AUY 922

Luminespib (NVP-AUY922) is a potent HSP90 inhibitor with IC50s of 7.8 and 21 nM for HSP90α and HSP90β, respectively.

25 mol of NH2-Fx-r-Fx-r-Fx-r on resin was reacted with 3-[2-(2- (0143) Bromoethoxy)ethoxy]propanoic acid (4 eq, BroadPharm, San Diego CA), HBTU (4 eq), and DIPEA (8 eq) in 1 mL DMF. The peptide was washed (2 x DMF/MeOH/DCM), cleaved from resin using trifluoroacetic acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at - 20°C for 1 hour. The precipitate was purified by RP-HPLC, lyophilized, and dissolved in 1 mL DMF. Luminespib (2 eq), and solid potassium carbonate (10 eq, Sigma-Aldrich) was added to the reaction mixture. The suspension was stirred overnight, filtered, then precipitated in ether and purified by HPLC. Two isomers were identified during HPLC purification, likely due to attachment to either of the two resorcinol hydroxyls. The earlier eluting isomer was purified and tested due to its higher relative abundance. The peptide was identified by ESI mass spectrometry, expected m/z = 1551.97, found m/z = 1551.97. The absorbance profile of Compound 6 was not found to be shifted as compared to Luminespib itself, therefore the peptide was quantified by measuring Luminespib absorbance at 305 nm with an extinction coefficient of 8520 M'1cm'1.25 muetaetaomicronIota of NH2-Fx-r-Fx-r-Fx-r on resin was reacted with S-trityl-2- mercaptoproprionic acid (4 eq), PyBOP (4 eq), and DIPEA (8 eq) in 1 mL DMF. The peptide was washed (2 x DMF/MeOH/DCM), cleaved from resin using trifluoroacetic (0139) acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at -20°C for 1 hour. The precipitate was purified by RP-HPLC, dried under vacuum and dissolved in 0.5 mL (0140) acetonitrile:water (5:1). 2-mercpatoethanol (20 eq, Sigma-Aldrich) was added to the reaction mixture followed by iodine (10 eq) and the reaction was stirred for 30 minutes. The peptide was purified by HPLC and lyophilized. 5-(2, 4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4- (morpholinomethyl)phenyl)isoxazole-3-carboxamide (Luminespib, 3 eq, Adooq Bioscience, Irvine CA) was reacted with Nu, Nu'-Disuccinimidyl carbonate (3 eq, Sigma-Aldrich) and 4- (Dimethylamino)pyridine (12 eq, Sigma-Aldrich) in 0.4 mL DMF for 1 hour. The peptide was dissolved in 0.1 mL DMF and added to the reaction mixture and the solution was left stirring overnight. The peptide was precipitated in ether and purified by HPLC. Two isomers were identified during HPLC purification, likely due to attachment to either of the two resorcinol hydroxyls. The earlier eluting isomer was purified and tested due to its higher relative abundance. The solution was frozen in dry ice as the compound eluted from the column and lyophilized. The peptide was identified by ESI mass spectrometry, expected m/z = 1599.88, found m/z = 1599.88. The peptide was quantified via absorbance spectrophotometry using a SpectraMax M5 spectrophotometer. The absorbance profile of Compound 5 was found to be shifted as compared to Luminespib itself, therefore the peptide was quantified by cleavage in 25 mM TCEP in PBS pH 7.4 for 10 minutes, then measuring free Luminespib absorbance at 305 nm with an extinction coefficient of 8520 M'1cm'1. TCEP was not found to affect the extinction coefficient of Luminespib.25 pmol of NH2-Fx-r-Fx-r-Fx-r-K(Mtt) on resin was reacted with S-trityl-2- mercaptoproprionic acid (4 eq), PyBOP (4 eq), and DIPEA (8 eq) in 1 mL DMF. The peptide was washed (2 x DMF/MeOH/DCM) and deprotected with trifluoroacetic (0146) acid:triisopropylsilane:DCM (5:3:92, 2 x 15 minutes). The peptide washed and equilibrated in acetonitrile:water (5: 1 ). Cysteamine (20 eq) was dissolved in 1 mL acetonitrile:water (5: 1 ) and added to the reaction mixture followed by iodine (10 eq). The reaction was stirred for 30 minutes. The peptide was washed (2 x DMF:MeOH:DCM) and reacted with 5- Carboxytetramethylrhodamine (2 eq), HBTU (2 eq), and DIPEA (4 eq) in 0.5 mL DMF for 2 hours. The peptide was washed, cleaved from resin using trifluoroacetic (0147) acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at -20°C for 1 hour. The precipitate was purified by HPLC and lyophilized. 5-(2, 4-Dihydroxy-5-isopropylphenyl)-N-ethyl- 4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (Luminespib, 3 eq, Adooq Bioscience, Irvine CA) was reacted with Nu, Nu'-Disuccinimidyl carbonate (3 eq, Sigma-Aldrich) and 4- (Dimethylamino)pyridine (12 eq, Sigma-Aldrich) in 0.4 mL DMF for 1 hour. The peptide was dissolved in 0.1 mL DMF and added to the reaction mixture and the solution was left stirring overnight. The peptide was precipitated in ether and purified by HPLC. The earlier eluting isomer was purified and tested due to its higher relative abundance. The solution was frozen in dry ice as the compound eluted from the column and lyophilized. The peptide was identified by ESI mass spectrometry, expected m/z = 2140.12, found m/z = 2140.12. The peptide was quantified using the 5-Carboxytetramethylrhodamine absorbance at 547 nm with an extinction coefficient of 92000 M'1cm'1.Step 2: Synthesis of AUY922-diglycolate-cholesterol: Weighed amounts of cholesterol-diglycolic acid (2, 12.0 g, 23.86 mmol), AUY922 (3, 10.0 g, 21.48 mmol) and N, N-dimethylamino pyridine (3.0 g, 24.55 mmol) were stored under high vacuum for 10 minutes and purged with nitrogen. The contents were then dissolved in anhydrous dichloromethane (200 mL) and stirred at 19°C under nitrogen atmosphere. To this, a solution diisopropyl carbodiimide (6.0 mL, 38.98 mmol) in dichloromethane (25 mL) was introduced over a period of 15 minutes and the mixture was stirred at 19°C for 4 hours. Reaction was monitored by TLC using 10percent methanol in dichloromethane as eluent and spray dried with p-anisaldehyde staining solution or observed under UV light. After complete consumption of cholesterol-diglycolic acid (2), the resultant hazy solution was cooled to -15°C and the solid separated was removed by filtration through a cotton pad and filtrates from this batch and recycled AUY922 (2.0 g) batch were combined and concentrated to a thick solution. [0093] Purification: The thick solution was loaded on a pre-packed silica gel column in dichloromethane (1500 g). Eluted with 1 -5percent methanol in dichloromethane. Fractions containing major product were concentrated to get pale yellow solid foam (11.50 g). The solid foam was subjected to another silica gel chromatographic purification by loading on a pre-packed silica gel column in dichloromethane (1500 g). The column was eluted with an isocratic eluent system with a solvent ratio of 3/0.5/96.5 methanol isopropanol/ dichloromethane. This process of purification on silica gel column was repeated until >95percent purity by HPLC and nmR was achieved. Alternatively, the product was passed through a reverse phase column (RP, C-18) in neat methanol with a loading ratio of 0.5 g per 240 g of C-18 column. Pure product fractions were collected and concentrated. Dried in vacuum oven for 18 hours to get pure AUY922-diglycolate-cholesterol (4) as off-white powder (4.95 g, 24percent· yield, HPLC purity 98percent at 230 nm). nmR (600 MHz, DMSO-de) : delta 10.42 (s, 1 H), 8.86 (t, J = 4 Hz, 1H), 7.23 (ABq, J = 8, 40 Hz, 4H), 6.96 (s, 1 H), 6.72 (s, 1H), 5.37 (bs, IH), 4.58-4.53 (m, 1H), 4.29 (s, 2H), 4.17 (s, 2H), 3.60-3.53 (m, 4H), 2.08 (bs, 2H), 3.33 (bs, 2H), 3.26-3.17 (m, 2H), 3.12-3.00 (m, IH), 2, 40-2, 30 (m, 6H), 2.05-1.90 (m, 2H), 1.90-1.75 (m, 3H), 1.65-1.45 (m, 5H), 1.44-1.20 (m, 6H), 1.19-0.95 (m, 17H), 0.94-0.91 (m, 9H), 0.88 (d, J = 4 Hz, 3H), 0.70 (s, 3H) ppm. ESI-MS Data: m/z = 950.59 [M+H].

NVP-AUY 922 is a potent inhibitor of heat shock protein 90 (Hsp90) that prevents the proliferation of a range of human cancer cell lines. NVP-AUY 922 has been shown to enhance the radiation sensitivity of tumor cell lines under hypoxia.