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Synthesis of 2-chloro-6-(2-hydrazinyl(4-nitrobenzylidene)pyrazine)

Method:

2-chloro-6-hydrazinopyrazine (100.0mg, 0.69 mmol, 1.00 equiv.) was partially dissolved in EtOH (~12 mL) forming a yellow suspension. 4-nitrobenzaldehyde (117.0 mg, 0.77 mmol, 1.1 equiv.) was then added partial dissolution. The mixture was stirred for 10 minutes before being heated to 30°C to aid solubility. The yellow suspension was stirred and heated for a further 2 hours and 40 minutes and became progressively deeper orange coloured; it was monitored by TLC (9:1 PET ether: Ethyl acetate) until completion. The solid was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(4-nitrobenzylidene)pyrazine) as a yellow powder (105.6 mg, 55%). This was characterised by IR and 1H NMR. 


Synthesis of 2-chloro-6-(2-hydrazinyl(2-hydroxybenzylidene)pyrazine)

Method:

2-chloro-6-hydrazinopyrazine (100.0mg, 0.69 mmol, 1.00 equiv.) was partially dissolved in EtOH (~10 mL) forming a yellow solution. 2-hydroxybenzaldehyde (0.1 mL, 0.958 mmol, 1.2 equiv.) was then added giving a clear yellow solution. The mixture was stirred for 2 hours before being heated to 72°C for 5 minutes and became progressively slightly darker in colour; it was monitored by TLC (9:1 PET ether: Ethyl acetate) until completion. The solid was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(2-hydroxybenzylidene)pyrazine) as yellow crystals (20.6 mg, 12%). This was characterised by IR and 1HNMR. The filtrate was also recovered in vacuo to give an orange powder (142.6 mg, 83%) and characterised by IR and 1H NMR.

Synthesis of 2-chloro-6-(2-hydrazinyl(2,4-dimethoxybenzylidene)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 2,4-dimethoxybenzaldehyde (B) (126 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(2,4-dimethoxybenzylidene))pyrazine) as a yellow powder (59.1 mg, 29.2%) which was characterised by IR and 1H NMR. Remaining product in the filtrate, a dark orange oil, was also recovered in vacuo (98.1 mg, 48.4%). TLC of the filtrate showed a mixture of many compounds which proved very difficult to separate using a silica column, so the filtrate was discarded.

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

2-chloro-6-(2-hydrazinyl(4-(dimethylamino)benzylidene))pyrazine) (51 mg, 0.18 mmol, 1.00 equiv.) was dissolved in DCM (20 mL) forming a yellow solution. PIDA (62 mg, 0.19 mmol, 1.1 equiv.) was then added. The yellow solution became progressively orange within two hours. The reaction was stirred for 2.5 hours at room temperature before heating to 30 °C for a further two hours. The reaction was monitored by TLC (3:7 Ethyl acetate : PET ether) however starting material remained so it was left stirring at room temperature overnight. After 22.5 hours the solution was a darker orange and TLC showed 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 brown crude oil. The crude product was characterised by IR and 1HNMR before purification by silica column (5 g silica) with a 1:1 Ethyl acetate : PET ether solvent system. A dark brown powder (13.2 g, 26 %) was obtained and the pure product, 5-chloro-3-(4-(dimethylamino)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine, was then characterised by IR, 1HNMR, and 13CNMR, DEPT and MS.

 *Combined fractions 10-23 as pure product, fractions 3-9 were combined and retained in case of later analysis.

Synthesis of 2-chloro-6-(2-hydrazinyl(4-chlorobenzylidene))pyrazine)

Method

2-chloro-6-hydrazinopyrazine (A) (101.0 mg, 0.70 mmol, 1.00 equiv.) was partially dissolved in EtOH (10 mL) forming a yellow solution. 4-chlorobenzaldehyde (B) (105.0 mg, 0.75 mmol, 1.1 equiv.) was then added causing full dissolution. The yellow solution was stirred for five minutes at room temperature before a pale yellow solid precipitated out of the reaction mixture. The reaction was stirred for a further 2.5 hours and was monitored by TLC (3:7 Ethyl acetate : PET ether)* until completion. The solid was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(4-chlorobenzylidene))pyrazine) as a pale yellow powder (103.9 mg, 56.5 %). This was characterised by IR and 1HNMR. The filtrate was also recovered in vacuo and characterised by IR and 1HNMR but excess 4-chlorobenzaldehyde starting material was also present so an accurate yield could not be determined.

*4-chlorobenzaldehyde is unstable to TLC (see 2DTLC).


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

Method

2-chloro-6-hydrazinopyrazine (A) (100.7mg, 0.70 mmol, 1.00 equiv.) was partially dissolved in EtOH (10 mL) forming a yellow solution. 4-(dimethylamino)benzaldehyde (B) (115.3 mg, 0.77 mmol, 1.1 equiv.) was then added causing full dissolution. The orange-yellow solution was stirred for five minutes at room temperature before an orange solid precipitated out of the reaction mixture. The reaction was stirred for a further 2.5 hours and became progressively more red-orange; it was monitored by TLC (3:7 Ethyl acetate : PET ether) until completion. The crude solid was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(4-(dimethylamino)benzylidene))pyrazine) as an orange powder (129.2 mg / 67.3 %). This was characterised by IR and 1HNMR. The filtrate was also recovered in vacuo to give a deep red powder and characterised by IR and 1HNMR.


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

Method:

2-chloro-6-hydrazinopyrazine (A) (100.7 mg, 0.70 mmol, 1.00 equiv.) was partially dissolved in EtOH (10 mL) after it was diluted by a factor of 10, forming a white solution. Benzaldehyde (B) (81.0 mg, 0.77 mmol, 1.1 equiv.) was then added causing full dissolution. The solution was stirred for five minutes at room temperature before a solid precipitated out of the reaction mixture. The reaction was stirred for a further 2.5 hours with no significant change in colour observed; it was monitored by TLC (1:9 Ethyl acetate : PET ether for half the reaction and  3:7 Ethyl acetate : PET ether for the other half) until completion. The solid was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(benzylidene)pyrazine) as a white powder (71.3 mg,44.5%). This was characterised by IR and 1HNMR. 

 

 

Synthesis of benzaldehyde (6-chloro-pyrazin-2-yl)-hydrazone

Method:

2-chloro-6-hydrazinopyrazine (A) (100.7 mg, 0.70 mmol, 1.00 equiv.) was partially dissolved in EtOH (10 mL) after it was diluted by a factor of 10, forming a white solution. Benzaldehyde (B) (81.0 mg, 0.77 mmol, 1.1 equiv.) was then added causing full dissolution. The solution was stirred for five minutes at room temperature before a solid precipitated out of the reaction mixture. The reaction was stirred for a further 2.5 hours with no significant change in colour observed; it was monitored by TLC (1:9 Ethyl acetate : PET ether for half the reaction and  3:7 Ethyl acetate : PET ether for the other half) until completion. The solid was filtered under vacuum to give benzaldehyde (6-chloro-pyrazin-2-yl)-hydrazone as a white powder (71.3 mg,44.5%). This was characterised by IR and 1HNMR. 

 

 

Synthesis of 2-chloro-6-(2-hydrazinyl(4-carboxybenzylidene)Pyrazine)

2-chloro-6-hydrazinopyrazine (A) (100.00 mg, 0.70 mmol, 1.00 equiv.) was dissolved in EtOH (10 mL) forming a yellow solution, to which 4-carboxybenzaldehyde (B) (113.5.0 mg, 0.76 mmol, 1.1 equiv.) was added. The yellow solution was stirred for five minutes at room temperature, with B not seeming to dissolve to any appreciable degree. The reaction was stirred for a further 2.5 hours and was monitored by TLC. Solvent systems included 1:9 and 3:7 Ethyl acetate: PET ether, as well as dichloromethane with all of them little to no evidence of product forming – it could be that TLC is not an appropriate method for monitoring this reaction. The reaction mixture was filtered under vacuum to give 2-chloro-6-(2-hydrazinyl(4-carboxybenzylidene))pyrazine) as a pale pink powder (108.6 mg, 56.9%) which was characterised by IR and 1H NMR. Remaining product in the filtrate was also recovered in vacuo, with no appreciable yield. The product was characterised by IR and 1H NMR, the latter displaying a clear and large excess of 4-carboxybenzaldehyde starting material.

 

 


Step 5 – synthesis of 4-(4-(5-(3,4-difluorophenethoxy)-[1,2,4]triazolo[4,3,a]pyrizin-3-yl))morpholine

Masses and measurements:

SnAr product - 0.05g, 0.116mmol

NaOtBu - 0.0156g, 0.162mmol

Morpholine - 0.02mL, 0.139mmol

Pd2(dba)2 - 0.53mg, 0.583μmol

John Phos- 1.158mg, 5.22μmol

Toluene - 2mL


Procedure:


(SnAr product) 3-(4-bromophenethoxy)-[1,2,4]triazolo[4,3,a]pyrazine (0.05g, 0.116mmol) was dissolved in toluene (2mL) in a RBF and the base, NaOtBu, (0.0516g, 0.162mmol) was added. The ligand, John Phos (1.158mg, 5.22μmol) and the amine, morpholine (0.02mL, 0.139mmol) were added to the reaction mixture. Lastly, the palladium source, Pd2(dba)(0.53mg, 0.58μmol) was added to the RBF and the mixture was left to stir and heat to 80°C until TLC analysis showed any sign of reaction. 

TLC step 5 3 cropped 1.jpg
               
TLC step 5 3 cropped 2.jpg
                       
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 TLC after 1 day                     TLC after 1 week                          TLC with 100% DCM                   

The reaction mixture was seperated using 2M HCl givng an orange layer and a pale yellow layer. TLC analysis was done using the organic top layer. After 1 day TLC analysis (98% DCM, 2% methanol) still showed presence of the starting material. After 1 week TLC analysis confirmed less starting material and possibly a new product. Next the whole top organic layer wasextracted and rotary evapoaretd leaving a dark green crude product. The crude product was run on a flash column (2% methanol, 98% DCM) and TLC analysis (2% methanol, 98% DCM) confirmed the presence of a possible product in test tube 16-25. These test tubes were collected and rotary evapoarted, however H NMR analysis confirmed no presence of the desired final product. 

                             

TLC step 5 2.jpg
                       

                    TLC analysis Test tubes 21-35