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We all know about the recreational use of cannabis.

After all, with roughly 230 million users worldwide,


it’s the world’s most used drug. However, recreational drug use is not what we’re going to talk about in
this video because this channel is about actual science. Most people heard about the use of cannabis for
medicinal purposes. The underlying system in our body that cannabinoids like THC from cannabis
interact with, is involved in almost all diseases. This means that there is huge potential for application of
cannabinoids in medicine, but this doesn’t mean that smoking some weed will solve all your problems. In
actuality, research in this field is only really starting to uncover the complex biochemistry that is behind
the interaction of cannabinoids with the human body. We will learn much more about this later in the
video.

One case that exemplifies the medicinal potential of cannabinoids is the molecule cannabidiol, or CBD.
CBD has been approved by the FDA this year for treatment of two rare epilepsy disorders in infants.
These patients usually have to rely on combinations of pretty strong anticonvulsants to get some relief. A
study in 2017 looked at cases of drug-resistant seizures of the Dravet-Syndrome, so cases, were standard
medication didn’t really help. They showed that the frequency of seizures per month could be decreased
from 12 to 6 with the use of CBD in addition to other medication. Moreover, 5% of patients remained
completely seizure-free. The treatment is however not completely risk free as they observed some side
effects such as loss of appetite, fatigue or diarrhea. Here in Switzerland, they even started selling
cigarettes containing CBD for recreational use. Even though CBD is not psychoactive and is legal, you’re
actually not allowed to smoke CBD cigarettes in Switzerland during your military service, which I find
pretty funny.

CBD’s infamous psychoactive cousin THC on the other hand is also used for treatment of chronic pain
and MS. Until recently, it was thought that cannabis was the only plant that produces THC. In 1994, it
was shown that liverwort, which sounds like something taken straight out of a Harry Potter book,
produces a cannabinoid that’s structurally very similar to THC. Now, in 2018, latest research
demonstrated that this cannabinoid, called PET, displays some pretty interesting pharmacological
characteristics and the whole story has led to some silly headlines. We’ll talk about this and of course
some synthesis in more depth later in the video, so be sure to watch everything.

This is going to be quite a detailed and interdisciplinary video. It looks like most of you guys enjoy the
history behind the molecules and would like me to not rush through it, so we’re going to talk about a lot
of stuff and take our time. This video took me eons to make, so please show your support and subscribe
to my channel if you liked it. The video consists of three parts:
Firstly, we’ll learn more about the history of cannabinoids and how the father of THC got away with
taking 5 kilograms of cannabis from the police. We’ll see how THC was essential for the discovery of
various biochemical processes and pathways in our body and also why cannabis users have stimulated
appetite, called the munchies. We’ll briefly talk about the first total synthesis of THC as well.

Secondly, we’ll talk about whether Switzerland really wants to get high on moss, as some like to call it.
This is based on work of Professor Gertsch and co-workers at the University of Bern. We’ll talk about
their findings, how PET compares to THC and what this means for the future of cannabinoids in
medicine. By the end of these two parts, you’ll probably think that I’ve become a biochemist but have no
worries.

In the third part of the video, we’ll talk about organic synthesis. You see, to enable the biochemical
assessment of PET and other unnatural cannabinoids, the scientists at Bern were supported by synthetic
chemists at ETH Zurich. Ultimately, a lot of the biochemical groundwork we’ll cover relied on the use of
highly potent, unnatural and radiolabeled cannabinoids. Of course, good old organic chemistry was there
to help out. We’ll also contrast this modern synthesis of PET with an approach from 2008.

You already know which part I’m looking forward to the most. Nevertheless, this video is super diverse
and I’m sure you’ll find something you like regardless of your background.

Cannabis has been used recreationally for millennia. We’ll focus on actual scientific developments from
the 20th century, because I don’t really care whether some weird Chinese doctors calmed down patients
with wine and cannabis before surgeries.

So up until the 1960s, no one had ever isolated the active compound in cannabis. In 1964, the Jewish
chemists Raphael Mechoulam and his colleague Yechiel Gaoni first isolated and characterized THC in
1964. Just one year before, in 1963, they had already isolated and established the structure of CBD. The
cannabis they used for the extraction of cannabinoids was actually supplied from the police. The Israeli
police at that time had enough hashish that was being smuggled from the Lebanon. The director at the
Weizmann Institute, where they were researchers at, had some friends in the police and after some
persuasion, Mechoulam was allowed to pick up some kilograms. There’s a nice interview that talks about
the story in more depth, I’ll include a link in the description.

One year later, in 1965, Mechoulam and Gaoni published the first total synthesis of THC. We’ll have a
brief look at key steps from the synthesis. They started with reaction of the terpene (E)-citral with this
aryl lithium species. After nucleophilic addition on to the aldehyde, the free alcohol was obtained. Now,
the alcohol was tosylated by treatment with TsCl in pyridine at room temperature. Interestingly, this
cyclic compound, which is basically methyl-protected CBD, was obtained in low yield after
chromatography. How is this possible? Mechoulam postulated that there’s an allylic rearrangement
happening, converting the starting material to this conjugated olefin. Then, intramolecular SN2 prime
substitution leads to the formation of the 6-membered ring, now with a (Z) double bond in place. Just a
few additional steps completed the total syntheses of CBD and THC.

Quite a bunch of approaches towards THC have been produced in the 20 th century. Many of them rely on
the use of terpenes as starting materials and include quite a lot of interesting rearrangements. Another
approach published 5 years after the first total synthesis of Mechoulam is actually just a one-pot
sequence. 2-carene-epoxide is reacted with this dihydroxybenzene in the presence of boron trifluoride.
In a first step, one of the free phenol groups attacks the cyclopropane which is ruptured due to Lewis-
acid mediated opening of the epoxide. This installs the ether linkage. The second step is basically an
electrophilic aromatic substitution. Here, both substituents are strongly electron donating and activate
the ortho position for the intramolecular allylation.

The synthesis, structural elucidation and new isolation procedures facilitated research into the
pharmacology of THC and other cannabinoids in the 1960s and 1970s. This enabled SAR studies, so
structure-activity relationships with un-natural variants of THC or CBD and other research into the
mechanism, function and degradation of cannabinoids in humans.

Up to that point, there were some anecdotal reports and medieval manuscripts that described cannabis
as a treatment for epilepsy. In 1980, Mechoulam and co-workers from Sao Paolo tested this in a scientific
study on patients that did not respond to traditional medication. The results were quite astonishing;
almost all participants showed improved condition without any harsh side effects.

At this point, researchers knew that cannabinoids were beneficial for multiple conditions. However,
nobody knew why cannabinoids had such profound therapeutic effects. In the mid-1980s, groundbreaking
findings were made in Allyn Howlett's laboratory at St Louis University. This research provided evidence
for a type of G-protein coupled receptor in the plasma membrane which specifically interacted with
cannabinoids. This CB1 receptor is actually one of the most abundant receptors in the central nervous
system. Howlett also demonstrated that CB1 is involved in a highly complex signalling pathway which
influences many enzymes that are key in regulating many biochemical processes. To give you an idea of
just how complex these signalling cascades are, here’s a modern overview of pathways of the antitumor
effects of cannabinoids in cancer that affect cell cycle, proliferation, cell death and tumor progression.
CB1 is involved in pain, inflammation, motor control, olfaction, neural plasticity but also in the regulation
of appetite and food intake. It’s well known that THC increases appetite in users. There are a lot of different
ways by which THC is believed to do this. THC increases the incentive-motivational properties of food, so
essentially eating pleasure, and the palatability, so the tastiness of food. In addition, THC also increases
ghrelin levels, which is an appetite-stimulating hormone.

Since activating CB1 increases appetite, it shouldn’t be surprising that blocking CB1 has the opposite effect.
This is the story behind Rimonabant, a CB1 antagonist, which was sold as an antiobesity drug. The emphasis
is on the “was sold”; just 2 years after first approval, Rimonabant was withdrawn worldwide. Although it
was effective for weight loss, serious psychiatric side effects, including suicide, outweighed its benefits. By
now we know that CB1 is involved in various processes, so specificity is pretty difficult and this can lead to
severe side effects. Nevertheless, further studies into CB antagonists might lead to a breakthrough in
obesity research. Novel antagonists with selectivity for CB1 receptors in peripheral organs may achieve
similar metabolic results with decreased psychiatric adverse effects because they’re not hitting CB1
receptors in the brain. Rimonabant is still pretty useful for pharmacological studies as we’ll see later.

In 1993, Sean Munro at Cambridge University identified a second type of cannabinoid receptor called CB2,
which can be found in the peripheral nervous system, organs and the immune system. Although their
functions have similarities, the CB2 receptor shares only about 45% of homology with CB1. It’s important
to note that expression of the CB receptors highly varies between different locations within a given tissue.
A particularly interesting feature CB2 receptors is that they appear to be highly inducible, with expression
in CB2 increasing up to 100 fold following tissue injury or during inflammation. So, it’s not as simple as
having just two distinct receptors. There are also different isoforms that drive specificity, so the
endocannabinoid receptors can exert a wide variety of different cell-specific effects.

Of course now the question you might ask yourself is, why are there receptors for compounds that come
from plants? A friend of mine once told me it’s because humans were meant to smoke cannabis and get
high, but these receptors actually exist for endogenous compounds we produce in the human body. This
system started evolving over 600 million years ago, so the plant didn’t come first.

The first example of such an endocannabinoid is anandamide, which was discovered in 1992. Again, good
old Mechoulam was involved. The isolation of 0.6mg of anandamide was the result of 2 years of
chromatography and binding assays, starting from 4.5kg of pig brains. Anandamide interacts with both
CB1 as well as CB2 and plays a role in multiple processes. An interesting one is the following: Moderate
intensity exercise increases anandamide levels significantly. It’s believed that anandamide induces the
mood-lifting effects of exercise, for example the runners high, by interacting with CB receptors in the
brains reward centres and leading to release of dopamine.

Three years later, in 1995, Mechoulam and co-workers found another endocannabinoid,
arachidonylglyceride or 2-AG. Most people talk about more about anandamide than 2-AG. I think it’s
probably because anandamide was named after the Sanskrit word “bliss”, so people call it the “bliss
molecule” and that makes it more appealing. In actuality, 2-AG is present at much higher levels than
anandamide in the CNS. The affinity of 2-AG for the CB receptors is much higher and it’s suggested that 2-
AG is the more important natural ligand.

Given that we have two similar but also different receptors as well as endocannabinoids, the topic of
specificity comes up again. Let’s first think of the how the body does this. There are some other
endocannabinoids than the two ones we mentioned, but the toolbox is pretty limited. By highly complex
combination and regulation of dozens of factors, our body somehow still is able to elicit highly specific
effects. Well, when you’re trying to develop compounds that interact with the cannabinoid system in a
specific manner, you can’t just throw swallow or inject something that hits both receptors. So, that’s why
people have investigated specific binders. In some cases like JWH-133, the structure still looks like a
cannabinoid. Others, like GW-842 look like different though. Why should we care? Well, this compound
exhibits potent analgesic and anti-inflammatory properties but without the cannabis-like behavioural
effects, as it has a low affinity for the CB1 receptor. So you might treat things like chronic pain with it
without getting high as a kite.

So in summary, the research motivated by the cannabis plant has uncovered the vital endocannabinoid
system. This system consists of the CB receptors and endocannabinoid messenger molecules we’ve talked
about, as well as enzymes that are responsible for the synthesis and degradation of endocannabinoids.
The endocannabinoid system influences a myriad of processes and some people call it the most important
physiologic system involved in establishing and maintaining human health.

Alright, so let’s talk about getting high on moss. What is liverwort and is it actually superior to medicinal
cannabis? Because this video has been a total freestyle so far anyways, let’s involve even more scientific
disciplines and talk about evolutionary biology. I’m not an expert on this and I absolutely hated learning
about taxonomies and plants during my studies. On a high level though, the key takeaway is that liverwort
and cannabis are two totally different plants. Bryophytes are non-vascular plants, they lack supporting and
nutrient-transporting tissues, which limits how tall they can grow amongst other things. Angiosperms, the
vast majority of plants, do have such tissues and reproduce with flowers, seeds and fruits. On the
evolutionary timescale, bryophytes and angiosperms have been separated for 300 million years. The
remarkable result of convergent evolution is that these two different plants both produce very similar
cannabinoids. Like THC, cis-PET occurs in the plant as the aryl carboxylic acid, which is decarboxylated
during smoking or drying.

So, you can find liverwort species that produce cis-perrottetinene in Japan, New Zealand, Costa Rica and
Tasmania. Liverwort preparations are purchasable online and sold as a legal high. The online community
reporting about legal highs both affirms and refutes the anecdotes regarding the cannabis-like effects.
Now, Professor Gertsch from the University of Bern wanted to evaluate perrottetinene pharmacologically
for the first time. In the next part, I’m going to summarize the key findings of his research. If you want to
know more than just my dumbed down version, feel free to read the paper.

In mice, the team demonstrated that PET reaches the brain relatively easily. PET binds to CB1 and CB2
receptors but to a much weaker degree compared to THC. This means that liverwort doesn’t give you the
same euphoric high as cannabis, making it more attractive for medicinal purposes. The lowered potency
also decreases the risk of overdose. The team also investigated the unnatural diastereomer, trans-PET. As
in the case of the THC scaffold, the cis-PET isomer is a less potent binder than the trans isomer.

So far, we’ve only seen a quantification of the binding interaction. What is the macroscopic result of this
CB receptor binding? There is a behavioral test called the tetrad test, which is employed to screen drugs
that induce CB receptor-mediated effects in rodents. This tetrad test, as the name suggests, consists of
four components.

The first experiment is a measurement of body temperature, which can be decreased dose-dependently
by cannabinoids. To get a reference, let’s first focus on THC. At a relatively low dose of 10 mg/kg, THC
decreases body temperature, okay. The same result can be observed for cis-PET, albeit at higher doses.
We’ve seen that PET is a less potent CB receptor agonist than THC so this makes sense. The unnatural
trans-diastereomer has a similar effect. As a kind of control experiment if you will, they also tested the PET
isomers in combination with another compound SR1. Pretreatment of the mice with this compound fully
abolished the pharmacological effect of PET. Can you guess which compound this is? I’ll give you a hint,
we’ve already seen it in this video.

It’s our good friend Rimonabant again. We might not use it as a drug against diabetes, but in this case, the
CB1 antagonist is useful in demonstrating that the tetrad is dependent on CB1 receptor activation in the
brain. Because pretreatment with rimonabant occupies the CB1 receptors, pharmacological effects that
would arise from PET binding are not present.
The second experiment is the measurement of catalepsy. It’s measured by retaining the mouse in an
imposed position with their forelimbs resting on an elevated bar. Catalepsy is the amount of time that the
mouse stays immobile for. PET also passes this test.

The third experiment in the tetrad test is the measurement of hypomotility or in other words, decreased
spontaneous activity. You can measure hypomotility by placing the mouse in a cage with grid lines and
count how many lines it crosses during a certain time period. Essentially, you’re measuring how much the
mice move around. In this case, locomotion was tested with the rotarod test. You put the mouse on a
rotating metal rod and test the time, after which it falls off the rod. I know, all of this sounds really weird.
For PET, as for THC, the mice fall off the rod much faster compared to the control measurement.

The last component of the tetrad test is the measurement of analgesia or in other words, painlessness.
Here, the mouse is placed on a please that’s heated to about 55 °C. The experiment measures the time it
takes for the mouse to show a nociceptive response such as licking its paw, shaking the feet or jumping off
the plate. In this experiment, mice treated with PET show longer response times, indicating an increased
painlessness.

With this part, I wanted to give you offer you some insights into how the effects of cannabinoids are
actually measured. Long story short, cis-PET activates CB1 as well as CB2 receptors but to a weaker degree
than THC. So far, we haven’t really identified anything special though. You might think: so what?

It turns out that PET has a unique effects. You see, PET actually decreases levels of E2 and D2
prostaglandins. What does this mean? Well, prostaglandins are lipids with hormone-like effects that are
involved in a variety of biological processes. In this case, lowering prostaglandin levels could mean that
PET-like cannabinoids could have less side effects such as memory loss and less neuroinflammation.
Looking forward, this could make PET-based cannabinoids ideal candidates for treatment of chronic pain
or inflammation. However, all of this research is still in its early stages.

Alright, let’s move on to the third part of the video and finally talk about some organic chemistry. What
enabled the team in Bern to execute the pharmacological assessment was a supply of synthetic PET and
its unnatural diastereomer. This was the work of Professor Carreira and co-workers at ETH in Zurich. We’ll
immediately jump into the forward synthesis.

Their route didn’t start from a dihydroxybenzene like the other approaches towards cannabinoids we’ve
seen so far. Rather, they started from 3-bromo-5-fluoro anisole and subjected it to Suzuki coupling with
this boronic acid. This reaction proceeds under standard conditions with high yield and installs the side
chain containing the phenyl ring. Next, the crucial C-C bond was formed via lithiation and quenching with
acrolein. The allylic alcohol is obtained in excellent yield, as the lithiation is directed by both the fluoro and
methoxy substituents in a synergistic manner. Also, there nucleophilic addition selectively occurs in a 1,2
fashion. Next, the product was used in an alpha-allylation of this aldehyde. This is the key step of this
synthesis. This reaction employs two chiral catalysts and installs the two cis-stereocenters that are found
in cis-PET. The aldehyde also contains a terminal alkene, which underwent intramolecular ring-closing
metathesis with use of Grubbs II catalyst to forge the cyclohexene.

Let’s dive deeper on the allylation as it’s really fascinating. This reaction is an example of stereodivergent
dual catalysis. What does that mean? Two catalysts are employed and they generate two activated
intermediates; in this case is the electrophilic Iridium-allyl complex and the enamine derived from the
aldehyde. These intermediates are both chiral, so this enables you to access all possible stereoisomers
from the same achiral starting materials by changing the stereochemistry of your catalysts. Remember
trans-PET, the unnatural diastereomer the team also tested pharmacologically? Well, simply taking the
enantiomer of the amine catalyst is what allowed them to synthesize it. This really highlights the utility of
stereodivergence, in this case for the preparation of unnatural analogues of biologically active molecules.

Next, the oxidation stage at the carbonyl carbon was adjusted by converting the aldehyde to the methyl
ester by Pinnick oxidation and esterification. Now, both methyl groups were introduced by Grignard
reaction. You might not be very familiar with the Pinnick oxidation, so as a little exercise, take a minute
and write down a plausible mechanism. It looks like we’re throwing in a lot of stuff here, so what’s actually
going on? Chlorous acid, so chlorine with an oxidation state of plus 3, is the active oxidant. The chlorite
anion attacks the aldehyde and subsequently undergoes a pericyclic fragmentation. Essentially, you’re
abstracting a hydride from the aldehyde, thus oxidizing it to the acid. This reduces chlorous acid to
hypochlorous acid which itself is still quite reactive. To consume it, high excess, in this case 30 equivalents,
of a scavenger such as 2-methyl-2-butene is added. You might have guessed that for substrates that
contain other functional groups that are susceptible to oxidation, this reaction isn’t the most selective.

To complete the synthesis, the aryl ether bond was constructed via intramolecular nucleophilic aromatic
substitution. Interestingly, the outcome of the cyclization was highly dependent on the choice of base.
While employing lithium or sodium HMDS in refluxing THF only led to recovery of starting material,
potassium HMDS afforded the SnAR product in 78% yield. Finally, the product was demethylated by
treatment with excess sodium ethanethiolate in refluxing DMF to give cis-PET.
The Carreira synthesis of PET wasn’t the first one though. In 2008, Professor Kim and co-workers at Seoul
University reported a synthesis of cis-PET. We’ll also look at this one in the forward sense to have a nice
contrast to the modern approach discussed before. Their route started with a three-step sequence from
this aldehyde in order to introduce the sidechain. Wittig-reaction and subsequent hydrogenation of the
olefin installed the saturated sidechain present in PET. After demethylation of the phenol groups with
boron tribromide, this dihydroxybenzene was obtained. The Kim group also used the inherent reactivity
of the aryl ring to introduce a substituent in ortho-position, namely an iodine via electrophilic aromatic
substitution. After protection of the phenol groups, the aryl iodide was subjected to a Stille-coupling with
this organotin compound.

So, after formation of the C-C bonds with the aryl ring, the stage was set for the construction of the cis-
bicyclic system. The team accomplished this with a highly useful reaction, the Ireland-Claisen
rearrangement. First, the secondary alcohol used in esterification of this carboxylic acid. It also contains a
terminal olefin, so you might already be able to guess which reaction this will enable later on in the
synthesis. Now, the key Ireland-Claisen rearrangement installed both stereocenters with high
diastereoselectivity. As a last exercise, think of a mechanism and transition state model to rationalize the
diastereoselectivity. First, treatment of the allylic ester with LDA and TBSCl with HMPA as an additive,
forms the silyl ketene acetal with high Z-selectivity. The ensuing sigmatropic rearrangement proceeds via
this 6-membered chair-like transition state. This closed transition state is why Claisen rearrangements are
really useful in diastereoselective synthesis.

We’re already quite familiar with the underlying strategy of the finishing touches. The carboxylic acid was
converted to the methyl ester to allow a double Grignard reaction. Note that this also removes the benzoyl
protecting groups, which makes sense because they’re also esters. Then, Grubbs metathesis forged the
cyclohexene ring, just like we’ve seen it in the Carreira synthesis. The ring-closure did not require any
protection of the two phenol groups, which is kind of impressive as these can often bind to and deactivate
the ruthenium catalyst. Lastly, exposure of this product to acid resulted in formation of an intermediary
carbocation, which could be intercepted by the phenol group to afford the natural product.

This concludes this video on cannabinoid chemistry. If you stayed up until now, you’re a real MVP. There’s
more to cover on this topic if you found this interesting and want to see more. Also, please give me your
feedback regarding the format of the video. To be honest, the non-synthesis part is probably a bit longer
than I expected but I wanted to produce a more detailed video and keep it interdisciplinary. If you have
any suggestions for topics you’d like to see covered, you can also comment them below. Please don’t
forget to like the video and subscribe to my channel. As always, until next time.