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16th January 2012 @ 00:10
*PS = Pictet-Spengler

Experiment concluded on 25/01/12.

Scheme
Scheme%20KAB8-2.png
Table%20KAB8-2%20(fixed).jpg

Hazard and Risk Assessment
As for KAB8-1 (here) and KAB1-1 (here).

Previous Related Experiments
Attempts to imrove the acid-catalyzed Pictet-Spengler of MNR10-1 using binaphthalenedisulfonic acid to give MNR12
Acid-mediated PS cyclisation to give racemic PZQ (KAB3-3), the dimethoxy N-benzoyl PZQ analogue (KAB8-1), the dimethoxy PZQ analogue (KAB1-2) and the N-benzoyl PZQ analogue (KAB7-1)
Acid-catalyzed Pictet-Spengler of MNR8-1 using binapthalenedisulfonic acid to give KAB1-1

Following Experiments
Enediamide intermediate cyclisation to give the dimethoxy N-benzoyl PZQ analogue (KAB12-1)

Procedure

Start: 16/01/12
Finish: 20/01/12

MNR10-1 (226.2 mg) was dissolved in toluene (2 mL), then added dropwise to a a suspension of MW45-3 (38.1 mg) in toluene (3 mL) at 0 oC, with vigorous stirring. The reaction mixture was then heated to 70 oC and the reaction progress monitored by TLC against the MNR10-1 starting material and the expected product (KAB8-1).

Acid Added: 38.1 mg
MNR10-1 Added: 226.2 mg
Toluene: ~5 mL

16/01/12
12:05 - MNR10-1 added to acid/toluene mixture.
12:07 - (+ 2 min) Started heating to 70 oC.
12:20 - (+ 15 min) Aliquot 1
13:07 - (+ 1 h 2 min) Aliquot 2
13:58 - (+ 1 h 53 min) Aliquot 3
17:49 - (+ 5 h 44 min) Aliquot 4

17/01/12
08:16 - (+ 20 h 11 min) Aliquot 5
18:10 - (+ 1 d 6 h 5 min) Aliquot 6

18/01/12
09:30 - (+ 1 d 21 h 25 min) Aliquot 7
17:15 - (+ 2 d 5 h 10 min) Aliquot 8
17:50 - (+ 2 d 5 h 45 min) Turned up setpoint to 80 oC
18:30 - (+ 2 d 6 h 25 min) Aliquot 9

19/01/12
18:30 - (+ 3 d 6 h 25 min) Aliquot 10
18:30 - heat and stirring turned off

20/01/12
13:30 - (+ 4 day 1 h 25 min) Aliquot 11
13:30 - Reaction terminated, diluted and quenched

Reaction mixture at 15 min
Reaction mixture at 1 h
Reaction mixture (20 h)
Reaction mixture (2 d)


Monitoring the Reaction Progress by TLC

All TLCs were run in 100% EtOAc with a KMnO4 stain, with the exception of TLC 1, 7-9 which were run in EtOAc/hexane, 3:1. NOTE: TLC 7-9 was also run twice to the solvent line.

TLC of reaction mixture (1)
TLC of reaction mixture (2)
TLC of reaction mixture (3)
TLC of reaction mixture (4)
TLC of reaction mixture (5)
TLC of reaction mixture (6)
TLC of reaction mixture (7)
TLC of reaction mixture (7) - under short wave UV
TLC of reaction mixture (8)
TLC of reaction mixture (9)

NOTE: The lane "8-2" in TLC 9 contains aliquot 8.

TLC of reaction mixture (10)

NOTE TLC 10: The two close spots in the aliquot 9 lane were clearly resolved under short wave UV light. The plate was run 3 times to the solvent line in EtOAc/hexane, 3:1, v/v, in an attempt to resolve the close spots in the reaction mixture.

TLC of reaction mixture (11)
TLC of reaction mixture (12)

TLC 11 and 12 eluent: EtOAc/hexane, 4:1, v/v, with KMnO4 stain. The high Rf spot (the enediamide intermediate) turned intense yellow without heating.

Monitoring the Reaction Progress by LCMS
Expected MS:
MS%20expected%20KAB8-2.jpg

LCMS of Aliquot 8
LCMS of Aliquot 8 (annotated)

There were two LCMS peaks that give major MS peaks of m/z 367.2. Both the expected product and the known stable enediamide intermediate have mw of 366, suggesting the reaction mixture contains both the expected product and the intermediate (see table of values above).
The peak with retention time: 24.450 min is most likely the KAB8-2 peak. The low intensity of both the UV and MS trace, compared to that of the signal with retention time of 27.817 agrees with the relative intensity of the product and intermediate TLC spots.
Annotated PDF Legend: (i) UV absorption (210 nm) of KAB8-2 product, (ii) UV absorption (210 nm) of enediamide intermediate, (iii) UV absorption (254 nm) of enediamide intermediate, the KAB8-2 was too low in concentration or conjugation to show an absorption signal at this wavelength, (iii) MS detection of the enediamide, (iv) MS detection of the KAB8-2 product. Note the relative intensities of (iii) and (iv)

The peaks at m/z = 285 could correspond to the daughter ions flying with Na+, both of which have the same mw:
Notes.png

19/01/12
The heat was turned off at 18:30, and the reaction was left to stand (no stirring) overnight.

20/01/12

At 13:30 (4 days), after reaction mixture was left to stand at room temperature for 19 hours, the solution appeared to consist of to phases, the top, clear and colourless and the bottom, a pale brown colour. The solution was diluted with EtOAc (30 mL) and the the entire reaction mixture turned beige and cloudy.
After dilution with EtOAc
First extraction with EtOAc
Second extraction with EtOAc

After quenching with saturated sodium bicarbonate (30 mL), during which a minute amount of gas was released, the organic EtOAc layer was set aside. The aqueous layer was further extracted with EtOAc (2 x 20 mL) and the combined EtOAc fractions were dried over magnesium sulfate, filtered, then concentrated under reduced pressure to give crude KAB8-2 as a yellow oil (116 mg).
Crude KAB8-3


24/01/12
Murray purified the crude product by silica gel flash chromatography in 50-60% EtOAc/hexane, v/v. The suspected enediamide intermediate was eluted with fractions 3-10, which were combined then concentrated under reduced pressure to give KAB8-2 as the dimethoxy N-benzoyl PZQ analogue enediamide intermediate as an off-white, cloudy solid/oil (65 mg, see expected product of KAB11-1, 11-2 and 11-3). The product showed some signs of crystallising/solidifying and was placed in the freezer overnight.
Total mass recovery for the purification was 55%. The remainder of mass was a mix of highly polar, baseline products (assumed the difference of mass ~40 mg) and some of the completely PS cyclised product (<10 mg).
KAB8-2, enediamide intermediate


25/01/12
Proton NMR (CDCl3)of the suspected enediamide intermediate (major spot), which was isolated by silica gel column chromatography confirmed its identity as the intermediate. The two alkene were clearly visible as broad peaks (X and X ppm).
The remainder of the isolated intermediate (~57 mg) was immediately used in the following experiment: Enediamide intermediate cyclisation to give the dimethoxy N-benzoyl PZQ analogue (KAB12-1).

Conclusion



To Do:
- Upload NMR
- Assign alkene peaks.
- Write conclusion.


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NOTES 16/01/12
- Starting material almost completely consumed after 1 hour.
- Possibly fragmented reaction mixture contents ~2 hr mark. Will wait a bit longer to see if product starts to appear.
- It almost appeared as if product was forming in Aliquots 2 and 3, however, after taking multiple TLCs, it appears the Rf of the supposed product spot, was slightly lower than the expected product Rf.

NOTES 17/01/12
- 08:15: Noticed volume is slightly less, not sure if it's just removal of aliquots.
- TLC 5 has a spot with an Rf between the starting material (SM, MNR10-1) and the expected product (previously made KAB8-1). The intense high spot is presumably the enediamide intermediate, which is typically has higher Rf values than the starting material or expected products with PZQ and its analogues.
- It looks like there are two spots in the KAB8-2 aliquot 6 in TLC 7. They are very close together, so it's difficult to tell.

NOTES 18/01/12
- No obvious changes in the reaction either visually or by TLC. Turned up the heat to try and drive the cyclisation.
- TLC 10: I'm now fairly confident that there is product in the reaction mixture. Will leave it at 80oC overnight and assess any changes in the morning.
- LCMS of Aliquot 8: Now I'm definitely sure there is product. With the LCMS fixed, we may have a way of assaying the reaction progress against relative peak intensity of the MS traces.
- Could this be why it's so difficult to get past the intermediate? Aromatic? --> No. 4n + 2 does not equal 8.
Explanantion?.png

NOTES 19/01/12
- Lab had scheduled powerdown, so the reaction mixture was left to stand.
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Linked Posts
Attached Files
Scheme KAB8-2
Table KAB8-2
Reaction mixture at 15 min
Reaction mixture at 1 h
TLC of reaction mixture (1)
TLC of reaction mixture (2)
TLC of reaction mixture (3)
TLC of reaction mixture (4)
Reaction mixture (6 h)
TLC of reaction mixture (5)
Reaction mixture (20 h)
TLC of reaction mixture (6)
TLC of reaction mixture (7)
TLC of reaction mixture (7) - under short wave UV
TLC of reaction mixture (8)
TLC of reaction mixture (9)
TLC of reaction mixture (10)
LCMS of Aliquot 8
LCMS of Aliquot 8 (annotated)
Expected MS
Reaction mixture (2 d)
Tautomer
Daughter ions (1)
After dilution with EtOAc
First extraction with EtOAc
Second extraction with EtOAc
TLC of reaction mixture (11)
TLC of reaction mixture (12)
Crude KAB8-3
Table KAB8-2 (fixed)
KAB8-2, enediamide intermediate