Tag Archives: Malaria

Synthesis of 3-oxo-2-(3-(trifluoromethyl)phenyl)pentanenitrile (SGS18-3-1)

This reaction was performed by Heath, Ronan, Francis, Nathan, Jonah, Oliver, Daniel, Stuart, Jacky and Erin.

Synthesis of 18-3-1

3-(Trifluoromethyl)phenylacetonitrile (11.4g, 0.0617 mol) was added to ethyl propanoate (5.35 g, 0.0524 mol) and THF (100 mL). Potassium tert-butoxide (17.0 g, 0.152 mol) was added with stirring. The reaction mixture immediately turned red and warmed. The reaction was then placed in a water bath and the temperature maintained at 40°C with stirring for 7 h. The potassium tert-butoxide dissolved after 30 min of stirring.

HCl (80 mL, 1 M) was added to stop the reaction, then DCM (60 mL) was added. The intense red organic layer was collected, and the aqueous layer extracted with DCM (30 mL x 2). The organic layer was dried over sodium sulfate and then filtered.

A TLC was run in 25:75 EtOAc:hex, and showed that the crude mixture contains some starting material and two coloured spots. This is consistent with the expected keto and enol tautomers.

 

TLC 75:25 hexane:EtOAc

Synthesis of 3-oxo-2-(4-(trifluoromethyl)phenyl)pentanenitrile, SGS18-2-1

Reaction performed by Heath, Jonah, Ronan, Francis, Stuart, Daniel, Oliver, Jacky, Nathan and Erin!

SGS18-2-1

4-(trifluoromethyl)phenylacetonitrile (10 g, 0.054 mol) was added to ethyl propanoate (5.52 g, 0.055 mol) and THF (100 mL), and stirred until dissolved. Potassium tert-butoxide (12.5 g, 0.112 mol) was added with stirring.  The reaction immediately turned red and warmed to ~40 oC.  The reaction was kept at this temperature and stirred for 7 h.

HCl (80 mL, 1 M) was added to stop the reaction, then DCM (40 mL) was added. The intense red organic layer was collected, and the aqueous layer extracted with DCM (20 mL x 2). The organic layer was dried over sodium sulfate and then filtered.

TLCs were run in 10:90 EtOAc:hex and 25:75 EtOAc:hex, and both showed that the crude mixture contains some starting material and two coloured spots.  This is consistent with the expected keto and enol tautomers. 

After addition of potassium tert-butoxide

Synthesis of 2-(4-chlorophenyl)-3-oxopentanenitrile (SGS18-1-1)

Reaction performed by Stuart, Heath and Francis (contributions by Jonah, Ronan, Nathan, Jacky, Daniel, Oliver and Erin) 

Synthesis of SGS18-1

This is a repeat of SGS 10-2 to verify synthesis and yield of Daraprim.

 (4-chloropenyl)acetonitrile (11.426 g, 75 mmol) was added to ethyl propanoate (7.698 g, 75 mmol) and THF (50 mL). Potassium tert-butoxide (24 g, 214 mmol) was added with stirring. The reaction mixture immediately turned red and warmed. The reaction was then placed in a water bath and the temperature maintained at 40°C with stirring for 7 h. The potassium tert-butoxide dissolved after 30 min of stirring.

HCl (80 mL, 1 M) was added to stop the reaction, then DCM (60 mL) was added. The intense red organic layer was collected, and the aqueous layer extracted with DCM (30 mL x 2). The organic layer was dried over sodium sulfate and then filtered.

TLCs were run in 10:90 EtOAc:hex and 25:75 EtOAc:hex, and both showed that the crude mixture contains some starting material and two coloured spots.  This is consistent with the expected keto and enol tautomers. 

 

TLC 10:90 EtOAc:hex

TLC 10:90 EtOAc:hex. Rf values of major spots are 0.1 and 0.52 (of darker and lighter respectively).

TLC 25:75 EtOAc:hex

TLC 25:75 EtOAc:hex. Rf values of the largest spots are 0.3 and 0.74 (darker and lighter respectively).

QSAR modeling for Series 4

I tried a ligand-centric, QSAR approach to model activity for Series 4. I took the S4 compounds on the April 7, 2018 Master List and separated them into two .csv files, one containing compounds with measured activities and one with unmeasured compounds.

For both data sets, I used the SMILES strings to calculate 1D and 2D molecular descriptors using PaDEL (http://www.yapcwsoft.com/dd/padeldescriptor/), which is freely available. PaDEL calculates 1,444 1D and 2D descriptors. It was not obvious how to proceed with putting together a single activity value for the compounds, as they were measured in different assays, and many had values such as >10 or >50. For values such as >X, I entered a value that was 2X. I tried various methods to perform regression in Weka (https://www.cs.waikato.ac.nz/~ml/weka/), freely available, focusing on the more interesting machine learning methods. 20% of the data was set aside as a test set for cross-validation. Random forest performed by far the best, using 136 trees, with a max depth of 16, and max features of 6 per tree.

Statistics calculated for the training vs test predictions:

Correlation coefficient 0.7178
Kendall's tau 0.4993
Spearman's rho 0.647
Mean absolute error 7.1934
Root mean squared error 12.3386
Relative absolute error 78.7046 %
Root relative squared error 70.3855 %

 

Using xgboost (extreme gradient boosted trees, https://github.com/dmlc/xgboost) through python, I got better results (with 120 estimators, max depth=3, learning rate =0.1, subsample = 0.9).

R2: 0.91

Kendall's tau: 0.49

Spearman's rho: 0.62

Mean squared error: 49.7.

 

I then trained xgboost on the total set of measured compounds (training + test). The model was saved as are the predictions made for the training and test sets. I applied the final model to the set of untested compounds. The most promising compounds are in order, OSM-S-486, OSM-S-433, OSM-S-536, OSM-S-538, OSM-S-204.

 

Synthesis of 5-chloro-3-(2-hydroxyphenyl)-[1,2,4]triazolo[4,3-a]pyrazine

Method:

2-chloro-6-(2-hydrazinyl(2-hydroxybenzylidene))pyrazine (134 mg, 0.54 mmol, 1.00 equiv.) was dissolved in dry DCM (30 mL) forming an orange solution. Diacetoxyiodobenzene (174 mg, 0.54 mmol, 1.00 equiv.) was added and the solution stirred at room temperature under an inert atmosphere for approximately 3.5 hours upon which it slowly turned a dark red colour. TLC analysis showed the reaction had not reached completion, so the solution was heated to ~35 ˚C for ~1 hour forming a near black solution. The solution removed from stirring and exposed to the atmosphere overnight, forming a pale yellow solution. The solution was quenched with saturated aqueous Na2CO3 solution (20 mL) and extracted with DCM (2 × 10 mL). The combined organic layers were washed with saturated brine solution (20 mL), dried over MgSO4, filtered and concentrated to give a crude product as a brown oil. The crude product was analysed by 1H NMR and what was thought to be the desired product was purified by flash column chromatography (EtOAc) and isolated as a brown oil (7 mg). NMR analysis of this sample showed no presence of any product, only many peaks due to impurities and was therefore inconclusive as to the identity of the product.


Synthesis of 5-chloro-3-(phenyl)-[1,2,4]triazolo[4,3-a]pyrazine

Method:

2-chloro-6-(2-hydrazinyl(benzylidene))pyrazine) (40.0 mg, 0.172 mmol, 1.0 equiv.) was dissolved in dry dichloromethane (15 mL) forming a yellow solution. Diacetoxyiodobenzene (PIDA) (55.4 mg, 0.172 mmol, 1.0 equiv.) was then added and the solution was stirred at room temperature under nitrogen for approximately 16 hours. After this time, the reaction mixture was exposed to air and analysed by TLC (1:1 EtOAc: PET ether) showing a clear product peak. The reaction mixture was quenched by the addition of a saturated aqueous solution of sodium hydrogen carbonate (~15 mL). Aqueous layers were separated and then extracted with DCM (2 x 15 mL). The organic layers were combined and washed with brine (1 x 20 mL), dried with MgSO4, filtered and evaporated in vacuo, yielding the crude product as a fine yellow powder (22.5 mg, 56.5%). The crude product was characterised using 1H NMR and IR. The product was purified using a silica column and the pure product (yellow solid) was obtained in vacuo (19.7 mg, 49.5%). The product was characterised by 1H NMR, 13C NMR, DEPT 90 + 135, GC-MS and IR.


Synthesis of 5-chloro-3-(4-bromophenyl)-[1,2,4]triazolo[4,3-a]pyrazine

Method:

2-chloro-6-(2-hydrazinyl(4-bromobenzylidene))pyrazine) (C) (100.2, 0.322 mmol, 1.0 equiv) was dissolved in dry dichloromethane (15 mL) forming a pale yellow solution. Diacetoxyiodobenzene (PIDA) (114 mg, 0.354 mmol, 1.1 equiv) was then added and the solution was stirred at room temperature under nitrogen for approximately 16 hours. After this time, the reaction mixture was exposed to air and analysed by TLC (pure EtOAc) showing a clear product peak as well as some residual PIDA. The reaction mixture was quenched by the addition of a saturated aqueous solution of sodium hydrogen carbonate (~15 mL). Aqueous layers were separated and then extracted with DCM (2 x 15 mL). The organic layers were combined and washed with brine (1 x 20 mL), dried with MgSO4, filtered and evaporated in vacuo, yielding the crude product as pale yellow crystals (69.6 mg, 70.2%). The crude product was characterised using 1H NMR, 13C NMR, DEPT 90 + 135, GC-MS and IR. The purified product was isolated using a silica plug since TLC showed the presence of the product and PIDA with significant separation.


Synthesis of 2-chloro-6-(2-hydrazinyl(4-bromo)benzylidene))pyrazine

Method:

2-chloro-6-hydrazinopyrazine (A) (100.00 mg, 0.70 mmol, 1.00 equiv.) was dissolved in EtOH (10 mL) forming a bright yellow solution, to which 4 bromobenzaldehyde (B) (141 mg, 0.76 mmol, 1.1 equiv.) was added. The reaction was stirred for a further 3.5 hours and was monitored by TLC. During this time, a yellow precipitate formed. The reaction mixture was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(4-bromobenzylidene))pyrazine) as a yellow powder (111.3 mg, 51.6%) which was characterised by IR and 1H NMR.TLC of the showed the reaction had gone to completion.


Synthesis of 2-chloro-6-(2-hydrazinyl(2-hydroxybenzylidene)pyrazine) (Repeat with different conditions)

Method:

2-chloro-6-hydrazinopyrazine (202.0 mg, 1.39 mmol, 1.00 equiv.) was partially dissolved in EtOAc (30 mL) forming a yellow solution before addition of 2-hydroxybenzaldehyde (0.2 mL, 1.92 mmol, 1.2 equiv.). The reaction was heated under reflux under a Dean-Stark trap for 1 hour, darkening the solution slightly, however no water was collected in the trap. After 1 hour at reflux, a TLC of the reaction mixture revealed that the reaction had reached completion. The solvent was removed in vacuo giving an orange powder which was shown to contain 2-hydroxybenzaldehyde, desired product, and an unknown side product. The powder was recrystallized from boiling ethanol to give yellow needle like crystals (123 mg). The crystals were analysed by NMR and IR spectroscopy and mass spectrometry to determine that they were salicylaldehyde azine formed by a side reaction with free hydrazine impurities in the 2-chloro-6-hydrazinopyrazine. The filtrate was therefore collected and concentrated in vacuo giving an orange powder (209 mg) analysed by NMR and IR spectroscopy to contain 2-chloro-6-(2-hydrazinyl(2-hydroxybenzylidene))pyrazine and salicylaldehyde azine in a 2:1 molar ratio.


Synthesis of 5-chloro-3-(4-nitrophenyl)-[1,2,4]triazolo[4,3-a]pyrazine

Method:

2-chloro-6-(2-hydrazinyl(4-nitrobenzylidene))pyrazine) (106 mg, 0.34 mmol, 1.0 equiv.) was dissolved in DCM (20 mL) forming a yellow solution. PIDA (109 mg, 0.34 mmol, 1.0 equiv.) was then added. The reaction was stirred for 2 hours at room temperature before heating to 30 °C for a further hour, yielding a yellow solution with a small amount of precipitation. The reaction was monitored by TLC (Ethyl acetate) showing that only a small amount of starting material remained. The reaction was quenched with saturated sodium hydrogen carbonate solution. The organic layer was isolated by separation with brine before drying over MgSO4. The dried filtrate was concentrated in vacuo to a dark yellow solid. The crude product was characterised by IR and 1HNMR before purification by silica column with ethyl acetate, followed by 5% MeOH in DCM as the solvent system. A bright yellow powder (34 mg, 35.7 %) was obtained and the pure product, 5-chloro-3-(4-nitrophenyl)-[1,2,4]triazolo[4,3-a]pyrazine, was then characterised by IR, 1H NMR, and 13C NMR , DEPT, 1H-13C HSQC and MS.