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Unlocking tools in chemistry to facilitate progress in drug discovery

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Unlocking tools in chemistry to facilitate progress in drug discovery

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UNLOCKING TOOLS IN CHEMISTRY TO FACILITATE PROGRESS IN DRUG
DISCOVERY

by

Katharina Grotsch

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
CHEMISTRY

May 2022

Copyright 2022 Katharina Grotsch

i
Acknowledgements

First of all, thank you to my mom, Barbara, and dad, Jürgen, for your endless support. Through all
the ups and downs over the past 25 years, I could always count on you to lend an open ear. Thank
you for providing me with the best support I could have asked for, even though it wasn’t always
easy. This is just as much your achievement as it is mine.

To Valery – thank you for providing me with an enormous amount of intellectual freedom, and for
supporting all of my endeavors, no matter how ridiculous they may have seemed. I truly admire
your creativity and pleasure in risk-taking, something that I aspire to do more of. Thank you, Prof.
Vsevolod Katritch, for your positive attitude, patience, and support throughout the years. I
appreciate your trust, and thank you for always including me and the other trainees. Thank you
also to the rest of my committee, Prof. Surya Prakash, Prof. Barry Thompson, and Prof. Nicos
Petasis, for providing me with the knowledge that propelled me through this PhD. A special thank
you goes to Prof. Thorsten Bach, without whom I would not be here.

As much as I am grateful for those that guided me through my academic journey, I could not be
more thankful for everyone I met along the way. Adam – you have a most admirable ability to find
light in even the darkest times, an incredibly rare trait that has truly changed my life. You always
supported me when I needed support, and challenged me when I needed to be challenged. I am so
happy I met you.

ii
Thank you also to my lab mates Shubhangi Aggarwal, Sydney Hiller, Shelby Hill, Will Richards,
Kevin Vargas, Rudra Parsaud, Jose Ricardo Moreno, and Dr. Dmitry Eremin. I am in awe of your
independent work-ethic and admire all of your individual pursuits for the future. I wish you all
nothing but the best! Thank you also to Dr. Saheem Zaidi, Dr. Anastasiia Sadybekov, Dr. Joice
Thomas, and Dr. Jeff Celaje for being fantastic co-workers throughout the years.

Thank you also to Justin, Kris, Allie, Emily, Maddy, and Mike (yes, running those 26.2 miles was
one of the hardest things I’ve ever done and I will never forget that day). A special shoutout goes
to my time with the BCLA family, which turned out to be one of the best decisions I’ve ever made.
Thank you, Adhi, for inspiring me to take this leap, and thank you Carolina, Stefanie, Valentina,
and Katrina, for always having my back.

Last but not least, thank you to my chicas. You all inspire me every day, and I’m so grateful for
our friendship through the best and worst times of our lives. Carmen, your passion and love for
adventure makes every day more exciting. Mich, you are never afraid of a challenge and have
courage that I can only aspire to. Jess, you have an incredible mind and I hope that never changes.

Lucia – this thesis is for you. You are the strongest person I’ve ever met and you inspire me every
day.

iii
Table of Contents
Acknowledgements i
List of Tables vi
List of Schemes vi
List of Figures viii
List of Abbreviations ix
Abstract x
Introduction 1
Chapter 1. Synthesis of Drug-like Heterocycle Moieties 5
1.1 Problems Inspiring the Studies 5
1.2 Bromoethene Sulfonyl Fluoride 6
1.3 Initial Developments: Sulfonamide Functionalized Triazoles 8
1.4 Sultam Synthesis 10
1.5 Experimental Data and Spectra 15
1.6 Distribution of Credit 25
Chapter 2. Exploring the Limits of what is Tangible and what is Feasible: Searching for
Drug Candidates in an Unknown Chemical Space 27
2.1 Strategy and Overview 27

iv
2.2 Creation of On-Demand Virtual Library 29
2.3 Receptor model preparation and optimization 32
2.4 Virtual Ligand Screening 34
2.5 Selection and Synthesis of Drug Candidates 35
2.6 Experimental identification and validation of CB2 binders 36
2.7 Discussion 39
2.8 Conclusion 40
2.9 Synthetic Procedures and Spectra 41
2.9 Distribution of Credit 74
Chapter 3. Making Lemonade out of Lemons: The Unmet Potential of Cannabinoids and
the Endocannabinoid System 75
3.1 Introduction 75
3.2 The Endocannabinoid System 76
3.3 Phytocannabinoids 84
3.4 The War on Drugs 94
3.5 Outlook 98
Chapter 4. Taking Inspiration from Nature: Strategies towards the Facile Synthesis of
Mycophenolic Acid and its analogues 100
4.1 Introduction 100

v
4.2 Previous Synthetic Strategies 103
4.3 Retrosynthetic Strategy 104
4.4 Synthetic Progress and Challenges 106
4.5 Synthetic Procedures and Spectra 108
Bibliography 113

vi
List of Tables

Table 1.1: Optimization of reaction conditions. ............................................................................. 9
Table 1.2: Optimized reaction conditions for sultam synthesis in organic media. All reactions
were carried out on a 1 mmol scale. ............................................................................................. 13
Table 1.3: Observed reactions in aqueous media. ........................................................................ 14
Table 2.1: Results of testing of hits from virtual ligand screening in binding and functional
assays. ........................................................................................................................................... 38

List of Schemes

Scheme 1.1: Basic Mechanism of the Sulfur Fluoride Exchange Reation ..................................... 6
Scheme 1.2: a) Two-step synthesis of ESF and b) subsequent bromination and elimination of
ESF to form Br-ESF........................................................................................................................ 7
Scheme 1.3: Synthesis of sulfonamide functionalized a) isoxazoles and b) triazoles from Br-ESF.
......................................................................................................................................................... 7
Scheme 1.4: Previous work leading to the synthesis of sulfonamide functionalized triazoles. a)
BnBr, EtOH, 0˚C to rt, 20 min, 87%, b) MeI, K
2
CO
3
, DMF, 0˚C to rt, 1 h, 22%, c) NCS,
AcOH/water, rt, 2 h, 64%, d) Cl
2
, DCM/water, 0˚C, 5 min, 90%. ................................................ 8
Scheme 1.5: Substrate scope for triazole synthesis under the aforementioned conditions. ......... 10
Scheme 1.6: Functionalization of 1,2,3-substituted Triazoles to generate the respective sulfonate
esters and sulfonamides. ............................................................................................................... 10

vii
Scheme 1.7: Formation of a) double-addition, b) single-addition and c) sultam products upon
reaction of primary amines with Br-ESF. ..................................................................................... 11
Scheme 1.8: Sultam product formation by rapid Michael addition and subsequent cyclization.. 12
Scheme 2.1: Combinatorial strategy for creation of a) triazole library and b) isoxazole library
from available building blocks...................................................................................................... 32
Scheme 4.1: Trimerization of halogenated alkyne (2 eq.) with 2-butyne (1.5 eq.) with high
selectivity in an overall yield of 84%. ........................................................................................ 102
Scheme 4.2: Synthesis of phthalide by trimerization and subsequent cyclization via acid
catalysis. ...................................................................................................................................... 102
Scheme 4.3: Retrosynthetic strategy for the total synthesis of Mycophenolic acid. .................. 105
Scheme 4.4: Synthesis of benzothiazole sulfone to carry out the Julia-Kociensky olefination by
a) substation of the thiol and b) oxidation with mCBPA. ........................................................... 107

viii
List of Figures

Figure 1.1: Prior examples of pendant functionalization with SO2F. Reaction conditions:
a) ESF, solvent, 5 mins; b) ESF, solvent, 5 mins; c) ESF, PR3 (10 mol %), CH2Cl2, 24 h;
d) ESF, AcOH, reflux, 2 h. ........................................................................................................... 11
Figure 1.2: Substrate scope of sultam synthesis in aqueous media. ............................................ 14
Figure 2.1: Performance evaluation for CB2 crystal structure and ligand-guided optimized
models. a) ROC plots for CB2 crystal structure and ligand-based optimized models.
Examples of predicted binding poses of selected known high-affinity CB2 ligands in
optimized models with agonist (b) and antagonist (c) molecules. ................................................ 34
Figure 2.2: Synthesized compounds that were identified as hits for CB1 and CB2. .................... 36
Figure 2.3: Characterization of best CB2 hits. ............................................................................. 37
Figure 3.1: a) Structures of Δ9-THC, CBD, AEA, and 2-AG and b) Schematic
representation of the main components of the endocannabinoid system within the central
nervous system (CNS). ................................................................................................................. 80
Figure 3.2: Chemical structures of common phytocannabinoids. ................................................ 85
Figure 3.3: Some known isomers of THC. .................................................................................. 86
Figure 3.4: Some possible transformations for CBD and related compounds. ............................ 87
Figure 3.5: A select timeline of the history of cannabis as medicine. ......................................... 95
Figure 4.1: Structure of Mycophenolic acid. ............................................................................. 103
Figure 4.2: Previous retrosynthetic methods for the total synthesis of Mycophenolic Acid
by a) Birch, b) Danheiser, c) De La Cruz, d) Covarrubias-Zuñiga, and e) Brookes. ................. 104
Figure 4.3: a) Representation of both possible isomers and the interaction ovserved by
ROE b) ratio of Z/E products based on 1H NMR by integration of the methoxy peak.............. 108

ix
List of Abbreviations
˚C = degrees Celcius
s = singlet
d = doublet
t = triplet
m = multiplet
DCM = dichloromethane
DMF = N,N’-dimethylformamide
DMSO = dimethylsulfoxide
EtOAc = ethyl acetate
DCE = dichloroethane
EtOH = ethanol
DEE = diethyl ether
THF = tetrahydrofouran
MeOH = methanol
eq. = equivalents
g = gram
LC/MS = liquid chromatography/ mass spectrometry
GC/MS = gas chromatography/ mass spectrometry
NMR = nuclear magnetic resonance
TLC = thin layer chromatography

x
Abstract

Chemistry is often regarded as the central science, providing a link between the study of life at the
atomic and molecular level. As such, it plays a central role in our understanding of how the human
body functions. Given the many problems surrounding the drug development cycle today, this
thesis is a continued attempt to return to the chemical drawing board in an effort to provide new
tools and machinery for the development of successful therapeutics. In Chapter 1, we explore a
new synthetic methodology that has the potential to open the path to the discovery of new
molecular entities. In Chapter 2, we harness this synthetic strategy in combination with new tools
in computational biology to explore new chemical space in the hope of finding successful ligands
for important biological targets. In Chapter 3, we create a bridge between Big Pharma and ancient
herbal medicine, addressing the unexplored polypharmacological powers of plants and addressing
the various regulatory hurdles associated with their use to treat ailements. Lastly, in Chapter 4, we
return to the tedious tradition of natural product synthesis, paying homage to nature and her awe-
inspiring acuity.

1
Introduction

At the beginning of every scientific discovery lies a quest in search of a larger truth. If the purpose
of science is to march towards a greater and greater understanding of how the world works, the
question is: how do we get there? Introducing the much over-used “paradigm shift”, philosopher
Thomas Kuhn defines scientific progress as “a series of peaceful interludes punctuated by
intellectually violent revolutions”. Here, he differentiates between normal science, a steady-state
attempt at clearing up the status quo, and revolutionary science, which is a “proliferation of
compelling articulations” to address the anomalies of a given world-view in a non-linear fashion.

An interdisciplinary endeavor, drug research has contributed to the progress of medicine more than
any other factor over the last century.
1
Its humble beginnings point to a time in which chemistry
had reached a degree of maturity and accuracy that allowed for the pursuit of applications based
on accepted principles and methods.
2
Namely, Kekulé’s theory on the structure of aromatic
molecules catalyzed research on coal-tar derivatives, particularly on dyes, which had a profound
influence on medicine.
1, 2
New analytical techniques allowed for the isolation and purification of
targeted compounds, and pharmacology claimed its place amongst the medical disciplines. Then,
in the first half of the 20
th
century, a series of emerging technologies allowed for the study of
penicillin by Chain and Florey which opened a new door for the treatment of bacterial infections.
3

As a result, many pharmaceutical companies invested in microbiology to find drugs with
interesting pharmacological properties. With the influence of Biochemistry, enzymes and
receptors were introduced as drug targets and the characterization of these receptors soon became
the basis for the discovery of a large number of drugs including beta-blockers and
benzodiazepines.
1

2

At this stage, the combined influences of chemistry, pharmacology, and molecular biology, led to
the emergence of a field in which the development of new pharmaceuticals was based largely on
the understanding of relevant biological structures and mechanisms. Technologies like rapid DNA
sequencing and high throughput screening (HTS) have since become the go-to method of finding
new hits and discovering more lead compounds. Though this progress is undoubtedly remarkable,
the output in recent years does not mirror this success.

New molecular entities (NME) are produced at the same rate today as they were 50 years ago, with
the industry averaging about one NME every six years despite unprecedented pharmaceutical
spending.
4, 5
Over 96% of drug development efforts result in failure, with especially high rates of
failure for diseases with a poorly understood pathophysiology.
6
The burden of this expensive and
time-consuming R&D process often results in site closures, job losses, and inflated prices of the
few drugs that surmount the demands of regulatory approval.
6
Perhaps even more troubling is the
fact that it often discourages scientific innovation in favor of compounds with identical
mechanisms of action to existing drugs (also known as “me too drugs”), and deters efforts to
develop therapies for treatment-resistant conditions. Although such compounds have several other
justifiable aims like improved pharmacokinetics, improved specificity, and fewer drug-drug
interactions, they provide little new knowledge to the scientific community.
7
But what exactly are
the current shortcomings of the drug development process? And how can we as chemists make an
effort to minimize cost and maximize progress?

3
It seems that at this point, the organic chemist serves only as an engineer to the molecular biologist
where output skews towards applied science in lieu of pure science. Therefore we ask ourselves –
how can we as chemists possibly contribute? Assuming that the goal of organic synthesis is to start
with a target and ask how it could be engineered, we have largely succeeded at this. From peptides,
to steroids, to macromolecules and complex natural products, there is not much we cannot make.
However, it is evident that the field of chemistry has been advancing far slower than its biological
counterpart. As stated by Sharpless et. al. in the highly cited 2001 review introducing Click
Chemistry:

“With a few billion years and a planet at her disposal, nature has had both time and resources to
spare, but we, as chemists on a human timescale, do not… The long and admirable history of
natural products synthesis, culminating as it has in the protection-laden schemes of kinetic
carbonyl chemistry, perhaps blinds us to the possibility of developing synthetic strategies that
enable much more rapid discovery and production of molecules with a desired profile of
properties. If useful properties are our goal, for example, better pharmaceuticals, then the use of
complicated synthetic strategies is justified only if they provide the best way to achieve those
properties.”
8

Inspired by our lab’s rich history of moving beyond the “paradigm of carbonyl chemistry”, this
thesis is a continued attempt to return to the chemical drawing board, pursuing the idea that it
should be our goal as chemists not to imitate nature, but to draw inspiration from her efficiency.
8

Here we will explore (1) synthetic strategies to access vast areas of uncovered chemical space, (2)
the promise of finding new therapeutic strategies by fusing age-old practices with modern science,

4
and (3) improving the efficiency of a given synthetic strategy only where replicating nature is the
most favorable solution.

5
Chapter 1. Synthesis of Drug-like Heterocycle Moieties

1.1 Problems Inspiring the Studies
Given the vast chemical space that is estimated to exist, it makes sense to use only the most
practical and reliable methods available in synthesis to enumerate a given scaffold. Inspired by
nature, “click-chemistry” is the modular congregation of small molecular entities or “building
blocks” by carbon-heteroatom links to accelerate the discovery of new molecules with useful
properties.
8
Ever since the term was coined by Sharpless in 2001, click-chemistry, specifically the
copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) has been used liberally for the
generation of combinatorial libraries.
9-13

Recently, the chemistry of organic sulfur(VI)fluorides has been rediscovered and appreciated as a
new “clickable” scaffold due to the high stability and selective reactivity of the -SO2F functional
group.
14
As with all “click chemistry”, Sulfur Flouride Exchange (SuFEx) reactions are governed
by the philosophy of “near perfect reactivity”, making them highly suitable for the rapid synthesis
of functional molecules.
15
The rich reactivity of sulfur (VI) fluorides was discovered a long time
ago in Germany, as the result of an investigation into dye properties of sulfur (VI) derivatives
made available from coal tar.
16
After this reactivity disappeared from the view of the synthetic
chemist for several decades, it was picked up again in the mid- 20
th
century.
14
As summarized by
Dong and coworkers, the unique reactivity of sulfonyl fluorides can be summarized by four key
points.

(1) Resistance to reduction: sulfonyl-fluoride bond cleavage is exclusively heterolytic due to
Fluorine’s high electronegativity, whereas homolytic scission is common for its S-Cl counterpart.

6
(2) Thermodynamic stability: the stability of sulfur(VI) centers is far higher as compared to
sulfur(IV), making it a superior connecter under harsh reaction conditions. (3) Exclusive reaction
at sulfur: the high polarizability of the chlorine center on an -SO2Cl moiety makes it subject to
reductive attack, often resulting in mixtures of sulfonylated and chlorinated products upon reaction
with carbon nucleophiles. (4) The special nature of fluoride-proton interaction: the fluoride ion is
highly stabilized by its interaction with H
+
and SiR3
+
counterions.

Scheme 1.1: Basic Mechanism of the Sulfur Fluoride Exchange Reation
Due to the popularity SuFEx chemistry has gained in applications ranging from materials
chemistry to drug discovery, there is an increasing need for scalable synthetic methodologies to
introduce the -SO2F functional group.
15

1.2 Bromoethene Sulfonyl Fluoride
One of the most powerful reagents for the introduction of the SO2F group, is the use of
ethenesulfonyl fluoride (ESF). Described as “the most perfect Michael Accepter ever found”, ESF
is synthesizable on a large scale and reacts cleanly with primary and secondary amines, amongst
others (Scheme 1.2a).
14, 17
Later the synthesis of 1-bromoethene-1-sulfonyl fluoride (Br-ESF), a
related molecule with a bromide group attached to the vinyl group adjacent to the sulfonyl fluoride,
was reported by Leng and co-workers (Scheme 1.2b).
18

7

Scheme 1.2: a) Two-step synthesis of ESF and b) subsequent bromination and elimination of
ESF to form Br-ESF.
Introducing an additional reactive site, Br-ESF is tris-electrophile, and is considered an even
stronger Michael acceptor than ESF.
18
Although its synthesis has been known since 1985, its
synthetic applications were limited due to inefficient preparation on a large scale.
19
We have since
developed a procedure that allows for the addition of Bromine to the double bond under
illumination with a simple 40-W bulb in as little as 10 h.
20
Initially, Br-ESF was reported for use
in the synthesis of regioselective 5-sulfonylfluoro isoxazoles via a 1,3 dipolar cycloaddition. This
strategy is of interest as it yields functionalized isoxazoles, a family of heterocycles that have been
applied for use in pharmaceuticals, natural products, and materials science.
18
Most importantly,
the reaction sequence generates a promising drug-like scaffold that has room for functionalization
at three positions which has, to the best of our knowledge, not been enumerated on a large scale.

Scheme 1.3: Synthesis of sulfonamide functionalized a) isoxazoles and b) triazoles from Br-ESF.
Here, we go on to explore the further capabilities of this fascinating building block in the synthesis
of drug-like heterocycle moieties.

8
1.3 Initial Developments: Sulfonamide Functionalized Triazoles
1,2,3-subsituted triazoles have found many applications in the molecular sciences, specifically in
drug discovery. Notably, however, sulfonyl fluoride substituted triazole moieties are
conspicuously missing in the literature. Until now, synthesis of sulfonamide functionalized
triazoles was a multi-step process that was unselective and only possible in low yields (<10%).
20,
21

Scheme 1.4: Previous work leading to the synthesis of sulfonamide functionalized triazoles. a)
BnBr, EtOH, 0˚C to rt, 20 min, 87%, b) MeI, K
2
CO
3
, DMF, 0˚C to rt, 1 h, 22%, c) NCS,
AcOH/water, rt, 2 h, 64%, d) Cl
2
, DCM/water, 0˚C, 5 min, 90%.

In 2018, our lab published a new protocol for the regioselective synthesis of fluorosulfonyl 1,2,3-
triazoles and isoxazoles from bromosulfonyl fluoride (Br-ESF), generating a synthetic pathway to
the highly inaccessible sulfonamide functionalized heterocycles (Scheme 1.3).
20
This can be
performed under mild conditions, has good functional-group tolerance, and is regiospecific and
metal-free. In our initial studies, a variety of reaction conditions was screened by reacting Br-ESF
with benzyl azide with a variety of solvents (Table 1.1). It was found that the reaction consistently
took place in good yields at 50˚C in DMF.

9
Table 1.1: Optimization of reaction conditions.

Entry Base eq. Br-
ESF
eq. Azide Solvent Time (h) Yield
(%)
1 NEt3 1 1 CHCl3 24 12
2 DIPEA 1 1 CHCl3 24 17
3 none 1 1 CHCl3 24 61
4 none 1 1 toluene 24 32
5 none 1 1 DMF 24 76
6 none 1 1 CH3CN 24 22
7 none 1 1 MeOH 24 18
8 none 1.2 1 DMF 14 83
9 none 1.4 1 DMF 14 89

To further examine the scope of the reaction, these conditions were tested on a variety of substrates
(Scheme 1.5). Product yields ranged from 65%-88% and were achieved within 24h, further
demonstrating the applicability of this reaction.

10

Scheme 1.5: Substrate scope for triazole synthesis under the aforementioned conditions.
Importantly, these frameworks can easily undergo SuFEx reactions with silyl ethers to form the
respective sulfonate esters, or with amines to form the respective sulfonamides. Henceforth, they
provide an interesting opportunity to explore a wide range of scaffolds upon enumeration, a
concept that will be explored further in Chapter 2 of this thesis.

Scheme 1.6: Functionalization of 1,2,3-substituted Triazoles to generate the respective sulfonate
esters and sulfonamides.

1.4 Sultam Synthesis
Previously, the Michael Acceptor properties of ESF have been utilized to decorate nitrogen,
oxygen, and carbon nucleophiles with the SO2F functional group.
22
Here, the use of N-

11
nucleophiles as reactive agents is of particular interest, where examples of both mono-substitution
and di-substitution have been observed (Figure 1.1).

Figure 1.1: Prior examples of pendant functionalization with SO2F. Reaction conditions: a)
ESF, solvent, 5 mins; b) ESF, solvent, 5 mins; c) ESF, PR3 (10 mol %), CH2Cl2, 24 h; d) ESF,
AcOH, reflux, 2 h.
Given its similar Michael Acceptor properties, we investigated the reactivity of N-nucleophiles
with Br-ESF. The reaction of Br-ESF with a variety of primary amines yielded a mixture of the
mono-addition and double-addition products, as well as small amounts of a cyclized sulfonamide.
This was of particular interest as cyclic sulfonamides, also known as sultams, have a rich and
varied history of applications in drug discovery, mainly due to their use as stable lactam
equivalents.
23
In addition, this strategy yields a complex heterocyclic product in one step with
room for further functionalization at the bromine-handle.

Scheme 1.7: Formation of a) double-addition, b) single-addition and c) sultam products upon
reaction of primary amines with Br-ESF.
In order to test the applicability of this procedure, we reacted benzylamine with Br-ESF under a
variety of conditions. Product and side-product formation was monitored using GC-MS.

12
According to our data (Table 1.2) entry 6 provided the highest ratio of sultam-formation, and
resulted in an isolated product yield of 59%. It was found that excess of Br-ESF and concentrated
reaction conditions generally led to the formation of the double-addition product in favor of the
sultam. This data suggests that the reaction takes place in two steps: (1) a rapid nucleophilic attack
on the Michael Acceptor on the time-scale of several minutes, and (2) a slow SuFEx reaction to
form the cyclic sulfonamide on the time scale of several hours. These observations were confirmed
by GC-MS, where the mono-addition product appeared almost instantaneously and was
consistently cyclized after 24 hours.

Scheme 1.8: Sultam product formation by rapid Michael addition and subsequent cyclization.

13
Table 1.2: Optimized reaction conditions for sultam synthesis in organic media. All reactions
were carried out on a 1 mmol scale.
Entry Solvent Solvent
Volume
(mL)
Eq.
Br-
ESF
Eq.
Amine
Base T
[˚C ]
Main Product
1 MeCN 0.3 mL 1.20 1.00 none rt

2 MeCN 0.3 mL 1.20 1.00 2,6-di-tert-
butyl-4-
methylpyridine
rt

3 CHCl3 0.3 mL 1.00 1.00 none rt

4 CHCl3 0.3 mL 1.00 1.00 2,6-di-tert-
butyl-4-
methylpyridine
rt

5 CHCl3 3.0 mL 1.00 1.00 none 60

6 CHCl3 3.0 mL 1.00 1.00 DIPEA rt

In order to further investigate the application of this strategy, we attempted to conduct the reaction
in aqueous media (Table 1.3). Here, we noticed that the product was formed in only one hour (as
compared to 24 h in organic media), but decomposed over time to form two unknown side-products
after 24 hours of reaction time. Given that the SuFEx reaction is the limiting step, these

14
observations are likely a result of hydrogen-bonding between a water molecule and the fluorine
leaving group, facilitating the nucleophilic attack on the sulfur-center.

Table 1.3: Observed reactions in aqueous media.
Entry Solvent Solvent
Volume (mL)
Eq.
Br-ESF
Eq.
Amine
Time T
[˚C ]
Main Product
1 H2O 3 mL 1.00 1.30 1 h rt

2 H2O 3 mL 1.00 1.30 24 h rt no product

Given this, we examined the utility of our conditions on a broader substrate scope, including
aromatic amines, short chain amines, and complex natural products. The reactions were conducted
in water with excess amine to yield the respective sultam products.

Figure 1.2: Substrate scope of sultam synthesis in aqueous media.

15
1.5 Experimental Data and Spectra
1.5.1 General Information
1
H,
13
C, and
19
F NMR spectra were recorded on Varian 400-MR, Varian VNMRS-500, and Varian
VNMRS-600 instruments at 295K unless otherwise noted. Proton magnetic resonance spectra
(
1
H NMR) were recorded at 500 MHz, and carbon magnetic resonance spectra (
13
C NMR) were
recorded at 125 MHz, unless otherwise mentioned. Chemical shifts (δ) are expressed in parts per
million, relative to the residual solvent signals as internal standards. Multiplicities are noted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; sex, sextet; sept, septet. High resolution
mass spectra were measured using Agilent 6545XT qToF instrument coupled with 1290 LC
system. QToF mass spectrometer is equipped with an atmospheric pressure chemical ionization
source. Measurements were performed in positive ion mode with following ionization parameters:
Capillary Voltage –3.5 kV, Corona current 4 µA, vaporization at 350 °C, nitrogen was applied as
a nebulizer gas 35 psi, dry gas 13 L × min
−1
, 325 °C, and collision gas. Spectra were recorded in
m/z 100 – 1700 range. For external calibration and tuning, a low-concentration tuning mix solution
by Agilent Technologies was utilized. For sample injection (1 µL injection of ca. 10
–4
M solution
in MeOH) LC system was used. Injected compounds were passed through XDB-C18, 2.1 × 50
mm, 1.8 µm column at 40 °C with gradient H2O/MeOH (0.1% formic acid) elution. UV/Vis flow
detection was also applied (190 – 900 nm). All the MS spectra were recorded at 1 Hz. Spectra were
processed using Agilent MassHunter 10.0 software package. Precoated Merk F-254 silica gel
plates were used for analysis by thin layer chromatography (TLC), visualized with short wave UV
light, and stained with KMnO4 and PPh3/Ninhydrin. Column chromatography was carried out
employing EMD (Merk) Silica Gel 60 (40-63 μm). Reagents were obtained from AA BLOCKS,
Enamine, or Sigma Aldrich, and used without further purification, unless otherwise noted.

16
1.5.2 Synthesis of Br-ESF from Chloroethane Sulfonyl Chloride

Chloroethane sulfonyl fluoride
K(FHF) (119 g, 1.53 mol, 2.5 eq.) was dissolved in 250 mL H2O. After 1 h, chloroethane sulfonyl
chloride (100 mg, 613 mmol, 1.0 eq.) was added and the reaction was stirred at rt for 2 h. Upon
completion, the reaction mixture was extracted with DCM (3x 100 mL) and dried over MgSO4.
After rotary evaporation of the solvent, chloroethane sulfonyl fluoride was obtained as a off-white
liquid in a yield of 98% (88g g, 600 mmol).

Ethenesulfonyl fluoride (ESF)
Chloroethane sulfonyl chloride (88 g, 600 mmol, 1.0 eq.) was dissolved in 80 mL H2O and 80 g
of ice. After 15 minutes, MgO (16 g, mmol, eq.) was added and the reaction was stirred at rt for
20 h. Upon completion, the reaction mixture was extracted with DCM (3x 100 mL) and brine (1 x
100 mL) and then dried over MgSO4. After rotary evaporation of the solvent, ethanesulfonyl
fluoride was obtained as a yellow liquid in a yield of 78% (468 g, 83.6 mmol).

1,2-dibromoethane-1-sulfonyl fluoride
To begin with, 6.05 mL bromine (18.9 g, 118 mmol, 1.30 eq.) was added to a round-bottom flask
containing 7.52 mL ethene sulfonyl fluoride (10.0 g, 90.9 mmol, 1.00 eq.) dissolved in chloroform

17
(40 mL) and stirred at room temperature with a table lamp for 24 hours. After extracting with
dichloromethane (1 x 30 mL), the organic layer was washed with NaS2O3 until clear (3 x 30 mL)
and dried with MgSO4. After evaporation of the solvent, 1,2-dibromoethane-1-sulfonyl fluoride
was obtained as a yellow liquid in a yield of 92% (22.49 g, 83.6 mmol). H NMR (600 MHz,
CDCl3) δ 5.62 – 4.81 (m, 1H), 4.16 (dd, J = 11.9, 4.8 Hz, 1H), 3.84 (dd, J = 11.9, 8.4Hz, 1H); 13C
NMR (151 MHz, CDCl3) δ 57.5 (d, J = 21.1 Hz), 28.4. 19F NMR (470 MHz, CDCl33) δ 46.7.

1-bromoethene-1-sulfonyl fluoride (Br-ESF)
To begin with, 11.3 mL triethylamine (8.25 g, 81.5 mmol, 1.00 eq.) were added dropwise to a
solution of 22.0 g 1,2-dibromoethane-1-sulfonyl fluoride (81.5 mmol, 1.00 eq.) dissolved in 350
mL diethyl ether at -78˚C, and stirred for 20 minutes. After washing with ice water and extracting
with diethyl ether (3 x 30 mL), the organic layer was dried with MgSO4. After evaporation of the
solvent, 1-bromoethene-1-sulfonyl fluoride was obtained as a dark-yellow liquid in a yield of 77%
(11.9 g, 62.8 mmol). H NMR (600 MHz, CDCl3) δ 7.20 – 7.16 (m, 1H), 6.58 (dd, J = 5.4, 3.8 Hz,
1H); 13C NMR (151 MHz, CDCl3) δ 134.7(d, J = 2.0 Hz), 119.8 (d, J = 34.3 Hz); 19F NMR (470
MHz, CDCl3) δ 48.8; HRMS (m/z): [M+H]+ calcd for C2H3Br1F1O2S1: 188.9016; found
188.9012.

18
1.5.3 Synthetic protocol for Sulfonyl-Fluoride Functionalized Triazole Synthesis

1-Benzyl-1H-1,2,3-triazole-4-sulfonyl fluoride
1-Bromoethene-1-sulfonyl fluoride 3 (2.63 g, 14 mmol), benzyl azide (1.33 g, 10 mmol), DMF (6
mL), reaction time (14 h) and temperature (50 °C). The product was purified by flash column
chromatography (hexane/EtOAc = 6:4) affording 5a (2.14 g, 89% yield) as an off white solid.
1
H
NMR (600 MHz, CDCl3) δ 8.14 (s, 1H), 7.44 – 7.44 (m, 3H), 7.35 (s, 2H), 5.64 (s, 2H);
13
C NMR
(151 MHz, CDCl3) δ 140.3 (d, J = 36.7 Hz), 132.4, 129.9, 128.7, 128.3, 128.3, 55.5; 19F NMR
(564 MHz, CDCl3) δ 65.9; HRMS (m/z): [M+H]+ calcd for C9H9F1N3O2S1: 242.0394; found
242.0394.

1-(4-methoxyphenyl)-1H-1,2,3-triazole-4-sulfonyl fluoride
In a screw cap tube, 164 mg 1-azido-4-methoxybenzene (1.10 mmol, 1.00 eq.) and 312 mg 1-
bromoethene-1-sulfonyl fluoride (1.65 mmol, 1.50 eq.) were dissolved in dimethylformamide
(1.00 mL) and stirred for 20 hours at 80˚C. After extraction with ethyl acetate (3 x 5 mL), the
organic layer was dried with MgSO4 and the solvent was evaporated. Subsequent purification by
column chromatography (hexanes/dichloromethane = 2/1) resulted in 1-(4-methoxyphenyl)-1H-
1,2,3-triazole-4-sulfonyl fluoride as a beige solid in a yield of 81% (0.228 g, 0.891 mmol). H NMR

19
(600 MHz, CDCl3) δ 8.51 (s, 1H), 7.58 (d, J = 9.0 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 3.83 (s, 3H);
13C NMR (151 MHz, CDCl3) δ 161.3, 140.8 (d, J = 36.9 Hz), 128.8, 126.5, 122.9, 115.4, 55.9;
19F NMR (564 MHz, CDCl3) δ 66.62; HRMS (m/z): [M+Na]+ calcd for C9H8FN3O3SNa1:
280.0162, found 280.0162.

1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-sulfonyl fluoride
In a screw cap tube, 206 mg 1-azido-4-(trifluoromethyl)benzene (1.10 mmol, 1.00 eq.) and 312
mg 1-bromoethene-1-sulfonyl fluoride (1.65 mmol, 1.50 eq.) were dissolved in
dimethylformamide (1.00 mL) and stirred for 20 hours at 80˚C. After extraction with ethyl acetate
(3 x 5 mL), the organic layer was dried with MgSO4 and the solvent was evaporated. Subsequent
purification by column chromatography (hexanes/dichloromethane = 2/1) resulted in 1-(4-
(trifluoromethyl)phenyl)-1H-1,2,3-triazole-4-sulfonyl fluoride as a beige solid in a yield of 69%
(0.225 g, 0.759 mmol). H NMR (600 MHz, acetoneD6) δ 9.69 (s, 1H), 8.22 (dd, J = 8.4, 2.1 Hz,
2H), 7.95 (dd, J = 8.6, 2.0 Hz, 2H); 13C NMR (151 MHz, acetone-D6) δ 140.2 (d, J = 37.1 Hz),
138.8, 131.4 (q, J = 33.1 Hz), 129.4, 127.3, 127.3, 122.9, 122.1; 19F NMR (564 MHz, acetone-
D6) δ 65.28, -63.31; HRMS (m/z): [M+Na]+ calcd for C9H5F4N3O2SNa+ : 317.9936, found
317.9935.

20

1-mesityl-1H-1,2,3-triazole-4-sulfonyl fluoride
In a screw cap tube, 2-azido-1,3,5-trimethylbenzene (177 mg, 1.10 mmol, 1.00 eq.) and 1-
bromoethene-1-sulfonyl fluoride (312 mg, 1.65 mmol, 1.50 eq.) were dissolved in
dimethylformamide (1.00 mL) and stirred for 20 hours at 80˚C. After extraction with ethyl acetate
(3 x 5 mL), the organic layer was dried with MgSO4 and the solvent was evaporated. Subsequent
purification by column chromatography (hexanes/dichloromethane = 2/1) resulted in 1-mesityl-
1H-1,2,3-triazole-4-sulfonyl fluoride as a beige solid in a yield of 86% (0.254 g, 0.946 mmol). H
NMR (600 MHz, CDCl3) δ 8.32 (s, 1H), 7.05 (s, 2H), 2.38 (s, 3H), 1.98 (s, 6H); 13C NMR (151
MHz, CDCl3 ) δ 141.8, 140.3 (d, J = 36.9 Hz), 134.9, 132.0, 130.4, 129.8, 21.4, 17.5; 19F NMR
(564 MHz, CDCl3) δ 66.1; HRMS (m/z): [M+H]+ calcd for C11H13FN3O2S: 270.0707, found
270.0705.

1-(4-Bromophenyl)-1H-1,2,3-triazole-4-sulfonyl fluoride (5o)
1-Bromoethene-1-sulfonyl fluoride (375 mg, 2 mmol), 1-azido-4-bromobenzene (196 mg, 1
mmol), DMF (1 mL), reaction time (24h) and temperature (80 °C). The product was purified by
flash column chromatography (hexane/EtOAc = 6:4) affording 5p (247 mg, 81% yield) as an off
white solid. 1 H NMR (600 MHz, CDCl3) δ 8.68 (s, 1H), 7.79 – 7.75 (m, 2H), 7.69 – 7.66 (m,
2H); 13C NMR (151 MHz, CDCl3) δ 141.4(d, J = 37.3 Hz), 134.6, 133.7, 126.5, 24.9, 122.6; 19F

21
NMR (564 MHz, CDCl3) δ 66.66; HRMS (m/z): [M+H] + calcd for C8H6BrFN3O2S: 305.9343,
found 305.9341.

1-(Naphthalen-1-ylmethyl)-1H-1,2,3-triazole-4-sulfonyl fluoride (5b):
1-Bromoethene-1-sulfonyl fluoride (263 mg, 1.4 mmol), 1-(azidomethyl)naphthalene (183 mg, 1
mmol), DMF (1 mL), reaction time (14 h) and temperature (50 °C). The product was purified by
flash column chromatography (hexane/EtOAc = 6:4) affording 5b (257 mg, 88% yield) as an off
white solid. 1 H NMR (600 MHz, CDCl3) 1 H NMR (600 MHz, CDCl3) δ 8.00 (d, J = 8.0 Hz,
1H), 7.96 (dd, J = 6.1, 3.3 Hz, 1H), 7.94 (s, 1H), 7.87 (dd, J = 6.0, 3.3 7 Hz, 1H), 7.65 – 7.49 (m,
4H), 6.08 (s, 2H); 13C NMR (151 MHz, CDCl3)) δ 140.4 (d, J = 36.8 Hz), 134.3, 131.4, 131.0,
129.5, 129.1, 128.1, 127.6, 127.0, 125.6, 122.3, 53.49; 19F NMR (564 MHz, CDCl3) δ 66.3;
HRMS (m/z): [M+Na] + calcd for C13H10F1N3O2S1Na1: 314.0370; found 314.0374.

1-(2-(3-Cyclohexylureido)ethyl)-1H-1,2,3-triazole-4-sulfonyl fluoride (5c)
1-Bromoethene-1-sulfonyl fluoride (263 mg, 1.4 mmol), 1-(2-azidoethyl)-3-cyclohexylurea (211
mg, 1 mmol), DMF (1 mL), reaction time (14 h) and temperature (50 °C). The product was purified

22
by flash column chromatography (hexane/EtOAc = 1:9) affording 5c (268 mg, 84% yield) as an
off white solid. 1 H NMR (400 MHz, acetoneD6:CDCl3) δ 9.04 (d, J = 1.0 Hz, 1H), 5.68 (s, 1H),
5.46 (s, 1H), 4.86 – 4.52 (m, 2H), 3.70 (dd, J = 11.5, 5.9 Hz, 2H), 3.55 – 3.28 (m, 1H), 1.84 – 1.75
(m, 2H), 1.73 – 1.60 (m, 2H), 1.59 – 1.43 (m, 1H), 1.40 – 1.18 (m, 2H), 1.26 – 0.91 (m, 3H); 13C
NMR (100 MHz, acetone-D6:CDCl3) δ 158.1, 139.8 (d, J = 35.9Hz), 131.7, 52.8, 49.5, 40.4, 34.4,
26.5, 25.8.; δ 65.08 19F NMR (564 MHz, acetone-D6) δ 65.08 (d, J = 0.9 Hz); HRMS (m/z):
[M+H] + calcd for C11H19F1N5O3S1: 320.1187, found 320.1187.

1.5.4 Synthetic protocols for Sultam Synthesis

2-benzyl-4-bromo-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and benzylamine (147 mg, 1.21 mmol, 1.30 eq.) was added rapidly. The reaction
mixture was stirred at room temperature for 1h. After extraction with diethyl ether (3 x 15 mL)
and subsequent purification by column chromatography (hexanes/dichloromethane = 1/1), 2-
benzyl-4-bromo-1,2-thiazetidine 1,1-dioxide was obtained as a beige solid in a yield of 68% (200
mg, 0.72 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 7.42- 7.31 (m, 5H), 5.65 (dd, J = 7.1, 4.7
Hz, 1H), 4.36-4.22 (m, 2H), 3.68 (dd, J = 7.1, 7.1 Hz, 1H), 3.17 (dd, J = 7.1, 4.7 Hz, 1H)

23

2-amino-4-bromo-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and a hydrazine solution (64%, 68 mg, 1.38 mmol, 1.30 eq.) was added rapidly at
0˚C. The reaction mixture was stirred at room temperature for 1 h. After extraction with diethyl
ether (3 x 15 mL) and subsequent purification by column chromatography
(hexanes/dichloromethane = 1/1), 2-amino-4-bromo-1,2-thiazetidine 1,1-dioxide was obtained as
a beige solid in a yield of 47% (99 mg, 0.50 mmol).
1
H NMR (400 MHz, Acetonitrile-d3) δ 5.04
(dd, J = 7.5, 6.5 Hz, 1H), 4.28 (dd, J = 13.7, 7.5 Hz, 1H), 3.64 (dd, J = 13.6, 6.5 Hz, 1H).

4-bromo-2-ethyl-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and a ethylamine solution (70%, 88 mg, 1.38 mmol, 1.30 eq.) was added rapidly at
0˚C. The reaction mixture was stirred at room temperature for 3 h. After extraction with diethyl
ether (3 x 15 mL) and subsequent purification by column chromatography
(hexanes/dichloromethane = 1/1), 4-bromo-2-ethyl-1,2-thiazetidine 1,1-dioxide was obtained as a
beige solid in a yield of 23% (52 mg, 0.24 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 5.18 –
4.99 (m, 1H), 3.80 – 3.64 (m, 1H), 3.50 – 3.30 (m, 1H), 2.87 (q, 2H), 1.15 (t, J = 7.2, 0.7 Hz, 3H).

24

4-bromo-2-(4-phenylbutyl)-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and 4-phenylbutan-1-amine (236 mg, 1.59 mmol, 1.50 eq.) was added rapidly. The
reaction mixture was stirred at room temperature for 2 h. After extraction with diethyl ether (3 x
15 mL) and subsequent purification by column chromatography (hexanes/dichloromethane = 1/1),
4-bromo-2-(4-phenylbutyl)-1,2-thiazetidine 1,1-dioxide was obtained as an off-white solid in a
yield of 50 % (169 mg, 0.53 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 7.28 – 7.17 (m, 2H),
7.17 – 7.04 (m, 3H), 5.64 – 5.45 (m, 1H), 3.67 – 3.56 (m, 1H), 3.17 – 3.06 (m, 1H), 3.06 – 2.94
(m, 2H), 2.62 – 2.48 (m, 2H), 1.71 – 1.51 (m, 4H).

4-bromo-2-(3,4,5-trimethoxybenzyl)-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and (3,4,5-trimethoxyphenyl)methanamine (313 mg, 1.59 mmol, 1.50 eq.) was
dissolved in 1 mL toluene and added rapidly. The reaction mixture was stirred at room temperature
for 1h. After extraction with diethyl ether (3 x 15 mL) and subsequent purification by column
chromatography (hexanes/EtOAC = 1/2), 4-bromo-2-(3,4,5-trimethoxybenzyl)-1,2-thiazetidine
1,1-dioxide was obtained as an off-white solid in a yield of 66% (257 mg, 70 mmol).
1
H NMR

25
(500 MHz, Chloroform-d) δ 6.40 – 6.29 (m, 2H), 5.56 – 5.41 (m, 1H), 3.89 – 3.78 (m, 2H), 3.63 –
3.51 (m, 9H), 3.51 – 3.41 (m, 1H), 2.96 – 2.83 (m, 1H).

4-bromo-2-(((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthren-1-yl)methyl)-1,2-thiazetidine 1,1-dioxide
In a screw cap tube, 1-bromoethene-1-sulfonyl fluoride (200 mg, 1.06 mmol, 1.00 eq.) was added
to 3 mL H2O and ((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthren-1-yl)methanamine (695 mg, 1.21 mmol, 1.30 eq.) was added rapidly. The
reaction mixture was stirred at room temperature for 2 h. After extraction with diethyl ether (3 x
15 mL) and subsequent purification by column chromatography (hexanes/dichloromethane = 1/1),
4-bromo-2-(((1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthren-1-yl)methyl)-1,2-thiazetidine 1,1-dioxide was obtained as a beige solid in
a yield of 55% (264 mg, 87 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 7.22 – 7.14 (m, 1H),
7.02 – 6.99 (m, 1H), 6.90 (s, 1H), 5.76 – 5.61 (m, 1H), 3.94 – 3.77 (m, 1H), 3.43 – 3.23 (m, 1H),
3.14 – 2.75 (m, 5H), 1.82 – 1.65 (m, 4H), 1.62 – 1.49 (m, 1H), 1.49 – 1.38 (m, 3H), 1.24 (s, 3H),
1.22 (d, J = 2.9 Hz, 6H), 0.99 – 0.96 (m, 1H), 0.94 (s, 3H).

1.6 Distribution of Credit
Chapter 1 is a result of a collaborative effort with Dr. Joice Thomas. The majority of the text herein
was written by the author (Katharina Grotsch). Table 1.1 was adapted from Dr. Thomas and used

26
with his permission. Specific contributions are as follows: all synthetic procedures were carried
out by the author, except for the synthesis of 1-(4-Bromophenyl)-1H-1,2,3-triazole-4-sulfonyl
fluoride (5o), 1-(Naphthalen-1-ylmethyl)-1H-1,2,3-triazole-4-sulfonyl fluoride (5b), and 1-(2-(3-
Cyclohexylureido)ethyl)-1H-1,2,3-triazole-4-sulfonyl fluoride (5c), which were carried out by Dr.
Thomas.

27
Chapter 2. Exploring the Limits of what is Tangible and what is Feasible:
Searching for Drug Candidates in an Unknown Chemical Space

2.1 Strategy and Overview
Early successes in drug discovery relied on serendipitous chemical findings. These successes
played a large role in directing the future of drug discovery and remain the basis for many of the
most well-known drugs to date. Nonetheless, the field of drug discovery has become stagnant.
With the emergence of new computational technologies, we now have the opportunity to lend
serendipity a helping hand in the search for new drug candidates.

It is estimated that the number of realistic drug-like molecules that could ever be synthesized is
anywhere between 10
23
and 10
63
, a chemical space only a small fraction of which has been
explored.
24-26
Even on the lower end, this number is entirely unfathomable for the synthetic
chemist to traverse. Now, computer-assisted molecular design allows us to overcome this
bottleneck and synthesize hundreds of millions of compounds in silico, to explore a quantity and
diversity of compounds that is otherwise inaccessible.
25

Ideally, structure-based docking of large virtual compound libraries is an excellent time- and cost-
effective way to yield new leads for important drug targets.
27
The recent explosion of available G-
protein coupled receptor (GPCR) structures and the availability of inexpensive cloud computing
resources provides a strong template for this application.
28, 29
Nonetheless, the success of virtual
ligand screening (VLS) is inherently limited – at this time, there is no way to accurately predict
the affinity of a given compound, and the method can generate false positive hits.
30
Consequently,

28
the practicality of docking ultra large virtual libraries is restricted by the accessibility of the
compound library itself.
28, 31
In the past this accessibility has been problematic, especially for large
compound libraries. However, recent advances in synthetic chemistry and the ever-increasing
number of available reagents has allowed vendors to offer on-demand compound databases
ranging in the billions. Based on this, Lyu and co-workers were able to find promising new
chemotypes based on the structure-based docking of 170 million make-on-demand compounds
generated from 130 distinct chemical reactions.
31

In order to verify our approach, the compounds were docked against the cannabinoid type II
receptor (CB2R). The endogenous cannabinoid system (ECS) consists of two identified GPCRs,
cannabinoid receptor type I (CB1) and CB2, that were named after their affinity for the agonist ∆
9
–
THC.
32, 33
Although selectively targeting one half of the endogenous cannabinoid system (ECS)
has been challenging due to the high degree of homology and sequence identity between the two
receptors, the recently published crystal structure of CB2 should aid in facilitating the precise
modulation of the ECS.
32, 34, 35
This is significant because CB2 has recently gained traction as an
important target for neurodegenerative disorders, neuropathic pain and inflammation, as well as
fibrotic conditions and cancer.
36-40
It should be noted that, unlike its illustrious counterpart, CB2
does not produce any psychotropic side-effects, making it ever-more attractive for therapeutic
purposes.
34
We will examine the role of the type I and II cannabinoid receptors, as well as the
respective endogenous and exogenous cannabinoids that interact with it more closely in Chapter 3.

Despite tremendously advancing the state of healthcare over the past decades, it is possible that
we have maximized the potential of rational drug discovery by deliberately reconnoitering and

29
refining a known drug-class. Given the recent advances in biology, chemistry, and computational
science, we now have better tools than ever to expand the known chemical space in a time- and
cost-effective manner by generating and docking ultra-large libraries of compounds which are
generated by a sequence of orthogonal chemical reactions. Here, we suggest that even the
enumeration of a single “superscaffold” can generate a library with enough chemical diversity to
lead to the discovery of new ligands for important drug targets.

2.2 Creation of On-Demand Virtual Library
For the creation of the on-demand virtual triazole library, building block libraries were
downloaded and searched for alcohols and halides as azide precursors. The compounds from this
search were enumerated and exclusions were applied to ensure synthetic feasibility based on
laboratory evidence and chemical intuition. Next, these libraries were combined with existing
azide building blocks from vendor libraries and exclusions based on functional group presence,
molecular weight, and heteroatom count were applied to ensure appropriate safety and reactivity.
The remaining compounds were reacted with Br-ESF to generate the sulfonyl-fluoride
functionalized triazole library. The vendor libraries were searched for primary and secondary
amines and, after applying the appropriate exclusions, reacted with the triazoles to generate the
final compound library. After filtering the compounds according to Lipinski’s rule of 5, 70 million
readily synthesizeable compounds remained for VLS screening (Scheme 2.1 a).

Steps for Triazole reaction:
1. Download building block library
2. Search for precursors to azides in the building block library:

30
a) halides
b) alcohol
3. Make reaction (enumeration/generation of compounds based on reaction):
a) Halide-azide (1st row in Rxn1Reaction table)
b) Alcohol-azide (2
nd
row in Rxn1Reaction table)
4. Exclusion of compounds containing certain moieties from:
a) Halide-azide
b) Alcohol-azides
5. Search for azides in building block library with certain exclusions
6. Append azides from all three sources, and delete all azides with Mol weight > 350 and
atom count < 5
7. Make reaction/enumeration/generation of intermediate compounds (3
rd
row in
Rxn1Reaction table)
8. Search for primary and secondary amines with respective exclusions in the building block
library
9. Append primary and secondary amines and delete amines with Mol weight > 350
10. Final make reaction/enumeration/generation of final compounds and filter all molecules
>500 Mol weight (4th row in Rxn1Reaction table)

Similarly, for the creation of the on-demand virtual isoxazole library, vendor libraries were
searched for aldehyde precursors from which the respective oximes were formed. The compounds
from this search were filtered by molecular weight, enumerated, and exclusions applied to ensure
synthetic feasibility based on laboratory evidence and chemical intuition. The remaining

31
compounds were again reacted with Br-ESF to generate the sulfonyl-fluoride functionalized
isoxazole library. Sourcing the same library of primary and secondary amines based on vendor
availability and reacting these with the computationally derived SO2F-isoxazoles, the
sulfonamide-functionalized isoxazoles were generated. After filtering the compounds according
to Lipinski’s rule of 5, 70 million readily synthesizable compounds remained for VLS screening
(Scheme 2.1 b).

Steps for Isoxazole reaction:
1. Download building block library
2. Search for precursor aldehydes and filter out aldehydes with Mol weight > 350
3. Make reaction/enumeration/generation of oxime intermediate compounds (1
st
row in
Rxn2Reaction table)
4. Make reaction/enumeration/generation of isoxazole intermediate compounds (2
nd
row in
Rxn2Reaction table)
5. Search for primary and secondary amines with respective exclusions in the building block
library
6. Append primary and secondary amines and delete amines with Mol weight > 350
7. Final make reaction/enumeration/generation of final compounds and filter all molecules
>500 Mol weight (3
rd
row in Rxn2Reaction table)

32

Scheme 2.1: Combinatorial strategy for creation of a) triazole library and b) isoxazole library
from available building blocks.

2.3 Receptor model preparation and optimization
CB2R crystal structure and ligand-optimized structural models were employed for prospective
virtual ligand screening. The virtual screening models were prepared using the X-ray structure of
CB2 receptor with a rationally designed antagonist AM10257 at 2.8 Å resolution (PDB ID
5ZTY).
41
The structure was converted from PDB coordinates to ICM object using ICM-Pro
conversion algorithm.
42
The conversion involves building of the hydrogen and missing heavy
atoms, local minimization of polar hydrogens, optimization of His, Asn and Gln side chain
rotamers and protonation state and assigning secondary structure.
To account for binding pocket flexibility upon binding of different ligand scaffolds, ligand-guided
receptor optimization algorithm (LiBERO) was used to refine the sidechains and water molecules
in 8 Å radius from the orthosteric binding pocket.
43
Two binding modes for the CB2 receptor
binding pocket were prepared: one for antagonists and another for agonists. For validation of each
of the models, the diverse sets (20 molecules each) of known high-affinity agonists (ChEMBL,
pAct > 9) and antagonists (ChEMBL, pAct > 8) were docked into the corresponding models along
with 200 decoy molecules selected from CB2 receptor decoy database (GDD).
44
The AUC (Area

33
Under The Curve) ROC (Receiver Operating Characteristics) curves were used to quantitatively
evaluate the models, where ROC curves were plotted based on the True Positive Rates (TPR) and
False Positive Rates (FPR) from the docking of true binders and decoys.
We employed the crystal structure of CB2R with antagonist AM10257
49
for prospective virtual
ligand screening. To account for binding site flexibility during the binding of different CB2
ligands, we employed ligand-based optimization of the CB2 binding pocket as described in.
43
Two
diverse sets of compounds were used as seed compounds for optimization of the binding site:
known high-affinity agonists (ChEMBL, pAct > 9) and antagonists (ChEMBL, pAct > 8). The set
of high-affinity ligands and a CB2 specific decoy compound library [reference to GPCR-decoy
database] were used in benchmark docking to evaluate the performance of the structural models
to discriminate between CB2 binders and a decoy set. The receiver operating characteristic curve
ROC values were used as quantitative criteria for the selection of the best models. The two best
structural models, corresponding to antagonist- and agonist-bound state, showed improved values
of AUC in comparison to the CB2 crystal structure (Figure 2.1). Moreover, optimized models
showed better docking scores for 20 diverse high affinity ligands compared to the CB2 crystal
structure.
Docking poses of high-affinity ligands in the best models were similar to the conformation of the
co-crystallized ligand. The two best structural models, corresponding to antagonist- and agonist-
binding states, along with the crystal structure of CB2R were employed to generate a 4D structural
model to screen several receptor conformations in a single screening run. The performance of the
obtained 4D model was assessed in benchmark VLS and showed comparable docking scores and
ROC values to that of individual structural models.

34

Figure 2.1: Performance evaluation for CB2 crystal structure and ligand-guided optimized models.
a) ROC plots for CB2 crystal structure and ligand-based optimized models. Examples of predicted
binding poses of selected known high-affinity CB2 ligands in optimized models with agonist (b)
and antagonist (c) molecules.

2.4 Virtual Ligand Screening
Docking/VLS simulations were performed using ICM-Pro molecular modeling software (Molsoft
LLC). The CB2 model optimized with agonists, CB2 model optimized with antagonists, and
crystal structure of the CB2 receptor were employed in 4D docking to account for binding pocket
flexibility in a single docking run, as implemented in ICM-Pro.
42
Energy potential maps were
calculated for each model and stored in a single multi-dimensional map file (4D grid). During
docking ligands were given full torsion flexibility in internal coordinates. Docking simulations
used biased probability Monte Carlo (BPMC) optimization of the compound’s internal coordinates
in the pre-calculated 4D grid energy potentials. In VLS, the exhaustive sampling of the molecule

35
conformational space in the rectangular box of the CB2 orthosteric binding pocket was performed
and the best docking conformation of each molecule was stored with the corresponding predicted
binding score.
The initial screening of 140 million compounds was performed on Google Cloud Platform
using ̴100K cores and took around 24 hours to complete the screening with docking thoroughness
set to 1. The top 500 thousand compounds with the best scores were redocked twice with
thoroughness increased to 2. The chemical diversity of top hits was evaluated using the Tanimoto
coefficient calculated in ICM-Pro (http://www.molsoft.com/icm/fingerprints.html).

2.5 Selection and Synthesis of Drug Candidates
The top 100 scoring molecules from the Virtual Ligand Screening were sorted and scored based
on synthetic ease. In order to ensure the most efficient synthetic timeline, only the simplest
molecules were selected for the validation of this method. Some factors that were taken into
account are (a) azides synthesized from halogen precursors were preferred over alcohol precursors,
(b) primary amines received preferential treatment over secondary amines, and (c) possible
complications due to sterics were limited. Out of this batch, 14 compounds were selected by taking
price and vendor availability into account. Of the 14 selected compounds, 11 were fully
synthesized in house using the methods described in Chapter 1 of this thesis (Figure 2.2). Detailed
information on synthetic protocols and spectra for each compound can be found in Section 2.9.

36

Figure 2.2: Synthesized compounds that were identified as hits for CB1 and CB2.
2.6 Experimental identification and validation of CB2 binders
2.6.1 Experimental Procedures
Functional potency in CB1/CB2 Tango assays: The Tango arrestin recruitment assays were
performed as previously described
45
. Briefly, HTLA cells were transiently transfected with human
CB1 or CB2 Tango DNA construct overnight in DMEM supplemented with 10 % FBS, 100 µg/ml
streptomycin and 100 U/ml penicillin. The transfected cells were then plated into Poly-L-Lysine
coated 384-well white clear bottom cell culture plates in DMEM containing 1% dialyzed FBS at a
density of 10,000-15,000 cells/well. After 6 hours incubation, the plates were added with drug
solutions prepared in DMEM containing 1% dialyzed FBS for overnight incubation. Specially for
the antagonist assay, 100 nM of CP55940 was added after 30 minutes of incubation of the drugs.
On the day of assay, medium and drug solutions were removed and 20 µL/well of BrightGlo
reagent (Promega) was added. The plates were further incubated for 20 min at room temperature
and counted using a Wallac TriLux Microbeta counter (PerkinElmer). Results were analyzed using
GraphPad Prism 9.

37

Radioligand binding in CB1/CB2 binding assays: The affinities (Ki) of the new compounds for rat
CB1 receptor as well as for human CB2 receptors were obtained by using membrane preparations
from rat brain or HEK293 cells expressing hCB2 receptors, respectively, and [
3
H]CP-55,940 as the
radioligand, as previously described.
46, 47
Results from the competition assays were analyzed using
nonlinear regression to determine the IC50 values for the ligand; Ki values were calculated from
the IC50 using GraphPad Prism . Each experiment was performed in triplicate and Ki values
determined from three independent experiments and are expressed as the mean of the three values.

2.6.2 Results
Experimental testing identified 6 compounds with CB2R binding affinity below 10μM, of which 2
compounds were in the sub-micromolar range (Table 2.1 and Figure 2.3).

Figure 2.3: Characterization of best CB2 hits.

-14 -12 -10 -8 -6 -4
0
50
100
CB1 Tango Antagonist
log[drug], M
Emax (% Rimonabant)
59134
59135
59137
59141
61746
61747
Rimonabant
-14 -12 -10 -8 -6 -4
0
50
100
CB2 Tango Antagonist
log[drug], M
Emax (% SR144528)
59134
59135
59137
59141
61746
61747
SR144528

38
Table 2.1: Results of testing of hits from virtual ligand screening in binding and functional
assays.
BRI-ID PDSP
ID
CB1 Antagonist
potency
CB2 Antagonist
potency
Model Tanimoto
Ki, uM Ki, uM
13900 59134 2.34 1.71-2.92 3.05 1.68-3.31 1 0.51
13901 59135 1.08 0.77-1.42 2.03 1.59-2.53 2 0.49
13903 59137 2.23 1.30-2.96 6.22 3.79-7.68 1 0.51
13907 59141 0.40 0.25-0.59 0.60 0.34-1.04 0.50
13911 61746 2.42 1.68-3.64 8.11 4.24-22.45 2 0.44
13912 61747 0.18 0.14-0.23 0.32 0.21-0.46 1 0.53

39
2.7 Discussion
Here, we demonstrate that even the enumeration of one single scaffold can generate enough
chemical diversity to lead to the serendipitous discovery of promising leads for important targets.
With Tanimoto coefficients >0.3 for the majority of compounds, we were able to generate a
chemically diverse library based on a new synthetic strategy developed in our lab. Previous
attempts of the characterization of new ligands for CB2 resulted in hit rates between 15-33%.
48, 49

Given this value, which is on the higher end of the range observed for GPCRs, we can conclude
that the receptor model at hand provides a strong basis for our work. In addition, the experimental
hit rate of 55% for this library suggests that we could be working with a privileged scaffold, or
“superscaffold”. Based on chemical intuition, this would appear logical as the scaffold used for
the design of this library is privileged by the presence of two entities that appear abundantly in
small molecule drugs: a) an electron deficient sulfonamide and b) a 5-membered heterocycle
moiety.

Sulfonamides have been long known to possess interesting properties and are present in more than
30 drugs in clinical use.
50
The electron density is localized on the oxygen atoms, essentially
creating an “O
-
charge”, making the nitrogen atom inductively electron-deficient and weakly basic.
Similarly, triazoles have gained increasing popularity in drug discovery and have been used as
antifungal, antiepileptic, and anticonvulsant agents.
51
In part, this is due to their facile synthesis
and the popularity of CuAAC in HTS. However, they also possess interesting electronic properties,
including two hydrogen bond donors and one hydrogen bond acceptor.
52, 53
Due to the smaller size
of 5-membered heterocycles in comparison to their 6-membered counterparts, the heterocycle lone
pairs are more splayed, but still able to part-take in hydrogen bonding. Similar properties are

40
observed for the isoxazole moiety, with the exception that the oxygen atom is more polarized and
more basic than the nitrogen on its triazole counterpart. The combination of these effects could
provide a rational explanation for the existence of a privileged scaffold, suggesting that the library
might provide useful ligands for more than one receptor.
54

Albeit that these observations are indicative of a privileged scaffold, further investigations are
needed to verify this assumption. The sulfonamide-functionalized triazole CORT113176 has
previously been identified as a selective glucocorticoid receptor (GR) antagonist, but other targets
should be investigated to validate the broad applicability of this scaffold.
21
Over 35% of drugs
acting on more than one target and the increasing emergence of polypharmacoligical ligands as
treatments for complex neurological diseases, infectious diseases, or cancer.
55-57
Nonetheless, it is
important to differentiate between privilege and promiscuity, keeping in mind that a certain degree
of selectivity remains crucial in the effective treatment of a given disease.
58

2.8 Conclusion
Historically, libraries for high-throughput screening (HTS) and VLS have been limited to a size
range of several million compounds.
59, 60
Now we can access more chemical space than ever, using
vendor libraries such as the enamine REAL library which now comprises over 11 billion
compounds.
48
In addition, new computational methods have been developed to rapidly screen
libraries of this size. Although these tools have proven to be of great value in identifying new leads
for important biological targets, our results demonstrate that not only the size of such libraries
plays a role in the search for new drug-like scaffolds and stresses the need intelligent design
towards this approach.

41
2.9 Synthetic Procedures and Spectra
2.9.1 General Information
1
H,
13
C, and
19
F NMR spectra were recorded on Varian 400-MR, Varian VNMRS-500, and Varian
VNMRS-600 instruments at 295K unless otherwise noted. Proton magnetic resonance spectra
(
1
H NMR) were recorded at 500 MHz, and carbon magnetic resonance spectra (
13
C NMR) were
recorded at 125 MHz, unless otherwise mentioned. Chemical shifts (δ) are expressed in parts per
million, relative to the residual solvent signals as internal standards. Multiplicities are noted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; sex, sextet; sept, septet. High resolution
mass spectra were measured using Agilent 6545XT qToF instrument coupled with 1290 LC
system. QToF mass spectrometer is equipped with an atmospheric pressure chemical ionization
source. Measurements were performed in positive ion mode with following ionization parameters:
Capillary Voltage –3.5 kV, Corona current 4 µA, vaporization at 350 °C, nitrogen was applied as
a nebulizer gas 35 psi, dry gas 13 L × min
−1
, 325 °C, and collision gas. Spectra were recorded in
m/z 100 – 1700 range. For external calibration and tuning, a low-concentration tuning mix solution
by Agilent Technologies was utilized. For sample injection (1 µL injection of ca. 10
–4
M solution
in MeOH) LC system was used. Injected compounds were passed through XDB-C18, 2.1 × 50
mm, 1.8 µm column at 40 °C with gradient H2O/MeOH (0.1% formic acid) elution. UV/Vis flow
detection was also applied (190 – 900 nm). All the MS spectra were recorded at 1 Hz. Spectra were
processed using Agilent MassHunter 10.0 software package. Precoated Merk F-254 silica gel
plates were used for analysis by thin layer chromatography (TLC), visualized with short wave UV
light, and stained with KMnO4 and PPh3/Ninhydrin. Column chromatography was carried out
employing EMD (Merk) Silica Gel 60 (40-63 μm). Reagents were obtained from AA BLOCKS,
Enamine, or Sigma Aldrich, and used without further purification, unless otherwise noted.

42
2.9.2 Synthetic Procedures
Synthesis of BRI-13900

A 50 mL round-bottom flask was charged with 1-([1,1'-biphenyl]-4-yl)ethan-1-ol (1.0 eq., 500 mg,
2.52 mmol) and Cu(ClO4)2
.
6H2O (0.1 eq., 92 mg, 0.14 mmol). The reagents were dissolved in
3 mL dichloromethane and TMSN3 (1.2 eq., 0.4 mL, 3.03 mmol) was added dropwise at room
temperature. The reaction mixture was stirred for 1 h and extracted with DCM (3x10 mL) and
dried over sodium sulfate. After rotary evaporation of the solvent, the crude reaction mixture was
purified by column chromatography (SiO2, 30% EtOAc in Hexanes). The azide (2.7) was obtained
as a clear oil in (96%, 272 mg) yield.
1
H

NMR (400 MHz, Chloroform-d) δ 7.65 – 7.56 (m, 1H),
7.54 – 7.31 (m, 1H), 4.67 (d, J = 6.8 Hz, 0H), 1.58 (d, J = 6.8 Hz, 1H).

In a 10 mL reaction tube, 2.7 (1.0 eq., 250 mg, 1.12 mmol) and Br-ESF (3.0 eq., 635 mg, 3.36
mmol) were suspended in 1 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 10 mL) and washed with brine. The organic
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
2.7
2.8

43
mixture. Subsequent purification by column chromatography (SiO2, 40% → 60% DCM in
Hexanes) yielded the triazole (2.8) as an off-white solid (80%, 296 mg).
1
H NMR (400 MHz,
Chloroform-d) δ 8.09 (d, J = 1.2 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.60 – 7.56 (m, 2H), 7.50 –
7.36 (m, 5H), 5.96 (q, J = 7.1 Hz, 1H), 2.11 (d, J = 7.1 Hz, 3H).
19
F NMR (376 MHz, Chloroform-
d) δ 66.29.

To begin with, a 10 mL reaction tube was charged with 2.8 (1.0 eq., 100 mg, 0.30 mmol), 4-
(pyrrolidin-2-yl)pyrimidine (2.0 eq., 90 mg, 0.60 mmol), and triethylamine (2.0 eq., 0.9 mL,
0.60 mmol). After addition of 1 mL acetonitrile, the reaction mixture was stirred for 24 h at 80˚C.
The crude mixture was obtained by rotary evaporation of the solvent, and subjected to purification
by column chromatography (SiO2, 5% MeOH in DCM) to obtain the product (2.1, BRI-13900) as
an off-white solid (32%, 44 mg).
1
H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 9.11 (d, J = 1.3
Hz, 1H), 8.81 (d, J = 5.3, 0.8 Hz, 1H), 7.71 – 7.62 (m, 5H), 7.49 – 7.43 (m, 4H), 7.40 – 7.33 (m,
1H), 6.12 (q, J = 7.1 Hz, 1H), 4.87 (apparent ddd, 1H), 3.68 – 3.59 (m, 1H), 3.51 – 3.39 (m, 1H),
2.09 – 1.97 (m, 4H), 1.95 – 1.86 (m, 1H), 1.85 – 1.76 (m, 1H), 1.69 – 1.58 (m, 1H).
13
C NMR (101
MHz, DMSO-d6) δ 170.52, 158.52, 158.10, 144.33, 140.67, 139.90, 139.72, 129.38, 128.10,
127.69, 127.61, 127.46, 127.34, 127.17, 118.97, 64.12, 60.55, 50.18, 33.56, 24.30, 21.09. APCI -
MS (TOF): measured m/z 461.1773, calcd for C24H24N6O2S [M+H]
+
m/z 461.1754 (Δ = 4.1 ppm).

2.1
2.8

44
Synthesis of BRI-13901

A 50 mL round bottomed flask was flame-dried and charged with 1-chloro-4-
(chloro(phenyl)methyl)benzene (1.0 eq., 1.00 g, 4.22 mmol) in 10 mL dimethyl formamide.
Sodium azide (2.0 eq., 548 mg, 8.43 mmol) was added to the solution at room temperature and
stirred for 10 h. Upon completion, the reaction mixture was extracted with Et2O (3x20 mL) and
dried over sodium sulfate. The crude mixture was obtained by rotary evaporation of the solvent
and subjected to purification by column chromatography (SiO2, 30% DCM in Hexanes) to obtain
the product (2.9) as a yellow oil (75%, 770 mg).
1
H NMR (400 MHz, Chloroform-d) δ 7.55 – 7.06
(m, 9H), 5.68 (s, 1H).

In a 10 mL reaction tube, 2.9 (1.0 eq., 770 mg, 3.16 mmol) and Br-ESF (3.0 eq., 1.79 g, 9.48
mmol) were suspended in 4 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 20 mL) and washed with brine. The organic
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
mixture. Subsequent purification by column chromatography (SiO2, 40% DCM in Hexanes)
yielded the triazole (2.10) as a yellow oil (81%, 1.17 g).
1
H NMR (400 MHz, Chloroform-d) δ
2.9
2.10
2.9

45
8.13 (s, 1H), 7.44 – 7.39 (m, 3H), 7.39 – 7.35 (m, 2H), 7.18 – 7.12 (m, 3H), 7.11 – 7.03 (m, 2H).

19
F NMR (376 MHz, Chloroform-d) δ 66.58.

To begin with, a 10 mL reaction tube was charged with 2.10 (1.0 eq., 100 mg, 0.28 mmol),
(octahydro-1H-indol-2-yl)methanol (2.0 eq., 88 mg, 0.57 mmol), and triethylamine (2.0 eq.,
0.8 mL, 0.57 mmol). After addition of 1 mL acetonitrile, the reaction mixture was stirred for 20 h
at 80˚C. The crude mixture was obtained by rotary evaporation of the solvent, and subjected to
purification by column chromatography (SiO2, 5% → 10% MeOH in DCM) to obtain the product
(2.2, BRI 13901) as an off-white solid (23%, 32 mg).
1
H NMR (400 MHz, DMSO-d6) δ 8.91 (s,
1H), 7.52 – 7.47 (m, 2H), 7.46 – 7.38 (m, 3H), 7.33 – 7.26 (m, 2H), 7.26 – 7.19 (m, 2H), 3.73 –
3.60 (m, 4H), 3.49 (dd, J = 10.4, 6.5 Hz, 1H), 2.03 – 1.85 (m, 1H), 1.78 – 1.65 (m, 2H), 1.63 –
1.52 (m, 2H), 1.51 – 1.30 (m, 4H), 1.27 – 1.05 (m, 2H).
13
C NMR (101 MHz, DMSO-d6) δ 145.10,
137.98, 137.14, 133.78, 130.54, 129.38, 129.31, 129.09, 128.28, 128.12, 67.08, 64.93, 62.18,
60.78, 36.31, 30.95, 30.19, 25.70, 24.26, 20.10. APCI -MS (TOF): measured m/z 487.1578, calcd
for C24H27ClN4O3S [M+H]
+
m/z 487.1565 (Δ = 2.7 ppm).

2.2
2.10

46
Synthesis of BRI-13902

A 50 mL round bottomed flask was flame-dried and charged with 1-chloro-4-
(chloro(phenyl)methyl)benzene (1.0 eq., 1.00 g, 4.22 mmol) in 10 mL dimethyl formamide.
Sodium azide (2.0 eq., 548 mg, 8.43 mmol) was added to the solution at room temperature and
stirred for 10 h. Upon completion, the reaction mixture was extracted with Et2O (3x20 mL) and
dried over sodium sulfate. The crude mixture was obtained by rotary evaporation of the solvent
and subjected to purification by column chromatography (SiO2, 30% DCM in Hexanes) to obtain
the product (2.9) as a yellow oil (75%, 770 mg).
1
H NMR (400 MHz, Chloroform-d) δ 7.55 – 7.06
(m, 9H), 5.68 (s, 1H).

In a 10 mL reaction tube, 2.9 (1.0 eq., 770 mg, 3.16 mmol) and Br-ESF (3.0 eq., 1.79 g, 9.48
mmol) were suspended in 4 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 20 mL) and washed with brine. The organic
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
mixture. Subsequent purification by column chromatography (SiO2, 40% DCM in Hexanes)
yielded the triazole (2.10) as a yellow oil (81%, 1.17 g).
1
H NMR (400 MHz, Chloroform-d) δ
2.10
2.9
8 0
0 0
0 0
0
09
99
V2
00
0
-
11.
18
28
2.6
55
7
0.0
00
0 N
0 0
2.9
8 0
0 0
0 0
0
09
99
V2
00
0
-
11.
18
28
2.6
55
7
0.0
00
0 N
0 0
0 0
0 0
0 0
0 0
0 0
-
11.
89
73
2.2
43
2
0.0
00
0
R#
0 0
0 0
0 0
0 0
0 0
0 0
-

47
8.13 (s, 1H), 7.44 – 7.39 (m, 3H), 7.39 – 7.35 (m, 2H), 7.18 – 7.12 (m, 3H), 7.11 – 7.03 (m, 2H).
19
F NMR (376 MHz, Chloroform-d) δ 66.58.

In a 10 mL reaction tube, 2.10 (1.0 eq., 100 mg, 0.28 mmol) was dissolved in 1 mL acetonitrile
and 2-amino-2-phenylethan-1-ol (2.0 eq., 78 mg, 0.57 mmol) was added. DBU (2.0 eq., 87 mg,
0.57 mmol) was added and the reaction mixture was stirred at rt for 24 hours. After evaporation of
the solvent, the crude mixture was purified by column chromatography (SiO2, 5% → 10% MeOH
in DCM) to obtain the product (2.11, BRI 13902) as an off-white solid (15%, 19 mg).
1
H NMR
(600 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.53 – 7.19 (m, 14H), 3.57 – 3.52 (m, 1H), 3.47 (dd, J = 5.9
Hz, 1H), 3.32 (s, OH), 3.24 (dd, J = 6.5, 5.0 Hz, 1H). HR APCI -MS (TOF): measured
m/z 201.0467, calcd for C13H10Cl [M]
•+
m/z 201.0466 (Δ = 0.5 ppm).

Synthesis of BRI-13903

A 50 mL round bottomed flask was flame-dried and charged with 4-(bromomethyl)-1,1'-biphenyl
(1.0 eq., 1.00 g, 4.05 mmol) in 10 mL dimethyl formamide. Sodium azide (2.0 eq., 526 mg,
2.11
2.12
2.10

48
8.09 mmol) was added to the solution at room temperature and stirred for 24 h. Upon completion,
the reaction mixture was extracted with Et2O (3x20 mL) and dried over sodium sulfate. The crude
mixture was obtained by rotary evaporation of the solvent and purified by column chromatography
(SiO2, 20% DCM in Hexanes) to obtain the product (2.12) as a clear oil (67%, 758 mg).
1
H NMR
(400 MHz, Chloroform-d) δ 7.67 – 7.60 (m, 4H), 7.53 – 7.45 (m, 2H), 7.43 – 7.37 (m, 3H), 4.38
(s, 2H).

In a 10 mL reaction tube, 2.12 (1.0 eq., 300 mg, 1.34 mmol) and Br-ESF (3.0 eq., 762 mg, 4.03
mmol) were suspended in 2 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 20 mL) and washed with brine. The organic
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
mixture. Purification by column chromatography (SiO2, 50% DCM in Hexanes) yielded the
triazole (2.13) as a yellow oil (87%, 387 mg).
1
H NMR (400 MHz, Chloroform-d) δ 8.13 (s, 1H),
7.74 – 7.64 (m, 2H), 7.64 – 7.55 (m, 2H), 7.54 – 7.36 (m, 5H), 5.68 (s, 2H).
19
F NMR (376 MHz,
Chloroform-d) δ 66.33.

2.13
2.12
2.3
2.13

49
In a 10 mL reaction tube, 2.13 (1.0 eq., 100 mg, 0.32 mmol) was dissolved in 1 mL acetonitrile
and 4-(pyrrolidin-2-yl)pyrimidine (2.0 eq., 94 mg, 0.63 mmol) was added. Triethylamine (2.0 eq.,
64 mg, 0.63 mmol) was added and the reaction mixture was stirred at 80˚C for 24 hours. After
evaporation of the solvent, the crude mixture was purified by column chromatography (SiO2,
5% MeOH in DCM) to obtain the product (2.3, BRI 13903) as an off-white solid (49%, 68 mg).
1
H NMR (600 MHz, DMSO-d6) δ 9.12 (d, J = 1.4 Hz, 1H), 9.06 (s, 1H), 8.81 (d, J = 5.2 Hz, 1H),
7.73 – 7.69 (m, 2H), 7.68 – 7.64 (m, 3H), 7.49 – 7.44 (m, 4H), 7.39 – 7.35 (m, 1H), 5.76 (s, 2H),
4.86 (dd, J = 8.5, 4.3 Hz, 1H), 3.65 (ddd, J = 10.0, 7.1, 5.0 Hz, 1H), 3.51 – 3.41 (m, 1H), 2.13 –
2.02 (m, 1H), 1.95 – 1.88 (m, 1H), 1.85 – 1.76 (m, 1H), 1.69 – 1.61 (m, 1H).
13
C NMR (151 MHz,
DMSO-d6) δ 170.54, 158.53, 158.11, 144.37, 140.77, 139.91, 134.69, 129.51, 129.30, 129.09,
128.86, 128.67, 127.63, 127.17, 64.16, 53.70, 50.21, 33.62, 24.31. APCI -MS (TOF): measured
m/z 447.1610, calcd for C23H22N6O2S [M+H]
+
m/z 447.1598 (Δ = 2.7 ppm).

Synthesis of BRI-13904

Sodium borohydride (0.35 eq., 22 mg, 0.59 mmol) was added to a solution of 4-
(diethylamino)benzaldehyde (1.0 eq., 300 mg, 1.69 mmol) in 3 mL ethanol and stirred at room
temperature for 1.5 h. The reaction mixture was extracted with Et2O (3x10 mL) and the combined
organic layers were dried over sodium sulfate. After rotary evaporation of the solvent, the crude
mixture was purified by column chromatography (SiO2, 5% MeOH in DCM) to obtain the product
2.14

50
(2.14) as a white solid (99%, 299 mg).
1
H NMR (400 MHz, Chloroform-d) δ 7.24 – 7.19 (m, 2H),
6.69 – 6.65 (m, 2H), 4.55 (d, J = 5.7 Hz, 2H), 3.36 (q, J = 7.1 Hz, 4H), 1.16 (d, J = 7.1 Hz, 6H).

In a 50 mL round bottom flask, 2.14 (1.0 eq., 100 mg, 0.56 mmol) was dissolved in 3 mL DCM
and cooled down in an ice bath. At 0˚C, SO2Cl (10.0 eq., 663 mg, 5.58 mmol) was added slowly
and the reaction mixture was warmed to rt and stirred for 16 h. After rotary evaporation and
complete removal of leftover SO2Cl, the chlorinated intermediate was resuspended in DMF. NaN3
(2.5 eq., 91 mg, 1.39 mmol) was added to this solution and the reaction mixture was stirred for 4
h at room temperature. Subsequently, the mixture was extracted with Et2O, and purified by column
chromatography (SiO2, 20% EtOAc in Hexanes) to afford 2.15 as a yellow oil (75%, 258 mg).
1
H
NMR (400 MHz, Acetonitrile-d3) δ 7.17 – 7.05 (m, 2H), 6.70 – 6.62 (m, 2H), 4.16 (s, 2H), 3.32
(q, J = 7.0 Hz, 4H), 1.09 (t, J = 7.0 Hz, 6H).

In a 10 mL reaction tube, 2.15 (1.0 eq., 150 mg, 0.73 mmol) and Br-ESF (3.0 eq., 416 mg, 2.20
mmol) were suspended in 1 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 20 mL) and washed with brine. The organic
2.14
2.15
2.15
2.16

51
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
mixture. Purification by column chromatography (SiO2, 50% DCM in Hexanes → 60% DCM in
Hexanes) yielded the triazole (2.16) as a yellow oil (63%, 146 mg).
1
H NMR (400 MHz,
Chloroform-d) δ 7.97 (s, 1H), 7.14 – 7.05 (m, 2H), 6.67 – 6.48 (m, 2H), 5.39 (s, 2H), 3.28 (q, J =
7.3, 6.7 Hz, 4H), 1.08 (t, J = 7.1 Hz, 6H).
19
F NMR (376 MHz, Chloroform-d) δ 66.28.

In a 10 mL reaction tube, 2.16 (1.0 eq., 100 mg, 0.32 mmol) was dissolved in 1 mL acetonitrile,
and (1H-pyrazol-5-yl)methanamine (2.0 eq., 62 mg, 0.64 mmol) and DBU (2.0 eq., 97 mg, 0.64
mmol) were added. DMAP (0.2 eq., 7.82 mg, 0.064 mmol) was added and the reaction mixture
was stirred at rt for 3 h. After evaporation of the solvent, the crude mixture was purified by column
chromatography (SiO2, 2% MeOH in DCM) to obtain the product (2.17, BRI 13904) as an off-
white solid (49%, 68 mg).
1
H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.39 (d, J = 2.8 Hz, 1H),
7.31 – 7.16 (m, 2H), 6.71 – 6.47 (m, 3H), 5.49 (s, 2H), 3.67 (s, 2H), 3.29 (q, J = 7.0 Hz, 4H), 1.05
(t, J = 7.0 Hz, 6H).
13
C NMR (101 MHz, DMSO-d6) δ 161.83, 147.99, 143.43, 133.99, 130.40,
129.48, 120.76, 111.70, 109.38, 54.27, 44.01, 39.44, 12.74. APCI -MS (TOF): measured
m/z 390.1703, calcd for C17H23N7O2S [M+H]
+
m/z 390.1707 (Δ = 1.0 ppm).

2.16
2.17

52
Synthesis of BRI-13905

In a 50 mL round bottom flask, (4-benzylphenyl)methanol (1.0 eq., 250 mg, 1.26 mmol) was
dissolved in 4 mL toluene. DPPA (1.2 eq., 416 mg, 1.51 mmol) and DBU (1.5 eq., 288 mg, 1.89
mmol) were added and the reaction was stirred at room temperature for 3 h. After extraction with
EtAOc (3x 20 mL) and rotary evaporation of the solvent, the crude mixture was purified by column
chromatography (SiO2, 5% EtOAc in Hex) to obtain the product (2.18) as an off-white solid (71%,
201 mg).
1
H NMR (400 MHz, Chloroform-d) δ 7.39 – 6.85 (m, 8H), 4.29 (s, 2H), 3.99 (s, 2H).

In a 10 mL reaction tube, 2.18 (1.0 eq., 200 mg, 0.89 mmol) and Br-ESF (3.0 eq., 508 mg, 2.69
mmol) were suspended in 2 mL dimethyl formamide and stirred at 80˚C for 4 h. Subsequently, the
reaction mixture was extracted with DCM (3x 20 mL) and washed with brine. The organic layer
was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude mixture.
Purification by column chromatography (SiO2, 50% DCM in Hexanes) yielded the triazole (2.19)
as a yellow oil (96%, 284 mg).
1
H NMR (400 MHz, Methanol-d4) δ 8.99 (s, 1H), 7.34 – 7.29 (m,
2H), 7.28 – 7.21 (m, 4H), 7.19 – 7.13 (m, 3H), 5.67 (s, 2H), 3.96 (s, 2H).
19
F NMR (376 MHz,
Methanol-d4) δ 64.49.

2.18
2.18 2.19

53

In a 10 mL reaction tube, 2.19 (1.0 eq., 50 mg, 0.15 mmol) was dissolved in 1 mL acetonitrile, and
(1H-pyrazol-5-yl)methanamine (2.0 eq., 29 mg, 0.30 mmol) and DBU (2.0 eq., 45 mg, 0.30 mmol)
were added. DMAP (0.2 eq., 3.69 mg, 0.030 mmol) was added and the reaction mixture was stirred
at rt for 4 h. After evaporation of the solvent, the crude mixture was purified by column
chromatography (SiO2, 10% MeOH in DCM) to obtain the product (2.20, BRI 13905) as an off-
white solid (38%, 47 mg).
1
H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.34 (d, J = 2.8 Hz, 1H),
7.41 – 7.11 (m, 9H), 6.60 (d, J = 2.8 Hz, 1H), 5.60 (s, 2H), 3.89 (s, 2H), 3.60 (s, 2H).
13
C NMR
(101 MHz, DMSO-d6) δ 162.48, 143.53, 142.32, 141.33, 133.97, 132.77, 130.07, 129.63, 129.12,
128.94, 128.88, 126.47, 109.39, 54.01, 41.12, 39.65. APCI -MS (TOF): measured m/z 409.1459,
calcd for C20H20N6O2S [M+H]
+
m/z 409.1441 (Δ = 4.4 ppm).

Synthesis of BRI-13906

In a 50 mL round bottom flask, 3-(methoxymethyl)aniline (1.0 eq., 1.11 g, 8.12 mmol) was
dissolved in 5 mL 5 M HCl. A solution of sodium nitrite (1.5 eq., 840 mg, 12.18 mmol) in 20 mL
H2O was added dropwise at 0˚C and stirred at room temperature for 20 h. Upon completion, the
reaction mixture was quenched with NaHCO3 and extracted with EtOAC (3x 100 mL). After
evaporation of the solvent, the crude mixture was purified by column chromatography (SiO2,
2.19
2.20
2.21

54
10% EtOAc in Hexanes) to obtain the product (2.21) as a yellow oil (99%, 1.32 g).
1
H NMR (400
MHz, Chloroform-d) δ 7.39 – 7.30 (m, 1H), 7.14 – 7.08 (m, 1H), 7.08 – 7.02 (m, 1H), 6.99 – 6.93
(m, 1H), 4.45 (s, 2H), 3.40 (s, 3H).

In a 10 mL reaction tube, 2.21 (1.0 eq., 500 mg, 3.06 mmol) and Br-ESF (3.0 eq., 1.74 g, 9.19
mmol) were suspended in 5 mL dimethyl formamide and stirred at 80˚C for 24 h. Subsequently,
the reaction mixture was extracted with DCM (3x 50 mL) and washed with brine. The organic
layer was dried over sodium sulfate, and rotary evaporation of the solvent yielded the crude
mixture. Purification by column chromatography (SiO2, 10% EtOAc in Hexanes → 50% EtOAc
in Hexanes) yielded the triazole (2.22) as a yellow oil (59%, 520 mg).
1
H NMR (400 MHz,
Chloroform-d) δ 8.72 (s, 1H), 7.77 – 7.74 (m, 1H), 7.71 – 7.67 (m, 1H), 7.60 – 7.55 (m, 1H), 7.52
– 7.49 (m, 1H), 4.56 (s, 2H), 3.46 (s, 3H).
19
F NMR (376 MHz, Chloroform-d) δ 66.64.

In a 10 mL reaction tube, 2.22 (1.0 eq., 100 mg, 0.35 mmol) was dissolved in 1 mL acetonitrile,
and (4-chloro-6-(piperazin-1-yl)pyrimidine (2.0 eq., 139 mg, 0.70 mmol) and DBU (2.0 eq., 106
mg, 0.70 mmol) were added. DMAP (0.2 eq., 8.56 mg, 0.07 mmol) was added and the reaction
mixture was stirred at rt for 4 h. After evaporation of the solvent, the crude mixture was purified
2.21
2.22
2.22 2.23

55
by column chromatography (SiO2, 5% MeOH in DCM) to obtain the product (2.23, BRI 13906)
as an off-white solid (56%, 90 mg).
1
H NMR (600 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.31 (s, 1H),
7.93 – 7.91 (m, 1H), 7.90 – 7.86 (m, 1H), 7.61 – 7.56 (m, 1H), 7.50 – 7.46 (m, 1H), 6.96 (s, 1H),
4.51 (s, 2H), 3.83 – 3.77 (m, 4H), 3.33 (s, 3H), 3.24 – 3.19 (m, 4H).
13
C NMR (151 MHz, DMSO-
d6) δ 162.37, 159.67, 158.34, 143.96, 141.03, 136.38, 130.25, 128.78, 126.89, 120.28, 120.12,
119.96, 102.50, 73.20, 58.21, 45.65. APCI -MS (TOF): measured m/z 450.1129, calcd for
C18H20ClN7O3S [M+H]
+
m/z 450.1110 (Δ = 4.2 ppm).

Synthesis of BRI-13907

A 50 mL dry round bottom flask was charged with 3-methoxy-2-napthaldehyde (1.0 equiv., 500
mg, 2.68 mmol) and was dissolved in 5 mL ethanol. Triethylamine (2.0 equiv., 750 µL, 5.37 mmol)
and hydroxylamine.HCl (2.0 equiv., 373 mg, 5.37 mmol) were disdsolved in 5 mL water and added
dropwise to aldehyde solution. The reaction was stirred at r.t. for 5 h and extracted with EtOAc
(3X10) to obtain 2.24 as an off-white solid (85%, 425 mg).
1
H NMR (400 MHz, CDCl3): δ 8.60
(s, 1H), 8.17 (d, J = 2.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.47 (ddt, J =
8.3, 6.8, 1.6 Hz, 1H), 7.35 (ddt, J = 8.1, 6.9, 1.1 Hz, 1H), 7.16 (s, 1H), 3.99 (s, 3H).

2.24
2.24 2.25

56
A 100 mL dry round bottom flask was charged with 2.24 (1.0 equiv., 337 mg, 1.67 mmol) and was
dissolved in 22 mL DMF. The flask was covered with aluminuum foil and N-Chlorosuccinimide
(1.05 equiv., 235 mg, 1.75 mmol) was added portionwise over 30 min. The reaction mixture was
stirred at r.t. for 48 h and then extracted with diethyl ether (20 mL). The organic layer was extracted
with water (10 mL x 3) and brine (10mL). The organic layer was concentrated in vacuo to obtain
the desired product (2.25) as a pale yellow solid (99%, 390 mg).
1
H NMR (400 MHz, DMSO-d6)
δ 11.65 (s, 1H), 8.36 (s, 1H), 8.31 (s, 1H), 8.14 (dq, J = 8.5, 0.9 Hz, 1H), 8.09 – 8.05 (m, 1H), 7.71
– 7.66 (m, 1H), 7.58 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 3.88 (s, 3H).

A 25 mL dry round bottom flask was charged with 2.25 (1.0 equiv., 150 mg, 1.67 mmol) and was
dissolved in 3 mL tert-butanol and DIPEA (2.5 equiv., 277 µL, 1.59 mmol) was added. The
reaction mixture was vigorously stirred. Alongside, 1-bromoethene-1-sulfonyl fluoride (4 equiv.,
481mg, 2.55mmol) was dissolved in 3 mL tert-butanol and was added dropwise to the reaction
mixture over a period of 20 min. The solution ws stirred for 2 h. The reaction mixture was washed
with water (10 mL). The aqeoeus layer was washed with DCM (3 x 10mL) and finally the organic
layer was washed with brine (10 mL). The organic layer was dried The residue was purified though
silica gel chromatography with 0% to 3% ethyl acetate in hexanes to obtain the desired product
(2.26) as an off white white solid (18%, 27 mg)
1
H NMR (600 MHz, cdcl3) δ 8.36 (d, J = 3.0 Hz,
1H), 8.29 (dd, J = 8.5, 1.0 Hz, 1H), 7.95 – 7.92 (m, 1H), 7.75 (d, J = 1.3 Hz, 1H), 7.71 (ddd, J =
2.25
2.26

57
8.4, 6.9, 1.3 Hz, 1H), 7.58 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 3.90 (s, 3H).
19
F NMR (376 MHz,
CDCl3) δ 64.70.

In a 10 mL reaction tube, 2.26 (1.0 eq., 20 mg, 0.06 mmol) was dissolved in 0.5 mL acetonitrile
and piperazin-1-yl(1H-pyrrol-2-yl)methanone (3.0 eq., 31 mg, 0.18 mmol) was added.
Triethylamine (2.0 eq., 12 mg, 0.12 mmol) was added and the reaction mixture was stirred at 80˚C
for 6 hours. After evaporation of the solvent, the crude mixture was purified by column
chromatography (SiO2, 5% MeOH in DCM) to obtain the product (2.4, BRI 13907) as an off-
white solid (49%, 68 mg).
1
H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 8.52 (s, 1H), 8.39 –
8.18 (m, 1H), 8.18 – 8.07 (m, 1H), 7.83 – 7.77 (m, 1H), 7.72 (s, 1H), 7.71 – 7.64 (m, 1H), 7.06 –
6.78 (m, 1H), 6.73 – 6.52 (m, 1H), 6.26 – 5.92 (m, 1H), 3.94 – 3.73 (m, 7H), 3.40 – 3.35 (m, 4H).
13
C NMR (101 MHz, DMSO-d6) δ 163.77, 162.10, 160.58, 151.45, 132.24, 130.76, 130.16,
129.92, 129.59, 127.37, 124.17, 124.07, 123.77, 122.03, 121.88, 112.74, 109.93, 108.93, 61.88,
46.17, 29.4 (HSQC). APCI-MS (TOF): measured m/z 501.1005, calcd for C23H23ClN4O5S
Subtract [M+H]
+
m/z 501.0994 (Δ = 1.8 ppm).

Synthesis of BRI-13910

2.26
2.4
2.27

58
A 50 mL round bottom flask was charged with hydroxylamine hydrochloride (1.2 eq., 148 mg,
2.1 mmol) and sodium carbonate (1.2 eq., 226 mg, 2.1 mmol) and dissolved in 2.5 mL of a 5:1
water:ethanol mixture and the solution was mixed for 15 min. The aldehyde (1.0 eq., 500 mg,
1.8 mmol) was added portionwise over 5 minutes, 10 mL more of EtOH were added and the
reaction was stirred for 45 min. Reaction progress was monitored via LC-DAD. After 72 h the
reaction was complete. Product was extracted in EtOAc (3 × 10 mL), washed with water
(3 × 10 mL) and brine (2 × 10 mL), and dried over sodium sulfate. Solvents were evaporated under
vacuum and the product, initially an oil, was cooled crashed out with hexanes to afford white solid.
Solvent was removed under vacuum, yielding the product (2.27) as a white solid in 94% (504 mg).
Purity was confirmed by LC-UV-APCI-MS.
1
H NMR (499.8 MHz, DMSO-d6; δ, ppm): 11.25 (s,
1H), 8.10 (s, 1H), 7.73 (s, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.32 (t,
J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 5.14 (s, 2H).
13
C
NMR (125.7 MHz, DMSO-d6; δ, ppm): 158.1, 147.9, 138.2, 134.5, 131.1, 130.7, 130.4, 129.9,
129.4, 127.8, 119.5, 115.9, 112.2, 67.6. APCI-MS (TOF): exact for C14H12Cl2NO2 [M + H]
+

m/z 296.0240, accurate m/z 296.0242 (Δ = 0.7 ppm).

In a 20 mL scintillation vial, the oxime (1.0 eq., 496 mg, 1.7 mmol) was dissolved in 2 mL DMF
and placed in a 15 °C water bath with continuous stirring. NCS (1.05 eq, 235 mg, 1.8 mmol) was
added portionwise over 30 minutes to the stirring reaction. The mixture was stirred in the dark at
15 °C. Reaction progress was monitored via LC-DAD. After 2 h the reaction was complete. The
2.27 2.28

59
reaction was quenched with 2 mL of water and the product was extracted to DCM (3 × 10 mL).
The combined organic layer was washed with a 5:1 brine:water mixture (3 × 10 mL), and brine (1
× 10 mL), and dried over sodium sulfate. Solvents were evaporated under vacuum to yield the
product (2.28) as a light yellow solid in 98% (545 mg). Purity was confirmed by LC-UV-APCI-
MS.
1
H NMR (499.8 MHz, DMSO-d6; δ, ppm): 12.42 (s, 1H), 7.75 (s, 1H), 7.67 (d, J = 8.3 Hz,
1H), 7.59 – 7.34 (m, 4H), 7.25 – 7.12 (m, 1H), 5.18 (s, 2H).
13
C NMR (125.7 MHz, DMSO-d6;
δ, ppm): 158.0, 138.0, 135.1, 134.0, 131.1, 130.7, 130.4, 130.0, 129.5, 127.8, 119.5, 116.9, 112.8,
67.8. APCI-MS (TOF): exact for C14H11Cl3NO2 [M + H]
+
m/z 329.9850, accurate m/z 329.9844
(Δ = 1.8 ppm).

In a 20 mL scintillation vial, the chloro-oxime (1.0 eq., 331 mg, 1 mmol) were dissolved in 10 mL
of DCM. Br-ESF (2.0 eq., 378 mg, 2 mmol) was added and the mixture was stirred at r.t. for 5
min. While the mixture was stirring, triethylamine (2.0 eq., 202 mg, 2.0 mmol) was slowly added
dropwise over 1 min (white fume formed immediately above the solution and the solution
gradually changed color to dark orange). After 2 h of stirring, the reaction was quenched with 10
mL of water. The product was extracted to DCM (3 x 10 mL), washed with water (3 × 10 mL) and
brine (2 × 10 mL), and dried over sodium sulfate. The solution was passed through a short pad of
silica and solvents were evaporated under vacuum, until the product (2.29) crystalized as beige
crystals in a yield of 78% (316 mg). Purity was confirmed by LC-UV-APCI-MS.
1
H NMR (499.8
MHz, DMSO-d6; δ, ppm): 8.71 (s, 1H), 7.76 (s, 1H), 7.69 – 7.65 (m, 2H), 7.64 – 7.60 (m, 1H),
2.28 2.29

60
7.52 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 8.3 Hz, 1H), 5.22 (s, 2H)
13
C NMR
(125.7 MHz, DMSO-d6; δ, ppm): 163.3, 158.6, 137.9, 131.7, 131.2, 130.8, 130.7, 130.5, 129.6,
127.9, 127.3, 119.8, 118.3, 113.4, 112.8 (d, J = 3.5 Hz), 67.9.
19
F NMR (470.3 MHz, DMSO-d6;
δ, ppm): 65.5. APCI-MS (TOF): exact for C16H11Cl2FNO4S [M + H]
+
m/z 401.9764, accurate
m/z 401.9771 (Δ = 1.7 ppm).

In a 20 mL scintillation vial, sulfonyl fluoride (1.0 eq., 201 mg, 0.5 mmol) was dissolved in 5 mL
MeCN, and methyl(1H-pyrazol-3-yl)methylamine (2.0 eq., 111 mg, 1 mmol) and DBU (2.0 eq.,
152 mg, 1 mmol) were added. DMAP (0.2 eq., 12 mg, 0.1 mmol) was added and the reaction
mixture was stirred at r.t. for 3 h. Solvent was evaporated until 3 mL total volume and product was
purified by MS-guided HPLC in MeCN/H2O/formic acid system. Removal of solvent resulted in
formate salt of the product (2.30, BRI 13910) as an off-white solid in a yield of 8% (22 mg). Purity
was confirmed by LC-UV-APCI-MS.
1
H NMR (499.8 MHz, DMSO-d6; δ, ppm): 8.61 (s, 1H),
8.39 (s, 1H), 8.22 (br s, 3H), 7.75 (s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.62 (s, 1H), 7.57 (d, J = 7.7
Hz, 1H), 7.50 – 7.45 (m, 2H), 7.23 (d, J = 8.2 Hz, 1H), 6.81 (s, 1H), 5.21 (s, 2H), 3.87 (s, 2H),
2.33 (s, 3H).
13
C NMR (125.7 MHz, DMSO-d6; δ, ppm): 163.9, 163.0, 161.9, 158.5, 157.7, 138.0,
134.8, 131.2, 130.8, 130.7, 130.5, 129.5, 127.9, 127.5, 119.7, 118.2, 113.3, 111.2, 110.2, 67.9,
46.4, 34.1. APCI-MS (TOF): exact for C21H19Cl2N4O4S [M + H]
+
m/z 493.0499, accurate
m/z 493.0501 (Δ = 0.4 ppm).
2.29
2.30

61
Synthesis of BRI-13911

A round bottom flask (50 mL) was charged with hydroxylamine hydrochloride (1.2 eq., 80 mg
1.15 mmol) and sodium carbonate (1.2 eq., 122 mg, 1.15 mmol) and dissolved in 6 mL of a 5:1
water: ethanol mixture and the solution was mixed for 15 min. The aldehyde (1.0 eq., 250 mg, 961
mmol) was added portion wise over 5 minutes. Reaction progress was monitored via TLC in 3:1
hexanes: EtOAc. After 120 h the reaction was complete. Product was extracted in EtOAc (3 × 10
mL), washed with water (3 × 10 mL) and brine (2 × 10 mL), and dried over sodium sulfate.
Solvents were evaporated under vacuum and the remaining solvent was removed under vacuum
and product dried overnight to yield the product (2.31) as an off-white solid in 98% (259 mg).
1
H
NMR (600 MHz, cdcl3) δ 9.83 (s, 1H), 8.04 (s, 1H), 7.44 – 7.40 (m, 2H), 7.10 – 7.02 (m, 4H), 6.88
(d, J = 8.3 Hz, 1H), 5.11 (s, 2H), 3.90 (s, 3H).
19
F NMR (564 MHz, cdcl3) δ -113.74 (ddd, J = 13.8,
8.9, 5.3 Hz), -114.27 (ddd, J = 14.0, 8.9, 5.4 Hz).

In a 50 mL round bottom flask oxime (1.0 eq., 254 mg, 923 mmol) was dissolved in 10 mL DMF
and placed in a 15 °C water bath covered in foil with continuous stirring. NCS (1.05 eq., 129 mg,
969 mmol) was added portion wise over 30 minutes to the stirring reaction. The mixture was stirred
in the dark at 15 °C. Reaction progress was monitored via TLC in 3:1 hexanes: EtOAc. After 24 h
2.31
2.32
2.31

62
13 mg of NCS was added to push the reaction to completion. After an additional 5 h the reaction
was complete. The reaction was quenched with 5 mL of water and the product was extracted to
ether (3 × 10 mL). The combined organic layer was washed with a 5:1 brine: water mixture (3 ×
10 mL), and brine (1 × 10 mL), and dried over sodium sulfate. Solvents were evaporated under
vacuum and the product was left on a vacuum line overnight to yield the product (2.32) as an off-
white solid in 99% (282 mg).
1
H NMR (600 MHz, cdcl3) δ 7.47 – 7.40 (m, 4H), 7.08 – 7.04 (m,
3H), 6.93 – 6.89 (m, 1H), 5.12 (s, 2H), 3.91 (s, 3H).
19
F NMR (564 MHz, cdcl3) δ -113.79 (td, J =
8.7, 4.3 Hz), -114.16 (ddd, J = 14.1, 8.8, 5.4 Hz).

In a 20 mL scintillation vial chloro-oxime (1.0 eq., 310 mg, 1 mmol) was dissolved in 20 mL of
DCM. Br-ESF (2.0 eq., 378 mg, 2 mmol) was added and the mixture was stirred at r.t. for 5 min.
While the mixture was stirring, triethylamine (2.0 eq., 202 mg, 2 mmol) was slowly added
dropwise over 1 min (white fumes formed immediately above the solution and the solution
gradually changed color). After 4 h of stirring, the reaction was complete and quenched with 10
mL of water. The product was extracted to DCM 3×10 mL, washed with water (3 × 10 mL) and
brine (2 × 10 mL), dried over sodium sulfate. Solution was passed through a short pad of silica
and solvents were evaporated under vacuum to yield the product (2.33) as an off-white solid in
62% (236 mg).
1
H NMR (600 MHz, cdcl3) δ 7.48 – 7.42 (m, 3H), 7.37 (d, J = 1.3 Hz, 1H), 7.33
(dd, J = 8.3, 2.1 Hz, 1H), 7.11 – 7.05 (m, 2H), 6.99 (d, J = 8.4 Hz, 3H).
19
F NMR (564 MHz, cdcl3)
δ 64.29 (d, J = 1.5 Hz), -113.88 (ddd, J = 14.0, 8.6, 5.3 Hz).
2.33
2.32

63

In a 10 mL reaction tube, the isoxazole (1.0 eq., 130 mg, 341 mmol) was dissolved in 0.5 mL
DMSO. Subsequently, the amine (2.0 eq., 29 mg 284 mmol), HOBt (1 mol%), 1,1,3,3-
tetramethyldisiloxane (2.0 eq., 76 mg, 568 mmol), and DIPEA (2.0 eq., 73 mg, 568 mmol) were
added and the reaction was stirred at rt and the reaction was monitored by LCMS. After 24 h the
reaction was complete and diluted with 10 mL of EtOAc. The product was washed with water (2
x 10 mL) and 1M HCl (1 x 1 mL) then brine (1 x 10 mL) After rotary evaporation of the solvent,
the crude mixture was purified by Semi-prep to obtain the final product (2.5, BRI 13911) as an
off-white solid in 10% (13 mg).
1
H NMR (600 MHz, cd2cl2) δ 7.61 – 7.57 (m, 3H), 7.50 (dd, J =
8.3, 2.1 Hz, 1H), 7.25 – 7.20 (m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 5.45 – 5.44 (m, 1H), 5.39 (d, J =
6.7 Hz, 1H), 5.23 (s, 2H), 4.20 – 4.15 (m, 1H), 4.03 (s, 3H), 3.67 (tt, J = 7.9, 6.3 Hz, 1H), 2.22
(dtd, J = 13.1, 8.2, 4.8 Hz, 1H), 2.16 – 2.11 (m, 1H), 1.88 – 1.77 (m, 2H), 1.69 (ddt, J = 13.1, 9.5,
6.5 Hz, 1H), 1.60 (ddt, J = 13.4, 9.2, 7.9 Hz, 1H).
13
C NMR (151 MHz, cd2cl2) δ 166.09, 163.40,
162.30, 161.77, 152.16, 148.53, 132.55 (d, J = 2.9 Hz), 129.74, 129.69, 120.91, 119.72, 115.43,
115.29, 111.79 (d, J = 2.6 Hz), 106.00, 77.98, 70.43, 62.46, 55.89, 31.58, 30.30, 19.89.
19
F NMR
(564 MHz, cd2cl2) δ -114.70 (tt, J = 8.9, 5.4 Hz).

2.5
2.33

64
Synthesis of BRI-13912

In a 50 mL round bottom flask, dibenzo[b,d]thiophene-4-carbaldehyde (1.0 eq., 500 mg, 2.36
mmol) was dissolved in 8 mL ethanol. Potassium carbonate (1.2 eq., 390 mg, 2.83 mmol) and
hydroxylamine (2.0 eq., 327 mg, 4.71 mmol) were dissolved in 2 mL water and added dropwise
to the aldehyde solution. After stirring at room temperature for 24 h, the reaction was quenched
with cold water and dilute hydrochloric acid was added until the solution was brought down to a
pH of 6-7. After extraction to dichloromethane (3x 10), the combined organic layers were washed
with brine and dried with sodium sulfate to obtain the product (2.34) as an off-white solid (92%,
490 mg) that was used for chlorination without further purification.
1
H NMR (600 MHz,
Chloroform-d) δ 8.47 (s, 1H), 8.27 – 8.22 (m, 1H), 8.22 – 8.19 (m, 1H), 7.94 – 7.90 (m, 1H), 7.56
– 7.53 (m, 2H), 7.51 – 7.48 (m, 2H)

In a 50 mL round bottom flask, 2.34 (1.0 eq., 200 mg, 0.49 mmol) was dissolved in 6 mL DMF.
NCS (1.1 eq., 109 mg, 1.07 mmol) was added at 0˚C and the reaction was warmed up to room
temperature and stirred at room temperature for 5 h. The resulting mixture was extracted with
DCM (3x 10) and washed with water to obtain the product (2.35) as a yellow solid (86%, 101 mg).
2.34
2.34
2.35

65
1
H NMR (600 MHz, Chloroform-d) δ 8.23 (apparent dt, J = 7.8, 1.2 Hz, 1H), 8.18 – 8.15 (m, 1H),
8.08 (apparent dt, J = 7.7, 1.2 Hz, 1H), 7.58 – 7.54 (m, 1H), 7.48 – 7.45 (m, 2H).

In a 20 mL scintillation vial, 2.35 (1.0 eq., 70 mg, 0.27 mmol) was dissolved in 7 mL DCM and
cooled to 0˚C. NEt3 (1.0 eq., 19 mg, 0.19 mmol) was added and the solution was stirred for 20
minutes. Br-ESF (2.0 eq., 72 mg, 0.38 mmol) was added dropwise at 0˚C and the solution was
warmed to rt and stirred. After 1 hour, another equivalent of NEt3 was added (1.0 eq., 19 mg, 0.19
mmol), and the reaction was stirred for 20 hours. After rotary evaporation of the solvent, the crude
mixture was purified by column chromatography (SiO2, 25% DCM in Hexanes) to obtain the
product (2.36) as a white crystalline solid (12%, 7.3 mg).
1
H NMR (600 MHz, Chloroform-d) δ
8.39 – 8.34 (m, 1H), 8.29 – 8.20 (m, 1H), 7.98 – 7.92 (m, 1H), 7.87 – 7.80 (m, 1H), 7.72 (s, 1H),
7.65 (t, J = 7.8 Hz, 1H), 7.55 (ddd, J = 6.7, 4.8, 1.6 Hz, 2H).
19
F NMR (564 MHz, Chloroform-d)
δ 64.74.

In a 10 mL reaction tube, 2.36 (1.2 eq., 7.3 mg, 0.02 mmol) was dissolved in 0.5 mL DMSO.
Subsequently, 4-(pyrrolidin-2-yl)pyrimidine (1.0 eq., 2.7 mg, 0.018 mmol), HOBt (1 mol%),
1,1,3,3-tetramethyldisiloxane (2.0 eq., 4.9 mg, 0.036 mmol), and DIPEA (2.0 eq., 4.7 mg, 0.036
2.35
2.36
2.36
2.6

66
mmol) were added and the reaction was stirred at rt for 24 hours. After rotary evaporation of the
solvent, the crude mixture was purified by column chromatography (SiO2, DCM → 5% MeOH in
DCM) to obtain the product (2.6, BRI 13912) as an off-white solid (12%, 7.3 mg).
1
H NMR (600
MHz, Chloroform-d) δ 9.11 (d, J = 1.4 Hz, 1H), 8.75 (d, J = 5.1 Hz, 1H), 8.39 (dd, J = 7.9, 1.1 Hz,
1H), 8.33 – 8.25 (m, 1H), 8.08 – 7.93 (m, 1H), 7.87 (dd, J = 7.5, 1.1 Hz, 1H), 7.67 (t, J = 7.7 Hz,
1H), 7.62 – 7.51 (m, 3H), 7.38 (s, 1H), 5.03 (dd, J = 8.5, 3.8 Hz, 1H), 3.94 – 3.79 (m, 1H), 3.79 –
3.62 (m, 1H), 2.41 – 2.24 (m, 1H), 2.24 – 2.09 (m, 1H), 2.09 – 1.98 (m, 1H), 1.98 – 1.87 (m, 1H).
13
C NMR (151 MHz, Methylene Chloride-d2) δ 169.20, 165.05, 161.73, 158.50, 157.55, 140.41,
137.40, 137.21, 134.62, 127.43, 126.85, 124.80, 124.69, 123.95, 122.68, 121.84, 121.69, 118.40,
107.14, 64.32, 50.13, 33.55, 24.23.

2.9.3 Characterization

67
BRI-13900

68
BRI-13901

69

BRI-13902

70
BRI-13903

71
BRI-13904

72
BRI-13905

73
BRI-13906

74
2.9 Distribution of Credit

Chapter 2 is a result of a collaborative effort between the Katritch group, namely Prof. Katritch,
Dr. Saheem Zaiidi and Dr. Anastasiia Sadybekov, and the Fokin Group, namely Prof. Fokin,
Katharina Grotsch (myself), Sydney Hiller and Dr. Dmitry Eremin. Specific contributions are as
follows. The majority of the text herein was written by the author (Katharina Grotsch). Section 2.2
was adapted from a procedure written by Dr. Saheem Zaiidi, and sections 2.3 and 2.4 were adapted
from Dr. Anastasiia Sadybekov and used with her permission. Synthetic contributions were made
by Sydney Hiller (BRI 13911), Dr. Dmitry Eremin (BRI 13910), and Shubanghi Aggarwal (BRI
13107).

75
Chapter 3. Making Lemonade out of Lemons: The Unmet Potential of
Cannabinoids and the Endocannabinoid System

3.1 Introduction
As one of the oldest plants cultivated by man, cannabis has played an important role in many
ancient civilizations ranging all the way from China, to India, and the Middle East.
61-64
The world’s
oldest pharmacopoeia, the pen-ts’ao ching, reported its use for rheumatic pain, constipation, and
disorders of the female reproductive system in as early as 2,700 B.C.
65
The use of cannabis for
mind-altering and medicinal purposes was explored by the Assyrians around the second
millennium B.C., where it was referred to as ganzi-gun-nu (“the drug that takes away the mind”),
and illustrated a central theme in Arab poetry of the middle ages.
66, 67
In Europe, Cannabis was
introduced by Napoleonic soldiers returning from Egypt and British soldiers returning from
India.
61
Famous intellectuals of the era described the “groundless gaiety” and “distortion of colors
and sounds”, as well as dissociation of ideas, errors of time and space, and fluctuation of emotions,
associated with smoking cannabis.
68
However, the inception of a rampant political movement that
originated at the beginning of the 20
th
century led to prohibition of cannabis throughout western
civilization.
69
Concurrently, regional medical practices became reliant on a heavily regulated
system comprised mainly of single-molecule therapeutics, creating the highly competitive drug
marketplace we know today.
69

In the mid-20
th
century, a multitude of scientific discoveries shed light on the quintessential role
of the endogenous cannabinoid system (ECS) in maintaining homeostasis in the human body.
35, 70-
75
It is now known that the ECS is responsible for regulating sleep, appetite, stress, and memory

76
among other things.
62
Unsurprisingly, it is an attractive target for the cure of various diseases,
especially of the central nervous system (CNS).
74, 76-78
As with opium poppies before, the study of
an active component in cannabis has shed light on an endogenous system that controls various
neurobiological functions, indicating significant promise for the development of novel
pharmaceuticals.
79, 80
Yet, relatively little progress has been made on exploring cannabinoids as
therapeutic agents – despite their well-established safety profile. Is this a result of lacking scientific
promise? Or is it simply a result of the multitude of social, political, economic, and technological
developments that have shaped the world as it is today?

Here, we cursorily outline the role of the endocannabinoid system in regulating physiological
functions to underline its importance, and summarize the biological activity of known
phytocannabinoids. With this background in mind, we attempt to understand to what extent the
convoluted interplay of government regulations, economic developments, and shifts in the
sociopolitical climate have influenced scientific progress. In doing so, we aim to highlight this
underdeveloped area of research, and propose a new outlook that amalgamates modern science
with the empirical knowledge gathered over centuries, challenging the field of drug discovery as
a whole.

3.2 The Endocannabinoid System
The endogenous cannabinoid system (ECS) in its most rudimentary form is comprised of (a) the
cannabinoid type I (CB1) and cannabinoid type II (CB2) cannabinoid receptors, (b)
arachidonoylethanolamide (anandamide or AEA) and 2-arachidonyl glycerol (2-AG) as
endogenous ligands, and (c) the enzymes involved in cannabinoid synthesis and degradation.
81, 82

77
Its nomenclature is derived from the finding that various endocannabinoids and constituents of
cannabis sativa act on the same receptor targets.
61
In essence, the ECS provides protection against
inflammatory and neuropathic stress, making it an attractive target for the treatment of chronic
stress of the brain and body as a whole.
83
Given the dearth of effective medications for both chronic
inflammation and neurological stress, there is a clear need for the development of new therapeutics
for treating these conditions. For the purposes of this review, we will be focusing mainly on the
endogenous cannabinoid system in the CNS. Importantly, alterations in the ECS are found in
patients with most neurological diseases, outlining the critical role it plays and endorsing it as an
important target for the development of new therapeutic agents for various CNS diseases including
Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), multiple
sclerosis (MS), epilepsy, generalized anxiety disorder (GAD), obsessive compulsive disorder
(OCD), social anxiety disorder (SAD), and post-traumatic stress disorder (PTSD).
84-87

3.2.1 Cannabinoid Receptors
The endogenous cannabinoid system consists of so far two identified G-protein coupled receptors
(GPCRs), CB1 and CB2 that were named after their affinity for the agonist ∆
9
–THC.
32, 33
Both CB1
and CB2 are coupled through the Gi/o family of proteins, and are expressed both in the CNS and
the immune system.
33, 88, 89

CB1 was first cloned by Tom Bonner’s lab in 1990.
73
Autoradiographic studies have shown that
CB1 can be found mainly in the cerebral cortex, hippocampus, basal ganglia, and cerebellum –
regions that are consistent with the known effects of cannabinoids on motivation and cognition.
73,
88, 90
Indeed, the physiological responses generally associated with ∆
9
–THC consumption such as

78
reduced stress, increased appetite, and euphoria, are generally attributed to activation of CB1
receptors.
91
In essence, these effects can be explained by the expression of CB1 at GABAergic,
glutamatergic, serotonergic, noradrenergic, and dopaminergic terminals, where they are localized
at axons and axon terminals of presynaptic neurons, and act to suppress the release of these
neurotransmitters, thereby controlling synaptic transmission and neuronal excitability.
74, 92-94

However, precise mechanisms of action are still subject to heavy debate.

The CB2 receptor was first cloned in 1993 at the MRC Laboratory of Molecular Biology in
Cambridge, England, and has a 44% sequence identity with CB1.
32, 35
Immunocytochemical
evidence has identified the presence of CB2 in spleen, thymus, tonsils, bone marrow, pancreas,
mast cells, peripheral blood leukocytes, and several cultured immune cell models.
33, 95
Although
CB2 is expressed mainly in the immune system, it is also present in the CNS, where it has been
shown to control synaptic function and regulate synaptic plasticity, making it highly relevant target
for many neurological disorders.
96, 97
It should be noted that the high sequence identity between
CB1 and CB2 makes it difficult to selectively target one of these receptors over the other.

3.2.2 Endocannabinoids
By inference, the presence of cannabinoid receptors indicates the existence of endogenous
molecules that have the ability to modulate those receptors. These effects are mainly attributed to
the two eicosanoids, AEA and 2-AG (Figure 3.1a).
98-101
The endocannabinoids (eCBs) are
lipophilic and, unlike most neurotransmitters, they are not stored in vesicles but rather synthesized
“on demand” from membrane phospholipids as a result of increased intracellular Ca
2+
levels at the
post-synaptic site.
96, 102
Unlike most neurotransmitters, their action is generally presynaptic rather

79
than postsynaptic, meaning that once at their target site, eCBs bind to CB1 receptors located at the
pre-synaptic site in a retrograde manner, suppressing neurotransmitter release (Figure 3.1b).
33, 102

Although they are inherently quite similar, the two ligands exhibit distinct functions in the ECS.
While both AEA and 2-AG regulate presynaptic neurotransmitter release, the molecules mediate
short-term and long-term synaptic plasticity in the brain by operating in phasic and tonic modes.
103

The available evidence suggests that AEA acts as the tonic signaling molecule, adapting slowly to
stimulus and firing a sustained response, whereas 2-AG represents the phasic signal, adapting
rapidly to stimulus and producing a more transient response during neuronal depolarization.
103

After the desired homeostatic response has been achieved, both AEA and 2-AG are removed from
the synapse and degraded by their respective hydrolytic enzymes.
103

80

Figure 3.1: a) Structures of Δ9-THC, CBD, AEA, and 2-AG and b) Schematic representation of
the main components of the endocannabinoid system within the central nervous system (CNS).
3.2.3 Enzymes
Synthesis of 2-AG and other monoacylglycerols is catalyzed by diacylglycerol lipase α (DAGLα),
and synthesis of anandamide and other N-acylethanolamines is catalyzed by N-
acylphosphatidylethanolamine (NAPE)-specific phospholipase D-like hydrolase (NAPE-PLD).
104,
105
The most notable and well-understood degradation enzymes in the endocannabinoid system are
fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), which hydrolyze AEA
and 2-AG, respectively.
106, 107
Experimental evidence indicates that FAAH is located primarily on
the intracellular membrane of postsynaptic cells, whereas MAGL is generally located in
presynaptic terminals in the vicinity of CB1 receptors.
108, 109

81
3.2.4 The role of the Endocannabinoid System in the CNS
Entering the neurochemical, psychological, and philosophical realm of discussion, we are faced
with three important questions about the endocannabinoid system– ‘how do these components
interact with each other to produce a physiological response?’, ‘why, from an evolutionary
standpoint, do they work in this manner?’, and ‘to what effect do they influence our behavior?’.

To answer these questions, we must closely examine the known mechanisms of endocannabinoid
signaling (Figure 3.1b). As previously mentioned, endogenous cannabinoids act as retrograde
messengers to suppress neurotransmitter release. In other words, endocannabinoids are
synthesized “on-demand” in response to neuronal stimulation and suppress the release of
chemicals such as glutamate and GABA.
110, 111
In essence, this molecular mechanism outlines the
process of endocannabinoid-mediated synaptic plasticity.
112
The evidence for this is
overwhelming, as three independent research groups in the early 2000s reported that postsynaptic
depolarization-induced Ca
2+
elevation in the hippocampus and cerebellar cortex triggers the
postsynaptic synthesis of endogenous cannabinoids, which proceed to inhibit CB1-mediated
neurotransmitter release at the presynaptic site.
113-115
Since the early 2000s, eCBs have been shown
to activate both short-term (depolarization-induced suppression of inhibition/excitation, or
DSI/DSE) and long-term plasticity (long-term depression, or LTD) at synapses throughout the
brain.
112, 116
The most important and well-explored of these phenomena is LTD, which is defined
by the reduction in neurotransmitter release upon binding of eCBs to CB1 and has been reported
in the dorsal striatum, nucleus accumbens, amygdala, and hippocampus among others.
117-122
The
exact mechanisms underlying these changes are highly complex and still not fully understood.
However, it is known that endocannabinoid-mediated LTD is a fundamental mechanism for

82
inducing long-term changes to neural circuits and behavior.
116
Simply put, the endocannabinoid
system exists to provide on-demand protection against excitotoxicity in CNS neurons.
123

This brings us to the second question regarding the evolutionary purpose of the endocannabinoid
system as a protective mechanism against fear, anxiety and stress. Fear and anxiety are natural
phenomena that occur as a result of a real or perceived threat, or the possibility of such a threat
arising in the future.
74, 94
Similarly, the stress response is a bodily reaction to this challenge in order
to prepare it for upcoming danger, functioning as a protective mechanism that is essential to an
organism’s survival.
94
The body’s response to stress consists of an autonomic and a
neuroendocrine responses that are activated in parallel.
74
The autonomic nervous system consists
of the sympathetic and parasympathetic nervous system, and functions mainly by using
catecholamines like norepinephrine and acetylcholine as neurotransmitters.
124
In contrast, the
neuroendocrine system is mediated by activation of the hypothalamic pituitary adrenal (HPA) axis,
releasing cortisol, corticotropin, and other corticosteroids.
124
Although these mechanisms are
integral in delegating the basic survival instinct, superfluous response to external stressors,
especially when chronic, can prove detrimental to cognitive health and incite a shift in several
neurobehavioral responses including anxiety, memory, pain sensitivity, and coping behaviors and
lead to a range of neuropsychiatric disorders.
74, 125, 126
Therefore, it is vital that the domains of fear,
anxiety, and stress are regulated by the endocannabinoid system in an effort to maintain
homeostasis in a healthy brain.
94

Lastly, it is important to touch upon the effects of endocannabinoid mediated synaptic plasticity
on human behavior. Several clinical and preclinical studies have been conducted in an effort to

83
explore how the ECS acts as a buffer against the effects of stress. As previously discussed, the
ECS controls several brain regions related to fear and anxiety, generally regulating overactivation.
Acute exposure to stress results in an increase of FAAH activity, and thus a reduction of AEA
levels in the amygdala and prefrontal cortex, leading to activation of the HPA axis and an increase
in the concentration of 2-AG, which in turn inhibits the release of glutamate and GABA in the
hypothalamus and prefrontal cortex, respectively.
94, 127, 128
However, the repeated exposure of the
brain to non-habituating, chronic stress results in desensitization of CB1 receptor signaling.
94, 129

This becomes important as chronic stress can trigger or exacerbate a variety of psychiatric
disorders including schizophrenia and major depressive disorder (MDD).
130
Importantly, the
endogenous cannabinoid system is also present in fear-related brain areas and plays a central role
in the regulation of fear-memory processing.
94, 119, 131

3.2.5 The Endocannabidiome
The endocannabinoid system, as currently defined, is an oversimplification of the complex action
of mediators and alternative metabolic processes. The modulation of its components is part of a
larger network known as the endocannabidiome.
132
This system spans from GPCRs (GPR55,
GPR119), to ion channel receptors (TRPV1) and nuclear receptors (PPAR-γ), and includes
mediators such as N-acyl amino acids and N-acyl neurotransmitters.
132-136
Notably, the existence
of the endocannabidiome exposes the flaws of reducing a physiological response to confined
ligand-target interactions. Despite the ever-evolving progress in science that has allowed us to
“zoom in” on explicit mechanisms of interest, we mustn’t forget that the human body is not
composed of a combination of isolated systems but should instead be thought of as a complex web
of highly intertwined molecular entities.

84
Particularly interesting is the interplay between the eicosanoid and endocannabinoid signaling
systems. Although the two have traditionally been investigated separately, there are a multitude of
factors pointing towards a potential biological dialogue.
137
Both the endogenous cannabinoids 2-
AG and AEA, as well as other eicosanoids such as prostaglandins, thromboxanes, and leukotrienes
are synthesized from arachidonic acid (AA).
138
In addition, the lipases that initiate both pathways
respond to some of the same secondary messengers, meaning that they will be activated together,
and some of the enzymes involved in eicosanoid biosynthesis can metabolize both AA and
endogenous cannabinoids.
137
Interestingly, endocannabinoids can also be converted to a number
of prostanoids - both prostaglandin (PG)-glyceryl esters as well as PG-ethanolamides
(prostamides) can be formed from 2-AG and AEA, respectively.
139, 140
Despite the mounting
evidence that these two systems are deeply entangled, not much research has been done on the role
of these pathways in human health and wellbeing.

3.3 Phytocannabinoids
Having elucidated the function of the endocannabinoid system and explored the role of
endogenous cannabinoids, it is more than fitting to take a closer look at their illustrious namesakes.
The Cannabis sativa plant is distributed as Hashish (resin from upper leaves and flower buds) and
Marijuana (dried leaves and flowering heads), which both contain a variety of cannabinoids and
non-cannabinoids.
141
There are over 500 known compounds and at least 120 unique
phytocannabinoids that have been identified as of today. These can be divided into 10 subclasses;
∆
9
– and ∆
8
–tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), cannabinol
(CBN), cannabinodiol (CBND), cannabielsoin (CBE), cannabicyclol (CBL), cannabitriol (CBT),
and miscellaneous type (Figure 3.2).
69
Additionally, there are several other constituents in the

85
plant that may or may not contribute to the overall pharmacological effect, including terpenes,
nitrogenous compounds, amino acids, proteins, enzymes and glycoproteins, sugars, hydrocarbons,
simple alcohols and aldehydes, and steroids, amongst others.
69
Although many of the natural
products in cannabis have been synthesized, isolated, and characterized, several questions remain
open about the activity of these molecules and their possible synergistic interactions.

Figure 3.2: Chemical structures of common phytocannabinoids.
3.3.1 
9
-Tetrahydrocannabinol ( 
9
-THC)
In 1964 Gaoni and Mechoulam reported the isolation of ∆
9
–THC as the first structurally elucidated
active component of Cannabis sativa.
70
There are several constitutional- and stereoisomers of
THC, but (-)-trans-∆
9
–tetrahydrocannabinol or (6aR, 10aR)-delta-9-tetrahydrocannabinol is the
main plant-derived isomer and, by extension, also the most well explored. Interestingly, it is far
less stable than its ∆
8
and ∆
10
analogs, with ∆
10
being the most stable as a result of the double bond
in conjugation with the aromatic ring (Figure 3.3).

86

Figure 3.3: Some known isomers of THC.
∆
9
–THC acts as a partial agonist on both CB1 and CB2, with Ki values in the low nanomolar
range.
142
The psychoactive effects of ∆
9
–THC are mediated by CB1 and its potential
immunological or anti-inflammatory effects are thought to be a result of CB2 receptor agonism.
143

The effects of this molecule are fairly well studied, but the complexity of the interactions leaves
several questions open. Effectively, it is known that ∆
9
–THC perturbs GABA and glutamatergic
neurotransmission in a similar fashion to endogenous cannabinoids, producing many of the
common effects associated with consumption of cannabis.
142, 143
Notably, however, neuronal CB1
receptors are targeted in a less selective manner by phytocannabinoids than the respective
endogenous cannabinoids.
143
Emerging evidence over the last two decades has shown that in vivo
administration of ∆
9
–THC can actually increase the release of certain neurotransmitters, i.e.
acetylcholine in rat hippocampus, acetylcholine, glutamate and dopamine in rat prefrontal cortex,
and dopamine in mouse and rat nucleus accumbens.
143-145
These combined stimulatory-inhibitory
influences could be responsible for the excitant and depressant effects of ∆
9
–THC.
146, 147

The implications of ∆
9
–THC administration on psychosis, addiction, and memory and cognition
remain controversial. Generally, cognitive deficits observed from acute exposure to cannabis are
transient.
142, 148
In contrast, prolonged use is associated with more pronounced chronic deficits in
learning and memory.
149
It is worth noting that more recent studies have not replicated this

87
conclusion.
150, 151
Given this controversy, further research will be required to facilitate the approval
of ∆
9
–THC for medicinal purposes on a global scale.

3.3.2 Cannabidiol (CBD)
(-)-Cannabidiol (CBD) is the second major constituent of Cannabis sativa. It was first isolated in
1940 by Adams and coworkers, but its structure was not fully elucidated until almost thirty years
later when Mechoulam’s group was able to isolate CBD from Lebanese hashish and establish its
structure and stereochemistry.
71, 72, 152
It differs from the THC in that it has a pyran ring, and can
easily undergo acid- and base-catalyzed transformations to produce ∆
9
–THC and ∆
6
–CBD,
respectively (Figure 3.4).
152

Figure 3.4: Some possible transformations for CBD and related compounds.
Although structurally similar to ∆
9
–THC, CBD exhibits none of the addictive or psychoactive
properties associated with its infamous relative and is known to have very low affinity to both
known cannabinoid receptors. This lack of affinity seems to be a result of the two rings in CBD
being oriented in a perpendicular fashion, as compared to the planar conformation of ∆
9
–THC.
153

Unlike the endogenous cannabinoids and ∆
9
–THC, CBD possesses a highly complex and diverse
pharmacological profile, relying on interactions with a myriad of receptors. Here, we will highlight
only the most important interactions. One known mechanism of cannabidiol action is its function
as an antagonist of cannabinoid receptor agonists.
154
It was able to block the effects of CB1 agonists

88
WIN55212 and CP55940 at a far lower dose than is required for receptor activation by CBD.
Studies have also shown that it enhances endogenous adenosine signaling through inhibition of
uptake, providing an explanation for its anti-inflammatory properties.
155, 156
In addition, CBD is a
modest agonist of the serotonin (5-HT2A) receptor, which may be responsible for its analgesic and
anxiolytic effects.
157
It is also a potent antioxidant, as studies by Hampson et. al. have shown that
CBD prevents hydrogen peroxide-induced oxidative damage as well as or better than vitamin C
and vitamin E.
158
Furthermore, there is evidence for activity at the δ- and μ- opioid receptors and
TRPV1 cation channels.
143

CBD has a well-established safety profile and generally is well tolerated in doses up to 1,500
mg/day orally, without any reported negative effects on mood or motor skills.
159
Evidence from
human studies has highlighted the potential of CBD for treatment against anxiety at 300 to 600 mg
PO daily.
76
With this in mind, interest in the therapeutic potential of cannabidiol has skyrocketed
over the past decades. Increasing amounts of preclinical and clinical data have been gathered to
support the application of CBD as an antipsychotic, analgesic, antiemetic, antioxidant, anti-
epileptic, anti-inflammatory, and anti-convulsant.
76, 142

3.3.3 Approved Cannabinoids
The only two pharmaceutical forms of ∆
9
–THC on the US market are nabilone (a synthetic
derivative of ∆
9
–THC) and dronabinol (synthetic ∆
9
–THC).
160
Both medications are used in the
treatment of chemotherapy-related nausea and AIDS-associated weight loss and anorexia.
142
On
June 25
th
, 2018, the FDA approved Epidiolex, a highly purified botanical CBD extract, for the
treatment of Dravet syndrome and Lenox Gastaut syndrome, two forms of childhood-onset

89
epilepsy.
69, 161
Almost a decade after a study conducted by the lab of Ben Whalley highlighted the
antiseizure properties of cannabidiol, Epidiolex is the first cannabis-derived medicine approved
for clinical use. The only currently approved combined formulation, Sativex, contains a 1:1 ratio
of CBD:∆
9
–THC.
69
Interestingly, users have often times described vastly different sensations
based on whether the administered drug was synthetic or plant-derived, although the two were
chemically identical.
69

3.3.4 Entourage Effect
Here lies the pressing question: how can two chemically identical compounds produce different
effects based purely on the method of their isolation? Given that chemistry is an exact science and
spectroscopic methods can confirm the identity of the molecules in question, there are only two
scenarios that could explain this phenomenon - (a) one of the substance was mistakenly identified,
or (b) one of the substances contains an impurity that contributes to the overall pharmacological
profile.
69
The so-called “entourage effect” provides a strong case for the latter, and was first
described by Ben-Shabat in 1998 with reference to the enhanced activity of the endogenous
cannabinoid 2-AG by inactive fatty acid glycerol esters.
162
Since then, the term has been extended
to incorporate other cannabinoids and non-cannabinoids that enhance the activity of cannabis
preparations.
163
As stated by Mechoulam, “this type of synergism may play a role in the widely
held view that in some cases, plants are better drugs than the natural products isolated from
them”.
164

At this stage, it is only logical to ask: what therapeutic advantage, if any, does utilizing the entire
cannabis plant provide as opposed to the government-approved synthetic formulations like

90
dronabinol? One indication that the non-psychoactive components of the cannabis plant alter the
physiological response is demonstrated by the markedly different effects of the Cannabis sativa
and Cannabis indica chemovars.
165
Although both contain ∆
9
–THC, the former tends to enhance
creativity and productivity, whilst the latter is known to induce relaxation. As a matter of fact, the
disparities between the different chemovars are so significant that the species assignation of
cannabis itself is subject to heavy debate.
165
The question remains – why do cannabis users
experience such divergent strain-dependent sensations if the main active ingredient is the same?

Since the original discovery of the entourage effect, it has been shown on several occasions that
THC monotherapy is not as effective as the dual administration of THC in combination with CBD
or terpenoids.
163
In 2010, Johnson and coworkers conducted a multicenter, double-blind,
randomized placebo-controlled study of cannabis-based extracts in patients with cancer-related
pain.
166
In their findings, the THC-predominant extract produced similar results to the placebo,
whereas a plant extract containing a mixture of CBD and THC was statistically significantly better
than both.
165, 166
In another study, researchers found that small doses of pure CBD reduce pain until
a peak is reached, after which further increases are ineffective.
167
This bell-shaped dose-response
curve was, however, not observed for a full-spectrum cannabis extract with equivalent doses of
CBD, which resulted in a linear dose-response curve with no observed ceiling effect.
167
Thus,
counterintuitively, higher purity formulations of the active ingredients in cannabis did not
guarantee higher therapeutic efficacy. Further evidence for the entourage effect was provided by
a study conducted in 2018, which employed five distinct cannabis extracts with a uniform
concentration of CBD on mice with induced seizures.
168
The results of this study showed that all
five extracts were beneficial when compared to the control, but there were pronounced differences

91
between the number of mice developing tonic-clonic seizures (21.5-66.7%) as a result of the
varying amounts of the “minor” components in each extract.
165, 168
In summary, these findings
show that isolating or synthesizing only the active components of Marijuana may significantly
limit the plant’s therapeutic potential and, by extension, limit the variability of interbreeding and
hybridization within the highly versatile cannabis genome.

Currently, the lack of hard scientific evidence to back these empirical findings limits the utility of
the entourage effect in therapeutic applications.
169
Neither the effects of cannabinoid-cannabinoid
interactions, nor the effects of cannabinoid-terpenoid interactions have been clearly elucidated. If
there are to be significant advances in cannabinoid-based drug discovery, it is quintessential that
more comprehensive studies are designed and performed to gather conclusive scientific evidence.

4.3.5 Cannabis as a Medical Armory of Weapons
For most of modern history, efforts in the field of drug discovery have centered around the idea of
creating highly potent and highly specific molecules to treat diseases, with the aim of avoiding
unwanted side-effects. As we move into the third decade of the 21st century, it becomes clear that
this reductionist approach hasn’t even begun to unravel the multifarious mysteries of medicine. In
cases where the underlying disease pathophysiology is more complex than the dysfunction or
dysregulation of a single target, enzyme, or receptor, there is no hope of developing a single drug
with a single target to treat that condition. Specifically psychiatric and neurodegenerative diseases
seem to be polygenic in origin, given that the most effective medications on the market have
complex pharmacology and ill-defined mechanisms of action.
56
This empirical observation, in
combination with the repeated failure of using highly potent and target-specific drugs in clinical

92
development, allows us to infer retrospectively that treatment of CNS diseases is highly convoluted
and requires the modulation of multiple biological targets.
170
It seems that the development of
antipsychotic and antidepressant medications should be approached with the prospect of restoring
physiological balance by administering drugs with pleiotypic actions, rather than by aggressively
pursuing a specific target.

The term combination therapy, or polypharmacy, refers to the combined administration of two or
more single-target molecules to yield a more favorable outcome. As such, it is the simplest
approach to circumventing the limitations of single molecule-defined target drug discovery.
Although it does account for an added layer of complexity, the efficacy of combining two or more
single-target drugs is limited by pharmacokinetic properties such as half-life and distribution, as
well as unwanted drug-drug interactions.
170
In contrast, the development of one multitarget drug
that address several biological targets as “magic shotguns” instead of “magic bullets”, is known as
polypharmacology.
56, 170
This approach provides the added promise of reducing treatment
complexity and lowering drug dosage to produce adequate pharmacological effects due to
synergistic multi-target modulation, without the aforementioned complications. Since the
introduction of the term by Bryan Roth in 2004, several developments in machine learning,
statistical analysis, network analysis, and in silico/in vitro approaches have facilitated the inception
of de novo methods to evaluate and rationally design multitarget compounds. Notably, in 2012
Besnard et. al. described an automated approach for the rational design of polypharmacological
ligands by designing focused libraries of analogues of an initial compound through machine
learning, and built Bayesian models to prioritize these compounds according to a multi-
dimensional set of objectives.
55
Other approaches include Keiser’s similarity ensemble approach

93
(SEA) and Reker’s self-organizing map-based prediction of drug equivalence relationships
(SPiDER).
171, 172
In addition, an increasing number of chemical probes and empirical models are
being developed to facilitate the experimental validation of target synergies. For example, the
‘Therapeutic Handshake’ has been successfully applied to explain the efficacy of the combination
of CBD and THC in Sativex.
173
Albeit that these developments have facilitated the rational design
of new polypharmacological ligands, safety issues surrounding multi-target interactions remain
the biggest limitation of this approach.

With the outlook of building on the well-established safety profile of plants like cannabis sativa
with modern scientific discoveries, we propose an extension of the “multi compound-single target”
and “single compound-multi target” approaches in the form of a “multi compound-multi target”
approach. Rather than attempting to find a “magic bullet” or a “magic shotgun” to treat complex
diseases, we suggest gathering an “armory of weapons” that consists of multiple compounds with
multiple targets, and can be combined and administered as necessary. Not only does this
significantly reduce the time spent on rational design of novel ligands for each specific condition,
it also has the potential to reduce the rate of failure in clinical trials because of unwanted side-
effects, making the process both faster and more cost-efficient. James Black famously proposed
that “the most fruitful basis for the discovery of a new drug is to start with an old drug”.
55
Perhaps
it is time to take this one step further from the discovery of new drugs to new drug combinations.
Here, we find ourselves at the intersection of modern drug discovery and ancient herbal medicine,
with the prospect of building on our empirical knowledge of plant material with modern scientific
methods. By doing so, we hope to gather concrete data to support and evaluate these complex
natural compounds, the multitude of targets they interact with, and the physiological responses

94
they produce. Not only will this enable us to fine-tune formulations of multiple compounds to elicit
a specific desired effect, it also has the potential to enhance our understanding of the nature of
CNS diseases as a whole.

3.4 The War on Drugs
Cannabis sativa is one of the oldest plants known to man, and yet there is a shocking lack of
conclusive knowledge on its individual constituents, their mechanisms of action, the physiological
responses they evoke, and their possible synergistic interactions. While multiple studies
demonstrated that marijuana smokers have impaired cognitive performance, just as many failed to
observe such effects.
174-178
While there is evidence that combined administration of cannabinoids
can result in an “entourage effect”, the few reported small-scale studies that were conducted did
not confirm such interactions.
165, 179, 180
In summary – the lack of decisive and replicable evidence
leaves many open-ended questions making it difficult to build on the vast empirical knowledge
that has been gathered over centuries.

3.4.1 The Tangled History of Cannabis
What is the reason for this lack of progress? And why has cannabinoid-based drug development
been so stagnant in comparison to opioids? With the introduction of cannabis, opium, and coca
into western culture at a time of rapid technological and scientific developments, the blurred lines
between religious, social, and medicinal use of these plants became ever more defined.
181

However, early efforts to identify and isolate the active components of cannabis for medicinal
purposes proved to be too big of a challenge for the state of knowledge at the time.
182
Availability
of other therapeutics discouraged physicians from prescribing such preparations, and cannabis was

95
swept under the rug as a useless remedy. Henceforth, cannabis became looped into the efforts to
eliminate illegitimate use of drugs under a series of international drug conventions in the early 20
th

century. The results of this have shaped the portrayal of cannabis in popular culture, as well as
efforts in science up to this day (Figure 3.5).

Figure 3.5: A select timeline of the history of cannabis as medicine.

3.4.2 Regulatory Status and Academic Research
In the US, federal law prohibits the possession, production, and distribution of cannabis. The
Controlled Substance Act (CSA) of 1970 lists cannabis (in the form of resin, extracts, tincture,
pure THC, and pure CBD) as a Schedule I drug with no medical use, in the same category as heroin
and worse than methamphetamine and cocaine.
181, 183, 184
As a consequence, obtaining permission
to conduct clinical research on cannabis is a lengthy process and requires approval from both the

96
FDA and the DEA.
185
In addition, all cannabis used for research purposes must be obtained
exclusively from the University of Mississippi, which inherently limits the quality and diversity
of samples.
161, 186
This is troubling on several accounts. First off, it is widely accepted that samples
obtained from this source have more resemblance with marijuana from the 80’s than the wide
variety and increased potency of cannabis products available on the commercial market today. In
addition, restricting research to samples from just one source neglects to acknowledge both the
biggest advantage and the greatest challenge associated with plant medicine – the idea that
different strains produce different effects. Without access to the wide variety of cannabis products
that are available to the consumer, the research becomes tenuous.

These are issues that researchers have faced for decades, but they become ever-more relevant as
both the medical and recreational use of cannabis is skyrocketing. At its core, the CSA provides a
legal foundation for the government’s fight against drugs with a high potential for abuse. But what
defines a “drug of abuse”, and why are some drugs viewed differently than others? To what extent
does the policy on drugs like alcohol, tobacco, marijuana, and several prescription drug families
reflect their true dangers? And how have these policies been modified and skewed in order to
facilitate the regulating body’s political agenda? Here, the lines between government policy and
scientific progress become blurred, especially as most academic research institutes rely heavily on
funding provided by government agencies.
187
Really, it’s a Catch-22 situation – as long as
academic research on cannabinoids remains so heavily restricted, efforts to enforce the appropriate
regulations on their consumption will remain futile.

97
3.4.3 Cannabis in Big Business
While federal regulations have not changed much since the 70’s, several states across the US have
loosened their restrictions on marijuana, creating a new legal cannabis market. In 1996 California
passed Prop 215, the country’s first medical marijuana law, in an effort to provide relief to patients
suffering from chronic illnesses. Since then, the movement has spread across the country in what
has been called “medicine by popular vote”.
69, 188
As of November 2021, medical marijuana is
legal in 36 states across the US, and 18 states as well as the district of Columbia have enacted
legislation to regulate the non-medical use of cannabis.
189
Consequently, the legal medical and
recreational cannabis market has become a multi-billion dollar industry and is expected to continue
growing at a compound annual rate of 26% per year.
190

In fact, Big Marijuana has become so powerful that indirect competitors in Big Tobacco, Big
Alcohol, and Big Pharma have recently announced deals with cannabis companies in response to
the plethora of social and political campaigns against opioid, alcohol, and tobacco use.
191
In past
years, the pharma giant Novartis, the alcohol firm Molson Coors Brewing, and several tobacco
companies have joined forces with marijuana businesses as in an effort to capitalize on this new
movement.
191

3.4.4 Dangers of Cannabis in a Free Market
This in and of itself should ring alarm bells, as each of the aforementioned industries have a history
of actively campaigning to change legislation, influence public opinion, and distort research in
their favor, demonstrating the dangers of leaving public health in the hands of Big Business. In
addition, large corporations have a monetary incentive to breed a steady population of heavy users

98
for their personal benefit - a concept known as the 80:20 rule where 20% of users account for 80%
of consumption. In a marketplace where profit is the driving factor, consumer welfare is secondary.

At this stage, Big Marijuana has an enormous amount of regulatory freedom, especially in
comparison to researchers at academic institutions. Transitioning from prohibition to becoming
one of the fastest-growing industries in North America in less than a decade, the cannabis industry
has expanded at a rate at which the scientific community is unable to keep up with.

3.5 Outlook
As one of the oldest plant remedies known to man, the potential of cannabis sativa to heal various
ailments is no secret. Despite having been subjected to a rollercoaster ride of regulations, it
continues to be the most commonly cultivated and consumed illicit drug in the world.
192, 193
The
cannabis plant has a well-established safety profile and a multitude of active and non-active natural
compounds that could contribute to its overall pharmacological effect. The “seed observations”,
in this case, have been gathered in an “exploratory phase” over centuries. As such, the plant, its
individual components, and their combinations have the potential to elucidate the relevant
mechanisms associated with complex diseases, making it the ideal starting point to explore the
entire endocannabidiome and modify it according to a desired therapeutic outcome.

Despite its enormous potential, the rules and regulations surrounding cannabis in research have
presented a major roadblock in this endeavor. Simultaneously, a unique patient-centric movement
propagating the legalization of cannabis across North America has created a new multi-billion
dollar industry that is continuing to grow exponentially. In this extraordinary situation, individual

99
commercial entities in several states across the US have the liberty to grow and distribute
marijuana and marijuana-based products without being subjected to the lengthy FDA-approval
process. As a result, the fate of millions of consumers is left in the hands of profit-oriented
corporations. This is not to discredit the use of marijuana on an individual level to relieve stress,
pain, or inflammation. However, if we have learned anything from the opioid crisis, it is to be
weary of simple solutions for complex problems. Unless there is an active effort to fund and
facilitate unbiased academic research on cannabinoids and the endocannabinoid system, the
cannabis industry could be setting itself up for its own downfall.

100
Chapter 4. Taking Inspiration from Nature: Strategies towards the Facile
Synthesis of Mycophenolic Acid and its analogues

4.1 Introduction
In the last chapter of this quest to understand the progress and developments in modern drug
discovery, we tackle one of the most quintessentially important drug classes: natural products
(NPs). Historically, natural products and their analogues have played a key role in drug discovery
for centuries. To this day, around 35% of medicines originated from natural products.
194
This only
seems logical, given that the pool of bioactive naturally occurring compounds is highly diverse
and full of structural complexity.
195
From an evolutionary standpoint, these compounds were
optimized by nature to serve a particular purpose, including the interaction and competition with
other organisms, which explains their relevance for cancer and infectious diseases in particular.
195

In addition, the rich history of plant-medicine provides on-hand safety and efficacy data that would
be difficult to obtain otherwise. From a synthetic standpoint, natural products also provide huge
scaffold diversity, a larger number of sp
3
carbon atoms, fewer heteroatoms, and greater molecular
rigidity, as compared to their synthetic counterparts.

Despite their obvious prevalence, there are several challenges in the development of
pharmaceuticals from NPs, which has led to a shift towards synthetic compound libraries, such as
the ones discussed in Chapter 2 of this thesis. Namely, natural product screens are often
incompatible with traditional target-based assays and identifying and isolating the bioactive
compound of interest can be challenging.
196
It can also be challenging to gain IP rights for these
compounds, given their historical and cultural associations.
197
From a practical standpoint, the

101
success of natural products in drug discovery is largely contingent upon the availability of
sufficient biological material to isolate and characterize. For this reason, the total synthesis of
natural products is still prevalent, in order to provide a sustainable and economically viable supply
of compound on the market.
195

Despite all of the synthetic advancements discussed in the introductory portion of this thesis,
natural product chemistry remains an art-form. Even with the synthetic tools and methodologies
we have at hand in the 21
st
century, multi-step syntheses challenge even the most well-versed
chemists. Therefore, from both an academic and an industrial standpoint, there will continue to be
progress towards developing faster and easier synthetic methods to reach a given target compound.
One such method, which is of interest here, is the Ru-catalyzed [2+2+2] trimerization for the
polyfunctionalization of benzenes and other cycloadducts.
198
Given that the synthesis of fully
functionalized aromatic systems provides an enormous synthetic challenge, this method could be
of tremendous value for the synthesis of a plethora of natural products. It was recently shown that
CpRuCl(cod) complexes can influence the regioselectivity of certain 1,3-dipolar cycloadditions.
199

Furthermore, it was shown that electron-deficient haloalkynes underwent very efficient
trimerizations in the presence of CpRuCl(cod) with high regio- and chemo-selectivity.
198

Specifically, Silvestri and co-workers were able to react a series of electron-deficient, halogenated
alkynes with 2-butyne to selectively generate only two of the seven possible trimerized products
(Scheme 4.1).

102

Scheme 4.1: Trimerization of halogenated alkyne (2 eq.) with 2-butyne (1.5 eq.) with high
selectivity in an overall yield of 84%.
It should be noted that only electron-deficient alkynes bearing a terminal halide substituent reacted
with measurable chemo- and regio-selectivity, but this strategy opens up a window for the
synthesis of complex natural products nonetheless. Specifically, Silvestri and co-workers
demonstrated that the use of appropriate starting materials, in this case but-2-yne-1,4-diol, could
generate a variety of fully-functionalized isobenzofuranones, which are promising scaffolds in the
synthesis of a variety of natural products including Mycophenolic acid, Acremonide,
Chrysoarticullin C, and Austalide Q (Scheme 4.2).
198

Scheme 4.2: Synthesis of phthalide by trimerization and subsequent cyclization via acid
catalysis.
Here, we will focus on strategies towards the synthesis of Mycophenolic acid, an bioactive natural
product that was first discovered on the hunt for new antibiotics as an alternative to Penicilin.
200
It
was found that Penicillium brevicompactum produced a material inhibiting the grown of
Staphylococcus aureus, which was later discovered to be Mycophenolic acid.
200, 201
Since then, it

103
has become somewhat of an all-rounder and is used in the treatment of various diseases.
Mycophenolate mofetil, the ester of mycophenolic acid, is widely used as an immunosuppressant
drug for kidney, liver, and heart transplantations.
200

Figure 4.1: Structure of Mycophenolic acid.
4.2 Previous Synthetic Strategies
Although the drug has been around for decades and is widely used to treat a plethora of ailments,
cost- and time-efficient strategies for the total synthesis of the compound are few and far between.
A presumably simple molecule with no stereocenters, this finding might be somewhat surprising,
but demonstrates just how challenging the synthesis of polyfunctionalized aromatic systems is.
The first synthesis of this challenging molecule was achieved by Birch in 1969, and where the
phthalic ester was generated from a conjugated diene, and underwent a series of Wittig reactions
followed by oxidation and reduction steps to yield the final product (Figure 4.2a).
202
In 1986,
Danheiser and co-workers utilized an aromatic annulation strategy based on the thermal
combination of heterosubstituted acetylenes and cyclobutanones as the key strategy (Figure
4.2b).
203
Later, De La Cruz followed with a route that centered around a Diels Alder reaction and
subsequent aromatization (Figure 4.2c), and Covarrubias-Zuñiga presented a ring annulation
sequence involving a Michael Addition reaction and intramolecular Diekman condensation
reaction in situ (Figure 4.2d).
204, 205
The most recent report of the total synthesis of Mycophenolic
acid was published in 2013 by Brookes and co-workers, and involved a Pd-catalyzed
decarboxylative alkylation and biomimetic cyclization sequence (Figure 4.2e).
206

104

Figure 4.2: Previous retrosynthetic methods for the total synthesis of Mycophenolic Acid by a)
Birch, b) Danheiser, c) De La Cruz, d) Covarrubias-Zuñiga, and e) Brookes.
While all of these strategies circumvent some of the problems with functionalizing existing
aromatic systems, they involve up to fifteen steps, require complex and costly starting materials,
and are not always applicable to other phthalide-containing natural products.

4.3 Retrosynthetic Strategy
In order to overcome these limitations, we propose a retrosynthetic strategy using a Ru-catalyzed
[2+2+2] cycloaddition reaction to yield Mycophenolic Acid in six steps. This method uses but-2-

105
yn-1-ol, propionic acid, 4-bromobut-1-yne, and methyl 4-oxopentanoate as starting materials,
providing a cost- and time-efficient alternative to the aforementioned routes. In addition, we
strategically employ a convergent approach, in which two separate building blocks, A and B, are
formed and combined via trimerization (Scheme 4.3). This is beneficial for synthetic ease and
yield optimization.

Scheme 4.3: Retrosynthetic strategy for the total synthesis of Mycophenolic acid.
As previously mentioned, this route centers around the synthesis of building blocks A and B, which
are subsequently trimerized. Compound A is formed by a Wittig reaction of the Wittig salt
generated from , 4-bromobut-1-yne with methyl 4-oxopentanoate. Subsequently, the compound is
iodinated to enable functionalization after trimerization. Compound B is generated by a simple
O O
I
Br
O O
+
A
O O
I
OMe O
B
Br
O O
HO
O
O
OH
HO
O
O
O
Br
I
O
O
O
O
HO
O O
+
OMe O
O
P
Br
+
4.1 4.2
4.3
4.4 4.5
4.6
4.7
4.5
4.2

106
esterification of propionic acid with but-2-yn-1-ol. The resulting compound is then brominated to
generate the halogenated alkyne, ensuring specificity in later functionalization.

Having formed both components, the trimerization reaction can be initiated based on the findings
discussed in section 4.1 of this thesis.
198
Subsequently, all that is left is functionalization of the
Iodine and Bromine handles, which is proposed to proceed via Ullmann coupling according to a
procedure by Majumder and coworkers, and hydroxylation according to a procedure by Xia and
co-workers, respectively.
207, 208

4.4 Synthetic Progress and Challenges
4.4.1 Synthesis of Compound A
Compound A was synthesized via esterification of propionic acid with DMAP and DCC. The
reaction was complete after 12 hours and resulted in a yield of 41%. Subsequent bromination of
the terminal alkyne (4.1) was achieved with NBS and AgNO3, in a yield of 56%. The resulting
compound (4.2) was stored at 0˚C, as decomposition of the halogenated alkyne was observed after
several hours at room temperature. It is important to note that the reaction must proceed in the
aforementioned sequence, as the halogenated alkyne decomposes during esterification. Detailed
synthetic procedures can be found in section 4.5 of this thesis.

4.4.2 Synthesis of Compound B
The central step for synthesis of compound B is olefination of methyl 4-oxopentanoate. Several
strategies were explored to form this C=C double bond, including a Wittig reaction, HWE reaction,

107
Julia-Lythgoe reaction, and Julia-Kociensky reaction. The main difficulty here lies in achieving
selectivity for the E-isomer that is present in Mycophenolic acid.

Initially, a Julia-Kociensky reaction was attempted due to its preferential selectivity for formation
of the E-olefin.
209
Here, introduction of a benzothiazole enables a pronounced complexation that
influences selectivity. The benzothiazole sulfone is generated by reacting a benzothiazole with the
compound of interest, in this case but-3-yn-1-ol, and subsequently oxidized with mCBPA (Scheme
4.4). Subsequent attempts at olefination using NaHMDS or KHMDS did not yield the desired
product. After a series of test reactions, it became evident that the terminal alkyne presented a
complication and prohibited product formation. Although there are strategies to overcome this
issue, these will add additional synthesis steps.

Scheme 4.4: Synthesis of benzothiazole sulfone to carry out the Julia-Kociensky olefination by a)
substation of the thiol and b) oxidation with mCBPA.
For this reason, a traditional Wittig olefination was attempted, which yielded in a mixture of the
Z- and E- isomer of the desired product in a ratio of 4/1 (Figure 4.3). Using ROE, it was confirmed
that the Z-isomer was the major product. This finding can be explained by the steric competition
between the methylene and methyl groups on the double bond, where the methylene-methylene
spatial interaction is favored, resulting in the Z-isomer as the major product. Subsequent

108
isomerization attempts were not successful. Iodination of the alkyne was carried out using Iodine
and N-Morpholine.

Figure 4.3: a) Representation of both possible isomers and the interaction ovserved by ROE b)
ratio of Z/E products based on 1H NMR by integration of the methoxy peak.
4.4.3 Future Directions
Given that both building blocks A and B have been synthesized, all that remains is trimerization
and subsequent functionalization of the halogenated positions. Lastly, the ester should be
hydrolyzed to yield Mycophenolic acid. These steps should be carried out using the procedures
described in section 4.3.

4.5 Synthetic Procedures and Spectra
4.5.1 General Information
1
H,
13
C, and
19
F NMR spectra were recorded on Varian 400-MR, Varian VNMRS-500, and Varian
VNMRS-600 instruments at 295K unless otherwise noted. Proton magnetic resonance spectra
(
1
H NMR) were recorded at 500 MHz, and carbon magnetic resonance spectra (
13
C NMR) were
recorded at 125 MHz, unless otherwise mentioned. Chemical shifts (δ) are expressed in parts per

109
million, relative to the residual solvent signals as internal standards. Multiplicities are noted as
follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; sex, sextet; sept, septet. High resolution
mass spectra were measured using Agilent 6545XT qToF instrument coupled with 1290 LC
system. QToF mass spectrometer is equipped with an atmospheric pressure chemical ionization
source. Measurements were performed in positive ion mode with following ionization parameters:
Capillary Voltage –3.5 kV, Corona current 4 µA, vaporization at 350 °C, nitrogen was applied as
a nebulizer gas 35 psi, dry gas 13 L × min
−1
, 325 °C, and collision gas. Spectra were recorded in
m/z 100 – 1700 range. For external calibration and tuning, a low-concentration tuning mix solution
by Agilent Technologies was utilized. For sample injection (1 µL injection of ca. 10
–4
M solution
in MeOH) LC system was used. Injected compounds were passed through XDB-C18, 2.1 × 50
mm, 1.8 µm column at 40 °C with gradient H2O/MeOH (0.1% formic acid) elution. UV/Vis flow
detection was also applied (190 – 900 nm). All the MS spectra were recorded at 1 Hz. Spectra were
processed using Agilent MassHunter 10.0 software package. Precoated Merk F-254 silica gel
plates were used for analysis by thin layer chromatography (TLC), visualized with short wave UV
light, and stained with KMnO4 and PPh3/Ninhydrin. Column chromatography was carried out
employing EMD (Merk) Silica Gel 60 (40-63 μm). Reagents were obtained from AA BLOCKS,
Enamine, or Sigma Aldrich, and used without further purification, unless otherwise noted.

4.5.2 Synthesis of Compound A

A 100 mL dry round bottom flask was charged with but-2-yne-1-ol (1.0 eq., 500 mg, 7.13 mmol),
propioinic acid (1.1 eq., 550 mg, 7.85 mmol), and DMAP (0.12 eq., 105 mg, 0.86 mmol), and was

110
dissolved in 20 mL dry DCM. After DCC was added slowly at 0˚C, the reaction mixture was stirred
at r.t. for 16 h and filtered through a celite column. Subsequent purification by column
chromatography (SiO2, 5% EtOAc in Hexanes) resulted in the product (4.1) as an off-white oil in
a yield of 41% (357 mg, 0.36 mmol).
1
H NMR (600 MHz, Chloroform-d) δ 4.73 (dd, J = 2.2, 1.0
Hz, 2H), 2.91 (s, 1H), 1.85 (s, 3H).
13
C NMR (151 MHz, Chloroform-d) δ 151.76, 84.45, 75.42,
74.11, 71.87, 54.40, 3.61.

In a 100 mL dry round bottom flask, 4.1 (1.0 eq., 400 mg, 3.28 mmol) and NBS (1.2 eq., 700 mg,
3.93 mmol) were suspended in 10 mL actone and AgNO3 (0.10 eq.., 56 mg, 0.33 mmol) was added
while stirring. The reaction mixture was stirred at r.t. for 14 h and filtered through a celite column.
After extraction with EtOAc (3 x 15 mL), the product (4.2) was obtained as an off-white oil in a
yield of 56% (368 mg, 1.83 mmol).
1
H NMR (600 MHz, Chloroform-d) δ 4.72 (s, 2H), 1.85 (s,
3H).
13
C NMR (151 MHz, Chloroform-d) δ 151.75, 84.50, 72.21, 71.87, 54.50, 53.68, 30.90, 3.62.

4.5.2 Synthesis of Compound B

In a 25 mL dry Schlenk tube, 4-bromobut-1-yne (1.0 eq., 300 mg, 2.26 mmol) and PPh3 (1.0 eq.,
591 mg, 2.26 mmol) were suspended in 3 mL dry acetonitrile. The reaction mixture was heated to

111
80˚C and stirred for 72 h. After rotary evaporation and addition of benzene, the mixture was cooled
to -20˚C filtration and filtered yielding the Wittig salt of 4-bromobut-1-yne was (4.3) obtained as
an off-white solid in 92% (833 mg, 2.07 mmol).
1
H NMR (600 MHz, Chloroform-d) δ 7.96 – 7.85
(m, 6H), 7.84 – 7.75 (m, 3H), 7.74 – 7.63 (m, 6H), 4.29 – 4.11 (m, 2H), 3.07 – 2.74 (m, 2H), 1.72
(s, 1H).
31
P NMR (243 MHz, Chloroform-d) δ 24.75.

A 50 mL dry round bottom flask was charged with 4.3 (1.1 eq., 123 mg, 0.32 mmol), and was
dissolved in 6 mL dry THF and cooled down to -78˚C. N-butyl lithium (1.1 eq., 0.32 mmol), was
added slowly and the reaction was stirred for 45 minutes at room temperature. A solution of methyl
4-oxopentanoate (1.0 eq., 38 mg, 0.29 mmol) in 3 mL dry THF was added dropwise at -78˚C and
then warmed stirred at room temperature for 18h. Subsequent purification by column
chromatography (SiO2, 25% EtOAc in Hexanes) resulted in a mixture of methyl (Z)-4-methyloct-
4-en-7-ynoate and methyl (E)-4-methyloct-4-en-7-ynoate (4.4) as an off-white oil in a yield of
71% (140 mg, 0.62 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 5.41 – 5.01 (m, 1H), 3.67 (s,
3H), 3.05 – 2.83 (m, 2H), 2.59 – 2.29 (m, 4H), 2.14 – 1.90 (m, 1H), 1.71 (q, J = 1.3 Hz, 3H).

112
A 25 mL dry Schlenk tube was charged with 4.4 (1.0 eq., 400 mg, 2.41 mmol), N-morpholine (5.0
eq., 1.05 g, 12.03 mmol), and I2 (1.5 eq., 916 mg, 3.61 mmol), and was dissolved in 10 mL
benzene. The reaction mixture was stirred at r.t. for 24 h, washed with NaS2O3, extracted with
diethyl ether (3 x 15 mL), and dried over magnesium sulfate. Subsequent purification by column
chromatography (SiO2, 20% DEE in Hexanes) resulted in the product (4.5) as an off-white oil in
a yield of 29% (357 mg, 0.68 mmol).
1
H NMR (400 MHz, Chloroform-d) δ 5.24 – 5.10 (m, 1H),
3.80 – 3.60 (m, 3H), 3.24 – 2.99 (m, 2H), 2.57 – 2.30 (m, 4H), 1.70 (s, 3H).

113
Bibliography

1. Drews, J., Drug Discovery: A Historical Perspective. Science 2000, 287 (5460), 1960-
1964.
2. Moore, F. J., A history of chemistry. 1918.
3. Chain, E.; Florey, H. W.; Gardner, A. D.; Heatley, N. G.; Jennings, M. A.; Orr-Ewing,
J.; Sanders, A. G., PENICILLIN AS A CHEMOTHERAPEUTIC AGENT. The Lancet 1940, 236
(6104), 226-228.
4. Munos, B., Lessons from 60 years of pharmaceutical innovation. Nat. Rev. Drug Discov.
2009, 8 (12), 959-968.
5. Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg,
S. R.; Schacht, A. L., How to improve R&D productivity: the pharmaceutical industry's grand
challenge. Nat. Rev. Drug Discov. 2010, 9 (3), 203-214.
6. Hingorani, A. D.; Kuan, V.; Finan, C.; Kruger, F. A.; Gaulton, A.; Chopade, S.; Sofat,
R.; Macallister, R. J.; Overington, J. P.; Hemingway, H.; Denaxas, S.; Prieto, D.; Casas, J. P.,
Improving the odds of drug development success through human genomics: modelling study. Sci.
Rep. 2019, 9 (1).
7. Aronson, J. K.; Green, A. R., Me‐too pharmaceutical products: History, definitions,
examples, and relevance to drug shortages and essential medicines lists. British Journal of Clinical
Pharmacology 2020, 86 (11), 2114-2122.
8. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse Chemical Function
from a Few Good Reactions. Angewandte Chemie International Edition 2001, 40 (11), 2004-2021.
9. Kolb, H. C.; Sharpless, K. B., The growing impact of click chemistry on drug discovery.
Drug Discovery Today 2003, 8 (24), 1128-1137.
10. Sharpless, K. B.; Manetsch, R., In situclick chemistry: a powerful means for lead
discovery. Expert Opinion on Drug Discovery 2006, 1 (6), 525-538.

114
11. Savonnet, M.; Kockrick, E.; Camarata, A.; Bazer-Bachi, D.; Bats, N.; Lecocq, V.; Pinel,
C.; Farrusseng, D., Combinatorial synthesis of metal–organic frameworks libraries by click-
chemistry. New Journal of Chemistry 2011, 35 (9), 1892.
12. Wang, X.; Huang, B.; Liu, X.; Zhan, P., Discovery of bioactive molecules from CuAAC
click-chemistry-based combinatorial libraries. Drug Discov Today 2016, 21 (1), 118-132.
13. Suzuki, T.; Kasuya, Y.; Itoh, Y.; Ota, Y.; Zhan, P.; Asamitsu, K.; Nakagawa, H.;
Okamoto, T.; Miyata, N., Identification of Highly Selective and Potent Histone Deacetylase 3
Inhibitors Using Click Chemistry-Based Combinatorial Fragment Assembly. PLoS ONE 2013, 8
(7), e68669.
14. Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B., Sulfur(VI) Fluoride Exchange
(SuFEx): Another Good Reaction for Click Chemistry. Angewandte Chemie International Edition
2014, 53 (36), 9430-9448.
15. Barrow, A. S.; Smedley, C. J.; Zheng, Q.; Li, S.; Dong, J.; Moses, J. E., The growing
applications of SuFEx click chemistry. Chemical Society Reviews 2019, 48 (17), 4731-4758.
16. Steinkopf, W., Über Aromatische Sulfofluoride. Journal für Praktische Chemie 1927, 117
(1), 1-82.
17. Zheng, Q.; Dong, J.; Sharpless, K. B., Ethenesulfonyl Fluoride (ESF): An On-Water
Procedure for the Kilogram-Scale Preparation. The Journal of Organic Chemistry 2016, 81 (22),
11360-11362.
18. Leng, J.; Qin, H.-L., 1-Bromoethene-1-sulfonyl fluoride (1-Br-ESF), a new SuFEx
clickable reagent, and its application for regioselective construction of 5-sulfonylfluoro isoxazoles.
Chemical Communications 2018, 54 (35), 4477-4480.
19. Champseix, A.; Chanet, J.; Etienne, A.; Le Berre, A.; Masson, J., Syntheses de β-sultames
(thiazétidines-1, 2 dioxyde-1, 1). Bulletin de la Société chimique de France 1985, (3), 463-472.
20. Thomas, J.; Fokin, V. V., Regioselective Synthesis of Fluorosulfonyl 1,2,3-Triazoles from
Bromovinylsulfonyl Fluoride. Organic Letters 2018, 20 (13), 3749-3752.
21. Hunt, H. J.; Belanoff, J. K.; Walters, I.; Gourdet, B.; Thomas, J.; Barton, N.; Unitt, J.;
Phillips, T.; Swift, D.; Eaton, E., Identification of the Clinical Candidate (R)-(1-(4-Fluorophenyl)-
6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-

115
4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone (CORT125134): A Selective Glucocorticoid
Receptor. Journal of Medicinal Chemistry 2017, 60 (8), 3405-3421.
22. Krutak, J. J.; Burpitt, R. D.; Moore, W. H.; Hyatt, J. A., Chemistry of ethenesulfonyl
fluoride. Fluorosulfonylethylation of organic compounds. The Journal of Organic Chemistry
1979, 44 (22), 3847-3858.
23. Mustafa, A., The Chemistry of Sultones and Sultams. Chemical Reviews 1954, 54 (2), 195-
223.
24. Bohacek, R. S.; McMartin, C.; Guida, W. C., The art and practice of structure‐based drug
design: a molecular modeling perspective. Medicinal research reviews 1996, 16 (1), 3-50.
25. Polishchuk, P. G.; Madzhidov, T. I.; Varnek, A., Estimation of the size of drug-like
chemical space based on GDB-17 data. Journal of Computer-Aided Molecular Design 2013, 27
(8), 675-679.
26. Ertl, P., Cheminformatics Analysis of Organic Substituents: Identification of the Most
Common Substituents, Calculation of Substituent Properties, and Automatic Identification of
Drug-like Bioisosteric Groups. Journal of Chemical Information and Computer Sciences 2003, 43
(2), 374-380.
27. Gentile, F.; Agrawal, V.; Hsing, M.; Ton, A.-T.; Ban, F.; Norinder, U.; Gleave, M. E.;
Cherkasov, A., Deep Docking: A Deep Learning Platform for Augmentation of Structure Based
Drug Discovery. ACS Central Science 2020, 6 (6), 939-949.
28. Walters, W. P.; Wang, R., New Trends in Virtual Screening. Journal of Chemical
Information and Modeling 2020, 60 (9), 4109-4111.
29. Liu, L.; Jockers, R., Structure-Based Virtual Screening Accelerates GPCR Drug
Discovery. Trends in Pharmacological Sciences 2020, 41 (6), 382-384.
30. Jorgensen, W. L., The many roles of computation in drug discovery. Science 2004, 303
(5665), 1813-8.
31. Lyu, J.; Wang, S.; Balius, T. E.; Singh, I.; Levit, A.; Moroz, Y. S.; O’Meara, M. J.;
Che, T.; Algaa, E.; Tolmachova, K.; Tolmachev, A. A.; Shoichet, B. K.; Roth, B. L.; Irwin, J.
J., Ultra-large library docking for discovering new chemotypes. Nature 2019, 566 (7743), 224-
229.

116
32. Zou, S.; Kumar, U., Cannabinoid Receptors and the Endocannabinoid System: Signaling
and Function in the Central Nervous System. Int. J. Mol. Sci. 2018, 19 (3), 833.
33. Howlett, A. C., The cannabinoid receptors. Prostaglandins & Other Lipid Mediators 2002,
68-69, 619-631.
34. Li, X.; Hua, T.; Vemuri, K.; Ho, J.-H.; Wu, Y.; Wu, L.; Popov, P.; Benchama, O.;
Zvonok, N.; Locke, K. A.; Qu, L.; Han, G. W.; Iyer, M. R.; Cinar, R.; Coffey, N. J.; Wang, J.;
Wu, M.; Katritch, V.; Zhao, S.; Kunos, G.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu,
Z.-J., Crystal Structure of the Human Cannabinoid Receptor CB2. Cell 2019, 176 (3), 459-467.e13.
35. Munro, S.; Thomas, K. L.; Abu-Shaar, M., Molecular characterization of a peripheral
receptor for cannabinoids. Nature 1993, 365 (6441), 61-65.
36. Contino, M.; Capparelli, E.; Colabufo, N. A.; Bush, A. I., Editorial: The CB2 Cannabinoid
System: A New Strategy in Neurodegenerative Disorder and Neuroinflammation. Front Neurosci
2017, 11, 196.
37. Guindon, J.; Hohmann, A. G., Cannabinoid CB2 receptors: a therapeutic target for the
treatment of inflammatory and neuropathic pain. British Journal of Pharmacology 2008, 153 (2),
319-334.
38. Lunn, C. A.; Reich, E. P.; Fine, J. S.; Lavey, B.; Kozlowski, J. A.; Hipkin, R. W.;
Lundell, D. J.; Bober, L., Biology and therapeutic potential of cannabinoid CB2 receptor inverse
agonists. British Journal of Pharmacology 2008, 153 (2), 226-239.
39. Zhou, L.; Zhou, S.; Yang, P.; Tian, Y.; Feng, Z.; Xie, X.-Q.; Liu, Y., Targeted inhibition
of the type 2 cannabinoid receptor is a novel approach to reduce renal fibrosis. Kidney
International 2018, 94 (4), 756-772.
40. Xiang, W.; Shi, R.; Kang, X.; Zhang, X.; Chen, P.; Zhang, L.; Hou, A.; Wang, R.;
Zhao, Y.; Zhao, K.; Liu, Y.; Ma, Y.; Luo, H.; Shang, S.; Zhang, J.; He, F.; Yu, S.; Gan, L.;
Shi, C.; Li, Y.; Yang, W.; Liang, H.; Miao, H., Monoacylglycerol lipase regulates cannabinoid
receptor 2-dependent macrophage activation and cancer progression. Nature Communications
2018, 9 (1).
41. Li, X.; Hua, T.; Makriyannis, A.; Stevens, R. C.; Correspondence, Z.-J. L.; Vemuri, K.;
Ho, J.-H.; Wu, Y.; Wu, L.; Popov, P.; Benchama, O.; Zvonok, N.; Locke, A.; Qu, L.; Han,
G. W.; Iyer, M. R.; Cinar, R.; Coffey, N. J.; Wang, J.; Wu, M.; Katritch, V.; Zhao, S.; Kunos,

117
G.; Bohn, L. M.; Liu, Z.-J., Crystal Structure of the Human Cannabinoid Receptor CB2 Data
Resources 5ZTY Article Crystal Structure of the Human Cannabinoid Receptor CB2. Cell 2019.
42. Abagyan, R.; Orry, A.; Raush, E.; Totrov, M. ICM Manual, version 3.8.
43. Orry, A. J.; Abagyan, R., Homology modeling: methods and protocols. Springer: 2012.
44. Gatica, E. A.; Cavasotto, C. N., Ligand and Decoy Sets for Docking to G Protein-Coupled
Receptors. Journal of Chemical Information and Modeling 2012, 52 (1), 1-6.
45. Kroeze, W. K.; Sassano, M. F.; Huang, X.-P.; Lansu, K.; McCorvy, J. D.; Giguère, P.
M.; Sciaky, N.; Roth, B. L., PRESTO-Tango as an open-source resource for interrogation of the
druggable human GPCRome. Nature Structural & Molecular Biology 2015, 22 (5), 362-369.
46. Nikas, S. P.; Alapafuja, S. O.; Papanastasiou, I.; Paronis, C. A.; Shukla, V. G.;
Papahatjis, D. P.; Bowman, A. L.; Halikhedkar, A.; Han, X.; Makriyannis, A., Novel 1′,1′-Chain
Substituted Hexahydrocannabinols: 9β-Hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol
(AM2389) a Highly Potent Cannabinoid Receptor 1 (CB1) Agonist. Journal of Medicinal
Chemistry 2010, 53 (19), 6996-7010.
47. Nikas, S. P.; Sharma, R.; Paronis, C. A.; Kulkarni, S.; Thakur, G. A.; Hurst, D.; Wood,
J. T.; Gifford, R. S.; Rajarshi, G.; Liu, Y.; Raghav, J. G.; Guo, J. J.; Järbe, T. U.; Reggio, P.
H.; Bergman, J.; Makriyannis, A., Probing the carboxyester side chain in controlled deactivation
(-)-δ(8)-tetrahydrocannabinols. J Med Chem 2015, 58 (2), 665-81.
48. Sadybekov, A. A.; Sadybekov, A. V.; Liu, Y.; Iliopoulos-Tsoutsouvas, C.; Huang, X.-
P.; Pickett, J.; Houser, B.; Patel, N.; Tran, N. K.; Tong, F.; Zvonok, N.; Jain, M. K.; Savych,
O.; Radchenko, D. S.; Nikas, S. P.; Petasis, N. A.; Moroz, Y. S.; Roth, B. L.; Makriyannis, A.;
Katritch, V., Synthon-based ligand discovery in virtual libraries of over 11 billion compounds.
Nature 2022, 601 (7893), 452-459.
49. Congreve, M.; De Graaf, C.; Swain, N. A.; Tate, C. G., Impact of GPCR Structures on
Drug Discovery. Cell 2020, 181 (1), 81-91.
50. Bharatam, P. V.; Amita; Gupta, A.; Kaur, D., Theoretical studies on S–N interactions in
sulfonamides. Tetrahedron 2002, 58 (9), 1759-1764.
51. Kharb, R.; Sharma, P. C.; Yar, M. S., Pharmacological significance of triazole scaffold.
Journal of Enzyme Inhibition and Medicinal Chemistry 2011, 26 (1), 1-21.

118
52. Muthuraja, P.; Beaula, T. J.; Sethuram, M.; Jothy, V. B.; Dhandapani, M., Hydrogen
bonding interactions on 1H-1,2,3-triazole based crystals: Featuring experimental and theoretical
analysis. Current Applied Physics 2018, 18 (6), 774-784.
53. Hua, Y.; Flood, A. H., Click chemistry generates privileged CH hydrogen-bonding
triazoles: the latest addition to anion supramolecular chemistry. Chemical Society Reviews 2010,
39 (4), 1262.
54. Evans, B. E.; Rittle, K. E.; Bock, M. G.; Dipardo, R. M.; Freidinger, R. M.; Whitter, W.
L.; Lundell, G. F.; Veber, D. F.; Anderson, P. S.; Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.;
Chen, T. B.; Kling, P. J.; Kunkel, K. A.; Springer, J. P.; Hirshfield, J., Methods for drug
discovery: development of potent, selective, orally effective cholecystokinin antagonists. Journal
of Medicinal Chemistry 1988, 31 (12), 2235-2246.
55. Besnard, J.; Ruda, G. F.; Setola, V.; Abecassis, K.; Rodriguiz, R. M.; Huang, X.-P.;
Norval, S.; Sassano, M. F.; Shin, A. I.; Webster, L. A.; Simeons, F. R. C.; Stojanovski, L.; Prat,
A.; Seidah, N. G.; Constam, D. B.; Bickerton, G. R.; Read, K. D.; Wetsel, W. C.; Gilbert, I.
H.; Roth, B. L.; Hopkins, A. L., Automated design of ligands to polypharmacological profiles.
Nature 2012, 492 (7428), 215-220.
56. Roth, B. L.; Sheffler, D. J.; Kroeze, W. K., Magic shotguns versus magic bullets:
selectively non-selective drugs for mood disorders and schizophrenia. Nature Reviews Drug
Discovery 2004, 3 (4), 353-359.
57. Grotsch, K.; Fokin, V. V., Between Science and Big Business: Tapping Mary Jane’s
Uncharted Potential. ACS Central Science 2022.
58. Zhao, H.; Dietrich, J., Privileged scaffolds in lead generation. Expert Opinion on Drug
Discovery 2015, 10 (7), 781-790.
59. Engels, M. F.; Venkatarangan, P., Smart screening: approaches to efficient HTS. Curr Opin
Drug Discov Devel 2001, 4 (3), 275-83.
60. Villoutreix, B. O.; Eudes, R.; Miteva, M. A., Structure-based virtual ligand screening:
recent success stories. Comb Chem High Throughput Screen 2009, 12 (10), 1000-16.
61. Mechoulam, R.; Parker, L. A., The Endocannabinoid System and the Brain. Annual Review
of Psychology 2013, 64 (1), 21-47.

119
62. Gonçalves, E. C. D.; Baldasso, G. M.; Bicca, M. A.; Paes, R. S.; Capasso, R.; Dutra, R.
C., Terpenoids, Cannabimimetic Ligands, beyond the Cannabis Plant. Molecules 2020, 25 (7),
1567.
63. Di Marzo, V.; Bifulco, M.; Petrocellis, L. D., The endocannabinoid system and its
therapeutic exploitation. Nat. Rev. Drug Discov. 2004, 3 (9), 771-784.
64. Zuardi, A. W., History of cannabis as a medicine: a review. Revista Brasileira de
Psiquiatria 2006, 28 (2), 153-157.
65. Touw, M., The Religious and Medicinal Uses ofCannabisin China, India and Tibet. J.
Psychoactive Drugs 1981, 13 (1), 23-34.
66. Thompson, R. C., A dictionary of Assyrian botany. British Academy: London, 1949.
67. Rosenthal, F., The Herb: Hashish versus Medeival Muslim Society. Bulletin of the School
of Oriental and African Studies, University of London 1972, 35 (3), 633-636.
68. Moreau, J. J., Hashish and Mental Illness. Raven Press: 1973.
69. Bonn-Miller, M. O.; Elsohly, M. A.; Loflin, M. J. E.; Chandra, S.; Vandrey, R., Cannabis
and cannabinoid drug development: evaluating botanical versus single molecule approaches. Int.
Rev. Psych. 2018, 30 (3), 277-284.
70. Gaoni, Y.; Mechoulam, R., Isolation, Structure, and Partial Synthesis of an Active
Constituent of Hashish. J. Am. Chem. Soc. 1964, 86 (8), 1646-1647.
71. Mechoulam, R.; Shvo, Y., Hashish—I: The Structure of Cannabidiol. Tetrahedron 1963,
19 (12), 2073-2078.
72. Adams, R.; Hunt, M.; Clark, J. H., Structure of Cannabidiol, a Product Isolated from the
Marihuana Extract of Minnesota Wild Hemp. I. J. Am. Chem. Soc. 1940, 62 (1), 196-200.
73. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I., Structure of
a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990, 346 (6284),
561-564.

120
74. Morena, M.; Patel, S.; Bains, J. S.; Hill, M. N., Neurobiological Interactions Between
Stress and the Endocannabinoid System. Neuropsychopharmacology 2016, 41 (1), 80-102.
75. Di Marzo, V., New approaches and challenges to targeting the endocannabinoid system.
Nature Reviews Drug Discovery 2018, 17 (9), 623-639.
76. Blessing, E. M.; Steenkamp, M. M.; Manzanares, J.; Marmar, C. R., Cannabidiol as a
Potential Treatment for Anxiety Disorders. Neurotherapeutics 2015, 12 (4), 825-836.
77. Campos, A. C.; Fogaça, M. V.; Sonego, A. B.; Guimarães, F. S., Cannabidiol,
neuroprotection and neuropsychiatric disorders. Pharmacol. Res. 2016, 112, 119-127.
78. Carlson, G.; Wang, Y.; Alger, B. E., Endocannabinoids facilitate the induction of LTP in
the hippocampus. Nat. Neurosci. 2002, 5 (8), 723-724.
79. Baker, D.; Pryce, G.; Giovannoni, G.; Thompson, A. J., The therapeutic potential of
cannabis. Lancet Neurol 2003, 2 (5), 291-8.
80. Bostwick, J. M., Blurred Boundaries: The Therapeutics and Politics of Medical Marijuana.
Mayo Clinic Proceedings 2012, 87 (2), 172-186.
81. Russo, E. B., Beyond Cannabis: Plants and the Endocannabinoid System. Trends in
Pharmacological Sciences 2016, 37 (7), 594-605.
82. Di Marzo, V.; Piscitelli, F., The Endocannabinoid System and its Modulation by
Phytocannabinoids. Neurotherapeutics 2015, 12 (4), 692-698.
83. Donvito, G.; Nass, S. R.; Wilkerson, J. L.; Curry, Z. A.; Schurman, L. D.; Kinsey, S.
G.; Lichtman, A. H., The Endogenous Cannabinoid System: A Budding Source of Targets for
Treating Inflammatory and Neuropathic Pain. Neuropsychopharmacology 2018, 43 (1), 52-79.
84. Aso, E.; Ferrer, I., Cannabinoids for treatment of Alzheimer’s disease: moving toward the
clinic. Frontiers in Pharmacology 2014, 5.
85. Chagas, M. H.; Zuardi, A. W.; Tumas, V.; Pena-Pereira, M. A.; Sobreira, E. T.;
Bergamaschi, M. M.; dos Santos, A. C.; Teixeira, A. L.; Hallak, J. E.; Crippa, J. A., Effects of
cannabidiol in the treatment of patients with Parkinson's disease: an exploratory double-blind trial.
J Psychopharmacol 2014, 28 (11), 1088-98.

121
86. Curtis, A.; Mitchell, I.; Patel, S.; Ives, N.; Rickards, H., A pilot study using nabilone for
symptomatic treatment in Huntington's disease. Movement Disorders 2009, 24 (15), 2254-9.
87. Billakota, S.; Devinsky, O.; Marsh, E., Cannabinoid therapy in epilepsy. Current Opinion
in Neurology 2019, 32 (2), 220-226.
88. Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Devane, W. A.; Felder, C.;
Herkenham, M.; Mackie, K.; Martin, B. R.; Mechoulam, R.; Pertwee, R. G., International Union
of Pharmacology. XXVII. Classification of Cannabinoid Receptors. Pharmacol. Rev. 2002, 54 (2),
161-202.
89. Solymosi, K.; Kofalvi, A., Cannabis: A Treasure Trove or Pandora's Box? Mini-Reviews
in Medicinal Chemistry 2017, 17 (13), 1-1.
90. Robson, P., Therapeutic aspects of cannabis and cannabinoids. Br. J. Psychiatry 2001, 178
(2), 107-115.
91. Hua, T.; Vemuri, K.; Pu, M.; Qu, L.; Han, G. W.; Wu, Y.; Zhao, S.; Shui, W.; Li, S.;
Korde, A.; Laprairie, R. B.; Stahl, E. L.; Ho, J.-H.; Zvonok, N.; Zhou, H.; Kufareva, I.; Wu,
B.; Zhao, Q.; Hanson, M. A.; Bohn, L. M.; Makriyannis, A.; Stevens, R. C.; Liu, Z.-J., Crystal
Structure of the Human Cannabinoid Receptor CB1. Cell 2016, 167 (3), 750-762.e14.
92. Tsou, K.; Brown, S.; Sañudo-Peña, M. C.; Mackie, K.; Walker, J. M.,
Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system.
Neuroscience 1998, 83 (2), 393-411.
93. Herkenham, M.; Lynn, A. B.; Little, M. D.; Johnson, M. R.; Melvin, L. S.; De Costa,
B. R.; Rice, K. C., Cannabinoid receptor localization in brain. Proceedings of the National
Academy of Sciences 1990, 87 (5), 1932-1936.
94. Lutz, B.; Marsicano, G.; Maldonado, R.; Hillard, C. J., The endocannabinoid system in
guarding against fear, anxiety and stress. Nat. Rev. Neurosc. 2015, 16 (12), 705-718.
95. Berdyshev, E. V., Cannabinoid receptors and the regulation of immune response. Chem.
Phys. Lipids 2000, 108 (1-2), 169-190.
96. Kendall, D. A.; Yudowski, G. A., Cannabinoid Receptors in the Central Nervous System:
Their Signaling and Roles in Disease. Frontiers in Cellular Neuroscience 2017, 10.

122
97. Stumpf, A.; Zhang, H.-Y.; Özdoğan, T.; Pannasch, U.; Theis, A.-K.; Otte, D.-M.;
Wojtalla, A.; Rácz, I.; Ponomarenko, A.; Xi, Z.-X.; Zimmer, A.; Schmitz, D., Cannabinoid Type
2 Receptors Mediate a Cell Type-Specific Plasticity in the Hippocampus. Neuron 2016, 90 (4),
795-809.
98. Devane, W. A.; Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.;
Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R., Isolation and structure of a brain
constituent that binds to the cannabinoid receptor. Science 1992, 258 (5090), 1946-9.
99. Mechoulam, R.; Ben-Shabat, S.; Hanus, L.; Ligumsky, M.; Kaminski, N. E.; Schatz, A.
R.; Gopher, A.; Almog, S.; Martin, B. R.; Compton, D. R.; Pertwee, R. G.; Griffin, G.;
Bayewitch, M.; Barg, J.; Vogel, Z., Identification of an endogenous 2-monoglyceride, present in
canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 1995, 50 (1), 83-90.
100. Devane, W. A.; Axelrod, J., Enzymatic synthesis of anandamide, an endogenous ligand for
the cannabinoid receptor, by brain membranes. Proceedings of the National Academy of Sciences
1994, 91 (14), 6698-6701.
101. Sugiura, T.; Kishimoto, S.; Oka, S.; Gokoh, M., Biochemistry, pharmacology and
physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand. Progress in
Lipid Research 2006, 45 (5), 405-446.
102. Basavarajappa, B. S.; Shivakumar, M.; Joshi, V.; Subbanna, S., Endocannabinoid system
in neurodegenerative disorders. J. Neurochem. 2017, 142 (5), 624-648.
103. Ahn, K.; McKinney, M. K.; Cravatt, B. F., Enzymatic Pathways That Regulate
Endocannabinoid Signaling in the Nervous System. Chem. Rev. 2008, 108 (5), 1687-1707.
104. Bisogno, T.; Howell, F.; Williams, G.; Minassi, A.; Cascio, M. G.; Ligresti, A.; Matias,
I.; Schiano-Moriello, A.; Paul, P.; Williams, E.-J.; Gangadharan, U.; Hobbs, C.; Di Marzo, V.;
Doherty, P., Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of
endocannabinoid signaling in the brain. J. Cell Biol. 2003, 163 (3), 463-468.
105. Okamoto, Y.; Morishita, J.; Tsuboi, K.; Tonai, T.; Ueda, N., Molecular Characterization
of a Phospholipase D Generating Anandamide and Its Congeners. J. Biol. Chem. 2004, 279 (7),
5298-5305.
106. Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.; Giang, D. K.; Martin, B.
R.; Lichtman, A. H., Supersensitivity to anandamide and enhanced endogenous cannabinoid

123
signaling in mice lacking fatty acid amide hydrolase. Proceedings of the National Academy of
Sciences 2001, 98 (16), 9371-9376.
107. Dinh, T. P.; Carpenter, D.; Leslie, F. M.; Freund, T. F.; Katona, I.; Sensi, S. L.; Kathuria,
S.; Piomelli, D., Brain monoglyceride lipase participating in endocannabinoid inactivation.
Proceedings of the National Academy of Sciences 2002, 99 (16), 10819-10824.
108. Ueda, N.; Tsuboi, K.; Uyama, T., Metabolism of endocannabinoids and relatedN-
acylethanolamines: Canonical and alternative pathways. FEBS Journal 2013, 280 (9), 1874-1894.
109. Katona, I.; Freund, T. F., Multiple Functions of Endocannabinoid Signaling in the Brain.
Annu. Rev. Neurosci. 2012, 35 (1), 529-558.
110. Egertova, M.; Giang, D. K.; Cravatt, B. F.; Elphick, M. R., A new perspective on
cannabinoid signalling: complimentary localization of fatty acid amide hydrolase and the CB1
receptor in rat brain. Proceedings of the Royal Society of London B 1998, 265 (1410), 2081-2085.
111. Elphick, M. R., The evolution and comparative neurobiology of endocannabinoid
signalling. Philosophical Transactions 2012, 367 (1607), 3201-3215.
112. Chevaleyre, V.; Takahashi, K. A.; Castillo, P. E., ENDOCANNABINOID-MEDIATED
SYNAPTIC PLASTICITY IN THE CNS. Annu. Rev. Neurosci. 2006, 29 (1), 37-76.
113. Wilson, R. I.; Nicoll, R. A., Endogenous cannabinoids mediate retrograde signalling at
hippocampal synapses. Nature 2001, 410 (6828), 588-592.
114. Kreitzer, A. C.; Regehr, W. G., Retrograde Inhibition of Presynaptic Calcium Influx by
Endogenous Cannabinoids at Excitatory Synapses onto Purkinje Cells. Neuron 2001, 29 (3), 717-
727.
115. Ohno-Shosaku, T.; Maejima, T.; Kano, M., Endogenous Cannabinoids Mediate
Retrograde Signals from Depolarized Postsynaptic Neurons to Presynaptic Terminals. Neuron
2001, 29 (3), 729-738.
116. Heifets, B. D.; Castillo, P. E., Endocannabinoid Signaling and Long-Term Synaptic
Plasticity. Annu. Rev. Physiol. 2009, 71 (1), 283-306.

124
117. Gerdeman, G. L.; Ronesi, J.; Lovinger, D. M., Postsynaptic endocannabinoid release is
critical to long-term depression in the striatum. Nat. Neurosci. 2002, 5 (5), 446-451.
118. Robbe, D.; Kopf, M.; Remaury, A.; Bockaert, J.; Manzoni, O. J., Endogenous
cannabinoids mediate long-term synaptic depression in the nucleus accumbens. Proceedings of the
National Academy of Sciences 2002, 99 (12), 8384-8388.
119. Marsicano, G.; Wotjak, C. T.; Azad, S. C.; Bisogno, T.; Rammes, G.; Cascio, M. G.;
Hermann, H.; Tang, J.; Hofmann, C.; Zieglgänsberger, W.; Di Marzo, V.; Lutz, B., The
endogenous cannabinoid system controls extinction of aversive memories. Nature 2002, 418
(6897), 530-534.
120. Chevaleyre, V.; Heifets, B. D.; Kaeser, P. S.; Südhof, T. C.; Castillo, P. E.,
Endocannabinoid-mediated long-term plasticity requires cAMP/PKA signaling and RIM1alpha.
Neuron 2007, 54 (5), 801-12.
121. Chevaleyre, V.; Castillo, P. E., Heterosynaptic LTD of Hippocampal GABAergic
Synapses. Neuron 2003, 38 (3), 461-472.
122. Yasuda, H.; Huang, Y.; Tsumoto, T., Regulation of excitability and plasticity by
endocannabinoids and PKA in developing hippocampus. Proceedings of the National Academy of
Sciences 2008, 105 (8), 3106-3111.
123. Marsicano, G.; Goodenough, S.; Monory, K.; Hermann, H.; Eder, M.; Cannich, A.;
Azad, S. C.; Cascio, M. G.; Gutiérrez, S. O.; van der Stelt, M.; López-Rodriguez, M. L.;
Casanova, E.; Schütz, G.; Zieglgänsberger, W.; Di Marzo, V.; Behl, C.; Lutz, B., CB1
cannabinoid receptors and on-demand defense against excitotoxicity. Science 2003, 302 (5642),
84-8.
124. Larkin, K. T.; Brown, L. A.; Tiani, A. G., Autonomic and neuroendocrine response to
stress. In Cardiovascular Implications of Stress and Depression, Elsevier: 2020; pp 87-110.
125. Graham, B. M.; Langton, J. M.; Richardson, R., Pharmacological enhancement of fear
reduction: preclinical models. British Journal of Pharmacology 2011, 164 (4), 1230-1247.
126. McEwen, B. S., Brain on stress: How the social environment gets under the skin.
Proceedings of the National Academy of Sciences 2012, 109 (4), 17180-17185.

125
127. Di, S.; Malcher-Lopes, R.; Marcheselli, V. L.; Bazan, N. G.; Tasker, J. G., Rapid
Glucocorticoid-Mediated Endocannabinoid Release and Opposing Regulation of Glutamate and
γ-Aminobutyric Acid Inputs to Hypothalamic Magnocellular Neurons. Endocrinology 2005, 146
(10), 4292-4301.
128. Hill, M. N.; McLaughlin, R. J.; Pan, B.; Fitzgerald, M. L.; Roberts, C. J.; Lee, T. T. Y.;
Karatsoreos, I. N.; Mackie, K.; Viau, V.; Pickel, V. M.; McEwen, B. S.; Liu, Q. S.; Gorzalka,
B. B.; Hillard, C. J., Recruitment of Prefrontal Cortical Endocannabinoid Signaling by
Glucocorticoids Contributes to Termination of the Stress Response. The Journal of Neuroscience
2011, 31 (29), 10506-10515.
129. Hill, M. N.; Kumar, S. A.; Filipski, S. B.; Iverson, M.; Stuhr, K. L.; Keith, J. M.; Cravatt,
B. F.; Hillard, C. J.; Chattarji, S.; McEwen, B. S., Disruption of fatty acid amide hydrolase activity
prevents the effects of chronic stress on anxiety and amygdalar microstructure. Molecular
Psychiatry 2013, 18 (10), 1125-1135.
130. Kopp, B. L.; Wick, D.; Herman, J. P., Differential effects of homotypic vs. heterotypic
chronic stress regimens on microglial activation in the prefrontal cortex. Physiol. Behav. 2013,
122, 246-252.
131. Ruehle, S.; Rey, A. A.; Remmers, F.; Lutz, B., The endocannabinoid system in anxiety,
fear memory and habituation. J. Psychopharm. 2012, 26 (1), 23-39.
132. Cristino, L.; Bisogno, T.; Di Marzo, V., Cannabinoids and the expanded endocannabinoid
system in neurological disorders. Nat. Rev. Neurology 2020, 16 (1), 9-29.
133. O'Sullivan, S. E., An update on PPAR activation by cannabinoids. Br. J. Pharmacol. 2016,
173 (12), 1899-1910.
134. De Petrocellis, L.; Orlando, P.; Moriello, A. S.; Aviello, G.; Stott, C.; Izzo, A. A.; Di
Marzo, V., Cannabinoid actions at TRPV channels: effects on TRPV3 and TRPV4 and their
potential relevance to gastrointestinal inflammation. Acta Physiologica 2012, 204 (2), 255-266.
135. Begg, M.; Pacher, P.; Bátkai, S.; Osei-Hyiaman, D.; Offertáler, L.; Mo, F. M.; Liu, J.;
Kunos, G., Evidence for novel cannabinoid receptors. Pharmacology & Therapeutics 2005, 106
(2), 133-45.
136. Pertwee, R. G., GPR55: a new member of the cannabinoid receptor clan? Br. J. Pharmacol.
2007, 152 (7), 984-986.

126
137. Rouzer, C. A.; Marnett, L. J., Endocannabinoid Oxygenation by Cyclooxygenases,
Lipoxygenases, and Cytochromes P450: Cross-Talk between the Eicosanoid and Endocannabinoid
Signaling Pathways. Chemical Reviews 2011, 111 (10), 5899-5921.
138. McPartland, J. M., Cannabis and Eicosanoids. Journal of Cannabis Therapeutics 2001, 1
(1), 71-83.
139. Yu, M.; Ives, D.; Ramesha, C. S., Synthesis of Prostaglandin E2 Ethanolamide from
Anandamide by Cyclooxygenase-2. Journal of Biological Chemistry 1997, 272 (34), 21181-
21186.
140. Kozak, K. R.; Crews, B. C.; Morrow, J. D.; Wang, L.-H.; Ma, Y. H.; Weinander, R.;
Jakobsson, P.-J.; Marnett, L. J., Metabolism of the Endocannabinoids, 2-Arachidonylglycerol and
Anandamide, into Prostaglandin, Thromboxane, and Prostacyclin Glycerol Esters and
Ethanolamides. Journal of Biological Chemistry 2002, 277 (47), 44877-44885.
141. Kalant, H., Medicinal Use of Cannabis: History and Current Status. Pain Research and
Management 2001, 6 (2), 80-91.
142. Boggs, D. L.; Nguyen, J. D.; Morgenson, D.; Taffe, M. A.; Ranganathan, M., Clinical
and Preclinical Evidence for Functional Interactions of Cannabidiol and Δ9-
Tetrahydrocannabinol. Neuropsychopharmacology 2018, 43 (1), 142-154.
143. Pertwee, R. G., The diverse CB1and CB2 receptor pharmacology of three plant
cannabinoids: Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabivarin. Br. J.
Pharmacol. 2008, 153 (2), 199-215.
144. Pertwee, R. G.; Ross, R. A., Cannabinoid receptors and their ligands. Prostaglandins,
Leukotrienes and Essential Fatty Acids (PLEFA) 2002, 66 (2-3), 101-121.
145. Pistis, M.; Ferraro, L.; Pira, L.; Flore, G.; Tanganelli, S.; Gessa, G. L.; Devoto, P., Δ9-
Tetrahydrocannabinol decreases extracellular GABA and increases extracellular glutamate and
dopamine levels in the rat prefrontal cortex: an in vivo microdialysis study. Brain Research 2002,
948 (1-2), 155-158.
146. Patel, S.; Hillard, C. J., Pharmacological Evaluation of Cannabinoid Receptor Ligands in
a Mouse Model of Anxiety: Further Evidence for an Anxiolytic Role for Endogenous Cannabinoid
Signaling. J. Pharmacol. Exp. Ther. 2006, 318 (1), 304-311.

127
147. Schramm-Sapyta, N. L.; Cha, Y. M.; Chaudhry, S.; Wilson, W. A.; Swartzwelder, H. S.;
Kuhn, C. M., Differential anxiogenic, aversive, and locomotor effects of THC in adolescent and
adult rats. Psychopharmacology 2007, 191 (4), 867-77.
148. Ranganathan, M.; D’Souza, D. C., The acute effects of cannabinoids on memory in
humans: a review. Psychopharmacology 2006, 188 (4), 425-444.
149. Meier, M. H.; Caspi, A.; Ambler, A.; Harrington, H.; Houts, R.; Keefe, R. S. E.;
McDonald, K.; Ward, A.; Poulton, R.; Moffitt, T. E., Persistent cannabis users show
neuropsychological decline from childhood to midlife. Proc. Natl. Acad. Sci. USA 2012, 109 (40),
E2657-E2664.
150. Jackson, N. J.; Isen, J. D.; Khoddam, R.; Irons, D.; Tuvblad, C.; Iacono, W. G.; McGue,
M.; Raine, A.; Baker, L. A., Impact of adolescent marijuana use on intelligence: Results from two
longitudinal twin studies. Proc. Natl. Acad. Sci. USA 2016, 113 (5), E500-E508.
151. Mokrysz, C.; Freeman, T. P.; Korkki, S.; Griffiths, K.; Curran, H. V., Are adolescents
more vulnerable to the harmful effects of cannabis than adults? A placebo-controlled study in
human males. Translational Psychiatry 2016, 6 (11), e961-e961.
152. Mechoulam, R.; Hanuš, L. R., Cannabidiol: an overview of some chemical and
pharmacological aspects. Part I: chemical aspects. Chem. Phys. Lipids 2002, 121 (1-2), 35-43.
153. Burstein, S., Cannabidiol (CBD) and its analogs: a review of their effects on inflammation.
Biorg. Med. Chem. 2015, 23 (7), 1377-1385.
154. Thomas, A.; Baillie, G. L.; Phillips, A. M.; Razdan, R. K.; Ross, R. A.; Pertwee, R. G.,
Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor
agonists in vitro. Br. J. Pharmacol. 2007, 150 (5), 613-623.
155. Carrier, E. J.; Auchampach, J. A.; Hillard, C. J., Inhibition of an equilibrative nucleoside
transporter by cannabidiol: A mechanism of cannabinoid immunosuppression. Proceedings of the
National Academy of Sciences 2006, 103 (20), 7895-7900.
156. Malfait, A. M.; Gallily, R.; Sumariwalla, P. F.; Malik, A. S.; Andreakos, E.; Mechoulam,
R.; Feldmann, M., The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic
therapeutic in murine collagen-induced arthritis. Proceedings of the National Academy of Sciences
2000, 97 (17), 9561-9566.

128
157. De Gregorio, D.; McLaughlin, R. J.; Posa, L.; Ochoa-Sanchez, R.; Enns, J.; Lopez-
Canul, M.; Aboud, M.; Maione, S.; Comai, S.; Gobbi, G., Cannabidiol modulates serotonergic
transmission and reverses both allodynia and anxiety-like behavior in a model of neuropathic pain.
Pain 2019, 160 (1), 136-150.
158. Hampson, A. J.; Grimaldi, M.; Axelrod, J.; Wink, D., Cannabidiol and (-) 9-
tetrahydrocannabinol are neuroprotective antioxidants. Proc. Natl. Acad. Sci. USA 1998, 95 (14),
8268-8273.
159. Iffland, K.; Grotenhermen, F., An Update on Safety and Side Effects of Cannabidiol: A
Review of Clinical Data and Relevant Animal Studies. Cannabis and Cannabinoid Research 2017,
2 (1), 139-154.
160. Todaro, B., Cannabinoids in the Treatment of Chemotherapy-Induced Nausea and
Vomiting. Journal of the National Comprehensive Cancer Network 2012, 10 (4), 487-492.
161. Mead, A., Legal and Regulatory Issues Governing Cannabis and Cannabis-Derived
Products in the United States. Frontiers in Plant Science 2019, 10.
162. Ben-Shabat, S.; Fride, E.; Sheskin, T.; Tamiri, T.; Rhee, M.-H.; Vogel, Z.; Bisogno,
T.; De Petrocellis, L.; Di Marzo, V.; Mechoulam, R., An entourage effect: inactive endogenous
fatty acid glycerol esters enhance 2-arachidonoyl-glycerol cannabinoid activity. Eur. J.
Pharmacol. 1998, 353 (1), 23-31.
163. Sanchez-Ramos, J., The entourage effect of the phytocannabinoids. Ann. Neurol. 2015, 77
(6), 1083-1083.
164. Mechoulam, R.; Ben-Shabat, S., From gan-zi-gun-nu to anandamide and 2-
arachidonoylglycerol: the ongoing story of cannabis. Natural Product Reports 1999, 16 (2), 131-
143.
165. Russo, E. B., The Case for the Entourage Effect and Conventional Breeding of Clinical
Cannabis: No “Strain,” No Gain. Frontiers in Plant Science 2019, 9.
166. Johnson, J. R.; Burnell-Nugent, M.; Lossignol, D.; Ganae-Motan, E. D.; Potts, R.; Fallon,
M. T., Multicenter, Double-Blind, Randomized, Placebo-Controlled, Parallel-Group Study of the
Efficacy, Safety, and Tolerability of THC:CBD Extract and THC Extract in Patients with
Intractable Cancer-Related Pain. J. Pain Symptom Manag. 2010, 39 (2), 167-179.

129
167. Gallily, R.; Yekhtin, Z.; Hanuš, L. O., Overcoming the Bell-Shaped Dose-Response of
Cannabidiol by Using Cannabis Extract Enriched in Cannabidiol. Phamracol. Pharm. 2015, 06
(02), 75-85.
168. Berman, P.; Futoran, K.; Lewitus, G. M.; Mukha, D.; Benami, M.; Shlomi, T.; Meiri,
D., A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in
Cannabis. Sci. Rep. 2018, 8 (1).
169. Worth, T., Cannabis’s chemical synergies. Nature 2019, 572 (7771), S12-S13.
170. Proschak, E.; Stark, H.; Merk, D., Polypharmacology by Design: A Medicinal Chemist’s
Perspective on Multitargeting Compounds. J. Med. Chem. 2019, 62 (2), 420-444.
171. Keiser, M. J.; Roth, B. L.; Armbruster, B. N.; Ernsberger, P.; Irwin, J. J.; Shoichet, B.
K., Relating protein pharmacology by ligand chemistry. Nature Biotechnology 2007, 25 (2), 197-
206.
172. Reker, D.; Rodrigues, T.; Schneider, P.; Schneider, G., Identifying the macromolecular
targets of de novo-designed chemical entities through self-organizing map consensus. Proceedings
of the National Academy of Sciences 2014, 111 (11), 4067-4072.
173. Brodie, J. S.; Di Marzo, V.; Guy, G. W., Polypharmacology Shakes Hands with Complex
Aetiopathology. Trends in Pharmacological Sciences 2015, 36 (12), 802-821.
174. Lane, S. D.; Cherek, D. R.; Lieving, L. M.; Tcheremissine, O. V., Marijuana effects on
human forgetting functions. J Exp Anal Behav 2005, 83 (1), 67-83.
175. Ramaekers, J. G.; Moeller, M. R.; van Ruitenbeek, P.; Theunissen, E. L.; Schneider, E.;
Kauert, G., Cognition and motor control as a function of Delta9-THC concentration in serum and
oral fluid: limits of impairment. Drug Alcohol Depend. 2006, 85 (2), 114-22.
176. Ramaekers, J. G.; Kauert, G.; Theunissen, E. L.; Toennes, S. W.; Moeller, M. R.,
Neurocognitive performance during acute THC intoxication in heavy and occasional cannabis
users. Journal of Psychopharmacoly 2009, 23 (3), 266-77.
177. Hart, C. L., Effects of Acute Smoked Marijuana on Complex Cognitive Performance.
Neuropsychopharmacology 2001, 25 (5), 757-765.

130
178. D'Souza, D. C.; Ranganathan, M.; Braley, G.; Gueorguieva, R.; Zimolo, Z.; Cooper, T.;
Perry, E.; Krystal, J., Blunted psychotomimetic and amnestic effects of delta-9-
tetrahydrocannabinol in frequent users of cannabis. Neuropsychopharmacology 2008, 33 (10),
2505-16.
179. Russo, E. B., Taming THC: potential cannabis synergy and phytocannabinoid-terpenoid
entourage effects. Br. J. Pharmacol. 2011, 163 (7), 1344-1364.
180. Finlay, D. B.; Sircombe, K. J.; Nimick, M.; Jones, C.; Glass, M., Terpenoids From
Cannabis Do Not Mediate an Entourage Effect by Acting at Cannabinoid Receptors. Frontiers in
Pharmacology 2020, 11.
181. Pertwee, R. G., Handbook of cannabis. Oxford University Press: 2016; Vol. 1.
182. Mechoulam, R.; Burstein, S. H., Marijuana; chemistry, pharmacology, metabolism and
clinical effects. Academic Press: New York, 1973.
183. Marihuana Tax Act. United States of America, 1937; Vol. 50.
184. The Controlled Substances Act. Administration, U. S. D. E., Ed. 1970; Vol. 84.
185. Zarrabi, A. J.; Frediani, J. K.; Levy, J. M., The State of Cannabis Research Legislation in
2020. New England Journal of Medicine 2020, 382 (20), 1876-1877.
186. Solomon, R., Racism and Its Effect on Cannabis Research. Cannabis and Cannabinoid
Research 2020, 5 (1), 2-5.
187. Hart, C. L., Exaggerating Harmful Drug Effects on the Brain Is Killing Black People.
Neuron 2020, 107 (2), 215-218.
188. Voth, E. A., Guidelines for prescribing medical marijuana. West. J. Med. 2001, 175 (5),
305-6.
189. State Medical Marijuana Laws. https://www.ncsl.org/research/health/state-medical-
marijuana-laws.aspx (accessed 11/5/2021).

131
190. Piomelli, D.; Solomon, R.; Abrams, D.; Balla, A.; Grant, I.; Marcotte, T.; Yoder, J.,
Regulatory Barriers to Research on Cannabis and Cannabinoids: A Proposed Path Forward.
Cannabis and Cannabinoid Research 2019, 4 (1), 21-32.
191. Young, D., Big cannabis is rising high. New Zealand Drug Foundation: 2018; Vol. 29, p
14–17.
192. Windle, S. B.; Wade, K.; Filion, K. B.; Kimmelman, J.; Thombs, B. D.; Eisenberg, M.
J., Potential harms from legalization of recreational cannabis use in Canada. Canadian Journal of
Public Health 2019, 110 (2), 222-226.
193. Leggett, T., A review of the world cannabis situation. Bull Narc 2006, 58 (1-2), 1-155.
194. Harvey, A. L.; Edrada-Ebel, R.; Quinn, R. J., The re-emergence of natural products for
drug discovery in the genomics era. Nature Reviews Drug Discovery 2015, 14 (2), 111-129.
195. Atanasov, A. G.; Zotchev, S. B.; Dirsch, V. M.; Supuran, C. T., Natural products in drug
discovery: advances and opportunities. Nature Reviews Drug Discovery 2021, 20 (3), 200-216.
196. Henrich, C. J.; Beutler, J. A., Matching the power of high throughput screening to the
chemical diversity of natural products. Natural Product Reports 2013, 30 (10), 1284.
197. Harrison, C., Patenting natural products just got harder. Nat Biotechnol 2014, 32 (5), 403-
4.
198. Silvestri, A. P.; Oakdale, J. S., Ruthenium(II)-Catalyzed Intermolecular
Cyclo(co)trimerization of 3-Halopropiolamides with Internal Alkynes. American Chemical
Society (ACS): 2018.
199. Oakdale, J. S.; Sit, R. K.; Fokin, V. V., Ruthenium-Catalyzed Cycloadditions of 1-
Haloalkynes with Nitrile Oxides and Organic Azides: Synthesis of 4-Haloisoxazoles and 5-
Halotriazoles. Chemistry - A European Journal 2014, 20 (35), 11101-11110.
200. Bentley, R., Mycophenolic Acid: A One Hundred Year Odyssey from Antibiotic to
Immunosuppressant. Chemical Reviews 2000, 100 (10), 3801-3826.
201. Florey, H. W.; Jennings, M. A.; et al., Mycophenolic acid; an antibiotic from Penicillium
brevicompactum Dlerckx. Lancet 1946, 1 (6385), 46-9.

132
202. Birch, A. J.; Wright, J. J., A total synthesis of mycophenolic acid. Journal of the Chemical
Society D: Chemical Communications 1969, (14), 788.
203. Danheiser, R. L.; Gee, S. K.; Perez, J. J., Total synthesis of mycophenolic acid. Journal of
the American Chemical Society 1986, 108 (4), 806-810.
204. DE LA CRUZ, R. A.; TALAMAS, F. X.; VAZQUEZ, A.; MUCHOWSKI, J. M.,
ChemInform Abstract: Total Synthesis of Mycophenolic Acid. ChemInform 1997, 28 (52).
205. Covarrubias-Zúñiga, A.; Gonzalez-Lucas, A.; Domı
́ nguez, M. M., Total synthesis of
mycophenolic acid. Tetrahedron 2003, 59 (11), 1989-1994.
206. Brookes, P. A.; Cordes, J.; White, A. J. P.; Barrett, A. G. M., Total Synthesis of
Mycophenolic Acid by a Palladium-Catalyzed Decarboxylative Allylation and Biomimetic
Aromatization Sequence. European Journal of Organic Chemistry 2013, 2013 (32), 7313-7319.
207. Xia, S.; Gan, L.; Wang, K.; Li, Z.; Ma, D., Copper-Catalyzed Hydroxylation of
(Hetero)aryl Halides under Mild Conditions. Journal of the American Chemical Society 2016, 138
(41), 13493-13496.
208. Majumder, A.; Gupta, R.; Mandal, M.; Babu, M.; Chakraborty, D., Air-stable
palladium(0) phosphine sulfide catalysts for Ullmann-type C–N and C–O coupling reactions.
Journal of Organometallic Chemistry 2015, 781, 23-34.
209. Kocienski, P.; Bell, A.; Blakemore, P., 1-tert-Butyl-1H-tetrazol-5-yl sulfones in the
modified Julia olefination. Synlett 2000, 365-366.

Abstract (if available)

Abstract Chemistry is often regarded as the central science, providing a link between the study of life at the atomic and molecular level. As such, it plays a central role in our understanding of how the human body functions. Given the many problems surrounding the drug development cycle today, this thesis is a continued attempt to return to the chemical drawing board in an effort to provide new tools and machinery for the development of successful therapeutics. In Chapter 1, we explore a new synthetic methodology that has the potential to open the path to the discovery of new molecular entities. In Chapter 2, we harness this synthetic strategy in combination with new tools in computational biology to explore new chemical space in the hope of finding successful ligands for important biological targets. In Chapter 3, we create a bridge between Big Pharma and ancient herbal medicine, addressing the unexplored polypharmacological powers of plants and addressing the various regulatory hurdles associated with their use to treat ailments. Lastly, in Chapter 4, we return to the tedious tradition of natural product synthesis, paying homage to nature and her awe- inspiring acuity.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Grotsch, Katharina (author)

Core Title Unlocking tools in chemistry to facilitate progress in drug discovery

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2022-05

Publication Date 05/10/2024

Defense Date 05/10/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cannabinoid receptors,click chemistry,combinatorial chemistry,drug discovery,endogenous cannabinoid system,isoxazoles,OAI-PMH Harvest,synthetic methods,triazoles

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Fokin, Valery (committee chair), Katritch, Vsevolod (committee member), Prakash, Surya (committee member)

Creator Email grotsch@usc.edu,katharinagrotsch@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111313289

Unique identifier UC111313289

Document Type Dissertation

Format application/pdf (imt)

Rights Grotsch, Katharina

Type texts

Source 20220517-usctheses-batch-942 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu