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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2010 April 9.
Published in final edited form as:
PMCID: PMC2665923
NIHMSID: NIHMS103387

From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3β Inhibitors that Suppress Proliferation and Survival of Pancreatic Cancer Cells

Abstract

Recent studies have demonstrated that Glycogen Synthase Kinase 3β (GSK-3β) is overexpressed in human colon and pancreatic carcinomas contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3β inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3β and an enhanced selectivity against Cyclin-dependent Kinase 2 (CDK-2). Selected GSK-3β inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20 and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3β inhibitors 5 and 26 resulted in suppression of GSK-3β activity and a distinct decrease of the X-linked Inhibitor of Apoptosis (XIAP) expression leading to significant apoptosis. The present data suggest a possible role for GSK-3β inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.

Introduction

Glycogen Synthase Kinase 3 (GSK-3a) was identified in the late 1970s and originally found to regulate glycogen metabolism.1 Later, this enzyme has attracted immense interest due to its diverse roles in cellular events. It is well established that GSK-3 affects a variety of biological processes such as cell cycle progression, proliferation, apoptosis, signaling, and transcription by phosphorylation of many different substrates. In mammals, GSK-3 consists of two distinct isoforms, α and β,2 which are highly homologous within their ATP binding domain, whereas they are significantly different in their terminal regions. This serine/threonine kinase is constitutively active in resting cells and physiologically inhibited by distinct signaling pathways at multiple levels in stimulated cells. GSK-3β is negatively regulated, for example, by post-translational phosphorylation of Ser-9 located in the N-terminal domain.3 GSK-3β is also phosphorylated constitutively at Tyr-216; this phosphorylation step appears to take place through autophosphorylation and plays a role in stabilizing the enzyme. In addition, there are different extracellular stimuli and proteins that can regulate this enzyme. For example, GSK-3β is inhibited by the presence of secreted glycoproteins, the so-called Wnts, that function in a pathway is crucial for the determination of the cell's fate during embryonic development.4 Wnt signaling targets a particular subcellular pool of GSK-3β, in which it is complexed to axin, β-catenin, and the adenomatous polyposis coli (APC) proteins. This protein complex is disrupted during Wnt signaling as a result of the displacement of axin by other proteins, such as disheveled and FRAT (frequently rearranged during advanced T-cell lymphomas, also called GBP (GSK-3-binding protein)). The resulting dephosphorylation of β-catenin leads to its accumulation and translocation to the nucleus where it forms a transcriptional complex with Tcf and Lef family proteins subsequently stimulating activation of several oncogenes. In earlier publications, it was indicated that GSK-3β plays the role of tumor suppressor by down-regulating various proto-oncoproteins.5-7 Consequently, GSK-3β inhibitors have been considered to possibly mimic the Wnt signaling pathway and to be potentially oncogenic.3 However, in spite of these concerns, it is well known that long-term use of lithium, a non-specific GSK-3β inhibitor, for the treatment of bipolar disorder is not associated with an increased risk of cancer.8 Moreover, lithium actually increases survival rates of patients with adenocarcinomas.9 The administration of the GSK-3β inhibitor 6-{2-[4-(2,4-Dichloro-phenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino]-ethylamino}-nicotinonitrile (CHIR 99021)10 in Zucker Diabetic Fatty (ZDF) rats for up to 20 h was found not to cause an observable increase in β-catenin or cyclin D1 mRNA in brain, liver, lung, adipose tissue, or colon. Thus, it is possible that the inhibition of GSK-3β by itself may be unable to elevate β-catenin levels in primary cells; such elevations may require that the cell lines have already undergone some prior transforming events such as APC protein truncation.11 Recently, a line of evidence led to an indication that GSK-3β is pathologically active in different types of gastrointestinal cancer.12,13 It was shown that this enzyme is overexpressed in colon and pancreatic cancer cells where it has been implicated in NFκB (nuclear factor kappa B) activation.14,15 In fact, GSK-3β is found to accumulate in the nucleus of cancer cells and has recently been shown to regulate chromatin structure and the binding of NFκB to its target gene promoters in chronic lymphocytic leukemia cells.16 Importantly, inhibition of GSK-3β activity in several cancer cell types results in diminished NFκB transcriptional activity of anti-apoptotic genes resulting in apoptosis.15,16 Thus, GSK-3β has emerged as a promising target in development of new drugs for the treatment of chronic and progressive diseases.3,17 Over the past decades, a number of small molecule GSK-3 inhibitors have been designed from natural products or known kinase inhibitors17 for diabetes and neurodegenerative disorders, but only a few of these were tested in cancer cell lines. In the present work we have directed our attention to the possibility to use inhibitors of GSK-3β in the treatment of pancreatic cancer. Our work in this area was influenced by the maleimide-bearing natural product staurosporine first identified as protein kinase C (PKC) inhibitor, although now known to be able to target other protein kinases, including GSK-3β. From our original screening studies of staurosporine and its analogs, we were led to the design of benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3β inhibitors.18 In continuation of this work, and to gain better insights into the SAR of this benzofuran containing scaffold, a small library of substituted maleimides has been generated. Using structure-based design methods, we have optimized our lead compounds so as to arrive at subnanomolar potency GSK-3β inhibitors that are relatively selective for GSK-3β versus homologous kinases.

Results and Discussion

Chemical Synthesis

The synthesis of the benzofuran-3-yl-(indol-3-yl)maleimides (1-38) (Table 1) is straightforward and based on the condensation of the appropriately substituted 3-indolylglyoxylic acid esters and benzofuranyl-3-acetamides. The general method is shown in Scheme 1 and follows our previously published work.18 Preparation of indolyl-based glyoxalates commences with N-alkylation of the indole 39 followed by acylation of the resulting indole 40 with ethyl oxalyl chloride to afford the precursor 41. The required benzofuranyl-3-acetamides 44 were prepared from the appropriately substituted 3-benzofuranones 42. A Horner-Emmons reaction of 42 with (carbethoxymethylene)triphenylphosphorane afforded ethyl (1-benzofuran-3-yl)acetates 43, which was subsequently converted into the acetamides 44. Condensation of the acetamides 44 with the glyoxilic esters resulted in formation of maleimide core.

Scheme 1
Reagents and conditions: (a) MeI, NaH, DMF, (b) EtO2CCOCl, Et2O; (c) Ph3P=CH2CO2Et, toluene, 110 °C; (d) NH3/MeOH; (e) t-BuOK, THF.
Table 1
GSK-3 β Inhibition by Substituted Benzofuran-3-yl-(indol-3-yl)maleimides and Compounds 77, 78.

As we have already synthesized a small set of compounds substituted in the 5-position,18 it was important to explore the extent to which the introduction of different functionalities, including H-bond donor and acceptor groups to the 6- or 7-position of the indole or benzofuran ring, could be used to adjust kinase selectivity and potency. Based upon the above considerations, we investigated the effects of adding a hydroxy, alkoxy, hydroxymethyl, methoxymethyl and a carboxylic acid group. Accordingly, commercially available 6-hydroxybenzofuran-3-one 45 was protected as its silyl ether 46 (Scheme 2). Treatment of the 46 with (carbethoxymethylene)triphenylphosphorane and subsequent amidation of the intermediate ester provided the acetamide 47. The alcohol functionality was protected as its tert-butyldiphenylsilyl ether (compound 48), or alkylated with different alkyl and aryl halides (compounds 49-54). Finally, condensation with the appropriate indolylglyoxylic acid esters followed by deprotection of the silyl ether if needed gave a variety of 6-hydroxy substituted maleimides (4, 9, 10, 16-18) as shown in Table 1.

Scheme 2
Reagents and conditions: (a) TBDPSCl, Imidazole, DMF-CH2Cl2; (b) Ph3P=CH2CO2Et, toluene, 110 °C; (c) NH3/MeOH; (d) TBDPSCl, Imidazole, DMF-CH2Cl2; (e) CeCO3, Na2CO3, RHal, DMF, 55 °C.

For the introduction of hydroxymethyl and methoxymethyl functionality at the 6-position of the benzofuran moiety, the 3-methoxybenzyl alcohol 55 was converted into the acetate 56 (Scheme 3). Chloroacetylation of 56 and subsequent cyclization of the corresponding aryl ketone 57 yielded the 6-substituted 3-benzofuranone 58. The general procedure was followed to obtain the 6-hydroxymethylbenzofuranyl-3-acetamide 59. The acetamide 59 was converted in turn to the methyl ether 60, or silyl ether 61 and then to the maleimides 2, 3, 7, 15, 23, 32 and 34 (Table 1).

Scheme 3
Reagents and conditions: (a) Ac2O, Et3N, DMAP, CH2Cl2; (b) ClCH2COCl, AlCl3, CH2Cl2, reflux; (c) NaOAc, MeOH; (d) Ph3P=CH2CO2Et, toluene, 110 °C; (e) NH3/MeOH; (f) NaH, MeI, THF, 20 °C; (g) TBDPSCl, Imidazole, DMF-CH2Cl2.

Next, our effort was directed to the introduction of hydroxymethyl, methoxymethyl, and carboxylic acid group at the 7-position of the indole moiety (Scheme 4). Accordingly, the (1H-indol-7-yl)methanol 62 was used to synthesize glyoxalates 63 and 64. The general procedure to form the maleimide was followed to obtain the desired compound 31 and 33. The oxidation of the alcohol 33 led to the corresponding aldehyde 65, which was then subjected to a Horner-Emmons reaction to give the ester 66 (Scheme 5). Treatment with nickel boride, which was generated in situ, resulted in the selective reduction of the double bond and formation of the ester 35. The latter was converted into the acid 36 by the treatment with sodium hydroxide.

Scheme 4
Reagents and conditions: (a) MeI, NaH, DMF; (b) EtO2CCOCl, Et2O; (c) TBDPSCl, Imidazole, DMF-CH2Cl2; (d) t-BuOK, THF; (e) TBAF, THF; (f) PDC, THF; (g) Ph3P=CH2CO2Et, THF, reflux; (h) NiCl2·6H2O, NaBH4, THF-MeOH; (i) 1M NaOH, EtOH.
Scheme 5
Reagents and conditions: (a) DMF-DMA, 125 °C; (b) DMF-DIPA, 125 °C; (c) Fe, AcOH, EtOH; (d) Pd(PPh3)4, 4-chloro-phenylboronic acid, K2CO3, DME/H2O, 85 °C.

The SAR work was further extended by exploring the effect of poly-substitution of the aromatic rings. Disubstituted indoles were prepared from the corresponding dihalogenated nitrotoluenes 67 and 68 by using a known protocol (Scheme 5).19 The reaction of 67 and 68 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) afforded a mixture of the enamine 69 with the methoxy-substituted side-product 70. In this step, nucleophilic aromatic substitution can be avoided by replacement of DMFDMA with N,N-dimethylformamide di-iso-propyl acetal (DMF-DIPA).20 A reductive cyclization provided the indoles 71 and 72.21 A variety of acetamides prepared from commercially available or made in-house benzofuranones were used in a coupling reaction with oxalates 73-76 to prepare the maleimides 5-11. To attach an aromatic ring to the indole, a Suzuki coupling reaction of the appropriate precursor, such as 5-fluoro-6-iodo-indole (5) with p-chlorophenyl boronic acid was employed to afford the final maleimide 12. In addition, 5,7-dibromo, 5,6-methylenedioxy, and 1H-benzo[g]indole were used to synthesize compounds 20, 26, and 38, respectively.

Evaluation of the Maleimides to in vitro Inhibit GSK-3β

The new benzofuran-3-yl-(indol-3-yl)maleimides were screened for their potency to inhibit GSK-3β. Commercially available human GSK-3β was assayed for its ability to phosphorylate the primed peptide substrate (RRRPASVPPSPSLSRHSS(P)HQRR; 10 μM) in the presence of 0-30 μM of the maleimides. For comparison purposes, we also determined the IC50 values of the known GSK-3β inhibitor 77 (ARA014418)22 and 78 (SB-216763)23 in the same assay. As presented in Table 1, the IC50 values vary from poor to excellent (less than 1 nM).

Many GSK-3β inhibitors are also quite potent toward CDK-2 since the ATP binding pockets of these two kinases are very similar.24 To measure the selectivity of new maleimides several compounds with different structural features were tested against CDK-2/CyclinE. The compounds 2, 7, 15 and 33 were found to be selective toward GSK-3β, based on the ratios of IC50s found for these two kinases (Table 2). From this set, compounds 7, 11, 15 and 33 were selected for more extensive evaluation against a panel of CDKs, and these exhibited a significant level of selectivity (Table 1 in the Supporting information).

Table 2
CDK-2/CyclinE Inhibition by Substituted Maleimides.[a]

SAR Analysis and Molecular Modeling Studies

Our SAR studies revealed that the introduction of hydrophilic substituents at position 6 or 7 of the indole part of our core scaffold is favorable. Indeed, compounds 29, and 31-36 demonstrated very good GSK-3β inhibitory activity in an in vitro assay. Compound 33, bearing a methoxymethyl group, was found to be the best inhibitor with an IC50 0.23 ± 0.04 nM. Previously, we discovered that the presence of a halogen in the 5-position of the indole is favorable.18 This finding was confirmed by the remarkably high activities of compounds 2-4, and 13-15. On the other hand, the introduction of an additional halogen led to a reduction in the inhibitory activity of the corresponding compounds. Thus, almost all of the 5,6 and 5,7 disubstituted indoles (compounds 6, 8-11, and 20) were found to be less potent than their monosubstituted analogs. The same effect occurred with 5-methoxy-substituted indolylmaleimides. The introduction of either Cl or I in the 6-position resulted in a ca. 2 (compound 28) or 3.5 (compound 27) fold of decrease in activity relative to the monosubstituted 5-methoxyindolylmaleimide (IC50 125 nM).18 It should be noted that an even more significant drop in activity was observed for compound 12 (IC50 > 7μM) which contains a larger p-chlorophenyl substituent in the 6-position. The SAR of the benzofuran component is as follows: the hydroxymethyl group attached to the position 6 dramatically improved the potency of this scaffold, thus the 6-hydroxymethylbenzofuranyl analogues 2, 7 and 15 were found to be the most potent among all 6-substituted benzofuranylmaleimides. This improvement is especially remarkable in the polysubstituted series as can be seen by comparing maleimides 6 and 7. Also, the maleimide 2 was 10-fold more potent than its 6-hydroxysubstituted analogue 4. In contrast, the potency was significantly lower for compounds having methoxy (compound 8, IC50 870 nM), cyclopropylmethoxy (compound 9, IC50 > 1 μM), or cyclobutylmethoxy (compound 10, IC50 > 4 μM) attachments. Another observation related to the size and nature of the benzofuran counterpart substitution is that the incorporation of side chains with terminal double (compound 17, IC50 48.3 nM) or triple bonds (compound 16, IC50 25.3 nM) generated compounds with comparable activities, while the p-methoxybenzyl derivative 18 turned out to be ca. 10-fold less potent. Compound 34 with a methoxymethyl and a hydroxymethyl groups attached to the indole and benzofuran rings, respectively, that was synthesized to further improve the potency of the maleimide-containing scaffold showed IC50 at 0.73 ± 0.10 nM. It was even slightly less potent than 33 (IC50 0.23 ± 0.04 nM).

In addition, the hydroxymethyl substituent at the 6-position of the benzofuran moiety provided an enhanced selectivity against CDK-2 as can be seen upon comparing the pairs of compounds, 15 and 14, or 7 and 11 (Table 2). By contrast, the same group attached to the 7-position of the indolyl counterpart caused a considerable drop in selectivity (compounds 31 and 32).

To ascertain whether molecular modeling could possibly explain the observed SAR results, compounds 2 and 31 were docked into the ATP binding site of GSK-3β (1R0E) (Figure 1(A-D)).25 Their binding poses were governed largely by two adjacent intermolecular hydrogen bonds: the NH group of the maleimide ring forms a hydrogen bond with the backbone carbonyl oxygen of Asp133 and one of the carbonyl oxygen atoms of the maleimide forms a hydrogen bond with the backbone NH of Val135. In addition to these main interactions, the 7-hydroxymethyl group of compound 31 appears to be within hydrogen bonding distance of the side chain of Arg141 (IC50 = 5.4 nM), whereas the 6-hydroxymethyl group of compound 2 is within hydrogen bonding distance of the side chains of Gln185, Asn186, and Asp200 (IC50 = 0.35 nM). In the case of compound 33 having a 7-methoxymethyl group at the 7-position of the indole ring, its IC50 of 0.23 nM was better than that found for 31. While both the hydroxymethyl and methoxymethyl groups at the 7-position of the indole ring are able to H-bond with the nearby Arg residue, it is only the methoxymethyl group (as in 33) that can help drive the inhibitor into its binding site, whereas the hydroxymethyl group of 31 may be less favorably oriented as it points into the open area where it is accessible to solvent molecules. The modeling studies will find more solid ground once we complete X-ray co-crystal structures of some of these inhibitors bound to human GSK-3β.

Figure 1Figure 1
(A, B) Views of compound 31 docked into the GSK-3β binding site in which the backbone is shown as ribbons (A) and the protein surface is colored by lipophilic potential (blue = hydrophilic, brown = lipophilic) (B); (C) Docked pose of compound ...

GSK-3 Inhibitors Decrease Proliferation and Survival of Pancreatic Cancer Cells

A select few of the GSK-3β inhibitors that have different structural features and demonstrate varying potencies in the in vitro assay were examined for their capacity to decrease pancreatic cancer cell proliferation and survival. It was shown earlier that treatment of BXPC3, HupT3 and MiaPaCa-2 cells with the known GSK-3β inhibitors 77 and 78, which we used as reference compounds, led to a significant decrease in pancreatic cancer cell proliferation.16 Some of our new compounds, such as 5, 6, 11, 20 and 26 showed activity similar to the reference compounds but exhibited better efficacy (Table 3).

Table 3
Growth Inhibition of Pancreatic Cancer Cells by Selected Maleimides

The two most potent compounds, 5 and 26, and the inactive compound 27 (Table 3) were tested for their ability to inhibit GSK-3β in cell lines. Using Western immunoblotting, we estimated GSK-3β activity by monitoring the level of phosphorylation of glycogen synthase (GS), a substrate of GSK-3β. The two most potent compounds, 5 and 26, effectively inhibited GSK-3β activity as shown by a decrease in GS phosphorylation (Fig. 2A) leading to decreased proliferation and survival of BXPC3 and HupT3 pancreatic cancer cells (Table 3; Fig. 2A, B, D). Compound 27, however, did not affect GSK-3β activity, proliferation or survival of pancreatic cancer cells (Table 3; Fig. 2B, C). Consistent with the idea that GSK-3β positively regulates NFκB-mediated pancreatic cancer cell survival,16 we found that treatment with the GSK-3β inhibitors 5 and 26 resulted in a pronounced decrease in NFκB-mediated expression of XIAP, the most potent antiapoptotic protein, leading to subsequent apoptosis in pancreatic cancer cells (Fig. 2A, B, D). Again, compound 27 had no such effect (Fig. 2B, C) even at concentrations up to 50 μM (Table 3). As expected, compound 5 showed significant anticancer activity at concentrations of 1-5 μM (Table 3). In addition, compound 5 was evaluated against a panel of kinases and found to be selective (Table 2 in Supporting Information).

Figure 2
Treatment with GSK-3β inhibitors induces apoptosis in pancreatic cancer cells. (A) BXPC3 pancreatic cancer cells were treated with GSK-3β inhibitors 5 (5 μM) and 26 (5 μM) for 6, 12, 24, and 36 h as indicated. Whole cell ...

Our results demonstrated that although some of these compounds are potent GSK-3β inhibitors, as shown by the in vitro kinase assay, they did not inhibit GSK-3β activity in cell culture and therefore, showed no anticancer effect in pancreatic cancer cells. The discrepancy regarding the effect in the in vitro kinase assay compared to the cell culture experiments is not surprising. Chemoresistance is intrinsic to cancer. The common reason for resistance to a broad range of drugs is overexpression of permeability-glycoprotein (P-gp) and other drug transporters (ATP-binding cassette) that detect and eject anticancer drugs from cancer cells. This could result in failure to achieve effective intracellular concentrations for some compounds that are lipophilic and should show excellent ability to cross biological membrane. In summary, because the maleimides 13, 21, 22, 25, 27 and 33 (Table 3) showed little or no inhibitory activity in the cell proliferation studies, it is possible that these compounds are being recognized and ejected by energy-dependent transporters or detoxified by a CYP-mediated mechanism in the pancreatic cancer cells, a point worthy of further investigation.

Conclusion

A library of benzofuran-3-yl-(indol-3-yl)maleimides was designed, synthesized, and tested for their selectivity and potency as GSK-3β inhibitors relative to other homologous kinases. Selected compounds were examined in turn for their ability to suppress pancreatic cancer cell proliferation and survival in vitro. We have been able to show that some of these compounds, namely compounds 5, 6, 11, 20 and 26, demonstrate good antiproliferative activity against a panel of pancreatic cancer cell lines, which consisted of MiaPaCa-2, BXPC3, and HupT3. Most notable was our finding that the treatment of BXPC3 with inhibitors 5 and 26 resulted in a distinct decrease in NFκB-mediated expression of XIAP, the most potent antiapoptotic protein, leading to subsequent apoptosis in these pancreatic cancer cells. Because of these valuable results, we plan to prepare a wider variety of GSK-3β inhibitors implementing modifications aimed at improving compound druggability (aqueous solubility and metabolic stability) through both substitutent modifications and alteration in the scaffold in order to arrive at possible clinical candidates for pancreatic cancer therapy.

Experimental Section

Synthesis of benzofuran-3-yl-(indol-3-yl)maleimides

1H NMR and 13C NMR spectra were recorded on Bruker spectrometer at 400 MHz and 100 MHz respectively with TMS as an internal standard. Standard abbreviations indicating multiplicity were used as follows: s = singlet, b.s = broad singlet, d = doublet, t = triplet, q = quadruplet, and m = multiplet. HRMS experiments were performed on LTO-FTICR or Shimadzu IT-TOF Mass Spectrometers. TLC was performed with Merck 250-mm 60F254 silica gel plates. Preparative TLC was performed with Analtech 1000-mm silica gel GF plates. Column chromatography was performed using Merck silica gel (40-60 mesh). Analytical HPLC was carried out on an Ace 3AQ column (100 × 4.6 mm), with a Shimadzu 10 VP Series HPLC with a diode array detector; flow rate = 2.0 mL/min; from 10% acetonitrile in water to 50% in 10 min and to 100% acetonitrile in 5 min with 0.05% TFA (Method A), or from 30% acetonitrile in water to 100% of acetonitrile in 15 min with 0.05% TFA (Method B) or from 30% acetonitrile in water to 100% of acetonitrile in 30 min with 0.05% TFA (Method C), a column Ace AQ5 (250 × 10 mm) was used with this last method. The purity of key target compounds determined by HPLC (methods A, B, or C) was higher than 95% (Table 3 in Supporting Information).

Compounds 1, 13, 19, 24, 29 and 37 were described in our previous publication.18

General procedures for the preparation of maleimides

The following methods represent the typical procedures for the synthesis of the benzofuranylindolylmaleimide-based compounds.

Synthesis of the indole moiety

To a solution of substituted indole (1 eq) in dry DMF (2 mL/mmol) cooled with an ice bath was added NaH (55% suspension in mineral oil, 1.5 eq), followed by MeI (1.2 eq) after which the reaction mixture was allowed to warm to rt. The reaction was monitored by TLC. After completion the reaction mixture was poured into ice-water and the solution was extracted with EtOAc. The organic phase was washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude was purified by column chromatography (from hexane to EtOAchexane; 1:3) or filtered through silica gel (EtOAc-hexane; 1:3) and subjected to further reaction without additional purification. To a solution of substituted 1-methyl-1H-indole in Et2O (5mL/mmol) cooled to 0 °C, ethyl oxalyl chloride (6 eq) was added dropwise. The reaction mixture was stirred for 0.5 h at 0 °C, allowed to warm to rt and stirred until completion. The reaction was quenched by addition of water and diluted with EtOAc. The organic layer was washed with saturated NaHCO3, water and brine, dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography (EtOAc-hexane; 1:3) to give the substituted (1-methyl-1H-indol-3-yl)-oxo-acetic acid ethyl ester.

Formation of the benzofuran counterpart

Benzofuran-3-yl-acetic acid ethyl esters

To a solution of substituted or unsubstituted benzofuran-3-one (1 eq) in toluene (35 mL/mmol) was added (carbethoxymethylene)triphenylphosphorane (4 eq), and the mixture was refluxed overnight or until completion. When needed an additional amount (1 or 2 eq) of (carbethoxymethylene)triphenylphosphorane was added and the mixture was refluxed until completion. The reaction mixture was cooled to rt and concentrated. The residue was purified by column chromatography (hexane to ethyl acetate:hexane; 1:3) to give the product.

2-Benzofuran-3-yl-acetamides

Product obtained from the previous step was added to liquid ammonia at −78 °C, the reaction flask was sealed and heated at 50-60 °C until completion. The reaction mixture was cooled to −78 °C, allowed to reach rt slowly so that the excess of ammonia was evaporated and residue was washed with hexane to afford the substituted or unsubstituted 2-benzofuran-3-yl-acetamide used without any further purification.

Formation of the benzofuran-3-yl-(indol-3-yl)maleimides

To a suspension of substituted or unsubstituted 2-benzofuran-3-yl-acetamide (1 eq) and substituted or unsubstituted indolyl-3-glyoxylate (1 eq) in dry THF (10 mL/mmol) at 0 °C, a 1.0 M solution of tert-BuOK in THF (4eq) was added dropwise, and the reaction mixture was stirred at rt for several hours until completion. The reaction mixture was quenched with water and diluted with EtOAc. The organic phase was washed with brine then dried over Na2SO4, and evaporated in vacuo. When deprotection of the silyl ether was required, the residue was dissolved in THF (20 mL/mmol) and a 1.0 M solution of TBAF in THF (1.5 eq) was added at rt. The mixture was stirred for 2 h. The reaction mixture was quenched with NH4Cl and stirred for 5 min. The solvent was removed and the residue was dissolved in CH2Cl2 and washed twice with water and brine. The organic phase was dried over Na2SO4 and concentrated. The residue was purified by preparative TLC (EtOAc-hexane; 1:1 or MeOH-CH2Cl2; 5:95) or column chromatography (EtOAchexane; 1:9 to 1:1 or MeOH-CH2Cl2; 1:99 to 1:9) to afford product. The yields were in the range of 20 to 72%.

3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxymethyl-benzofuran-3-yl)-pyrrole-2,5-dione (2)

1H NMR (DMSO-d6, 400 MHz) δ 3.88 (s, 3H), 4.52 (d, J = 5.7 Hz, 2H), 5.24 (t, J = 5.7 Hz, 1H), 6.53 (dd, J = 2.3, 10.3 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.95 (dt, J = 2.4, 9.1 Hz, 1H), 7.49 (dd, J = 4.5, 9.5 Hz, 1H), 7.54 (s, 1H), 8.02 (s,1H), 8.24 (s,1H), 11.19 (s,1H); 13C NMR (DMSO-d6, 100 MHz) 33.7, 63.1, 104.27, 104.31, 103.1, 106.3, 109.4, 110.5, 110.8, 111.7, 112.2, 112.3, 121.5, 121.9, 122.9, 124.5, 126.4, 126.5, 132.6, 133.9, 136.2, 140.6, 147.4, 154.9, 156.5, 172.2, 172.5; FAB-HRMS calcd for C22H15FN2O4 [M + H]+: 391.1089; found: 391.1089, calcd for C22H15FN2O4 [M + Na]+: 413.0908; found: 413.0907.

3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-methoxymethyl-benzofuran-3-yl)-pyrrole-2,5-dione (3)

1H NMR (CDCl3, 400 MHz) δ 3.33 (s, 3H), 3.86 (s, 3H), 4.48 (s, 2H), 6.64 (dd, J = 2.2, 9.8 Hz, 1H), 6.81-6.90 (m, 3H), 7.21 (dd, J = 4.5, 8.9 Hz, 1H), 7.39 (brs, 1H), 7.49 (s,1H), 7.79 (s,1H), 8.12 (s,1H); FAB-HRMS calcd for C23H17FN2O4 [M + H]+: 405.12451; found: 405.12427, calcd for C22H15FN2O4 [M + Na]+: 427.1065; found: 427.1061.

3-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxy-benzofuran-3-yl)-pyrrole-2,5-dione (4)

1H NMR (DMSO-d6, 400 MHz) δ 3.88 (s, 3H), 6.39 (dd, J = 2.0, 8.5 Hz, 1H), 6.54 (dd, J = 2.2, 10.5 Hz, 1H), 6.61 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 6.95 (dt, J = 2.0, 8.5 Hz, 1H), 7.49 (dd, J = 4.5, 9.0 Hz, 1H), 7.98 (s, 1H), 8.09 (s,1H), 9.58 (s, 1H), 11.16 (s,1H); FAB-HRMS calcd for C21H13N2O4F [M − H]: 375.0781; found: 375.0773. (Partially degradated after few weeks at rt).

3-(5-Fluoro-6-iodo-1-methyl-1H-indol-3-yl)-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (5)

1H NMR (CDCl3, 400 MHz,) δ 3.83 (s, 3H), 4.02 (s, 3H), 6.42 (d, J = 7.9 Hz, 1H), 6.73 (d, J = 7.5 Hz, 1H), 6.76 (d, J = 9.0 Hz, 1H), 6.83 (t, J = 7.9 Hz, 1H), 7.67 (m, 2H), 7.73 (s, 1H), 8.16 (s, 1H); FAB-HRMS calcd for C22H14N2O4FI [M + Na]+: 538.9875; found: 538.9866.

3-Benzofuran-3-yl-4-(6-chloro-5-fluoro-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (6)

1H NMR (CDCl3, 400 MHz) δ 3.81 (s, 3H), 6.75-6.80 (m, 2H), 6.87 (t, J = 4.7 Hz, 1H), 7.20 (t, J = 4.5 Hz, 1H), 7.31 (m, 2H), 7.50 (d, J = 5.0 Hz, 1H), 7.73 (s, 1H), 8.14 (s, 1H); FAB-HRMS calcd for C21H12N2O3ClF [M + H]+: 395.0599; found: 395.0617.

3-(6-Chloro-5-fluoro-1-methyl-1H-indol-3-yl)-4-(6-hydroxymethyl-benzofuran-3-yl)-pyrrole-2,5-dione (7)

1H NMR (CDCl3, 400 MHz) δ 3.83 (s, 3H), 4.67 (s, 2H), 6.75 (d, J = 10 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 6.0 Hz, 1H), 7.52 (s, 1H), 7.77 (s, 1H), 8.14 (s, 1H); FAB-HRMS calcd for C22H14N2O4FCl [M + H]+: 425.0699; found: 425.0713.

3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (8)

1H NMR (CD3CN, 400 MHz) δ 4.33 (s, 3H), 4.39 (s, 3H), 7.07 (dd, J = 1.2, 5.2 Hz, 1H), 7.21 (d, J = 5.2 Hz, 1H), 7.34 (d, J = 6.5 Hz, 1H), 7.67 (d, J = 1.2 Hz, 1H), 8.12 (d, J = 3.7 Hz, 1H), 8.37 (s, 1H), 8.64 (s, 1H), 9.25 (s, 1H); FAB-HRMS calcd for C22H14N2O4ClF [M − H]: 423.0553; found: 423.0559.

3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-cyclopropylmethoxy-benzofuran-3-yl)-pyrrole-2,5-dione (9)

1H NMR (CD3CN, 400 MHz) δ 0.32 (m, 2H), 0.60 (m, 2H), 1.21 (m, 1H), 3.80 (d, J = 7.0 Hz, 2H), 3.84 (s, 3H), 6.48 (dd, J = 2.2, 8.7 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 6.70 (d, J = 10.5 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 7.56 (d, J = 6.3 Hz, 1H), 7.82 (s, 1H), 8.08 (s, 1H), 8.70 (s, 1H); FAB-HRMS calcd for C25H18N2O4ClF [M + H]+: 465.1012; found: 465.1010.

3-(6-Chloro-5-methoxy-1-methyl-1H-indol-3-yl)-4-(6-cyclobutylmethoxy-benzofuran-3-yl)-pyrrole-2,5-dione (10)

1H NMR (CD3CN, 400 MHz) δ 1.84-1.88 (m, 4H), 1.95-1.99 (m, 2H), 2.75 (m, 1H), 3.83 (s, 3H), 3.93 (d, J = 7.1 Hz, 2H), 6.47 (dd, J = 2.1, 8.8 Hz, 1H), 6.62 (d, J = 8.8 Hz, 1H), 6.77 (d, J = 10.6 Hz, 1H), 7.08 (d, J = 2.1 Hz, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.81 (s, 1H), 7.81 (s, 1H), 8.08 (s, 1H), 8.7 (s, 1H); FAB-HRMS calcd for C26H20N2O4ClF [M − H]: 477.1023; found: 477.1024.

3-(6-Chloro-5-fluoro-1-methyl-1H-indol-3-yl)-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (11)

1H NMR (CDCl3, 400 MHz) δ 3.82 (s, 3H), 4.00 (s, 3H), 6.38 (d, J = 7.7 Hz, 1H), 6.71 (d, J = 7.5 Hz, 1H), 6.80 (m, 2H), 7.31 (d, 1H), 7.52 (s, 1H), 7.74 (s, 1H), 8.16 (s, 1H); FAB-HRMS calcd for C22H14N2O4ClF [M − H]: 423.0553; found: 423.0557.

3-[6-(4-Chlorophenyl)-5-fluoro-1-methyl-1H-indol-3-yl]-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (12)

3-(5-Fluoro-6-iodo-1-methyl-1H-indol-3-yl)-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (5) (0.020 g, 0.039 mmol), Pd(PPh3)4 (4.5 mg, 0.004 mmol), and 4-chloro-phenylboronic acid (15.1 mg, 0.097 mmol) were dissolved in dimethoxy ethane (DME) (4 mL), and the mixture was degassed for 1 min and stirred for 10 min at rt. A 2M K2CO3 (2 M, 0.049 mL, 0.098 mmol) was added. The mixture was degassed again for 1 min, and stirred at 85 °C overnight. The resulting mixture was cooled to ambient temperature and poured into a mixture of 0.1 N HCl/EtOAc (15 mL/15 mL). After partition, the organic layer was washed with water, filtered, and concentrated. The residue was purified by preparative TLC using (EtOAc-hexane, 1:1) to afford 12 as an orange solid (10 mg, 51%). The sample was further purified by HPLC for biological test. 1H NMR (CDCl3, 400 MHz,) δ 3.89 (s, 3 H), 4.02 (s, 3 H), 6.53 (d, 1 H, J = 7.8 Hz), 6.74 (d, 1 H, J = 7.8 Hz), 6.90-6.82 (m, 2 H), 7.26 (m, 1 H), 7.48-7.39 (m, 4 H), 7.53 (s, 1 H), 7.79 (s, 1 H), 8.15 (s, 1H); FAB-HRMS calcd for C28H18N2O4FCl [M + Na]+: 523.0832; found: 523.0824.

3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (14)

1H NMR (CDCl3, 400 MHz) δ 3.82 (s, 1H), 4.00 (s, 3H), 6.37 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.78 (t, J = 7.8 Hz, 1H), 7.14-7.23 (m, 3H), 7.37 (b.s, 1H), 7.70 (3, 1H), 8.14 (s, 1H); FAB-HRMS calcd for C22H15N2O4Br [M + H]+: 451.0293; found: 451.0279.

3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(6-hydroxymethyl-benzofuran-3-yl)-pyrrole-2,5-dione (15)

1H NMR (DMSO-d6, 400 MHz) δ 3.86 (s, 3H), 4.52 (d, J = 5.6 Hz, 2H), 5.26 (t, J = 6.0 Hz, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 7.1 (d, J = 2.0 Hz, 1H), 7.21 (dd, J = 1.6, 8.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.55 (s, 1H), 7.96 (s,1H), 8.26 (s,1H), 11.20 (s, 1H); FAB-HRMS calcd for C25H18BrN2O4 [M + H]+: 451.0288; found: 451.0288.

3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(6-prop-2-ynyloxy-benzofuran-3-yl)-pyrrole-2,5-dione (16)

1H NMR (CDCl3, 400 MHz) δ 3.83 (s, 3H), 4.67 (s, 1H), 5.42 (d, J = 3.9 Hz, 1H), 6.53-6.69 (m, 3H), 6.81 (t, J = 4.5 Hz, 1H), 7.22-7.12 (m, 6H), 7.71 (s, 1H), 8.08 (s, 1H); FAB-HRMS calcd for C24H15BrN2O4 [M + H]+:475.02880; found: 475.02966; calcd for C24H15BrN2O4 [M + Na]+: 497.0108; found: 497.0119. (Partially degradated after few weeks at rt).

3-(6-Allyloxy-benzofuran-3-yl)-4-(5-bromo-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (17)

1H NMR (DMSO-d6, 400 MHz) δ 3.84 (s, 3H), 4.99 (s, 2H), 6.59 (dd, J = 1.8, 8.7 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 7.20 (dd, J = 1.5, 8.5 Hz, 1H), 7.31-7.34 (m, 2H), 7.44 (d, J = 8.5 Hz, 1H), 7.94 (s, 1H), 8.17 (s, 1H), 11.20 (s, 1H); FAB-HRMS calcd for C24H17N2O4Br [M + H]+: 477.0445; found: 477.0445.

3-(5-Bromo-1-methyl-1H-indol-3-yl)-4-[6-(4-methoxy-benzyloxy)-benzofuran-3-yl]-pyrrole-2,5-dione (18)

1H NMR (DMSO-d6, 400 MHz) δ 3.75 (s, 3H), 3.84 (s, 3H), 4.99 (s, 2H), 6.61 (dd J = 2.0, 8.7 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.91 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 1.5 Hz, 1H), 7.04 (dd, J = 1.5, 8.6 Hz, 1H), 7.31-7.36 (m, 3H), 7.45 (d, J = 8.6 Hz, 1H), 7.94 (1s, 1H), 8.17 (s, 1H); FAB-HRMS calcd for C29H21N2O5Br [M − H]: 579.0526; found: 579.0521.

3-(5,7-Dibromo-1-methyl-1H-indol-3-yl)-4-(7-methoxy-benzofuran-3-yl)-pyrrole-2,5-dione (20)

1H NMR (CDCl3, 400 MHz) δ 4.00 (s, 3H), 4.17 (s, 3H), 6.30 (d, J = 7.8 Hz, 1H), 6.73 (d, J = 7.8 Hz, 1H), 6.82 (t, J = 7.8 Hz, 1H), 7.12 (d, J = 1.7 Hz, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.55 (s, 1H), 7.61 (s, 1H), 8.93 (s, 1H); FAB-HRMS calcd for C22H14N2O4Br2 [M − H]: 526.9248; found: 526.9247.

3-Benzofuran-3-yl-4-(5-iodo-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (21)

1H NMR (DMSO-d6, 400 MHz) δ 3.87 (s, 3H), 6.88 (d, J = 7.8 Hz, 1H), 6.94 (t, J = 7.8 Hz, 1H), 7.14 (s, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 8.26 (s, 1H), 11.20 (s, 1H); FAB-HRMS calcd for C21H13N2O3I [M + H]+: 469.0044; found: 469.0045.

3-(5-Fluoro-benzofuran-3-yl)-4-(5-iodo-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (22)

1H NMR (DMSO-d6, 400 MHz) δ 3.87 (s, 3H), 6.65 (dd, J = 2.5, 9.0 Hz, 1H), 7.12 (m, 1H), 7.39 (m, 2H), 7.68 (dd, J = 4.0, 9.0 Hz, 1H), 7.98 (s, 1H), 8.31 (s, 1H), 11.18 (s, 1H); FAB-HRMS calcd for C21H12N2O3FI [M − H]: 484.9804; found: 484.9815.

3-[4-(6-Hydroxymethyl-benzofuran-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-1-methyl-1H-indole-5-carbonitrile (23)

1H NMR (DMSO-d6, 400 MHz) δ 3.91 (s, 3H), 4.51 (d, J = 5.7 Hz, 2H), 5.25 (t, J = 5.8 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 6.85 (d, J = 8.2 Hz, 1H), 7.40 (s, 1H), 7.47 (dd, J = 1.2, 8.5 Hz, 1H), 7.55 (s, 1H), 7.68 (d, J = 8.6 Hz, 1H), 8.05 (s,1H), 8.33 (s,1H), 11.27 (s,1H); FAB-HRMS calcd for C23H15N3O4 [M + Na]+: 420.0955; found: 420.0952.

3-(5-Cyclopropylethynyl-1-methyl-1H-indol-3-yl)-4-(5-fluoro-benzofuran-3-yl)-pyrrole-2,5-dione (25)

1H NMR (CDCl3, 400 MHz,) δ 0.67 (m, 2H), 0.82 (m, 2H), 1.34 (m, 1H), 3.87 (s, 3H), 6.59 (d, J = 8.7 Hz, 1H), 6.97-6.93 (m, 2H), 7.17 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.46-7.43 (m, 1H), 7.53 (br. s, 1H), 7.81 (s, 1H), 8.10 (s, 1H); FAB-HRMS calcd for C26H16N2O3F [M − H]: 423.1150; found: 423.1147.

3-(5-Fluoro-benzofuran-3-yl)-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)-pyrrole-2,5-dione (26)

1H NMR (CDCl3, 400 MHz) δ 3.79 (s, 3H), 5.80 (m, 2H), 6.29 (s, 1H), 6.59 (dd J = 2.4, 9.0 Hz, 1H), 6.74 (s, 1H), 6.91 (td, J = 2.5, 9.0 Hz, 1H), 7.41 (dd, J = 4.1, 8.0 Hz, 1H), 7.65 (s, 1H), 7.95 (s, 1H), 8.13 (s, 1H); FAB-HRMS calcd for C22H13N2O5F [M + H]+: 405.0881; found: 405.0880.

3-Benzofuran-3-yl-4-(6-chloro-5-methoxy-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (27)

1H NMR (DMSO-d6, 400 MHz) δ 3.11 (s, 3H), 3.83 (s, 3H), 6.30 (s, 1H), 6.93 (t, J = 4.7 Hz, 1H), 7.04 (d, J = 4.7 Hz, 1H), 7.22 (t, J = 5.2 Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.86 (s, 1H), 8.01 (s, 1H); FAB-HRMS calcd for C22H15N2O4Cl [M + H]+: 407.0793; found: 407.0793.

3-Benzofuran-3-yl-4-(6-iodo-5-methoxy-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (28)

1H NMR (CDCl3, 400 MHz) δ 3.09 (s, 3H), 3.83 (s, 3H), 6.29 (s, 1H), 6.96 (t, J = 4.7 Hz, 1H), 7.08 (d, J = 4.7 Hz, 1H), 7.21 (m, 2H), 7.47 (m, 2H), 7.69 (s, 1H), 7.86 (s, 1H), 8.00 (s, 1H); FAB-HRMS calcd for C22H15N2O4I [M + H]+: 441.0149; found: 441.0149.

3-(7-Methoxy-benzofuran-3-yl)-4-(1-methyl-6-trifluoromethyl-1H-indol-3-yl)-pyrrole-2,5-dione (30)

1H NMR (CDCl3, 400 MHz) δ 3.94 (s, 3H), 4.02 (s, 3H), 6.43 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.81 (t, J = 8.0 Hz, 1H), 7.12 (m, 2H), 7.62 (s, 1H), 7.70 (s, 1H), 7.88 (s, 1H), 8.14 (s, 1H); FAB-HRMS calcd for C23H15N2O4F3 [M + H]+: 441.1062; found: 441.1057.

3-Benzofuran-3-yl-4-(7-hydroxymethyl-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (31)

1H NMR (CDCl3, 400 MHz) δ 1.77 (t, J = 6.0 Hz, 1H), 4.22 (s, 3H), 4.98 (d, J = 6.0 Hz, 2H), 6.72 (t, J = 7.6 Hz, 1H), 6.86-6.95 (m, 2H), 6.98 (t, J = 7.2 Hz, 2H), 7.19 (dt, J = 1.4, 7.5 Hz, 1H), 7.47 (t, J = 8.3 Hz, 1H), 7.56 (brs, 1H), 7.72 (s,1H), 8.09 (s,1H); FAB-HRMS calcd for C22H16N2O4 [M − H]: 371.1037; found: 371.1038.

3-(6-Hydroxymethyl-benzofuran-3-yl)-4-(7-hydroxymethyl-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (32)

1H NMR (DMSO-d6, 400 MHz) δ 4.18 (s, 3H), 4.52 (d, J = 5.6 Hz, 2H), 4.84 (d, J = 5.6 Hz, 2H), 5.25 (t, J = 6.0 Hz, 1H), 5.36 (t, J = 6.0 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.90 (s, 2H), 6.97 (d, J = 6.8 Hz, 1H), 7.51 (s, 1H), 7.89 (s,1H), 8.18 (s,1H), 11.18 (s,1H); FABHRMS calcd for C23H18N2O5 [M + H]+: 403.12885; found: 403.12867; calcd for C23H18N2O5 [M + Na]+: 425.1108; found: 425.1107.

3-Benzofuran-3-yl-4-(7-methoxymethyl-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (33)

1H NMR (DMSO-d6, 400 MHz) δ 3.26 (s, 3H), 4.10 (s, 3H), 4.73 (s, 2H), 6.63 (t, J = 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.86-6.93 (m, 2H), 6.96 (d, J = 7.2 Hz, 1H), 7.20 (dt, J = 1.4, 7.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.88 (s,1H), 8.25 (s,1H), 11.19 (brs, 1H); FAB-HRMS calcd for C23H18N2O4 [M + H]+: 387.13393; found: 387.13419; calcd for C23H18N2O4 [M + Na]+: 409.1159; found: 409.1161.

3-(6-Hydroxymethyl-benzofuran-3-yl)-4-(7-methoxymethyl-1-methyl-1H-indol-3-yl)-pyrrole-2,5-dione (34)

1H NMR (DMSO-d6, 400 MHz) δ 3.27 (s, 3H), 4.11 (s, 3H), 4.50 (d, J = 8.0 Hz, 2H), 4.73 (s, 2H), 5.23 (t, J = 8.0 Hz, 1H), 6.66 (t, J = 7.6 Hz, 1H), 6.80-6.89 (m, 3H), 6.97 (d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.88 (s, 1H), 8.21 (s, 1H), 11.19 (brs, 1H); FAB-HRMS calcd for C24H20N2O5 [M − H]: 415.1294; found: 415.1297.

3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1-methyl-1H-indol-7-yl]-propionic acid ethyl ester (35)

To a solution of 3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1-methyl-1H-indol-7-yl]-acrylic acid ethyl ester (68) (45 mg, 0.10 mmol) in (MeOH-THF, 3:1) (20 mL) was added successively at 0°C NiCl2·6H2O (24 mg, 0.10 mmol) and NaBH4 (7.7 mg, 0.20 mmol) and the mixture was stirred at this temperature for 15 min and then at rt for 2 h. The resulting mixture was diluted with EtOAc and washed with saturated aqueous NaHCO3 solution. The aqueous phase was extracted with EtOAc and the combined organic phases were dried over Na2SO4, and evaporated in vacuo. The residue was purified by preparative TLC (EtOAc-hexane; 4:6) followed by a purification by HPLC with the method C to yield the product (20 mg, 45%). 1H NMR (CDCl3, 400 MHz) δ 1.25 (t, J = 7.2 Hz, 3H), 2.67 (t, J = 8.2 Hz, 2H), 3.40 (t, J = 8.2 Hz, 2H), 4.08-4.18 (m, 5H), 6.68 (t, J = 7.6 Hz, 1H), 6.81-6.93 (m, 4H), 7.18 (dt, J = 1.5, 7.4 Hz, 3H), 7.45 (d, J = 8.3 Hz, 1H), 7.66 (s, 1H), 7.79 (s, 1H), 8.08 (s, 1H); FAB-HRMS calcd for C26H22N2O5 [M + H]+: 443.1602 ; found: 443.1613.

3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1-methyl-1H-indol-7-yl]-propionic acid (36)

The 3-[3-(4-Benzofuran-3-yl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1-methyl-1H-indol-7-yl]-propionic acid ethyl ester (35) (8 mg, 0.018 mmol) was dissolved in EtOH (2 mL) and 1N NaOH (0.072 mL, 0.072 mmol) was added to the solution. The mixture was stirred at rt overnight. The solvent was evaporated in vacuo, water and 1N HCl were added to reach pH 2. The acidic solution was extracted with EtOAc three times. The combined organic layers were washed with water, brine, dried over Na2SO4 and evaporated. The residue was first purified by preparative TLC (MeOH-CH2Cl2; 1:9) followed by a purification by HPLC with the method C to yield the product (3 mg, 39%). 1H NMR (CD3OD, 400 MHz) δ 2.66 (t, J = 7.8 Hz, 2H), 3.40 (t, J = 7.8 Hz, 2H), 4.15 (s, 3H), 6.60 (t, J = 7.8 Hz, 1H), 6.61-6.82 (m, 3H), 6.87 (d, J = 7.0 Hz, 3H), 7.15 (m, 1H), 7.45 (d, J = 8.3 Hz, 3H),7.73 (s, 1H), 8.13 (s, 1H); FAB-HRMS calcd for C24H18N2O5 [M + H]+: 415.1289 ; found: 415.1304.

3-(5,6-Difluoro-benzofuran-3-yl)-4-(1-methyl-1H-benzo[g]indol-3-yl)-pyrrole-2,5-dione (38)

1H NMR (CDCl3, 400 MHz) δ 4.41 (s, 3H), 6.66 (dd, J = 7.8, 10.2 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 7.27 (m, 2H), 7.44 (t, J = 7.8 Hz, 1H), 7.56 (t, J = 7.0 Hz, 1H), 7.70 (s, 1H), 7.82 (d, J = 7.8 hz, 1H), 8.17 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H); FAB-HRMS calcd for C25H14N2O3F2 [M + H]+: 429.1050; found: 429.1059.

GSK-3β in vitro kinase assay

The in vitro kinase assay was performed in a 40 μL reaction volume containing 250 ng GSK-3β Calbiochem (EMD Biosciences, La Jolla, CA), 10 μM pGS peptide (RRRPASVPPSPSLRHSS(P)HQRR, where the priming phosphoserine is underlined), 10 μM γ32P ATP (specific activity equal to), and increasing concentrations of the inhibitors in kinase buffer (20 mM MOPS pH 7.2, 15 mM MgCl2, 5 mM EGTA, 25 mM β–glycerol phosphate, 1 mM Na3VO4, 1 mM DTT). The inhibitors were allowed to bind GSK-3β for 10 min prior to the addition of peptide. The reaction was started by the addition of the ATP solution and was carried out at 30 °C for 30 min after which 25 μl of the reaction mixture was spotted on P81 Whatman filters (Florham Park, NJ), dried, washed twice with 0.75% H3PO4, and rinsed with acetone. The dried filters were placed in scintillation vials containing 2ml of Scinti-safe Econo 2 solution (Fisher Scientific, Pittsburgh, PA) and read in a Beckman Coulter LS 600IC (Fullerton, CA) scintillation counter.

IC50 determination

The data were plotted as average percent activity versus the log of the concentration of inhibitor. The IC50 was determined according to NIH guidelines (http://www.ncgc.nih.gov/guidance/section3.html) and the 4-parameter logistic plot (4PL), which calculates the relative IC50 based on the maximum (top) and minimum (bottom) percent activity as well as the Hill slope. In some cases the 4PL did not give an accurate fitting of the data (error fit above 40%) and thus a 3-parameter logistic fit top (3PLFT) or 3-parameter logistic fit bottom (3PLFB) were used to obtain the IC50 using the equation y = Bottom + (Top−Bottom)/(1+10^((LogIC50−x)*HillSlope)) from Graphpad Prism 5. This program displays the LogIC50 as well as the IC50 and gives the standard error of the LogIC50, this was converted back to the % fitting error of the IC50 by using the equation %FE(IC50) = FE(LogIC50)*ln(10)*100.

Cytotoxicity Assay. Materials and Methods

Immunoblot analysis and antibodies

For immunoblots, cells were lysed as described previously.15 Protein sample concentration was quantified and equal amount (50 μg whole protein extract) of protein was loaded in each well of SDS-polyacrylamide (PAGE) gel. Cell or tissue extracts were separated by 10% SDS-PAGE, transferred to polyvinylidene diflouride membrane (PVDF), and probed as indicated. Antibodies for immunoblot analysis were obtained from the following suppliers: PARP and XIAP from BD Biosciences Pharmingen (San Diego, CA); phospho-Glycogen Synthase (Ser641) and Glycogen Synthase from Cell Signaling Technologies (Beverly, MA); β-actin from Novus (Littleton, CO). Bound antibodies were detected as previously described.

MTS assay

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay from Promega (Madison, WI) was carried out in the presence or absence of the potential GSK-3 inhibitors according to the manufacturer protocol. 72 Hours post-treatment, cell viability was measured and compounds IC50 values were determined.

Supplementary Material

1_si_001

Acknowledgment

This work was supported in part by NIH grant (1R01 MH072940-01, Grantee Alan P. Kozikowski), SPORE grant (P50 CA102701, Grantee Daniel D. Billadeau), and by the Mayo Foundation (D.D.B.). We thank Drs. Arsen Gaysin and Rong He for technical assistance, and Dr. Aruna Mahesh for assistance in the GSK-3β kinase assay.

Footnotes

aAbbreviations : APC, adenomatous polyposis coli; CDK-2, Cyclin-dependent Kinase 2; CDKs, Cyclin-dependent Kinases; DMF-DMA, N,N-dimethylformamide dimethyl acetal; DMF-DIPA, N,N-dimethylformamide di-iso-propyl acetal; FRAT, frequently rearranged in advanced T-cell lymphoma; GBP, GSK-3-binding protein; GS, Glycogen Synthase; GSK-3, Glycogen Synthase Kinase 3; GSK-3β, Glycogen Synthase Kinase 3 isoform type β; NFκB, nuclear factor kappa B; pGS : phosphorylated Glycogen Synthase; P-gp, permeability-glycoprotein; PKC, Protein Kinase C; TPSA, total polar surface area; XIAP, X-linked Inhibitor of Apoptosis; ZDF rat, Zucker Diabetic Fatty rat.

Supporting Information Available: Molecular modeling methods, experimental procedures for some intermediates, 13C NMR data, HPLC purity data for all final compounds, and selectivity data for compounds 5, 7, 11, 15, and 33. These materials are available free of charge via the Internet at http://pubs.acs.org.

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