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

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.


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 pyrrolidonebased 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. Scheme 3. Synthesis of (R)-tryptophanol-derived oxazolopyrrolidone lactams 7n-u. Reaction conditions: (a) Pd(PPh 3 ) 2 Cl 2 , aq. sol. Na 2 CO 3 (1 M), 1,4-dioxane, 100 • C, 2-5 h.
The absolute configuration of the new formed stereogenic center C-7a was established by X-ray analysis of compound 8b (Figure 3). The 13 C 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 1 H NMR spectra as a doublet at 1.12 ppm for 7j and at 0.60 ppm for 7j', and in the 13 C 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). . X-ray crystallographic structures of compounds 8b, 7j, and 7j' (crystallographic information file (CIF) data can be found in the Supplementary Materials Tables S1-S15).

Effect of Tryptophanol-Derived Oxazolopyrrolidone Lactams on Cell Viability and on Apoptosis
To perform a structure-activity relationship (SAR) study, a first series of tryptophanolderived oxazolopyrrolidone lactams containing different substituents on the phenyl ring (R 1 ) at position C-7a was synthesized (compounds 7a-g and 8a-g, Table 1). In the design of this new compounds series, a diversity of substituents with electron donating properties (-CH 3 and -OCH 3 groups) and electron withdrawing properties (-F, -Cl, -Br, and -SO 2 CH 3 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 (R 1 = H), 7b (R 1 = F), and 8e (R 1 = CH 3 ) showed moderate antiproliferative activity, while compounds 7g and 8g (R 1 = SO 2 CH 3 ) 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 R 1 had a positive impact on the antiproliferative activity, for both enantiomers (compounds 7c-d and 8c-d). The derivative 7c (R 1 = Cl) exhibited the highest activity and was selected for chemical derivatizations to improve the antiproliferative activity of this scaffold in AGS cells. Table 1. Screening of (R) and (S)-tryptophanol-derived oxazolopyrrolidone lactams 7a-g and 8a-g in AGS cell line. Four sites were identified for suitable structural modifications in compound 7c: metaposition 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).
(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 (R 1 = p-Cl-Ph), 7p (R 1 = p-OH-Ph), and 7q (R 1 = p-CH 2 OH-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. Table 2. Screening of (R)-tryptophanol-derived oxazolopyrrolidone lactams 7c, 7h-u, and 7j' in AGS cell line.
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 IC 50 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). Trifluoromethyl derivative 7o (R 1 = p-CF 3 -Ph) and di-halogenated derivative 7s (R 1 = 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 (IC 50 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).

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 monoand 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 monohydroxylated 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 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.

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 1 H 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 NaHCO 3 (15 mL) and brine (15 mL), dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel flash chromatography using a mixture of EtOAc/n-hexane as eluent. ( 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%); α 25 D = −54.7 • (c = 2.0, MeOH); 1 H NMR spectra was found to be identical to the one reported [15] and obtained for compound 8a. Anal. Calcd. for C 21  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 N 2 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 MgSO 4 , and concentrated in vacuo. The residue was purified by silica gel flash chromatography using EtOAc/n-hexane as eluent.

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).

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.
Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824 7/14/3/208/s1: crystallographic information for compounds 7j, 7j', and 8b; LC-HRMS/MS data for compound 7s and its metabolites; NMR spectra of compounds 7h, 7j, 7j', 7o, and 7s.  Institutional Review Board Statement: Ethical review and approval were waived for this study. This plasma was obtained from "Instituto Português do Sangue" that is the Portuguese institute of blood. The plasma was obtained from blood that was already out of date for use in medical procedures. This blood was to be destroyed if it were not used by us. The IPS makes agreements with the institutions so that it can be used for research purposes.

Informed Consent Statement: Not applicable.
Data Availability Statement: CCDC 2050433-2050435 contains the supplementary crystallographic data for this paper. These data are provided free of charge by The Cambridge Crystallographic Data Centre.