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20th October 2016 @ 22:12

10/18

MRLS 4-2 (0.473 g, 1.4 mmol, 1 equiv) was added to toluene (10 mL) along with MRLS 8-1 (fraction 1, 0.2761 g, 1.5 mmol, 1 equiv), potassium hydroxide (0.3281 g, 5.8 mmol, 4.2 equiv) and 18-crown-6 (0.032 g, 0.12 mmol, 0.09 equiv).

The reaction was stirred at room temp for 30 minutes and then heated to 40°C (bath temperature) for 4 h.

Planned on leaving stirring at room temperature for only 10 minutes, but problems with hot plate meant lukewarm water for 15 minutes until I got one that worked properly.

Came back later that night (11:30) to monitor reaction via TLC. Also noticed that hot plate had stopped heating. TLC showed almost no starting material—I decided to replace the hot plate and keep it stirring over night (~10 more hours) to make sure all starting material had completely reacted.

10/19

TLC showed no SM and baseline spots (tested w/ 60% EtOAc/Hexanes, KMnO4 stain) (shown below).

The sample was cooled to room temperature and diluted with 8 mL of water. The mixture was extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with water (2 x 8 mL) until the aqueous layer became neutral. The organic layer was then washed with brine (7 mL) and dried over Na2SO4. The brown/red solution was gravity filtered and dried via rotovap to yield a red brown oil. It was placed under high vac over night.

10/20

Yield: 0.55 g/.6811

80.75% yield

Took NMR.

Unfortunately, ran out of time so could not run a column on the crude product to verify a successful synthesis. Hopefully, another group will pick up this project!

 

 

Attached Files
13th October 2016 @ 20:02

Started 10/4

 

 Based on AEW 221-2.

Procedure:

Dried 3-necked round bottom flask+stir bar on high-vacuum w/ heat gun. Immediately placed under Argon. (attempting to conduct reaction under anhydrous conditions)

Transferred LiAlH4 (about 0.5 g) to the round bottom flask. Added 12 mL Et2O (anhydrous) via syringe. Stirred in ice bath (~0 C).

Dissolved MRLS 6-1 (4.57 g, 18.26 mmol) in anhydrous Et2O (8 mL). Transferred this mixture to the LiAlH4 suspension via syringe. Added dropwise over ~5 minutes. Stirred in ice bath for about 1 hour. Removed the round bottom from the ice bath and stirred at room temperature for 3 hours. Then, refluxed the mixture for about 16 hours. 

10/5

Quenched the LAH by stirring with rochelle salts and Et2O overnight.

10/6

Filtered the reaction mixture through celite. Washed round bottom with EtOAc. Performed extraction (3x30 mL EtOAc) of the mixture, and combined the organic layers. Dried the layers with sodium sulfate, followed by gravity filtration and rotovap. Yield: 3.39 g, 83.5%. Took NMR of the crude.

10/11

Attempted to purify the crude with the Biotage Isolera. 

Used KP-Sil 100 g column. Loaded sample by dissolving in DCM, adding silica gel, and then rotovap (dry loading instead of using a samplet). Unfortunately, had issue with attaching solvent lines to the column, resulting in a spill (possibly affecting sample?). Collected 6 fractions.

10/13

Rotovapped fractions. Although I had expected the product to be in the second fraction, there was almost no product after rotovapping. The other fractions, however, did have some product (test tubes 20-29 and 30-40).

 

10/14

Took proton NMR of fractions 20-29 and 30-40. Ethyl acetate solvent impurities near 1.25 ppm (t), 2.03 ppm (s), 4.11 ppm (q). Will attempt to run NMR again after removing solvents.

Redid NMR (removed EtOAc/Hexanes by rotovapping CDCl3 mixture, then adding CDCl3, rotovapping once more, and then dissolving that sample in CDCl3). Attached below. The NMRs seem to be very similar, but unsure why there are differences. I will try asking for assistance on our GitHub post (issue #430).

10/17

It seems like the fractions are diastereomers of one another (thanks to Chase Smith (@MedChemProf) for his help here!) .

stereoisomers

I've also added GC-MS results (TIC and MS) for both fractions. The mass peak matches that of the structure above with a cleaved CH2OH (M+ of ~191).

 

 

Attached Files
13th October 2016 @ 19:48

Synthesis begun on 10/3

Reaction Scheme:

 

Compound

MW (g/mol)

Mol (mmol)

g

Density (g/mL)

Volume (mL)

eqv

2-chloro-6-hydrazinylpyrazine

(MRLS 1-2)

144.56

1.435

0.2075

 

 

1

Tertbutyl 3-formylpiperidine-1-carboxylate

213.27

1.48

.3157

 

 

1

EtOH (190 proof)

60.05

 

 

 

5

 

   

 

 

 

 

 

Product

339.82

1.435

0.488 

 

 

 1

Procedure

MRLS 1-3 (0.2075 g, 1.435 mmol)) was stirred in EtOH (190 proof, 5 mL). Note that the MRLS 1-3 was added slowly to make sure it all went into the mixture.

Tertbutyl 3-formylpiperidine-1-carboxylate (0.3157 g, 1.48 mmol) was added to the suspension and the reaction mixture was stirred at room temperature for 12 hours. Once it appeared the starting material was consumed, the solvent was removed under reduced pressure to yield a yellow pale solid. Dried under high vac, and the product was analyzed via NMR (0.25 g, 51% yield).

(Compared to the MRLS 3-1, this product was much paler and drier, resembling the 3-2. However, the NMRs look similar. I'm having difficulties analyzing using GC-MS, as no peaks appear on the TIC. Will update if I make any progress. Reminder: The only difference between the procedures for the 3-1 and 3-3 was the addition of acetonitrile as a solvent.)

 

Attached Files
13th October 2016 @ 19:25

Synthesis begun on 9/29

Bringing through more material to continue primary synthesis. This time, we are not using acetonitrile, and instead just using 3% EtOH.

Reaction Scheme:

 

Compound

MW (g/mol)

Mol (mmol)

g

Density (g/mL)

Volume (mL)

eqv

2-chloro-6-hydrazinylpyrazine

(MRLS 1-4)

144.56

3.46 mmol

0.5

 

 

1

Tertbutyl 3-formylpiperidine-1-carboxylate

213.27

3.5 mmol

0.746 g

 

 

1

3% EtOH in H2O

60.05

 

 

 

10

 

   

 

 

 

 

 

Product

339.82

3.46

1.176 g

 

 

 1

Procedure: 

MRLS 1-4 (0.5 g, 3.46 mmol) was stirred in 3% EtOH (10 mL). After stirring for 1 hour, the MRLS 1-4 had yet to completely dissolve. 

Tertbutyl 3-formylpiperidine-1-carboxylate (0.8519 g, 4 mol) was added to the suspension and the reaction mixture was stirred at room temperature under Argon for 90 minutes. Unlike the last attempt (MRLS 3-1), an orange, sticky material formed that gathered on the stir bar. The stir bar was removed and cleaned, and the reaction was left stirring under Argon for another 48 hours. 

10/1

Checked reaction via TLC, but the starting material had yet to be consumed (perhaps some of the undissolved MRLS 1-4 from earlier). I decided to rotovap what was left, but the spinning of the round bottom caused the reaction mixture to foam up and collect in the bump trap. Although rotovapping had worked for MRLS 3-1, the solvent had not evaporated. Instead, proceeded with vacuum filtration. Collected 0.3448 g of material (yield: 29.3%). Unlike the yellow fluffy solid from the last attempt, the product was a pale, peach color.

10/3

Filtered the solution that was in the filter flask from 10/1. Collected 0.2683 g of material (yield:22.8%). Verified via TLC that this material was identical to product from 10/1, so the two were combined (total:0.6131 g, 52 % yield). Prepared NMR sample (will update).

10/4

After checking NMR of MRLS 1-4, we noticed a strange peak around 2 ppm (also seen in NMR below). I decided to run this reaction once more with MRLS 1-3, which had a cleaner NMR. 

Attached Files
12th October 2016 @ 05:09

Started 10-6-2016

This is different than previous attempts in that we're using polymer bound p-tsoh in hopes of having a cleaner reaction mixture. We also used a microwave reactor to run the reaction at 140C.

scheme

compound

MW (g/mol)

Mol (mmol)

g

Density (g/mL)

Volume (mL)

eqv

2 chloro 6 hydrazinylpyrazine

144.56

4.80

0.70g

 

 

1

toluene

 

 

 

 

19 mL

 

Triethyl orthoformate

148.20

9.70

1.42 g

0.891

1.60

2

TsOH (poly bound)

190.2

0.614

0.33g 

 

 

0.2

Product

 154.56

 3.47

 0.74

 

 

 1

 

2-chloro-6-hydrazinylpyrazine (from Synthesis of 2-Chloro-6-hydrazinylpyrazine (MRLS 1-1) (0.70 g, 4.80 mmol) was dissolved in dry toluene (19 mL). Dry Triethyl orthoformate (1.60 mL, 6.94 mmol) and polymer bound TsOH (0.33g g, 2.5-3 mmol active ingredient/1g material) were added, and the resulting reaction mixture was put into a microwave reaction vial and run at 140C for 3.5 hours.

Procedure based on HM 1-1.

Log

10-6 3:55 pm

Starting material dissolved in toluene, which didn't dissolve until about 60C as expected. Triethyl orthoformate added after starting material was dissolved, along with 0.33g polymer bound p-TsOH. This mixture was added to a 20 mL microwave reaction flask and put into the microwave reactor for 3.5 hours at 140C. Pressure did not exceet 2.23 bar. 

8:50 pm

flask was removed from reactor and taken back to the hood to cool. TLC tomorrow.

10-7

TLC performed, 60% etoac. product is believed to be the bottom spot that shows up under long-wave UV. s=starting mat, c=co-spot, p=product

image

 

Reaction mixture after reaction, p-TsOH polymer filtered off. very clear.

Displaying IMG_1424.JPG

The reaction mixture was filtered off and left an oil. NMR was performed on this oil, and the lines we want are definitely there (8. 26, 9.42, 9.68ppm) Column will be run after break.

10-18

Oil dissolved in DCM and put into a samplet to use in the biotage. 4 fractions were spotted. The fourth fraction was isolated and the solvent was removed under reduced pressure. TLC and NMR (calling it 2-4-1 was performed on this, which showed it to indeed be the core, but a very small amout (about 40 mg). 

In the crude NMR, it was noted that there seemed to be a lot of this compound in the reaction mixture:

 intermediate

It was thought that isolating this and submitting it back to acidic conditions would force the ring to close, so the solvent from this fraction was removed under reduced pressure and the resulting oil was dissolved in toluene, and 0.25g of polymer bound p-tsoh was added after it was dissolved. This was brought to reflux for 24 hours, after which most of the unclosed structure was gone from the TLC. 

Displaying IMG_1443.JPG

10-19

The toluene was removed under reduced pressure, which again left an oil. This oil was dissolved in DCM and submitted to the biotage. Four fractions were spotted, and the fourth was the probable product. Unfortunately, when the solvent was removed under reduced pressure, an oil resulted (not the crystal from the previous procedures). TLC was performed on this oil, which certainly seemed to be the core. NMR will be performed on this oil tomorrow.

.Displaying IMG_1442.JPG

Attached Files