Warning: The chemicals used in this video are toxic and carcinogenic.

Sulfuric acid is highly corrosive.

Sodium is a reactive and pyrophoric metal.

Solutions of sodium methoxide are caustic and corrosive.

Diethyl ether is highly flammable.

Wear gloves and work outside or in a fume hood.

And fire safety protocols must be in place.

Greetings fellow nerds.

In previous videos we've been making the precursors to pyrimethamine

and have so far gotten up to step 5 on the main route.

In this video we're pulling out all the stops and ploughing through both steps 6 and 7 to finish

and finally produce pyrimethamine after two years of work.

Let's get started.

First we take our 2-(p-chlorophenyl)-3-oxopentanenitrile we made in a previous video.

We've shown by NMR it's reasonably pure but it has water in it.

Water is detrimental to the next steps so we have to remove as much as we can.

We take our entire 41g stock of crude 2-(p-chlorophenyl)-3-oxopentanenitrile and place it in a flask.

Then we add in 50mL of toluene solvent.

Be sure to add in a stir bar.

We set up on top a dean stark apparatus and reflux condenser.

Turning on the heating we raise the temperature until the mixture starts to reflux.

What we're doing is removing the water in the mixture by distillation.

Water forms an azeotrope with toluene and the dean stark apparatus lets us separate the toluene from the water

and return the toluene to the mixture, continuing the cycle.

For further information about the dean stark trap you can check the video description.

Eventually, all the water will be removed.

Turn off the heating and let the mixture cool.

Now remove the dean stark apparatus and assemble back on the reflux condenser.

Through the top of the condenser we add in 30 mL of trimethyl orthoformate that we made in a previous video.

Then we add in 75 mL of methanol that was dried over molecular sieves.

Finally we add in 0.5mL of concentrated sulfuric acid that i distilled from drain cleaner.

Now heat the mixture until it refluxes.

What's happening is the trimethyl orthoformate is reacting with the 2-(p-chlorophenyl)-3-oxopentanenitrile to form an enol ether.

The full name of our target compound is 2-(4-chlorophenyl)-3-methoxypent-2-enenitrile, but i'm just going to call it the enol ether to save time.

Interestingly enough, if you watch closely, you'll see the liquid level decreasing despite efficient refluxing.

What's happening is we're losing methyl formate as it boils out.

Methyl formate has a boiling point of just 32 degrees celsius which is too low for my reflux condenser to efficiently condense.

So it boils out and is lost in my fume hood.

Anyway, keep refluxing the mixture for 3-4 hours.

Then turn off the heating and let it cool.

We have now cleared step 6 of the main pathway.

At this point we need a source of guanidine.

I'm going to use the guanidinium chloride we made in a previous video.

Now we want free based guanidine so we have to make it just before use.

Swap out the flask of enol ether mixture and put the reflux condenser on another larger flask

containing 200mL of methanol dried with molecular sieves and then distilled.

Now get 15g of sodium metal that we made in a previous video and roll it into wires.

Add them through the top of reflux condensor a little at time and keep the refluxing under control.

What we're making is sodium methoxide in methanol solution.

Once it's all added and cooled, take off the reflux condenser.

Now add in 20g of guanidinium chloride that we made in a previous video.

We're reacting the sodium methoxide with the guanidinium chloride to make guanidine and sodium chloride.

For those of you who have been closely examining my pathway you'll notice for the last year

i've been planning to use guanidinium carbonate rather than guanidinium chloride.

I was hoping to use guanidine straight from hair relaxers but i was worried they weren't pure enough so i converted to guanidinium chloride.

But both compounds will work.

You can use whichever one you have access to.

Anyway, now that we've produced guanidine in solution, we have now cleared the last precursor step.

Only step 7 remains.

To do it, we add in the entirety of our enol ether reaction mixture.

The excess sodium methoxide will react with the sulfuric acid and turn it into sodium sulfate.

The excess beyond that will flip the acidic conditions of the enol ether formation to the strongly alkaline conditions we need for the guanidine reaction.

Now put the reflux condenser back on and start refluxing the mixture.

What we're doing here is reacting guanidine with the enol ether to form a pyrimidine ring.

This is the final reaction in making pyrimethamine.

The reaction is slow so you'll need to reflux for a long time.

I left this going for 36 hours.

And here we are 36 hours later.

Turn off the heating and let it cool.

Now we need to start the complicated process of separating out the pyrimethamine.

Take off the reflux condenser and assemble a distillation setup around the mixture.

Now distill off about 100mL of liquid or so.

I want to add water later on but i'm worried about overfilling the flask,

so i'm removing some solvent first before i add in an equal amount of water.

I'll be honest i'm just making this process up as i go along.

I got no procedure how to get pyrimethamine out of the reaction mixture.

All i'm going on is my own years of lab experience.

I want to reduce the volume of the mixture so getting rid of the solvents seems the most logical place to start.

Now turn off heating and add in 100mL of water.

Turn the heating back on and keep boiling off more solvent.

I want to essentially remove as much of the organic solvents as i can and replace them with water.

I didn't simply boil down dry earlier because i don't know how sensitive pyrimethamine is to heat.

If i boil dry i could destroy the pyrimethamine.

Adding in water first means we can remove the solvents and still remain below the boiling point of water.

Keep boiling until the distillate temperature goes past 90 celsius.

At this temperature most of the methanol will have boiled off and conveniently,

toluene and dioxane will also boil off since they form azeotropes with water below this temperature.

No turn off the heating and let it cool.

Now we add in 23g of sodium hydroxide and stir.

I want to make the water even more polar so it will absorb all the highly polar impurities but reject the non-polar impurities.

Now place the mixture in an ice bath and let it cool.

Once it's cold, we add in 150mL of diethyl ether that i distilled from starter fluid.

I wanted it cold to minimizes losses of the volatile ether.

It looks like i don't have enough water.

The aqueous layer still has large undissolved chunks that haven't dissolve.

Add in another 200mL of water and let it stir for half an hour.

turn off stirring and let it settle.

There we go this is much better.

We have a clean aqueous layer that dissolves all the salts and other polar side products and contaminants.

And in the upper ether layer is where all the non-polar impurities collect.

In the middle is a layer of pyrimethamine.

It has only minor solubility in both so it separates into its own layer.

Now we need to filter this.

I first collected the middle pyrimethamine with a separatory funnel but this is optional.

Suction filter the mixture to get crude pyrimethamine.

Wash it once with water and twice with ether to remove more of the impurities.

Then let it dry on the filter.

Scrap it off, and here is our crude yield of pyrimethamine at 5.5g.

To purify it further we recrystallize from ethanol.

Put in on gentle heating and adding ethanol while boiling until it dissolves.

I found i needed about 90mL of ethanol to fully dissolve my pyrimethamine.

Once it's all dissolved, turn off the heating and let it crystallize back out.

Most of it will still be dissolved in the ethanol but what we get will be exceptionally pure.

And here we go.

Now filter the crystals and wash them with some ethanol.

Then wash them with diethyl ether.

Now leave under the vacuum stream to dry.

And there we have it, two years of work.

Although i admit the work was not continuous.

Nonetheless we finally have pyrimethamine.

I sent a sample off for NMR spectroscopy analysis and got back this spectrum.

Now this spectrum was taken in chloroform-d solvent.

If you search the literature for pyrimethamine you'll find most literature spectra are taken in dimethyl sulfoxide d6 solvent.

So my spectrum actually looks different then what you'll find in the literature.

I asked the NMR lab to take another spectrum in dimethyl sulfoxide and this is what i got.

This matches what you'll find in the literature.

Now i can analyze both spectra but personally i'm going to analyze the chloroform-d version

since it's less well known as the dimethyl sulfoxide d6 version.

These peaks here are the aromatic protons.

And this spike here is the chloroform-d solvent peak.

This peak here is the amine on the guanidine structural element.

This peak here is the amine on the hydrocarbon backbone.

This peak here is the CH2 on the ethyl group and that peak there is the CH3.

This broad peak here is water contamination for obvious reasons.

These very tiny peaks here and here are diethyl ether.

This is leftover contamination from when i washed the final product with diethyl ether.

This tiny little peak here is not supposed to be there.

It's some sort of contaminant.

It looks like acetone but i can't be sure.

It also doesn't make very much sense to be acetone since i never used acetone in the final steps.

Nonetheless we have made pyrimethamine, finally.

We have now cleared step 7 and have confirmed our product.

Time to break out the champagne.

Our total yield was 1.8g.

This is about 72 pills worth.

Although most of it is still in the ethanol filtrate and with further purification i'm sure it can be recovered.

You're probably asking what the overall cost and yield is right from the beginning.

Unfortunately because of the huge number of failures, repeats and do overs along the way the numbers don't have any meaning anymore.

The total cost spent to do all the research was about $22000 over the course of two years.

Keep in mind this is not the cost of the actual process.

This is the total cost of all of the work and huge amount of failure that went into research, designing, experimenting, and verifying this pathway.

The cost of chemicals, broken equipment as well as my own time as a Ph.D. chemist is also in there.

Going on the raw cost this batch right here of 72 pills worth is about $300 dollars per pill.

I know it's stupidly expensive but that's to be expected for a first time laying out the pathway.

But now that we have it.

To repeat it would cost a tiny fraction of that.

Anyone copying me would not have to go through all the failures i did.

As a rough conservative estimate i think this can be fully repeated with a thousand dollars, for a projected cost of $15 a pill.

And this pathway isn't even optimized yet.

There are huge improvements available at each step as i just eyeballed the procedure for the most part.

Now that we have a starting point, I'm sure with some refinements we can get a much better yield than 1.8g for an even better cost ratio.

For example, just getting the full 5g from the ethanol solution would get us to $5 a pill.

Optimizing to less than a dollar a pill should be very straightforward.

But i never meant for this to be a viable option.

Even if this could be made completely free the process is far too complicated for anyone to perform.

If you actually need it you have far more pressing concerns on your mind about your health than how to manufacture a drug at home.

Additionally, this is not medical grade.

To get medical grade certification i'd need a huge amount of purification and testing every step of the way to ensure absolute purity and quality.

I don't think reactions performed in a soup can would pass any sort of medical certification.

Finally, for all the work we've put into this, it would probably have been cheaper

just to go through the whole licensing and business startup procedure and order all the direct precursors.

You could make kilograms for pennies.

Making it from domestically available chemicals makes about as much sense as building a computer from sand.

It can be done, but you don't really need to.

This was more of a challenge like climbing mount everest.

Nonetheless, i have proven that you can make anything from anything else, as long as you have the right elements, knowledge and work.

And in doing this research we also have a spin-off benefit.

The major breakthrough for amateur chemistry was the discovery of how to make sodium metal without electrolysis using domestically available chemicals.

This wasn't a research objective but was accidentally discovered as part of the pyrimethamine research.

Even if we had failed completely in making pyrimethamine, we nonetheless have made a valuable discovery.

The fact that we succeeded in making pyrimethamine as well only affirms how highly successful our research has been.

And it underscores why all research is valuable.

Even if it doesn't appear to have any directly useful or profitable results.

Because sometimes when you actually do it you find other things that are directly profitable or useful.

At this point I'd like to thank you my viewers for pushing me through this when i myself had my doubts and was ready to give up.

I'd also like to extend a special thanks to my patreon contributors both past and present.

I wish i had all your names but patreon doesn't list former contributors.

Nonetheless if you contributed at any point than this thanks goes to you.

You know who you are.

There was no way i would have been able to fund a $22000 amateur research project.

It was because of you that this happened.

This channel honestly would have died two years ago if it wasn't for you.

Thank you.

At this point i'd like to extend an invitation to anyone who wishes to be a contributor.

A project like this is dependent on outside support.

And I hope I've proven that I can get them done.

Now i'll probably make a few more videos related to pyrimethamine.

For example i intend to make a compilation movie combining all the steps in one video.

But after that i'm not sure what's next in terms of big projects, or if we should even have them.

But i'll make another video where we can discuss that and plan for the future.

So until then think about some big projects as well as the future directions for this channel.

But for now, let's just appreciate a goal accomplished with plenty of trials and failures as well as great and successful detours and stops along the way.

If there was ever a time to break out the champagne, it's now.

Thanks for watching.