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24th November 2011 @ 01:04
Slight confusion over the labelling of sample vials. Both MW36 and MW29 are compound 1a (Scheme 1).

Scheme 1


Procedure


Similar reaction conditions for the preparation of MNR11-9 were applied to MW29-4.

Related Experiments
Acid-mediated Pictet-Spengler of MW29 to give rac-PZQ MNR13-1
Attempts to the acid-mediated Pictet-Spengler cyclization of the ‘Ugi intermediate’ MW29 (MW31)

Hazard and Risk Assessment
HIRAS 3-1



Preparation of KAB3-1:

MW29-4 (42 mg) was dissolved in toluene (0.6 mL) before the addition of methanesulfonic acid (0.001 mL) to give a clear, colourless solution. The reaction mixture was heated in an oil bath, 116C at 3:00 pm. The temperature of the oil bath fluctuated between 113-120C until the reaction mixture was removed from heat at 4:00 pm - total 1 hour at reflux. The solution had turned yellow accompanied by a dark red, non-miscible substance.

A sample of KAB3-1 was extracted from the reaction mixture then quenched with saturated sodium bicarbonate. TLC was performed on the KAB3-1 extraction against the starting material MW29-4, in ethylacetate:hexane, 3:1 with a KMnO4 stain (Photo 2).

KAB3-1 1st TLC 75%

Photo 2. From top to bottom: (1)MW29-4, (2)MW29-4 and KAB3-1, (3)KAB3-1


It was difficult to see what was product, starting material or byproduct. The TLC was repeated (ethylacetate:hexane, 3:1; KMnO4 stain) with the addition of racemic praziquantel (PZQ) (Photo 3).

KAB3-1 TLC 75 %

Photo 3. From left to right: (1)MW29-4, (2)MW29-4, KAB3-1 and PZQ, (3)KAB3-1, (4)PZQ

It was still difficult to determine the progress of the reaction based on this TLC. The TLC was repeated, reducing polarity of the mobile phase, to promote separation of any close eluting spots. Another TLC was performed in ethylacetate:hexane, 1:1 then stained with KMnO4 (Photo 4).

50% TLC

Photo 4. From left to right: (1)MW29-4, (2)MW29-4, KAB3-1 and PZQ, (3)KAB3-1, (4)PZQ

The TLC in 50% ethylacetate indicated consumption of the MW29-4 starting material and suggested the formation of the PZQ. The second fastest spot in KAB3-1 was unknown.

The KAB3-1 reaction mixture was quenched with saturated sodium bicarbonate (10 mL) then extracted with ethylacetate (3 x 15 mL) (Photos 5-7). The organic layers were combined then dried over magnesium sufate to give a clear yellow solution. The solvent was removed under reduced pressure to give crude KAB3-1 (PZQ) as a dark brown-red oil (28.6 mg)

Quenching KAB3-1

Photo 5: Quenching of KAB3-1 with sodium bicarbonate before the first extraction. The non-miscible red layer was clearly visible at the organic-aqueous interface.
KAB3-1 2nd Extraction

Photo 6: The aqueous layer of KAB3-1 just before the second extraction with ethylacetate.
Final KAB3-1 extraction

Photo 7: The less coloured organic layer above the orange-brown aqueous layer, just after the final extraction

Empty flask: 33.4408 g
Flask + sample: 33.4694 g

CRUDE YIELD of KAB3-1 (PZQ): 28.6 mg

25/11/11
Attempt at purification by flash column chromatography resulted in poor separation (Ethylacetate in hexane 20-100%). Monitoring separation by TLC was difficult due to high dilution of KAB3-1.
The column was flushed with neat methanol and all fractions combined. The solvent was removed to give crude KAB3-1 (~30 mg). Little to no product was lost.

28/11/11 - Second attempt at purification of KAB3-1
The recovered KAB3-1 was purified by flash column chromatography (silica gel 6 cm, diameter 1 cm in ethylacetate:hexane, 1:1).
Fourteen fractions were collected. Thin Layer Chromatography (in ethylacetate:hexane, 1:1) indicated elution of product in fractions 1-3 and 5-8. Fractions 1-3 and 5-8 were combined to give KAB3-1a and KAB3-1b, respectively. Both solutions were concentrated under reduced pressure.

Concentrated KAB3-1b was a thick yellow oil. TLC of KAB3-1b was performed against racemic PZQ and both had Rf of ~0.4.
KAB3-1b was put under high vacuum for 3 hours before storage in the freezer overnight.

KAB3-1a was concentrated under reduced pressure to give a yellow oil.

NMR Data
KAB3-1a NMR



29/11/11

KAB3-1b was removed from the freezer - no crystallisation. The solvent was removed from KAB3-1a under reduced pressure.

YIELD
Fractions 1-3 KAB3-1a: 10 mg
Fractions 5-8 KAB3-1b: 3 mg

TLC fractions

Repeat TLC of KAB3-1a and KAB3-1b. From left to right: (1) KAB3-1b, (2)KAB3-1b duplicate, (3)racemic PZQ, (4)KAB3-1b, rac-PZQ and KAB3-1a, (5)KAB3-1a

The solvent was removed from KAB3-1a under reduced pressure and the sample placed under high vacuum. NMR analysis of KAB3-1a and KAB3-1b pending.


Preparation of KAB3-2

24/11/11

The reaction was prepared as above, MW29-4 (24 mg), toluene (0.6 mL) and methanesulfonic acid (0.001 mL). The clear, colourless mixture was stirred at room temperature from 4:15 pm (Photo 8).

KAB3-2 at time zero

Photo 8. KAB3-2 at time zero

At 1.5 hours after the reaction start, a sample of KAB3-2 was extracted and quenched in saturated sodium carbonate before TLC analysis in ethylacetate:hexane, 1:1. The TLC showed no spots corresponding to the starting material (MW29-4) or the expected product KAB3-X (PZQ). The large, fast eluting smear present in the KAB3-1 and all MW29-4 TLCs was present in the KAB3-2 TLC. The reaction mixture was left to stir at room temperature overnight.
Unlike KAB3-1, the reaction mixture of KAB3-2 at 1.5 remained mostly clear and colourless. However, there was some formation of a brown precipitate.

KAB3-2 after 1.5 hours

Photo 9: KAB3-2 after 1.5 hours.

29/11/11

The reaction mixture removed from heat, diluted with EtOAc (5 mL) then quenched with saturated sodium bicarbonate (6 mL). Following separation of the organic fraction, the aqueous layer was extracted with EtOAc (2 x 5 mL). The combined organic fractions were dried over magnesium sulfate, before removal of the solvent under reduced pressure. Crude yield: 13.1 mg.


***Procedure finished. Difficult to make any conclusions from this experiment due to the low mass of product obtained (I'm still getting used to working with tiny amounts). However, procedure was utilised in future experiment KAB3-5 in attempt to push the peptide acetal Ugi intermediate to PZQ product.

Following experiments
The reflux conditions were adapted in:
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-mediated Pictet-Spengler reaction to give PZQ (KAB3-4) and the N-benzoyl PZQ analogue (KAB7-2)
Acid-mediated Pictet-Spengler reaction to give PZQ (KAB3-5)


No further characterisation of the product has been performed as of 08/12/11 due to low quantity of product. The NMR recorded of the samples were too dilute. It is unknown what the outcome of the experiment was.
Linked Posts
Attached Files
HIRAS 3-1
Scheme KAB3-1
Scheme KAB3-X
50% TLC
Quenching KAB3-1
Final KAB3-1 extraction
KAB3-1 @ 4:00 pm
KAB3-2 after 1.5 hours
KAB3-1 TLC 75 %
KAB3-1 1st TLC 75%
KAB3-1 2nd Extraction
KAB3-2 at time zero
KAB3-1 Table
2nd Column
TLC fractions
KAB3-1a NMR
KAB3-1a NMR