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23rd September 2016 @ 18:25

 20 September 2016

Synthesis of EVKCN_Prod3.png

Reference

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


Data Table
Substance Quant. Mol. wt. mmoles Equiv.
methyl 6-chloropyrazine-2-carboxylate 0.58 g 172.57 3.36 1
dimethoxy ethane 10 mL 90.12     
trimethylsilyltrifluoro-methane 0.70 g  142.2 4.92 1.5 
caesium fluoride 0.051 151.9 3.36  1
THF 4.0 mL 72.11     
acetic acid 2.0 mL 60.05     
tertbutylammoniun fluoride 0.90 g 261.46   3.4 1
ethyl acetate 50 mL 88.11     
1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one

0.71 g (theoretical)

0.07 g (actual)

209.98

3.36 (theoretical)

0.33 (actual)

1 (theoretical)

0.1 (actual)


Procedure
  1. Dissolved starting material in dimethoxy ethane (10 mL). 
  2. Added trimethylsilyltrifluoro-methane (0.70 mL) and caesium fluoride (0.051 g). 
  3. Stirred at room temperature overnight. Solution turned a dark yellow, then brown color. 
  4. Concentrated mixture under reduced pressure, resulting in a dark brown oil. 
    Step 3 brown oil.jpg
     
  5. Dissolved resulting residue in THF (4.0 mL) and acetic acid (2.0 mL) 
  6. Added tertbutylammonium fluoride (0.867 g) 
  7. Allowed reaction to stir overnight. 
  8. Diluted reaction with ethyl acetate (50 mL) 
  9. Washed three times with dilute aqueous sodium bicarbonate solution (0.25M, 100 mL portions). 
  10. Washed with brine (100 mL) 
  11. Dried over anhydrous magnesium sulfate, then filtered. 
  12. Removed solvent under reduced pressure. 
  13. Triturated with a 1:1 dichloromethane/hexanes solution (50 mL), causing a dark brown semi-solid to precipitate from solution. Results were not consistent with literature precedent.  
  14. Removed solvent under reduced pressure, concentrating product back into a dark brown oil. 
    Dark brown oil re-rotovap.jpg
  15. Crude product (dark brown oil) was tested via TLC.
    Left = SM, Center = cospot, Right = product
    TLC Crude Product 3.jpg

    1:1 EtOAc:Hexanes solvent system; silica gel plate; UV visualization
    Rf(bottom) = 0.25, Rf(middle) = 0.42, Rf(top) = 0.50
  16. Purified solution using Biotage Isolera. Fractions 2, 3, 6 and 7 were shown to most likely contain the purified product.
    Step 3 Biotage Screen.JPG
  17. Fractions were analyzed using TLC, GCMS, and NMR. Analysis of the results showed that fractions 6 and 7 contained the most pure sample of product 3, 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one.
  18. Fractions 6 and 7 were combined and solvents removed in vacuo in preparation for step 4. 
Observations and Analysis

TLC: 
From bottom to top: Fraction 2, 3, 6, 7
TLC Product 3 Fractions.jpg
1:1 EtOAc:Hexanes solvent system; silica gel plate; UV visualization
Rf(2, 3) = 0.50, Rf(6, 7) = 0.24
Fraction 2 potentially contained an impurity in addition to the less polar compound in Fraction 3. 
Fraction 3 contained one less polar compound.
Fractions 6 and 7 contained a second more polar compound.
The fractions were further analyzed by GCMS to determine which was our compound of interest. 
 
GCMS:
Fraction 2 - visualized 2 distinct retention times, indicative of an impurity
EVKCN_Step3_Frac2_GCMS-1.jpg
EVKCN_Step3_Frac2_GCMS-2.jpg
 
 
Fraction 3 
EVKCN_Step3_Frac3_GCMS-1.jpg
EVKCN_Step3_Frac3_GCMS-2.jpg
 
 
Fractions 6 and 7 (combined)
EVKCN_Step3_Frac6_GCMS-1.jpg
EVKCN_Step3_Frac6_GCMS-2.jpg

NMR: 1H-NMR (DMSO, 500MHz), 13C-NMR (DMSO, 126 MHz) measured in ppm
1H and 13C-NMR for starting material, methyl 6-chloropyrazine-2-carboxylate, can be found in Synthesis of methyl 6-chloropyrazine-2-carboxylate (EVKCN 1-2).

1H-NMR for product, 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one
Wideview
EVKCN_Prod3_1HNMR_Wide_Revised.jpeg
 
Closeup 
EVKCN_Prod3_1HNMR_Close_Revised.jpeg
∂ 8.90 (s, 1H), 8.88 (s, 1H)
 
13C-NMR for product, 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one.
Wideview
EVKCN_Prod3_13CNMR_Wide_Revised.jpeg
Closeup
EVKCN_Prod3_13CNMR_Close_Revised.jpeg
∂ 152.56, 150.13, 147.54, 145.54, 142.29, 124.45 

Conclusions and Next Steps 

     From our TLC, GCMS, and NMR data, we have concluded that we produced some of our desired product, 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one. TLC of the crude product indicated an incomplete reaction as some UV-visualizable spots characteristic of the starting material were present in the product. However, after submitting the crude product to the Biotage Isolera, it appeared that we acheived some separation upon TLC and GCMS analysis. Although Fraction 2 appeared to contain some impurity in the TLC, fractions 3, 6, and 7 appeared to be fairly pure (with 6 and 7 containing the same product). We combined fractions 6 and 7, then submitted the 3 samples to GCMS. We found the combined fractions 6 and 7 appeared to be out desired product as we observed the M+ of m/z 209.98, which is characteristic of 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one. However, upon analysis by NMR, we found that our product also likely contained the more reduced version of the product, 2-(6-chloropyrazin-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol. This is evidenced by by the m/z 279.9, 281.9 in GCMS, as well as the peaks at 8.07 and 5.73 ppm in the 1HNMR. 

2-(6-chloropyrazin-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol. .png

2-(6-chloropyrazin-2-yl)-1,1,1,3,3,3-hexafluoropropan-2-ol (Image courtesy of Chase Smith via Github)

In any repeat trials, shortening the reaction, running it at 0 deg. C, and/or reducing the amount of trimethylsilyltrifluoro-methane to 1 equivalent, may help to increase the yield of our desired product (advice courtesy of Chase Smith via Github).

     Because we produced primarily the desired product, we continued on with step 4 in which we will perform a reductive amination in an attempt to produce N-benzyl-1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-amine. However, for future trials, it may be more effective to perform another separation using Biotage Isolera in order to achieve a more pure product.  

 

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20th September 2016 @ 19:32

8 September 2016

Synthesis of methyl 6-chloropyrazine-2-carboxylate.jpg

References

  1. Nikishkin et al. New J Chem. 2013. 37, 394.
  2. Scanio, M. J. C.; Shi, L.; Bunnelle, W. H.; Anderson, D. J.; Helfrich, R. J.; Malysz, J.; Thorin-Hagene, K. K.; Handel, C. E. V.; Marsh, K. C.; Lee, C.-H.; Gopalakrishnan, M. J. Med. Chem. Journal of Medicinal Chemistry 2011, 54(21), 7678–7692.
 
Data Table
 

Substance

Quant.

Mol. wt.

mmol

Equiv.

2-(6-chloropyrazin-2(1H)-ylidene)malononitrile

0.92 g

178.58

5.15

1

MeOH

54.2 mL

32.04

   

Li2CO3

0.571 g

73.891

7.72

 1.5

magnesium monoperoxyphthalate hexahydrate (80% tech)

1.911 g

494.64

3.86

 0.75

H2O

21 mL

     

CHCl3

21 mL

     

Methyl 6-chloropyrazine-2-carboxylate

0.89 g (theoretical)

0.60 g (actual)

172.57

5.15 (theoretical)

3.48 (actual)

1

 
Procedure
  1. Dissolved 2-(6-chloropyrazin-2(1I)-ylidene) malnonitrile (0.92 g, 5.15 mmol) in MeOH (54.2 mL).

  2. Added Li2CO3 (0.571 g, 7.72 mmol) to this solution.

  3. Stirred mixture for 30 min.

  4. Added magnesium monoperoxyphthalate hexahydrate (1.911 g, 3.86 mmol) in small portions at 0 deg. C.

  5. Allowed mixture to stir overnight in ice bath. 

  6. The solution was dry filtrated in vacuo, resulting in a dark yellow residue. (9/13/2016)

    Step 2 yellow residue.jpg
  7. Residue was partitioned between H2O (21 mL) and CHCl3 (21 mL).

  8. Dried organic phase over MgSO4, and filtered through a short plug of silica.

  9. Removed all volatiles in vacuo.

  10. Product was a pale yellow oil that crystallized upon standing (0.60 g, 3.48 mmol, 67% yield). 

    Product 2.jpg
 
Observations and Analysis
 
TLC:
Used previously dissolved starting material, 2-(6-chloropyrazin-2(1I)-ylidene) malnonitrile, in methanol. 
Dissolved product, methyl 6-chloropyrazine-2-carboxylate, in DCM. 
Left = starting material, center = cospot, right = product
TLC Product 2.jpg
1:1 EtOAc:Hexanes solvent system; silica gel plate; UV visualization
Rf(SM): 0.38, Rf(Prod): 0.62
 
 NMR: 1H-NMR (DMSO, 500 MHz), 13C-NMR (DMSO, 126 MHz) measured in ppm
Product was dissolved in DMSO.
Product 1H-NMR 
EVKCN_Prod2_1HNMR_Revised.jpeg
∂ 9.16 (s, 1H), 9.04 (s, 1H), 3.92 (s, 3H)
Product 13C-NMR 
EVKCN_Prod2_13CNMR_Revised.jpeg
∂ 163.29, 148.44, 148.66, 144.14, 142.58, 53.44
 
TRIAL #2

This procedure was repeated on 9/15/16 with the second batch of starting material, with amounts adjusted for 0.75 grams of starting material. 0.29 g (39.9 % yield) of product was collected.

Observations and Analysis

TLC:
Used previously dissolved starting material, 2-(6-chloropyrazin-2(1I)-ylidene) malnonitrile, in methanol. 
Dissolved product, methyl 6-chloropyrazine-2-carboxylate, in DCM. 
Left = starting material, center = cospot, right = product
TLC Product 2 Attempt 2.jpg
1:1 EtOAc:Hexanes solvent system; silica gel plate; UV visualization
Rf(SM): 0.38, Rf(Prod): 0.62
 
NMR: 
1H-NMR (DMSO, 500 MHz), 13C-NMR (DMSO, 126 MHz) measured in ppm
Product was dissolved in DMSO.
Product 1H-NMR 
EVKCN_Prod2_Attempt2_1HNMR.jpeg
∂ 9.16 (s, 1H), 9.04 (s, 1H), 3.92 (s, 3H)
Product 13C-NMR
EVKCN_Prod2_Attempt2_13CNMR.jpeg
∂ 163.30, 148.66, 144.14, 142.70, 142.58, 53.44 
 
Conclusions and Next Steps
     Based on our TLC and NMR data, we can conclude that we achieved a successful reaction in which all the starting material, 2-(6-chloropyrazin-2(1H)-ylidene)malononitrile, was consumed to produce methyl 6-chloropyrazine-2-carboxylate. TLC showed that there was no spot characteristic of the starting material present in the product. Additionally, NMR peaks were consistent with the literature values (2). 
     Moving forward, we plan to continue on to step 3, in which we will use the product from this reaction and treat it with trimethylsilyltrifluoro-methane and caesium fluoride in order to produce 1-(6-chloropyrazin-2-yl)-2,2,2-trifluoroethan-1-one. 
 
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5th September 2016 @ 21:21

6 September 2016

EVKCN_Prod1_Scheme.png

References

  1. Nikishkin et al. New J Chem. 2013, 37, 394.

  2. P. J. J. Colbon, A. C. Foster and M. E. Giles, J. Heterocycl. Chem., 2008, 45, 1451–1456.

Data Table

Substance

Quant.

Mol. wt.

mmoles

Equiv.

2,6-dichloropyrazine

1.00 g

148.98

6.71

1

malononitrile

0.887 g

66.06

13.42

2

NaH (in mineral oil suspension, 60% by mass)

0.27 g (pure)

0.45 g (in oil)

23.99771

   

THF

109 mL

72.1

   

HCl (1 M)

15 mL

36.46094

   

2-(6-chloropyrazin-2(1H)-ylidene)malononitrile

1.20 g (theoretical yield)

1.14 g (95% yield according to precedent)

0.95 g (79% actual yield)

178.58

6.71 (theoretical)

5.32 (actual)

1 (theoretical)

0.79 (actual)


Procedure

  1. Malononitrile (0.887 g, 13.42 mmol, 2 equiv.), a brown crystalline solid, was dissolved in dry THF (84 mL) and placed in a system under argon.

  2. NaH in mineral oil suspension (0.45 g, 60% NaH by mass) was added to the mixture in small portions, and the mixture was stirred for 30 minutes.

  3. 2,6-dichloropyrazine (1.00 g, 6.71 mmol, 1 equiv.) was dissolved in dry THF (25 mL). A small amount of a white solid impurity remained undissolved. The solution was added dropwise to the existing mixture under argon.

  4. The resulting mixture was bright yellow. It was allowed to reflux overnight for 24 hours.

    Reflux conditions
    Reflux conditions
  5. HCl (1M, 20 mL) was used to acidify the solution. The solution turned a deep red color. (9/8/2016)

    Product 1 before rotovap .jpg
  6. The organic phase was separated and volatiles removed in vacuo.

  7. The residue was partitioned between H2O (150 mL) and Et2O (150 mL) to wash away inorganic salts and excess malononitrile.

  8. Et2O was removed under vacuum.

  9. Product was a dark red crystalline solid (0.95 g, 5.32 mmol, 79% yield). 

    Product 1.jpg
 
Observations and Analysis
 
TLC:
Attemped to dissolve starting material (2,6-dichloropyrazine) and product in DCM --> starting material (SM) dissolves well, product does not dissolve well
 
Solvent system attempt #1: 1:1 EtOAc:Hex
  • SM visualized by UV, not visualized by KMnO4 or ninhydrin
  • Product not visualized by UV, KMnO4, or ninhydrin
  • SM spots travelled too far up plate

Solvent system attempt #2: 1:2 EtOAc:Hex

  • Same results as solvent system attempt #1

Attempted to redissolve product in methanol --> dissolved much more successfully

Solvent system attempt #4: 1:4 EtOAc:Hex

  • SM visualized by UV, not visualized by KMnO4 or ninhydrin
  • Product not visualized by any methods
    • Shows up as yellow-orange spot near bottom of plate (at starting point)

Solvent system attempt #5: 10% methanol in EtOAc

  • SM and product visualized by UV
  • Potentially trace amounts of SM in product

Product 1 TLC (10% methanol in EtOAc).jpg

10% methanol in EtOAc solvent system, silica gel plate, UV visualization

Left = SM, Center = cospot, Right = product

Rf(SM) = 0.763, Rf(Prod)= 0.289

NMR: 1H-NMR (DMSO, 500MHz), 13C-NMR (DMSO, 126 MHZ) measured in ppm
10 mg product dissolved in methanol-d4, but did not dissolve properly --> weak NMR peaks
Reattempt: dissolved 10 mg product in DMSO
10 mg SM dissolved in deuterated chloroform
Starting Material 1H-NMR
EVKCN Starting Material H1NMR.jpeg
∂ 8.51 (s, 2H)
Starting Material 13C-NMR
EVKCN Starting Material C13NMR.jpeg
∂ 147.80, 142.39 
Product 1H-NMR
EVKCN_Prod1_1HNMR_Revised.jpeg
∂ 9.07 (bs, 1H), 7.82 (s, 1H), 7.55 (s, 1H)
Product 13C-NMR
EVKCN_Prod1_13CNMR_Revised.jpeg
∂ 158.79, 147.71, 134.19, 128.54, 65.85
 
Conclusions and Next Steps 
     Based on the TLC and NMR data, we concluded that there was a complete reaction of the starting material, 2,6-dichloropyrazine, to produce the desired product, 2-(6-chloropyrazin-2(1H)-ylidene)malononitrile. TLC indicated a complete reaction as there was no UV-visualizable spot characteristic of the starting material present in the product. Additionally, both 1H- and 13C-NMR data were consistent with the literature values (1). 
     For our next step, we will proceed to step 2 in which we plan to use the product from this step to react with magnesium monopreoxyphthalate in the presence of Li2CO3 and methanol to produce methyl 6-chloropyrazine-2-carboxylate.
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