 28.23 (s). White solid (64% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₉H₂₂N₂O₆ 375.1556, found 375.1553.

Methyl benzoylglycyl-l-valinate (6b). ¹H NMR (400 MHz, CDCl₃) δ 7.85 (d, J = 8.0 Hz, 2H), 7.56–7.38 (m, 4H), 7.27 (s, 1H), 4.55 (d, J = 4.0 Hz, 1H), 4.26 (d, J = 4.0 Hz, 2H), 3.73 (s, 3H), 2.25–2.13 (m, 1H), 0.94 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.21 (s), 169.34 (s), 167.93 (s), 133.53 (s), 131.86 (s), 128.59 (s), 127.21 (s), 57.52 (s), 52.25 (s), 43.78 (s), 31.07 (s), 19.05 (s), 17.79 (s). White solid (62% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₅H₂₀N₂O₄ 293.1501, found 293.1498.

Methyl (Z)-(2-(1-(2-methoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetyl)-L-valinate (9a). ¹H NMR (400 MHz, CDCl₃) δ 9.14–9.05 (m, 1H), 7.90 (d, 1H), 7.71 (t, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.78 (s, 1H), 4.61–4.49 (m, 3H), 3.81 (s, 3H), 3.70 (s, 3H), 2.16 (d, J = 8.0 Hz, 1H), 0.88 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.91 (s), 167.26 (s), 166.45 (s), 166.08 (s), 147.77 (s), 133.82 (s), 133.77 (s), 131.55 (s), 129.33 (s), 128.29 (s), 123.60 (s), 99.53 (s), 57.27 (s), 52.32 (s), 51.80 (s), 43.57 (s), 31.22 (s), 18.93 (s), 17.73 (s). White solid (71% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₉H₂₂N₂O₆ 375.1556, found 375.1553.

Methyl (Z)-(2-(1-(2-ethoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetyl)-L-valinate (9b). ¹H NMR (400 MHz, CDCl₃) δ 9.09 (d, J = 4.0 Hz, 1H), 7.89 (d, J = 4.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.62 (t, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.77 (d, J = 4.0 Hz, 1H), 4.60–4.49 (m, 3H), 4.32–4.18 (m, 2H), 3.70 (s, 3H), 2.21–2.09 (m, 1H), 1.37–1.27 (m, 3H), 0.88 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.90 (s), 167.26 (s), 166.50 (s), 165.63 (s), 133.82 (s), 133.73 (s), 131.48 (s), 129.33 (s), 128.32 (s), 123.58 (s), 100.13 (s), 60.67 (s), 57.26 (s), 52.31 (s), 43.59 (s), 31.23 (s), 18.95 (s), 17.72 (s), 14.28 (s). White solid (75% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₀H₂₄N₂O₆ 389.1713, found 389.1714.

Methyl (Z)-(2-(1-(2-butoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetyl)-L-valinate (9c). ¹H NMR (400 MHz, CDCl₃) δ 9.10 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 6.36 (d, J = 8.0 Hz, 1H), 5.77 (s, 1H), 4.59–4.49 (m, 3H), 4.21 (d, J = 8.0 Hz, 2H), 3.70 (s, 3H), 2.20–2.11 (m, 1H), 1.67 (d, J = 7.9 Hz, 2H), 1.45–1.39 (m, 2H), 0.96 (d, J = 4.0 Hz, 3H), 0.93–0.83 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.81 (s), 167.23 (s), 166.46 (s), 165.72 (s), 147.47 (s), 133.87 (s), 133.73 (s), 131.47 (s), 129.34 (s), 128.36 (s), 123.59 (s), 100.13 (s), 64.60 (s), 57.25 (s), 52.26 (s), 43.68 (s), 31.24 (s), 30.70 (s), 19.16 (s), 18.93 (s), 17.69 (s), 13.71 (s). White solid (62% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₈N₂O₆ 417.2026, found 417.2060.

Methyl (Z)-(2-(1-(2-(tert-butoxy)-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetyl)-L-valinate (9d). ¹H NMR (400 MHz, CDCl₃) δ 9.04 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 6.41 (d, J = 8.7 Hz, 1H), 5.71 (s, 1H), 4.60–4.46 (m, 3H), 3.69 (s, 3H), 2.20–2.11 (m, 1H), 1.53 (s, 9H), 1.02–0.75 (m, 6H). ¹³C NMR (176 MHz, CDCl₃) δ 171.83 (s), 167.23 (s), 166.62 (s), 164.93 (s), 146.44 (s), 133.94 (s), 133.62 (s), 131.26 (s), 129.37 (s), 128.28 (s), 123.50 (s), 102.42 (s), 57.24 (s), 52.26 (s), 43.66 (s), 31.25 (s), 28.21 (s), 18.98 (s), 17.68 (s). White solid (67% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₈N₂O₆ 417.2026, found 417.2019.

Methyl (S)-2-(2-benzamidoacetamido)-2-phenylacetate (6c). ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 6.7 Hz, 1H), 7.52–7.45 (m, 1H), 7.43–7.30 (m, 7H), 7.19 (s, 1H), 5.57 (d, J = 8.0 Hz, 1H), 4.32–4.13 (m, 2H), 3.70 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.00 (s), 168.57 (s), 167.76 (s), 135.93 (s), 133.51 (s), 131.83 (s), 129.05 (s), 128.71 (s), 128.64 (d, J = 12.4 Hz), 128.58 (s), 127.36 (s), 127.18 (s), 56.77 (s), 52.89 (s), 43.57 (s). White solid (65% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₈H₁₈N₂O₄ 327.1345, found 327.1369.

Methyl-(S,Z)-2-(2-(1-(2-methoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetamido)-2-phenylacetate (10a). ¹H NMR (400 MHz, CDCl₃) δ 9.10 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.35–7.32 (m, 5H), 6.99 (d, J = 8.0 Hz, 1H), 5.75 (s, 1H), 5.57 (d, J = 8.0 Hz, 1H), 4.53 (s, 2H), 3.81 (s, 3H), 3.71 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.77 (s), 167.21 (s), 166.03 (s), 165.90 (s), 147.74 (s), 135.85 (s), 133.82 (s), 133.75 (s), 131.51 (s), 129.36 (s), 129.02 (s), 128.67 (s), 128.28 (s), 127.23 (s), 123.62 (s), 99.57 (s), 56.58 (s), 52.94 (s), 51.74 (s), 43.43 (s). White solid (75% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₀N₂O₆ 409.1400, found 409.1439.

Methyl (S,Z)-2-(2-(1-(2-ethoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetamido)-2-phenylacetate (10b). ¹H NMR (400 MHz, CDCl₃) δ 9.08 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.37–7.28 (m, 5H), 6.95 (d, J = 8.0 Hz, 1H), 5.73 (s, 1H), 5.55 (d, J = 7.0 Hz, 1H), 4.51 (d, J = 4.0 Hz, 2H), 4.26 (d, J = 8.0 Hz, 2H), 3.69 (s, 3H), 1.34 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.80 (s), 167.24 (s), 166.03 (s), 165.64 (s), 147.55 (s), 133.85 (s), 133.69 (s), 131.44 (s), 129.37 (s), 129.00 (s), 128.85–128.75 (m), 128.66 (s), 128.30 (s), 127.24 (s), 123.57 (s), 100.13 (s), 60.67 (s), 56.57 (s), 52.95 (s), 43.36 (s), 14.31 (s). White solid (78% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₅H₂₆N₂O₆ 423.1556, found 423.1570.

Methyl (S,Z)-2-(2-(1-(2-butoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetamido)-2-phenylacetate (10c). ¹H NMR (400 MHz, CDCl₃) δ 9.09 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.40–7.29 (m, 5H), 7.09–6.98 (m, 1H), 5.74 (s, 1H), 5.57 (d, J = 8.0 Hz, 1H), 4.53 (d, J = 4.0 Hz, 2H), 4.21 (t, J = 8.0 Hz, 2H), 3.69 (s, 3H), 1.74–1.64 (m, 2H), 1.48–1.39 (m, 2H), 0.98 (t, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.83 (s), 167.31 (s), 166.08 (s), 165.81 (s), 147.56 (s), 135.93 (s), 135.93 (s), 133.96 (s), 133.76 (s), 131.49 (s), 129.43 (s), 129.07 (s), 128.91 (s), 128.72 (s), 128.39 (s), 127.29 (s), 100.21 (s), 64.68 (s), 56.64 (s), 52.98 (s), 43.51 (s), 30.79 (s), 19.25 (s), 13.81 (s). White solid (67% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₅H₂₆N₂O₆ 451.1869, found 451.1873.

Methyl (S,Z)-2-(2-(1-(2-(tert-butoxy)-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetamido)-2-phenylacetate (10d). ¹H NMR (400 MHz, CDCl₃) δ 9.03 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.36–7.28 (m, 5H), 6.95 (d, J = 8.0 Hz, 1H), 5.69 (s, 1H), 5.55 (d, J = 8.0 Hz, 1H), 4.50 (m, 2H), 3.69 (s, 3H), 1.54 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 170.76 (s), 167.31 (s), 166.16 (s), 165.00 (s), 146.47 (s), 135.92 (s), 134.03 (s), 133.66 (s), 131.27 (s), 129.39 (s), 129.06 (s), 128.90 (s), 128.70 (s), 128.30 (s), 127.22 (s), 123.56 (s), 102.48 (s), 81.14 (s), 56.58 (s), 52.96 (s), 43.60 (s), 28.28 (s). White solid (64% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₅H₂₆N₂O₆ 451.1869, found 451.1873.

Methyl benzoyl-L-phenylalanyl-L-valinate (6d). ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 4.0 Hz, 2H), 7.32–7.21 (m, 5H), 6.95 (d, J = 8.0 Hz, 1H), 6.50 (d, J = 8.0 Hz, 1H), 4.94 (q, J = 8.0 Hz, 1H), 4.44 (dd, J = 4.0 Hz, 1H), 3.71 (s, 3H), 3.29–3.06 (m, 2H), 2.19–1.93 (m, 1H), 0.83 (d, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.66 (s), 170.93 (s), 167.30 (s), 136.52 (s), 133.80 (s), 131.82 (s), 129.41 (s), 128.71 (s), 128.61 (s), 127.06 (s), 57.52 (s), 54.83 (s), 52.14 (s), 38.25 (s), 31.08 (s), 18.85 (s), 17.74 (s). White solid (60% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₆N₂O₄ 383.1971, found 383.1970.

Methyl ((S)-2-((Z)-1-(2-methoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (11a). ¹H NMR (400 MHz, CDCl₃) δ 9.05 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.22–7.09 (m, 5H), 6.38 (s, 1H), 5.98 (s, 1H), 5.43 (s, 1H), 4.59 (dd, J = 4.0 Hz, 1H), 3.84 (s, 3H), 3.71 (s, 3H), 3.67–3.60 (m, 1H), 3.44–3.34 (m, 1H), 2.21–2.07 (m, 1H), 0.83 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.78 (s), 168.59 (s), 167.36 (s), 166.04 (s), 136.70 (s), 133.69 (s), 133.55 (s), 131.43 (s), 128.97 (s), 128.72 (s), 128.55 (s), 128.03 (s), 126.91 (s), 101.06 (s), 57.53 (s), 52.24 (s), 51.80 (s), 34.05 (s), 31.14 (s), 29.21 (s), 18.88 (s), 17.76 (s). White solid (67% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₆H₂₈N₂O₆ 464.1947, found 464.1995.

Methyl ((S)-2-((Z)-1-(2-ethoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (11b). ¹H NMR (400 MHz, CDCl₃) δ 9.05 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.23–7.08 (m, 4H), 6.40 (s, 1H), 5.94 (s, 1H), 5.45 (s, 1H), 4.60 (d, J = 4.0 Hz, 1H), 4.38–4.20 (m, 2H), 3.71 (s, 2H), 3.68–3.58 (m, 1H), 3.45–3.32 (m, 1H), 2.20–2.10 (m, 1H), 1.36 (t, J = 8.0 Hz, 3H), 0.87 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.78 (s), 168.59 (s), 167.36 (s), 166.04 (s), 136.70 (s), 133.69 (s), 133.55 (s), 131.43 (s), 128.97 (s), 128.72 (s), 128.55 (s), 128.03 (s), 126.91 (s), 101.06 (s), 57.53 (s), 52.24 (s), 51.80 (s), 34.05 (s), 31.14 (s), 21.96 (s), 18.88 (s), 17.76 (s). White solid (51% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₇H₃₀N₂O₆ 479.2182, found 479.2165.

Methyl ((S)-2-((Z)-1-(2-butoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (11c). ¹H NMR (400 MHz, CDCl₃) δ 9.01 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.67–7.61 (t, J = 8.0 Hz, 1H), 7.55–7.51 (t, J = 8.0 Hz, 1H), 7.16–7.06 (m, 5H), 6.49–6.30 (s, 1H), 5.89 (s, 1H), 5.42 (s, 1H), 4.57 (d, J = 8.0 Hz, 1H), 4.30–4.10 (m, 2H), 3.64 (s, 3H), 3.62–3.59 (m, 1H), 3.35 (m, 1H), 2.16–2.06 (m, 1H), 1.71–1.63 (m, 2H), 1.46–1.37 (m, 2H), 0.96 (t, J = 8.0 Hz, 3H), 0.80 (d, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.86 (s), 169.10–168.50 (m), 167.40 (s), 165.77 (s), 136.84 (s), 133.68 (s), 131.41 (s), 129.06 (s), 128.83 (s), 128.56 (s), 128.13 (s), 126.93 (s), 123.55 (s), 101.64 (s), 64.72 (s), 57.57 (s), 52.27 (s), 34.09 (s), 31.29 (s), 30.78 (s), 29.76 (s), 19.24 (s), 19.04 (s), 17.82 (s), 13.79 (s). White solid (58% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₉H₃₄N₂O₆ 507.2498, found 507.2518.

Methyl ((S)-2-((Z)-1-(2-(tert-butoxy)-2-oxoethylidene)-3-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (11d). ¹H NMR (400 MHz, CDCl₃) δ 8.97 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.66 (t, J = 8.0 Hz 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.17–7.09 (m, 5H), 6.37 (s, 1H), 5.81 (s, 1H), 5.41 (s, 1H), 4.60 (dd, J = 8.0 Hz, 1H), 3.69 (s, 3H), 3.68–3.60 (m, 1H), 3.40–3.30 (m, 1H), 2.21–2.05 (m, 1H), 1.55 (s, 9H), 0.98–0.67 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 170.76 (s), 167.25 (s), 165.95 (s), 165.62 (s), 147.49 (s), 135.85 (s), 133.87 (s), 133.74 (s), 131.47 (s), 129.34 (s), 129.03 (s), 128.87 (s), 128.69 (s), 128.34 (s), 127.22 (s), 123.62 (s), 100.19 (s), 60.68 (s), 56.57 (s), 52.97 (s), 45.71 (s), 43.50 (s), 29.71 (s), 14.30 (s), 14.17 (s). White solid (55% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₉H₃₄N₂O₆ 507.2498, found 507.2518.

Methyl (2-naphthoyl)glycyl-L-valinate (13a). ¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 8.07 (s, 1H), 7.89 (d, J = 4.0 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.55–7.41 (m, 1H), 4.60–4.54 (m, 1H), 4.43–4.28 (m, 2H), 3.70 (s, 3H), 2.32–2.08 (m, 1H), 0.95 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.39 (s), 170.15 (s), 168.16 (s), 134.76 (s), 132.48 (s), 130.62 (s), 129.02 (s), 128.21 (s), 128.08 (s), 127.66 (s), 127.60 (s), 126.56 (s), 123.72 (s), 57.73 (s), 52.14 (s), 43.86 (s), 30.98 (s), 19.08 (s), 17.92 (s). Yellow solid (66% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₁₉H₂₂N₂O₄ 343.1658, found 343.1665.

Methyl (Z)-(2-(1-(2-methoxy-2-oxoethylidene)-3-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)acetyl)-L-valinate (14a). ¹H NMR (400 MHz, CDCl₃) δ 9.70 (s, 1H), 8.40 (s, 1H), 8.12–8.06 (m, 1H), 8.06–7.98 (m, 1H), 7.78–7.49 (m, 2H), 6.46 (d, J = 8.0 Hz, 1H), 5.78 (s, 1H), 4.68–4.45 (m, 3H), 3.84 (s, 3H), 3.69 (s, 4H), 2.23–2.09 (m, 1H), 0.88 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.83 (s), 167.17 (s), 166.50 (s), 166.34 (s), 148.26 (s), 136.02 (s), 133.87 (s), 130.52 (s), 129.96 (s), 129.65 (s), 128.64 (s), 128.54 (s), 126.11 (s), 125.20 (s), 124.73 (s), 97.97 (s), 57.32 (s), 52.26 (s), 51.69 (s), 43.93 (s), 31.20 (s), 18.91 (s), 17.72 (s). Yellow solid (60% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₃H₂₄N₂O₆ 425.1713, found 425.1697.

Methyl (Z)-(2-(1-(2-ethoxy-2-oxoethylidene)-3-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)acetyl)-L-valinate (14b). ¹H NMR (400 MHz, CDCl₃) δ 9.71 (s, 1H), 8.41 (s, 1H), 8.12–8.06 (m, 1H), 8.05–7.99 (m, 1H), 7.71–7.57 (m, 2H), 6.47 (d, J = 8.7 Hz, 1H), 5.77 (s, 1H), 4.61–4.52 (m, 2H), 4.30 (q, J = 8.0 Hz, 2H), 3.70 (s, 2H), 2.23–2.12 (m, 1H), 1.37 (t, J = 8.0 Hz, 3H), 0.88 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.82 (s), 167.19 (s), 166.38 (s), 166.05 (s), 147.98 (s), 136.03 (s), 133.86 (s), 130.52 (s), 129.98 (s), 129.65 (s), 128.69 (s), 128.62 (s), 128.51 (s), 126.15 (s), 124.72 (s), 98.61 (s), 60.53 (s), 57.31 (s), 52.27 (s), 43.98 (s), 31.22 (s), 18.94 (s), 17.71 (s), 14.35 (s). Yellow solid (57% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₃H₂₄N₂O₆ 439.1869, found 439.1867.

Methyl ([1,1′-biphenyl]-4-carbonyl)glycyl-L-valinate (16a). ¹H NMR (400 MHz, CDCl₃) δ 8.07–8.01 (m, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 1H), 7.55 (dd, J = 8.0 Hz, 4H), 7.44–7.31 (m, 3H), 4.57 (dd, J = 8.0 Hz, 1H), 4.31 (d, J = 4.0 Hz, 2H), 3.70 (s, 3H), 2.31–2.09 (m, 1H), 0.95 (dd, J = 6.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.33 (s), 170.04 (s), 167.85 (s), 144.40 (s), 139.92 (s), 132.18 (s), 128.89 (s), 127.99 (s), 127.89 (s), 127.15 (s), 127.08 (s), 57.69 (s), 52.15 (s), 43.83 (s), 31.03 (s), 19.08 (s), 17.91 (s). Yellow solid (62% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₃H₂₄N₂O₆ 369.1814, found 369.1799.

Methyl (Z)-(2-(3-(2-methoxy-2-oxoethylidene)-1-oxo-5-phenylisoindolin-2-yl)acetyl)-L-valinate (17a). ¹H NMR (400 MHz, CDCl₃) δ 9.41 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 4.0 Hz, 1H), 7.71 (d, J = 4.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 6.36 (d, J = 1.0 Hz, 1H), 5.81 (s, 1H), 4.60–4.44 (m, 3H), 3.81 (s, 3H), 3.71 (s, 3H), 2.24–2.08 (m, 1H), 0.89 (dd, J = 6.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.93 (s), 167.20 (s), 166.49 (s), 166.12 (s), 147.82 (s), 147.14 (s), 140.09 (s), 134.59 (s), 130.46 (s), 129.17 (s), 128.47 (s), 127.97 (s), 127.65 (s), 127.25 (s), 124.04 (s), 99.77 (s), 57.34 (s), 52.41 (s), 51.96 (s), 43.81 (s), 31.31 (s), 19.01 (s), 17.81 (s). Yellow solid (64% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₅H₂₆N₂O₆ 451.1869, found 451.1879.

Methyl (Z)-(2-(3-(2-ethoxy-2-oxoethylidene)-1-oxo-5-phenylisoindolin-2-yl)acetyl)-L-valinate (17b). ¹H NMR (400 MHz, CDCl₃) δ 9.43 (d, J = 4.0 Hz, 1H), 7.99 (d, J = 4.0 Hz, 1H), 7.87 (d, J = 4.0 Hz, 1H), 7.73 (d, J = 4.0 Hz, 2H), 7.53 (t, J = 8.0 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H), 6.37 (d, J = 8.0 Hz, 1H), 5.83 (s, 1H), 4.63–4.52 (m, 3H), 4.29 (d, J = 8.0 Hz, 2H), 3.73 (s, 3H), 2.25–2.12 (m, 1H), 1.35 (t, J = 8.0 Hz, 3H), 0.91 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.91 (s), 167.20 (s), 166.54 (s), 165.66 (s), 147.52 (s), 147.12 (s), 140.12 (s), 134.63 (s), 130.41 (s), 129.16 (s), 128.45 (s), 127.99 (s), 127.66 (s), 127.14 (s), 124.02 (s), 100.40 (s), 60.79 (s), 57.33 (s), 52.40 (s), 43.84 (s), 31.33 (s), 19.03 (s), 17.80 (s), 14.38 (s). Yellow solid (60% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₆H₂₈N₂O₆ 465.2026 found 465.2043.

Methyl (S)-2-(2-(2-naphthamido)acetamido)-2-phenylacetate (13b). ¹H NMR (400 MHz, CDCl₃) δ 8.33 (s, 1H), 7.87–7.78 (m, 5H), 7.58–7.47 (m, 3H), 7.42–7.37 (m, 2H), 7.35–7.28 (m, 3H), 5.61 (d, J = 8.0 Hz, 1H), 4.46–4.22 (m, 2H), 3.70 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.14 (s), 168.90 (s), 167.93 (s), 136.01 (s), 134.94 (s), 132.62 (s), 130.72 (s), 129.13 (s), 128.78 (s), 128.50 (s), 128.00 (s), 127.87 (s), 127.79 (s), 127.47 (s), 126.81 (s), 123.74 (s), 56.91 (s), 52.99 (s), 43.78 (s). Yellow solid (60% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₀N₂O₄ 377.1501, found 377.1418.

Methyl-(S,Z)-2-(2-(1-(2-methoxy-2-oxoethylidene)-3-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)acetamido)-2-phenylacetate (14c). ¹H NMR (400 MHz, CDCl₃) δ 9.70 (s, 1H), 8.39 (s, 1H), 8.12–8.07 (m, 1H), 8.04–7.98 (m, 1H), 7.67–7.61 (m, 2H), 7.37–7.29 (m, 5H), 7.00 (d, J = 8.0 Hz, 1H), 5.73 (s, 1H), 5.63–5.50 (m, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.68 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.76 (s), 167.15 (s), 166.47 (s), 165.80 (s), 148.27 (s), 136.02 (s), 135.86 (s), 133.87 (s), 130.53 (s), 129.96 (s), 129.66 (s), 129.02 (s), 128.88 (s), 128.67 (s), 128.64 (s), 128.54 (s), 127.24 (s), 126.12 (s), 124.77 (s), 98.02 (s), 56.60 (s), 52.95 (s), 51.67 (s), 43.75 (s). Yellow solid (62% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₆H₂₂N₂O₆ 459.1556, found 459.1564.

Methyl(S,Z)-2-(2-(1-(2-ethoxy-2-oxoethylidene)-3-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)acetamido)-2-phenylacetate (14d). ¹H NMR (400 MHz, CDCl₃) δ 9.75 (s, 1H), 8.44 (s, 1H), 8.17–8.11 (m, 1H), 8.07–8.02 (m, 1H), 7.71–7.64 (m, 2H), 7.42–7.30 (m, 7H), 6.97 (d, J = 6.8 Hz, 1H), 5.76 (s, 1H), 5.60 (d, J = 8.0 Hz, 1H), 4.60 (d, J = 2.7 Hz, 2H), 4.32 (d, J = 8.0 Hz, 2H), 3.71 (s, 2H), 1.38 (t, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.75 (s), 167.21 (s), 166.07 (s), 165.83 (s), 148.01 (s), 136.05 (s), 135.86 (s), 133.87 (s), 130.54 (s), 130.02 (s), 129.67 (s), 129.21 (s), 129.04 (s), 128.89–128.87 (m), 128.69 (s), 128.65 (s), 127.22 (s), 126.14 (s), 124.79 (s), 98.66 (s), 60.57 (s), 56.58 (s), 52.98 (s), 43.83 (s), 14.38 (s). Yellow solid (58% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₇H₂₄N₂O₆ 473.1713, found 473.1757.

Methyl (S)-2-(2-([1,1′-biphenyl]-4-carboxamido)acetamido)-2-phenylacetate (16b). ¹H NMR (400 MHz, CDCl₃) δ 7.88 (d, J = 8.0 Hz, 2H), 7.70–7.58 (m, 5H), 7.52–7.45 (m, 2H), 7.45–7.33 (m, 6H), 7.31 (s, 1H), 5.62 (d, J = 8.0 Hz, 1H), 4.39–4.14 (m, 2H), 3.74 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.12 (s), 168.75 (s), 167.55 (s), 144.66 (s), 140.01 (s), 135.98 (s), 132.16 (s), 129.16 (s), 129.01 (s), 128.81 (s), 128.13 (s), 127.81 (s), 127.47 (s), 127.30 (s), 56.87 (s), 53.02 (s), 43.67 (s). Yellow solid (55% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₄H₂₂N₂O₄ 403.1658, found 403.1653.

Methyl (S,Z)-2-(2-(3-(2-methoxy-2-oxoethylidene)-1-oxo-5-phenylisoindolin-2-yl)acetamido)-2-phenylacetate (17c). ¹H NMR (400 MHz, CDCl₃) δ 9.39 (d, J = 0.8 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.86–7.80 (m, J = 7.9, 1.4 Hz, 1H), 7.73–7.66 (m, 2H), 7.50 (t, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.35–7.29 (m, 5H), 6.96 (d, J = 6.9 Hz, 1H), 5.76 (s, 1H), 5.56 (d, J = 8.0 Hz, 1H), 4.53 (d, J = 4.0 Hz, 2H), 3.80 (s, 3H), 3.69 (s, 3H).¹³C NMR (101 MHz, CDCl₃) δ 170.78 (s), 167.08 (s), 166.02 (s), 165.92 (s), 147.75 (s), 147.05 (s), 140.04 (s), 135.86 (s), 134.55 (s), 130.34 (s), 129.09 (s), 129.03 (s), 128.69 (s), 128.38 (s), 127.94 (s), 127.57 (s), 127.24 (s), 127.15 (s), 123.96 (s), 99.72 (s), 56.58 (s), 52.96 (s), 51.82 (s), 43.55 (s). Yellow solid (58% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₈H₂₄N₂O₆ 485.1713, found 485.1740.

Methyl (S,Z)-2-(2-(3-(2-ethoxy-2-oxoethylidene)-1-oxo-5-phenylisoindolin-2-yl)acetamido)-2-phenylacetate (17d). ¹H NMR (400 MHz, CDCl₃) δ 9.39 (d, J = 0.9 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.86–7.80 (m, 1H), 7.72–7.66 (m, 2H), 7.53–7.46 (m, 2H), 7.46–7.39 (m, 1H), 7.38–7.27 (m, 5H), 6.96 (d, J = 6.9 Hz, 1H), 5.76 (s, 1H), 5.56 (d, J = 8.0 Hz, 1H), 4.59–4.44 (m, 2H), 4.26 (d, J = 8.0 Hz, 2H), 3.70 (s, 3H), 1.33 (d, J = 8.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 170.77 (s), 167.11 (s), 165.99 (s), 165.59 (s), 147.47 (s), 147.03 (s), 140.08 (s), 135.88 (s), 134.60 (s), 130.30 (s), 129.08 (s), 129.03 (s), 128.69 (s), 128.36 (s), 127.95 (s), 127.58 (s), 127.23 (s), 127.18 (s), 123.94 (s), 100.33 (s), 60.70 (s), 56.58 (s), 52.96 (s), 43.58 (s), 14.32 (s). Yellow solid (56% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₉H₂₆N₂O₆ 498.1869, found 498.1845.

Methyl (4-methylbenzoyl)-L-phenylalanylvalinate (19a). ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8.1 Hz, 2H), 7.32–7.18 (m, 7H), 6.87 (d, J = 7.1 Hz, 1H), 6.53 (d, J = 7.9 Hz, 1H), 5.00–4.85 (m, 1H), 4.43 (d, J = 8.0 Hz, 1H), 3.71 (s, 3H), 3.28–3.11 (m, J = 8.0 Hz, 2H), 2.38 (s, 3H), 2.12–2.04 (m, 1H), 0.98–0.71 (m, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.68 (s), 170.99 (s), 167.26 (s), 142.31 (s), 136.59 (s), 130.95 (s), 129.41 (s), 129.27 (s), 128.69 (s), 127.06 (s), 127.03 (s), 57.51 (s), 54.74 (s), 52.12 (s), 38.19 (s), 31.07 (s), 21.46 (s), 18.85 (s), 17.74 (s). White solid (40% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₃H₂₈N₂O₄ 397.2127, found 397.2145.

Methyl ((S)-2-((Z)-3-(2-methoxy-2-oxoethylidene)-5-methyl-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (20a). ¹H NMR (400 MHz, CDCl₃) δ 8.85 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.19–7.02 (m, 5H), 6.40 (s, 1H), 5.92 (s, 1H), 5.41 (s, 1H), 4.56 (dd, J = 8.0 Hz, 1H), 3.81 (s, 3H), 3.67 (s, 3H), 3.64–3.56 (m, 1H), 3.42–3.30 (m, 1H), 2.50 (s, 3H), 2.16–1.98 (m, 1H), 0.82 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.87 (s), 168.81 (s), 167.41 (s), 166.20 (s), 144.34 (s), 136.86 (s), 133.93 (s), 132.34 (s), 129.04 (s), 128.56 (d, J = 3.0 Hz), 126.91 (s), 126.31 (s), 123.42 (s), 100.40 (s), 57.59 (s), 52.27 (s), 51.83 (s), 34.03 (s), 31.19 (s), 29.72 (s), 22.42 (s), 18.96 (s), 17.84 (s). White solid (40% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₇H₃₀N₂O₆ 479.2182, found 479.2209.

Methyl ((S)-2-((Z)-3-(2-ethoxy-2-oxoethylidene)-5-methyl-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (20b). ¹H NMR (400 MHz,) δ 8.83 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.18–7.05 (m, 5H), 6.40 (s, 1H), 5.86 (s, 1H), 5.39 (s, 1H), 4.56 (dd, J = 8.0 Hz, 1H), 4.29–4.21 (m, 2H), 3.65 (s, 3H), 3.64–3.57 (m, 1H), 3.41–3.26 (m, 1H), 2.49 (s,3H), 2.17–2.03 (m,1H), 1.33 (t, J = 8.0 Hz, 3H), 0.80 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.86 (s), 168.88 (s), 167.40 (s), 165.74 (s), 144.73 (s), 136.96 (s), 133.99 (s), 132.29 (s), 129.06 (s), 128.55 (s), 126.90 (s), 126.38 (s), 123.41 (s), 101.26 (s), 60.72 (s), 57.58 (s), 52.21 (s), 34.03 (s), 31.24 (s), 29.81 (s), 22.39 (s), 19.04 (s), 17.83 (s), 14.38 (s). White solid (40% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₈H₃₂N₂O₆ 492.2339, found 492.2367.

Methyl (4-(tert-butyl)benzoyl)-L-phenylalanyl-L-valinate (19b). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J = 8.0 Hz, 2H), 7.43–7.39 (m, 2H), 7.30–7.19 (m, 6H), 6.98 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 8.1 Hz, 1H), 4.96 (q, J = 8.0 Hz, 1H), 4.45 (dd, J = 8.0 Hz, 1H), 3.71 (s, 3H), 3.28–3.13 (m, 2H), 2.14–2.00 (m, 1H), 1.32 (s, 9H), 0.83 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.70 (s), 171.12 (s), 167.28 (s), 155.35 (s), 136.62 (s), 130.88 (s), 129.41 (s), 128.67 (s), 126.97 (d, J = 4.6 Hz), 125.53 (s), 125.22 (s), 57.50 (s), 54.73 (s), 52.11 (s), 38.13 (s), 34.94 (s), 31.14 (s), 31.08 (s), 18.86 (s), 17.75 (s). White solid (55% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₆H₃₄N₂O₄ 439.2597, found 439.2552.

Methyl ((S)-2-((Z)-5-(tert-butyl)-3-(2-methoxy-2-oxoethylidene)-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (21a). ¹H NMR (400 MHz, CDCl₃) δ 9.18 (d, J = 4.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.20–7.05 (m, 5H), 6.34 (s, 1H), 5.91 (s, 1H), 5.38 (s, 1H), 4.60–4.52 (m, 1H), 3.82 (s, 3H), 3.69 (s, 3H), 3.66–3.59 (m, 1H), 3.45–3.28 (m, 1H), 2.16–2.00 (m, 1H), 1.41 (s, 9H), 0.82 (d, J = 8.0 Hz, 7H). ¹³C NMR (101 MHz, CDCl₃) δ 171.79 (s), 168.72 (s), 166.08 (s), 157.92 (s), 146.94 (s), 136.89 (s), 133.75 (s), 129.34 (s), 128.99 (s), 128.75 (s), 128.54 (s), 126.84 (s), 126.12 (s), 125.31 (s), 123.13 (s), 100.68 (s), 57.55 (s), 52.22 (s), 51.80 (s), 35.87 (s), 34.09 (s), 31.34 (s), 31.19 (s), 29.70 (s), 18.89 (s), 17.84 (s). White solid (35% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₃₀H₃₆N₂O₆ 521.2651, found 521.2672.

Methyl ((S)-2-((Z)-5-(tert-butyl)-3-(2-ethoxy-2-oxoethylidene)-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (21b). ¹H NMR (400 MHz, CDCl₃) δ 9.17 (d, J = 1.1 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.60 (dd, J = 8.0, 1.6 Hz, 1H), 7.21–7.08 (m, 1H), 6.37 (s, 1H), 5.88 (s, 1H), 5.40 (s, 1H), 4.58 (dd, J = 8.6, 5.0 Hz, 1H), 4.35–4.20 (m, 1H), 3.68 (s, 1H), 3.67–3.61 (m, 1H), 3.42–3.30 (m, 1H), 2.20–2.07 (m, 1H), 1.40 (s, 9H), 1.34 (t, J = 8.0 Hz, 3H), 0.84 (d, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.80 (s), 168.80 (s), 165.62 (s), 157.86 (s), 136.94 (s), 133.79 (s), 129.00 (s), 128.96 (s), 128.69 (s), 128.51 (s), 126.82 (s), 126.14 (s), 125.31 (s), 123.10 (s), 101.15 (s), 60.62 (s), 57.56 (s), 52.22 (s), 35.86 (s), 34.09 (s), 31.34 (s), 31.24 (s), 29.70 (s), 18.97 (s), 17.86 (s), 14.34 (s). White solid (32% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₃₁H₃₈N₂O₆ 535.2730, found 535.2773.

Methyl ((S)-2-((Z)-3-(2-butoxy-2-oxoethylidene)-5-(tert-butyl)-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (21c). ¹H NMR (400 MHz, CDCl₃) δ 9.19 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 9.4 Hz, 1H), 7.21–7.10 (m, 3H), 6.41 (s, 1H), 5.89 (s, 1H), 5.43 (s, 1H), 4.60 (dd, J = 8.6, 4.9 Hz, 1H), 4.31–4.16 (m, 2H), 3.69 (s, 2H), 3.67–3.62 (m, 1H), 3.44–3.30 (m, 1H), 2.19–2.10 (m, 1H), 1.74–1.65 (m, 2H), 1.49–1.44 (m, 2H), 1.42 (s, 9H), 0.99 (t, J = 8.0 Hz, 3H), 0.86 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.79 (s), 168.81 (s), 165.74 (s), 157.85 (s), 150.88 (s), 136.95 (s), 133.81 (s), 129.00 (s), 128.68 (s), 128.50 (s), 128.25 (s), 126.81 (s), 126.14 (s), 125.31 (s), 123.09 (s), 101.17 (s), 64.58 (s), 57.55 (s), 52.21 (s), 35.86 (s), 34.07 (s), 31.34 (s), 31.27 (s), 30.75 (s), 29.70 (s), 19.18 (s), 18.97 (s), 17.85 (s), 13.75 (s). White solid (30% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₃₃H₄₂N₂O₆ 563.3121, found 563.3135.

Methyl ((S)-2-((Z)-3-(2-(tert-butoxy)-2-oxoethylidene)-5-(tert-butyl)-1-oxoisoindolin-2-yl)-3-phenylpropanoyl)-L-valinate (21d). ¹H NMR (400 MHz, CDCl₃) δ 9.03 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.62–7.55 (m, 1H), 7.21–7.08 (m, 5H), 6.40 (s, 1H), 5.78 (s, 1H), 5.39 (s, 1H), 4.63–4.55 (m, 1H), 3.68 (s, 3H), 3.66–3.62 (m, 1H), 3.42–3.26 (m, 1H), 2.13 (m, 1H), 1.55 (s, 9H), 1.39 (s, 9H), 0.86 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.78 (s), 168.95 (s), 165.01 (s), 157.60 (s), 137.08 (s), 133.91 (s), 129.03 (s), 128.95 (s), 128.47 (s), 128.28 (s), 126.77 (s), 126.17 (s), 124.83 (s), 123.01 (s), 103.33 (s), 81.00 (s), 57.57 (s), 52.21 (s), 35.81 (s), 34.08 (s), 31.30 (s), 31.24 (s), 29.70 (s), 28.21 (s), 19.10 (s), 17.87 (s). White solid (32% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₃₃H₄₂N₂O₆ 562.3043, found 562.3095.

Methyl (3-nitrobenzoyl)-L-phenylalanyl-L-valinate (19c). ¹H NMR (400 MHz, CDCl₃) δ 8.59 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.36–7.25 (m, 5H), 6.46 (d, J = 8.0 Hz, 1H), 4.97 (q, J = 8.0 Hz, 1H), 4.48 (dd, J = 8.0 Hz, 1H), 3.75 (s, 3H), 3.23 (m, 1H), 2.21–2.09 (m, 1H), 0.88 (dd, J = 8.0 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.61 (s), 170.89 (s), 164.79 (s), 148.27 (s), 136.21 (s), 135.38 (s), 132.92 (s), 129.73 (s), 129.37 (s), 128.78 (s), 127.25 (s), 126.27 (s), 122.36 (s), 57.64 (s), 55.23 (s), 52.22 (s), 38.47 (s), 31.08 (s), 18.87 (s), 17.77 (s). White solid (53% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₅N₂O₄ 428.1822, found 428.1808.

Methyl benzoylglycyl-L-alanyl-L-phenylalaninate (22a). ¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J = 7.3 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.45–7.38 (m, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.24–7.15 (m, 1H), 7.10–7.02 (m, 1H), 4.88–4.79 (m, 1H), 4.58 (t, J = 8.0 Hz, 1H), 4.12–4.07 (m, 2H), 3.68 (s, 3H), 3.13 (m, 1H), 3.04 (m, 1H), 1.34 (d, J = 8.0 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) δ 171.82 (s), 168.98 (s), 167.88 (s), 166.79 (s), 135.82 (s), 133.50 (s), 131.89 (s), 129.23 (s), 128.58 (s), 128.51 (s), 127.28 (s), 127.09 (s), 53.31 (s), 52.39 (s), 48.98 (s), 43.47 (s), 37.80 (s), 18.18 (s). White solid (50% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₂H₂₆N₃O₅ 412.1873, found 412.1873.

Methyl (2-((Z)-1-(2-methoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)acetyl)-L-alanyl-L-phenylalaninate (23). ¹H NMR (400 MHz, CDCl₃) δ 9.05 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.71–7.63 (m, J = 9.6, 5.7 Hz, 1H), 7.61–7.54 (m, J = 13.2, 5.9 Hz, 1H), 7.29–7.17 (m, 1H), 7.07 (d, J = 6.8 Hz, 1H), 6.98 (t, J = 8.8 Hz, 1H), 6.74 (t, J = 7.9 Hz, 1H), 5.68 (s, 1H), 4.78 (m, 1H), 4.59–4.49 (m, 1H), 4.44 (s, 2H), 3.75 (s, 3H), 3.68 (s, 3H), 3.16–2.97 (m, 2H), 1.32 (d, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.62 (s), 171.53 (s), 167.20 (s), 166.33 (s), 166.04 (s), 147.96 (s), 135.60 (s), 133.78 (s), 133.66 (s), 131.48 (s), 129.42 (s), 129.22 (s), 128.60 (s), 128.25 (s), 127.19 (s), 123.51 (s), 99.13 (s), 53.43 (s), 52.43 (s), 51.73 (s), 48.91 (s), 43.06 (s), 37.74 (s), 18.37 (s). White solid (28% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₆H₂₇N₃O₇ 493.1849, found 493.4983.

Methyl benzoyl-L-valyl-L-leucyl-L-phenylalaninate (22b). ¹H NMR (400 MHz, DMSO) δ 8.17 (d, J = 8.1 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.90–7.82 (m, 1H), 7.55–7.48 (m, 1H), 7.44 (dd, J = 9.2, 4.1 Hz, 1H), 7.23 (t, J = 7.1 Hz, 1H), 7.17 (d, J = 5.8 Hz, 1H), 4.53 (d, J = 8.0 Hz, 1H), 4.41 (q, J = 8.0 Hz, 1H), 4.32 (t, J = 8.0 Hz, 1H), 3.58 (s, 3H), 2.99 (m, 2H), 2.13 (m, 1H), 1.59 (m, 1H), 1.43 (t, J = 8.0 Hz, 2H), 0.94–0.79 (m, 12H). ¹³C NMR (101 MHz, DMSO) δ 172.32 (s), 171.91 (s), 171.14 (s), 167.07 (s), 137.21 (s), 134.86 (s), 131.48 (s), 129.30 (s), 128.50 (s), 128.44 (s), 127.82 (s), 126.82 (s), 59.56 (s), 53.62 (s), 52.05 (s), 51.67–51.39 (m), 41.45 (s), 37.16 (s), 30.59 (s), 24.49 (s), 23.28 (s), 22.11 (s), 19.76 (s), 19.06 (s). White solid (42% yield). ESI-HRMS m/z [M + H]⁺ calcd for C₂₈H₃₈N₃O₅ 496.2812, found 496.3018.

Methyl ((S)-2-((Z)-1-(2-methoxy-2-oxoethylidene)-3-oxoisoindolin-2-yl)-3-methylbutanoyl)-L-leucyl-L-phenylalaninate (24). ¹H NMR (400 MHz, CDCl₃) δ 9.06 (d, J = 7.9 Hz, 1H), 7.95 (d, J = 15.8 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 7.69 (td, J = 7.8, 1.1 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.29 (dd, J = 16.1, 6.9 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1H), 6.41 (d, J = 15.7 Hz, 1H), 6.26 (s, 1H), 4.78 (d, J = 8.0 Hz, 1H), 4.46–4.38 (m, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.65 (m, 2H), 3.34–3.16 (m, 2H), 1.62–1.53 (m, 1H), 1.48–1.38 (m, 2H), 1.13–0.67 (m, 12H). ¹³C NMR (101 MHz, CDCl₃) δ 171.29 (s), 171.25 (s), 168.93 (s), 167.58 (s), 166.27 (s), 141.77 (s), 135.84 (s), 133.73 (s), 133.52 (s), 131.34 (s), 131.14 (s), 130.22 (s), 128.83 (s), 128.02 (s), 127.72 (s), 126.74 (s), 123.46 (s), 119.69 (s), 101.51 (s), 53.14 (s), 52.45 (s), 51.83 (s), 51.73 (s), 39.94 (s), 34.87 (s), 31.92 (s), 29.70 (s), 24.69 (s), 22.80 (s), 21.40 (s), 20.84 (s), 14.13 (s). White solid (25% yield). ESI-HRMS m/z [M + Na]⁺ calcd for C₃₂H₃₉N₃O₇ 601.2686, found 601.4069.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

MKG thanks UGC for JRF/SRF. NKS thanks SERB-New Delhi Core Research Grant with Grant No. CRG/2020/001028.

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Footnotes

† Electronic supplementary information (ESI) available: NMR and mass data of all new compounds are provided. 2D NMR of compound 19a and DMSO-titration spectra are also provided. Computational study outcomes (energy vs. conformation profiles are also provided. See DOI: https://doi.org/10.1039/d3ob00742a

‡ These authors are equally contributed.

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