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Author: Subject: Preparation of Thalidomide
karlos³
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[*] posted on 6-3-2020 at 19:51
Preparation of Thalidomide


I want to present you guys my first attempt at the preparation of this infamous medication, a known teratogenic which caused one of the largest scandals in the pharmaceutical industry ever.
It causes horrible birth defects, but is still in use nowadays against leprosy, certain types of cancer, and even a harmless but ugly skin condition called psoriasis.
Its intended use was originally as a sedative and then it was found to be effective against pregnancy induced morning sickness, which lead to increased prescription especially for pregnant women, and thus the whole misery had started.
Interestingly, it is actually even a strong sedative, as it is structually somewhat related to barbiturates.

A. Reagents
- L-Glutamic acid
- Phthalic acid anhydride
- DMF
- Pyridine
- PEG-400
- Ammonium acetate
- HCl N6
- aqueous Ammonia 25%

B. Equipment
- heated Magneticstirplate
- Oilbath
- Erlenmeyer flask
- 30cm Liebig condenser with moderately cold water running through
- Vacuum filtration setup

C. Procedure

Preparation of N-Phthaloyl-DL-glutamic Acid

- 14,95g of phthalic anhydride and
- 14,74g of L-glutamic acid were given together into
- 40ml of pyridine, then put into a preheated oilbath and the temperature increased until at 115°C.
It was stirred at this temperature for 100min, then the heat was turned off and the oilbath removed.
When the temperature fell to ca. 70°C, 150ml of cold water were added to the stirred mixture in the flask.
Enough N6 HCl was added until the mixture was strongly acidic, then the clear solution was left on the stirrer for a few hours, standing in a cold water bath.
When the mixture was checked the next time, circa 3-4h later, a very fine powdery precipitate was visible in the lower half of the flask.
This precipitate was vacuum filtered and washed with 3x20ml of water, then dried with moderate heat.
A sample of the dried powder melted at 192-194°C.
The total weight was 20,5g, which corresponds to 73,9%(also, 73,9mmol of the theory).

Synthesis of Thalidomide

- 10,25g of N-Phthaloyl-DL-glutamic Acid(37mmol) and
- 10g of anhydrous ammonium acetate(129,5mmol, 3,5eq) were heated in the oil bath on the stirrer, in
- 25ml PEG-400.
That is were my thinking went wrong though.
The reagents still hadn't satisfyingly dissolved in my chosen solvent when around 100°C, so I pondered a bit and tried to make at least up for some yield opposed to no yield.
I added 25ml of DMF to the mixture, which is a known working solvent for this reaction, just not very effective.
So, it was heated further until the mixture reached 152-155°C and was kept there for 50min, then the oilbath was removed and the heat turned off.
At around 95°C, 200ml of cold water were added to the mixture, which immediately resulted in a few very tiny precipitates.
The mixture was then put into the ice-bath and left there for an hour.
More precipitated, and I had the suspicion some of it could be unreacted substrate, so I added a bit of 25% ammonia solution to keep any unreacted substrate in solution and to have only product as the precipitate.
The solid was filtered out and washed with 3x50ml of cold water and 2x20ml of cold ethanol, then dried using moderate heat.
The dried substance was weighed and it was not surprising that it amounted to only 1,1g of an off-white powder.
Then it was recrystallised, dissolved in DMF and precipitated with ethanol and the next day filtered, washed with cold ethanol(2x20ml), and dried again.
The resulting substance, now really white, amounts now to 930mg, or 3,6mmol, which corresponds to 10,27% of the theory.
A melting point test resulted in a temperature of 268-270°C.


D. Discussion

The second reaction to form the gluthethimide ring was totally crap.
I had the idea that the reaction could be done in PEG-400, since it is so high boiling(ideally, diphenyl ether at a temperature of 175°C showed the best results).
But the solubility issues crushed my hope about that.
So I had to use the much lower yield giving DMF(it results in around half as much product as the diphenyl ether).
The advantage I had with the DMF/PEG-400 mixture though was, that I had a simpler workup, since the solvents are soluble in water, unlike diphenyl ether.
Possible yield with diphenyl ether is around 65%, using DMF they haven't even reached 40%, merely 38% of the theory.
This was the reason why I thought PEG-400 would be a good choice, table S7. on page, that I was under the impression the temperature of the reaction correlates with the yield, and diphenyl ether was both the highest boiling and yielding of the ones they tried, with the yield somewhat increasing together with the temperature.

As I still have some precursor, and as this one was easily made, I will try the second reaction again without making such stupid mistakes and then trying to save everything with a not very smart decision.
But at least I got still a bit out of it instead of nothing :)

E. References

Vu, B. D., Ho Ba, N. M., & Phan, D. C. (2019). A FACILE SYNTHESIS OF THALIDOMIDE.
https://sci-hub.se/10.1021/acs.oprd.9b00122

SUPPORTING INFORMATION
A FACILE SYNTHESIS OF THALIDOMIDE
Binh Duong Vu, Ngoc Minh HoBa1, Dinh Chau Phan
https://pubs.acs.org/doi/suppl/10.1021/acs.oprd.9b00122/supp...
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[*] posted on 7-3-2020 at 06:32


Interestingly, it sometimes causes psoriasis.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078429/
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[*] posted on 7-3-2020 at 07:57


Carl sir, respect from me.
A very nice write up.
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[*] posted on 8-3-2020 at 04:25


Very nice write up! Thalidomide is nice stuff as long as it is not used by pregnant women.

Maybe you could try this with urea in a microwave? As soon as the water is gone no more radiation is absorbed, so once the reaction is done the reaction stops. No need for a solvent nor temperature control. I did this with a different subtrate.

Source:
https://www.mdpi.org/ecsoc/ecsoc-5/Papers/e0008/e0008.htm
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[*] posted on 8-3-2020 at 18:53


Karlos:

Interesting report. Finally some real chemistry being posted - thank you. Here are a couple of comments.

If you use L-glutamine in your first reaction in place of L-glutamic acid, the product (N-phthaloyl-L-glutamine) can be cyclized directly to thalidomide using acetic anhydride.

See: F.E King et al., J. Chem. Soc., 1959, 873-880.

Carbonyldiimidazole (CDI) and cat DMAP in THF with N-phthaloyl-L-glutamine can also be used for the final step in place of acetic anhydride.

See: G.W. Muller et al., Org. Process Res. Dev., 3, 139-140 (1999).

AvB

[Edited on 9-3-2020 by AvBaeyer]
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[*] posted on 9-3-2020 at 00:09


This is a good example of nice amateur organic chemistry at a good level. Nice to see this kind of results.
Doing organic chemistry in a home lab at a decent level is quite hard, but as this demonstrates, it can be done!
Thumbs up!




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karlos³
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[*] posted on 26-3-2020 at 16:44


Thank you all for the replies, but lets be honest, despite working this first trial was really half-assed!
Usually I do better, this was just something really unusual to me.

@Tsjerk, Thanks for the suggestion with the microwave method, I just haven't chosen this route because they used a sealed vessel, this was the reason not to choose this method.
I assumed it is something special similar to a bomb for high pressurized reactions, able to withstand quite some pressure and this was not something I would like to do in the homelab.
But you have simply made a premixed solution of the reagents, evaporated that dry and nuked it in the microwave and nothing more? That is impressive and something I would have chosen too.
I am sure that the method of your baclofen synthesis will be better than what I did here...
Just like this: http://www.sciencemadness.org/talk/viewthread.php?tid=104964...
Or do you any better and more specific suggestions?
If you could give me some tips besides that I would be very grateful!

@AvBaeyer, thank you for the suggestion of alternative routes, I've seen them actually beforehand already and my choice was the glutamic acid route because of the reagents on hand already.
While it may not be the best or highest yielding method, the advantage was for me that I only needed to purchase two cheap reagents, and no need to use my last little bit of Ac2O, or CDI which I do not have(sadly).
Specifically, I chose this route because it doesn't uses the nasty 4-DMAP but simply cheap pyridine for the synthesis of the intermediate.

But regarding one-pot conditions, there is also a way directly to the product with glutamic acid, under microwave conditions, with phthalic anhydride and the glutamic acid, and preferably thiourea as nitrogen source, giving somewhat around 50-60% yield this way.
Ammonium acetate, urea, were tried by them too, but this increases the quantity of the side product phthalimide, while thiourea seemingly favors the glutarimide formation.
While interesting, the use of microwave conditions, specifically these ominous special-microwave-able and sealed vessels were what made it unattractive to me, as I have almost no experience with reactions in the microwave.
Also the thiourea had to be bought, only for that one reaction, while the ammonium acetate was already there.

Anyways, I'll try with urea the next time, still have some intermediate and plan to make some more too, so I can try the one or other glutarimide preparation, hopefully with better results :)
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[*] posted on 27-3-2020 at 06:20


Quote: Originally posted by karlos³  
@Tsjerk, Thanks for the suggestion with the microwave method, I just haven't chosen this route because they used a sealed vessel, this was the reason not to choose this method.
I assumed it is something special similar to a bomb for high pressurized reactions, able to withstand quite some pressure and this was not something I would like to do in the homelab.
But you have simply made a premixed solution of the reagents, evaporated that dry and nuked it in the microwave and nothing more? That is impressive and something I would have chosen too.
I am sure that the method of your baclofen synthesis will be better than what I did here...
Just like this: http://www.sciencemadness.org/talk/viewthread.php?tid=104964...
Or do you any better and more specific suggestions?
If you could give me some tips besides that I would be very grateful!


I never noticed they used any kind of pressure vessel. I just put everything in a beaker, with a bigger beaker upside down over it, and nuke it as you say. It takes a while to get started if the material is too dry, maybe a drop of water would help. After that, you will get a disgusting brown boiling mass, wait until it stops boiling. As in my earlier write up linked above I used hot 33% ethanol to dissolve everything before it solidifies... If it solidifies you will have to break the beaker or have a lot of patience. In my case the product dissolves in hot diluted ethanol, while the gummy side-product doesn't. The 33% ethanol will still boil and splash as you add it but it will make a sort of a foam of your mess. Now stir hard, hot filter and you will have a clear solution that crystallizes upon cooling.

The microwave step is a bit dirty, it fumes of NH3 like mad and the up-side down beaker is definitely a recommendation if you don't want to clean all the inside of your microwave. Now you only have to clean the bottom plate.

I think this reaction is quite endothermic, it doesn't go very fast and seems to require quite some microwave power, possibly because of the water that has to evaporate. It is just a sticky mass that splashes. I got far better results when first dissolving the stoichiometric amounts (little excess urea) of reactants in water and then boil that to dryness than by mixing the two and use that directly.


Edit: and don't put more than 10 grams in a 100ml beaker.


[Edited on 27-3-2020 by Tsjerk]
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[*] posted on 29-9-2020 at 10:23


I think I actually managed to ruin the microwave glutarimide formation :o
I ran that at 20mmol substrate, with 24mmol urea, and formed the mono-urea salt just as you describe.
Then I reacted that at a lower wattage though(600W) and I did so for almost six minutes.
It had stopped foaming mostly after 4min(circa) and at 5min I thought it did again, and close at 6min I realised that this isn't the reaction anymore.
Well, what can I say, I think I carbonised most of the product.

I tried then immediately to dissolve whatever is possible, and added 12ml of MeOH carefully under stirring.
But the complete bottom of the flask is full with tarry soot that didn't wanted to dissolve.
And the beaker cracked due to the temperatures, but luckily did not break.
So I filtered that red-brown MeOH and added some water to it, it is now being cooled and will hopefully show a little bit of product anyways....

But this synthesis, I will not do that again, thats for sure!
The remaining precursor will be reacted in PEG-400 with urea, and then it is over with me and the glutarimide ring formations!

I found a variation of the alternative route using glutamine, one that uses tosyl chloride for the cyclisation, and thats a perfect alternative for me, I will attempt this.
AvBaeyer was right, and I should have turned my brain on earlier to think what suitable reagents can catalyse the cyclisation else.
Here is it: https://patents.google.com/patent/US20050272934A1/en
Nice how they are running that one-pot, in example 2&3, thats definitely an attractive way for me.
I will just carefully add the tosyl chloride in little portions to the pyridine solution of the glutamine+phthalic anhdyride reaction mixture.
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[*] posted on 1-11-2020 at 04:14


I've tried a little bit around, also attempted the one-pot synthesis.

What I tried was the amidation in PEG-400, using urea as nitrogen source, that worked fine and fast, the yield was after purification 28,59%.
Could have worked better, but since I pushed the temperature rising pretty strong, I accidentally got over 200° and the PEG decided to get discolored together with my product.
Nonetheless, the advantage is that its a pretty fast process.

Tried the one-pot synthesis twice, far from being optimised though.
I used to little pyridine as solvent, and both times issues when I cooled the reaction to add the tosyl chloride, it quickly became thick and viscous and resulted in hot-spots causing discoloration.
The first time I was more careful and so it had worked better, but also wasted product because I didn't heated well enough after the tosyl chloride was in the reaction, only kept it somewhat warm,
In turn, only 11,5% yield, although it was directly of an impressive white and did not require much purification, only washed it twice in ethanol and then recrystallised it from EtOH/DMF with a fitting melting point.
The second trial one-pot was even worse, nothing to say about it, discolored and I haven't even calculated the yield, not to add further frustration.

A friend has attempted this method likewise, but he used acetic anhydride instead of tosyl chloride, with a similar outcome(including discoloration and not really an optimal yield).

Another attempt according to the attached document and the following patent: https://patents.google.com/patent/WO2009083724A1/en
has been made.

The attached paper claims a good yield of N-phthaloyl L-glutamine by the reaction of phthalic anhydride and glutamine in DMF, it sure looks good.
Although I couldn't replicate that outcome, actually, it was even far from it... and I wasted my last 17g of phthalic anhydride on it, yet still don't know what happened.

But the good news is, that I tried the cyclisation with Ac2O nonetheless on the, and yes that was all of it, 1,9g of the phthaloyl precursor.
I reacted it in a solution of 10ml DMF(5 would have sufficed) with 2ml of acetic anhydride, a 3,2eq. to the N-phthaloylglutamine, for 3h and kept it at 110-120°C the whole time.

When it had cooled down it was put in a bath of cold water, then I've used a 25% solution of ethanol in water and added it to the stirred mixture, at RT, in total 40ml but already after half of it had been added it became turbid and the first thalidomide crystals show up on the walls from the stirring.
After all of it had been added, I stirred an hour further and cooled it then down much.
Few hours later it was filtered and washed in the filter with lots of cold water, and then dried.
The next day 1,372mg were the result, or 5,31mmol corresponding to a yield of 77,3% of the theory.
The appearance was very impressive I had to show a picture to some friends because it looked was just so clean and white.
Today I took a melting point and it came out at 269-270°, and that without even doing a single recrystallisation, unbelievable that only water was used to purify and wash everything unwanted out of the product :o
Although, for a substance that barely fits a half gram into a liter of water at RT, one can probably clean this stuff up very well with only water if the purity was high enough to begin with.

There is still room for improvement left, but so far it is quite a lot of fun.
Luckily, I could enjoy that a friend was joining in with a likewise large interest in this substance and its preparation :)
Together we put much research and experiments in it and tried several approaches using both glutamine and glutamic acid with phthalic anhydride from various patents and papers.
And I can proudly say, so far we probably found out almost everything that does not work as claimed :D
But not only because of this, but also because of the occasional satisfying result it was a very entertaining experience.
I really enjoyed the exchange of ideas and findings, then getting back to the project and later on present each other the results followed by speculating how that came to be :D

Attachment: 37(234-237) JMCT11.pdf (175kB)
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[*] posted on 1-11-2020 at 04:51


Thalidomide is pretty interesting biologically, since its effects are due to altering the specificity of E3 ubiquitin ligases, thus causing certain proteins to be degraded. This can be exploited: if you link a thalidomide derivative with a compound that binds a protein, the target protein will be ubiquitylated and degraded. These chimeric compounds are called PROTACs.



As below, so above.
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[*] posted on 1-11-2020 at 17:18


I can just say that the sheer quantity of articles about this substance is really impressively large.
That has definitely been very helpful to prepare it.
Its pharmacology is most interesting as well, again, making it probably one of the best researched substances existing.
At least, as I have read, it is the one holding the record for the most publications published about it.

But I have to admit my interest in it stems from another pharmacologically unique property of it, as in the attached paper.
In that respect it turned out to be outstanding, and I'm not repeating what that paper claims but confirming it.

It is in general a rather strange substance with its effects, sharing most of the properties of related substances, while on the other hand being seemingly free from the disadvantages that are ubiquitous in almost every other compound with the same, or better, a similar mode of action.
Definitely another remarkable and interesting property of it.

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[*] posted on 1-11-2020 at 20:57


Hi karlos³, thx for sharing the doc. These sleep phases are very important. A lot of psychiatric substances damage them even those used for curing sleeping disorders. Agomelatine is an exception as it is similar to melatonin, but agomelatine could damage liver. There is very likely no remedy without adverse effect.



If there is a heaven, it seems not to be materially based. Does chemistry exist there and if yes, how does it look like? Are there good souls well supplied with laboratory equipment, glass, chemicals and information?
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[*] posted on 14-1-2021 at 09:39


I prepared N-Phthaloyl-DL-glutamic acid without solvent. Only water and HCl were used in the workup. Yield: 54%

Also I got the microwave preperation of thalidomide to work, but I'm still working on optimizing this. For now I screwed up one run by burning it like karlos, and in a second run I got a (crude) 28% yield. When I was preparing new phthaloyl glutamate I screwed up because my glutamic acid was not dry enough.

Billman and Harting showed phthaloyl glutamate could be made without a solvent at 185oC in 1948.

Attachment: billman1948.pdf (223kB)
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A couple of years later, Sheehan and Bolhofer showed this reaction also proceeds at 145oC, but they do not isolate phthaloyl glutamate and go straight for the anhydride.

Attachment: sheehan1950.pdf (509kB)
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In 1956 Tipson shows the reaction run at 145oC (although run in a Dean Stark with xylene) gives a separable mixture.

Attachment: tipson1956.pdf (484kB)
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In 2001 there was a publication on the microwave assisted synthesis of thalidomide with phthaloyl glutamate and urea or thiourea. Thiourea is supposed to give yields up to 85%. In a one-pot reaction phthalic anhydride, glutamic acid and thiourea are supposed to give a 60% yield of thalidomide.

Attachment: seijas2001.pdf (31kB)
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Phthaloyl glutamate: Phthalic anhydride (10 gr, 68 mmol) and glutamic acid (10 gr, 68 mmol) were mixed and finely ground with mortar and pestle. The powder was loaded in a 100 ml beaker, covered with aluminium foil, and placed in a 145oC oil bath. After five minutes the reaction mixture begins to melt and after a few more minutes starts to bubble. The bubbles are water which is generated in the reaction. Magnetic stirring was used to keep the foam down to prevent the mixture from boiling over. Manual stirring would be impractical as the phthalic anhydride sublimes.
After 25 minutes the last white solid particles react, and the bubbling ceases. At this point the oil bath is removed and while stirring, water and 5 ml of 30% HCl* is added to a total volume of 75 ml. The reaction is heated until all solids dissolve. The mixture is cooled to 4oC and the precipitated product is filtered and rinsed with cold water. After recrystallization from water (50 ml) the product is filtered over vacuum and dried in an oven at 150oC. When almost dry, the powder is cooled and powdered with mortar and pestle and dried at 150oC for another hour. This final step makes there is no residual phthalic acid left, as this would sublime. No weight loss was noticed, but maybe the phthalic acid already sublimed in the first round. Yield: 10.17 gr, 54%.

*Glutamic acid left in the reaction mixture is very soluble as the hydrochloride.


Preliminary:
Thalidomide was made by dissolving 8,5 gr phthaloyl glutamate and 1.93 gr (1.05 eq.) in 25 ml of hot water and boiling to almost dryness (monitored by weighing the beaker) in a 100 ml beaker. The beaker was placed in an oven at 100o until dry. A 400 ml beaker was then placed upside down over the smaller beaker and the microwave function was then turned on at 500 Watt. The phthaloyl glutamate mono urea salt starts to boil gives off water and ammonia fumes. Care must be taken not to let the mixture get too hot. When it becomes orange it is enough. This was done by doing short microwave bursts of 10-30 seconds. Methanol was added to the still hot mixture which makes it stirrable. The reaction was heated to a boil and stirred until the crystals are almost colorless. This is filtered and rinsed with a little cold methanol. Yield: 2.2 gr, 28%

[Edited on 14-1-2021 by Tsjerk]
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