Tag Archives: Malaria

Step Four: Synthesis of (E)-2-chloro-6-(2-(4-(difluoromethoxy)benzylidene)hydrazineyl)-3,5-dimethylpyrazine

Reaction run on 12/8/16

CC1=NC(C)=C(N=C1Cl)N/N=C/C2=CC=C(OC(F)F)C=C2

Procedure:

4-(difluoromethoxy) benzaldehyde (1.5mL) was added to the 2-chloro-6-hydrazineyl-3,5-dimethylpyrazine (0.8g) produced in the last reaction. Ethanol (20mL) was also added with vigorous stirring. The product will be left to stir under an argon atmosphere overnight and TLC will be used to monitor the completeness of the reaction.



Third Step: Synthesis of 2-chloro-6-hydrazineyl-3,5-dimethylpyrazine (HASJK-3-3)

Reaction taken place on 12/5

CC1=NC(C)=C(N=C1Cl)NN

Procedure:

2,6 dichloro-3,5 dimethylpyrazine (0.6g), ethanol (6mL) and hydrazine (0.15 mL) were combined in a round bottom flask and stirred under argon atmosphere. It was then heated in an 80oC oil bath overnight. After, the solvent will be removed under vacuum. Yellow/gold crystals (0.8g) resulted NMR shown below.

 


Second Step: Synthesis of 2,6-dichloro-3,5-dimethylpyrazine Column Chromatography (HASJK-3-2)

Columned on 11/28/16

The product was columned by the Biotage. The fractions 5-7 (high spots) were combined. The product was a white crystal. Fractions 19-30 were also combined (low spot). NMR was run on the high spot. Initial look suggests that the chlorinated dimethyl product was synthesized with traces of starting material. Ran NMR/GCMS on both high and low spots, shown below.

 

Silica gel plates. 50/50 EtoAc/Hexane. Active under UV light.


Second Step: Synthesis of 2,6-dichloro-3,5-dimethylpyrazine (HASJK-3-1)

Date of Reaction: 11/23

CC1=NC(C)=C(N=C1Cl)Cl

 

Procedure:

Excess phosphoryl chloride (5.0mL, 0.0535 mol) was added to the oxidized dimethyl product (1.2g, 0.008 mol). The mixture was stirred and refluxed for 1.5 hours at 70oC. The reaction mixture was then alkalized using 20% NaOH. An extraction was carried out with CHCl3 (3 x 20 mL). Next steps will be to run a column chromatography on that product and then to get NMR data.

 

Table of Reagents (Rxn2) DiMethyl

 
 
 
 
 

Reagent

mass (g)

mols

Molecular Weight (g/mol)

Volume (mL)

Density (g/mL)

Product RxN1

1.200

0.008

158.590

 
 

Phosphoryl Chloride

3.481

0.023

153.333

2.122

1.640

Product RxN2 Dimethyl

1.347

0.008

178.040

 
 
 
 
 
 

*Used 5 mL of POCl3

 

Attached are TLCs 

Synthesis of 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one (BPVR 1-1) Attempt 2

O=C(C1=NC(Cl)=CN=C1)C(F)(F)F

 

Reference

Holsinger, L.J., Compounds that Inhibit Protease Cathepsins and HCV Replication. U.S. Patent 2008080785, April 30, 2009.

 UNSUCCESSFUL
 

Substance

Amount

Mol. wt.

mmols

Equiv.

Starting Material

1 g

172.57

5.79

1

trimethylsilyltrifluoro-methane

0.70 g

142.2

4.92

0.85

caesium fluoride

0.880

151.9

5.79

1

tetra-butylammoniun fluoride (1M in THF)

7.25 mL

261.46

7.25

1.25

Concentrated Acetic Acid (17.4 M)

2.8 mL

 

49.2

8.5

Product

 

1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one

1.033 g (Theoretical)

 

0.1127 mg

(Actual)

209.98

4.92 (Theoretical)

 

0.54

(Actual)

0.85 (Theoretical)

 

11% Yield

(Actual)

 

Procedure:

(11/30/16)

 
  1. Dissolved starting material in dimethoxy ethane (15 mL).

  2. Dried CsF (0.88g) under vacuum while heating with a heat gun.

  3. Add trimethylsilyltrifluoro-methane (0.82 g) and CsF (0.880 g) to the starting material solution.

  4. Stirred at 4 degrees celsius for 36 hours.
    (12/2/16)

  5. A TLC was taken to see if the reaction was complete.

  6. The reaction was then concentrated at room temperature in high vac to a brown oil.



    (12/7/16)

  7. The concentrated product was then dissolved in 10 mL of THF, 2.8 mL acetic acid, and treated with 7.25 mL of tetrabutylammonium fluoride.

  8. Reaction was aged for 3 hours and then stopped by diluting the reaction with ethyl acetate (20 mL). A TLC was taken after 3 hours but no change was observed between the starting material before the workup and the product after the workup.

  9. The product was then gravity filtered to remove excess CsF.

  10. Product was washed three times with dilute aqueous sodium bicarbonate solution (0.25M, 100 mL portions).

  11. Washed with brine (100 mL).

  12. Dried over anhydrous magnesium sulfate, then filtered.

  13. Concentrated in vacuo.

1;1 Ethyl Acetate : Hexanes; S → Starting Material, C→ Co-Spot, P → Product
(12/8/16)

  1. Triturated brown oil product in a minimal amount of dichloromethane and hexanes to yield a brown solid.

    1. Yield: 0.1127 g (11%)

 

Analysis: In order to determine the identity of the product, GCMS and NMR were run.

 

GCMS:

 
[Untitled].pdf

The Peak at 207 m/z is the same that was observed last attempt. The mass of the expected product is 209, thus this data suggests that the synthesis was not successful.

 

NMR:

 

Both Carbon and PRoton NMR do not match any the expected spectra for the desired product, which further suggests that the synthesis was not successful.

 

Conclusions:

 

Decreasing the temperature of the reaction and decreasing the molar equivalents of the reactants did not improve the outcome of the reaction. Looking back at the previous group that worked on this project, their data suggests that they too were not successful in making this compound. We would suggest that future attempts to synthesize “The Burns” take another synthesis route. One possible synthesis is proposed below:

 

other thing.jpg

 

The reference for this reaction didn’t perform this reaction exactly; however, it might still work. Another option is to come up with a complete new total synthesis for “The Burns” compound.

 

Deprotection attempt 2 of tert-butyl 3-(5-(2-phenyl-2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-[1,2,4]triazolo[4,3-a]pyrazin-3-yl)piperidine-1-carboxylate (MRLS 9-1)


deprotect.jpg

Compound

Mw (g/mol)

mmol

mL

g

equiv

Starting Material

526.6501

0.09494

 

50mg

1

HCl-Ether

38

0.0312

0.156 (2M HCl)

 

3.3

Product

Theoretical Values

339.17

0.09494

 

32.2mg

1

dioxane

88.11

 

2 mL (4M)

 

3.3

Product Actual Value

339.17

0.07371

 

25.0 mg

78% Yield

 

November 30, 2016

Procedure:

  1. Dissolve 50 mg of the starting material in 500 uL of anhydrous diethyl ether and place system under argon.

  2. Add 2M HCl-Ether (0.1 mL) dropwise to the solution while stirring.

  3. Age solution for 2 hours at room temperature and the reaction was monitored with TLC.

  4. Left in fridge overnight.

the nemesis deprot round 2.jpg

100% EtOAc solvent system, silica gel plate, UV visualization

S → Starting Material; C → Co-spot; P → Product.

This TLC is similar to the first deprotection we did, which we deemed unsuccessful in taking out that BOC group, though there was success in knocking out the THP. This can be determined by comparing the S and C to the P. The S and C show a dark spot higher up on the plate; P shows one lower with a faint smear above it. This suggests that the THP group was successfully knocked out and the BOC group only partially (this agrees with our NMR and GCMS results from the first deprotection). Another TLC was done to confirm this hypothesis and determine if the reaction should be hit harder to take out both the THP and BOC group.

  the nemesisdeproround2.jpg

100% EtOAc solvent system, silica gel plate, dipped in ninhydrin and treated with heat

S1→ Original Purified Starting Material; S2→ Starting Material from First Deprotection; C → Co-spot; P → Product.

Treating the TLC plate with ninhydrin and heat results in two dark spots on the base line of S2, and C and a faint brown spot on the base line of P. The difference in intensity can be attributed to concentration. The presence of these spots after being treated with heat suggest that the BOC group was successfully knocked off, and resulted in the formation of an amine, which is the desired product. The BOC was only successful knocked off with heat on the plate. To do this on a larger scale, these results suggests the deprotection reaction must be hit harder; the reaction must be done under heat with the addition of dioxane.

 

December 5, 2016

We were unsuccessful in removing the BOC with extra time . This suggests the reaction needs to be hit harder, specifically with the addition of dioxane and ran in an oil bath.

  1. Added 2 ml of 4M Dioxane and 0.1 mL of HCl  dropwise to the product while stirring and place in an oil bath at 50 degrees Celsius. System was placed under argon.  The reaction was monitored with TLC.

  2. After 2 hours at 50 degrees, TLC indicated that there was still starting material. The temperature was raised to 102 degrees to induce reflux. The reaction was then left to reflux for 45 minutes.

  3. After 45 minutes the reaction turned brown and a TLC was taken. The results of the TLC and a ninhydrin test suggest that the complete deprotection was successful due to the disappearance of the high Rf spot observed in previous attempts.

 

December 7, 2016

Mass Spec:

 
Deprotection.pdf
 

The peak at m/z 340 suggests that we successfully removed the Boc protecting group that we had trouble removing in the past. It is possible, however, some residual Boc protecting groups were removed in the mass spec instrument, so the product was also analyzed with proton and carbon NMR.

 
December 8, 2016 

NMR:

Still to be determined

 

First Step Synthesis of 2-chloro-3,5-dimethylpyrazine 1-oxide. (HASJK-3-0)

CC1=NC(C)=C(Cl)[N+]([O-])=C1

ImageIMG_9860.JPG

SM/P 

2-chloro-3,5-dimethylpyrazine 

sulfuric acid, conc. 

potassium persulfate 

2-chloro-3,5-dimethylpyrazine 1-oxide 

mw 

142.59 

  

270.31 

158.59 

Equiv 

1.00 

  

1.07 

1.00 

mmol 

14.00 

  

15.00 

14.00 

mg 

2000 

  

4000 

2220 

mL 

  

14.00 

  

  

11/8/2016.  To a 1-neck 14/20 50 mL RB were added 14 mL of concentrated H2SO4, which was cooled to approximately 0 C with ice bath.  The pyrazine was then added, followed by potassium persulfate.  The resulting suspension was allowed to stir with warming to room temperature overnight. 

11/9/16.  The resulting clear solution was poured into a separatory funnel with distilled water and ice and extracted 3 x ~ 90 mL CHCl3.  The combined organic layer was washed with saturated aqueous NaHCO3, water, and brine and dried over MgSO4

11/13/16.  The resulting clear, colorless solution was concentrated with rotary evaporation to afford 2.5 g of a clear, colorless liquid from which a colorless solid formed upon cooling. 

11/14/16.  The Round bottom flask was placed under high vacuum for approximately 1-1.5 hours to afford 1.9 g of a colorless crystalline solid after removal of high vacuum volatiles.  

Crude yield 1900/2200 x 100 = 86%

Synthesis of (E)-2-chloro-6-(2-(4(pyrrolindin-1-yl)benzylidene)hydrazine)pyrazine from 4-(pyrrolindin-1-yl)benzaldehyde and 2-chloro-6-hydrazinylpyrazine in a condensation reaction #2

AT-1-023 #2

Synthesis of (E)-2-chloro-6-(2-(4(pyrrolindin-1-yl)benzylidene)hydrazine)pyrazine from 4-(pyrrolindin-1-yl)benzaldehyde and 2-chloro-6-hydrazinylpyrazine in a condensation reaction #2

 

Both Reactions were carried out using a microscale to minimalize waste of intermediate product used as reactant.

Reaction 1: Repeat of original condensation reaction

 

 

reaction scheme 

 

 

 

Reagent

Eq

Mw

mmol

w/vol

2-chloro-6-hydrazinylpyrazine

1

144.56

0.2853

41mg

4-(pyrrolindin-1-yl)benzaldehyde

1

175.23

0.2853

50mg

ethanol

~

46.07

~

4ml

 

Experimental Procedure

2-chloro-6-hydrazinylpyrazine (41mg, 0.2853mmol) and 4-(pyrrolindin-1-yl)benzaldehyde (50mg, 0.2853mmol) were added to ethanol (4ml) in a glass vial. The glass vial had a lid put on it and shaken and left to react for 4 hours.

produt.png

TLC analysis (70:30, Pet. Ether/ EtOAc and the final TLC was 1:1 Pet. Ether/ EtOAc) was taken after two hours, showing it hadn’t gone to completion. After 4 hours TLC analysis showed it had.

tlcs.png

In the TLC, on the spots for the product, several isomers had the same r.f. value so it is impossible to tell them apart from TLC. Crude NMR taken and shown to be the same as reaction 2, so the crude products (78.6mg) were combined to column together.

Reaction 2: Acetonitrile and acetic acid

 

 reaction scheme

 

 

Reagent

Eq

Mw

mmol

w/vol

2-chloro-6-hydrazinylpyrazine

1

144.56

0.2853

41mg

4-(pyrrolindin-1-yl)benzaldehyde

1

175.23

0.2853

50mg

Acetonitrile

1

41.05

~

1ml

Glacial acetic acid

~

60.05

~

0.045ml

Experimental Procedure

2-chloro-6-hydrazinylpyrazine (41mg, 0.2853mmol) and 4-(pyrrolindin-1-yl)benzaldehyde (50mg, 0.2853mmol) were added to acetonitrile (1ml) and glacial acetic acid (0.045ml in a glass vial. The glass vial had a lid put on it and shaken and left to react for 4 hours.

produt.png

TLC analysis (70:30, Pet. Ether/ EtOAc and the final TLC was 1:1 Pet. Ether/ EtOAc) was taken after two hours, showing it hadn’t gone to completion. After 4 hours TLC analysis showed it had.

tlcs.png

In the TLC, on the spots for the product, several isomers had the same r.f. value so it is impossible to tell them apart from TLC. Crude NMR taken and shown to be the same as reaction 2, so the crude products (75.5mg) were combined to column together.

Mixed reactions for isomer separation

The acetonitrile (75.5mg) and ethanol (78.6mg) crude products were mixed together (154.1mg). The crude NMR’s had shown the same product produced for both reactions, which contained the correct isomer. The products were dissolved in DCM and TLC systems for the best separation was tested. TLC tank A contained 80:20 Pet. Ether: EtOAc and tank B contained 90:10 Pet. Ether: EtOAc.

Tank C contained 80:20 Pet. Ether: diethyl ether and tank D contained 70:30 Pet. Ether: Diethyl ether.

Finally Tank E contained 70:30 Toluene: acetone.

tlc.png

The best tank for the separation of my crude product was a mixture between tank A and B, with 85:15 Pet. Ether and EtOAc. The crude product was purified via flash chromatography and 50 vials were taken off.

column tlc.png

A single product was present between vials 5-7, 8-12 had a mixture of isomers and 13-48 had a mixture of at least 2 isomers. The pure product betweek vials 5-7 was mostly clean but still contained some water, EtOAc and Pet. Ether. The correct isomer was not isolated however. It was still contained within the mass between vials 13-48.

fractions.png

Due to the very close r.f. values it was very difficult to separate out the isomers (all yellow solids), which meant that this step could not be moved forward because the correct product was not isolated.

 Reference:

  1. Open source malaria: our experiment, http://malaria.ourexperiment.org/triazolopyrazine_se/9268/Synthesis_of_E2chloro62naphthalen2ylmethylenehydrazinylpyrazine_TY_21.html ,date accessed: 04/11/16

 

Cyclisation of (E)-2-chloro-6-(2-(4(pyrrolindin-1-yl)benzylidene)hydrazine)pyrazine (AT-01-23) to form 5-chloro-3-(4-(pyrronlidin-1-yl)phenyl)-[4,3-a]pyrazine (AT-01-25)

AT-1-025

Cyclisation of (E)-2-chloro-6-(2-(4(pyrrolindin-1-yl)benzylidene)hydrazine)pyrazine (AT-01-23) to form 5-chloro-3-(4-(pyrronlidin-1-yl)phenyl)-[4,3-a]pyrazine (AT-01-25) 

reaction scheme 

Reagent

Eq

Mw

mmol

w/vol

AT-01-23

1

300.79

0.7989

0.2403g

DCM

~

84.39

~

15ml

PIDA

1

322.1

0.7989

0.2573g

NaHCO3

~

~

~

30ml

Mg2CO3

~

~

~

~

 

Experimental Procedure

 

(E)-2-chloro-6-(2-(4(pyrrolindin-1-yl)benzylidene)hydrazine)pyrazine (240.3mg, 0.7989mmol) was added to DCM (15ml) in a round bottom flask. PIDA (257.3mg, 0.7989mmol) was also added and the reaction was left to stir at room temperature. On addition to the PIDA, the reaction changed from a bright red solution to a rusty red colour.

IMG_2841.png

The reaction was stirred at room temperature for 7 days, over which time the reaction changed from pale yellow to orange.

IMG_2848 (1).png

Upon completion of the reaction, which was determined by TLC analysis, (5:3 Pet. Ether: Diethyl ether for first TLC, 1:1 hexane: EtOAc for the second TLC plate and 70:30 EtOAc/ Pet. Ether for the third)

IMG_2847.png

The mixture was placed into a separating funnel and the solution was separated into the inorganic and organic layers by the addition of NaHCO3 (30ml). The organic layer was tapped off and the inorganic layer was put to one side. The  organic layer was then washed with DCM (4x30ml). The mixture was then dried by Mg2CO3 and filtered. From the resulting solution a crude NMR was taken.

The solvent from the crude was in vacuo. Silica and EtOAc were added, solvent removed via in vacuo again and then Flash columned, to purify the product.

column.png

Solvent system for the column started at 1:1 Pet. Ether/ EtOAc, but was changed to 70/30 EtOAc/ Pet. Ether to increase the mobile phase. 57 Test tubes were taken. Products appeared in test tubes 3-6, 9-11, 17-20, 21-23, 24, 27-29, 30-33 and 37-57.  

TLC of column.png

The Final product was given as a yellow solid for each of the fractions. None of the products produced were pure, they were all a mix of isomers. No yield was recorded but each fraction from 21-23 onwards had a yield. (0.0202-0.0277g, 9-10%).

 1H NMR showed that the material contained many different isomers of the correct product in the same fractions. Most of them also had water, EtOAc and Pet. Ether impurities.

 

A-1-025.

 

Reference:

  1. Open source malaria: our experiment, http://malaria.ourexperiment.org/triazolopyrazine_se/9259/Synthesis_of_5Chloro124triazolo43apyrazine_TZ_61.html, date accessed: 04/11/16