8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction

Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.


General considerations
All commercial chemicals were used without prior purification. Polymer supported fluoride (2.0-3.0 mmol/g, 20-50 mesh) was purchased from Sigma Aldrich. Polymer supported base Amberlite IRA-67 (5.6 mmol/g) was purchased from Sigma Aldrich. CH 2 Cl 2 was distilled from calcium hydride and THF was distilled from sodium and benzophenone when dry solvents were used. All reactions were monitored by TLC (Merck silica gel 60 F 254 ) and analysed under UV (254 nm). Microwave reactions were performed in a Biotage Initiator reactor with fixed hold time. Column chromatography was performed by flash chromatography (wet-packed silica, 0.04-0.063 mm) or by automated column chromatography on a Biotage SP-4 instrument using pre-packed silica columns. 1 H and 13 C NMR spectra were obtained at 400 and 100 MHz respectively, using a Varian 400/54 spectrometer. The solvent peak was used as reference. LCMS analysis was performed on a API SCIEX 150 EX Perkin Elmer ESI-MS (30 eV) connected to a Perkin Elmer gradient pump system and a C8 column (Gemini) using acetonitrile and MilliQ-water with 1% formic acid as mobile phases with a gradient of 5 to 95% acetonitrile over 4 min. Analytical highperformance liquid chromatography (HPLC) analysis was carried out on a Waters separation module 2690 connected to a Waters photodiode array detector 996 using an Atlantis ® 5 μm C18 AQ (250×4.6 mm) column. Preparative HPLC was carried out on a Waters 600 controller connected to a Waters 2487 Dual λ Absorbance detector using an Atlantis ® Prep T3 5 μm C-18 (250×19 mm) column. HRMS analysis was performed on a Waters LCTp XE mass spectrometer with an Acquity UPLC BEH C18 (pH 10) or an Acquity UPLC CSH C18 (pH 3) column eluting with a gradient of 5-95% acetonitrile in MQ-water. Synthesis of 5a, 4a, 3a and 2a have recently been published and can be found in reference [1].

General procedure A: Mitsunobu reaction in the 9-position
The alkylation was performed following a published procedure [2] with minor modifications. 1 (1.0 eq.) was dissolved in dry THF under nitrogen in oven-dried round-bottomed flask and alcohol (1.1-2.5 eq.) and PPh 3 (1.1-2.5 eq.) was added. The nitrogen flow was temporarily suspended when solid alcohols were added. When all of the PPh 3 was dissolved, DIAD (1.0-2.5 eq.), was added drop wise. The reaction was stirred at room temperature under nitrogen, until TLC indicated full consumption of the starting material, unless otherwise noted. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography or automated flash column chromatography.

General procedure B: Mitsunobu reaction in the 2-position
The alkylation was performed following a published procedure [2] with minor modifications. The purine was dissolved in dry THF under nitrogen in an oven-dried round-bottomed flask, PBu 3 (2.4-2.6 eq.), alcohol (2.4-2.6 eq.) and ADDP (2.5-2.6 eq.) were added in that order. The nitrogen flow was temporarily removed when solid alcohols and ADDP were added. The reaction was stirred at room temperature, until TLC indicated full consumption of the starting material, unless otherwise noted. The obtained white precipitate was filtered off, washed with THF and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography or automated flash column chromatography.

General procedure C: Substitution of chloride with dimethylamine
Dimethylamine substitution of 6-chloropurines was performed following a published procedure [3] with modifications. The 6-chloropurines (1.0 eq.) were dissolved in dry DMF in a dry microwave vial without a magnetic stirring bar. The vial was capped and nitrogen was bubbled through the solution. The reaction mixture was heated in a microwave reactor at 180°C until the starting material was consumed according to TLC. The solvent was removed under reduced pressure, co-evaporated with toluene when needed and the crude product was purified by flash column chromatography.
General procedure D: 8-bromination of 2,6,9-substituted purines Purine (1.0 eq.) was dissolved in dry CH 2 Cl 2 in oven-dried glassware under nitrogen. The nitrogen flow was temporarily removed when pyridinium tribromide (PyrBr 3 ) (1.1-1.4 eq.) was added in one portion, the reaction mixture was stirred at room temperature under nitrogen until full conversion was observed by TLC or LCMS. The reaction was quenched with 10% Na 2 S 2 O 3 (aq.); the colour changed from yellow to colourless. The pH was adjusted to 9-12 with 15% NaOH (aq.). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (x 3). The organic phases were pooled, washed with brine, dried over Na 2 SO 4 and filtered. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography.

2-Amino-9-tert-butoxycarbonyl-6-chloropurine
Following a published procedure [2] with minor modifications, 2-amino-6chloropurine (2.50 g, 14.7 mmol) and di-tert-butyl dicarbonate (3.21 g, 14.7 mmol) were dissolved in anhydrous DMSO (50 ml). The pale yellow solution was cooled in an ice bath with vigorous stirring and the ice bath was removed just as the DMSO started to freeze. DMAP (90 mg, 0.74 mmol) was added and the solution was stirred under nitrogen at room temperature for 8 h. Additional tbocanhydride (220 mg, 1.00 mmol) was added and the reaction mixture was stirred at room temperature overnight. Full consumption of starting material and formation of one new spot was confirmed by TLC (10% methanol in CHCl 3 ).The reaction mixture was diluted with water (450 ml) and extracted with ethyl acetate (3 x 150 ml). The organic phases were washed with water (5 x 100 ml), dried over Na 2 SO 4 , filtered and evaporated to give the expected product as a white solid (3640 mg, 92%) which was used without further purification in in the next step. 1 H and 13 C NMR corresponds with previously published results [1]. 1

2-tert-Butoxycarbonylamino-6-chloropurine (1)
Sodium hydride (715 mg, 17.9 mmol, 60 % mineral oil dispersion) was added to a stirred solution of 1-amino-9-tert-butoxycarbonyl-6-chloropurine (3490 mg, 12.9 mmol) in dry THF (120 ml) at room temperature under nitrogen. The white suspension was stirred at room temperature for 2 h. Full conversion of starting material was confirmed by TLC (70% ethyl acetate in pentane). The reaction was cooled to 0°C and quenched with brine (5 ml), a white precipitate was observed after the addition. The mixture was then allowed to reach room temperature. The THF was removed under reduced pressure (note: save 10-20 ml THF, simplifies the extraction). CHCl 3 (200 ml) and distilled water (800 ml) were added (everything did not dissolve). The pH on the aqueous phase was determined to 14. The phases were separated and extracted with CHCl 3 (3 x 100 ml, note: in between every extraction wait approximately 10 min) and these organic phases were discarded. Sat. NaHCO 3 (aq.) was added to the aqueous phase whereupon a white precipitate was observed and the aqueous phase was extracted with CHCl 3 (3 x 100 ml). The combined organic phases were washed with brine. The solvent was removed under reduced pressure to yield 1 as a white solid in quantitative yield. 1 H and 13 C NMR corresponds with previously published results [1]. 1  6-Chloro-2-tert-butoxycarbonylamino-9-isobutylpurine (2b) Compound 2b was synthesised following general procedure A from 1 (1500 mg, 5.56 mmol) isobutyl alcohol (0.55 ml, 5.94 mmol), PPh 3 (1605 mg, 6.12 mmol) and DIAD (1.1 ml, 5.6 mmol) in dry THF (75 ml). Full consumption of starting material was confirmed by TLC (eluting first with 50% ethyl acetate in heptane and then with 10% methanol in CHCl 3 ) after 1.5 h. The crude was purified by flash column chromatography (dryloaded from CH 2 Cl 2 on Celite®, 40% ethyl acetate in heptane) to provide 2b as a white solid (1646 mg, 91%). 1

2-Benzylamino-6-dimethylamino-9-cyclopentylmethyl-8-ethynylpurine (6c)
Amberlite IRA-67 (619 mg, 3.47 mmol), CuI (22 mg, 0.12 mmol) and PdCl 2 (PPh 3 ) 2 (21 mg, 0.030 mmol) were added to a solution of 5c (310 mg, 0.722 mmol) in THF (9 ml). The capped microwave vial was bubbled with nitrogen and ethynyltrimethylsilane (0.30 ml, 2.2 mmol) was added. The reaction mixture was heated in the microwave reactor at 110 °C for 50 min. Reaction was monitored by TLC (15% ethyl acetate in pentane). The reaction mixture was filtered through a short plug of silica and eluted with THF. The solvent volume was adjusted to 12 ml, polymer supported fluoride (386 mg) was added and the reaction was stirred at room temperature overnight. Full consumption of starting material was indicated by TLC (15% ethyl acetate in pentane). The polymer was filtered off and washed with CH 2 Cl 2 and THF. The solvents were removed and the crude product was purified by automated flash column chromatography (0-15% ethyl acetate in pentane) to provide 6c as a brown solid (155 mg, 57%). 1

Benzyl azide[4]
NaN 3 (131 mg, 2.02 mmol), MQ-water (2 ml) and benzyl bromide (120 μl, 1.01 mmol) were added in that order to a 2-5 ml microwave vial which was capped, flushed with nitrogen and heated in a microwave reactor at 120°C for 40 min. The reaction mixture was allowed to cool to ambient temperature and was then extracted with DEE (3 x 5 ml) (extraction was performed with glass pipette in the reaction vial). The collected organic phases were dried over Na 2 SO 4 , gravity filtrated in to a plastic falcon vial, and the solvents were removed under reduced pressure to provide benzyl azide as a clear liquid (110 mg, 82%). 1

H NMR (CDCl 3 )
corresponded to the published data and the product was used in the cyclization reactions without further purification). 1

9-Benzyl-2-(N-benzyl-tert-butoxycarbonylamino)-6-chloro-purine (10d)
Benzyl alcohol (1.15 ml, 11.1 mmol) was added to a stirred solution of 1 (1.00 g, 3.72 mmol) in dry THF (50 ml) followed by addition of PBu 3 (2.80 ml, 11.2 mmol) under nitrogen at room temperature. The septum was removed and ADDP (2.82 g, 11.2 mmol) was added, the septum with nitrogen inlet was quickly replaced and the reaction mixture was stirred at room temperature. The orange colour faded within 20 min to a pale yellow. Precipitation formed after 1 h. The reaction mixture was stirred at room temperature for 5 h after which time all starting material was consumed as observed by TLC (10% methanol in CHCl 3 ). The precipitate was filtered off and washed with small portions of cold THF (3 x 2 ml). The solvent was removed under reduced pressure, the crude was re-dissolved in ethyl acetate (180 ml), washed with sat. NaHCO 3 (aq., 3 x 30 ml), brine (30 ml) and dried over Na 2 SO 4 . The solvents were removed under reduced pressure and the crude was purified by automated flash column chromatography (0-25% ethyl acetate in pentane) to provide 10d as a pale yellow solid (1300 mg, 78%). 1

6-Dimetylamino-9-(2-indanyl)-2-(1-propylamino)purine (11b)
The reaction was run in two batches. 10b (511 mg, 1.19 mmol) and (200 mg, 0.47 mmol) were dissolved in a dry DMF (14 ml and 12 ml respectively) in two dry microwave vials. The reactions were flushed with nitrogen and then heated in a microwave reactor at 180°C for 60 min. The reactions were monitored by TLC (50% ethyl acetate in pentane). The solvents were removed under reduced pressure, co-evaporating with toluene (x 3). Trace amounts of starting material was still present after purification by automated flash column chromatography (10-30% ethyl acetate in pentane) and (20% ethyl acetate in pentane). The product could not be separated from the starting material. The obtained mixed fractions from the two batches were pooled, dissolved in dry DMF (15 ml) and heated in the microwave at 180 °C for additional 75 min. TLC (50% ethyl acetate in pentane) showed full consumption of the starting material. The solvent was removed under reduced pressure and the crude product was purified by automated flash column chromatography (15-20% ethyl acetate in pentane) to provide compound 11b as an colourless oil (318 mg, 57%). 1

6-Dimethylamino-9-(2-indanyl)-2-(p-chloro-benzylamino)purine (11c)
To 10c (743 mg, 1.46 mmol) was added 5.6 M dimethylamine in ethanol (15 ml, 230 mmol) and the reaction mixture was heated in a microwave reactor at 80°C for 20 min. TLC (30% ethyl acetate in pentane) indicated complete conversion of starting material and the solution was bubbled with nitrogen to remove excess dimethylamine prior to removal of the solvents under reduced pressure. The crude was dissolved in CH 2 Cl 2 (10 ml) and TFA (4 ml, 50 mmol) was added. TLC (50% ethyl acetate in pentane) indicated full conversion of starting material after 4 h, the solution was bubbled with nitrogen to remove excess TFA and the solvent was removed under reduced pressure. The crude was dissolved in CH 2 Cl 2 (30 ml), water was added (10 ml) and the solution basified by addition of 6 M NaOH (aq.). The phases were separated, the aqueous phase was extracted with CH 2 Cl 2 (4 x 40 ml) and the combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and the crude product purified by flash column chromatography (28% ethyl acetate in pentane) to provide 11c as an off white solid (579 mg, 95%). 1

2-Benzylamino-6-(ethoxy-carbonyl-methylamino)-9-(2-indanyl)purine (11d)
Glycine ethyl ester hydrochloride (191 mg, 1.37 mmol) and triethylamine (0.32 ml, 2.3 mmol) were added to 10a (218 mg, 0.458 mmol) in ethanol (18 ml). The reaction mixture was heated at 100°C overnight. Full consumption of starting material was confirmed by TLC (60% ethyl acetate in pentane). The solvent was removed under reduced pressure, the crude product was dissolved in CH 2 Cl 2 (7 ml) and TFA (3 ml, 40 mmol) was added. The reaction mixture was stirred at room temperature for 4 h. Full consumption of starting material was confirmed by TLC (60% ethyl acetate in pentane), the reaction mixture was cooled on ice, basified by addition of 3 M NaOH (aq.) and diluted with distilled water (20 ml) and CH 2 Cl 2 (35 ml). The aqueous phase was extracted with CH 2 Cl 2 (4 x 35 ml) and the combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and purification by automated flash column chromatography (2.5% methanol in CH 2 Cl 2 ) provided 11d as a white solid (184 mg, 91%). 1

6-(Ethoxy-carbonyl-methylamino)-9-(2-indanyl)-2-(1-propylamino)purine (11e)
The reaction was run in two batches, which was then pooled prior to work-up and purification. 10b (250 mg, 1.17 mmol) was suspended in ethanol (15 ml, 99.7%) in two oven-dried microwave vials. Glycine ethyl ester hydrochloride (245 mg, 1.76 mmol) and triethyl amine (0.41 ml, 2.94 mmol) were added and the reactions were heated to 100ºC, whereupon the starting materials dissolved. The reaction was left at 100ºC overnight. TLC (5% methanol in CH 2 Cl 2 ) indicated full consumption of the starting material. The two batches were pooled and the solvent was removed under reduced pressure. The crude product was dissolved in CH 2 Cl 2 (13 ml) and TFA (4 ml) was added. The reaction mixture was stirred at room temperature for 3 h after which full deprotection was confirmed by LCMS. The reaction mixture was cooled on ice and diluted with distilled water (20 ml) and then basified with 6 M NaOH (aq.). The aqueous phase was extracted with CH 2 Cl 2 (4 x 20 ml), the combined organic phases were washed with brine and dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. The crude product was purified by automated flash column chromatography (5% methanol in CH 2 Cl 2 ) to provide compound 11e as a light yellow oil/foam (451 mg, 98%). 1  9-Benzyl-2-benzylamino-6-(ethoxy-carbonyl-methylamino)purine (11f) K 2 CO 3 (350 mg, 2.53 mmol) and glycine ethyl ester hydrochloride (353 mg, 2.53 mmol) were added to a solution of 10d (380 mg, 0.845 mmol) in acetonitrile (19 ml, dried over molecular sieves). The solution was heated at 70 °C for 3 days after which full consumption of starting material was confirmed by TLC (50% ethyl acetate in pentane). The reaction mixture was diluted with water (20 ml) and extracted with CH 2 Cl 2 (5 x 20 ml). The combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvents were removed under reduced pressure, the crude was re-dissolved in CH 2 Cl 2 (6 ml) and TFA (2 ml, 26 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. Full conversion was confirmed by TLC (50% ethyl acetate in pentane). The reaction mixture was diluted with water (10 ml), basified (pH 12) with 6 M NaOH (aq.) and the aqueous phase was extracted with CH 2 Cl 2 (4 x 20 ml). The combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and purification by automated flash column chromatography (65-70% ethyl acetate in pentane) provided 11f as a white solid (217 mg, 62%). 1

9-Benzyl-2-benzylamino-6-(methoxy-carbonyl-methyl-thio)purine (11g)
A solution of 10d (452 mg, 26.0 mmol) and methyl thioglycolate (0.275 ml, 3.01 mmol) in dry toluene (13 ml) were added to NaH (125 mg, 3.13 mmol, 60% in mineral oil) in dry toluene (15 ml). The flask was fitted with a reflux condenser and heated at 70°C overnight. The reaction was monitored by TLC (40% ethyl acetate in pentane). The reaction was quenched with sat. NaHCO 3 (aq., 5 ml) and diluted with water (20 ml). The aqueous phase was extracted with CH 2 Cl 2 (6 x 15 ml) and the combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvents were removed under reduced pressure, the crude was re-dissolved in CH 2 Cl 2 (6 ml) and TFA (2 ml, 26.0 mmol) was added. The reaction mixture was stirred at room temperature for 5 h after which full consumption of starting material was confirmed by LC-MS. The reaction mixture was diluted with water (15 ml) and CH 2 Cl 2 (10 ml) and basified by addition of 6 M NaOH (aq.). The aqueous phase was extracted with CH 2 Cl 2 (6 x 15 ml) and the combined organic phases were washed with brine and dried over Na 2 SO 4 . The solvent was removed under reduced pressure and purification by automated flash column chromatography (50-65% ethyl acetate in pentane) provided 11g as a colourless oil (252 mg, 60%). 1

6-Amino-9-benzyl-2-benzylamino-8-bromopurine (12h)
A solution of NH 4 OH (aq. 28%, 7.5 ml) was added to a solution of 10d (703 mg, 1.56 mmol) in 1,4-dioxane (7.5 ml) in a 10-20 ml microwave vial. The vial was capped, flushed with nitrogen and heated in a microwave reactor at 100°C for 1 h. Excess NH 3 was bubbled off with nitrogen and the solvents were removed by evaporation on high vacuum. The white solid was taken up in CH 2 Cl 2 (4 ml), TFA (4 ml) was added and the reaction mixture was stirred at room temperature under nitrogen. Full consumption of the starting material and one new major spot was observed by TLC (10% methanol in CHCl 3 ) after 1 h. 3 M NaOH (aq.) was added to basify, CH 2 Cl 2 (200 ml) was added (the mixture was stirred until the white precipitation that had formed on basification was dissolved) followed by water (50 ml) and the phases were separated. The aqueous phase was extracted with CH 2 Cl 2 , the organic phases pooled, dried over Na 2 SO 4 and the solvents were removed under reduced pressure. The white crude was suspended in CH 2 Cl 2 (40 ml) and transferred to a three necked round-bottomed flask equipped with a nitrogen inlet. The mixture was heated at 40 °C to dissolve the material and PyrBr 3 (999 mg, 3.12 mmol) was added. The reaction was heated at 40 °C for 3 h and then at room temperature for 1 h after which TLC indicated full consumption of starting material (10% methanol in CHCl 3 ). The reaction was quenched with Na 2 S 2 O 3 (10% aq.), basified with 0.5 M NaOH (aq.) to pH 10-11 and diluted with CHCl 3 (300 ml). Water was added and the phases were separated, the aqueous phase extracted with CHCl 3 (2 x 50 ml), the organic phases were collected and the solvents were removed under reduced pressure. The crude was purified by flash column chromatography (2% methanol in CHCl 3 ) to provide 12h as a white solid (329 mg, 51% over 3 steps). 1

6-Amino-8-bromo-2-(1-propylamino)purine (12i)
A solution of NH 4 OH (aq. 28%, 7 ml) was added to a solution of 10b in 1,4dioxane (7 ml) in a 10-20 ml microwave vial. The vial was capped, flushed with nitrogen and heated in a microwave reactor at 100ºC for 1 h. Excess NH 3 was bubbled off with nitrogen and solvents were removed under reduced pressure. The resulting white solid was taken up in CH 2 Cl 2 (4 ml), TFA (4 ml) was added and the reaction mixture was stirred at room temperature under nitrogen for 1 h. Full consumption of starting material was observed by TLC (10% methanol in CHCl 3 ). The solution was concentrated by passing a stream of nitrogen over the solution. Water (50 ml) was added and the suspension was basified by addition of 3 M NaOH (aq.). The aqueous phase was extracted with CH 2 Cl 2 (3 x 100 ml), the organic phases were pooled, dried over Na 2 SO 4 and the solvents were removed under reduced pressure. The white crude was dissolved in CH 2 Cl 2 (30 ml) and transferred to a three necked round bottom flask equipped with a nitrogen inlet. PyrBr 3 (747 mg, 2.34 mmol) was added, the reaction mixture was stirred at room temperature for 15 min and then at 40 ºC for 26 h (precipitation formed after 3 min at room temperature and dissolved when heated). The reaction mixture was cooled to room temperature, quenched with 10% Na 2 S 2 O 3 (aq.), basified with 0.5 M NaOH (aq.) to pH 10-11 and diluted with CH 2 Cl 2 (150 ml) and water (50 ml). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (2 x 50 ml). The pooled organic phases were washed with brine, dried over Na 2 SO 4 and the solvents were removed under reduced pressure. Starting material was still present after purification by automated flash column chromatography (2% methanol in CHCl 3 ). The obtained mixed fractions were pooled and the solvents were removed under reduced pressure. The obtained solid was re-dissolved in dry CH 2 Cl 2 (30 ml) , PyrBr 3 (450 mg, 1.41 mmol) was added and the reaction mixture was stirred at room temperature for 19 h after which one major spot was observed on TLC (10% methanol in CHCl 3 ). The reaction was quenched with 10% Na 2 S 2 O 3 (aq.), basified with 0.5 M NaOH (aq.) to pH 9-10 and diluted with CH 2 Cl 2 (150 ml) and water (50 ml). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (2 x 50 ml). The pooled organic phases were washed with brine, dried over Na 2 SO 4 and the solvents were removed under reduced pressure. Purification by flash column chromatography (2% methanol in CHCl 3 ) provided 12i as an off white solid (328 mg, 73% over three steps). 1

2-Benzylamino-6-dimethylamino-8-ethynyl-9-(2-indanyl)purine (13a)
Amberlite IRA-67 (232 mg, 1.30 mmol), CuI (10 mg, 0.053 mmol) and Pd(PPh 3 ) 2 Cl 2 (7.5 mg, 0.011 mmol) were added to a solution of 12a (117 mg, 0.253 mmol) in THF (2 ml). The vial was capped, the reaction mixture was bubbled with nitrogen again and ethynyltrimethylsilane (0.11 ml, 0.80 mmol) was added. The vial was heated at 115°C for 50 min. TLC (35% ethyl acetate in pentane) showed only trace amounts of starting material and the reaction mixture was filtered through a short plug of silica and eluted with THF. The solvent volume was adjusted to 5 ml and polymer supported fluoride was added (134 mg). TLC (35% ethyl acetate in pentane) the following morning indicated full conversion of starting material. The polymers were filtered off and washed with a few ml of CH 2 Cl 2 and THF, the solvents were removed under reduced pressure. The crude product was purified by flash column chromatography (12-20% ethyl acetate in pentane) to provide 13a as a brown solid (72 mg, 70%). 1

6-Dimethylamino-8-ethynyl-9-(2-indanyl)-2-(p-chloro-benzylamino)purine (13c)
Amberlite IRA-67 (475 mg, 2.66 mmol), CuI (20 mg, 0.11 mmol) and Pd(PPh 3 ) 2 Cl 2 (16 mg, 0.023 mmol) were added to a solution of 12c (262 mg, 0.526 mmol) in dry THF (4.5 ml). The vial was capped, the reaction mixture was bubbled with nitrogen again and ethynyltrimethylsilane (0.22 ml, 1.6 mmol) was added. The reaction mixture was heated in a microwave reactor at 110°C for 50 min. TLC (20% ethyl acetate in pentane) indicated full conversion of starting material. The reaction mixture was filtered through a short plug of silica and eluted with THF. The solvent volume was adjusted to 5 ml, polymer supported fluoride (282 mg) was added and the reaction mixture was stirred at room temperature overnight. Full consumption of starting material was confirmed by LCMS. The polymer was filtered off and washed with CH 2 Cl 2 and THF. The solvents were removed under reduced pressure and the crude product was purified by flash column chromatography (91% ethyl acetate in pentane) to provide 13c as a brown solid (160 mg, 69%). 1

9-Benzyl-2-benzylamino-6-(ethoxy-carbonyl-methylamino)-8-ethynylpurine (13f)
Amberlite IRA-67 (356 mg, 1.99 mmol), CuI (16 mg, 0.084 mmol) and PdCl 2 (PPh 3 ) 2 (14 mg, 0.020 mmol) were added to a solution of 12f (198 mg, 0.400 mmol) in dry THF (1.5 ml). The vial was capped, nitrogen was bubbled through the reaction mixture and ethynyltrimethylsilane (0.17 ml, 1.2 mmol) was added. The reaction mixture was heated at 115°C for 50 min, whereupon full conversion of starting material was confirmed by TLC (30% ethyl acetate in pentane). The reaction mixture was filtered through a short plug of silica and eluted with THF. The solvent volume was adjusted to 6 ml, polymer supported fluoride (203 mg) was added and the reaction mixture was stirred at room temperature overnight. Full deprotection was confirmed by TLC (30% ethyl acetate in pentane), the polymer was filtered off and washed with CH 2 Cl 2 and THF. The solvents were removed under reduced pressure and purification by automated flash column chromatography (25% ethyl acetate in pentane) provided 13f as a sticky brown solid (203 mg, 69%). 1

9-Benzyl-2-benzylamino-8-ethynyl-6-(methoxy-carbonyl-methyl-thio)purine (13g)
Amberlite IRA-67 (160 mg, 0.896 mmol), CuI (7.5 mg, 0.039 mmol) and PdCl 2 (PPh 3 ) 2 (7.5 mg, 0.011 mmol) were added to a solution of 12g (90 mg, 0.18 mmol) in dry THF (1.5 ml). The vial was capped, nitrogen was bubbled through the reaction mixture and ethynyltrimethylsilane (0.075 ml, 0.54 mmol) was added. The vial was heated at 115°C for 50 min, trace amounts of starting material could be observed by TLC (30% ethyl acetate in pentane). The reaction mixture was filtered through a short plug of silica and eluted with THF. The solvent volume was adjusted to 3 ml, polymer supported fluoride (94 mg) was added and the reaction mixture was stirred at room temperature overnight. Full deprotection was confirmed by TLC (30% ethyl acetate in pentane). The polymer was filtered off and washed with CH 2 Cl 2 and THF. The solvents were removed under reduced pressure and the crude was passed through a silica column (20% ethyl acetate in pentane) which resulted in the isolation of a brown solid (40 mg, 50%). 1 H and 13 C NMR confirmed that 13g was the major compound with impurities and the material was used in the next step without further purification. 1

6-Amino-9-benzyl-2-benzylamino-8-ethynylpurine (13h)
PdCl 2 (PPh 3 ) 2 (26 mg, 0.04 mmol), CuI (28 mg, 0.15 mmol) and amberlite IRA-67 (646 mg, 3.62 mmol) were added to a solution of 12h (296 mg, 0.72 mmol) in dry THF (5 ml). The vial was capped, nitrogen was bubbled through the reaction mixture and ethynyltrimethylsilane (400 µL, 2.89 mmol) was added. The reaction mixture was heated in a microwave reactor at 110°C for 50 min, after which full consumption of starting material was confirmed by LCMS. The reaction mixture was filtered through a short plug of silica which was eluted with THF. The solvent volume was reduced to 15 ml and polymer supported fluoride (380 mg) was added. The reaction mixture was stirred at room temperature for 14 h. Full deprotection was confirmed by TLC (10% methanol in CHCl 3 ) and LCMS. The polymer was filtered off, washed with THF and CH 2 Cl 2 and the solvents were removed under reduced pressure. The crude product was passed through a silica column (0-5% methanol in CHCl 3 ) which provided 13h as a yellow solid (211 mg, 82%). The isolated material contained unidentified impurities and was used without further purification in the next step. 1 159.9, 155.4, 152.0, 139.8, 136.3, 130.7, 128.8

6-Amino-8-ethynyl-9-(2-indanyl)-2-(1-propylamino)purine (13i)
PdCl 2 (PPh 3 ) 2 (22 mg, 0.031 mmol), CuI (22 mg, 0.12 mmol), amberlite IRA-67 (543 mg, 3.04 mmol) were added to a solution of 12i (235 mg, 0.607 mmol) in dry THF (5 ml). The vial was capped, nitrogen was bubbled through the reaction mixture and ethynyltrimethylsilane (340 µl, 2.46 mmol) was added. The yellow reaction mixture was heated in a microwave reactor at 110°C for 50 min. Full consumption of 12i was confirmed by TLC (10% methanol in CHCl 3 ). The dark reaction mixture was filtered through a short plug of silica which was eluted with THF. The volume was adjusted to 7 ml and polymer supported fluoride (320 mg) was added. The reaction mixture was stirred at room temperature under nitrogen for 24 h. Full consumption of the starting material was confirmed by TLC (10% methanol in CHCl 3 ) after 24 h. The polymer was removed by filtration, washed with THF and CH 2 Cl 2 and the solvents were removed under reduced pressure. The crude was purified by flash column chromatography (3% methanol in CH 2 Cl 2 ) to provide 13i as a yellow solid (103 mg, 51%). 1

NOE build-up analysis
All NOESY spectra were recorded on Varian MR 400 MHz spectrometer. Compounds 8a and 7a (Fig. S1) were dissolved in DMSO-d 6 in a concentration of 0.011 M and 0.012M respectively. Chemical shifts are reported in parts per million with the solvent residual signal used as internal standard: DMSO-d 6 [δ H 2.50, δ C 39.52]. The NOE-build up was performed with six mixing times ranging from 80-1000 ms. The relaxation delay was set to 2.0 s, 16 scans were recorded with 4096 points in the direct and 128 points in the indirect dimension. Distances were calculated with the reference distance of 2.508 Å, measured for aromatic ortho protons. The NOE peak intensities were calculated using normalisation of both crosspeaks and both diagonalpeaks ([crosspeak 1 × crosspeak 2 ]/[diagonalpeak 1 × diagonalpeak 2 ]) 0.5 . At least three mixing times giving a linear (r 2 >0.95) initial NOE rate were used to determine σ ij build-up rates according to the equation r ij =r ref (σ ref /σ ij ) (1/6) , where r ij is the distance between protons i and j in Ångström and σ ij is the normalized intensity obtained from NOESY experiments. Calculated distances are shown in Table S1 and S2, for 8a and 7a, respectively. In addition, build-up curves for 8a and 7a are shown in Fig. S2 and S3, respectively.

Computational conformation analysis
All computational studies were performed using the software MacroModel [5]. Conformational searches for 8a and 7a were performed twice, using the OPLS-2005 and the Amber* force fields with several different solvation models. For OPLS-2005, water, chloroform and octanol were used and for Amber*, the water solvation model was used. The energy window for saving structures was set to 42 kJ/mol and RMSD cut off was set to 1-2 Å (1 Å for OPLS-2005 and water solvation for 8a, otherwise 2 Å). The torsional sampling mode (MCMM) was employed with 10000 steps per rotable bond. PRCG was used as the minimization method with a maximum of 2500 iterations. For 8a and 7a 963 and 711 conformations were obtained, respectively. The ensembles derived from calculations with different force fields and/or solvent models were combined followed by elimination of redundant conformations by comparison of the heavy atom coordinates applying a RMSD cutoff 2.5 Å. For 8a and 7a, 59 and 42 unique conformations were obtained, respectively.

Ensamble analysis using NAMFIS
Following a previously described protocol [6], ensemble calculation analysis was performed by fitting the experimentally measured distances to the computationally predicted conformations. Methyl, eqvivalent methylene and eqvivalent ortho protons were treated accoridng to the equation d= {[(d 1 ) -6 + (d 2 ) -6 + (d 3 ) -6 + (d n ) -6 ]/n -1/6 }, where n is the number of eqvivalnet protons e.g. for a methyl group n = 3 [7]. The experimental obtained distances is presented in Tabel S3 and S4 for 8a. The structures of the main conformers are shown in Fig. S4 and S5.    x The structures of the main conformers are shown in Fig. S4 and S5. where η MeOH and η water is the refractive index for methanol (1.333) and water (1.328), respectively. Absorption spectra were recorded on a Cary 5000 UV/Vis spectrometer. Emission spectra were recorded on a Horiba Spex fluorolog 3.    . For testing, a dilution series of small molecules (spanning 10 mM to 0.5 μM in DMSO, in 1:3-dilution) was added by direct addition to the assay plate by Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using pin transfer (100ss pins, V&P scientific) giving a test dilution series spanning 130 μM to 6.6 nM with a final concentration of 1.3 % DMSO. The assay mixture was incubated for 1 hour at room temperature and the fluorescence polarisation signal was measured on an EnVision multilabel plate reader fitted with a 555-nm excitation filter, 632-nm static and polarized filters, and a Texas Red FP dichroic mirror. Unlabelled WT-p53 peptide was used as a positive control, and DMSO was used as a negative control. Technical triplicate data was normalized to the positive (100% inhibition) and negative (0% inhibition) controls on the corresponding row of the 384-well plate (the percentage inhibition = 100 × (sample resultnegative control)/(positive control meannegative control)). Two to seven independent experiments of normalized data were combined into a data set, and then fit using a non-linear regression in GraphPad Prism with the formula log(inhibitor) vs. response -Variable slope (four parameters). Additionally the residuals of the curve fit were plotted to determine the fit of the theoretical curve. IC50 and 95% confidence intervals (CI) were determined from these graphs.

Solubility
Solubility assay was carried out on Biomek FX lab automation workstation (Beckman Coulter, Inc., Fullerton, CA) using SOL Evolution software (pION Inc., Woburn, MA). The detailed method is described as following. 10 L of 10 mM compound stock (in DMSO) was added to 190 L 1-propanol to make a reference stock plate. 5 L from this reference stock plate was mixed with 70 L 1-propanol and 75 L citrate phosphate buffered saline (isotonic) to make the reference plate, and the UV spectrum (250 nm -500 nm) of the reference plate was read. 6 L of 10 mM test compound stock was added to 594 L buffer in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate (pION Inc., Woburn, MA). 75 L filtrate was mixed with 75 L 1-propanol to make the sample plate, and the UV spectrum of the sample plate was read. Calculation was carried out by SOL Evolution software based on the AUC (area under curve) of UV spectrum of the sample plate and the reference plate. All compounds were tested in triplicates.

Molecular modelling
Conformational analysis was performed using MacroModel [9] in Maestro. The conformational search was performed using the OPLS-2005 force field with water as solvation model. The energy window for saving structures was set to 21 kJ/mol and a maximum atom deviation threshold of 0.5 Å. The torsional sampling mode (MCMM) was employed with 10000 steps per rotable bond. PRCG was used as the minimization method with a maximum of 10000 iterations. Protein crystal complexes were retrieved from the PDB database and prepared with the protein preparation wizard workflow in Maestro using default settings (minimization with OPLS2005 as forcefield converging heavy atoms to RMSD of 0.30Å) [10]. Grids for docking were prepared using Glide [11] in maestro. Grids were centred around the co-crystallized ligands with a box size of 20 Å. Prior to docking, all ligands were prepared using Ligprep [12] in maestro with OPLS_2005 as forcefield. Epik was used to generate possible ionization states at pH 7 ±2. Docking was performed in Glide with flexible ligands and added Epik penalties. Docking of structures generated by Ligprep was performed in Precision (XP) mode. Crystal complexes were compared using the protein structure alignment in Maestro. Alignments were made with protein backbone atoms and with all heavy atoms.