Chiral hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles: the search for selective p53-activating agents for colorectal cancer therapy

MANIO is an efficient p53-activating anticancer agent with remarkable selectivity to the p53 pathway and promising antitumor activity against colorectal cancer (CRC). Herein, a library of novel MANIO derivatives, including hydroxymethyl- and bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles, was synthesized by rational structural modulation. The antiproliferative activity of twenty derivatives was evaluated in a panel of human CRC cells with different p53 status. From this library, five compounds with R- and S-configuration and with aromatic or heteroaromatic groups at position 3, including the enantiomer of MANIO, were identified as selective towards p53-expressing cancer cells. On the other hand, two compounds with S-configuration, 6-hydroxymethyl- and 7-hydroxymethyl-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazoles, showed high cytotoxicity against WTp53-expressing HCT116 colon cells but, unlike MANIO, exhibited p53-independent inhibitory activity in CRC. The results described provide relevant structural and pharmacophoric data for the design of new p53-activating agents for precision therapy of CRC or other p53-related cancers harboring both wild-type or mutated p53 forms.


Introduction
Cancer is currently a major public health concern, with both incidence and mortality rates expected to increase in the coming decades. 1,2Among the various types of cancer, colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related deaths. 3,4The high mortality rates, often associated with treatment resistance and metastasization, highlight the limited effectiveness of existing therapies in treating CRC. 4 Progress in understanding the development of CRC has revealed that p53 dysfunction is a key event in both localized and advanced cases of CRC.Impairment of the p53 pathway, either by TP53 mutation or inhibition of p53 by its negative regulators, is a significant event in both localized and advanced CRC.Thus, restoration of p53 activity has emerged as one of the most attractive anticancer therapeutic strategies.
Recently, our research team disclosed a new p53-activating anticancer drug, (3S)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (MANIO) (Fig. 1). 5 MANIO represents a privileged anticancer drug compared to other currently available p53-activating agents regarding its antitumor activity in several cancer types, including CRC.MANIO showed a remarkable selectivity to the p53 pathway, activating wild-type (WT)p53 and restoring WT-like function to mutant (mut)p53 in human cancer cells.The half maximal inhibitory concentration (IC 50 ) value of MANIO in WTp53-expressing HCT116 colon cells, HCT116 p53 +/+ (0.97 μM), was approximately 50-fold lower than that obtained in HCT116 p53 −/− cells (48.25 μM), indicating a significant p53dependent growth inhibitory effect.MANIO has been shown to directly interact with the p53 DNA-binding domain (DBD), leading to activation of the p53 pathway.In fact, MANIO works as a bridging molecule between p53 and DNA.Ramos et al. have demonstrated that MANIO binds to a pocket formed between one dimer of the WTp53 DBD protein and the minor groove of the DNA molecule. 5ANIO interacts with the WTp53 DBD protein backbone through hydrogen bonds between its hydroxyl groups and the amide backbone groups of methionine 243 of chain A (M243A) and methionine 243 of chain B (M243B) (each belong to a different monomer).The remaining interactions of MANIO are stacking interactions made with the bases of DNA (DG8, DG12).In the specific case of mutp53 R248W, a highly frequent p53 mutation, in most clusters, the ligand makes a hydrogen bond between one of its hydroxyl oxygen atoms and the W248 side-chain nitrogen.The ligand phenyl group is close to the DNA bases adenine 6 and thymine 7.These interactions may eventually compensate for the loss of direct contacts between lysine 248 residue and the DNA.The latter interaction results in increased p53 stability, enhanced DNA-binding capacity and increased transcriptional activity.MANIO synergizes with conventional chemotherapeutics (e.g., doxorubicin (DOXO), cisplatin (CISP) and 5-fluorouracil (5-FU)) in patient-derived and immortalized CRC cells expressing WTp53 or mutp53.In addition, MANIO demonstrated in vivo p53-dependent antitumor activity in xenograft mouse models of CRC with no adverse side effects.It also demonstrated favorable druglikeness and pharmacokinetic (PK) properties for a clinical candidate. 5n this study, structural modulation of MANIO, a lead p53activating anticancer molecule, was performed with two main goals: to synthesize new bioactive 1H,3H-pyrrolo [1,2-c]  thiazoles and to gain in-depth knowledge of structure-activity relationships (SAR).

Synthesis of new MANIO-like derivatives
The structural modulation strategy focused on structural changes of MANIO at position 3 of the 1H,3H-pyrrolo [1,2-c]  thiazole core, namely the introduction of new aromatic, heteroaromatic, and alkyl substituents and the modification of the absolute configuration.These changes included the introduction of p-fluorophenyl, p-(trifluoromethoxy)phenyl, p-methoxyphenyl, bulky naphthyl and quinolinyl groups, and alkyl groups such as benzyl and methyl.Modifications in other positions of the bicyclic system involved the removal of one hydroxymethyl group or the oxidation of the sulphur atom.
Chiral 1H,3H-pyrrolo[1,2-c]thiazoles with R-configuration 3 were synthesized in moderate yields (35-69%) according to a known synthetic procedure (Scheme 1). 6Thiazolidines 2 were obtained from the reaction of L-cysteine with the corresponding aromatic aldehydes as mixtures of 2R,4R-and 2S,4R-diastereoisomers.The synthetic sequence proceeds via 1,3-dipolar cycloaddition of dimethyl acetylenedicarboxylate (DMAD) with the bicyclic münchnone generated in situ from thiazolidine 2, followed by elimination of carbon dioxide.][9] Thiazolidine 2 was heated in a solution of acetic anhydride in the presence of the dipolarophile.Under these reaction conditions the N-acylation occurs in situ giving selectively N-acetyl thiazolidine-4-carboxylic acids with 2R,4Rconfiguration.Thus, starting from thiazolidine-4-carboxylic acids 2 as a mixture of 2R,4R-and 2S,4R-diastereoisomers, chiral 1H-pyrrolo[1,2-c]thiazoles 3 were obtained as single enantiomer with R-configuration.In this process the chirality at C-4 of the thiazolidine is lost and the chirality at C-2 (C-3 in the product) is retained.The reduction of 3 was carried out with lithium aluminium hydride to afford the target 6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles 4 in moderate to good yields (34-75% for alcohols 4a, 4d and 4e).The formation of alcohols 4b and 4c bearing an OCF 3 or CF 3 group in the para position of the phenyl group was confirmed by proton NMR spectroscopy.However, we decided not to proceed with the biological evaluation studies due to the lack of stability of these compounds.
The sulfone functional group is known to impart polarity to molecules, reducing their lipophilicity and improving their solubility in aqueous media, and the cyclic sulfone nucleus in particular is commonly found in biologically active compounds. 12Thus, the sulfone of the enantiomer of MANIO was prepared starting from dimethyl (3R)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole-6,7-dicarboxylate (13), whose synthesis was previously described by our group. 13In this case, the reduction was carried out with a sodium borohydride-methanol system instead of LiAlH 4 , as the latter was found to reduce both the ester and the sulfone groups.The reaction of sulfone 13 with the NaBH 4 -methanol system in refluxing THF for 24 h allowed the synthesis of the target sulfone 15 in 52% yield, which was obtained together with the mono-reduced derivative, compound 14, in 31% yield (Scheme 4).Attempts to favor the exclusive formation of

Antiproliferative effect on CRC cells of MANIO-like derivatives
A small library of hydroxymethyl-and bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles and a bis(hydroxymethyl)-1H,3H-pyrazolo[1,5-c]thiazole were screened for p53dependent anticancer activity.These included 1H,3Hpyrrolo[1,2-c]thiazoles, the synthesis of which has been described above, as well as hydroxymethyl-and bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles 18 and 6,7-bis(hydroxymethyl)-1H,3H-pyrazolo[1,5-c]thiazole 19 previously described by our research group (Fig. 2). 10,11,16n vitro studies regarding the antiproliferative activity of compounds 18 and 19 have been carried out in a panel of human colorectal cancer cells with different p53 status, WTp53-expressing HCT116 colon cells (HCT116 p53 +/+ ) and p53 null isogenic derivative (HCT116 p53 −/− ).The comparison of the activity of the compounds was made by analyzing the corresponding IC 50 values (Table 1).1H,3H-Pyrrolo[1,2-c] thiazole 18a, the enantiomer of MANIO, was characterized by its high selectivity for HCT116 colon cells expressing p53 HCT116 p53 +/+ with IC 50 = 5.11 μM, whereas the IC 50 value obtained for the p53 null isogenic derivative (HCT116 p53 −/− ) was higher than 50 μM.Nevertheless, the IC 50 value for 18a against HCT116 p53 +/+ was 5-fold higher than that obtained for MANIO in the same cells.Compound 18b, containing a hydroxymethyl group at C-6, and 6,7-bis(hydroxymethyl) derivative 18g, containing a p-methoxyphenyl group at position 3, both with R-configuration, also showed a marked selectivity to p53-expressing cancer cells, with IC 50 values of 6.68 and 4.61 μM, respectively, compared to p53-null cells (>50 μM).Among the other compounds tested, none of them showed relevant selectivity for the WTp53-expressing HCT116 colon cells.Compound 18c, which differs from 18b only in the position of the hydroxymethyl group, shows a marked   10,11,16 loss of selectivity for HCT116 colon cells expressing p53 HCT116 p53 +/+ .A similar loss of selectivity was observed for compound 18d, which differs from 18b only in having no substituents at C-3. Compound 18e, without substituents at the 5-position, showed a loss of selectivity of the same order of magnitude compared to compound 18a.Although the introduction of a p-methoxyphenyl group at position 3 resulted in good performance, as observed for compound 18g, the introduction of a bulkier trimethoxyphenyl group (compound 18f) resulted in a complete loss of activity against HCT116 colon cells.On the other hand, compound 18h, with a p-hydroxyphenyl group at C-3, showed lower selectivity and an IC 50 against HCT116 p53 +/+ 3-fold higher than that observed for 18a.Finally, compound 19 with a 1H,3Hpyrazolo[1,5-c]thiazole core showed no activity against HCT116 colon cells.
SAR data allowed the identification of potential pharmacophores in the molecule and the definition of new MANIO-like derivatives to be studied (Fig. 3).It has been shown that the presence of an aryl group at C-3, a methyl group at C-5 and the presence of an hydroxymethyl group at position 6 may be crucial to ensure good antiproliferative activity in HCT116 colon cells expressing WTp53.Thus, the design of new 1H,3H-pyrazolo[1,5-c]thiazoles involved the structural modulation at position 3 through the introduction of fluorine containing aromatic substituents, naphthyl or quinolinyl groups.Among the new synthesized compounds, enantiomers of the most promising compounds studied (compounds 18b and 18g) were also prepared, as well as the sulfone of the enantiomer of MANIO.To confirm the importance of an aryl group at C-3 in ensuring good anticancer activity, the replacement of this type of functional group by alkyl groups was also carried out, resulting in novel 3-alkylated-6,7-bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c] thiazole derivatives.
3-Alkyl-1H,3H-pyrrolo[1,2-c]thiazoles 17 showed moderate p53 dependence, with IC 50 values in HCT116 p53 +/+ (16-33 μM) only about 10 μM lower than those in p53 null isogenic cells (29-46 μM).As mentioned above, MANIO interacts with the WTp53 DBD protein backbone through hydrogen bonds between its hydroxyl groups and the amide backbone groups of the methionine residues of two different monomers, and through stacking interactions made with the bases of the DNA. 5 This is in agreement with the results obtained for the alkylated derivatives, in particular for compounds 17b and

RSC Medicinal Chemistry Research Article
17c, where these aromatic stacking interactions are not possible.

Antiproliferative effect on MDA-MB-231, PANC-1 and A375 cells of MANIO-like derivatives
The antiproliferative activity of the most active compounds against HCT116 colon cells (HCT116 p53 +/+ ) was then evaluated in a panel of different human cancer cell lines, triple-negative breast cancer (MDA-MB-231), pancreatic adenocarcinoma (PANC-1) and melanoma (A375) (Table 3).However, none of the compounds showed relevant activity in any of the cell lines tested.Of note is the moderate activity of MANIO, its enantiomer and compound 12 in the human melanoma cell line (IC 50 < 15 μM).

Conclusion
Structural modulation of the lead compound MANIO was performed resulting in the synthesis of thirteen new 1H,3Hpyrrolo[1,2-c]thiazoles with R and S configuration.The antiproliferative activity of a library of twenty compounds was assessed in a panel of human colorectal cancer cells with different p53 status, providing relevant structural and pharmacophoric information.
Mono-hydroxymethyl derivatives with S-configuration stood out for their high antiproliferative activity in colon cancer cells expressing WTp53, with IC 50 < 1 μM, higher activity than the corresponding enantiomers and MANIO, but no p53-dependent growth-inhibitory effect.
Collectively, these results indicate that the presence of aromatic substituents at C-3 and a methyl group at C-5 are essential to ensure effective p53-dependent growth inhibitory activity.Furthermore, the absolute configuration at the chiral center was shown to play an important role in the antiproliferative activity in WTp53-expressing colon cancer cells.It was also observed that the position of the hydroxymethyl group in mono-hydroxymethyl derivatives affects also the inhibitory activity.Interestingly, while for 1H,3H-pyrrolo[1,2-c]thiazoles with R configuration the 6-hydroxymethyl derivative was significantly more active than the 7-hydroxymethyl derivative, in the case of 1H,3Hpyrrolo[1,2-c]thiazoles with S configuration, both 6-hydroxymethyl and 7-hydroxymethyl derivative show outstanding antiproliferative activity in WTp53-expressing HCT116 colon cells, but p53-independent inhibitory activity.Overall, the present work represents a successful strategy of modulation and rational structural optimization of a lead molecule, which allowed to gather pertinent structural and pharmacophoric data for the design of new p53-activating agents for precision therapy of CRC or other cancers harboring WT or mutp53.

Experimental
Chemistry Thin-layer chromatography (TLC) analyses were performed using precoated silica gel plates.Column chromatography was performed with silica gel 60 as the stationary phase. 1 H Nuclear magnetic resonance (NMR) spectra (400 MHz), 13 C NMR spectra (100 MHz) and 19 F spectra (376 MHz) were recorded in CDCl3, CD 3 OD or hexadeuterated dimethylsulfoxide (DMSO-d 6 ).Chemical shifts are expressed in parts per million (ppm) relatively to internal tetramethylsilane (TMS), and coupling constants ( J) are in hertz.Infrared (IR) spectra were recorded in a Fourier transform spectrometer using either a KBr matrix or diamond attenuated total reflectance (ATR) mode.Elemental analyses were carried out with an Elemental Vario Micro Cube analyser.High-resolution mass spectra (HRMS) were obtained on a TOF VG Autospect M spectrometer with electron impact (EI), on a Thermo Orbitrap Q-Exactive Focus spectrometer with electrospray ionization (ESI) or on a Bruker MicroTOF (APCI-FIA-TOF).Melting points were determined in open glass capillaries and are uncorrected.Optical rotations were measured on an Optical Activity AA-5 electrical polarimeter.
General procedure for the synthesis of 1,3-thiazolidine-4carboxylic acids 2. A solution of the corresponding aldehyde (29.0 mmol) in ethanol (22 mL) was added to a solution of L-cysteine (3.51 g, 29.0 mmol) in water (22 mL).The reaction was stirred for 5 h, the product was filtered and washed with diethyl ether.
General procedure for the synthesis of hydroxymethyl-1H,3H-pyrrolo[1,2-c]thiazoles.A solution containing the appropriate 1H,3H-pyrrolo[1,2-c]thiazole (2.30 mmol) in dry dichloromethane (30 mL) was added dropwise to a suspension of lithium aluminium hydride (2.2 equiv., 192 mg, 5.06 mmol) in anhydrous diethyl ether at 0 °C, unless otherwise stated.After completing the addition, the reaction mixture was refluxed under nitrogen for 1.5 h and then cooled on an ice bath.The excess of hydride was carefully decomposed by addition of ethyl acetate followed by slow addition of water (0.2 mL), NaOH 15% (0.2 mL) and water (0.6 mL).The mixture was filtered through celite and the inorganic residue was washed with several portions of hot dichloromethane.The filtrate was dried (Na 2 SO 4 ) and the solvent evaporated off.The crude product was purified by column chromatography [hexane-ethyl acetate] or recrystallisation and stored under nitrogen at −18 °C.
Sulforhodamine B (SRB) assay.5 × 10 3 cells per well were seeded in 96-well plates for 24 h and then treated with serial dilutions of the appropriate compound ranging from 0.2 to 50 μM and incubated for 48 h.IC 50 values for each cell line were determined by the sulforhodamine B (SRB) assay, as previously described. 20