Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites’ identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62–89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds’ metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.


Introduction
Colorectal cancer (CRC) figures currently as the third most common cancer and it ranks second as cause of cancer-related death globally [1]. Despite the latest progresses made in understanding CRC pathophysiology, poor therapeutic options are still available. To date, systemic treatment is based on combination therapies of DNA-damaging agents 5-fluorouracil (5-FU), oxaliplatin, and irinotecan with targeted drugs such as monoclonal anti-epidermal growth factor receptor (EGFR) antibodies and anti-angiogenic agents targeting the vascular endothelial growth factor (VEGF) pathway [2,3]. These combined treatment options have enhanced the median overall survival from about one year with 5-FU alone to up to three years with current standard therapies [4]. Nevertheless, patients develop resistance to available treatments [5] and chemotherapy-derived side-effects [6]. Therefore, the identification of novel therapeutics, with optimized pharmacological impact and minimized toxicity is urgently needed. Targeted therapies have gained importance in the field, and the tumor protein p53 is considered a relevant therapeutic target in CRC [7,8], continuing to figure as the most inactivated protein in spontaneous human cancers [9,10].
Inactivation of p53 can occur by mutation of the TP53 gene or by upregulation of its negative regulators MDM2 and/or MDM4 [11][12][13][14]. As guardian of the genome, p53 controls programmed cell death, tightly regulating apoptosis [15,16]. Protein p53 is the center of multiple essential cellular processes and under specific stress signals such DNA damage, oncogene activation and hypoxia, promotes cell cycle arrest coordinating DNA repair, senescence and autophagy [17][18][19][20]. Pharmacological restoration of tumor suppressor p53 function is a promising approach in cancer therapy. Although few small molecules have followed clinical development, p53-based therapy is still not clinically available for cancer treatment. Among the p53-activating small molecules in clinical trials for CRC (Figure 1), Idasanutlin (1, NCT03555149), Siremadlin (2, NCT03714958), and CGM097 (3, NCT01760525) effectiveness is limited only to wild-type (wt) p53 expressing CRC, and small molecule COTI-2 (4, NCT02433626) is a mutant p53 activator which mechanism of action results still unclear [21,22]. Another relevant clinical candidate, APR-246 (5), retains a p53-independent inhibitory activity in CRC [23]. Consequently, additional research is needed to discover novel p53-binding compounds to defeat CRC.
Pharmaceuticals 2023, 16, x FOR PEER REVIEW 2 of importance in the field, and the tumor protein p53 is considered a relevant therapeu target in CRC [7,8], continuing to figure as the most inactivated protein in spontaneo human cancers [9,10]. Inactivation of p53 can occur by mutation of the TP53 gene or upregulation of its negative regulators MDM2 and/or MDM4 [11][12][13][14]. As guardian of t genome, p53 controls programmed cell death, tightly regulating apoptosis [15,16]. Prote p53 is the center of multiple essential cellular processes and under specific stress sign such DNA damage, oncogene activation and hypoxia, promotes cell cycle arrest coor nating DNA repair, senescence and autophagy [17][18][19][20]. Pharmacological restoration of mor suppressor p53 function is a promising approach in cancer therapy. Although fe small molecules have followed clinical development, p53-based therapy is still not cli cally available for cancer treatment. Among the p53-activating small molecules in clini trials for CRC (Figure 1), Idasanutlin (1, NCT03555149), Siremadlin (2, NCT03714958), a CGM097 (3, NCT01760525) effectiveness is limited only to wild-type (wt) p53 expressi CRC, and small molecule COTI-2 (4, NCT02433626) is a mutant p53 activator which mec anism of action results still unclear [21,22]. Another relevant clinical candidate, APR-2 (5), retains a p53-independent inhibitory activity in CRC [23]. Consequently, addition research is needed to discover novel p53-binding compounds to defeat CRC. In previous work, enantiopure tryptophanol-derived isoindolinone SLMP53-1 was identified as a novel direct reactivator of wt p53 and DNA-contact mutant p53 R280 having p53-dependent in vivo antitumor activity [24]. Optimization of SLMP53-1 (6) l to the identification of SLMP53-2 (7), a small molecule that restores wt-like conformati and DNA-binding ability of mutp53-Y220C, by enhancing its interaction with the he shock protein 70 (Hsp70), in hepatocellular carcinoma [25]. This compound also show promising therapeutic potential in advanced melanoma and in vitro activity in HCT1 cells [26,27]. Due to the high failure rates in drug development, mainly related to po prediction of drug metabolism, development of new bioactive molecules with adequa metabolic properties is a basic requirement for preclinical success and developme [28,29]. For this reason, a preliminary study of the pharmacokinetic profile of SLMP53 (6) and SLMP53-2 (7) was conducted before proceeding with the optimization of try tophanol-derived isoindolinones as p53 activators. Determination of Phase I and Phase metabolites by LC-ESI-HRMS/MS and identification of the possible reactive metaboli allowed us to identify the metabolic liabilities of the tryptophanol-derived isoindolino scaffold. Biomimetic synthesis [30] of Phase I metabolites, identified in human liver m crosomes (HLM) incubations, was conducted and their anti-proliferative activity HCT116 cells evaluated. Subsequent scaffold structural derivatization was carried out blocking the 2-position of the indole with bromine ( Figure 2). The antiproliferative activ of the novel compounds and contribution to the p53 pathway was evaluated in hum colorectal carcinoma HCT116 cell lines. To evaluate the impact on the metabolic stabil of the performed structural optimizations, the pharmacokinetic (PK) profile of the mo In previous work, enantiopure tryptophanol-derived isoindolinone SLMP53-1 (6) was identified as a novel direct reactivator of wt p53 and DNA-contact mutant p53 R280K, having p53-dependent in vivo antitumor activity [24]. Optimization of SLMP53-1 (6) led to the identification of SLMP53-2 (7), a small molecule that restores wt-like conformation and DNA-binding ability of mutp53-Y220C, by enhancing its interaction with the heat shock protein 70 (Hsp70), in hepatocellular carcinoma [25]. This compound also showed promising therapeutic potential in advanced melanoma and in vitro activity in HCT116 cells [26,27]. Due to the high failure rates in drug development, mainly related to poor prediction of drug metabolism, development of new bioactive molecules with adequate metabolic properties is a basic requirement for preclinical success and development [28,29]. For this reason, a preliminary study of the pharmacokinetic profile of SLMP53-1 (6) and SLMP53-2 (7) was conducted before proceeding with the optimization of tryptophanolderived isoindolinones as p53 activators. Determination of Phase I and Phase II metabolites by LC-ESI-HRMS/MS and identification of the possible reactive metabolites allowed us to identify the metabolic liabilities of the tryptophanol-derived isoindolinone scaffold. Biomimetic synthesis [30] of Phase I metabolites, identified in human liver microsomes (HLM) incubations, was conducted and their anti-proliferative activity in HCT116 cells evaluated. Subsequent scaffold structural derivatization was carried out by blocking the 2-position of the indole with bromine ( Figure 2). The antiproliferative activity of the novel compounds and contribution to the p53 pathway was evaluated in human colorectal carcinoma HCT116 cell lines. To evaluate the impact on the metabolic stability of the performed structural optimizations, the pharmacokinetic (PK) profile of the most active compounds was also assessed. To assess the putative binding to p53, in vitro thermostability assays with the most promising compounds towards the wt p53 DNA-binding domain (p53DBD) active compounds was also assessed. To assess the putative binding to p53, in vitro thermostability assays with the most promising compounds towards the wt p53 DNA-binding domain (p53DBD) were performed, thus allowing us to extend our knowledge on the mode of action through which the scaffold promotes protein p53 reactivation.
Very similar metabolite profiles were identified for these two hit compounds by LC-ESI(+)-HRMS/MS. Similarly to what was observed in our previous PK studies with tryptophanol-derived oxazolopyrrolidone lactams [33], multiple isomeric metabolites stemming from the metabolic incorporation of one or two oxygens were identified ( Figure 3) (SI, Figure S4). Whereas the fragmentation pattern displayed by these ions do not allow us to assign the exact location of the metabolic transformation(s), the consistent presence of diagnostic fragment ions ( Figure 3) (SI, Figures S5-S13), attests the selective incorporation of 1 or 2 oxygens (mass increase of 16 and 32 u, respectively) at the indole moiety. Additionally, the metabolic product of indole ring cleavage, following initial oxidation of the parent drug, was selectively identified in SLMP53-1 (6) incubations (Figure 3 and SI, Figure S7). This suggests that the presence of the methyl group, at the indole nitrogen of SLMP53-2 (7), hampers this metabolic transformation. Nonetheless, SLMP53-2 (7) is prone to N-dealkylation pathways. In fact, three N-indole dealkylation products were identified for this hit compound ( Figure 3): one product from its direct N-dealkylation; and three products, presumably formed upon N-dealkylation of metabolites, stemming from oneoxygen incorporation (SI, Figures S12 and S13). Although observed, N-dealkylation is a minor metabolic transformation as shown by the plot of the relative intensities of each metabolite over reaction time. In fact, metabolites stemming from the incorporation of one oxygen are consistently the main Phase I metabolic transformation detected for SLMP53-1 (6) and SLMP53-2 (7) (SI, Figures S14 and S15). Noteworthy is the fact that the same metabolite profile was obtained for SLMP53-1 (6) incubations run in S9 rat liver fractions. This result suggests that specie-dependent dimorphic metabolite profiles are not expected for this scaffold, which is relevant since this hit was tested in vivo in a rodent model. Additionally, this result suggests that this scaffold is not prone to degradation by cytosolic metabolic enzymes.
Very similar metabolite profiles were identified for these two hit compounds by LC-ESI(+)-HRMS/MS. Similarly to what was observed in our previous PK studies with tryptophanol-derived oxazolopyrrolidone lactams [33], multiple isomeric metabolites stemming from the metabolic incorporation of one or two oxygens were identified ( Figure 3) (SI, Figure S4). Whereas the fragmentation pattern displayed by these ions do not allow us to assign the exact location of the metabolic transformation(s), the consistent presence of diagnostic fragment ions (Figure 3 and SI, Figures S5-S13), attests the selective incorporation of 1 or 2 oxygens (mass increase of 16 and 32 u, respectively) at the indole moiety. Additionally, the metabolic product of indole ring cleavage, following initial oxidation of the parent drug, was selectively identified in SLMP53-1 (6) incubations (Figure 3 and SI, Figure S7). This suggests that the presence of the methyl group, at the indole nitrogen of SLMP53-2 (7), hampers this metabolic transformation. Nonetheless, SLMP53-2 (7) is prone to N-dealkylation pathways. In fact, three N-indole dealkylation products were identified for this hit compound ( Figure 3): one product from its direct N-dealkylation; and three products, presumably formed upon N-dealkylation of metabolites, stemming from one-oxygen incorporation (SI, Figures S12 and S13). Although observed, N-dealkylation is a minor metabolic transformation as shown by the plot of the relative intensities of each metabolite over reaction time. In fact, metabolites stemming from the incorporation of one oxygen are consistently the main Phase I metabolic transformation detected for SLMP53-1 (6) and SLMP53-2 (7) (SI, Figures S14 and S15). Noteworthy is the fact that the same metabolite profile was obtained for SLMP53-1 (6) incubations run in S9 rat liver fractions. This result suggests that specie-dependent dimorphic metabolite profiles are not expected for this scaffold, which is relevant since this hit was tested in vivo in a rodent model. Additionally, this result suggests that this scaffold is not prone to degradation by cytosolic metabolic enzymes.
Glucuronidation is a key contributor for decreased drug bioavailability, by excretion [34,35]. To evaluate the ability of (S)-tryptophanol-derived isoindolinones to undergo this Phase II pathway, additional incubations were performed in alamethicin induced-HLM in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and uridine diphosphate glucuronic acid (UDPGA) co-factors [34]. While no Phase II metabolites were identified in SLMP53-2 (7) incubations, two glucuronic acid conjugates were identified for SLMP53-1 (6), by LC-ESI(-)-HRMS/MS: a Phase II metabolite compatible with direct conjugation of the SLMP53-1 (6) N-indole with glucuronic acid (SI, Figure S16); and one product formed upon glucuronidation of one of the Phase I metabolites, stemming from one-oxygen incorporation. The presence of the glucuronic acid moiety was attested by the observation of diagnostic neutral losses and fragment ions, [36] in the tandem mass spectra of these Phase II metabolites (SI, Figure S17). The fact that SLMP53-2 (7) is not prone to these Phase II pathways, suggests that the presence of the methyl group at the N-indole might be beneficial for increased bioavailability. Glucuronidation is a key contributor for decreased drug bioavailability, by excretion [34,35]. To evaluate the ability of (S)-tryptophanol-derived isoindolinones to undergo this Phase II pathway, additional incubations were performed in alamethicin induced-HLM in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and uridine diphosphate glucuronic acid (UDPGA) co-factors [34]. While no Phase II metabolites were identified in SLMP53-2 (7) incubations, two glucuronic acid conjugates were identified for SLMP53-1 (6), by LC-ESI(-)-HRMS/MS: a Phase II metabolite compatible with direct conjugation of the SLMP53-1 (6) N-indole with glucuronic acid (SI, Figure S16); and one product formed upon glucuronidation of one of the Phase I metabolites, stemming from oneoxygen incorporation. The presence of the glucuronic acid moiety was attested by the observation of diagnostic neutral losses and fragment ions, [36] in the tandem mass spectra of these Phase II metabolites (SI, Figure S17). The fact that SLMP53-2 (7) is not prone to these Phase II pathways, suggests that the presence of the methyl group at the N-indole might be beneficial for increased bioavailability.

Synthesis and Antiproliferative Activity of SLMP53-1 Phase I Metabolites
Since SLMP53-1 (6) and SLMP53-2 (7) showed very similar metabolite profiles, we decided to use SLMP53-1 (6) as a model for the subsequent studies involving the evaluation of the activity of the main Phase I metabolites, following their synthesis in a preparative scale. This also constituted an opportunity to perform a full structural characterization of the Phase I metabolites, whose preliminary LC-HRMS-MS characterization only attested the incorporation of one or two oxygens at the indole moiety but did not allow the identification of the exact location of these transformations.
The synthesis of main Phase I metabolites of the p53 activator SLMP53-1 (6) was . LC-HRMS/MS-based metabolite profile of (S)-tryptophanol-derived isoindolinones SLMP53-1 (6) (in orange) and SLMP53-2 (7) (in pink). In grey boxes are highlighted the diagnostic ions that attested for the metabolic transformation at the indole ring.

Synthesis and Antiproliferative Activity of SLMP53-1 Phase I Metabolites
Since SLMP53-1 (6) and SLMP53-2 (7) showed very similar metabolite profiles, we decided to use SLMP53-1 (6) as a model for the subsequent studies involving the evaluation of the activity of the main Phase I metabolites, following their synthesis in a preparative scale. This also constituted an opportunity to perform a full structural characterization of the Phase I metabolites, whose preliminary LC-HRMS-MS characterization only attested the incorporation of one or two oxygens at the indole moiety but did not allow the identification of the exact location of these transformations.
The synthesis of main Phase I metabolites of the p53 activator SLMP53-1 (6) was achieved by biomimetic oxidation catalyzed by the nonheme iron-based complex (2S, While metabolites SLMP53-1_M1, M2 and M6 were identified as reaction products by HRMS analysis (SI, Figures S20-S22) only vestigial amounts of these compounds were formed, which hampered their subsequent isolation. Of note is the fact that 10, the product of intramolecular aminoalkylation [37], was the main reaction product due to the acidic conditions needed for the catalyst system used. However, for the 5 h reaction the purification method was only focused on the isolation of SLMP53-1_M3a, M3b and M5. Following isolation of SLMP53-1_M3a, M3b, M4 and M5, NMR analysis unequivocally evidenced the metabolic oxidations at positions 2 and 3 of the indole core (SI, Figures S26 and S32). The main Phase I metabolites identified in HLM incubation correspond to the two diastereomeric oxindoles, SLMP53-1_M3 (Scheme 1). The two metabolites stemming from the incorporation of two oxygens (compatible with the 32 u mass increment) correspond to products of a subsequent metabolic hydroxylation at the C-3 indole position to SLMP53-1_M4, followed by the oxidative cleavage of the pyrrole ring which yield a formamide metabolite, SLMP53-1_M5, similar to L-formyl kynurenine, a catabolic product of L-tryptophan [38]. LC-ESI(+)-HRMS/MS analysis showed that the product profile obtained from biomimetic oxidation of SLMP53-1 (6) is similar to its respective metabolite profile, obtained from HLM incubations. Coherently, all products share compatible retention times and similar tandem HRMS profiles ( Figures S18-S25). Whereas very similar results were obtained using N,N′-Dimethyl-N,N′-bis(2-pyridylmethyl)ethane-1,2-diamine Iron(II) bis(triflate), [Fe(II)(bpmen)OTf2] (9) (results not shown), the commercial availability of White-Chen catalyst (8) justifies our selection for the scale-up synthesis of SLMP53-1 (6) metabolites. While metabolites SLMP53-1_M1, M2 and M6 were identified as reaction products by HRMS analysis (SI, Figures S20-S22) only vestigial amounts of these compounds were formed, which hampered their subsequent isolation. Of note is the fact that 10, the product of intramolecular aminoalkylation [37], was the main reaction product due to the acidic conditions needed for the catalyst system used. However, for the 5 h reaction the purification method was only focused on the isolation of SLMP53-1_M3a, M3b and M5. Following isolation of SLMP53-1_M3a, M3b, M4 and M5, NMR analysis unequivocally evidenced the metabolic oxidations at positions 2 and 3 of the indole core (SI, Figures S26  and S32). The main Phase I metabolites identified in HLM incubation correspond to the two diastereomeric oxindoles, SLMP53-1_M3 (Scheme 1). The two metabolites stemming from the incorporation of two oxygens (compatible with the 32 u mass increment) correspond to products of a subsequent metabolic hydroxylation at the C-3 indole position to SLMP53-1_M4, followed by the oxidative cleavage of the pyrrole ring which yield a formamide metabolite, SLMP53-1_M5, similar to L-formyl kynurenine, a catabolic prod-Scheme 1. Direct biomimetic oxidation of SLMP53-1 (6). i. White-Chen catalyst (8) 10 mol%, H 2 O 2 0.45 eq. (35% v/v), AcOH 5.00 eq., ACN at 40 • C.
The antiproliferative activity of SLMP53-1 Phase I metabolites was subsequently evaluated in human colon adenocarcinoma HCT116 cell line expressing wt p53 (HCT116 p53 +/+ ). To evaluate the selectivity for the p53 pathway, the metabolites were also screened in HCT116 cells in which p53 has been knocked out (HCT116 p53 −/− ). Growth inhibition values (GI 50 ) are listed in Table 1. All tested SLMP53-1 metabolites showed loss of potency and loss of selectivity towards the p53 pathway compared to SLMP53-1 (6). SLMP53-1_M4 was not tested due to its instability. With impact on decreased attrition rates [39,40], evaluation of bioactivation risk is considered a key issue during the drug development process. To investigate potential bioactivation pathways, SLMP53-1 (6) was incubated with S9 rat liver fraction and HLM in the presence of glutathione (GSH). Two GSH adducts were identified under these conditions (Figure 4): (1) SLMP53-1_GS1, plausibly stemming from the ring-opening of an arene oxide formed in the indole core, which are the expected CYP-mediated metabolic intermediates of the Phase I metabolites SLMP53-1_M1-M3; and (2) SLMP53-1_GS2, compatible with a cyclic adduct which might be formed from the initial thiol nucleophilic addition to the N-formyl amide carbonyl group of SLMP53-1_M5, followed by intramolecular addition of the α-nitrogen of the cysteine residue (SI, Figures S33 and S34) [41]. Interestingly, no reaction products were obtained upon L-lysine incubation with SLMP53-1_M5, followed by reductive stabilization. Whereas the GSH-derived adducts suggest potential bioactivation pathways, the SLMP53-1 in vivo assays were not associated with toxicity alerts [24]. Therefore, the formation of these adducts is likely to be more associated to excretion rather than toxicity pathways.

Metabolite
HCT116 p53 +/+ GI50 (µM) [24] 15.6 ± 1.6 34.0 ± 3.5 With impact on decreased attrition rates [39,40], evaluation of bioactivation risk is considered a key issue during the drug development process. To investigate potential bioactivation pathways, SLMP53-1 (6) was incubated with S9 rat liver fraction and HLM in the presence of glutathione (GSH). Two GSH adducts were identified under these conditions ( Figure 4): (1) SLMP53-1_GS1, plausibly stemming from the ring-opening of an arene oxide formed in the indole core, which are the expected CYP-mediated metabolic intermediates of the Phase I metabolites SLMP53-1_M1-M3; and (2) SLMP53-1_GS2, compatible with a cyclic adduct which might be formed from the initial thiol nucleophilic addition to the N-formyl amide carbonyl group of SLMP53-1_M5, followed by intramolecular addition of the α-nitrogen of the cysteine residue (SI, Figures S33 and S34) [41]. Interestingly, no reaction products were obtained upon L-lysine incubation with SLMP53-1_M5, followed by reductive stabilization. Whereas the GSH-derived adducts suggest potential bioactivation pathways, the SLMP53-1 in vivo assays were not associated with toxicity alerts [24]. Therefore, the formation of these adducts is likely to be more associated to excretion rather than toxicity pathways.

Synthesis of Bromine-Enriched Tryptophanol-Derived Isoindolinones
Based on the results obtained from the PK study on compounds SLMP53-1 (6) and SLMP53-2 (7), we searched for synthetic strategies aiming to optimize the robustness of the tryptophanol-derived isoindolinone scaffold towards CYPs oxidative metabolism. Bromine insertion at the indole C2 may electronically and metabolically promote the indole pyrrole ring stabilization due to its effect as deactivating group and to its steric hindrance [42]. Moreover, indole nitrogen protection was suggested to enhance the metabolic stability of this class of compounds. Therefore, a series of bromine tryptophanol-derived isoindolinones with phenyl or para-and meta-substituted phenyl groups in position C9b was prepared. Compounds 13c and 13d were obtained with yields of 92% and 84%, Figure 4. Possible SLMP53-1 (6) bioactivation pathways. In grey boxes are highlighted the diagnostic fragment ions that attested for the GSH addition at the indole ring.
Compounds 13a and 13c-o were analyzed by 1 H and 13 C nuclear magnetic resonance (NMR) experiments (SI, Figure S35-S38). Formation of the C-Br bound was confirmed by the disappearance of the H-2 indole singlet present in the 1 H NMR of the starting isoindolinones, the C-2 signal between 108.7-109.4 ppm for compounds 13a, 13e-i, 13m and 13n, and between 113.0-114.1 ppm for compounds 13c-d and 13j-k. The presence of benzoyl and tosyl groups induces a major shift for this signal appearing at chemical shifts 119.3 ppm (compound 13l) and at 121.6 ppm (compound 13o), respectively. The CH2-indole diastereotopic protons appear as two doublets of doublets at 2.69-2.97 ppm and 3.02-3.28 ppm. The stereogenic center C-3 signal appears in at 55.6-55.7 ppm, except for compounds 13i and 13o where this signal appears at chemical shift 57.1 and 54.9 ppm, respectively. The C-9b stereogenic center signal appears for all compounds between 100.0 and 101.2 Compounds 13j-o were prepared by indole nitrogen derivatization of bromine-enriched (S)-tryptophanol isoindolinone 13a, with yields of 62-84% (Scheme 3). The starting material was not totally consumed in the N-tosylation reaction, probably due to steric hindrance of the para-toluenesulfonate moiety leading to a lower yield of compound 13o.
Compounds 13a and 13c-o were analyzed by 1 H and 13 C nuclear magnetic resonance (NMR) experiments (SI, Figure S35-S38). Formation of the C-Br bound was confirmed by the disappearance of the H-2 indole singlet present in the 1 H NMR of the starting isoindo linones, the C-2 signal between 108.7-109.4 ppm for compounds 13a, 13e-i, 13m and 13n and between 113.0-114.1 ppm for compounds 13c-d and 13j-k. The presence of benzoy and tosyl groups induces a major shift for this signal appearing at chemical shifts 119.3 ppm (compound 13l) and at 121.6 ppm (compound 13o), respectively. The CH2-indole diastereotopic protons appear as two doublets of doublets at 2.69-2.97 ppm and 3.02-3.28 ppm. The stereogenic center C-3 signal appears in at 55.6-55.7 ppm, except for compound 13i and 13o where this signal appears at chemical shift 57.1 and 54.9 ppm, respectively The C-9b stereogenic center signal appears for all compounds between 100.0 and 101.2 Compounds 13a and 13c-o were analyzed by 1 H and 13 C nuclear magnetic resonance (NMR) experiments (SI, Figures S35-S38). Formation of the C-Br bound was confirmed by the disappearance of the H-2 indole singlet present in the 1 H NMR of the starting isoindolinones, the C-2 signal between 108.7-109.4 ppm for compounds 13a, 13e-i, 13m and 13n, and between 113.0-114.1 ppm for compounds 13c-d and 13j-k. The presence of benzoyl and tosyl groups induces a major shift for this signal appearing at chemical shifts 119.3 ppm (compound 13l) and at 121.6 ppm (compound 13o), respectively. The CH 2 -indole diastereotopic protons appear as two doublets of doublets at 2.69-2.97 ppm and 3.02-3.28 ppm. The stereogenic center C-3 signal appears in at 55.6-55.7 ppm, except for compounds 13i and 13o where this signal appears at chemical shift 57.1 and 54.9 ppm, respectively. The C-9b stereogenic center signal appears for all compounds between 100.0 and 101.2 ppm. These values are in agreement with NMR signals observed for the parent compounds 6 and 7, confirming the 3,9b-(S,R)-relationship for all compounds [26].

Structure-Activity Relationship (SAR) Studies
The antiproliferative activity of bromine-enriched tryptophanol-derived isoindolinones 13a and 13c-o was evaluated in human colon adenocarcinoma HCT116 cells (Table 2 and Figure 5). Four compounds were more active than hit SLMP53-2 (7). ppm. These values are in agreement with NMR signals observed for the parent c pounds 6 and 7, confirming the 3,9b-(S,R)-relationship for all compounds [26].

Structure-Activity Relationship (SAR) Studies
The antiproliferative activity of bromine-enriched tryptophanol-derived isoin linones 13a and 13c-o was evaluated in human colon adenocarcinoma HCT116 cells (T 2 and Figure 5). Four compounds were more active than hit SLMP53-2 (7).  The insertion of a bromine in the unprotected indole derivatives 13a and 13i le loss of the activity and selectivity toward the p53 pathway (compounds 13c-d and 13 A similar effect was observed when the bromo derivatives have an ethyl, an acetyl mesyl group in the indole nitrogen (compounds 13j, 13m and 13n). Whereas a loss o lectivity is observed for compounds 13j and 13n, introduction of an acetyl group (1 promotes 1.7-fold of selectivity in p53-expressing HCT116 cell line. Introduction of a zoyl group led to a derivative 2.8-fold less active than hit compound SLMP53-2 (8) bu derivative retains selectivity for the p53 pathway. Insertion of a bromine atom at the indole moiety of hit compound SLMP53-2 (7) resulted in a compound 3-fold less ac and less selective for the cell line with p53. The presence of a tosyl, a bulkier elect withdrawing group, led to a 4.0-fold less active derivative compared with a hydro (SLMP53-2 (7) vs. 13o). Introduction of a benzyl moiety at the indole nitrogen and in duction of the bromine at the C-2 indole led to compound 13k with compar The insertion of a bromine in the unprotected indole derivatives 13a and 13i led to loss of the activity and selectivity toward the p53 pathway (compounds 13c-d and 13j-o). A similar effect was observed when the bromo derivatives have an ethyl, an acetyl or a mesyl group in the indole nitrogen (compounds 13j, 13m and 13n). Whereas a loss of selectivity is observed for compounds 13j and 13n, introduction of an acetyl group (13m) promotes 1.7-fold of selectivity in p53-expressing HCT116 cell line. Introduction of a benzoyl group led to a derivative 2.8-fold less active than hit compound SLMP53-2 (8) but the derivative retains selectivity for the p53 pathway. Insertion of a bromine atom at the C2-indole moiety of hit compound SLMP53-2 (7) resulted in a compound 3-fold less active and less selective for the cell line with p53. The presence of a tosyl, a bulkier electron-withdrawing group, led to a 4.0-fold less active derivative compared with a hydrogen (SLMP53-2 (7) vs. 13o). Introduction of a benzyl moiety at the indole nitrogen and introduction of the bromine at the C-2 indole led to compound 13k with comparable antiproliferative activity to hit compound Pharmaceuticals 2023, 16, 146 9 of 26 SLMP53-2 (7). Interestingly, compound 13k showed 3.8-fold selectivity for the p53 pathway, being 1.8-fold more selective for the p53 pathway than SLMP53-2 (7) (3.8 for 13k versus 2.1 for SLMP53-2 (7)). Moreover, although bromine insertion at the C-2 indole led to 1.2-fold and 1.5-fold increase in the antiproliferative activity as in C9b para-Fluoro and C9b para-Chloro isoindolinones 13g and 13h, respectively, such optimization led to loss of selectivity for the p53 pathway. Additionally, the presence of a para-chloro and meta-nitro substituted phenyl moiety, together with insertion of the bromine at the C2-indole, was not beneficial for the antiproliferative activity and compound selectivity for the p53 pathway. The most promising derivative of this series was C-2-bromine-enriched (S)-tryptophanol-derived isoindolinone 13d with a GI 50 value of 4.0 µM in HCT116 p53 +/+ , being 2-fold more active than SLMP53-2 (7). In addition, compound 13d showed 1.9-fold selectivity for p53-expressing HCT116 cell line (13d, GI 50 p53 −/− = 7.5 µM). To evaluate the impact of stereochemistry on the antiproliferative activity, the enantiomer of 13d, compound 13d , was synthesized and tested against HCT116 cells with and without p53. However, a 3,9b-(R, S) stereochemistry induced an important loss of antiproliferative activity and selectivity for the p53 pathway (13d , HCT116 p53 +/+ GI 50 = 31.1 ± 4.0 µM; HCT116 p53 −/− GI 50 = 25.4 ± 1.2 µM).

Determination of the Metabolic Stability and Metabolite Profiling of 13d and 13k
While 13d and 13k can be classified as low clearance compounds [32], 13d was shown to be slightly less stable towards HLM enzymes than SLMP53-2 (8) ( Table 3) [32]. Nonetheless, 13k displayed not only an improvement in the antiproliferative activity but also equivalent metabolic stability, when compared with SLMP53-2 (7) ( Table 3). Notably, the C9b-phenyl moieties of 13d and 13k were not prone to metabolic degradation. The N-dealkylation and indole mono-oxidation were the sole metabolic pathways identified for 13k ( Figure 6, SI, Figures S44-S46). However, for 13d, in addition to these metabolic pathways, metabolites stemming from reduction in the N-propyl substituent followed by hydroxylation with bromine loss were also identified ( Figure 6, SI, Figures S47 and S48). Remarkably, metabolites stemming from the pyrrole ring opening were not detected for these compounds. This suggests that the co-existence of a bromine at C-2 and propyl or benzyl substituents at the indole nitrogen protects the indole ring from this catabolic pathway. This might be a relevant issue, since no antiproliferative activity was observed for SLMP53-1 indole-degradation products. However, a metabolic shift towards the formation of potential phenolic metabolites was observed. In fact, the plots of the relative intensities of each metabolite over time (SI, Figures S49 and S50) suggest that these metabolites (M1, M2 and M3) are consistently the main metabolic transformation detected for both compounds 13d and 13k.
The presence of a bromine substituent at the C-2 position of 13d and 13k, led us to investigate if it could constitute a metabolic liability towards the reaction with bionucleophiles. However, this hypothesis was discarded since no products compatible with the addition of GSH, and the concomitant loss of bromine, were detected following incubation of this bionucleophile with 13d and 13k, either with or without enzymatic conditions (in S9 rat liver incubations). Nonetheless, GSH adducts compatible with the ring-opening of arene oxides intermediates were identified in the S9 rat liver fraction incubation run in the presence of GSH (SI, Figures S51-S53). It should be noted, however, that 13k seems to be less prone to this type of bioactivation pathways. In fact, contrasting with 13d, for this derivative no adducts stemming from the bioactivation of Phase I metabolites were identi-fied. Therefore, despite displaying similar HLM intrinsic clearance to hit SLMP53-2, 13k shows less ability towards the catabolic pathways involving the indole ring opening (which are associated with activity loss) and more limited ability to yield reactive metabolites, when compared with its analogue 13d. The presence of a bromine substituent at the C-2 position of 13d and 13k, led us to investigate if it could constitute a metabolic liability towards the reaction with bionucleophiles. However, this hypothesis was discarded since no products compatible with the addition of GSH, and the concomitant loss of bromine, were detected following incubation of this bionucleophile with 13d and 13k, either with or without enzymatic conditions (in S9 rat liver incubations). Nonetheless, GSH adducts compatible with the ring-opening of arene oxides intermediates were identified in the S9 rat liver fraction incubation run in the presence of GSH (SI, Figures S51-S53). It should be noted, however, that 13k seems to be less prone to this type of bioactivation pathways. In fact, contrasting with 13d, for this derivative no adducts stemming from the bioactivation of Phase I metabolites were identified. Therefore, despite displaying similar HLM intrinsic clearance to hit SLMP53-2, 13k shows less ability towards the catabolic pathways involving the indole ring opening (which are associated with activity loss) and more limited ability to yield reactive metabolites, when compared with its analogue 13d.

Determination of the Thermostability of Wild-Type p53 DNA-Binding Domain by DSF Experiments
We next decided to evaluate whether 13d binds to protein p53 (Table 4). To this end the wt-p53 core domain (DNA Binding Domain; p53DBD; residues 94-312) [43] were expressed and purified, and using differential scanning fluorimetry (DSF) the protein melting temperature (Tm), in the absence and presence of tested compounds, was determined as an approach to confirm protein binding [44]. Methylene quinuclidinone (MQ) (2 mM) [45] was used as positive control. The assays were performed in the presence of dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In presence of DTT compound 13d did not increase the wt p53DBD Tm (Table 4; SI Figure S54). However, in presence of the reducing agent TCEP compound 13d promoted protein thermostability, increasing the Tm of the wt p53DBD from 40.99 ± 0.07 °C to 51.38 ± 0.38 °C (ΔTm = 10.39 °C). The Tm increase obtained in the presence of 2 mM MQ and 1 mM TCEP (ΔTm = 2.11 °C) or DTT (ΔTm = 2.28 °C) are in agreement with published data, thus validating the assay [45]. This first screening highlights that compound 13d interacts directly with wt-p53DBD (SI, Figure S54) and that stabilization of the p53 core domain is a putative mechanism for the observed effect in cellular assays. and potential bioactivation pathways. In the grey box is the diagnostic fragment ion that attested for the metabolic transformation at the indole ring.

Determination of the Thermostability of Wild-Type p53 DNA-Binding Domain by DSF Experiments
We next decided to evaluate whether 13d binds to protein p53 (Table 4). To this end the wt-p53 core domain (DNA Binding Domain; p53DBD; residues 94-312) [43] were expressed and purified, and using differential scanning fluorimetry (DSF) the protein melting temperature (T m ), in the absence and presence of tested compounds, was determined as an approach to confirm protein binding [44]. Methylene quinuclidinone (MQ) (2 mM) [45] was used as positive control. The assays were performed in the presence of dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In presence of DTT compound 13d did not increase the wt p53DBD T m (Table 4). However, in presence of the reducing agent TCEP compound 13d promoted protein thermostability, increasing the T m of the wt p53DBD from 40.99 ± 0.07 • C to 51.38 ± 0.38 • C (∆T m = 10.39 • C) ( Table 4; SI Figure S54). The T m increase obtained in the presence of 2 mM MQ and 1 mM TCEP (∆T m = 2.11 • C) or DTT (∆T m = 2.28 • C) are in agreement with published data, thus validating the assay [45]. This first screening highlights that compound 13d interacts directly with wt-p53DBD (SI, Figure S54) and that stabilization of the p53 core domain is a putative mechanism for the observed effect in cellular assays.

General Information
All reagents were purchased from commercial suppliers and used without further purification unless otherwise indicated. (S)-Tryptophanol-derived isoindolinones SLMP53-1 (6), SLMP53-2 (7) and compounds 11a-f and 13b were synthesized as previously reported [24][25][26]46]. (S)-and (R)-tryptophanol were synthesized by reduction in Land D-tryptophan with Lithium Aluminium hydride [47]. Except in the biomimetic reactions, all reaction solvents were distilled from appropriate drying agents prior to use. All reactions were performed under nitrogen atmosphere.
GIBCO TM Human liver microsomes (HLM), GIBCO TM rat S9 liver homogenate and Vivid ® regeneration system 100X were obtained from Thermo The pET15b expression construct to produce the human wt p53 core domain was acquired to Addgene [48] and sypro orange to Invitrogen (5000× stock solution).

Human Plasma Incubations
Human plasma incubations were performed in duplicate, at 37 • C, by standard methodology [49]. Specifically, incubations contained 40 µM of the test compound for a final volume of 1 mL. The mixtures were incubated for a total time of 24 h, and aliquots following 0, 30, 60, 120, 180 min and 24 h and quenched with a cold solution of reserpine 0.1 mM in acetonitrile. Following centrifugation (10,000 rpm, 10 min, RT), the clear supernatants were collected and analyzed by LC-ESI(+)-MS/MS. Additional control assays were conducted using PBS pH 7.4 instead of plasma solution and using procaine as a positive control.

S9 Rat Liver Fraction Incubations
Tested compounds at a 10 µM concentration (1 µL of 5 mM DMSO stock solution) were incubated with rat liver S9 fraction (2 mg protein/mL), Vivid ® regenerating system (2 µL) and NADPH (1mM) in ABIC 50 mM buffer pH 7.4, for a total incubation volume of 500 µL. Incubations were run in duplicate at 37 • C. Additional incubation was run using the same conditions in the presence of glutathione (1 mM

Half-Life Determination and Intrinsic Clearance Calculation
The in vitro depletion half-lives of tested compounds t 1/2 were calculated using Equation (1), assuming that testing compounds follow a first-order kinetic trend. The "slope" was determined from linear fitting of the natural logarithm of the concentration of drug remaining plotted against time: The intrinsic clearance in HLM (CLint, HLM) was calculated using Equation (2) [32,50]: x mL incubation mg microsomes x 45 mg microsomes g liver x 26 g liver Kg b.w.

Data Processing and Metabolite Identification
HLM incubations and rat S9 mix fractions incubations were analyzed by LC-HRMS/MS. All spectra corresponding to metabolites were then manually checked. Signals corresponding to ions identifying the appropriate (S)-tryptophanol-derived isoindolinone and Phase I and II metabolites were extracted from raw data by use of DataAnalysis program (Bruker Daltoniks, Mannheim, Germany). HPLC-MS signals of identified metabolites were also measured on the basis of chromatographic peak area normalized to the signal from an internal standard. The exact mass of each [M+H] + or [M-H]precursor and products ions were determined in accordance with a mass deviation from the accurate mass below ±5 ppm. After their detection, structural characterization of the potential metabolites was achieved by processing tandem MS/MS spectra.

Expression and Purification of Wild-Type p53 Core Domain
Expression of wt p53 core domain. The wt p53DBD was expressed in E. coli BL21 (DE3)-[pLys-S] in fusion with an N-terminal 21-residue tag containing the hexa-histidyl peptide (His 6 ) and the thrombin recognition sequence, using the pET15b-based expression construct [48].
For protein expression 10 mL of an overnight (ON) culture was used to inoculate 1 L of LB medium supplemented with 1 mL of ampicillin 100 mg/mL and 1mL of zinc sulphate 100 mM. Culture medium was incubated at 37 • C, 140 rpm till an OD 600nm of~0.5. Protein expression was then induced by adding 0.5 mL of isopropyl β-D-1-thiogalactopyranoside (IPTG) 1M (C final = 0.5 mM) and bacterial growth at 27 • C, 120 rpm ON. E. coli cells were pelleted by centrifugation for 10 min, 4000 rpm, at 4 • C. The cellular pellet was resuspended in 10 mL of Lysis buffer (Sodium phosphate 50 mM; NaCl 300 mM; glycerol 10%, pH 7.4) supplemented with 1mg/mL Lysozyme (Biochemica Applichem Panreac), 1 mM of phenylmethanesulfonyl fluoride (PMSF) (Merck), 5 µg of DNAse I (Panreac applichem) and 0.8% of Triton X-100 (Merck). After a first step of chemical lysis (30 min incubation, at 4 • C), cells were further disrupted by four cycles of sonication, at 4 • C, during 60 s at 50% duty free cycle on a Vibra Cell (Media Cibernetics). The lysate was subsequently cleared by centrifugation at 10,000 rpm, 40 min, at 4 • C and the supernatant containing the soluble proteins were collected.
Purification of human wt p53DBD and His 6 -tag removal. Immobilized Metal Affinity Chromatography (IMAC) was performed to purify the wt p53DBD using a nickelnitrilotriacetic acid (Ni-NTA) agarose resin (QIAGEN). Briefly, 1 mL of Ni 2+ resin was pre-equilibrated using MilliQ water (3 × 1 mL) and Lysis buffer containing 10 mM imidazole (3 × 1 mL). The soluble fraction of cellular lysate was supplemented with 6.8 µL of β-mercaptoethanol and 33.2 µL of imidazole 3 M to obtain the final concentrations of 10 µM and 10 mM, respectively. The lysate was added to the pre-equilibrated resin and protein binding was performed by incubation on an orbital shaker for one hour at 4 • C. The suspension was added to a 9 cm polypropylene conical column (Poly-Prep columns; BioRad) and protein purification was performed by gravity flow with an imidazole gradient consisting of three washing steps (2 × 5 mL of Lysis buffer, 20 mM imidazole; 2 × 5 mL of Lysis buffer, 50 mM imidazole; and 1 × 2.5 mL Lysis buffer, 75 mM imidazole) and an elution step with 5 × 500 µL of Lysis buffer, 250 mM imidazole. Protein eluted fractions were assessed by SDS-PAGE and Coomassie staining. Size exclusion chromatography was performed on the pooled protein eluted fractions, using an AKTA prime plus system (GE Healthcare) equipped with a UV detector (280 nm), a HiLoad 16/60, Superdex 200 prep grade column (GE Healthcare) and SEC buffer (25 mM potassium phosphate buffer, 150 mM NaCl, pH 7.5). The fractions corresponding to the wt p53DBD monomers were collected and treated with thrombin (8 units of thrombin/mg wt p53DBD). The thrombin cleavage was conducted for 18 h at rt at pH 7.4 in the assay buffer recommended by the supplier. Before and after digestion protein concentration was determined through Bradford method.

Differential Scanning Fluorimetry (DSF)
Thermostability of wt p53DBD core domain assessed by differential scanning fluorimetry (DSF). An amount of 5 µg of thrombin-cleaved wt p53DBD was incubated with testing compounds (2.5 mM) in 25 mM potassium phosphate buffer and 150 mM NaCl, pH 7.5 in a final volume of 50 µL containing 2 µL of 1 mM DTT or 1 mM TCEP to promote p53 core domain redox control, and Sypro Orange at 2.5x final concentration. The fluorescence was assessed on a C1000 Touch thermal cycler equipped with a CFX96 optical reaction module (BioRad) and the FRET channel (λexc 450-490 nm and λem 560-580 nm). Temperature was ramped from 20 to 90 • C with a rate of 0.2 • C/12 sec. The T m values were calculated using Origin 8 Software. Control assays included: (1) wt p53DBD in SEC buffer, in the absence of the compounds; (2) wt p53DBD in SEC buffer and in presence of DMSO 5%; (3) wt p53DBD in SEC buffer an in the presence of 1mM DTT or TCEP; (4) blank assays (testing compounds in absence of wt p53DBD and in the absence and presence of 1mM DTT and TCEP). In all assays 5% of DMSO was included. The compound MQ was used as a positive control according to the assay conditions described in the literature [45].

Conclusions
In this work, the pharmacokinetic profile of hit compounds SLMP53-1 (6) and SLMP53-2 (7) was studied. Moreover, a biomimetic synthesis of the SLMP53-1 phase I metabolites, identified in HLM incubations, was performed using a nonheme-containing iron(II) complex for the selective oxidation of the indole moiety. Whereas this class of catalysts was reported to promote oxidation of a wide range of chemically diverse substrates [30], this constitutes the first report on their efficient oxidation of indole rings and on their ability to mimic CYP-promoted indole ring metabolic degradation. Furthermore, based on the metabolic liabilities identified in SLMP53-1 (6), 16 enantiopure tryptophanol-derived isoindolinones were prepared. The antiproliferative activity of these compounds was evaluated in HCT116 cell lines with and without p53. Compared with hit compound SLMP53-2 (7), derivative 13d was 2-fold more active in HCT116 p53 +/+ cells, while derivative 13k was 2-fold selective for the p53 pathway. PK experiments showed that insertion of a bromine at the C2 indole minimizes the formation of catabolic products of C-2 oxidation and indole ring opening, which were shown to be associated with decreased activity. However, a metabolic switch towards the formation of potential phenolic metabolites at the indole moiety was observed. While the toxicology/activity impacts of these phenolic metabolites should be evaluated before more improvements in this scaffold, the C2-bromine per se did not constitute a metabolic lability, being inert towards the insertion of bionucleophiles. Whereas potential bioactivation pathways were identified following Phase I metabolism of these halogen-enriched compounds, 13k was suggested to be less prone to this type of biotransformation. Finally, DSF experiments showed that lead compound 13d interacts directly with wt-p53DBD promoting protein p53 thermostability, increasing wt-p53DBD melting temperature with ∆Tm 10.4 • C. Together, this study highlights the potential of the tryptophanol-derived isoindolinone scaffold as p53 activators in CRC.