Tryptophanol-Derived Oxazolopyrrolidone Lactams as Potential Anticancer Agents against Gastric Adenocarcinoma

Abstract

Gastric cancer is one of the deadliest cancers in modern societies, so there is a high level of interest in discovering new drugs for this malignancy. Previously, we demonstrated the ability of tryptophanol-derived polycyclic compounds to activate the tumor suppressor protein p53, a relevant therapeutic target in cancer. In this work, we developed a novel series of enantiomerically pure tryptophanol-derived small molecules to target human gastric adenocarcinoma (AGS) cells. From an initial screening of fourteen compounds in AGS cell line, a hit compound was selected for optimization, leading to two derivatives selective for AGS gastric cells over other types of cancer cells (MDA-MB-231, A-549, DU-145, and MG-63). More importantly, the compounds were non-toxic in normal cells (HEK 293T). Additionally, we show that the growth inhibition of AGS cells induced by these compounds is mediated by apoptosis. Stability studies in human plasma and human liver microsomes indicate that the compounds are stable, and that the major metabolic transformations of these molecules are mono- and di-hydroxylation of the indole ring.

1. Introduction

Cancer is considered a worldwide health problem, and its occurrence can be associated to a combination of environmental factors and genetic alterations [1]. According to the World Health Organization (WHO), it is estimated that in 2018, cancer contributed to 9.5 million deaths worldwide [2]. Gastric cancer (GC) ranks third in the list of deadliest cancers [1], and its occurrence and mortality are highly influenced by region and culture [3]. The survival rate of GC has not improved much over the last years. Patients with GC in early-stage, usually, do not have symptoms, which hinders the early detection of this cancer. For this reason, most patients present advanced GC and, in these cases, radical surgery is the first-line approach and the only curative treatment [4]. In the cases that surgery is not recommended, alternative treatments can be used, such as chemotherapy, radiotherapy, and immunotherapy. However, these therapeutic options only achieve modest results, and the poor response of this cancer to chemotherapy is, typically, associated to chemoresistance mechanisms [5,6]. Moreover, the severe side effects associated to drug-related toxicity are frequent [7,8]. Consequently, the discovery of new alternative therapeutics for the treatment of GC, with low cost and minimal side effects, is still urgently needed. In the last decades, the discovery of cellular mechanisms associated to malignancies has been intensive, and many anticancer agents were developed to disrupt specific biological pathways. With this, the discovery of new scaffolds increased, as well as the interest in new therapeutic applications to scaffolds already known. For example, the indole scaffold is associated to many pharmacological activities in medicinal chemistry, including antimicrobial, antioxidant, antiviral, and anticancer [9,10]. It is considered a privileged scaffold, commonly found in many natural products (e.g., alkaloids and microbial hormones) and synthetic molecules with medicinal value (e.g., compounds 1 and 2, Figure 1) [11].

Other examples are tryptophanol-based small molecules (e.g., compounds 3–6, Figure 2), reactivators of the p53 pathway, that showed in vitro antiproliferative activity in colon and breast cancer cells [12,13,14,15,16,17]. Specifically, tryptophanol-derived isoindolinones 4–5 presented promising in vivo antitumor results in xenograft mouse models, without cytotoxicity and genotoxicity [13,14,16]. Based on these results, and on reported results with pyrrolidone-based small molecules with anticancer activity [18,19], we envisioned that the merge of these two scaffolds could lead to compounds with interesting anticancer properties [15]. Herein, we report the synthesis of 29 enantiopure tryptophanol-derived oxazolopyrrolidone lactams (compounds 7 and 8, Figure 2), their antiproliferative activity in human gastric adenocarcinoma (AGS) cell line, and in vitro stability and metabolic studies with this scaffold.

2. Results and Discussion

2.1. Chemistry

Enantiopure bicyclic lactams 7a–j, 7j’, and 8a–g were easily accessed by a chiral-induced cyclocondensation reaction, starting from enantiopure tryptophanol and commercially available keto acids, in low to excellent yields (18–95%, Scheme 1) [20]. In almost all reactions, the formation of only one diastereomer was observed by thin-layer chromatography (TLC) and proton nuclear magnetic resonance (¹H NMR). In the cyclocondensation reaction of (R)-tryptophanol with 4-(4-chlorophenyl)-3-methyl-4-oxobutanoic acid, in which an additional chirality center is formed, diastereomer 7j (69% yield) was obtained, as well as the minor diastereoisomer 7j’ (18% yield).

Tryptophanol-derived oxazolopyrrolidone lactams 7k–m, with substituents on the indole nitrogen, were obtained in moderate to good yields (66–78%, Scheme 2). Specifically, compounds 7k–l were synthesized by treatment of 7c with sodium hydride in dimethylformamide, in the presence of iodoethane (compound 7k) or acetic anhydride (compound 7l). Compound 7m was prepared by reaction of 7c with di-tert-butyl dicarbonate, in the presence of 4-dimethylaminopyridine and triethylamine, in tetrahydrofuran.

Compounds 7n–u were obtained through Suzuki-Miyaura cross-coupling reaction between compound 7d and different aryl boronic acids, using Pd(PPh₃)₂Cl₂ as catalyst (Scheme 3). Except for compound 7u, which was obtained in low yield (28%) due to the low solubility of the boronic acid, all the other derivatives were obtained in high yields (71–97%).

The absolute configuration of the new formed stereogenic center C-7a was established by X-ray analysis of compound 8b (Figure 3). The ¹³C NMR spectroscopy data of compound 8b was used as reference to confirm the stereochemistry of the other derivatives. For compounds 7a–i and 8a–g, the signals of C-3, C-7a, and C-7 appear between 55.5–56.5, 101.7–102.6, and 35.0–35.4 ppm, respectively.

The spectral data obtained for compounds 7j and 7j’ indicate that the major diastereomer 7j has (3R, 7aR, 7S) configuration, while the minor diastereoisomer 7j’ has (3R, 7aR, 7R) configuration [21]. In particular, the methyl group appears in the ¹H NMR spectra as a doublet at 1.12 ppm for 7j and at 0.60 ppm for 7j’, and in the ¹³C NMR spectra at 13.96 ppm for 7j and at 16.40 ppm for 7j’. Moreover, the methyl group induces a shift in the C-7 that appears at 39.7 ppm for compound 7j and at 41.3 ppm for compound 7j’. The chemical shift of C-3 appears in a higher field for diastereoisomer 7j’ (54.8 ppm). The absolute configuration of diastereomers 7j and 7j’ was further confirmed by X-ray crystallography (Figure 3).

2.2. Effect of Tryptophanol-Derived Oxazolopyrrolidone Lactams on Cell Viability and on Apoptosis

To perform a structure–activity relationship (SAR) study, a first series of tryptophanol-derived oxazolopyrrolidone lactams containing different substituents on the phenyl ring (R¹) at position C-7a was synthesized (compounds 7a–g and 8a–g,

Table 1. Screening of (R) and (S)-tryptophanol-derived oxazolopyrrolidone lactams 7a–g and 8a–g in AGS cell line.

Compound	R1	R2	% CV at 100 µM 1
7a	H	H	50 ± 5
7b	F	H	50 ± 8
7c	Cl	H	6 ± 1
7d	Br	H	14 ± 3
7e	CH3	H	14 ± 2
7f	OCH3	H	18 ± 2
7g	SO2CH3	H	94 ± 12
8a	H	H	58 ± 4
8b	F	H	22 ± 4
8c	Cl	H	11 ± 1
8d	Br	H	25 ± 4
8e	CH3	H	60 ± 3
8f	OCH3	H	30 ± 4
8g	SO2CH3	H	91 ± 11

¹ Each % of CV (cell viability) value is the mean ± SD of triplicate of at least two different experiments. % CV determined by the MTT method after 48 h of compounds’ incubation.

). In the design of this new compounds series, a diversity of substituents with electron donating properties (–CH₃ and –OCH₃ groups) and electron withdrawing properties (–F, –Cl, –Br, and –SO₂CH₃ groups) were chosen. Both series of enantiomers, (S)- and (R)-tryptophanol derivatives, were synthesized to evaluate the impact of compound’s stereochemistry on the antiproliferative response of AGS cells. The activity of the target compounds was assessed using the MTT reduction assay. In general, (R)-tryptophanol-derived oxazolopyrrolidone lactams were more active than the corresponding enantiomers, except for derivative 8b with a para-fluoro substituent (7a–g vs. 8a–g). From the first screening at 100 µM, analogues 7a (R¹ = H), 7b (R¹ = F), and 8e (R¹ = CH₃) showed moderate antiproliferative activity, while compounds 7g and 8g (R¹ = SO₂CH₃) did not induce appreciable cytotoxicity. Remarkably, compounds 7c–e and 8c revealed an antiproliferative response higher than 85%. The presence of chlorine or bromine substituents at R¹ had a positive impact on the antiproliferative activity, for both enantiomers (compounds 7c–d and 8c–d). The derivative 7c (R¹ = Cl) exhibited the highest activity and was selected for chemical derivatizations to improve the antiproliferative activity of this scaffold in AGS cells.

Four sites were identified for suitable structural modifications in compound 7c: meta- position of the C-7a phenyl ring (compounds 7h and 7i, Scheme 1), position C-7 of the pyrrolidone ring (compounds 7j and 7j’, Scheme 1), alkylation of the N-indole (compounds 7k–m, Scheme 2) and C–C couplings in the C-7a phenyl ring (compounds 7n–u, Scheme 3).

The fifteen (R)-tryptophanol-derived oxazolopyrrolidone lactams 7h–u and 7j’ obtained, as well as 7c, were screened at 50 µM in AGS cell line (

Table 2. Screening of (R)-tryptophanol-derived oxazolopyrrolidone lactams 7c, 7h–u, and 7j’ in AGS cell line.

Compound	R1	R2	R3	R4	% CV at 50 µM 1
7c	Cl	H	H	H	11 ± 1
7h	CH3	Cl	H	H	11 ± 2
7i	OCH3	F	H	H	84 ± 7
7j	Cl	H	(S)-CH3	H	8 ± 0
7j’	Cl	H	(R)-CH3	H	81 ± 3
7k	Cl	H	H	CH2CH3	40 ± 5
7l	Cl	H	H	COCH3	63 ± 13
7m	Cl	H	H	CO2C(CH3)3	61 ± 9
7n	p-Cl-Ph	H	H	H	65 ± 13
7o	p-CF3-Ph	H	H	H	7 ± 1
7p	p-OH-Ph	H	H	H	56 ± 1
7q	p-CH2OH-Ph	H	H	H	55 ± 1
7r	m-Cl-Ph	H	H	H	17 ± 1
7s	3,4-Cl-Ph	H	H	H	8 ± 1
7t	pyridine	H	H	H	75 ± 14
7u		H	H	H	67 ± 6

¹ Each % of CV (cell viability) value is the mean ± SD of triplicate of at least two different experiments. % CV determined by the MTT method after 48 h of compounds’ incubation.

).

(R)-tryptophanol-derived oxazolopyrrolidones 7h and 7r showed similar antiproliferative activity to 7c, while 7j, 7o, and 7s were more active than the hit compound 7c. The presence of a pyridine (compound 7t) or a dioxane ring (compound 7u) led to a decrease of the antiproliferative effect in AGS cells. Additionally, meta-fluoro and para-methoxy substituents on the phenyl ring (compound 7i) resulted in a non-significant cell death. Compounds 7n (R¹ = p-Cl-Ph), 7p (R¹ = p-OH-Ph), and 7q (R¹ = p-CH₂OH-Ph), with bulky substituents, displayed moderate antiproliferative activity at 50 µM. The results also suggest that the presence of a meta-chloro substituent or electron withdrawing groups are important for the activity (7r and 7s vs. 7n and 7o, 7r, and 7s vs. 7p and 7q). Interestingly, the two diastereomers 7j and 7j’ had different effects in AGS cells. Diastereomer 7j, with (3R, 7R, 7aS) configuration, had a high antiproliferative effect, while diastereomer 7j’ (3R, 7R, 7aR) had almost no effect, suggesting that the C-7a stereochemistry is also decisive for the antiproliferative activity of tryptophanol-derived oxazolopyrrolidone lactams in AGS cells.

The substitution of the N-indole hydrogen (compound 7c) by ethyl (compound 7k), acetyl (compound 7l) or tert-butyloxycarbonyl (compound 7m) groups led to a decrease of activity, probably due to steric effects or because the establishment of a hydrogen bond might be important for the antiproliferative effect.

The IC₅₀ values of the most promising derivatives (7j, 7o, and 7s), as well as of the hit compound 7c, were determined in AGS cell line (

Table 3. IC₅₀ values ¹ in cancer cell lines of selected compounds.

Compound	AGS IC50 (µM)	MDA-MB-231 IC50 (µM)	A-549 IC50 (µM)	DU-145 IC50 (µM)	MG-63 IC50 (µM)	HEK 293T IC50 (µM)
7c	(3.4 ± 0.2) × 10	-	-	-	-	-
7j	(2.8 ± 0.4) × 10	-	-	-	-	-
7o	(1.5 ± 0.6) × 10	(2.8 ± 0.2) × 10	(6.3 ± 1.8) × 10	(2.4 ± 0.5) × 10	26.8 ± 0.4	(5.2 ± 0.2) × 10
7s	(1.3 ± 0.4) × 10	(6.8 ± 0.6) × 10	(8.8 ± 1.7) × 10	(2.1 ± 1.2) × 10	(5.6 ± 0.7) × 10	(11.7 ± 0.6) × 10

¹ IC₅₀ values determined by the MTT method after 48 h of compounds’ incubation. Each IC₅₀ value is the mean ± SD of, at least, three independent experiments with six replicates each.

). Trifluoromethyl derivative 7o (R¹ = p-CF₃-Ph) and di-halogenated derivative 7s (R¹ = 3,4-Cl-Ph) were the most active derivatives with 2.3 times more potency than the hit 7c, respectively. We then tested compounds 7o and 7s in four cancer cell lines of other tumor types (Table 3): MDA-MB-231 (breast adenocarcinoma), A-549 (lung carcinoma), DU-145 (prostate cancer), and MG-63 (osteosarcoma). Both compounds were much less potent in lung carcinoma cells (IC₅₀ higher than 60 µM) but presented moderate activity in prostate cancer cell line DU-145 (Table 3). In osteosarcoma and breast cells, compound 7o was around two times more active than compound 7s. Compounds 7o and 7s were then evaluated in HEK 293T normal cell line [22] and, except for A-549 cells, showed selectivity towards all cancer cell lines over the non-cancer derived cell line (Table 3).

The ability of compounds 7o and 7s to induce apoptosis was also explored by measuring caspase 3/7 activity in AGS cells. The assays showed that, after 48 h of compounds’ incubation at 12.5 µM, there was a significant increase of caspase 3/7 activity, indicating that the antiproliferative activity is associated with apoptosis induction (Figure 4).

2.3. Stability Studies in PBS, Human Plasma, and Human Liver Microsomes and Identification of Metabolites

Preliminary stability studies can provide useful information about possible liabilities of new drug candidates. Understanding possible clearance mechanisms and how to modulate the metabolism to reduce metabolic liability of a new bioactive chemical entity is a fundamental step in drug development that allows access to a hit compound with desirable ADME attributes [23]. The in vitro phosphate saline buffer (PBS), plasma, and metabolic stabilities for compound 7s were evaluated. This compound showed chemical stability in PBS conditions and under plasmatic enzyme activity after 24 h of incubation, at 37 °C (Figure 5A). The in vitro metabolic stability of compound 7s was determined upon incubation in human liver microsomes, in the presence of the Phase I cofactor NADPH (Figure 5B). This compound demonstrated to be moderately stable [24,25], with a half-life (t_1/2) of 45 min (see Supplementary Materials Figure S1) and an intrinsic hepatic clearance (CL_int) of 22.8 min⁻¹·mL⁻¹·Kg⁻¹. Three main Phase I metabolites, stemming from mono- and di-hydroxylation of the indole moiety, were identified by LC-HRMS/MS (liquid chromatography high resolution tandem mass spectrometry) analysis. The protonated molecule of the parent compound, 7s, is observed in the HRMS-ESI(+) full scan spectrum at m/z 477.1148 ± 3.6 ppm, with the characteristic dichlorine isotope cluster, and the base peak of the MS/MS spectrum is observed at m/z 304.0289 ± 0.3 ppm, which corresponds to the loss of the dichloro-biphenyl-dihydropyrrolone moiety from the protonated molecule (see Supplementary Materials Figure S2). A mass increment of 15.9944 u is observed for the protonated molecules of the two close eluting (major) metabolites at m/z 493.1100 ± 4.0 and m/z 493.1098 ± 3.7 ppm, which are, therefore, compatible with two isomer mono-hydroxylated metabolites of compound 7s, indicated with abbreviation mono-OH-7s (Figure 5C, see Supplementary Materials Figure S3). The structural similarity of these two Phase I metabolites was further confirmed by the similar fragmentation patterns observed in the tandem mass spectra (see Supplementary Materials Figure S3B,C), whose base peaks correspond to the loss the dichloro-biphenyl-dihydropyrrolone moiety, similarly to what is observed for 7s. Whereas the exact location of the hydroxyl group could not be determined, the hydroxylation of the indole moiety is suggested by the observation of the diagnostic fragment ion at m/z 146.0606 ± 4.1 ppm in the MS/MS spectra of the two mono-hydroxylated metabolites (Figure 5C, see Supplementary Materials Figure S3B,C). With retention time of 15.9 min, a minor di-hydroxilated metabolite was also identified based on the observation of the monoisotopic signal at m/z 509.1050 ± 4.1 ppm, in the full scan HRMS-ESI(+) spectra (see Supplementary Materials Figure S4B). Identification of the diagnostic fragment ion at m/z 150.0551 ± 1.3 ppm in the MS/MS spectrum confirms the di-hydroxylation on the indole ring (Figure 5C, see Supplementary Materials Figure S4B). The observation of the fragment ion at m/z 162.0551 ± 1.2 ppm (the di-hydroxylated version of the mentioned diagnostic fragment ion for mono-OH-7s metabolites), represents an additional evidence that the main site of Phase I biotransformation is the indole ring. This constitutes an expected metabolic transformation [26], which is not linked with drug bioactivation processes [27], and, therefore, is not anticipated to be a toxicity red flag alert. Nonetheless, taking into consideration the moderate metabolic stability of the parent compound, it might be relevant to assess the activity of hydroxylated metabolites, following further improvement of this scaffold.

3. Materials and Methods

3.1. Chemistry

General information: THF was dried using sodium wire and benzophenone as indicator. (R)-Tryptophanol was obtained by reduction of (R)-tryptophan using lithium aluminum hydride [28]. Other reagents were obtained from commercial suppliers (Sigma-Aldrich, Alfa Aesar, and Fluorochem). General information concerning the equipment used for the elucidation of the products’ chemical structures and product characterization (NMR, melting point, optical rotations, MS, and elemental analysis) are presented in our earlier publication [21]. Multiplicities in ¹H NMR spectra are given as: s (singlet), d (doublet), dd (double doublet), ddd (doublet of doublets of doublets), t (triplet), and m (multiplet). Compounds 7h, 7j, and 7j’ showed purity ≥ 95% by LC-MS, performed in a LaChrom HPLC constituted of a Merck Hitachi pump L-7100, Merck Hitachi autosampler L-7250, and a Merck Hitachi UV detector L-7400. Analyses were performed with a LiChrospher^®100 RP-8 (5 µm) LiChroCART^® 250-4 column at room temperature, using a mobile phase solution constituted of 65% acetonitrile and 35% Milli-Q water. Peaks were detected at λ = 254 nm.

General procedure for the synthesis of compounds 7a–j, 7j’, and 8a–g: To a suspension of enantiopure tryptophanol (0.53 mmol, 1.0 eq.) in toluene (5 mL) was added the appropriate oxocarboxylic acid (0.58 mmol, 1.1 eq.). The mixture was heated at reflux for 10–25 h in a Dean–Stark apparatus. The reaction mixture was concentrated in vacuo and the residue obtained was dissolved in EtOAc (10 mL). The organic phase was washed with saturated aqueous solution of NaHCO₃ (15 mL) and brine (15 mL), dried over Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified by silica gel flash chromatography using a mixture of EtOAc/n-hexane as eluent.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-phenyltetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7a): Following the general procedure, to a solution of (R)-tryptophanol (0.102 g, 0.536 mmol) in toluene (5 mL) was added 3-benzoyl propionic acid (0.105 g, 0.590 mmol). Reaction time: 19 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.166 g, 95%); ${\alpha }_D^{25}$ = −54.7° (c = 2.0, MeOH); ¹H NMR spectra was found to be identical to the one reported [15] and obtained for compound 8a. Anal. Calcd. for C₂₁H₂₀N₂O₂·0.05H₂O: C, 75.67%; H, 6.09%; N, 8.41%. Found C: 75.22%; H: 5.87%; N: 8.23%. The m.p. value was found to be identical to the one reported for compound 8a.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-phenyltetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8a): Following the general procedure, to a solution of (S)-tryptophanol (0.101 g, 0.529 mmol) in toluene (5 mL) was added 3-benzoyl propionic acid (0.104 g, 0.582 mmol). Reaction time: 24 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.127 g, 72%); ${\alpha }_D^{25 }$= +40.4° (c = 2.0, MeOH); m.p.: 153–156 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.99 (s, 1H, NH), 7.50 (d, J = 6.0 Hz, 2H, ArH), 7.46–7.29 (m, 5H, ArH), 7.17 (t, J = 7.5 Hz, 1H, ArH), 7.10–7.05 (m, 2H, ArH), 4.62–4.52 (m, 1H, H-3), 4.16 (t, J = 8.1 Hz, 1H, H-2), 3.63–3.58 (m, 1H, H-2), 3.07 (dd, J = 14.3, 6.2 Hz, 1H, indole-CH₂), 2.96–2.75 (m, 1H, CH₂), 2.68–2.35 (m, 3H, CH₂, and indole-CH₂), 2.34–2.14 (m, 1H, CH₂) ppm; Anal. Calcd. for C₂₁H₂₀N₂O₂: C: 75.88%; H: 6.06%; N: 8.43%. Found C: 75.95%; H: 5.76%; N: 8.36%. ¹H NMR spectra was found to be identical to the one reported [15].

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-fluorophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7b): Following the general procedure, to a solution of (R)-tryptophanol (0.100 g, 0.526 mmol) in toluene (5 mL) was added 3-(4-fluorobenzoyl) propionic acid (0.114 g, 0.581 mmol). Reaction time: 19 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale yellow crystalline solid (0.113 g, 70%); ${\alpha }_D^{25 }$ = −48.8° (c = 2.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8b. Anal. Calcd. for C₂₁H₁₉FN₂O₂: C: 71.98%; H: 5.47%; N: 8.00%. Found C: 72.09%; H: 5.49%; N: 7.94%. The m.p. value was found to be identical to the one reported for compound 8b.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(4-fluorophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8b): Following the general procedure, to a solution of (S)-tryptophanol (0.102 g, 0.535 mmol) in toluene (5 mL) was added 3-(4-fluorobenzoyl) propionic acid (0.115 g, 0.588 mmol). Reaction time: 21 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give orange crystalline solid (0.156 g, 83%); ${\alpha }_D^{25 }$ = +39.5° (c = 2.0, MeOH); m.p.: 197-198 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H, NH), 7.51-7.41 (m, 3H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.21–7.15 (m, 1H, ArH), 7.11–7.03 (m, 4H, ArH), 4.62–4.53 (m, 1H, H-3), 4.17 (dd, J = 8.8 Hz, 7.4 Hz, 1H, H-2), 3.59 (dd, J = 8.8 Hz, 6.9Hz, 1H, H-2), 3.05 (dd, J = 14.7 Hz, 6.2Hz, 1H, indole-CH₂), 2.90–2.78 (m, 1H, CH₂), 2.65–2.43 (m, 3H, CH₂, and indole-CH₂), 2.24 − 2.15 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 162.8 (d, Cq, J_C-F = 245.3 Hz), 138.8 (d, Cq, J = 3.1 Hz), 136.3 (Cq), 127.5 (Cq), 126.9 (d, ArCH, J = 8.1 Hz), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 118.9 (ArCH), 115.5 (d, ArCH, J = 21.5 Hz), 111.6 (Cq), 111.3 (ArCH), 102.2 (C-7a), 72.8 (C-2), 55.8 (C-3), 35.2 (CH₂), 32.7 (CH₂), 29.8 (indole-CH₂). Anal. Calcd. for C₂₁H₁₉FN₂O₂: C: 71.98%; H: 5.47%; N: 8.00%. Found C: 72.48%; H: 5.37%; N: 8.03%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-chlorophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7c): Following the general procedure, to a solution of (R)-tryptophanol (0.103 g, 0.541 mmol) in toluene (5 mL) was added 3-(4-chlorobenzoyl) propionic acid (0.127 g, 0.596 mmol). Reaction time: 18 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale yellow crystalline solid (0.133 g, 67%); ${\alpha }_D^{25 }$= −63.1° (c = 2.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8c. Anal. Calcd. for C₂₁H₁₉ClN₂O₂: C: 68.76%; H: 5.18%; N: 7.62%. Found C: 68.76%; H: 5.22%; N: 7.64%. The m.p. value was found to be identical to the one reported for compound 8c.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(4-chlorophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8c): Following the general procedure, to a solution of (S)-tryptophanol (0.104 g, 0.545 mmol) in toluene (5 mL) was added 3-(4-chlorobenzoyl) propionic acid (0.128 g, 0.600 mmol). Reaction time: 23 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1/1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.164 g, 82%); ${\alpha }_D^{25 }$= +54.5° (c = 2.0, MeOH); m.p.: 206–208 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.98 (s, 1H, NH), 7.47–7.32 (m, 6H, ArH), 7.21–7.16 (m, 1H, ArH), 7.12–7.06 (m, 2H, ArH), 4.62–4.52 (m, 1H, H-3), 4.17 (dd, J = 8.8 Hz, 7.5 Hz, 1H, H-2), 3.59 (dd, J = 8.8 Hz, 6.9 Hz, 1H, H-2), 3.05 (dd, J = 15.1 Hz, 7.5 Hz, 1H, indole-CH₂), 2.89–2.78 (m, 1H, CH₂), 2.65–2.44 (m, 3H, CH₂, and indole-CH₂), 2.22–2.14 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 141.5 (Cq), 136.3 (Cq), 134.3 (Cq), 129.0 (ArCH), 127.4 (Cq), 126.7 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.5 (ArCH), 118.8 (ArCH), 111.4 (Cq), 111.3 (ArCH), 102.1 (C-7a), 72.9 (C-2), 55.8 (C-3), 35.1 (CH2), 32.6 (CH2), 29.8 (indole-CH2). Anal. Calcd. for C₂₁H₁₉ClN₂O₂: C: 68.76%; H: 5.22%; N: 7.62%. Found C: 68.94%; H: 5.06%; N: 7.60%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-bromophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7d): Following the general procedure, to a solution of (R)-tryptophanol (0.102 g, 0.536 mmol) in toluene (5 mL) was added 3-(4-bromobenzoyl) propionic acid (0.153 g, 0.590 mmol). Reaction time: 18 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.182 g, 83%); ${\alpha }_D^{25 }$ = −53.6° (c = 2.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8d. Anal. Calcd. for C₂₁H₁₉BrN₂O₂·0.15H₂O: C: 60.92%; H: 4.71%; N: 6.77%. Found C: 60.47%; H: 4.55%; N: 6.55%. The m.p. value was found to be identical to the one reported for compound 8d.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(4-bromophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8d): Following the general procedure, to a solution of (S)-tryptophanol (0.102 g, 0.536 mmol) in toluene (5 mL) was added 3-(4-bromobenzoyl) propionic acid (0.151 g, 0.589 mmol). Reaction time: 18 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale yellow crystalline solid (0.159 g, 72%); ${\alpha }_D^{25 }$ = +52.3° (c = 2.0, MeOH); m.p.: 207-210 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.97 (s, 1H, NH), 7.52–7.45 (m, 3H, ArH), 7.37–7.32 (m, 3H, ArH), 7.21–7.05 (m, 3H, ArH), 4.62–4.52 (m, 1H, H-3), 4.17 (dd, J = 8.8 Hz, 7.4 Hz, 1H, H-2), 3.59 (dd, J = 8.8 Hz, 6.9 Hz, 1H, H-2), 3.05 (dd, J = 14.7 Hz, 6.1 Hz, 1H, indole-CH₂), 2.89–2.78 (m, 1H, CH2), 2.65–2.44 (m, 3H, CH₂, and indole-CH₂), 2.22–2.14 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 142.1 (Cq), 136.3 (Cq), 131.8 (ArCH), 127.5 (Cq), 127.1 (ArCH), 122.5 (Cq), 122.2 (ArCH), 122.1 (ArCH), 119.7 (ArCH), 118.9 (ArCH), 111.6 (Cq), 111.3 (ArCH), 102.1 (C-7a), 72.9 (C-2), 55.8 (C-3), 35.1 (CH2), 32.7 (CH₂), 29.8 (indole-CH₂). Anal. Calcd. for C₂₁H₁₉BrN₂O₂: C: 61.33%; H: 4.66%; N: 6.81%. Found C: 61.26%; H: 4.48%; N: 6.76%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(p-tolyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7e): Following the general procedure, to a solution of (R)-tryptophanol (0.103 g, 0.541 mmol) in toluene (5 mL) was added 3-(4-methylbenzoyl) propionic acid (0.114 g, 0.596 mmol). Reaction time: 19 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.160 g, 86%); ${\alpha }_D^{25 }$= −58.7° (c = 2.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8e. Anal. Calcd. for C₂₂H₂₂N₂O₂: C: 76.28%; H: 6.40%; N: 8.09%. Found C: 75.87%; H: 6.23%; N: 8.06%. The m.p. value was found to be identical to the one reported for compound 8e.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(p-tolyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8e): Following the general procedure, to a solution of (S)-tryptophanol (0.100 g, 0.526 mmol) in toluene (5 mL) was added 3-(4-methylbenzoyl) propionic acid (0.112 g, 0.583 mmol). Reaction time: 18 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale pink crystalline solid (0.097 g, 53%); ${\alpha }_D^{25 }$= +45.1° (c = 2.0, MeOH); m.p.: 210–213 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H, NH), 7.47–7.32 (m, 4H, ArH), 7.21–7.06 (m, 5H, ArH), 4.60–4.50 (m, 1H, H-3), 4.15 (dd, J = 8.7 Hz, 7.4 Hz, 1H, H-2), 3.61 (dd, J = 8.8 Hz, 6.9 Hz, 1H, H-2), 3.09 (dd, J = 14.7 Hz, 6.1 Hz, 1H, indole-CH₂), 2.90–2.79 (m, 1H, CH₂), 2.64-2.43 (m, 3H, CH₂, and indole-CH₂), 2.39 (s, 3H, CH₃), 2.26-2.17 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 139.9 (Cq), 138.2 (Cq), 136.3 (Cq), 129.3 (ArCH), 127.5 (Cq), 125.2 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 119.0 (ArCH), 111.9 (Cq), 111.2 (ArCH), 102.6 (C-7a), 72.9 (C-2), 55.7 (C-3), 35.4 (CH₂), 32.9 (CH₂), 29.9 (indole-CH₂), 21.4 (CH₃). Anal. Calcd. for C₂₂H₂₂N₂O₂: C: 76.28%; H: 6.40%; N: 8.09%. Found C: 76.49%; H: 6.27%; N: 8.16%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-methoxyphenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7f): Following the general procedure, to a solution of (R)-tryptophanol (0.100 g, 0.526 mmol) in toluene (5 mL) was added 3-(4-methoxybenzoyl) propionic acid (0.121 g, 0.581 mmol). Reaction time: 24 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale yellow crystalline solid (0.106 g, 56%); ${\alpha }_D^{25 }$= −43.0° (c = 1.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8f. Anal. Calcd. for C₂₂H₂₂N₂O₃·0.15H₂O: C: 72.36%; H: 6.17%; N: 7.67%. Found C: 72.22%; H: 6.21%; N: 7.53%. The m.p. value was found to be identical to the one reported for compound 8f.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(4-methoxyphenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8f): Following the general procedure, to a solution of (S)-tryptophanol (0.101 g, 0.533 mmol) in toluene (5 mL) was added 3-(4-methoxybenzoyl) propionic acid (0.122 g, 0.586 mmol). Reaction time: 25 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give pale yellow crystalline solid (0.134 g, 69%); ${\alpha }_D^{25 }$ = +48.1° (c = 1.0, MeOH); m.p.: 185–187 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H, NH), 7.47–7.32 (m, 4H, ArH), 7.20–7.05 (m, 3H, ArH), 6.92–6.89 (m, 2H, ArH), 4.61–4.50 (m, 1H, H-3), 4.15 (dd, J = 8.4 Hz, 7.7 Hz, 1H, H-2), 3.84 (s, 3H, O-CH₃), 3.61 (dd, J = 8.7 Hz, 7.0 Hz, 1H, H-2), 3.08 (dd, J = 14.7 Hz, 6.2 Hz, 1H, indole-CH₂), 2.90–2.79 (m, 1H, CH₂), 2.64–2.44 (m, 3H, CH₂, and indole-CH₂), 2.25–2.17 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl3) δ 180.2 (C=O), 159.7 (Cq), 136.3 (Cq), 134.9 (Cq), 127.5 (Cq), 126.6 (ArCH), 122.2 (ArCH), 119.5 (ArCH), 119.0 (ArCH), 114.1 (ArCH), 111.8 (Cq), 111.2 (ArCH), 102.5 (C-7a), 72.8 (C-2), 55.7 (OCH₃), 55.5 (C-3), 35.3 (CH₂), 32.8 (CH₂), 29.8 (indole-CH₂). Anal. Calcd. for C₂₂H₂₂N₂O₃: C: 72.91%; H: 6.12%; N: 7.73%. Found C: 72.73%; H: 5.76%; N: 7.73%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-(methylsulfonyl)phenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7g): Following the general procedure, to a solution of (R)-tryptophanol (0.106 g, 0.557 mmol) in toluene (5 mL) was added 3-(4-methylsulfonylbenzoyl) propionic acid (0.157 g, 0.613 mmol). Reaction time: 22 h. The compound was purified by flash chromatography (EtOAc/n-hexane 7:3) and recrystallized from EtOAc/n-hexane to give a pale yellow crystalline solid (0.171g, 75%); ${\alpha }_D^{25 }$= −57.2° (c = 2.0, MeOH); ¹H NMR was found to be identical to the one obtained for compound 8g. Anal. Calcd. for C₂₂H₂₂N₂O₄S: C: 64.37%; H: 5.40%; N: 6.82%. Found C: 64.31%; H: 5.32%; N: 6.81%. The m.p. value was found to be identical to the one reported for compound 8g.

(3S,7aS)-3-((1H-indol-3-yl)methyl)-7a-(4-(methylsulfonyl)phenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (8g): Following the general procedure, to a solution of (S)-tryptophanol (0.100 g, 0.526 mmol) in toluene (5 mL) was added 3-(4-methylsulfonylbenzoyl) propionic acid (0.148 g, 0.578 mmol). Reaction time: 23 h. The compound was purified by flash chromatography (EtOAc/n-hexane 7:3) and recrystallized from EtOAc/n-hexane to give yellow crystalline solid (0.131 g, 61%); ${\alpha }_D^{25 }$= +66.9 (c = 2.0, MeOH); m.p.: 205–207 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.01 (s, 1H, NH), 7.90–7.88 (m, 2H, ArH), 7.62–7.59 (m, 2H, ArH), 7.43 (d, J = 7.9 Hz, 1H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.21–7.16 (m, 1H, ArH), 7.11–7.06 (m, 1H, ArH), 7.00 (d, J = 2.3 Hz, 1H, ArH), 4.66–4.56 (m, 1H, H-3), 4.22 (dd, J = 8.9, 7.4 Hz, 1H, H-2), 3.61 (dd, J = 8.9, 7.0 Hz, 1H, H-2), 3.10 (s, 3H, SO₂CH₃), 3.00 (dd, J = 15.5, 5.9 Hz, 1H, indole-CH₂), 2.92–2.75 (m, 1H, CH₂), 2.70–2.45 (m, 3H, CH₂, and indole-CH₂), 2.21–2.13 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.1 (C=O), 149.2 (Cq), 140.5 (Cq), 136.3 (Cq), 128.0 (ArCH), 127.5 (Cq), 126.2 (ArCH), 122.4 (ArCH), 119.7 (ArCH), 118.7 (ArCH), 111.4 (Cq), 111.1 (ArCH), 101.8 (C-7a), 72.9 (C-2), 56.0 (C-3), 44.6 (SO₂CH₃), 35.1 (CH₂), 32.6 (CH₂), 29.5 (indole-CH₂). Anal. Calcd. for C₂₂H₂₂N₂O₄S: C: 64.37%; H: 5.40%; N: 6.82%. Found C: 64.59%; H: 5.51%; N: 6.69%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(3-chloro-4-methylphenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7h): Following the general procedure, to a solution of (R)-tryptophanol (0.041 g, 0.218 mmol) in toluene (2 mL) was added 4-(3-chloro-4-methylphenyl)-4-oxobutanoic acid (0.058 g, 0.254 mmol). Reaction time: 10 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from CH₂Cl₂/n-hexane to give a white solid (0.077 g, 93%); ${\alpha }_D^{25 }$= −29.6° (c = 1.0, CH₂Cl₂); m.p.: 171–172 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.97 (s, 1H, NH), 7.50 (d, J = 1.4 Hz, 1H, ArH), 7.48 (d, J = 8.1 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.28–7.23 (m, 2H, ArH), 7.21–7.15 (m, 1H, ArH), 7.13–7.09 (m, 1H, ArH), 7.06 (d, J = 2.4 Hz, 1H, ArH), 4.62–4.50 (m, 1H, H-3), 4.16 (dd, J = 8.8, 7.4 Hz, 1H, H-2), 3.61 (dd, J = 8.8, 6.9 Hz, 1H, H-2), 3.10 (dd, J = 14.6, 6.0 Hz, 1H, indole-CH₂), 2.85 (ddd, J = 16.3, 9.8, 8.1 Hz, 1H, CH₂), 2.62 (dd, J = 10.1, 3.3 Hz, 1H, CH₂), 2.50 (ddd, J = 18.4, 9.6, 6.1 Hz, 2H, CH₂, and indole-CH₂), 2.41 (s, 3H, CH₃), 2.25–2.15 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.1 (C=O), 160.6 (Cq), 142.3 (Cq), 136.2 (Cq), 134.9 (Cq), 131.4 (ArCH), 127.4 (Cq), 125.9 (ArCH), 123.5 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.6 (Cq), 118.9 (ArCH), 111.6 (ArCH), 111.2 (ArCH), 101.8 (C-7a), 72.9 (C-2), 55.7 (C-3), 35.1 (CH₂), 32.7 (CH₂), 29.8 (indole-CH₂), 20.0 (CH₃). LRMS (ESI) m/z calcd for C₂₂H₂₁ClN₂O₂: 380, found 381 [M+H]⁺.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(3-fluoro-4-methoxyphenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7i): Following the general procedure, to a solution of (R)-tryptophanol (0.100 g, 0.526 mmol) in toluene (5 mL) was added 3-(3-fluoro-4-methoxybenzoyl) propionic acid (0.131 g, 0.578 mmol). Reaction time: 22 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) and recrystallized from EtOAc/n-hexane to give a pale yellow crystalline solid (0.139 g, 69%); ${\alpha }_D^{25 }$ = −51.3° (c = 2.0, MeOH); m.p.: 131–132 °C; ¹H NMR (300 MHz, (CDCl₃) δ 8.01 (s, 1H, NH), 7.47 (d, J = 7.9 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.23–7.04 (m, 5H, ArH), 6.93 (t, J = 8.4 Hz, 1H, ArH), 4.61–4.41 (m, 1H, H-3), 4.16 (dd, J = 8.7, 7.4 Hz, 1H, H-2), 3.92 (s, 3H, O-CH₃), 3.61 (dd, J = 8.8, 6.9 Hz, 1H, H-2), 3.08 (dd, J = 13.9, 6.0 Hz, 1H, indole-CH₂), 2.90–2.83 (m, 1H, CH₂), 2.64–2.43 (m, 3H, CH_{2, and indole}-CH₂), 2.24–2.15 (m, 1H, CH₂) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 180.2 (C=O), 152.2 (d, Cq, J_C-F = 245.2 Hz), 147.5 (Cq), 147.3 (Cq), 136.3 (Cq), 135.7 (d, Cq, J = 5.2 Hz), 127.4 (Cq), 122.0 (d, ArCH, J = 6.3 Hz), 120.8 (d, ArCH, J = 3.5 Hz), 119.5 (ArCH), 118.8 (ArCH), 113.2 (d, ArCH, J = 1.6 Hz), 113.2 (ArCH), 111.5 (Cq), 111.3 (ArCH), 101.9 (C-7a), 72.9 (C-2), 56.5 (C-3), 55.8 (OCH₃), 35.2 (CH₂), 32.9 (CH₂), 29.8 (indole-CH₂). Anal. Calcd. for C₂₂H₂₁FN₂O₃: C: 69.46%; H: 5.56%; N: 7.36%. Found C: 69.49%; H: 5.76%; N: 7.12%.

(3R,7aR,7S)-3-((1H-indol-3-yl)methyl)-7a-(4-chlorophenyl)-7-methyltetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7j) and (3R,7aR,7R)-3-((1H-indol-3-yl)methyl)-7a-(4-chlorophenyl)-7-methyltetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7j‘): Following the general procedure, to a solution of (R)-tryptophanol (0.039 g, 0.207 mmol) in toluene (2 mL) was added 4-(4-chlorophenyl)-3-methyl-4-oxobutanoic acid (0.057 g, 0.239 mmol). Reaction time: 17 h. Two compounds were purified by flash chromatography (EtOAc/n-hexane 2:3) and recrystallized from CH₂Cl₂/n-hexane.

(7j): The product was obtained as a white solid (0.055 g, 69%); ${\alpha }_D^{25 }$= −30.5° (c = 1.0, CH₂Cl₂); m.p.: 201–202 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.00 (s, 1H, NH), 7.41 (d, J = 8.6 Hz, 3H, ArH), 7.34 (d, J = 8.8 Hz, 3H, ArH), 7.18 (t, J = 7.4 Hz, 1H, ArH), 7.11 (s, 1H, ArH), 7.07 (d, J = 7.4 Hz, 1H, ArH), 4.67–4.56 (m, 1H, H-3), 4.13 (t, J = 8.0 Hz, 1H, H-2), 3.58 (dd, J = 8.5, 6.6 Hz, 1H, H-2), 3.04–2.85 (m, 2H, CH₂, and indole-CH₂), 2.44 (td, J = 15.1, 8.1 Hz, 2H, CH₂, and indole-CH₂), 2.18 (dd, J = 17.3, 5.6 Hz, 1H, CH₂), 1.12 (d, J = 7.1 Hz, 3H, CH₃); ¹³C NMR (75 MHz, CDCl₃) δ 181.1 (C=O), 141.4 (Cq), 136.2 (Cq), 134.2 (Cq), 128.9 (ArCH), 127.5 (Cq), 127.0 (ArCH), 122.3 (ArCH), 122.1 (ArCH), 119.6 (ArCH), 118.8 (ArCH), 111.8 (Cq), 111.2 (ArCH), 103.0 (C-7a), 72.3 (C-2), 56.5 (C-3), 40.0 (CH₂), 39.7 (CH₂), 29.7 (indole-CH₂), 14.0 (CH₃). LRMS (ESI) m/z calcd for C₂₂H₂₁ClN₂O₂: 380, found 381 [M+H]⁺.

(7j’): The product was obtained as white solid (0.014 g, 18%); ${\alpha }_D^{25 }$ = −45.7° (c = 1.0, CH₂Cl₂); m.p.: 205-206 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.95 (s, 1H, NH), 7.48 (d, J = 7.9 Hz, 1H, ArH), 7.34 (t, J = 8.7 Hz, 5H, ArH), 7.19 (t, J = 7.3 Hz, 1H, ArH), 7.10 (t, J = 7.3 Hz, 1H, ArH), 6.99 (s, 1H, ArH), 4.55 (td, J = 12.4, 6.8 Hz, 1H, H-3), 4.24–4.17 (m, 1H, H-2), 3.64 (dd, J = 8.6, 7.1 Hz, 1H, H-2), 3.10 (dd, J = 14.7, 5.3 Hz, 1H, indole-CH₂), 2.78–2.63 (m, 2H, CH₂), 2.48 (dt, J = 15.8, 8.1 Hz, 2H, CH₂, and indole-CH₂), 0.65 (d, J = 6.5 Hz, 3H, CH₃); ¹³C NMR (75 MHz, CDCl₃) δ 177.1 (C=O), 137.9 (Cq), 136.3 (Cq), 134.4 (Cq), 128.7 (ArCH), 127.9 (ArCH), 127.5 (Cq), 122.4 (ArCH), 122.2 (ArCH), 119.7 (ArCH), 119.0 (ArCH), 111.6 (Cq), 111.2 (ArCH), 104.1 (C-7a), 73.5 (C-2), 54.8 (C-3), 42.1 (CH₂), 41.3 (CH₂), 29.6 (indole-CH₂), 16.4 (CH₃). LRMS (ESI) m/z calcd for C₂₂H₂₁ClN₂O₂: 380, found 381 [M+H]⁺.

General procedure for the synthesis of 7k–l: The (R)-tryptophanol-derived oxazolopyrrolidone lactam (0.129 mmol) was dissolved in dry DMF (5 mL), and the solution was cooled to 0 °C, under N₂ atmosphere. Sodium hydride (NaH) in 60% dispersion in mineral oil (0.250 mmol, 2.0 eq.) was added portion wise and the mixture stirred for 15 min. The appropriate protecting reagent (0.320 mmol, 2.5 eq.) was added and the reaction mixture stirred at room temperature for 3–6 h. After reaction completion, water (10 mL) was added followed by EtOAc (10 mL). The aqueous phase was washed with EtOAc (2x10 mL); the combined organic phases were washed with brine (10 mL), dried with MgSO₄, and concentrated in vacuo. The residue was purified by silica gel flash chromatography using EtOAc/n-hexane as eluent.

(3R,7aR)-7a-(4-chlorophenyl)-3-((1-ethyl-1H-indol-3-yl)methyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7k): Following the general procedure, to a solution of 7c (0.120 g, 0.327 mmol) in DMF (13.5 mL) was added NaH (0.016 g, 0.654 mmol) and ethyl iodide (65.4 µL, 0.818 mmol). Reaction time: 3 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:2) to afford the title compound as a pale yellow oil (0.101 g, 78%); ¹H NMR (300 MHz, CDCl₃) δ 7.39 (d, J = 7.9 Hz, 1H. ArH), 7.36–7.17 (m, 5H, ArH), 7.16–7.08 (m, 1H, ArH), 7.04–6.97 (m, 1H, ArH), 6.87 (s, 1H, ArH), 4.48 (m, 1H, H-3), 4.08 (dd, J = 8.8, 7.5 Hz, 1H, H-2), 4.01 (q, J = 7.3 Hz, 2H, CH₂CH₃), 3.52 (dd, J = 8.8, 7.0 Hz, 1H, H-2), 3.01 (dd, J = 14.6, 5.3 Hz, 1H, indole-CH₂), 2.84–2.70 (m, 1H, CH₂), 2.58–2.36 (m, 3H, CH₂, and indole-CH₂), 2.15–2.07 (m, 1H, CH₂), 1.33 (t, J = 7.3 Hz, 3H, CH₂CH₃); ¹³C NMR (75 MHz, CDCl₃) δ 180.2 (C=O), 141.6 (Cq), 136.1 (Cq), 134.3 (Cq), 129.0 (ArCH), 128.1 (Cq), 126.8 (ArCH), 125.3 (ArCH), 121.7 (ArCH), 119.2 (ArCH), 119.1 (ArCH), 110.0 (Cq), 109.4 (ArCH), 102.1 (C-7a), 72.9 (C-2), 55.9 (C-3), 40.9 (CH₂CH₃), 35.2 (CH₂), 32.8 (CH₂), 29.7 (indole-CH₂), 15.6 (CH₂CH₃). Anal. Calcd. for C₂₃H₂₃ClN₂O₂: C: 69.96%; H: 5.87%; N: 7.09%. Found C: 70.12%; H: 6.40%; N: 6.95%.

(3R,7aR)-3-((1-acetyl-1H-indol-3-yl)methyl)-7a-(4-chlorophenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7l): Following the general procedure, to a solution of 7c (0.094 g, 0.256 mmol) in DMF (9.5 mL) was added NaH (12.3 mg, 0.512 mmol) and acetic anhydride (60.6 µL, 0.641 mmol). Reaction time: 6 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) to afford the title compound as a white powder (0.072 g, 69%); m.p.: 66–67 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.32 (d, J = 7.8 Hz, 1H, ArH), 7.56 (s, 1H, ArH), 7.28–7.11 (m, 7H, ArH), 4.61–4.45 (m, 1H, H-3), 4.20 (dd, J = 8.7, 7.6 Hz, 1H, H-2), 3.49 (dd, J = 8.7, 6.5 Hz, 1H, H-2), 2.80–2.52 (m, 3H, CH₂, and indole-CH₂), 2.52 (s, 3H, CH₃), 2.48–2.35 (m, 2H, CH₂, and indole-CH₂), 2.16–2.02 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.9 (C=O), 168.9 (C=O), 141.2 (Cq), 135.9 (Cq), 134.5 (Cq), 130.6 (Cq), 129.1 (ArCH), 126.6 (ArCH), 125.5 (ArCH), 123.7 (ArCH), 123.3 (ArCH), 118.7 (ArCH), 118.3 (Cq), 116.8 (ArCH), 102.4 (C-7a), 72.6 (C-2), 54.9 (C-3), 34.7 (CH₂), 32.4 (CH₂), 29.6 (indole-CH₂), 24.2 (CH₃). Anal. Calcd. for C₂₃H₂₁ClN₂O₃: C: 67.56%; H: 5.18%; N: 6.85%. Found C: 67.37%; H: 5.47%; N: 6.72%.

Procedure for the synthesis of tert-butyl 3-(((3R,7aR)-7a-(4-chlorophenyl)-5-oxohexahydropyrrolo[2,1-b]oxazol-3-yl)methyl)-1H-indole-1-carboxylate (7m): To a solution of 7c (0.070 g, 0.191 mmol) in THF (7.0 mL) was added anhydrous Et₃N (58.6 µL, 0.420 mmol), DMAP (0.006 g, 0.048 mmol), and Boc₂O (0.054 g, 0.248 mmol). The reaction mixture was stirred at room temperature for 3 h. After reaction completion, the mixture was concentrated in vacuo and the crude was dissolved in EtOAc (20 mL). The organic phase was washed with a sat. sol. of NH₄Cl (2 × 15 mL), a sat. sol. of NaHCO₃ (2 × 15 mL) and brine (15 mL). The combined organic phases were dried with MgSO₄, concentrated in vacuo and the compound was purified by flash chromatography (EtOAc/n-hexane 2:3) to afford the title compound as a pale yellow powder (0.059 g, 66%); m.p.: 163–165 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J = 7.6 Hz, 1H, ArH), 7.37–7.30 (m, 4H, ArH), 7.29–7.18 (m, 3H, ArH), 7.14 (td, J = 7.5, 1.1 Hz, 1H, ArH), 4.57–4.40 (m, 1H, H-3), 4.14 (dd, J = 8.8, 7.5 Hz, 1H, H-2), 3.51 (dd, J = 8.9, 7.0 Hz, 1H, H-2), 2.90 (ddd, J = 14.7, 5.7, 1.2 Hz, 1H, indole-CH₂), 2.85–2.70 (m, 1H, CH₂), 2.59–2.30 (m, 3H, CH₂, and indole-CH₂), 2.16–2.05 (m, 1H, CH₂), 1.58 (s, 9H, C(CH₃)₃); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 149.8 (C=O), 141.4 (Cq), 134.4 (Cq), 130.4 (Cq), 129.1 (ArCH), 126.6 (ArCH), 124.7 (ArCH), 123.5 (ArCH), 122.7 (ArCH), 119.1 (ArCH), 116.1 (Cq), 115.4 (ArCH), 102.1 (C-7a), 83.8 (C(CH₃)₃), 72.8 (C-2), 55.2 (C-3), 35.2 (CH₂), 32.7 (CH₂), 29.5 (indole-CH₂), 28.4 (C(CH₃)₃); Anal. Calcd. for C₂₆H₂₇ClN₂O₄: C: 66.88%; H: 5.83%; N: 6.00%. Found C: 66.90%; H: 6.16%; N: 5.89%.

General procedure for the synthesis of 7n–u: To a solution of the appropriate tryptophanol-derived oxazolopiperidone lactams (0.230 mmol) in dioxane (2.3 mL) was added Pd(PPh₃)₂Cl₂ (0.023 mmol, 0.1 eq) and 1 M aq. sol. of Na₂CO₃ (690 µL), followed by the appropriate boronic acid (0.280 mmol,1.2 eq.). The resulting mixture was degassed and stirred at 100 °C, under N₂ atmosphere, for 2–5 h. After cooling to room temperature, the reaction mixture was diluted with CH₂Cl₂, filtered in celite, and concentrated in vacuo. The residue was purified by silica gel flash chromatography using EtOAc/N-hexane as eluent.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4’-chloro-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7n): Following the general procedure, to a solution of 7d (0.036 g, 0.088 mmol) in dioxane (1.0 mL) was added Pd(PPh₃)₂Cl₂ (0.003 g, 8.8 µmol), 1 M aq. sol. of Na₂CO₃ (266 µL), and 4-chlorophenylboronic acid (0.017 g, 0.107 mmol). Reaction time: 4 h. The compound was purified by flash chromatography (EtOAc/n-hexane 2:3) to afford the title compound as a white solid (0.036 g, 94%); m.p.: 201–204 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.08 (s, 1H, NH), 7.63–7.50 (m, 6H, ArH), 7.48–7.39 (m, 3H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.22–7.12 (m, 1H, ArH), 7.12–7.01 (m, 2H, ArH), 4.70–4.52 (m, 1H, H-3), 4.20 (dd, J = 8.7, 7.4 Hz, 1H, H-2), 3.66 (dd, J = 8.8, 6.8 Hz, 1H, H-2), 3.11 (dd, J = 15.1, 6.6 Hz, 1H, indole-CH₂), 3.01–2.78 (m, 1H, CH₂), 2.73–2.44 (m, 3H, CH₂, and indole-CH₂), 2.38–2.17 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 142.3 (Cq), 140.1 (Cq), 139.1 (Cq), 136.3 (Cq), 133.9 (Cq), 129.2 (ArCH), 128.5 (ArCH), 127.5 (Cq), 127.4 (ArCH), 125.9 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 118.9 (ArCH), 111.8 (Cq), 111.3 (ArCH), 102.4 (C-7a), 73.0 (C-2), 55.8 (C-3), 35.2 (CH₂), 32.8 (CH₂), 29.9 (indole-CH₂); Anal. Calcd. for C₂₇H₂₃ClN₂O₂: C: 73.21%; H: 5.23%; N: 6.32%. Found C: 73.56%; H: 5.83%; N: 5.92%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4’-(trifluoromethyl)-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7o): Following the general procedure, to a solution of 7d (0.050 g, 0.122 mmol) in dioxane (1.4 mL) was added Pd(PPh₃)₂Cl₂ (0.004 g, 12.2 µmol), 1 M aq. sol. of Na₂CO₃ (370 µL), and 4-(trifluoromethyl)phenylboronic acid (0.028 g, 0.148 mmol). Reaction time: 3 h. The compound was purified by flash chromatography (EtOAc/n-hexane 2:3) to afford the title compound as a white solid (0.050 g, 86%); m.p.: 201–203 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.99 (s, 1H, NH), 7.72 (s, 4H, ArH), 7.64–7.55 (m, 4H, ArH), 7.43 (d, J = 7.5 Hz, 1H, ArH), 7.33 (d, J = 8.1 Hz, 1H, ArH), 7.21–7.14 (m, 1H, ArH), 7.12–7.03 (m, 2H, ArH), 4.67–4.53 (m, 1H, H-3), 4.20 (dd, J = 8.8, 7.4 Hz, 1H, H-2), 3.65 (dd, J = 8.8, 6.8 Hz, 1H, H-2), 3.10 (dd, J = 15.0, 6.5 Hz, 1H, indole-CH₂), 2.91–2.83 (m, 1H, CH₂), 2.67–2.48 (m, 3H, CH₂, and indole-CH₂), 2.33–2.22 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 143.9 (Cq), 142.7 (Cq), 139.8 (Cq), 136.0 (Cq), 129.5 (q, Cq, J = 32.3 Hz), 127.8 (ArCH), 127.6 (ArCH), 127.1 (Cq), 125.8 (q, ArCH, J = 3.7 Hz), 125.7 (ArCH), 124.2 (q, Cq, J_C-F = 270.2 Hz), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 118.1 (ArCH), 111.7 (Cq), 111.2 (ArCH), 102.4 (C-7a), 72.9 (C-2), 55.8 (C-3), 35.2 (CH₂), 32.8 (CH₂), 29.9 (indole-CH₂); Anal. Calcd. for C₂₈H₂₃F₃N₂O₂: C: 70.58%; H: 4.87%; N: 5.88%. Found C: 70.09%; H: 5.19%; N: 5.83%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4’-hydroxy-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7p): Following the general procedure, to a solution of 7d (0.050 g, 0.122 mmol) in dioxane (1.4 mL) was added Pd(PPh₃)₂Cl₂ (0.004 g, 12.2 µmol), 1 M aq. sol. of Na₂CO₃ (370 µL), and 4-hydroxyphenylboronic acid (0.021 g, 0.148 mmol). Reaction time: 2 h. The compound was purified by flash chromatography (EtOAc/n-hexane 3:2) to afford the title compound as a white solid (0.044 g, 85%); m.p.: 223–225 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.98 (s, 1H, NH), 7.62–7.43 (m, 7H, ArH), 7.34 (d, J = 7.8 Hz, 1H, ArH), 7.18 (t, J = 7.6 Hz, 1H, ArH), 7.10–7.05 (m, 2H, ArH), 6.95 (d, J = 8.5 Hz, 2H, ArH), 5.21 (s, 1H, OH), 4.68–4.51 (m, 1H, H-3), 4.20 (dd, J = 8.5, 7.8 Hz, 1H, H-2), 3.66 (dd, J = 8.6, 6.8 Hz, 1H, H-2), 3.13 (dd, J = 14.9, 6.3 Hz, 1H, indole-CH₂), 2.98–2.76 (m, 1H, CH₂), 2.75–2.46 (m, 3H, CH₂, and indole-CH₂), 2.33–2.24 (m, 1H, CH₂); ¹³C NMR (75 MHz, (CD₃)₂SO) δ 179.5 (C=O), 157.2 (Cq), 140.8 (Cq), 139.9 (Cq), 136.0 (Cq), 130.1 (Cq), 127.7 (ArCH), 126.9 (Cq), 126.0 (ArCH), 125.5 (ArCH), 122.9 (ArCH), 121.0 (ArCH), 118.3 (ArCH), 117.9 (ArCH), 115.8 (ArCH), 111.4 (ArCH), 109.9 (Cq), 101.7 (C-7a), 72.3 (C-2), 55.3 (C-3), 40.4 (CH₂), 32.8 (CH₂), 29.8 (indole-CH₂); Anal. Calcd. for C₂₇H₂₄N₂O₃·0.15H₂O: C: 75.91%; H: 5.75%; N: 6.56%. Found C: 75.74%; H: 5.85%; N: 6.57%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4’-(hydroxymethyl)-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7q): Following the general procedure, to a solution of 7d (0.070 g, 0.170 mmol) in dioxane (2.0 mL) was added Pd(PPh₃)₂Cl₂ (0.005 g, 17.0 µmol), 1 M aq. sol. of Na₂CO₃ (520 µL), and 4-(hydroxymethyl)phenylboronic acid (0.032 g, 0.208 mmol). Reaction time: 3 h. The compound was purified by flash chromatography (EtOAc/n-hexane 3:2) to afford the title compound as a pale yellow solid (0.053 g, 71%); m.p.: 213–215 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H, NH), 7.65–7.44 (m, 9H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.17 (t, J = 7.3 Hz, 1H, ArH), 7.11–7.02 (m, 2H, ArH), 4.77 (s, 2H, CH₂), 4.67–4.53 (m, 1H, H-3), 4.19 (t, J = 8.0 Hz, 1H, H-2), 3.66 (t, J = 8.0 Hz, 1H, H-2), 3.11 (dd, J = 14.7, 6.0 Hz, 1H, indole-CH₂), 2.95–2.78 (m, 1H, CH₂), 2.69–2.47 (m, 3H, CH₂, and indole-CH₂), 2.38–2.20 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 142.0 (Cq), 140.9 (Cq), 140.4 (Cq), 140.1 (Cq), 136.3 (Cq), 127.5 (ArCH), 127.4 (Cq), 127.3 (ArCH), 127.2 (ArCH), 125.8 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 119.0 (ArCH), 111.9 (Cq), 111.3 (ArCH), 102.5 (C-7a), 73.0 (C-2), 65.3 (CH₂OH), 55.8 (C-3), 35.3 (CH₂), 32.8 (CH₂), 29.9 (indole-CH₂); Anal. Calcd. (C₂₈H₂₆N₂O₃·0.40H₂O): C: 75.44%; H: 6.07%; N: 6.29%. Found C: 75.18%; H: 6.21%; N: 6.14%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(3’-chloro-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7r): Following the general procedure, to a solution of 7d (0.070 g, 0.170 mmol) in dioxane (2.0 mL) was added Pd(PPh₃)₂Cl₂ (0.005 g, 17.0 µmol), 1 M aq. sol. of Na₂CO₃ (520 µL), and 3-chlorophenylboronic acid (0.033 g, 0.208 mmol). Reaction time: 4 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) to afford the title compound as a pale yellow solid (0.059 g, 78%); m.p.: 204–206 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.98 (s, 1H, NH), 7.47 (t, J = 1.6 Hz, 1H, ArH), 7.43 (s, 4H, ArH), 7.36 (dt, J = 7.4 Hz, 1.6 Hz, 1H, ArH), 7.30 (d, J = 7.7 Hz, 1H, ArH), 7.25–7.17 (m, 3H, ArH), 7.06–7.00 (m, 1H, ArH), 6.95–6.91 (m, 2H, ArH), 4.51–4.42 (m, 1H, H-3), 4.05 (dd, J = 8.9, 6.9 Hz, 1H, H-2), 3.51 (dd, J = 8.8, 6.8 Hz, 1H, H-2), 2.95 (dd, J = 14.4, 6.6 Hz, 1H, indole-CH₂), 2.80–2.66 (m, 1H, CH₂), 2.53–2.34 (m, 3H, CH₂, and indole-CH₂), 2.22–2.08 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 142.51 (Cq), 142.46 (Cq), 139.9 (Cq), 136.3 (Cq), 134.9 (Cq), 130.3 (ArCH), 127.8 (ArCH), 127.6 (ArCH), 127.6 (Cq), 127.5 (ArCH), 125.9 (ArCH), 125.4 (ArCH), 122.3 (ArCH), 119.6 (ArCH), 118.9 (ArCH), 111.7 (Cq), 111.3 (ArCH), 102.4 (C-7a), 72.9 (C-2), 55.7 (C-3), 35.2 (CH₂), 32.8 (CH₂), 29.8 (indole-CH₂); Anal. Calcd. for C₂₇H₂₃ClN₂O₂: C: 73.21%; H: 5.23%; N: 6.32%. Found C: 72.91%; H: 5.70%; N: 6.24%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(3’,4’-dichloro-[1,1’-biphenyl]-4-yl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7s): Following the general procedure, to a solution of 7d (0.070 g, 0.170 mmol) in dioxane (2.0 mL) was added Pd(PPh₃)₂Cl₂ (0.005 g, 17.0 µmol), 1 M aq. sol. of Na₂CO₃ (520 µL), and 3,4-dichlorophenylboronic acid (0.040 g, 0.208 mmol). Reaction time: 4 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) to afford the title compound as a pale yellow solid (0.069 g, 85%); m.p.: 176–178 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.98 (s, 1H, NH), 7.71 (d, J = 2.1 Hz, 1H, ArH), 7.49 (s, 5H, ArH), 7.38 (dd, J = 8.4 Hz, 2.2 Hz, 2H, ArH), 7.27 (d, J = 8.1 Hz, 1H, ArH), 7.11 (d, J = 8.1 Hz, 1H, ArH), 7.06–6.97 (m, 2H, ArH), 4.65–4.45 (m, 1H, H-3), 4.14 (dd, J = 8.7, 7.4 Hz, 1H, H-2), 3.58 (dd, J = 8.8, 6.8 Hz, 1H, H-2), 3.02 (dd, J = 15.0, 6.6 Hz, 1H, indole-CH₂), 2.87–2.76 (m, 1H, CH₂), 2.65–2.38 (m, 3H, CH₂,, and indole-CH₂), 2.26–2.15 (m, 1H, CH₂); ¹³C NMR (75 MHz, (CD₃)₂SO) δ 180.3 (C=O), 142.9 (Cq), 140.7 (Cq), 138.9 (Cq), 136.2 (Cq), 132.9 (Cq), 131.7 (Cq), 131.0 (ArCH), 128.9 (ArCH), 127.5 (Cq), 127.4 (ArCH), 126.3 (ArCH), 126.0 (ArCH), 122.3 (ArCH), 122.2 (ArCH), 119.6 (ArCH), 118.8 (ArCH), 111.7 (Cq), 111.3 (ArCH), 102.3 (C-7a), 72.8 (C-2), 55.8 (C-3), 35.2 (CH₂), 32.9 (CH₂), 29.9 (indole-CH₂); Anal. Calcd. for C₂₇H₂₂Cl₂N₂O₂: C: 67.93%; H: 4.65%; N: 5.87%. Found C: 67.96%; H: 4.90%; N: 5.73%. HRMS-ESI m/z calcd for C₂₇H₂₂Cl₂N₂O₂: 476.1058, found 477.1143 ± 3.6 ppm [M+H]+.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-(pyridin-4-yl)phenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7t): Following the general procedure, to a solution of 7d (0.070 g, 0.170 mmol) in dioxane (2.0 mL) was added Pd(PPh₃)₂Cl₂ (0.005 g, 17.0 µmol), 1 M aq. sol. of Na₂CO₃ (520 µL), and 4-pyridinylboronic acid (0.026 g, 0.208 mmol). Reaction time: 2 h. The compound was purified by flash chromatography (EtOAc/n-hexane 3:1) to afford the title compound as a pale yellow solid (0.068 g, 97%); m.p.: 214–215 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.69 (d, J = 5.6 Hz, 2H, ArH), 8.23 (s, 1H, NH), 7.63 (q, J = 8.4 Hz, 4H, ArH), 7.54 (d, J = 5.9 Hz, 2H, ArH), 7.43 (d, J = 7.8 Hz, 1H, ArH), 7.32 (d, J = 8.0 Hz, 1H, ArH), 7.17 (t, J = 7.4 Hz, 1H, ArH), 7.12–7.01 (m, 2H, ArH), 4.71–4.55 (m, 1H, H-3), 4.21 (t, J = 8.1 Hz, 1H, H-2), 3.65 (dd, J = 8.6, 7.0 Hz, 1H, H-2), 3.09 (dd, J = 14.7, 6.1 Hz, 1H, indole-CH₂), 2.88 (ddd, J = 24.1, 12.1, 6.3 Hz, 1H, CH₂), 2.72–2.45 (m, 3H, CH₂, and indole-CH₂), 2.37–2.20 (m, 1H, CH₂); ¹³C NMR (75 MHz, CDCl₃) δ 180.3 (C=O), 150.5 (ArCH), 147.9 (Cq), 144.0 (Cq), 138.2 (Cq), 136.3 (Cq), 127.5 (ArCH), 126.1 (ArCH), 122.4 (ArCH), 122.3 (ArCH), 121.8 (ArCH), 119.6 (ArCH), 118.9 (ArCH), 111.6 (Cq), 111.3 (ArCH), 102.3 (C-7a), 73.0 (C-2), 55.81 (C-3), 35.19 (CH₂), 32.75 (CH₂), 29.85 (indole-CH₂). Anal. Calcd. for C₂₆H₂₃N₃O₂·0.20H₂O: C: 75.59%; H: 5.72%; N: 10.17%. Found C: 75.90%; H: 5.81%; N: 9.73%.

(3R,7aR)-3-((1H-indol-3-yl)methyl)-7a-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (7u): Following the general procedure, to a solution of 7d (0.070 g, 0.170 mmol) in dioxane (2.0 mL) was added Pd(PPh₃)₂Cl₂ (0.005 g, 17.0 µmol), 1 M aq. sol. of Na₂CO₃ (520 µL), and 1,4-benzodioxane-6-boronic acid (0.037 g, 0.208 mmol). Reaction time: 5 h. The compound was purified by flash chromatography (EtOAc/n-hexane 1:1) to afford the title compound as a pale yellow solid (0.022 g, 28%); m.p.: 286–288 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.97 (s, 1H, NH), 7.56–7.44 (m, 5H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.19–7.08 (m, 5H, ArH), 6.95 (d, J = 8.3 Hz, 1H, ArH), 4.64–4.54 (m, 1H, H-3), 4.31 (s, 4H, CH2), 4.18 (dd, J = 8.7, 7.5 Hz, 1H, H-2), 3.65 (dd, J = 8.8, 6.9 Hz, 1H, H-2), 3.11 (dd, J = 14.7, 6.1 Hz, 1H, indole-CH2), 2.93–2.82 (m, 1H, CH2), 2.67–2.45 (m, 3H, CH2, and indole-CH2), 2.37–2.19 (m, 1H, CH2); ¹³C NMR (75 MHz, CDCl₃) δ 180.4 (C=O), 143.9 (Cq), 143.6 (Cq), 141.4 (Cq), 140.7 (Cq), 136.3 (Cq), 134.2 (Cq), 127.3 (Cq), 126.9 (ArCH), 125.4 (ArCH), 122.3 (ArCH), 122.0 (ArCH), 120.4 (ArCH), 119.4 (ArCH), 118.8 (ArCH), 117.6 (ArCH), 115.8 (ArCH), 111.8 (Cq), 111.2 (ArCH), 102.4 (C-7a), 72.9 (C-2), 64.7 (CH₂), 55.7 (C-3), 35.2 (CH₂), 32.9 (CH₂), 29.9 (indole-CH₂). Anal. Calcd. for C₂₉H₂₆N₂O₄: C: 74.66%; H: 5.68%; N: 6.00%. Found C: 74.65%; H: 5.70%; N: 5.67%.

3.2. Biological Assays

3.2.1. Cytotoxicity Assays

The cytotoxicity was assessed in different cell lines with the endpoint MTT, using previously reported procedure [29,30,31]. The following cells were obtained from the American Type Culture Collection: human embryonic kidney epithelial cell line (HEK 293T, ATCC HBT-22™), breast cancer cell line (MDA-MB-231, ATCC HTB-26™), osteosarcoma cell line (MG-63, ATCC CRL-1427™), gastric adenocarcinoma cell line (AGS, ATTC CRL-1739TM), prostate cancer cell line (DU-145, ATTC HTB-81™), and lung carcinoma cell line (A-549, ATCC CCL-185™). All cell lines were seeded at 2 × 10⁴ cells/well with exception of A-549 cell line, which was seeded at 5 × 10³ cells/well.

3.2.2. Caspase 3/7 Activity Assay

The activity of caspase 3/7 was determined by fluorimetric assay based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-ValAsp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase 3/7 using a previously reported procedure [32].

3.3. In Vitro Stability Assays

3.3.1. Buffer and Human Plasma Stabilities for Compound 7s

Human plasma was obtained from healthy volunteers and provided by Instituto Português do Sangue, Lisbon, Portugal. Buffer and human plasma stabilities were determined by standard methodology [33]. Specifically, human plasma was centrifuged (5 min, 2000× g, room temperature) and, then, diluted 50% in PBS buffer (pH 7.4). The reactions were initiated by the addition of a solution of compound 7s (4 mM in DMSO, 25 µL) to 975 µL of plasma solution, at 37 °C, obtaining a final concentration of 100 µM. Solutions were stirred at 37 °C and 100 µL aliquots were collected at different time points: 0, 30, 60, 120, and 180 min (one additional aliquot was collected at 24 h). A cold reserpine solution (internal standard, 5 µM in acetonitrile, 300 µL) was then added to quench the reactions. Following centrifugation (10 min, 10,000× g, room temperature), the clear supernatants were stored at −20 °C until further analysis by HPLC-DAD. Assays were run in duplicate and procaine was used as a positive control for plasma stability. Additional control assays were conducted using PBS (pH 7.4) instead of a plasma solution. HPLC-DAD analysis was performed as previously described [34].

3.3.2. Metabolic Stability for Compound 7s

The evaluation of the metabolic stability of compound 7s was conducted in human liver microsomes (GIBCO^TM, 20 donors) by a previously reported procedure [35]. Specifically, for a total incubation volume of 1 mL, in 100 mM phosphate buffer at pH 7.4, 7s (10 μM), human liver microsomes (0.8 mg protein/mL), NADPH (1 mM), and NADPH regeneration system (10 µL, Vivid^® Regeneration System, 100×) were used. Nevirapine was used as a positive control. Additional control incubations were performed in the absence of 7s or NADPH, and using heat-denatured (90 °C, 15 min) microsomes. The resulting mixtures were incubated at 37 °C, and all assays were run in duplicate. Aliquots (50 µL) were collected at different time points (0, 5, 10, 20, 30, 40, 50, 60, 75, 90, 120, and 180 min and 24 h) and 50 µL of cold reserpine solution (2.5 µM in acetonitrile) was then added to quench the reactions. Following centrifugation (10 min, 10,000× g, 4 °C), the supernatants were stored at −20 °C until LC-MS and LC-HRMS/MS analysis.

3.3.3. Half-Life t_1/2 Determination

Samples from the metabolic stability assay were analyzed by LC-MS using the experimental conditions previously described [36]. The in vitro depletion half-life of 7s, t_1/2, was calculated using Equation (1), assuming that the compound follows a first-order kinetic trend (see Supplementary Materials Figure S2). The “slope” was determined from linear fitting of the natural logarithm of the concentration of drug remaining plotted against time.

$$t_{1/2}=\frac{ln2}{slope}$$

(1)

The intrinsic clearance was calculated using Equation (2) [24,25]

$$C{L}_{int } = \frac{0.693}{ t_{\frac{1}{2}} } \times \frac{mL incubation}{mg microsomes} \times \frac{45 mg microsomes}{g liver} \times \frac{26 g liver}{Kg b.w.}$$

(2)

3.3.4. Metabolite Identification

The 60 min aliquot was analyzed by LC-HRMS/MS, as previously described [36]. All spectra corresponding to metabolites were then manually checked. The mass deviation from the accurate mass of the identified metabolites remained below 5 ppm for the precursor and product ions. After their detection, structural characterization of the potential metabolites was based on tandem mass data (see Supplementary Materials Figures S2–S4).

4. Conclusions

A series of enantiopure tryptophanol-derived bicyclic lactams was prepared, and its antiproliferative activity was evaluated in AGS cells. From the first screening emerged compound 7c, a (R)-tryptophanol derivative with a para-chloro phenyl substituent, which was selected for further optimization. Introduction of an additional di-halogenated aromatic ring in 7c structure led to two derivatives 2.3- to 2.7-fold more active in AGS cells. These compounds also showed moderate activity in prostate cancer cells, representing useful hit compounds for further optimization in this type of cancer. More importantly, additional assays with the two compounds showed they are not toxic in normal HEK 293T cells, and that the antiproliferative activity in AGS cells occurs through apoptosis. Stability studies with the most potent derivative, compound 7s, showed that the compound is stable in PBS and human plasma. Moreover, incubation assays in human liver microsomes, followed by LC-HRMS/MS analysis, showed that this compound is moderately metabolically stable and that the major metabolites stem from mono-hydroxylation of the indole ring, which is not anticipated to be a toxicity red flag alert.