1,4-Thienodiazepine-2,5-diones via MCR (I): Synthesis, Virtual Space and p53-Mdm2 Activity

Abstract

1,4-Thienodiazepine-2,5-diones have been synthesized via the Ugi-Deprotection-Cyclization (UDC) approach starting from Gewald 2-aminothiophenes i n a convergent and versatile manner. The resulting scaffold is unprecedented, cyclic and peptidomimetic with four points of diversity introduced from readily available starting materials. In addition to eighteen synthesized and characterized compounds, a virtual compound library was generated and evaluated for chemical space distribution and drug-like properties. A small focused compound library of 1,4-thienodiazepine-2,5-diones has been screened for the activity against p53-Mdm2 interaction. Biological evaluations demonstrated that some compounds exhibited promising antagonistic activity.

The development of new synthetic scaffolds is of uttermost importance for identifying new lead structures during the drug discovery process. Seven-membered 1,4-diazepine ring based drugs have been found repeatedly as the prototype of a privileged structure with a broad range of biological activities and applications in human medicine (1). Over the last decades, 1,4-benzodiazepines have emerged as a particularly fascinating class of scaffolds in medicinal chemistry because they hit various classes of pharmacologically relevant targets such as GPCRs, ion channels and enzymes (2). For example, anthramycin is a broad-spectrum antineoplastic antibiotic with low toxicity, which binds irreversibly to DNA and inhibits the synthesis of RNA and DNA (3). Recently, 1,4-benzodiazepin-2,5-diones (BDZs) have been widely investigated as potential medicinal agents (4,5). Therefore, preparation and evaluation of libraries based on privileged structures become an important part of the drug discovery process. Thiophene as an effective bioisostere of phenyl ring led to the discovery of a series of 1,4-thienodiazepine drugs, such as the block buster family of olanzapine, clotiazepam, and brotizolam. For example, clotiazepam is structurally similar to diazepam, which is one of the most frequently prescribed medications in the world during the past 40 years (6). Thus, the development of new thiophene scaffolds based on privileged structures, especially 1,4-benzodiazepine family is of great interest.

Multicomponent reaction (MCR) is an alternative strategy in different drug discovery stages including lead discovery and pre-clinical process development (7). MCRs allow the resource and cost effective, fast, and convergent synthesis of diverse compound libraries, and highly improve the efficiency to explore the chemical space with limited synthetic effort (8). The Gewald three-component reaction (G-3CR) is a unique method using elemental sulfur to yield thiophene ring, which builds a platform for the synthesis of new thiophene scaffolds (9). For example, olanzapine is manufactured by Eli Lilly via the formation of its thiophene ring through G-3CR (10). G-3CR provides a convenient way to synthesize 2-aminothiophene 2, which is bioisostere to anthranilic acid 1 (Figure 1) (11). According to the substructure search in currently published protein-ligand cocrystal structures (PDB query and Relibase), most of the retrieved ligands are nonsubstituted anthranilic acid derivatives. Advantageously, the G-3CR accessible thiophene scaffold allows for many more synthetic variations than the substituted anthranilic acids derivatives.

Figure 1.

Gewald aminothiophene as a bioisostere of anthranilic acids

Isocyanide-based MCRs (IMCR), such as the Ugi and Passerini reactions, provide a powerful tool for producing arrays of compounds based on multiple scaffolds and with high atom economy (12). The Ugi-Deprotection-Cyclization (UDC) approach generates very useful classes of scaffolds amenable by an initial Ugi reaction of two bifunctional orthogonally protected starting material classes and a secondary reaction to form heterocyclic rings, such as benzimidazole, quinoxalinone, imidazoline, γ-lactame, ketopiperazine and diketopiperazine (13). Notably, UDC strategy is particularly useful for the synthesis of 1,4-benzodiazepine-2,5-diones (14-17). The further enlargement of UDC amenable scaffold classes is highly desirable, and therefore we investigated the development of synthetic routes to 1,4-thienodiazepine-2,5-diones. Herein, we report the preparation of a new 1,4-thienodiazepine-2,5-dione scaffold by a union of Gewald and UDC MCRs (Figure 2).

Figure 2.

Synthesis of the 1,4-thienodiazepine-2,5-dione scaffold via the UDC approach (R¹, R², R³, R⁴: points of diversity)

Materials and Methods

General

All reagents were purchased from commercial sources and used without further purification. The reactions were conducted under air atmosphere unless otherwise indicated. Analytical thin-layer chromatography (TLC) was performed on SiO₂ plates on Alumina available from Whatman. Visualization was accomplished by UV irradiation at 254 nm. Preparative TLC was conducted using Preparative Silica gel TLC plates (1000 μm, 20cm×20cm). Flash column chromatography was performed using SiO₂ 60 (particle size 0.040-0.055 mm, 230-400 mesh, EM science distributed by Bioman). Proton and carbon NMR spectra were determined on Bruker Avance™ 600 MHz NMR spectrometer. Chemical shifts are reported as δ values in parts per million (ppm) as referenced to residual solvent. ¹H NMR spectra are tabulated as follows: chemical shift, number of protons, multiplicity (s = singlet, br.s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and coupling constant. High Resolution Mass spectra were obtained at the University of Pittsburgh Mass Spectrometry facility. LC-MS analysis was performed on an SHIMADZU instrument, using an analytical C18 column (Dionex Acclaim 120 Å, 2.1 × 50 mm, 3.0 μm, 0.2 mL/min). Acetonitrile/water mixtures were used as mobile phase for reverse-phase HPLC coupled to electrospray ionization-mass spectrometry (ESI-MS).

Methyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2a)

To a mixture of cyclohexanone (0.45 mol, 44.10 g), methyl cyanoacetate (0.3 mol, 28.77 g), sulfur (0.3 mol, 9.60 g), and 50 mL of ethanol, the solution of diethylamine (0.15 mol, 12.75 g) in 25 mL of ethanol was added dropwise with magnetic stirring. After the completion of the addition, the reaction mixture was kept stirring under room temperate overnight. The reaction flask was kept in the freezer for crystallization. Then the precipitate was collected by vacuum filtration, and washed by cold ethanol. After air dry overnight, 42.20 g of yellow solids were obtained (yield: 67%). HPLC/MS: t_R = 10.58 min; m/z = 212.1 [M+H]^{+ 1}H NMR (600 MHz, CDCl₃): 1.73-1.78 (4H, m), 2.49-2.51 (2H, m), 2.67-2.69 (2H, m), 3.79 (3H, s, OMe), 5.93 (2H, br.s, NH₂). ¹³C NMR (150 MHz, CDCl₃): 22.8, 23.3, 24.5, 26.9, 50.6, 105.6, 117.7, 132.4, 161.8, 166.5.

2-(Boc-amino)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (3a)

The mixture of 2a (10 mmol, 2.11 g), Boc₂O (12.5 mmol, 2.56 g), DMAP (1 mmol, 122 mg) in 25 mL of THF was stirring under reflux overnight. The additional 0.5 g of Boc₂O was added, and stirring under reflux overnight. After cooling to RT, the mixture was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (2.24 g) and 20 mL of water was added. The mixture was reflux for 5 hours. The reaction was quenched by water, the aqueous mixture was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration as yellow solids (1.42 g, 48%). HPLC/MS: t_R = 7.93 min, m/z = 296.0 [M-H]^- HRMS: 297.102519 (found); C₁₄H₁₉NO₄S, 297.1035 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.57 (9H, s), 1.79-1.82 (4H, m), 2.62-2.63 (2H, m), 2.82-2.83 (2H, m), 10.09 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 22.7, 23.0, 24.2, 26.3, 28.2, 82.3, 109.2, 125.3, 131.7, 151.8, 170.7.

Methyl 2-aminothiophene-3-carboxylate (2b)

Triethylamine (50 mmol, 5.0 mL) was added dropwise to a mixture of 1,4-dithiane-2,5-diol (7.60 g, 50 mmol), methyl cyanoacetate (9.59 g, 100 mmol), and DMF (40 mL). The mixture was stirred at 45 °C for 30 min. After cooled to RT, the reaction mixture was diluted with aqueous acetic acid (0.4 M, 200 mL). The mixture was extracted with ether (4 × 40 mL), and the combined organic layer was washed with water (2 × 40 mL). After dried over sodium sulfate, the organic layer was filtered through silica gel pad. After evaporation, 8.70 g of yellow solids were obtained (yield: 55%). HPLC/MS: t_R = 9.01 min; m/z = 158.2 [M+H]^{+ 1}H NMR (600 MHz, CDCl₃): 3.83 (3H, s), 5.94 (2H, br.s), 6.20 (1H, d, J = 5.4 Hz), 6.98 (1H, d, J = 6.0 Hz). ¹³C NMR (150 MHz, CDCl₃): 51.0, 106.9, 107.0, 125.8, 162.7, 165.8.

2-(Boc-amino)-3-thiophenecarboxylic acid (3b)

The mixture of 2b (20 mmol, 3.14 g), Boc₂O (25 mmol, 5.15 g), DMAP (2 mmol, 244 mg) in 30 mL of THF was stirring under reflux overnight. After evaporation of the solvent, the mixture was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (4.5 g) and 20 mL of water was added. The mixture was reflux for 5 hours. The reaction was quenched by water, the aqueous mixture was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration and further purified by flash chromatography with ethyl acetate, 1.52 g of brown solids were obtained (yield: 31%). HPLC/MS: t_R = 10.37 min; m/z = 242.0 [M-H]^- HRMS: 243.055945 (found); C₁₀H₁₃NO₄S, 243.05653 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.58 (9H, s), 6.71 (1H, d, J = 5.4 Hz), 7.23 (1H, d, J = 6.0 Hz), 9.88 (1H, br.s). ¹³C NMR (150 MHz, d⁶-DMSO): 28.2, 82.5, 112.5, 115.9, 125.0, 150.1, 151.8, 166.9.

Methyl 2-amino-5-phenylthiophene-3-carboxylate (2c)

Phenyl-acetaldehyde (0.1 mol, 12.0 g), sulfur (0.1 mol, 3.20 g), methyl cyanoacetate (0.1 mol, 8.57 mL) were stirred at RT in absolute ethanol (50 mL). Triethylamine (0.1 mol, 10.0 mL) was added slowly. After the addition was completed, the mixture was heated at reflux for 1 hour. After cooled to RT, the precipitate was collected by vacuum filtration, and washed by cold ethanol. After air dry overnight, 14.6 g of yellow solids were obtained (yield: 63%). HPLC/MS: t_R = 11.01 min; m/z = 234.3 [M+H]^{+ 1}H NMR (600 MHz, CDCl₃): 3.86 (3H, s), 6.02 (2H, br.s), 7.21-7.26 (2H, m), 7.33-7.46 (2H, m),7.45-7.46 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 51.1, 107.7, 124.7, 125.0, 126.6, 128.8, 133.9, 162.2, 165.8.

2-(Boc-amino)-5-phenylthiophene-3-carboxylic acid (3c)

The mixture of 2c (10 mmol, 2.33 g), Boc₂O (15 mmol, 3.10 g), DMAP (1 mmol, 122 mg) in 30 mL of THF was stirring under reflux overnight. After cooling to RT, the mixture was quenched by water, and extracted by DCM. The organic layer was dried, and concentrated. The residue was dissolved in 20 mL of ethanol, potassium hydroxide (2.24 g) and 20 mL of water was added. The mixture was reflux for 1 hour. The reaction was quenched by water, the aqueous mixture was washed by ether. The aqueous was the adjusted to pH = 6 with 1 M HCl aq. The product was collected by vacum filtration as yellow solids (1.50 g, 47%). HPLC/MS: t_R = 11.79 min; m/z = 320.1 [M+H]⁺ HRMS: 319.086854 (found); C₁₆H₁₇NO₄S, 319.08783 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.60 (9H, s), 7.29-7.31 (1H, m), 7.38- 7.40 (2H, m), 7.45 (1H, s), 7.59-7.60 (2H, m), 9.90 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 28.2, 82.9, 110.9, 119.7, 125.3, 127.4, 129.0, 132.7, 133.5, 152.0, 152.3, 169.1.

Ethyl 2-[[(2-tert-butoxycarbonyl-amino-2,3,4,5,6,7-hexahydro-1-benzothien-3-yl)carbonyl](benzyl)amino]-3-(tert-butyl-amino)-3-oxopropanoate (4a-1)

The mixture of 3a (74.3 mg, 0.25 mmol), benzylamine (0.25 mmol, 27.4 μL), ethyl glyoxylate (0.25 mmol, 49.6 μL), tert-butyl isocyanide (0.25 mmol, 28.3 μL) in 0.5 mL of methanol was stirring under RT for 2 days. The reaction was quenched by water, and extracted by DCM. The organic layer was washed by saturated potassium carbonate (aq), and dried over anhydrous sodium sulfate. After the evaporation of the solvent, the product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (93 mg, yield: 65%). HPLC/MS: t_R = 13.21 min; m/z = 572.2 [M+H]⁺ HRMS: 571.272879 (found); C₃₀H₄₁N₃O₆S, 571.27161 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.24 (3H, t, J = 7.2 Hz), 1.39 (9H, s), 1.55 (9H, s), 1.76-1.82 (4H, m), 2.31 (1H, m), 2.63-2.67 (2H, m), 3.48 (1H, s), 4.09 (2H, m), 4.21 (1H, m), 4.42 (1H, d, J = 15.0 Hz), 4.99 (1H, d, J = 15.0 Hz), 7.27-7.32 (5H, m), 8.19 (1H, s), 9.63 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 13.9, 22.6, 23.4, 23.8, 24.0, 28.3, 28.5, 52.1, 55.1, 61.4, 62.1, 80.7, 115.1, 126.6, 128.0, 128.4, 128.9, 129.4, 135.8, 137.2, 153.0, 164.6, 168.5, 169.0.

Method A for the synthesis of 5a

3,4,6,7,8,9-Hexahydro-3-(N-tert-butyl)-carboxamide-4-benzyl-1H-benzothieno-[2,3-e] -1,4-diazepine-2,5-dione (5a-1)

The mixture of 3a (74.3 mg, 0.25 mmol), benzylamine (0.25 mmol, 27.4 μL), ethyl glyoxylate (0.25 mmol, 49.6 μL), tert-butyl isocyanide (0.25 mmol, 28.3 μL) in 0.5 mL of methanol was stirring under RT for 2 days. The reaction was quenched by water, and extracted by DCM. The organic layer was washed by saturated potassium carbonate (aq), and dried over anhydrous sodium sulfate. After the evaporation of the solvent, the residue was treated by 0.5 mL of TFA, stirring under RT overnight. The reaction was added 10 mL of DCM, then neutralized by saturated potassium carbonate (aq). The mixture was extracted by DCM, the organic layer was combined and dried over anhydrous sodium sulfate. After the evaporation of the solvent, the residue was treated by triethylamine (50 μL) and TBD (10 mg) in 0.5 mL of THF, stirring overnight under 40 °C. 5a-1 was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (35 mg, yield: 33% over three steps). HPLC/MS: t_R = 10.95 min, m/z = 426.2 [M+H]⁺ HRMS: 425.176977 (found); C₂₃H₂₇N₃O₃S, 425.17731 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.86 (9H, s), 1.61 (1H, m), 1.79 (1H, m), 1.86 (1H, m), 1.94 (1H, m), 2.42 (1H, m), 2.60 (2H, m), 3.12 (1H, m), 3.99 (1H, d, J = 14.4 Hz), 4.62 (1H, s), 4.81 (1H, s), 5.63 (1H, d, J = 13.8 Hz), 7.39-7.43 (3H, m), 7.56-7.57 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 22.3, 23.0, 24.5, 26.1, 27.8, 51.3, 52.3, 67.1, 122.2, 128.7, 129.2, 129.6, 129.7, 134.1, 137.1, 104.8, 163.1, 163.7, 168.4.

3,4,6,7,8,9-Hexahydro-3-(N-tert-butyl)-carboxamide-4-phenethyl-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-2)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (58 mg, yield: 53% over three steps). HPLC/MS: t_R = 10.93 min, m/z = 440.2 [M+H]⁺ HRMS: 439.193180 (found); C₂₄H₂₉N₃O₃S, 439.19296 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.05 (9H, s), 1.61 (1H, m), 1.77 (1H, m), 1.83-1.90 (2H, m), 2.39 (1H, m), 2.58-2.59 (2H, m), 2.96-3.00 (1H, m), 3.01-3.07 (2H, m), 3.95-3.99 (2H, m), 4.60 (1H, s), 5.39 (1H, s), 7.22 (1H, m), 7.28-7.31 (4H, m). ¹³C NMR (150 MHz, CDCl₃): 22.3, 23.0, 24.5, 25.8, 28.0, 34.1, 50.3, 51.8, 68.2, 123.2, 126.8, 128.7, 128.8, 129.9, 134.0, 137.6, 139.7, 163.5, 163.6, 168.9.

3,4,6,7,8,9-Hexahydro-3-(N-tert-butyl)-carboxamide-4-(4-chlorobenzyl)-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-3)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (32 mg, yield: 28% over three steps). HPLC/MS: t_R = 11.32 min, m/z = 460.0 [M+H]⁺ HRMS: 459.136130 (found); C₂₃H₂₆ClN₃O₃S, 459.13834 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.92 (9H, s), 1.61 (1H, m), 1.78 (1H, m), 1.86 (1H, m), 1.93 (1H, m), 2.40 (1H, m), 2.58-2.60 (2H, m), 3.09 (1H, s), 4.19 (1H, d, J = 14.4 Hz), 4.58 (1H, s), 4.86 (1H, s), 5.37 (1H, d, J = 13.2 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.49 (1H, d, J = 8.4 Hz). ¹³C NMR (150 MHz, CDCl₃): 22.3, 22.9, 24.5, 26.0, 27.9, 51.6, 51.9, 67.3, 122.3, 129.6, 129.9, 130.5, 134.1, 134.6, 135.4, 140.3, 163.2, 163.3, 168.4.

3,4,6,7,8,9-Hexahydro-3-(N-cyclopropylmethyl)-carboxamide-4-(4-chlorophenyl)-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-4)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (18 mg, yield: 16% over three steps). HPLC/MS: t_R = 11.10 min, m/z = 446.0 [M+H]⁺ HRMS: 445.121614 (found); C₂₂H₂₄ClN₃O₃S, 445.12269 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.16 (9H, s), 1.61 (1H, m), 1.78 (1H, m), 1.86-1.88 (2H, m), 2.47 (1H, m), 2.61 (2H, m), 3.00 (1H, m), 4.88 (1H, s), 5.01 (1H, s), 7.33 (2H, d, J = 7.2 Hz), 7.39 (2H, d, J = 6.6 Hz).¹³C NMR (150 MHz, CDCl₃): 22.3, 23.0, 24.6, 25.9, 28.2, 29.7, 52.3, 70.3, 123.4, 127.7, 129.7, 130.2, 133.3, 135.0, 141.3, 162.7, 168.4, 170.7.

3,4,6,7,8,9-Hexahydro-3-(N-cyclopropylmethyl)-carboxamide-4-benzyl-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-5)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (27 mg, yield: 26% over three steps). HPLC/MS: t_R = 10.34 min, m/z = 424.3 [M+H]⁺ HRMS: 423.161189 (found); C₂₃H₂₅N₃O₃S, 423.16166 (calcd.). ¹H NMR (600 MHz, CDCl₃): -0.10 (2H, m), 0.32 (2H, m), 0.43 (1H, m), 1.76 (1H, m), 1.83 (1H, m), 1.89 (1H, m), 2.45 (1H, m), 2.51 (1H, m), 2.58-2.59 (2H, m), 2.77 (1H, m), 3.07 (1H, m), 4.22 (1H, d, J = 14.4 Hz), 4.63 (1H, s), 5.28 (1H, s), 5.45 (1H, d, J = 14.4 Hz), 7.35 (1H, m), 7.39-7.41 (2H, m), 7.53-7.54 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 3.29, 3.33, 10.1, 22.3, 23.0, 24.5, 25.9, 44.5, 52.4, 66.4, 122.1, 128.68, 128.74, 129.0, 129.49, 129.52, 134.3, 136.8, 140.5, 163.3, 164.2, 168.2.

3,4,6,7,8,9-Hexahydro-3-(N-mesityl)-carboxamide-4-(4-chlorobenzyl)-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-6)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (15 mg, yield: 12% over three steps). HPLC/MS: t_R = 11.63 min, m/z = 522.2 [M+H]⁺ HRMS: 521.153301 (found); C₂₈H₂₈ClN₃O₃S, 521.15399 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.26 (1H, m), 1.58 (6H, s), 1.77 (1H, m), 1.87-1.89 (2H, m), 2.17 (3H, m), 2.50-2.61 (3H, m), 3.08 (1H, m), 4.27 (1H, d, J = 14.4 Hz), 4.83 (1H, s), 5.35 (1H, d, J = 14.4 Hz), 6.58 (1H, s), 6.72 (2H, s), 7.32 (1H, d, J = 7.8 Hz), 7.45 (1H, d, J = 8.4 Hz). ¹³C NMR (150 MHz, CDCl₃): 17.1, 20.8, 22.2, 22.9, 24.6, 26.1, 52.3, 67.0, 121.8, 128.9, 129.4, 129.6, 129.7, 130.5, 134.6, 134.7, 135.3, 137.4, 162.7, 163.5, 167.7.

3,4,6,7,8,9-Hexahydro-3-(N-benzyl)-carboxamide-4-(4-chlorobenzyl)-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-7)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (20 mg, yield: 16% over three steps). HPLC/MS: t_R = 11.04 min, m/z = 494.0 [M+H]⁺ HRMS: 493.122043 (found); C₂₆H₂₄ClN₃O₃S, 493.12269 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.60 (1H, m), 1.77 (1H, m), 1.87 (2H, m), 2.29 (1H, m), 2.59 (1H, m), 2.65 (1H, m), 3.01 (1H, m), 3.94 (1H, m), 4.07 (1H, m), 4.29 (1H, d, J = 14.4 Hz), 4.63 (1H, s), 5.22 (1H, d, J = 14.4 Hz), 5.47 (1H, s), 6.86 (2H, m), 7.20-7.21 (2H, m), 7.26-7.29 (3H, m), 7.34-7.36 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 22.2, 22.9, 24.6, 25.7, 44.1, 51.9, 66.6, 122.1, 127.9, 128.7, 129.5, 130.2, 134.6, 135.0, 136.6, 139.8, 163.3, 163.9, 167.6.

3,4,6,7,8,9-Hexahydro-3-(N-3,4-dichlorobenzyl)-carboxamide-4-(4-chlorobenzyl)-1H-benzothieno-[2,3-e]-1,4-diazepine-2,5-dione (5a-8)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 1:1) as yellowish solids (18 mg, yield: 16% over three steps). HPLC/MS: t_R = 11.72 min; m/z = 561.9 [M+H]⁺ HRMS: 561.044289 (found); C₂₆H₂₂C_l3N₃O₃S, 561.04475 (calcd.). ¹H NMR (600 MHz, CD₃OD): 1.35-1.36 (1H, m), 1.66-1.77 (3H, m), 2.12-2.15 (1H, m), 2.51-2.54 (2H, m), 2.87 (1H, m), 4.00 (1H, d, J = 14.4 Hz), 4.27 (1H, d, J = 14.4 Hz), 4.34 (1H, d, J = 15.0 Hz), 7.43 (1H, d, J = 15.0 Hz), 6.98 (1H, m), 7.31-7.33 (5H, m), 7.37 (1H, m). ¹³C NMR (150 MHz, CD₃OD): 21.8, 22.6, 23.8, 25.0, 40.1, 42.0, 51.8, 121.5, 127.8, 128.4, 128.6, 129.6, 130.1, 130.2, 130.7, 131.6, 133.3, 133.9, 135.4, 139.4, 164.1, 165.8, 167.8.

Method B for the synthesis of 5b

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]thien-3-yl}carbonyl)(phenethyl)amino]-3-(tert-butyl-amino)-3-oxopropanoate (4b-1)

The mixture of 3b (74.3 mg, 0.25 mmol), 2-phenylethylamine (0.25 mmol, 31.4 μL), ethyl glyoxylate (0.25 mmol, 49.6 μL), tert-butyl isocyanide (0.25 mmol, 28.3 μL) in 0.5 mL of methanol was stirring under RT for 2 days. 4b-1 was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (74 mg, yield: 56%). HPLC/MS: t_R = 12.57 min; m/z = 532.2 [M+H]⁺ HRMS: 531.238491 (found); C₂₇H₃₇N₃O₆S, 531.24031 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.30 (3H, t, J = 7.2 Hz), 1.41 (9H, s), 1.50 (9H, s), 3.00 (2H, m), 3.81 (2H, m), 4.25-4.31 (2H, m), 4.49 (1H, s), 6.75 (1H, d, J = 5.4 Hz), 6.87 (1H, d, J = 6.0 Hz), 7.14-7.15 (2H, m), 7.20-7.23 (1H, m), 7.26-7.29 (2H, m), 7.84 (1H, br.s), 9.40 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 14.0, 28.2, 28.5, 35.4, 51.7, 62.3, 64.7, 73.0, 81.6, 114.1, 115.4, 122.7, 126.7, 128.6, 128.7, 137.8, 146.6, 152.3, 164.3, 168.4, 168.7.

3,4-Dihydro-3-(N-tert-butyl)-carboxamide-4-phenethyl-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5b-1)

The mixture of 4b-1 and 0.5 mL of DCM (10% TFA) was stirring under RT overnight. The reaction was added 10 mL of DCM, then neutralized by saturated potassium carbonate (aq). The mixture was extracted by DCM, the organic layer was combined and dried over anhydrous sodium sulfate. After the evaporation of the solvent, the residue was treated by triethylamine (50 μL) and TBD (10 mg) in 0.5 mL of THF, stirring overnight under 40 °C. 5b-1 was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (12 mg, yield: 22% over two steps). HPLC/MS: t_R = 9.79 min; m/z = 386.2 [M+H]⁺ HRMS: 385.145394 (found); C₂₀H₂₃N₃O₃S, 385.14601 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.05 (9H, s), 2.96-3.11 (2H, m), 3.92-4.05 (2H, m), 4.67 (1H, s), 5.35 (1H, br.s), 6.85 (1H, d, J = 5.4 Hz), 7.21- 7.24 (2H, m), 7.28-7.33 (4H, m). ¹³C NMR (150 MHz, CDCl₃): 28.1, 34.3, 51.0, 52.0, 68.4, 118.2, 124.6, 126.8, 127.6, 128.77, 128.79, 137.5, 163.5, 168.5.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]thien-3-yl}carbonyl)(2-(thiophen-3-yl)ethyl) amino]-3-(tert-butyl-amino)-3-oxopropanoate (4b-2)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (60 mg, yield: 45%). HPLC/MS: t_R = 12.44 min; m/z = 538.2 [M+H]⁺ HRMS: 537.194766 (found); C₂₅H₃₅N₃O₆S₂, 537.19673 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.30 (3H, t, J = 7.2 Hz), 1.41 (9H, s), 1.51 (9H, s), 3.24 (2H, m), 3.82-3.87 (2H, m), 4.25-4.32 (2H, m), 4.46 (1H, s), 6.75 (1H, m), 6.82 (1H, m), 6.86 (1H, m), 6.92 (1H, m), 7.15 (1H, m), 7.85 (1H, br.s), 9.44 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 14.0, 28.2, 28.5, 29.5, 51.7, 62.3, 64.6, 72.9, 81.6, 113.9, 115.5, 122.6, 124.1, 125.5, 127.1, 139.9, 146.9, 152.3, 164.1, 168.3, 168.8.

3,4-Dihydro-3-(N-tert-butyl)-carboxamide-4-(2-(thiophen-3-yl)ethyl)-1H-thieno[2,3-e] -1,4-diazepine-2,5-dione (5b-2)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (19 mg, yield: 43% over two steps). HPLC/MS: t_R = 9.64 min; m/z = 392.1 [M+H]⁺ HRMS: 391.101427 (found); C₁₈H₂₁N₃O₃S₂, 391.10243 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.04 (9H, s), 3.21-3.31 (2H, m), 3.99-4.02 (2H, m), 4.68 (1H, s), 5.40 (1H, br.s), 6.83 (1H, m), 6.93 (1H, m), 6.96 (1H, m), 7.18 (1H, m), 7.20 (1H, m). ¹³C NMR (150 MHz, CDCl₃): 28.0, 28.5, 51.0, 52.0, 68.5, 118.1, 124.4, 125.8, 127.2, 127.5, 140.0, 163.5, 163.7, 168.6.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]thien-3-yl}carbonyl)(phenyl)amino]-3-(cyclohexyl-amino)-3-oxopropanoate (4b-3)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (70 mg, yield: 52%). HPLC/MS: t_R = 12.75 min; m/z = 544.0 [M+H]⁺ HRMS: 543.238512 (found); C₂₈H₃₇N₃O₆S, 543.24031 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.27 (2H, m), 1.30 (3H, t, J = 7.2 Hz), 1.37-1.38 (1H, m), 1.39-1.41 (2H, m), 1.52 (9H, s), 1.58 (1H, m), 1.68-1.70 (2H, m), 1.93 (2H, m), 3.83 (1H, s), 4.21-4.24 (2H, m), 4.28-4.29 (1H, m), 4.81 (1H, d, J = 17.4 Hz), 5.08 (1H, d, J = 16.8 Hz), 6.60 (1H, d, J = 6.0 Hz), 6.87 (1H, d, J = 5.4 Hz), 7.32-7.35 (1H, m), 7.39-7.42 (2H, m), 7.51-7.52 (2H, m), 7.92 (1H, br.s), 9.79 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 14.0, 24.5, 25.6, 28.2, 32.4, 32.5, 48.4, 62.2, 63.4, 81.8, 112.9, 115.0, 122.8, 127.2, 127.9, 128.9, 135.9, 149.4, 152.3, 164.3, 168.3, 169.0.

3,4-Dihydro-3-(N-cyclohexyl)-carboxamide-4-benzyl-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5b-3)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (10 mg, yield: 20% over two steps). HPLC/MS: t_R = 10.01 min; m/z = 398.1 [M+H]⁺ HRMS: 397.145096 (found); C₂₁H₂₃N₃O₃S, 397.14601 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.49 (1H, m), 0.59 (1H, m), 0.98-1.02 (1H, m), 1.09-1.11 (2H, m), 1.24-1.27 (2H, m), 1.46 (3H, m), 3.25 (1H, m), 4.07 (1H, d, J = 13.8 Hz), 4.72 (1H, s), 4.84 (1H, br.s), 5.59 (1H, d, J = 14.4 Hz), 6.84 (1H, d, J = 5.4 Hz), 7.20 (1H, d, J = 5.4 Hz), 7.40-7.44 (3H, m), 7.59-7.60 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 24.4, 24.5, 25.2, 31.9, 32.2, 48.1, 52.8, 66.5, 118.0, 123.6, 127.8, 128.9, 129.4, 129.7, 136.7, 163.1, 163.3, 167.7.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]thien-3-yl}carbonyl)(3,4-dimethoxy phenethyl)amino]-3-(cyclopropylmethyl-amino)-3-oxopropanoate (4b-4)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (53 mg, yield: 36%). HPLC/MS: t_R = 11.70 min; m/z = 590.2 [M+H]⁺ HRMS: 589.248129 (found); C₂₉H₃₉N₃O₈S, 589.24579 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.24-0.26 (2H, m), 0.53-0.54 (2H, m), 1.01 (1H, m), 1.30 (3H, t, J = 7.2 Hz), 1.51 (9H, s), 2.95-2.99 (2H, m), 3.17-3.26 (2H, m), 3.84 (3H, s), 3.85 (3H, s), 4.25-4.34 (2H, m), 4.56 (1H, s), 6.65 (1H, m), 6.68-6.70 (1H, m), 6.75-6.78 (2H, m), 6.92 (1H, d, J = 5.4 Hz), 7.98 (1H, br.s), 9.40 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 3.3, 3.4, 10.5, 14.0, 28.2, 34.9, 44.6, 55.86, 55.90, 62.3, 64.2, 81.9, 111.3, 111.8, 113.8, 115.3, 120.6, 122.8, 130.2, 147.5, 147.8, 149.0, 152.3, 165.4, 168.4.

3,4-Dihydro-3-(N-cyclopropylmethyl)-carboxamide-4-(3,4-dimethoxyphenethyl)-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5b-4)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (10 mg, yield: 38% over two steps). HPLC/MS: t_R = 8.98 min; m/z = 444.0 [M+H]⁺ HRMS: 443.151569 (found); C₂₂H₂₅N₃O₅S, 443.15149 (calcd.). ¹H NMR (600 MHz, CDCl₃): -0.03 (2H, m), 0.35 (2H, m), 0.54 (1H, m), 2.65 (1H, m), 2.94-3.06 (3H, m), 3.85 (3H, s), 3.88 (3H, s), 3.96-4.11 (2H, m), 4.69 (1H, s), 5.60 (1H, br.s), 6.80-6.85 (4H, m), 7.17 (1H, d, J = 5.4 Hz). ¹³C NMR (150 MHz, CDCl₃): 3.35, 3.41, 10.4, 33.6, 44.7, 55.88, 55.92, 111.4, 111.9, 113.9, 118.2, 120.8, 127.7, 129.7, 141.7, 147.8, 149.0, 163.4, 163.9, 167.6.

Method C for the synthesis of 5c

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl) (cyclopropyl methyl)amino]-3-(tert-butyl-amino)-3-oxopropanoate (4c-1)

The mixture of 3c (0.2 mmol, 63.8 mg), cyclopropyl methamine (0.2 mmol, 17.3 μL), ethyl glyoxylate (0.2 mmol, 39.7 μL), tert-butyl isocyanide (0.2 mmol, 22.6 μL) in 0.5 mL of methanol was stirring under RT for 2 days. 4c-1 was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (46 mg, yield: 41%). HPLC/MS: t_R = 13.10 min; m/z = 558.2 [M+H]⁺ HRMS: 557.255949 (found); C₂₉H₃₉N₃O₆S, 557.25596 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.19-0.28 (2H, m), 0.60-0.65 (2H, m), 1.10-1.12 (1H, m), 1.31 (3H, t, J = 7.2 Hz), 1.41 (9H, s), 1.53 (9H, s), 3.38-3.42 (1H, m), 3.69-3.72 (1H, m), 4.29 (2H, q, J = 7.2 Hz), 4.65 (1H, s), 7.19 (1H, s), 7.25-7.27 (1H, m), 7.36-7.38 (2H, m), 7.56-7.57 (2H, m), 8.06 (1H, s), 9.53 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 3.4, 4.5, 10.0, 14.0, 28.2, 28.6, 51.6, 62.2, 63.4, 81.8, 115.2, 118.7, 125.2, 127.2, 129.0, 133.3, 133.9, 145.6, 152.4, 164.7, 167.8, 169.1.

7-Phenyl-3,4-dihydro-3-(N-cyclopropylmethyl)-carboxamide-4-cyclopropylmethyl-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5c-1)

The mixture of 4c-1 and 0.5 mL of DCM (10% TFA) was stirring under RT overnight. After the evaporation of the solvent, the residue was treated by triethylamine (100 μL) and TBD (10 mg) in 0.5 mL of THF, stirring overnight under 40 °C. 5c-1 was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (12 mg, yield: 35% over two steps). HPLC/MS: t_R = 10.45 min; m/z = 412.2 [M+H]⁺ HRMS: 411.161429 (found); C₂₂H₂₅N₃O₃S, 411.16166 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.47 (1H, m), 0.54 (1H, m), 0.62 (1H, m), 0.67 (1H, m), 1.15 (9H, s), 1.25 (1H, m), 3.33 (1H, m), 3.90 (1H, m), 4.81 (1H, s), 5.91 (1H, s), 7.29 (1H, m), 7.35-7.38 (2H, m), 7.43 (1H, s), 7.48-7.49 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 3.7, 4.4, 10.2, 28.2, 52.1, 53.7, 67.9, 122.6, 124.9, 125.5, 128.1, 129.1, 132.8, 136.7, 141.7, 163.2, 164.1, 168.2.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl)(2-methoxyethyl)amino]-3-(tert-butyl-amino)-3-oxopropanoate (4c-2)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellow oil (55 mg, yield: 49%). HPLC/MS: t_R = 12.81 min; m/z = 562.1 [M+H]⁺ HRMS: 561.250182 (found); C₂₈H₃₉N₃O₇S, 561.25087 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.31 (3H, t, J = 7.2 Hz), 1.41 (9H, s), 1.52 (9H, s), 3.36 (3H, s), 3.65-3.71 (2H, m), 3.81 (2H, m), 4.27 (2H, m), 4.72 (1H, s), 7.21 (1H, s), 7.23-7.26 (1H, m), 7.34-7.36 (2H, m), 7.53-7.55 (2H, m), 7.93 (1H, br.s), 9.54 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 14.1, 28.2, 28.5, 51.6, 59.0, 62.1, 64.5, 71.2, 81.8, 115.1, 119.0, 125.1, 127.2, 128.9, 133.2, 133.9, 145.6, 152.4, 164.3, 168.2, 168.9.

7-Phenyl-3,4-dihydro-3-(N-tert-butyl)-carboxamide-4-(2-methoxyethyl)-1H-thieno [2,3-e]-1,4-diazepine-2,5-dione (5c-2)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (11 mg, yield: 27% over two steps). HPLC/MS: t_R = 10.12 min; m/z = 416.0 [M+H]⁺ HRMS: 415.155023 (found); C₂₁H₂₅N₃O₄S, 415.15658 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.11 (9H, s), 3.20-3.24 (1H, m), 3.43 (3H, s), 3.63-3.66 (1H, m), 3.89-3.91 (1H, m), 4.53-4.57 (1H, m), 4.71 (1H, s), 6.91 (1H, s), 7.28-7.29 (2H, m), 7.33-7.35 (2H, m), 7.38 (1H, s), 7.45-7.47 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 28.2, 48.9, 51.4, 58.7, 66.4, 68.4, 122.5, 124.6, 125.5, 127.9, 129.0, 133.1, 136.2, 163.8, 164.7, 168.7.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl)(phenethyl) amino]-3-(tert-butyl-amino)-3-oxopropanoate (4c-3)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (63 mg, yield: 52%). HPLC/MS: t_R = 13.43 min; m/z = 608.1 [M+H]⁺ HRMS: 607.270091 (found); C₃₃H₄₁N₃O₆S, 607.27161 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.34 (3H, t, J = 7.2 Hz), 1.44 (9H, s), 1.53 (9H, s), 3.06 (2H, m), 3.89 (2H, m), 4.28-4.34 (2H, m), 4.53 (1H, s), 7.10 (1H, s), 7.19-7.20 (2H, m), 7.22-7.29 (4H, m), 7.35-7.37 (2H, m), 7.50-7.51 (2H, m), 7.88 (1H, br.s), 9.48 (1H, br.s).¹³C NMR (150 MHz, CDCl₃): 14.1, 28.2, 28.6, 35.4, 51.7, 62.4, 64.7, 81.9, 114.9, 118.4, 125.2, 126.8, 127.2, 128.7, 128.8, 128.9, 133.3, 133.8, 137.9, 145.8, 152.3, 168.2, 168.8.

7-Phenyl-3,4-dihydro-3-(N-tert-butyl)-carboxamide-4-phenethyl-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5c-3)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 3:1) as yellowish solids (18 mg, yield: 38% over two steps). HPLC/MS: t_R = 10.89 min; m/z = 462.0 [M+H]⁺ HRMS: 461.175973 (found); C₂₆H₂₇N₃O₃S, 461.17731 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.04 (9H, s), 2.95-3.10 (2H, m), 3.92-4.03 (2H, m), 4,71 (1H, s), 5.45 (1H, br.s), 7.19-7.21 (1H, m), 7.26-7.28 (5H, m), 7.30-7.32 (2H, m), 7.40-7.44 (3H, m). ¹³C NMR (150 MHz, CDCl₃): 28.1, 34.3, 50.9, 52.1, 68.5, 122.5, 125.1, 125.5, 126.8, 128.0, 128.8, 129.0, 132.9, 137.5, 163.6, 164.0, 168.8.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl)(2-methoxyethyl)amino]-3-(cyclopropylmethyl-amino)-3-oxopropanoate (4c-4)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (42 mg, yield: 38%). HPLC/MS: t_R = 12.47 min; m/z = 560.1 [M+H]⁺ HRMS: 559.236369 (found); C₂₈H₃₇N₃O₇S, 559.23522 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.27-0.29 (2H, m), 0.54-0.55 (2H, m), 1.04 (1H, m), 1.32 (3H, t, J = 7.2 Hz), 1.54 (9H, s), 3.22-3.28 (2H, m), 3.39 (3H, s), 3.70-3.75 (2H, m), 3.83-3.92 (2H, m), 4.27-4.32 (2H, m), 4.84 (1H, s), 7.24-7.28 (2H, m), 7.35-7.38 (2H, m), 7.54-7.55 (2H, m), 8.18 (1H, s), 9.51 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 3.37, 3.40, 10.5, 14.1, 28.2, 39.1, 44.5, 59.1, 62.2, 71.2, 82.0, 114.7, 119.1, 125.2, 127.2, 128.4, 128.9, 133.1, 133.9, 152.3, 165.3, 168.3, 168.7.

7-Phenyl-3,4-dihydro-3-(N-cyclopropylmethyl)-carboxamide-4-(2-methoxyethyl)-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5c-4)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 1:1) as yellowish solids (9 mg, yield: 29% over two steps). HPLC/MS: t_R = 9.69 min; m/z = 414.3 [M+H]⁺ HRMS: 413.140096 (found); C₂₁H₂₃N₃O₄S, 413.14093 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.01-0.05 (2H, m), 0.30-0.38 (2H, m), 0.69-0.71 (1H, m), 2.84-2.86 (1H, m), 2.94-2.98 (1H, m), 3.29-3.32 (1H, m), 3.45 (3H, s), 3.65-3.67 (1H, m), 3.94 (1H, m), 4.51-4.54 (1H, m), 4.80 (1H, s), 7.24-7.27 (1H, m), 7.30-7.32 (2H, m), 7.38 (2H, m), 7.43-7.45 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 3.37, 3.39, 10.5, 44.6, 49.1, 58.7, 67.9, 69.5, 122.4, 124.4, 125.4, 127.9, 129.0, 132.9, 136.2, 142.8, 163.8, 165.4, 168.3.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl)(2-methoxyethyl)amino]-3-(cyclohexyl-amino)-3-oxopropanoate (4c-5)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (82 mg, yield: 70%). HPLC/MS: t_R = 13.04 min; m/z = 588.3 [M+H]⁺ HRMS: 587.265023 (found); C₃₀H₄₁N₃O₇S, 587.26652 (calcd.). ¹H NMR (600 MHz, CDCl₃): 1.21- 1.25 (3H, m), 1.29 (3H, t, J = 7.2 Hz), 1.35-1.42 (2H, m), 1.52 (9H, s), 1.58-1.60 (2H, m), 1.71 (2H, m), 1.94-1.96 (2H, m), 3.36 (3H, s), 3.67 (1H, m), 3.73 (1H, m), 3.80-3.82 (3H, m), 4.27 (2H, m), 4.80 (1H, s), 7.21-7.25 (2H, m), 7.33-7.35 (2H, m), 7.52-7.53 (2H, m), 7.97 (1H, d, J = 7.2 Hz), 9.47 (1H, s). ¹³C NMR (150 MHz, CDCl₃): 14.1, 24.6, 25.6, 28.2, 32.5, 32.6, 48.6, 59.0, 62.1, 64.3, 71.0, 81.9, 114.9, 119.0, 125.1, 127.2, 128.9, 133.2, 133.9, 146.2, 152.3, 164.2, 168.4, 168.5.

7-Phenyl-3,4-dihydro-3-(N-cyclohexyl)-carboxamide-4-(2-methoxyethyl)-1H-thieno [2,3-e]-1,4-diazepine-2,5-dione (5c-5)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (12 mg, yield: 19% over two steps). HPLC/MS: t_R = 10.44 min; m/z = 442.1 [M+H]⁺ HRMS: 441.171348 (found); C₂₃H₂₇N₃O₄S, 441.17223 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.85-0.87 (1H, m), 1.06-1.16 (3H, m), 1.27-1.28 (1H, m), 1.44-1.51 (2H, m), 1.64-1.66 (1H, m), 1.76-1.77 (1H, m), 3.27-3.28 (1H, m), 3.45 (3H, s), 3.54-3.56 (1H, m), 3.64-3.66 (1H, m), 3.96 (1H, m), 4.54-4.57 (1H, m), 4.76 (1H, s), 7.07 (1H, d, J = 7.2 Hz), 7.28-7.30 (1H, m), 7.33- 7.36 (2H, m), 7.45-7.46 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 24.5, 24.7, 25.4, 32.4, 32.7, 48.3, 49.2, 58.7, 67.9, 69.5, 122.6, 124.5, 125.5, 127.9, 129.0, 133.0, 136.3, 151.6, 163.7, 164.5, 168.3.

Ethyl 2-[({2-[(tert-butoxycarbonyl)amino]-5-phenylthien-3-yl}carbonyl)(2-cyclopropylmethyl)amino]-3-(cyclohexyl-amino)-3-oxopropanoate (4c-6)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 5:1) as yellowish solids (70 mg, yield: 60%). HPLC/MS: t_R = 13.36 min; m/z = 584.2 [M+H]⁺ HRMS: 583.271707 (found); C₃₁H₄₁N₃O₆S, 583.27161 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.19-0.24 (2H, m), 0.62-0.63 (2H, m), 1.11 (1H, m), 1.25 (1H, m), 1.29 (3H, t, J = 7.2 Hz), 1.31-1.34 (2H, m), 1.38-1.42 (2H, m), 1.52 (9H, s), 1.55-1.59 (1H, m), 1.70 (2H, m), 1.93 (2H, m), 3.42-3.44 (1H, m), 3.67-3.70 (1H, m), 3.81-3.87 (1H, m), 4.28 (2H, q, J = 7.2 Hz), 4.74 (1H, s), 7.19 (1H, s), 7.24-7.27 (1H, m), 7.35-7.37 (2H, m), 7.54-7.56 (2H, m), 8.05 (1H, d, J = 7.2 Hz), 9.46 (1H, br.s). ¹³C NMR (150 MHz, CDCl₃): 3.6, 4.5, 10.0, 14.1, 24.5, 25.6, 28.2, 32.4, 32.6, 48.5, 55.5, 62.2, 63.2, 82.0, 115.0, 118.8, 125.2, 127.3, 129.0, 133.3, 133.9, 146.3, 152.3, 165.0, 168.0, 168.7.

7-Phenyl-3,4-dihydro-3-(N-cyclohexyl)-carboxamide-4-cyclopropylmethyl-1H-thieno[2,3-e]-1,4-diazepine-2,5-dione (5c-6)

The product was isolated by silica gel chromatography (petroleum ether/ ethyl acetate, 2:1) as yellowish solids (13 mg, yield: 25% over two steps). HPLC/MS: t_R = 10.67 min; m/z = 438.2 [M+H]⁺ HRMS: 437.176794 (found); C₂₄H₂₇N₃O₃S, 437.17731 (calcd.). ¹H NMR (600 MHz, CDCl₃): 0.46-0.53 (2H, m), 0.60-0.63 (2H, m), 0.87-0.91 (1H, m), 1.05-1.10 (2H, m), 1.15-1.32 (4H, m), 1.46-1.50 (2H, m), 1.61-1.63 (1H, m), 1.72-1.74 (1H, m), 3.31-3.34 (1H, m), 3.60-3.62 (1H, m), 3.88-3.92 (1H, m), 4.86 (1H, s), 6.02 (1H, d, J = 7.2 Hz), 7.29 (1H, m), 7.33-7.35 (2H, m), 7.39 (1H, s), 7.45-7.46 (2H, m). ¹³C NMR (150 MHz, CDCl₃): 3.7, 4.4, 10.3, 24.5, 24.7, 25.3, 32.5, 32.6, 48.5, 53.8, 67.6, 122.8, 124.4, 125.4, 127.9, 129.0, 133.0, 136.2, 163.4, 164.3, 168.2.

NMR and FP binding assays

The Human recombinant Mdm2 (residues 1-118) and p53 (residues 1-321) were expressed and purified as described in Bista et al., 2009 (18). Uniform ¹⁵N labeling was achieved by growing the Escherichia coli BL21(DE3) RIL in M9 minimal medium containing ¹⁵NH₄Cl as nitrogen source (19). The SEI-AIDA experiment was performed according to Bista et al., 2009 (18). 14 μM Mdm2/p53 complex was mixed with compounds in 1:1 molar ratio; amount of p53 released from the complex by the compound was estimated from 1D spectrum and K_d of Mdm2-inhibitor interaction was calculated according to Krajewski et al., 2007 (20). Fluorescence polarization (FP) binding assays were performed according to Czarna et al., 2009 (21). Binding constant and inhibition curves were fitted using the SigmaPlot (SPSS Science Software).

¹H-¹⁵N HSQC spectra were acquired using the fast-HSQC pulse sequence (Mori et al., 1995) (22). For the ¹H-¹⁵N HSQC spectrum, a total of 2048 complex points in t2 and 192 t1 increments were acquired. NMR data were processed using the Bruker program Xwin-NMR version 3.5. Titration experiments were performed using a series of ¹H-¹⁵N HSQC of labeled Mdm2 or unlabeled Mdm2 along with the unlabelled partner. 10% D₂O was added to NMR samples to provide lock signal. 100mM stock solutions of the compounds were prepared by dissolving them in perdeuterated DMSO. For HSQC titration, 0.2 mM ¹⁵N-labeled Mdm2 was mixed with compounds in 1:1 and 1:5 protein: compound molar ratio. Normalized chemical shift perturbations were calculated according to equation (Stoll et al., 2001) (23):

$$\mathrm{\Delta }\delta =\sqrt{0.04{\left({\delta }_N^{\mathit{bound}}-{\delta }_N^{\mathit{free}}\right)}^2+{\left({\delta }_H^{\mathit{bound}}-{\delta }_H^{\mathit{free}}\right)}^2}$$

Virtual Library Generation

The virtual library of Gewald-UDC thienodiazepinediones was created using the Reactor software as described in the manual (24). As starting materials, we used commercially available 2-aminothiophenes (N = 120), 349 primary amines and 322 isocyanides. The isocyanides are either commercially available or can be synthesized in 1-2 steps using the classical Hoffmann or Ugi syntheses (25). Consequently the theoretical chemical space of this virtual library is 120 × 349 × 322 = 13,485,360 (not including stereoisomers), which is almost unmanageable when calculating the physicochemical properties. As the father of modern MCR chemistry, Ivar Ugi pointed out in 1971 “if, for example, 40 each of the different components are reacted with one another, the result is 40⁴ = 2,560,000 reaction products, which is quite a high figure considering that it is the same order of magnitude as the total number of chemical compounds described to date” (25). Nowadays more than 1,000 isocyanides are commercially available, thus the U-4CR space increased to 1,000⁴ = 10¹¹ compounds which is out of reach of every current super computers. Thus, in order to investigate such large chemical spaces the program RandReactor was written to provide smaller random sublibraries of such very large chemical spaces. The program code is freely available and described in the Supplemental Information.

Results & Discussion

Synthesis of 1,4-thienodiazepine-2,5-dione scaffold

The synthesis of the thienodiazepinedione scaffold 5 was designed by employing the Ugi four-component reaction (U-4CR) and a subsequent deprotection and ring closure. In order to access a new scaffold with high variability the thiophene carboxylic acid was synthesized by another versatile MCR the Gewald-3CR. We synthesized three different exemplary substituted thiophene carboxylic acids to investigate scope and limitations and the influence of this particular starting material class in the subsequent UDC procedure. Cyclohexane-fused aminothiophene 2a was prepared via the G-3CR of cyclohexanone, sulfur and methyl cyanoacetate (26). 4,5-Unsubstituted aminothiophene 2b was synthesized from the Gewald reaction of 1,4-dithiane-2,5-diol and methyl cyanoacetate (27). Phenyl-substitued aminothiophene 2c was obtained from the G-3CR of phenylacetaldehyde, sulfur and methyl cyanoacetate (27). Then we initiated the preparation of thiophene carboxylic acid 3a-c by Boc-protection of 2a-c, followed by hydrolysis (Scheme 1). After pH adjustment to 6, compounds 3a-c were precipitated out and collected by filtration, which are ready to be applied according to UDC strategy.

Scheme 1.

Synthesis of 3a-c.

Therefore, N-Boc thiophene carboxylic acid 3a was employed as the bifunctional mono protected starting material for the synthesis of 1,4-thienodiazepine-2,5-diones 5a. Interestingly, there is no previous report on the compatibility of Gewald aminothiophene derivatives employed in the Ugi four-component reaction (U-4CR). A preliminary investigation by the reaction of thiophene carboxylic acid 3a, benzylamine, tert-butyl isocyanide, and ethyl glyoxalate, gave the desired condensation product 4a-1 in 65% yield (Scheme 2). The previous work in our lab demonstrated that TBD is an efficient organocatalyst for amidations of primary and secondary amines under mild conditions (28). As expected, compound 5a-1 was obtained by the deprotection of 4a-1 under a 10% solution of TFA in dichloromethane and following cyclization using catalytic amount of TBD in 62% yield over two steps.

Scheme 2.

Ugi four-component reaction of 3a. Regents and conditions: MeOH, RT, 2 days, 65%.

Under further investigation, we found that it is not necessary to isolate the Ugi product as the intermediate for next steps. The following deprotection of the crude Ugi product and subsequent cyclization gave the corresponding thienodiazepinedione 5a-1 in 33% yield over three steps. Encouraged by these results, a small focused library of thienodiazepinediones 5a was synthesized in order to enlarge the diversity of reactants (Table 1). The thienodiazepinedione products were isolated by chromatography without isolation of the intermediate. This protocol works well by employing different substituted amines and isocyanides as well. Overall, the yield is around 50-80% in average for each step of the transformation. Thus we predict that the procedure isolating and purifying (e.g. by mass directed HPLC) only the final product will be useful for automated library generation for the production of large libraries.

Table 1.

Synthesis of 1,4-thienodiazepine-2,5-diones 5a

entry	R1	R2	compound	yield (%)a
1	t-Bu	benzyl	5a-1	33
2	t-Bu	2-phenyl ethyl	5a-2	53
3	t-Bu	4-chlorobenzyl	5a-3	28
4	t-Bu	4-chlorophenyl	5a-4	16
5	cyclopropyl methyl	benzyl	5a-5	26
6	2,4,6-(CH3)3C6H2	4-chlorobenzyl	5a-6	12
7	benzyl	4-chlorobenzyl	5a-7	16
8	3,4-dichlorobenzyl	4-chlorobenzyl	5a-8	16

^aisolated yields, over three steps (Method A).

The Ugi reaction of N-Boc thiophene carboxylic acid 3b, 2-phenyl ethylamine, tert-butyl isocyanide, and ethyl glyoxalate, gave the corresponding condensation product 4b-1 in 56% yield, isolated by chromatography. A mild condition was adopted for the deprotection of Ugi product 5b-1 using a 10% solution of TFA in dichloromethane. However, the ‘three-step, one-separation’ procedure for the synthesis of 5b-1 is unsatisfactory by using the crude Ugi product 6a without separation. The transformation of 4b-1 after purification afforded the corresponding 1,4-thienodiazepine-2,5-dione 5b-1. N-Boc thiophene carboxylic acid 3b was employed as the bifunctional starting material for the synthesis of 1,4-thienodiazepine-2,5-diones 5b under Ugi-deprotection-cyclization sequence (Table 2). Meanwhile, 1,4-thienodiazepine-2,5-diones 5c were synthesized from N-Boc thiophene carboxylic acid 3c under similar conditions. The Ugi reaction of 3c and ethyl glyoxalate with different amines and isocyanides gave the condensation products 4c in 38-70% yield. Then, a series of 1,4-thienodiazepine-2,5-diones 5c were obtained by deprotection and subsequent cyclization of the corresponding Ugi products (Table 2).

Table 2.

UDC approach for the synthesis of 5b and 5c

entry	R1	R2	compound	yield (%)a
1	t-Bu	2-phenyl ethyl	4b-1	56
			5b-1	22b
2	t-Bu	3-thienyl ethyl	4b-2	45
			5b-2	43b
3	cyclohexyl	benzyl	4b-3	52
			5b-3	20b
4	cyclopropyl methyl	3,4-dimethoxy phenyl ethyl	4b-4	36
			5b-4	38b
5	t-Bu	cyclopropyl methyl	4c-1	41
			5c-1	35c
6	t-Bu	2-methoxy ethyl	4c-2	49
			5c-2	27c
7	t-Bu	phenyl ethyl	4c-3	52
			5c-3	38c
8	cyclopropyl methyl	2-methoxy ethyl	4c-4	38
			5c-4	29c
9	cyclohexyl	2-methoxy ethyl	4c-5	70
			5c-5	19c
10	cyclohexyl	cyclopropyl methyl	4c-6	60
			5c-6	25c

^aIsolated yields;

^bover two steps (Method B);

^cover two steps (Method C).

In summary, we have demonstrated that TDZ scaffold can be achieved by the union of two sequential multicomponent reactions (29). This approach takes advantages of the maximal points of diversity offered by the G-3CR and U-4CR. A wide range of amines and isocyanides, as well as thiophene carboxylic acids are tolerated under the reaction procedure. All compounds are racemic, because the Ugi reaction yields a new stereocenter at C-3 position. Our synthetic strategy allows convenient preparation of these compounds to avoid the limitations imposed by traditional methods involving the condensation of amino acid derivatives.

Cheminformatics of virtual libraries

In order to evaluate the chemical space of TDZ scaffold, a virtual library was created based on the synthetic route (refer to Figure 2). We developed a random module software for the program Reactor (JChem 5.2.2, 2009, www.chemaxon.com) allowing us to generate a random virtual library (N = 50,000), which is freely available and described in the Supplemental Information. The compounds from the virtual library were introduced into Instant JChem (Instant JChem 2.5.1, 2009, www.chemaxon.com) to calculate some main physical properties. In addition, the compound library of commercial available benzodiazepines (substructure search from eMolecules, N = 2,498) was also analyzed by Instant JChem for comparison. The distribution of benzodiazepine library and a random virtual library of 1,4-thienodiazepine-2,5-diones (N = 50,000) was presented in term of the major descriptors molecular weight, logP and TPSA (Figure 3). The virtual library based on this new scaffold enlarges the chemical space of the benzodiazepine family. We foresee this scaffold to be useful in virtual screening and lead discovery, due to its largely unexplored chemical space.

Figure 3.

The distribution of molecular weight, logP and TPSA: (A) benzodiazepine library of a substructure search from eMolecules (N = 2,498); (B) a random virtual library of 1,4-thienodiazepine-2,5-diones (N = 50,000).

The physical properties of the virtual library were analyzed by frequency distributions in PASW Statistics 18 (Figure S1 in Supplemental Information). The range of molecular weight (between 253.3 Da. and 569.7 Da. with a mean value of 469.7 Da.) is broad due to the diversity of reactant components. The molecular weight of the compounds we physically synthesized is less than 500 Da. The mean of logP is 2.1 with standard deviation 2.3 indicating the drug-like property. Total polar surface area (TPSA) is a major descriptor relevant for example for cell membrane permeability (30). TPSA of the majority of compounds is between 126.8 Ų and 190.4 Ų. The number of rotatable bonds (NRB), a very good descriptor of oral bioavailability, has a mean value of 7 with the standard deviation 2. The number of HBA (hydrogen bond acceptors) ranges from 4 to 13, with an average number of 7. The number of HBD (hydrogen bond donors) ranges from 2 to 8, with an average number of 3. In terms of drug likeness, 67.0% of 50,000 compounds obey Lipinski’s rule. And 85.8% of them are predicted to be bioavailable (mass ≤ 500, LogP ≤ 5, HBD ≤ 5, HBA ≤ 10, PSA ≤ 200, NRB ≤ 10, and fused aromatic rings ≤ 5). Noteworthy, the overall shape of the compounds based on the thienodiazepinedione scaffold is non-planar. The presence of stereochemistry and sp³ centers has been recognized to correlate with success as compounds transition from discovery, through clinical testing, to drugs (31). Based on the commonly accepted descriptors, it is likely that lead-like compounds with novel and potent biological activity will be obtained based on this scaffold.

We also create a virtual library of 3-D conformers, which can be used as an input for docking and other virtual analysis software (Supplemental Information). Omega (Omega 2.3.2, 2009, www.eyesopen.com) was used to generate 3-D structures of a random virtual library (N = 5,000). For example, the tricyclic backbone (6-, 5-, 7-membered rings) in 3-D structures of compound 5a-7 has the same conformation, while two side chains are oriented oppositely with certain rotation flexibility (Figure S2 in Supplemental Information).

Antagonist activities of p53-Mdm2 interaction

Since quite sometimes we are interested in the design of small molecular weight p53-Mdm2 antagonists and new scaffolds desirable for potent and selective candidates (32-34). The tumor suppressor p53 is well recognized as a therapeutic target for new anticancer interventions (35). The activation of wild-type p53 in human tumors by antagonizing murine double minute 2 (Mdm2) is a promising and potentially non toxic therapeutic strategy (36). The disruption of the p53-Mdm2 interaction can be accomplished by mimicking the p53 fragment with particular emphasis on the Mdm2 binding site using peptides, foldamers and peptoids (α-helical transactivation domain) (37). However, small-molecule libraries are favorable for the design of Mdm2 antagonists in terms of the desirable bioavailability and stability (33). The first class of potent and selective small-molecule Mdm2 antagonists, nutlins were identified from cis-imidazoline compounds (38). Next, benzodiazepinedione compound 6 has been identified as a potent Mdm2 antagonist (K_d = 80 nM) (39). The cocrystal structure of Hdm2 complex shows that compound 6 binds to the p53-binding site (side chains Phe 19, Trp 23, and Leu 26) of Hdm2 (Figure 4). Such benzodiazepinedione inhibitors were found to suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo (40). Further optimization and SAR were investigated in order to improve the potency and cellular activity of BDZ antagonists (41-44). Although bioisosterism is a lead modification approach to attenuate the biological properties of the lead compounds, 1,4-thienodiazepine-2,5-dione scaffold (TDZs) as a potential pharmacophore has been rarely investigated so far. Thus we hypothesized that the peptidomic 1,4-thienodiazepine-2,5-dione scaffold could also act as α-helix mimetics to disturb the p53-Mdm2 interaction.

Figure 4.

Cocrystal structure of benzodiazepinedione Hdm2 antagonist (PDB code: 1T4E). Compound 6 is shown in yellow sticks, three water molecules as turquoise balls, the receptor amino acid side chains are labeled around the binding site. The hydrogen bonding network is shown in red dash line.

Since thienodiazepinediones are bioisosteric to benzodiazepindiones, we screened the compound library by our recently developed fluorescence polarization (FP) assay.(21) This robust FP assay uses fluorescent p53-like peptide and recombinant Mdm2 to measure the potency of p53-Mdm2 antagonist. To our delight, 5a-7 and 5a-8 have shown antagonist activities of p53-Mdm2 interaction through FP assay screening. Both antagonists have dose dependent effect to compete with p53-like peptide, as shown in Figure 5. The measured inhibition constant (K_i) values of Mdm2 with 5a-7 and 5a-8 were determined as 40 μM and 45 μM, respectively.

Figure 5.

Some thienodiazepinediones are competitive inhibitors of the p53-Mdm2 interaction: (A) FP binding curve of compound 5a-7, (B) FP binding curve of compound 5a-8.

Compound 5a-7 was docked into the p53 binding site of Mdm2 in order to understand the possible binding mode of small molecular antagonists (Figure 6). We were using the modeling/docking software MOLOC (Gerber, P.; www.moloc.ch) (45,46). Compound 5a-6 also has weak interaction with Mdm2, which indicates that p-chlorobenzyl fragment might be crucial for the binding. This is consistent with other reported scaffolds, such as benzodiazepinedione 6, as well as nutlins, chromenones and spirooxindoles (23,38,47). Our recently described 3-finger pharmacophore model can be applied here and suggests that the compounds bind such that the p-chlorobenzyl group mimics Trp23, the cyclohexyl and the benzyl moiety mimics Leu26 and Phe19, respectively (33). This is consistent with the docking model suggesting that the 4-chlorobenzyl group points deeply into tryptophan binding site.

Figure 6.

Compound 5a-7 (yellow sticks) docked into p53 binding site of Mdm2 (PDB code: 1YCR). Two low energy poses are shown (yellow sticks) as well as the side chains of the p53 hot spot FWL (pink sticks). In the first model the thiophene annulated cyclohexyl ring and the benzyl group mimics Phe19 and Leu26, respectively; whereas in the second model the cyclohexylthiophene fragment lays on top of the Leu26 binding site and the benzyl group points into the Phe19 binding site. Both poses suggest that there is not perfect shape and electrostatic complementarity which accounts for the moderate affinity for Mdm2.

Next, we utilized NMR to investigate the antagonistic behavior of compounds 5a-7 and 5a-8 towards the p53-Mdm2 interaction. For this purpose, we used the NMR-based antagonist induced dissociation assay (AIDA) that we developed recently (18,20). This NMR competition assay is performed on the Mdm2/p53 complex and the release of p53 from the complex is monitored as a function of the increased addition of an inhibitor. Therefore, AIDA-NMR indicates not only whether the compound is able to inhibit the protein-protein interaction in vitro, but also the dissociation constant (K_d) of the compound-Mdm2 interaction. The assay shows that the compounds are able to dissociate the Mdm2/p53 complex, and K_d values are 30 ± 20 μM and 10 ± 6 μM for compound 5a-7 and 5a-8, respectively.

Furthermore, we used the heteronuclear single quantum coherence (HSQC) NMR spectroscopy, which has been intensively used to monitor the biophysical properties of p53-Mdm2 antagonists (23,48,49). 2D HSQC spectra, showing binary titrations of ¹⁵N-Mdm2 with compounds 5a-7 and 5a-8, are shown in Figure 7 and Figure 8, respectively. Although compound 5a-8 shows a stronger binding affinity, both antagonists have similar modes of binding to Mdm2. The biggest chemical shift perturbations occur in proximity of the p53 binding site, as shown in Figure 7.

Figure 7.

2D ¹H-¹⁵N HSQC spectrum of Mdm2 titrated with compound 5a-7. (Blue: human Mdm2(1-118) mixed molar with excess of compound 5a-7 (Mdm2:compound 5a-7 ratio 1:5); Red: the reference spectrum of free Mdm2)

Figure 8.

2D ¹H-¹⁵N HSQC spectrum of Mdm2 titrated with compound 5a-8. (Blue: human Mdm2(1-118) mixed molar with excess of compound 5a-8 (Mdm2:compound 5a-8 ratio 1:5); Red: the reference spectrum of free Mdm2)

Conclusions and Future Directions

We have described the synthesis of the new scaffold 1,4-thienodiazepine-2,5-dione using the unprecedented union of Gewald and Ugi multicomponent reactions and incorporating the Ugi-Deprotection-Cyclization (UDC) approach. The compounds accessible can be varied at four points in the molecular skeleton. The synthesis can be easily adapted for high throughput chemistry or medicinal chemistry for high-dimensional combinatorial libraries for screening purpose. The resulting compounds are in general drug-like by means of the Pfizer rules. Some compounds were shown to antagonize the p53-Mdm2 protein-protein interaction by two complementary screening techniques, and can provide a good starting point for further design and optimization. Moreover, we provide a useful program to generate the random virtual library for computational chemistry applications. Current efforts in our laboratories focus on the optimization of the potency of p53-Mdm2 antagonists based on this interesting new class of compounds.

Figure 9.

NMR mapping of binding site of compound 5a-8. Atoms experiencing very large (Δδ > 0.08 ppm) and large (Δδ > 0.04 ppm) differences in chemical shifts upon addition of molar excess of the compound are marked red and orange, respectively. Assignment of the N-terminal domain of Mdm2 was published previously (23).

Abbreviations

AIDA

antagonist induced dissociation assay

BDZ

1,4-benzodiazepin-2,5-dione

FP

fluorescence polarization

G-3CR

Gewald three-component reaction

HSQC

heteronuclear single quantum coherence

MCR

multicomponent reaction

Mdm2

murine double minute 2

TBD

1,5,7-triazabicyclo[4,4,0]dec-5-ene

TDZ

1,4-thienodiazepine-2,5-dione

UDC

Ugi-Deprotection-Cyclization

U-4CR

Ugi four-component reaction