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Development of selective covalent probes to identify and modulate protein targets

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Development of selective covalent probes to identify and modulate protein targets

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Content DEVELOPMENT OF SELECTIVE COVALENT PROBES TO IDENTIFY AND MODULATE
PROTEIN TARGETS

by

BIANCA A. ESPINOSA

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)

December 2022

Copyright 2022 Bianca A. Espinosa
ii

Table of Contents

List of Figures…………………………………………………………………………….............iv

Abstract………………………………………………………………………………….............viii

Chapter 1. Synthesis of electrophilic 4-anilino-quinazoline derivatives and characterization of
their covalent proteomic targets…………………………………………………………………...1

1.1 Introduction…………………………………………………………………………………....1
1.1.1 Covalent inhibitors…………………………………………………………………..1
1.1.2 Chemical proteomics………………………………………………………………..4

1.2 Investigate the target space of electrophilic 4-anilinoquinazoline derivatives using chemical
proteomics……………………………………………………………………………………..6
1.2.1 Design and synthesis of a panel of electrophilic 4-anilinoquinazoline
derivatives…………………………………………………………………………...7
1.2.2 Labeling of live cell proteomes…………………………………………………….10

1.3 Experimental Details………………………………………………………………………...14
1.3.1 Chemical synthesis…………………………………………………………………14

1.4 References……………………………………………………………………………………21

Chapter 2. Characterization of select covalent targets for electrophilic 4-anilinoquinazolines….22

2.1 Guanosine Monophosphate Synthase (GMPS)…………………………….…………………22
2.2 Designing Compound 1 competitors for future experiments…………………………………26
2.3 Investigating selectivity and labeling efficiency of 1 through competition and time-course
experiments……………………………………………………………………...……………….32
2.4 In vitro labeling of GAT domain of GMPS…………………………………………………...36
2.4.1 Generating WT GAT domain and C104A…………………………………………...36
2.4.2 In vitro labeling of GAT domain of GMPS…………………………………………38

2.5 In vitro labeling of 3x FLAG GMPS………………………………………………………….39
2.6 Biochemical IC50 …………………………………………………………………………….39
2.7 Quinazoline compound effects on GMPS activity in vivo …………………………………..41
2.8. Effects of 1 on cancer cell lines and viral infection………………………………………….42
2.9 Experimental Details…………………………………………………………….…………...45
2.9.1 Chemical Synthesis………………………………………………………………...45
2.9.2 Methods and materials for biological characterization…………………………….65

2.10 References…………………………………………………………………………………..75

Chapter 3. Novel covalent compounds with anti-COVID activities……………………………..76
iii

3.1 Generation of open-ring GMPS analogs 2-d and 2-c………………………………………….77
3.1.1 2-d-comp can increase IFN signaling activation……………………………………81
3.2 Design and synthesis of 1
st
generation panel………………………………………...……….82
3.2.1 IFN reporter assay of 1
st
generation panel………………………………..…………83
3.2.2 1
st
Gen treatment in SARS-CoV-2 infected Caco2 cells……………………………85
3.3 Design and synthesis of 2
nd
generation panel…………………………………………………88
3.3.1 2
nd
Gen treatment in SARS-CoV-2 infected Caco2 cells ………………………….89
3.4 Design and synthesis of 3
rd
generation panel…………………………………………………92
3.4.1 3
rd
Gen treatment in SARS-CoV-2 infected Caco2 cells ………………………….93
3.5 Best Performing Compounds in SARS-CoV-2 Infected Caco2 Cells
and Generation of C9-probe……………………………………………………………………...94
3.6 Target engagement of 2-d & C9-probe with SARS-CoV-2 viral proteins…………………...95
3.7 CTPS1 labeling……………………………………………………………………….............98
3.8 Target engagement of 2-d and C9-probe on Nsp12 …………………………………………99
3.9 Site identification of C9-probe on Nsp12…………………………………………………..102
3.10 C9 demonstrates anti-viral activity in vivo………………………………………………..105
3.13 Experimental details……………………………………………………………….............112
3.13.1 Chemical synthesis………………………………………………………………112
3.13.2 Methods and materials for biological characterization…………………………..160
3.14 References……………………………………………………………………..…………..165

iv

List of Figures
Figure 1. Covalent inhibitor kinetics…………………………………………………….………...2
Figure 2. Statistics on publications of covalent drugs……………………………………………...2
Figure 3. Approved covalent drugs by therapeutic indication……………………………………...3
Figure 4. Selected electrophiles and positions to be investigated for their dependence on target
space……………………………………………………………………………………………….6
Figure 5. Panel of quinazoline derivatives………………………………………………………..8
Figure 6. General synthetic scheme of the electrophile quinazolines……………………………....9
Figure 7. Electrophile quinazoline probes in HEK 293T cells……………………………………10
Figure 8. Dose-response labeling of VATPase, GMPS, and ALDH1A2…………………………13
Figure 10. Identification of GMPS via pull-down………………………………………………..23
Figure 11. De-novo purine synthetic pathway……………………………………………............24
Figure 12. GMP synthetase and glutaminase domains……………………………………………25
Figure 13. Compound 1 analogs looking at changes to electrophile type and core and aniline
modifications……………………………………………………………………………………..26
Figure 14. Labeling experiment in HEK 293 of Compound 1 analogs with altered
electrophiles……………………………………………………………………………………...27
Figure 15. Labeling experiment in HEK 293 of Compound 1 in competition with aniline
substituent modified analogs……………………………………………………………………..29
Figure 16. Dose-response competition of 1 against 1-g, 1-h, 4, and 1-j…………………………...30
Figure 17. Labeling of 1-c & 1-d in HEK293. Labeling of GMPS can be seen with only 1-
d………………………………………………………………………………………………..…31
v
Figure 18. Lead quinazoline compounds…………………………………………………………33
Figure 19. Labeling comparison of vATPase and GMPS………………………………………34
Figure 20. Dose-response labeling using 1 at various concentration points in HEK293
cells………………………………………………………………………………………............35
Figure 21. Competition labeling experiment between compounds 1 and 4………………..........36
Figure 22. Purification of GMPS WT GAT and C104A GAT…………………………………..37
Figure 23. In vitro labeling of WT GAT and C104A GAT………………………………………38
Figure 24. IC50 determination of GMPS………………………………………………………….40
Figure 25. IC50 values of selected GMPS compounds…………………………………………….41
Figure 26. Time-dependent inhibition of GMPS in cells…………………………………………42
Figure 27. NCI screening of 1…………………………………………………………………….43
Figure 28. Viral model to assess compound 1……………………………………………………44
Figure 29. Generation of open-ring analogs……………………………………………………...78
Figure 30. Treatment of 2-d and 2-c in cells and purified recombinant protein………………….79
Figure 31. Design of competitor 2-d-comp………………………………………………............80
Figure 32. IFN-B mRNA expression assay of 1, 2-d, 2-c, and 2-d-comp………………………....81
Figure 33. First generation Nsp12 panel…………………………………………………............82
Figure 34. IFN reporter assay of 1
st
generation panel……………………………………………84
Figure 35. IFN reporter assay repeated for best-performing drugs at lower
concentrations…………………………………………………………………………...……….85
Figure 36. SARS-CoV-2 infected Caco2 cells treatment………………………………..………86
Figure 37. 2
nd
generation Nsp12 panel …………………………………………………………..88
Figure 38. SARS-CoV-2 infected Caco2 cells treatment of 2
nd
gen panel………………………91
vi
Figure 39. 3
rd
generation Nsp12 panel……………………………………………………………92
Figure 40. SARS-CoV-2 infected Caco2 cells treatment of 3
rd
gen panel………………………93
Figure 41. SARS-CoV-2 infected Caco2 cells treatment of best performing compounds from all
three panels……………………………………………………………………………………...94
Figure 42. Treatment of 2-d and C9-probe on HEK293 cells transfected with various SARS-CoV-
2 proteins…………………………………………………………………………………………97
Figure 43. 2-d Labeling of IP FLAG-CTPS1…………………………………………………….98
Figure 44. Competition labeling experiment between 2-d (2uM) and the 22 compounds from the 3
generations of panels……………………………………………………………………………..99
Figure 45. Competition labeling experiment on cells transfected with Nsp12 and Nsp15………100
Figure 46. Competition labeling experiment on cells transfected with Nsp12…………………..101
Figure 47. Treatment and fluorescence visualization of 2-d against panel of alanine Nsp12
mutants………………………………………………………………………………….............103
Figure 48. Treatment and fluorescence visualization of C9-probe against panel of alanine Nsp12
mutants………………………………………………………………………………………….104
Figure 49. Workflow schematic to generate SARS-CoV-2-infected AAV-hACE2 mice for animal
studies…………………………………………………………………………………………..105
Figure 50. Relative mRNA abundance of IFNs in lungs harvested from SARS-CoV-2-infected
AAV-hACE2 mice treated with 2-d-comp and C9……………………………………………..106

Figure 51. Relative RNA abundance of SARS-CoV-2 viral proteins in lungs harvested from mice
treated with 2-d-comp and C9, and lung viral titer measurements………………………………107

Figure 52. C9 increases IFN-B and ISGs expression in the lungs of SARS-Co-V-2 infected AAV-
hACE2 delivered mice………………………………………………………………………….108

Figure 53. C9 inhibits SARS-CoV-2 gene expression and viral titers in lungs of AAV-hACE2
delivered mice…………………………………………………………………………………..109

vii
.
Figure 54. C9 helps reduce loss of body % weight and increases percent survival compared to
control…………………………………………………………………………………………..110

Figure 55. C9 reduces lung tissue inflammation in SARS-CoV-2 infected mice…….………..111
viii
Abstract

One of the most important aims of drug discovery is to develop ligands that are both
potent and selective for target proteins. Conventional medical chemistry methods rely on
having adequate prior knowledge of a target of interest, identifying a potential scaffold, and
modifying the scaffold to instigate selective inhibition. Although these traditional methods
have proven powerful in delivering potent ligands in many cases, they alone often do not
produce strong drug candidates in a timely or efficient manner. To meet this challenge, our
lab has developed a robust chemical proteomic method which allows us to develop potent
and selective covalent small molecules that can be used to rapidly identify and modulate
diverse protein targets. In this dissertation, I will discuss three projects in which this
approach was utilized to identify unique protein targets, and the design, synthesis, and
biological characterization of the electrophilic small molecule probes that were responsible
for their identification.

Chapter 1 details our efforts to investigate the dependence of electrophile position
and electrophile type on the ability of a pharmacophore to covalently bind to different
proteins. A panel of electrophilic probes was designed and developed in such a way that we
were able to study how minor changes in electrophilic position and type can dramatically
alter the range of possible targets of a conserved scaffold. We identified several unique
protein targets that were shown to be covalently labeled by our probes through a series of
Cu-catalyzed click-chemistry experiments that allowed for target visualization or
enrichment.

Chapter 2 focuses on one of the targets identified in the first chapter, Guanosine
Monophosphate Synthetase (GMPS). Structural activity relationship studies were performed
for optimization and a series of in vitro labeling experiments allowed for the visualization
of the covalent interaction of our hit compound with GMPS. We identify the site-of-
modification to be a single cysteine residue within the glutamine amido transferase domain
of the protein, and prove that mutagenesis of this residue abolishes labeling. The biochemical
effects of our compound against GMPS are demonstrated through mass spec analysis and
reveals that binding of our compound leads to an accumulation of substrate and subsequent
reduction of GMP production. Lastly, in vivo experimentation reveals both the anti-cancer
and anti-viral activity of our hit compound and provides compelling data for our
comprehensive study.

Chapter 3 looks at the design and synthesis of a covalent small molecule that was
inspired by our lead compound in Chapter 2. We utilize in vitro data of a SARS-CoV-2
cellular model to inform our design and synthesis of three generations of analogs. Of the
compounds identified to exhibit the strongest anti-viral activity in vitro, we synthesized
analogous probe versions, giving us the ability to perform visualization labeling
experiments. Through a series of pull-downs and enrichments, we identified nonstructural
protein 12 (Nsp12) to be the protein target being labeled, and believe the binding of our
compounds to this target is responsible for the observed anti-covid activity. Mass spec
analysis revealed several potential cysteine sites-of-modification, and site-directed
mutagenesis of these cysteines helped to reveal to be what we believe is the primary cysteine
ix
being labeled on Nsp12. Lastly, through collaboration efforts, we have demonstrated that
our lead compound exhibits anti-Covid activity in both cellular and animal models.

1
Chapter 1

1.1 Introduction

1.1.1 Covalent Inhibitors
Small molecules are chemical compounds that have been synthetically made to have
targeted effects when introduced into the body. They are typically administered orally, and
thus possess low molecular weight and good bioavailability. Generally speaking, small
molecules are utilized for their biological activities as therapeutics for the treatment and
prevention of conditions. Recent advancements in the design of chemical compounds has
led to the development of small molecule “tools” that have proven useful in biological
studies. Rather than relying on these tools for their therapeutic effects, these compounds are
used for their ability to probe systems and proteins, providing researchers with new
biological insights.
Though covalent small-molecule inhibitors have been historically met with
resistance from the pharmaceutical industry due to toxicity and safety concerns, recent
developments over the last decade have led to a reversal of this view (Fig 2)
1,2
. Traditionally,
small-molecules have been designed to interact with their protein targets relying on physical
forces such as hydrogen bonding and van der waals interactions.
3
The binding of such small-
molecules to their target proteins is rapid and reversible, requiring frequent dosing. Covalent
inhibitors on the other hand, due to their binding nature, result in a prolonged therapeutic
response from a longer drug-target interaction.

2

Figure 1. Covalent binding of a small-molecule inhibitor to an enzyme often involve an
initial reversible binding, followed by an irreversible step of a chemical bond formation
between the inhibitor and the enzyme.
3

There exists a two-step process for the binding of covalent inhibitors to their target (Fig 1).
The warhead chosen for any covalent inhibitor will dictate whether the reaction is reversible
or irreversible and what amino acid on the enzyme-target will be the site of modification.
Over the years, numerous warheads have been designed and implemented to target a range
of amino acids including cysteine, tyrosine, lysine, methionine, serine, glutamate and
asapartate.
3

Figure 2. Number of publications on covalent drugs per decade based on a key-word
search in SciFinder.
3

The resurgence of efforts to develop covalent inhibitors has resulted in nearly 50
FDA-approved covalent drugs with several more currently in trials.
3
Of the 10 best selling
3
drugs in the United States, three are covalent inhibitors.
1
The disease relevance of the current
FDA approved covalent inhibitors span across numerous therapeutic indications including
cancer, CNS, inflammation, and cardiovascular diseases.
4
The variety exemplifies the vast
therapeutic applications this classification of drug can have.

Figure 3. Approved covalent drugs by therapeutic indication. Among the covalent inhibitors
on the market, about 28% are used in oncology related targets, 23% are used in CNS and
cardiovascular disorders, 21% are anti-infectives (mostly theβ-lactam class of antibiotics).
3

Recent applications of covalent drugs have been applied to target Bruton’s tyrosine
kinase (BTK) and to the development of PROTACs in which covalent drugs such as
Ibrutinib were chosen to be implemented into bi-functional molecules.
5
Still, it is safe to say
much work can be done in the area of covalent drug development, both in terms of warhead
diversification efforts as well as expanding the types of proteins covalent drugs are designed
to target.
Conventionally, covalent compounds have been discovered through library
screening or structure-based design in what can be referred to as a target-to-compound
4
strategy. With these approaches, a protein target or family of proteins are chosen as the focus
of study. Small molecule libraries are then structurally designed to interact with the
protein(s) of interest using computational modeling, crystal structures, and medicinal
chemistry. This approach, while a traditional means of drug development, can sometimes be
extremely time-consuming, as the trial-and-error of structural activity relationship studies
can often span long periods of time. Alternatively, one can use a reversed method such as a
compound-to-target strategy, which can accelerate the development of promising drug
candidates while identifying potentially novel therapeutic targets.

1.1.2 Chemical Proteomics
Chemical proteomics is an area of chemical biology that utilizes synthetic chemistry
to generate small molecules called chemical probes. Upon binding to a protein, these
chemical probes can reveal the mode-of-action of the molecule in addition to elucidating the
protein’s function.
6
Chemical proteomics can be used to identify the targets of small
molecules in live cells, thus offering advantageous approach to target deconvolution. There
are several types of chemical proteomic approaches including affinity chromatography,
photoaffinity labeling, and activity-based protein profiling (ABPP). In all instances, a small
molecule is tethered to some other entity such as an immobilized bead, biotin, an alkyne, or
a photoaffinity group that is used for enrichement or detection.
7
Bioorthogonal chemistry
has emerged as a popular means of installing reporter tags to the small molecule of interest
due to the small size of bioorthogonal groups and the synthetic feasibility of their
incorporation.
5
Our lab utilizes an emerging chemical-proteomic method in order to selectively and
covalently target proteins without prior knowledge of their binding site, affinity, phenotype,
etc. Our approach involves designing probes containing an electrophile, or an electrophilic
“warhead”, that resides on a heterocyclic scaffold known to be a pharmacophore. These
probes are also equipped with a “clickable” tag, often a terminal alkyne or an azide group.
The electrophilic warhead is devised to target reactive, nucleophilic amino acid residues like
cysteine for covalent protein labeling when introduced into the proteome, while the clickable
tag can be subsequently conjugated with a fluorophore or biotin group with Cu-catalyzed
click-chemistry for target visualization or enrichment.
Using this emerging method, our lab previously synthesized an electrophilic 4
anilinoquinazoline probe that selectively and potently bound V-ATPase, a multisubunit
enzyme responsible for regulating acidity of various vesicles such as endosomes and
lysosomes in cells.
8
It was the first covalent small-molecule modulator of VATPase to be
reported, and because of our emerging method, we have been able to study what was once a
poorly understood mechanism of action owing to the limitations of conventional methods.
We have expanded our approach and synthesized a panel of analogous probes that contain
different electrophiles at different positions around the 4-anilinoquinazoline core to
investigate the dependence of target space on electrophilic position. We have introduced our
compounds into the proteome and have identified the major targets of each probe. We are
encouraged to observe that a few of them show distinct target profiles from that of our
original probe. We have begun characterizing the chemical nature of these probes, their
respective protein targets, and the inhibition-induced phenotypes they exhibit in regard to
disease relevance through both inter and trans-disciplinary collaboration efforts. With our
6
method, we anticipate being able to do the same with many more proteins in the future. We
believe that our chemical proteomic approach will result in the elucidation of new
mechanistic, physiological, and pathological information of diverse proteins. It is an exciting
and novel endeavor with great potential and promise that will employ the inter-disciplinary
methods of chemical biology, biochemistry, and organic synthesis, to ultimately contribute
to the larger chemical biology interface effort.

1.2 Investigate the target space of electrophilic 4-anilinoquinazoline derivatives
using chemical proteomics

We set out to utilize this new chemical genetic approach by selecting a pharmacophore
and investigating the dependence of electrophile position and type in regard to the scope of
proteins that are able to be labeled. The selected scaffold for this project was chose to be 4-
anilinoquinazoline for its prevalence in approved drugs as well as the thorough study
previously published in our group.
8

Figure 4. Selected electrophiles and positions to be investigated for their dependence on
target space.
7

1.2.1 Design and synthesis of electrophilic 4-anilinoquinazoline derivatives

Three cysteine-selective electrophiles were chosen for this endeavor including
chloracetamide, acrylamide, and isothiocyanide (Figure 4). These were chosen for both their
varied mild reactivity, with chloracetamide being the most of reactive of the three
9
, and for
their size. In comparing the chloroacetamide and acrylamide electrophiles, the presence of
an extra carbon from which the covalent interaction with the thiol cysteine would occur,
provides a structurally small but potentially significant binding effect on the probes within
the binding pocket of proteins. The positioning of these electrophiles, as shown in Figure 4,
would span across the entirety of the 4-anilinoquinazoline scaffold in order to fully
investigate the potential protein coverage of this pharmacophore relative to the limits of our
study. The term “electrophile walking” in which one can envision the selected electrophiles
being “walked” to different positions along the scaffold was coined to further describe our
rationale for this project. To implement our chemical-genetic approach, the handle selected
to be incorporated onto our probes was the meta-propargyloxy aniline due to its flexible
nature and it being synthetically feasible to incorporate onto various positions on the 4-
anilinoquinazoline.

8

Figure 5. A panel quinazoline derivatives harbor three different electrophilic warheads at
various positions on the quinazoline core or the anilino substituent.

At the time of joining this project, only the 8 probes with electrophile at positions 6,
7, and 8 (Q6-chloroacetamide, Q7- chloroacetamide, etc.), were yet to be synthesized. For
these probes, the propargyloxy aniline was synthesized separately from 3- amino phenol and
subsequently added to 4-chloro-6-nitroquinazoline. (Figure 6).

9

Figure 6. General synthetic scheme of the electrophile quinazolines.
The electrophile, to prevent decomposition during subsequent synthetic steps, was
the last to be added. The completed panel shown in Figure 5 covered the entirety of the
perimeter of the 4-anilinoquinazoline scaffold, as intended. The inclusion of the
electrophiles both on the quinazoline core as well as the phenyl group, provided two very
different binding configurations for these probes. On the other hand, changing each
electrophile to one carbon adject provided a small change of distance but, as we hoped would
be revealed in the binding assays, could have significant effects on the attenuation of these
probes to potential binding partners.

10
1.2.2 Labeling of live cell proteomes

To investigate the targets of the panel of quinazoline probes, Dr. Arunika Ekanayke,
a former graduate student in the Zhang lab, treated HEK 293T cells at three final
concentration points (01, 3, 1 uM) followed by lysis of the cells, protein normalization,
addition of “click” reagents tamra-N3 and Cu (II), 1-hr incubation, and lastly SDS-PAGE.

Figure 7. Initial treatment of probe in HEK 293 T cells. Only Probes 1,2,3 are shown as
they revealed distinct protein targets as highlighted in the figure.

11
The results for the initial treatment of these probes can be seen in Figure 7. Note that the
lanes for only probes 1, 2, and 3 are shown in the figure as these were the probes with the
most distinctly and uniquely labeled protein targets and will be the focus for the remainder
of this chapter.
We were pleasantly surprised to see that probes 1, 2, and 3 all had unique labeling
patterns, and noticeable labeling at 0.1 uM. It is important to note the existence of a
background, whether it be from characteristics of the gel or the labeling of high-abundance
cysteine-containing proteins. Additionally, it is important to point out that the intensity of
any specific band is not necessarily coordinated to labeling efficiency of these probes. The
intensity of any one band over another may indicate high endogenous levels of that specific
protein in a specific cell-line. Thus the intensity of a protein can vary significant from cell
line to cell line depending on endogenous protein levels. It is also possible that labeling
intensity can be the result of multiple cysteines being labeled on a single protein and not the
catalytic cysteine, which is the cysteine of most interest for the development of a small
molecule that can perturb protein function. When looking at the labeling patters seen in
Figure 7, there were specifics characteristics that stood out. One being the unique labeling
pattern as discussed previously. The other being the presence of three unique bands, each
labeled by a different probe as shown boxed in Figure 7, that reach what is commonly
referred to as “saturation” of the signal. In other words, the intensity of the fluorescence at
0.1 uM is similar if not identical, as this case for compound 1, to that of 1 uM. This feature
is indicative of the probe efficiently reacting with or occupying all the available cysteines
within that protein of interest. For all 3 probes, we see nearly complete saturation of the
signal at 0.1 uM.
12
As discussed early, the core structure of these probes was based off a previously
reported probe developed in our lab that was shown to selectively and potently bound V-
ATPase, a multisubunit enzyme responsible for regulating acidity of various vesicles such
as endosomes and lysosomes in cells.
8
Compound 2, with the chloroacetamide being at
position 7 on the quinazoline core, is structurally very similar to that of the V-ATPase probe
developed my Y. Chen. The difference in these probes is the handle where 2’s handle is
terminal alkyne attached to a propargyloxy aniline and Y. Chen’s probe is a direct alkyne to
the aniline. We were aware of the structural similarities when we were developing the panel
and saw compound 2 as a way to confirm a labeling profile. The bands shown boxed in
Figure 7 at around 70 kDa was consistent with what Y. Chen and others reported as being
the vacuolar ATPase subunit A.
What was most exciting to us, was the prominent band (p75) being labeled by
compound 1. At 75 kDa, the identity of the protein was unknown, but it was clear that 1
couple potently and selectively label a major target protein in the human proteome. To gain
a better understanding of the labeling efficiency of 1, 2, and 3, a dose-respose labeling
experiment, similar to the one performed in Figure 7 but with more concentration points,
was performed (Figure 8).

13

Figure 8. Dose-response labeling of VATPase, GMPS, and ALDH1A2 by compounds 2, 1
3.

The results of the dose-response labeling as show in Figure 8 were promising, as all 3 probes
demonstrated clear labeling of their protein targets. Compound 1 which quickly the target
we were most interested in identifying, showed near saturation of labeling with as little as
10 nM. Not only was saturation reached at such a low concentration, but the “cleanliness”
of the labeling is noteworthy. We then moved onto identifying the unknown target of
compound 1.

Ultimately, we were very pleased with the outcomes of this project. While
Compound 2, with its similar structure to that of Y. Chen’s probe, labeled V-ATPase, it is
noteworthy to point out that by moving the chloroacetamide one carbon over on the 4-
anilinoquinazoline (Compound 1) a new, distinct target was prominently labeled. Similarly,
moving the electrophile one carbon over in the other direction, compound 3 too labeled a
new protein target. Thus, our goal of investigating the dependence of electrophilic position
14
on the 4-anilinoquinazoline core in regards to human labeling in the human proteome was
successful.

1.3 Experimental Details
1.3.1 Chemical synthesis
Reagents and solvents were obtained from commercial suppliers and used without further
purification, unless otherwise stated. Flash column chromatography was carried out using
an automated system (Teledyne Isco CombiFlash. Reverse phase high performance liquid
chromatography (RP-HPLC) was carried out on a Shimadzu HPLC system. All anhydrous
reactions were carried out under nitrogen atmosphere. NMR spectra were obtained on
Varian VNMRS-500, VNMRS-600, or Mercury-400. The following abbreviations were
used to explain NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p
= pentet, m = multiplet, br = broad.

15
Preparation of probes and competitors for labeling experiments

3-(prop-2-yn-1-yloxy)aniline (1a)

NaOH pellets (1.49 g, 37.3 mmol) were weighed out and added to methanol (69.4 mL). 3-
aminophenol (3.82 g, 35 mmol) was then added and the solution was kept in an ultrasonic
bath for about 15 minutes until everything was completely dissolved. The volatiles were
evaporated by rotary evaporation and the residual water was removed by four successive co-
distillations with abs EtOH (27.8 mL, 4x). The obtained dried residue was dissolved in
CH3CN (69.4 mL) and propargyl bromide (80 wt. % solution in toluene, 4.62 mL, 41.56
mmol) was added in three portions over a 1hr period. The reaction mixture was allowed to
stir overnight. It was concentrated under rotary evaporation and the residue was partitioned
between aq NaOH (0.1 M, 69.4 mL) and diethyl ether (69.4 mL). The layers were separated
and the aqueous layer was extracted with diethyl ether (2x69.4 mL). The combined layers
were washed brine and dried over Na2SO4. Evaporation of the solvent gave a brownish oily
residue which was re-dissolved in MeOH (20.83 mL) and combined with in-situ generated
HCl (prepared from 104.2 mL of MeOH and 5.3 mL (74.76 mmol) of acetyl chloride). The
volatiles were evaporated resulting in a brownish solid which was resuspended in boiling
H
2
N OH
Br
+
1. NaOH, MeOH, rt
2. in-situ HCl, MeOH, rt
ClH
3
N O
1a
16
EtOAc (34.7 mL). After cooling the suspension to room temperature, white crystals were
collected by filtration and dried to afford compound 1a. (3.14 g, 49%).

1
H NMR (400 MHz, DMSO-d6) δ 9.73 (br s, 3H), 7.40 – 7.29 (m, 1H), 6.94 – 6.81 (m, 3H),
4.80 (d, J = 2.4 Hz, 2H), 3.60 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, DMSO-d6) δ 158.26, 134.14, 131.06, 116.19, 114.01, 110.46, 79.22,
79.13, 56.17.

6-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)quinazolin-4-amine (1)

4-chloro-6-nitroquinazoline (105 mg, 0.5 mmol) and 1a (101 mg, 0.55 mmol) were dissolved
in 2 mL iPrOH. Triethylamine (77 uL, 0.55 mmol) was added and the solution was allowed
17
to stir at room temperature for 4 hours. The reaction mixture was diluted with EtOAc and
washed with 10% Na2CO3 followed by brine. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation and the residue was purified using flash column
chromatography (EtOAc/Hex = 0-50%) to give 1b (139 mg, 87%) as a dark orange solid.

1
H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 9.81 (d, J = 2.3 Hz, 1H), 8.93 (s, 1H), 8.72
(dd, J = 9.2, 2.3 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H), 7.59 – 7.30 (m, 3H), 6.96 (d, J = 7.6 Hz,
1H), 4.84 (d, J = 2.4 Hz, 2H), 3.63 (t, J = 2.3 Hz, 1H).

N
4
-(3-(prop-2-yn-1-yloxy)phenyl)quinazoline-4,6-diamine (1c)

1b (139 mg, 0.43 mmol) was dissolved in 4 mL MeOH. Zn (140.6 mg, 2.15 mmol) and
NH4Cl (115 mg 2.15 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and concentrated by rotary evaporation to
yield 1c (111 mg, 89%) as a dark yellow oil, which was used in the next reaction without
further purification.

1
H NMR (400 MHz, Chloroform-d) δ 8.62 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.59 (s, 1H),
7.33 – 7.12 (m, 4H), 6.91 (s, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 4.72 (d, J = 2.4 Hz, 2H),
4.04 (br s, 2H), 2.54 (t, J = 2.4 Hz, 1H).

18

2-chloro-N-(4-((3-(prop-2-yn-1-yloxy)phenyl)amino)quinazolin-6-yl)acetamide (1)

55 mg of 1c (0.19 mmol) was dissolved in anhydrous DCM/THF (2.5 mL, 1:4) under
nitrogen gas. DIEA (73.1 uL, 0.42 mmol) was added via syringe. Chloroacetyl chloride (18.2
uL, 0.23 mmol) was added via syringe slowly dropwise. The reaction mixture was allowed
to stir overnight. The mixture was diluted with EtOAc and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and the residue was purified via flash chromatography
(1% TEA in EtOAc/Hexane, 0-15%) to yield 1 as a yellow solid (24 mg, 34%).

1
H NMR (600 MHz, Acetone-d6) δ 10.74 (s, 1H), 9.19 (s, 1H), 8.88 (s, 1H), 8.34 (dd, J =
9.0, 2.1 Hz, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.70 (t, J = 2.2 Hz, 1H), 7.64 – 7.54 (m, 1H), 7.39
(t, J = 8.2 Hz, 1H), 6.95 (ddd, J = 8.4, 2.5, 0.8 Hz, 1H), 6.18 (br s, 1H), 4.83 (d, J = 2.4 Hz,
2H), 4.35 (s, 2H), 3.11 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, Acetone-d6) δ 165.17, 159.51, 157.99, 150.07, 139.26, 138.40, 136.58
– 136.11 (m), 129.44, 128.21, 121.74, 116.70, 114.55, 112.66, 112.20, 110.66, 78.64, 76.28,
55.58, 43.17.
HRMS (ESI) m/z: [M+H]
+
calcd for C19H15ClN4O2 366.0884; Found 367.0976.

19

7-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)quinazolin-4-amine (2a)
4-chloro-7-nitroquinazoline (200 mg, 0.95 mmol) and 1a (192 mg, 1.045 mmol) were
dissolved in 3 mL iPrOH. Triethylamine (280 uL, 2 mmol) was added and the solution was
allowed to stir at room temperature for 4 hours. The reaction mixture was diluted with EtOAc
and washed with 10% Na2CO3 followed by brine. The organic layer was dried with Na2SO4
and concentrated by rotary evaporation and the residue was purified using flash column
chromatography (EtOAc/Hex = 0-50%) to give 2a (220 mg, 72%) as a dark orange solid.

N
4
-(3-(prop-2-yn-1-yloxy)phenyl)quinazoline-4,7-diamine (2b)
2a (220 mg, 0.68 mmol) was dissolved in 4 mL MeOH. Zn (224 mg, 3.4 mmol) and NH4Cl
(182 mg 3.4 mmol) were added, and the mixture was allowed to stir at room temperature for
N
N
Cl
+
TEA, iPrOH, rt
N
N
NH O
N
N
NH O
DIPEA, DCM/THF, rt
Cl
O
Cl
N
N
NH O
ClH
3
N O
Zn, NH
4
Cl MeOH, rt
1a
2a
2b
2
NO
2
NO
2
NH
2
N
H
O
Cl
20
16 hr. The reaction mixture was dissolved in EtOA and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and concentrated by rotary evaporation to yield 2b (190
mg, 96%) as a dark orange oil, which was used in the next reaction without further
purification.

2-chloro-N-(4-((3-(prop-2-yn-1-yloxy)phenyl)amino)quinazolin-7-yl)acetamide (2)
75 mg of 2b (0.26 mmol) was dissolved in anhydrous DCM/THF (2.5 mL, 1:4) under
nitrogen gas. DIPEA (99 uL, 0.57 mmol) was added via syringe. Chloroacetyl chloride (24.8
uL, 0.31 mmol) was added via syringe slowly dropwise. The reaction mixture was allowed
to stir overnight. The mixture was diluted with EtOAc and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and the residue was purified via flash chromatography
(1% TEA in EtOAc/Hexane, 0-15%) to yield 3 as a yellow solid (12 mg, 13%).

1
H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 2H), 7.95 (s, 1H), 7.72 (d, J = 9.4 Hz, 1H),
7.45 (d, J = 2.1 Hz, 1H), 6.87 (dd, J = 9.2, 2.1 Hz, 1H), 6.56 – 6.44 (m, 2H), 6.40 (d, J =
8.3 Hz, 1H), 6.08 – 5.98 (m, 1H), 3.91 (d, J = 2.4 Hz, 2H), 3.48 (s, 2H), 2.70 (t, J = 2.4 Hz,
1H).

13
C NMR (151 MHz, Methanol-d4) δ 166.92, 159.59, 158.12, 150.70, 145.46, 139.81,
137.36, 129.35, 125.08, 120.88, 117.17, 113.20, 111.46, 109.28, 106.77, 78.07, 75.67,
55.45, 42.64.

21
1.4 References

1. Singh, J. The resurgence of covalent drugs. (2011) doi:10.1038/nrd3410.

2. Gehringer, M. & Laufer, S. A. Emerging and Re-Emerging Warheads for Targeted
Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology. (2018)
doi:10.1021/acs.jmedchem.8b01153.

3. Sutanto, F., Konstantinidou, M. & Dömling, A. Covalent inhibitors: A rational approach to
drug discovery. RSC Medicinal Chemistry 11, 876–884 (2020).

4. Robertson, J. G. Current Topics Mechanistic Basis of Enzyme-Targeted Drugs. (2005)
doi:10.1021/bi050247e.

5. Xue, G. et al. Cite this. Chem. Commun 56, 1521 (2020).

6. Wright, M. H. & Sieber, S. A. Chemical proteomics approaches for identifying the cellular
targets of natural products. (2010) doi:10.1039/c6np00001k.

7. Chen, X. et al. Target identification of natural medicine with chemical proteomics
approach: probe synthesis, target fishing and protein identification. doi:10.1038/s41392-
020-0186-y.

8. Chen, Y. C. et al. Covalent modulators of the vacuolar ATPase. J Am Chem Soc 139, 639–
642 (2017).

9. Resnick, E. et al. Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.
(2019) doi:10.1021/jacs.9b02822.

22
Chapter 2. Characterization of select covalent targets for electrophilic 4-anilinoquinazolines

2.1 Introduction: Guanosine Monophosphate Synthase (GMPS)
2.2 Designing Compound 1 competitors for future experiments
2.3 Investigating selectivity and labeling efficiency of 1 through competition and time-
course experiments

2.4 In vitro labeling of GAT domain of GMPS
2.4.1 Generating WT GAT domain and C104A
2.4.2 In vitro labeling of GAT domain of GMPS

2.5 In vitro labeling of 3x FLAG GMPS
2.6 Biochemical IC50
2.7 Quinazoline compound effects on GMPS activity in vivo
2.8. Effects of 1 on cancer cell lines and viral infection
2.9 Experimental Details
2.9.1 Chemical Synthesis
2.9.2 Methods and materials for biological characterization
2.10 References

23
2.1 Guanosine Monophosphate Synthesis (GMPS)

We were eager to identify the unknown target and did so through a biotin-based pull
down and LC-MS/MS analysis which was performed by Dr. Ekanyake. HECK293 cells were
treated with probe , lysed, and biotin azide was subsequently added. Isolation of the beads
revealed the target band at 75 kDa to be Guanine monophosphate synthetase (GMPS)
(Figure 10).

Figure 10. Identification of p75. (A) Streptavidin pull-down enriched p75; (B) LC-MS/MS
analysis identified target protein as GMPS; (C) Immunoblot analysis using GMPS antibody
to confirm the target

Guanine monophosphate synthetase is involved in the de novo synthesis of the purine
nucleotide GMP. In this pathway, IMP is at the junction between AMP or GMP synthesis.
10

24
IMP dehydrogenase is responsible for oxidizing IMP into XMP, which is then aminated by
GMP synthetase using the amino group hydrolyzed from glutamine. Human GMPS consists
of 693 residues and contains two functional domains: the N-terminal glutamine
amidotransferase domain (GATase) and the C-terminal synthetase domain (ATPase).
11

Figure 11. De-novo purine synthetic pathway.

Within the amidotransferase domain, L-glutamine is utilized as the amino source and
undergoes hydrolysis to become L-glutamate. The removed amino group is then transferred
to the synthetase domain. Within the synthetase domain, ATP is hydrolyzed to give PPi and
AMP, which is used to create an unstable ATP-intermediate. The amino group generated
from the L-glutamine hydrolysis attacks this intermediate, releasing AMP and producing
GMP (Figure 11).
De-novo purine synthesis
25

Figure 12. GMP synthetase and glutaminase domains.

GMPS has been shown to be upregulated in and functionally important for cancer cell
growth.
10
Thus, GMPS has been identified as a potential anticancer target in addition to
having possible immunosuppressive applications. Given that cancer cells rely heavily on the
upregulation of nucleotide synthesis, the ability to inhibit the production of one of 4
nucleosides creates a potentially effective way to slow or even prevent cell proliferation.
Additionally, GMPS and other nucleotide synthesis enzymes play crucial roles in facilitating
viral replication during infection. Nucleotide components are critical for host cells as well
as the viral components that drive infection. Studies have shown that late-stage interference
of nucleotide synthesis is a more effective anti-viral strategy than early nucleotide synthesis
intervention. Inhibition of GMPS, a later nucleotide synthetic enzyme, may offer and
effective strategy for slowing viral infection. Hence, our compound 1 may offer both anti-
cancer and anti-viral strategies through the inhibition of GMPS.

26
2.2 Designing Compound 1 competitors for future experiments
To further confirm GMPS as the protein target of Compound 1, and in anticipation
of additional experiments needing to be conducted for this study, we looked to synthesizing
a series of potential competitors as well as control compounds. We synthesized a set of
compounds based on 1 but looking at changes in three area: electrophile type, core
modifications, and aniline substituents (Figure 13).

Figure 13. Compound 1 analogs looking at changes to electrophile type and core and aniline
modifications.

Most compounds were synthesized in using similar synthetic steps as previously reported in
Chapter 1, with the exception of the core compounds which required changes to the synthetic
route.
27
For 1-a and 1-b which focused on changing the electrophile to a chloro-methyl
acetamide and a fluroacetamide, labeling experiments in HEK 293 cells were conducted
(Figure 14). The initial experiment involved testing these compounds at 0.1 and 1 uM
respectively, however as seen on the in-gel fluorescence image, no labeling was seen at
around 75 kDa like the band that is prominently seen being labeled by 1. We decided to re-
test these compounds using higher concentrations, this time 1, 3, and 10 uM for 1-a and 10,
30, 100 uM for 1-b (selected given the reported reactivity of these electrophiles). The
increases in concentration allowed us to visualize faint labeling of 1-a at 10uM and of 1-b
at 30 uM. Though these experiments were informative in helping us to visualize the
reactivity differences of the chloroacetamide, chloromethyl acetamide, and fluoroacetamide
electrophiles, no improvements in labeling were seen and given these compounds could not
act as efficient competitors, they were not used for any additional follow up experiments.

Figure 14. Labeling experiment in HEK 293 of Compound 1 analogs with altered
electrophiles.
28

We then moved onto testing the aniline substituent modification compounds in HEK293
cells (Figure 15). To test these compounds, we performed a competition labeling experiment
in cells which involved first treating the cells with 0.1 uM of our main probe (1) for 30 min
followed by wash out and addition of the secondary compound for a 30 min treatment. We
observed that all but 1-f when treated at 1 uM was able to compete out 1. It is important to
note the 10x concentration difference when considering this data. 1-f contains an additional
carbon in the form of a benzylamine, which is distinctly different from the rest of these
compounds which do not contain an extra carbon between the amine and phenyl group of
the aniline indicating that this feature prevented GMPS binding. For the compounds that did
outcompete 1 at the much higher concentration of 1uM, we decided to repeat the competition
experiment using lower and more concentrations in order to accurately gauge the competitor
efficiency to that of 1.

29

Figure 15. Labeling experiment in HEK 293 of Compound 1 in competition with aniline
substituent modified analogs at 10x the concentration.

In the repeat experiment, we tested 1 at a fixed concentration of 100nM, and 1-g, 1-h, 4, and
1-j at 10, 30, 100, and 300 nM (Figure 16). By lowering the concentrations, we were able
to observe a dose-response competition effect between 1 and the potential competitors. All
4 showed a dose-response competition with 1-h showing the weakest and not able to out-
compete 1 even at 300 nM. 1-g showed relatively good competition at 100 and 300 nM,
however faint bands can still be seen in these lanes at 77 kDa that are not seen with 4 and 1-
j. Between these two, the competition appears to be comparable. This is not too surprising
30
given their similarity, with 4 having a meta-methoxy and 1-j having a para-methoxy. What
is surprising is that 10-h which performed the worst, contained an orth-methoxy. The
movement of this substituent around the aniline had varying effects on the compounds’
ability to compete off the signal, demonstrating the SAR that can be acquired with such small
modifications. Between 4 and 1-j, we selected 4 as a competitor to be used in future
experiments given that 1 contains a propargyloxy at the meta position. Keeping the position
on the aniline the same should give us a more reliable comparison.

Figure 16. Dose-response competition of 1 against 1-g, 1-h, 4, and 1-j. Competition can be
seen for all 4 competitors.

Finally, we moved on to looking at whether changes to the core of 1 (4-anilinoquinazoline)
would have any effects on the labeling ability of these compounds. We decided to investigate
whether the presence of both nitrogens in the quinazoline core were necessary for binding
31
to GMPS. We decided to synthesize two probes, a quinoline-based probe (1-c) and an
isoquinoline based-probe (1-d). Essentially, these two probes represented the individual
removal of each nitrogen in the quinazoline core. We tested these two probes in comparison
with 1 by treatment them in HEK293 followed by lysis , tamra-N3 incubation, and observing
the in-gel fluorescence (Figure 17).

Figure 17. Labeling of 1-c and 1-d in HEK293. Labeling of GMPS can be seen with only
1-d
The data we acquired was quite interesting. We were surprised to see that only 1-d, the
isoquinoline version of 1 was able to label GMPS at ~75 kDa while 1-c, the quinoline-
version, was unable to label GMPS. This indicated that the nitrogen at position 3 on the
quinazoline core was essential for GMPS binding. This nitrogen likely serves as a hydrogen
bond acceptor, and so removal of this site renders the probe unable to make an essential
32
bond. It is important to note however that the saturation of 1-d labeling of GMPS was not
achieved even at 1000 nM. This indicates that, while the 1’ nitrogen is not essential for
GMPS labeling, the presence of both nitrogens makes 1 a better overall probe for labeling
GMPS, and thus, 1 continues to be our primary probe for following experiments.

The SAR we obtained from these analogs of 1 provided us with valuable insight regarding
the structural components of 1 that allows it to bind so efficiently to its target GMPS. As
mentioned previously we selected 4 to the main competitor to be used in future experiments
due to how it performed in the competition with 1 as well as it’s structural similarity to 1.
We were also able to determine that only one nitrogen of the 4-anilino quinazoline was
necessary for probe binding to GMPS, however, the presence of both nitrogens allow 1 to
label and saturate the GMPS signal in as little as 100 nM.

2.3 Investigating selectivity and labeling efficiency of 1 through competition and time-
course experiments

Once GMPs was identified to be the target of our probe via biotin-pullodown, we
synthesized an analogous competitor, 4 as explained above, which contained a methoxy
group in place of the propargyloxy for follow-up experiments. Additionally, we synthesized
a control 5 containing the methoxy but lacking the chloroacetamide electrophile (Figure 18).
Having a control compound gives us the ability to confirm the necessity of the presence of
the electrophile for protein binding.
33

Figure 18. Compounds 1, the main GMPs probe, Compound 4, the main competitor, and
Compound 5, the control compound. All were used for subsequent experiments to confirm
binding to GMPS.

Given that Compound 1 was based off the previously reported V-ATPase covalent inhibitor
(Chapter 1), we checked the labeling patterns of 1 and 2 in HEK293 cells. Using the
previously described procedure for examining the proteomic labeling patterns using in-gel
fluorescence, we were able to confirm 2 continued to label a protein band around ~70kDa
while 1 appeared to label a distinct protein around ~75 kDa, with a saturation being reached
around 30nM compared to 100 nM of 2 (Figure 19).

34

Figure 19. Labeling comparison of 2 and 1. Compound 2 clearly labels a distinct band,
presumably vATPase at ~70 kDa, while compound 1 labels a distinct band, presumably
GMPS, at ~75 kDa.

As mentioned previously, we observed saturation of the GMPS signal with 30 nM of 1. To
verify that saturation of the signal was achieve at this concentration we performed a dose-
response labeling experiment using 1 at various concentration points. As seen in Figure 20,
dose-response labeling of the 75 kDa band can be seen until 30 nM when the intensity of the
signal stops increasing. Even as the concentration of 1 is increased until 1000 nM, the signal
remains consistently saturated.

35

Figure 20. Dose-response labeling using 1 at various concentration points in HEK293 cells
shows that saturation of the 75 kDa signal is reached at 30 nM of the probe.

We next wanted to see how rapid the saturation of this signal occurred. To do this we
performed a time course experiment in which we treated 1 at a fixed concentration of 100
nM for 1, 5, 10, 20, 30, 60, and 120 minutes. The bottom gel of Figure 21 shows that rapid
labeling of the band at 75 kDa (p75) occurred. By one min, the signal can be seen to be
saturated. This revealed that not only is 1 able to saturate the signal at p75 at a low nM
concentration, but it rapidly labels p75. We also performed a competition labeling
experiment between 1 and our competitor compound 4 in order to confirm selectivity of the
labeling achieved with 1.
36

Figure 21. Competition labeling experiment between 1 and 4 (top) and time course
experiment (bottom).

As seen in the top gel in Figure 21, pre-treatment with 4 was able to abolish the labeling of
1 at around 10 nM with the signal being entirely abolished at 100 nM. These two
experiments, the time-course and competition, helped to further confirm labeling of p75 by
1, and revealed 1 rapidly and selectively labeled its respective target at 100 nM.

2.4 In vitro labeling of GAT domain of GMPS
2.4.1 Generating WT GAT domain and C104A

As described earlier in this chapter, Human GMPS consists of 693 residues and
contains two functional domains: the N-terminal glutamine amidotransferase domain
(GATase) and the C-terminal synthetase domain (ATPase). GMPS belongs to the class I
glutamine amidotransferase-like domain family, which are characterized by their triad of
37
conserved Cys-His-Glu. In GMPS, the GAT domain contains a catalytic cysteine, C104. We
hypothesized that 1, given it contains the chloracetimide electrophile known to react
selectively to cysteine residues, was labeling this cysteine within the GAT domain of GMPS.

In order to confirm this, we needed to express the GAT domain of GMPS via the
E.coli bacterial expression vector pET28a. We truncated full-length GMPS via PCR to
obtain just the GAT domain insert, and cloned the fragment into the vector to obtain the
recombinant plasmid. Following transformation into GC5 E.coli cells, we were able to
inoculate and culture the desired quantity of cells to give us several ug of purified GAT
domain. The pET28a vector used for this contained a 6x-his tag, which allowed clean
isolation of the desired protein via NTA-agarose beads.

Figure 22. Purification of GMPS WT GAT and C104A GAT.

We also performed a mutagenesis of C104 using QuickChange II Site-Directed Mutagenesis.
We converted the GCC codon to a TGC to give us the C104A GAT mutant. The GAT
38
domain of GMPS is approximately 25 kDa, and after purification of the two domains, we
obtained very clean protein for both the WT and C104A (Figure 22).

2.4.2 In vitro labeling of GAT domain of GMPS

Once we had obtained the recombinant purified WT and C104A GAT domains, we
were able to proceed with additional experiments. We performed a dose-response labeling
experiment by treating 1 at various concentrations into 0.5 ug GAT protein/per sample.

Figure 23. In vitro labeling of WT GAT and C104A GAT. Only WT was able to be
labeled dose-dependently by 1.

As expected, only GAT WT was able to be labeled dose-dependently by 1. Mutated GAT
C104A showed no labeling by 1 (note slight band at 0.1 nM was due to sample leaking from
GAT WT 300 nM). This indicated that out hypothesis that 1 was forming a covalent bond
with the catalytic cysteine 104 within the GAT domain of GMPS was correct, and that by
mutating this cysteine to a non-nucleophilic residue like alanine, 1 was no longer able to
bind.

39

2.5 In vitro labeling of 3x FLAG GMPS

To further confirm that 1 was binding to C104, and to investigate the dependency of
binding on the GAT catalytic triad, the three amino acids of full-length hunan GMPS were
mutated individually (C104, H190, E192). 3x-FLAG GMPS was mutated through site-
directed mutagenesis to generate 3 mutants – C104A, H190A, E192Q. HEK293 cells were
transfected with the individual GMPS constructs. 1 was treated at 100nM into WT, C104A,
H190A, and E192Q, containing cells. Lysis and click with N3-azide followed and the in-gel
fluorescence was observed. Only WT and the two non-cysteine mutants were shown to be
labeled by 1. Abolishing C104, as demonstrated previously by the GAT C104A mutant,
preventing labeling of the protein by 1.

2.6 Biochemical IC50

Next, we assessed the biochemical implications of 1 on GMPS binding. Using 1 as
well as competitor 4 and control 5 (Figure 18), we sought out to perform an activity assay
that would analyze the activity of GMPS after treatment with each of these compounds. Full-
length human GMPS was cloned into pET28a vector and expressed in E.coli. An aborobance
assay monitoring the decrease of XMP levels at 340 nm following the addition of compound
was performed.
40

Figure 24. IC50 determination. Absorbance assay measuring XMP levels performed.
Inhibition observed with 1 but not with 5 which lacks chloroacetamide.

The reaction rates were monitored in the absence and presence of the compounds and
an IC50 was generated for each. We found that 1 inhibited GMPS activity with an IC50 value
of 4.9 nM while 4 inhibited activity with an IC50 of 23.5 nM (Figure 24). Control compound
5 which lacked the electrophile warhead showed no inhibition against recombinant GMPS,
indicating the necessity of the electrophilic warhead for not only binding to GMPS but
inhibition of protein activity. We also checked the activity of recombinant GMPS after
treatment with 1-d and 1-c, and observed that only the isoquinoline compound exhibited
inhibition while the quinoline compound did not, consistent with our previous experiments
that the isoquinoline nitrogen was important for probe binding (Figure 25).

41

Figure 25. IC50 values of selected GMPS compounds.

2.7 Quinazoline compound effects on GMPS activity in vivo

We next looked the effects of our compounds on GMPS activity in cells by
monitoring XMP and GMP levels after in situ inhibitor treatment. Following treatment of
HEK293 cells with 1, XMP accumulation was observed. Consequently, decreased levels of
GMP was also observed . XMP and GMP levels were monitored over 24 hours, with the
highest level of XMP being reached at 3 hours post-treatment. GMP levels continued to stay
at a lower concentration even after 24 hours post-treatment of 1. This data supported the
notion that 1 inhibits GMPS activity leading to an increase in the GMPS substrate (XMP)
and a decrease of the GMPS product (GMP) (Figure 26).

42

Figure 26. Time-dependent inhibition of GMPS in cells. Observed XMP accumulation and
decreased GMP output following treatment with 1.
2.8. Effects of 1 on cancer cell lines and viral infection

After confirming that 1 inhibited GMPS activity, we moved on to investigating the effects
of compound 1 in cancer cell lines. To do this, we utilized the National Cancer Institute’s 60
cancel line screening platform. Their initial single dose experiment of 1 demonstrated the
compound’s lethal effects on a series of cancer cell lines (Figure 27). As discussed earlier
in this chapter, GMPS plays a crucial role in purine biosynthesis and accordingly has been
identified as a potential anti-cancer target.

43

Figure 27. NCI screening of 1. Compound 1 demonstrated to be lethal against several
cancer cell lines.
Similarly, due to the dependence on nucleotide biosynthesis in viral replication, GMPS has
been identified as a potential anti-viral target to combat infection. To investigate this, we
used a Vesicular stomatitis virus (VSV) model in HK293 mammalian cells. Green
fluorescent protein (GFP) was used as the reporter, and monitored in response to increasing
concentrations of 1. A decrease in GFP, indicating a decrease in viral infection, was observed
as the concentration of 1 was increased. A viral plaque assay measuring viral titers in Vero
cells was also conducted, and an effective dose (ED50) was calculated for 1 (Figure 28).

44

Figure 28. Viral model to assess compound 1 effectiveness. VSV-GFP measured with
increasing concentration of 1 and calculated viral titer.
Through this work we identified a specific and covalent modulator of GMPS, a
protein involved in the de novo synthesis of the purine nucleotide GMP. We demonstrated
our lead compound is able to selectively and potently bind and label GMPS in cells lysates
and live cells. We identified and confirmed the site of modification to be C104, and
demonstrated that our compound to covalently bind to this cysteine. We derived the IC50
value of our compound on GMPS to be to very low at 4.9 nM. Our biochemical data of time-
dependent inhibition of GMPS in cells showed that compound 1 leads to XMP accumulation
and a decrease in GMPS levels. Screening by NCI revealed that Compound 1 was
demonstrated to be lethal against several cancer cell lines, and we also exemplified that 1
exhibits anti-viral activity against VSV.

45
2.9 Experimental details
2.9.1 Chemical Synthesis
Reagents and solvents were obtained from commercial suppliers and used without further
purification, unless otherwise stated. Flash column chromatography was carried out using
an automated system (Teledyne Isco CombiFlash. Reverse phase high performance liquid
chromatography (RP-HPLC) was carried out on a Shimadzu HPLC system. All anhydrous
reactions were carried out under nitrogen atmosphere. NMR spectra were obtained on Varian
VNMRS-500, VNMRS-600, or Mercury-400. The following abbreviations were used to
explain NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet,
m = multiplet, br = broad.

3-(prop-2-yn-1-yloxy)aniline (1a)

NaOH pellets (1.49 g, 37.3 mmol) were weighed out and added to methanol (69.4 mL). 3-
aminophenol (3.82 g, 35 mmol) was then added and the solution was kept in an ultrasonic
bath for about 15 minutes until everything was completely dissolved. The volatiles were
evaporated by rotary evaporation and the residual water was removed by four successive co-
distillations with abs EtOH (27.8 mL, 4x). The obtained dried residue was dissolved in
CH3CN (69.4 mL) and propargyl bromide (80 wt. % solution in toluene, 4.62 mL, 41.56
H
2
N OH
Br
+
1. NaOH, MeOH, rt
2. in-situ HCl, MeOH, rt
ClH
3
N O
1a
46
mmol) was added in three portions over a 1hr period. The reaction mixture was allowed to
stir overnight. It was concentrated under rotary evaporation and the residue was partitioned
between aq NaOH (0.1 M, 69.4 mL) and diethyl ether (69.4 mL). The layers were separated
and the aqueous layer was extracted with diethyl ether (2x69.4 mL). The combined layers
were washed brine and dried over Na2SO4. Evaporation of the solvent gave a brownish oily
residue which was re-dissolved in MeOH (20.83 mL) and combined with in-situ generated
HCl (prepared from 104.2 mL of MeOH and 5.3 mL (74.76 mmol) of acetyl chloride). The
volatiles were evaporated resulting in a brownish solid which was resuspended in boiling
EtOAc (34.7 mL). After cooling the suspension to room temperature, white crystals were
collected by filtration and dried to afford compound 1a. (3.14 g, 49%).

1
H NMR (400 MHz, DMSO-d6) δ 9.73 (br s, 3H), 7.40 – 7.29 (m, 1H), 6.94 – 6.81 (m, 3H),
4.80 (d, J = 2.4 Hz, 2H), 3.60 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, DMSO-d6) δ 158.26, 134.14, 131.06, 116.19, 114.01, 110.46, 79.22,
79.13, 56.17.
47

7-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)quinazolin-4-amine (2a)
4-chloro-7-nitroquinazoline (209 mg, 1.0 mmol) and 1a (201 mg, 0.55 mmol) were dissolved
in 6mL iPrOH. Triethylamine (155 uL, 1.1 mmol) was added and the solution was allowed
to stir at room temperature for 6 hours. The reaction mixture was diluted with EtOAc and
washed with 10% Na2CO3 followed by brine. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation and the residue was purified using flash column
chromatography (EtOAc/Hex = 0-50%) to give 2a (256mg, 85%) as a dark yellow solid.

1
H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 9.11 (d, J = 9.1 Hz, 1H), 8.96 (s, 1H), 8.68
(d, J = 2.3 Hz, 1H), 8.50 (dd, J = 9.1, 2.3 Hz, 1H), 7.59 – 7.46 (m, 1H), 7.46 – 7.31 (m, 2H),
6.95 (ddt, J = 6.7, 4.6, 2.8 Hz, 1H), 4.82 (d, J = 2.4 Hz, 2H), 3.61 (t, J = 2.3 Hz, 1H).

N
N
Cl
+
TEA, iPrOH, rt
N
N
NH O
N
N
NH O
DIPEA, DCM/THF, rt
Cl
O
Cl
N
N
NH O
ClH
3
N O
Zn, NH
4
Cl MeOH, rt
1a
2a
2b
2
NO
2
NO
2
NH
2
N
H
O
Cl
48
N
4
-(3-(prop-2-yn-1-yloxy)phenyl)quinazoline-4,7-diamine (2b)
2a (256 mg, 0.8 mmol) was dissolved in 10 mL MeOH. Zn (262 mg, 4.0 mmol) and NH4Cl
(213 mg 4.0 mmol) were added, and the mixture was allowed to stir at room temperature for
16 hr. The reaction mixture was dissolved in EtOA and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and concentrated by rotary evaporation to yield 2b (210
mg, 90%) as a light yellow solid, which was used in the next reaction without further
purification.

1
H NMR (400 MHz, DMSO-d6) δ 9.38 – 9.28 (m, 1H), 8.36 (s, 1H), 8.17 (d, J = 9.0 Hz,
1H), 7.57 (dt, J = 12.2, 2.2 Hz, 1H), 7.44 (ddd, J = 8.1, 2.1, 0.9 Hz, 1H), 7.24 (t, J = 8.2 Hz,
1H), 6.89 (dd, J = 9.0, 2.3 Hz, 1H), 6.78 – 6.64 (m, 2H), 6.05 (s, 2H), 4.76 (d, J = 2.4 Hz,
2H), 4.55 (dt, J = 5.2, 1.6 Hz, 1H), 3.56 (t, J = 2.4 Hz, 1H).

2-chloro-N-(4-((3-(prop-2-yn-1-yloxy)phenyl)amino)quinazolin-7-yl)acetamide (2)
145 mg of 2b (0.5 mmol) was dissolved in anhydrous DCM/THF (6 mL, 1:4) under nitrogen
gas. DIEA (190 uL, 1.1 mmol) was added via syringe. Chloroacetyl chloride (43.5 uL, 0.55
mmol) was added via syringe slowly dropwise. The reaction mixture was allowed to stir for
4 hours. The reaction mixture was concentrated by rotary evaporation and compound 2 was
purified using HPLC (isocratic 35% acetonitrile) to yield 36 mg (20%) of light yellow solid.

1
H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 2H), 7.95 (s, 1H), 7.72 (d, J = 9.4 Hz, 1H),
7.45 (d, J = 2.1 Hz, 1H), 6.87 (dd, J = 9.2, 2.1 Hz, 1H), 6.56 – 6.44 (m, 2H), 6.40 (d, J =
49
8.3 Hz, 1H), 6.08 – 5.98 (m, 1H), 3.91 (d, J = 2.4 Hz, 2H), 3.48 (s, 2H), 2.70 (t, J = 2.4 Hz,
1H).

13
C NMR (151 MHz, Methanol-d4) δ 166.92, 159.59, 158.12, 150.70, 145.46, 139.81,
137.36, 129.35, 125.08, 120.88, 117.17, 113.20, 111.46, 109.28, 106.77, 78.07, 75.67,
55.45, 42.64.

HRMS (ESI) m/z: [M+H]
+
calcd for C19H15ClN4O2 366.0884; Found 367.0970.

50
6-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)quinazolin-4-amine (1b)

4-chloro-6-nitroquinazoline (105 mg, 0.5 mmol) and 1a (101 mg, 0.55 mmol) were dissolved
in 2 mL iPrOH. Triethylamine (77 uL, 0.55 mmol) was added and the solution was allowed
to stir at room temperature for 4 hours. The reaction mixture was diluted with EtOAc and
washed with 10% Na2CO3 followed by brine. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation and the residue was purified using flash column
chromatography (EtOAc/Hex = 0-50%) to give 1b (139 mg, 87%) as a dark orange solid.

1
H NMR (500 MHz, DMSO-d6) δ 11.53 (s, 1H), 9.81 (d, J = 2.3 Hz, 1H), 8.93 (s, 1H), 8.72
(dd, J = 9.2, 2.3 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H), 7.59 – 7.30 (m, 3H), 6.96 (d, J = 7.6 Hz,
1H), 4.84 (d, J = 2.4 Hz, 2H), 3.63 (t, J = 2.3 Hz, 1H).

N
4
-(3-(prop-2-yn-1-yloxy)phenyl)quinazoline-4,6-diamine (1c)

1b (139 mg, 0.43 mmol) was dissolved in 4 mL MeOH. Zn (140.6 mg, 2.15 mmol) and
NH4Cl (115 mg 2.15 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and concentrated by rotary evaporation to
yield 1c (111 mg, 89%) as a dark yellow oil, which was used in the next reaction without
further purification.

51
1
H NMR (400 MHz, Chloroform-d) δ 8.62 (s, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.59 (s, 1H),
7.33 – 7.12 (m, 4H), 6.91 (s, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 4.72 (d, J = 2.4 Hz, 2H),
4.04 (br s, 2H), 2.54 (t, J = 2.4 Hz, 1H).

2-chloro-N-(4-((3-(prop-2-yn-1-yloxy)phenyl)amino)quinazolin-6-yl)acetamide (1)

55 mg of 1c (0.19 mmol) was dissolved in anhydrous DCM/THF (2.5 mL, 1:4) under
nitrogen gas. DIEA (73.1 uL, 0.42 mmol) was added via syringe. Chloroacetyl chloride (18.2
uL, 0.23 mmol) was added via syringe slowly dropwise. The reaction mixture was allowed
to stir overnight. The mixture was diluted with EtOAc and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and the residue was purified via flash chromatography
(1% TEA in EtOAc/Hexane, 0-15%) to yield 1 as a yellow solid (24 mg, 34%).

1
H NMR (600 MHz, Acetone-d6) δ 10.74 (s, 1H), 9.19 (s, 1H), 8.88 (s, 1H), 8.34 (dd, J =
9.0, 2.1 Hz, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.70 (t, J = 2.2 Hz, 1H), 7.64 – 7.54 (m, 1H), 7.39
(t, J = 8.2 Hz, 1H), 6.95 (ddd, J = 8.4, 2.5, 0.8 Hz, 1H), 6.18 (br s, 1H), 4.83 (d, J = 2.4 Hz,
2H), 4.35 (s, 2H), 3.11 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, Acetone-d6) δ 165.17, 159.51, 157.99, 150.07, 139.26, 138.40, 136.58
– 136.11 (m), 129.44, 128.21, 121.74, 116.70, 114.55, 112.66, 112.20, 110.66, 78.64, 76.28,
55.58, 43.17.
HRMS (ESI)m/z: [M+H]
+
calcd for C19H15ClN4O2 366.0884; Found 367.0976.
52

N-(3-methoxyphenyl)-6-nitroquinazolin-4-amine (4a)

4-chloro-6-nitroquinazoline (105 mg, 0.50 mmol) and m-anisidine (62 uL, 0.55 mmol) were
dissolved in 2 mL iPrOH. Triethylamine (77 uL, 0.55 mmol) was added and the solution was
allowed to stir at room temperature for 4 hours. The reaction mixture was concentrated by
rotary evaporation and the residue was purified using flash column chromatography
(EtOAc/Hex 0-50%) to give 4a (100 mg, 68% yield) as a yellow solid.

1
H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.67 (d, J = 2.5 Hz, 1H), 8.72 (s, 1H), 8.55
(dd, J = 9.2, 2.4 Hz, 1H), 7.93 (d, J = 9.2 Hz, 1H), 7.54 – 7.41 (m, 2H), 7.32 (t, J = 8.1 Hz,
1H), 6.77 (dd, J = 8.3, 2.6, 0.9 Hz, 1H), 3.78 (s, 3H).

N
N
Cl
NO
2
+
H
2
N
O
TEA, iPrOH, rt
N
N
NO
2
NH O
N
N
NH
2
NH O
DIPEA, DCM/THF, rt
Cl
O
Cl
N
N
H
N
NH O
O
Cl
Zn, NH
4
Cl MeOH, rt
4a
4b 4
53
N
4
-(3-methoxyphenyl)quinazoline-4,6-diamine (4b)

4a (50 mg, 0.17 mmol) was dissolved in 2 mL MeOH. Zn (55.6 mg, 0.85 mmol) and NH4Cl
(45.5 mg, 0.85 mmol) were added, and the mixture was allowed to stir at room temperature
for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and concentrated by rotary evaporation to yield 4b (40
mg, 88%) as an orange solid, which was used in the next reaction without further
purification.

1
H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.32 (s, 1H), 7.61 – 7.40 (m, 3H), 7.34 (d, J
= 2.4 Hz, 1H), 7.27 – 7.16 (m, 2H), 6.63 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 5.55 (s, 2H), 3.75
(s, 3H).

2-chloro-N-(4-((3-methoxyphenyl)amino)quinazolin-6-yl)acetamide (4)

43 mg of 4b (0.16 mmol) was dissolved in anhydrous DCM/THF (2.5 mL, 1:4) under
nitrogen gas. DIEA (61 uL, 0.35 mmol) was added via syringe. Chloroacetyl chloride (15.1
uL, 0.19 mmol) was added via syringe slowly dropwise. The reaction mixture was allowed
to stir overnight. The mixture was diluted with EtOAc and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and the residue was purified via flash chromatography
(EtOAc/Hexane, 0-50%) to yield 4 as a yellow solid (20 mg, 36%).

54
1
H NMR (400 MHz, Acetone-d6) δ 10.72 (s, 1H), 9.24 (s, 1H), 8.87 (s, 1H), 8.38 (d, J = 8.6
Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.75 – 7.55 (m, 2H), 7.37 (t, J = 8.2 Hz, 1H), 6.87 (dd, J
= 8.3, 2.5 Hz, 1H), 4.69 (br s, 1H), 4.36 (s, 2H), 3.85 (s, 3H).

13
C NMR (151 MHz, Acetonitrile-d3) δ 165.44, 159.98, 159.69, 149.80, 138.93, 137.84,
136.00, 129.65, 128.66, 121.42, 116.38, 114.37, 112.49, 112.17, 110.21, 55.11, 43.37.
HRMS (ESI) m/z: [M+H]
+
calcd for C17H15ClN4O2 342.0884; Found 343.0978.

N-(4-((3-methoxyphenyl)amino)quinazolin-6-yl)acetamide (5)
45 mg of 4b (0.17 mmol) was dissolved in anhydrous DCM/THF (2.5 mL, 1:4) under
nitrogen gas. DIEA (61 uL, 0.35 mmol) was added via syringe. Acetyl chloride (15.6 uL,
0.22 mmol) was added via syringe slowly dropwise. The reaction mixture was allowed to
stir overnight. The mixture was diluted with EtOAc and washed with 10% Na2CO3. The
organic layer was dried over NaSO4 and the residue was purified via flash chromatography
(EtOAc/Hexane, 0-50%) to yield 5 as a yellow solid (20 mg, 38%).

N
N
NH
2
NH O
4b
DIPEA, DCM/THF, rt
O
Cl
N
N
H
N
NH O
O
5
55
1
H NMR (400 MHz, Acetone-d6) δ 10.72 (s, 1H), 9.24 (s, 1H), 8.87 (s, 1H), 8.38 (d, J = 8.6
Hz, 1H), 8.06 (d, J = 9.0 Hz, 1H), 7.75 – 7.55 (m, 2H), 7.37 (t, J = 8.2 Hz, 1H), 6.87 (dd, J
= 8.3, 2.5 Hz, 1H), 4.69 (br s, 1H), 4.36 (s, 2H), 3.85 (s, 3H).

13
C NMR (151 MHz, Acetonitrile-d3) δ 165.44, 159.98, 159.69, 149.80, 138.93, 137.84,
136.00, 129.65, 128.66, 121.42, 116.38, 114.37, 112.49, 112.17, 110.21, 55.11, 43.37.
HRMS (ESI) m/z: [M+H]
+
calcd for C
17
H
16
N
4
O
2
308.1273; Found 309.1344

6-nitroquinolin-4-ol (1-c.1)

A mixture of concentrated sulfuric acid (6 mL) and concentrated nitric acid (6 mL) was made
and added to a solution of 4-hydroxyquinoline (5 g, 34.4 mmol) in sulfuric acid (6 mL). The
reaction mixture was allowed to stir at 0
o
C for 2 hr. Following completion of the reaction,
the mixture was added to ice-water (1:1) and the precipated crude product was collected by
vacuum filtration and dried to yield 1-c.1 (2g, 31% yield) as a white solid.
1

56

1
H NMR (500 MHz, DMSO-d6) δ 12.31 (s, 1H), 8.83 (d, J = 2.7 Hz, 1H), 8.42 (dd, J =
9.2, 2.7 Hz, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 6.19 (d, J = 7.5 Hz,
1H).

4-chloro-6-nitroquinoline (1-c.2)

2 g of 1-c.1 (10.50 mmol) was added to a 2-neck flask and flushed with nitrogen gas. POCl3
(10 mL, 107.00 mmol) was added and the reaction mixture was refluxed for 3h. Once
completed the excess POCl3 was removed via vacuum. The resulting solid was washed with
10% NaCO3 and extracted with EtOAc. The organic layer was washed with brine, dried with
NaSO4 and concentrated to yield 1-c.2 (1.1 g, 50% yield) as an off-white solid.
1

1
H NMR (400 MHz, Chloroform-d) δ 9.14 (d, J = 2.5 Hz, 1H), 8.90 (d, J = 4.7 Hz, 1H),
8.47 (dd, J = 9.2, 2.5 Hz, 1H), 8.22 (dd, J = 9.2, 0.5 Hz, 1H), 7.60 (d, J = 4.7 Hz, 1H).

57

6-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)quinolin-4-amine (1-c.3)

1-c.2 (104.3 mg, 0.50 mmol) and 1a (101 mg, 0.55 mmol) were dissolved in 2 mL iPrOH.
Triethylamine (77 uL, 0.55 mmol) was added and the solution was allowed to stir at 50
o
C
for 16 hours. The reaction mixture was diluted with EtOAc and washed with 10% Na2CO3
followed by brine. The organic layer was dried with Na2SO4 and concentrated by rotary
evaporation and the residue was purified using flash column chromatography (EtOAc/Hex
= 0-50%) to give 1-c.3 (90 mg, 56%) as a yellow solid.

58
1
H NMR (400 MHz, Chloroform-d) δ 8.88 (s, 1H), 8.60 (dd, J = 2.5, 0.5 Hz, 1H), 8.35 (dd,
J = 9.3, 2.5 Hz, 1H), 8.14 (d, J = 9.3 Hz, 1H), 7.11 – 7.01 (m, 1H), 6.82 (dd, J = 8.2, 2.2 Hz,
1H), 6.70 – 6.59 (m, 2H), 6.41 – 6.29 (m, 2H), 4.64 (d, J = 2.4 Hz, 2H), 2.44 (t, J = 2.4 Hz,
1H).

N
4
-(3-(prop-2-yn-1-yloxy)phenyl)quinoline-4,6-diamine (1-c.4)

50 mg of 1-c.3 (0.157 mmol), 51 mg of Zn (0.78 mmol), and 45 mg of NH4Cl (0.827 mmol)
were dissolved in 2 mL MeOH. The reaction was stirred at room temperature for 16 hr. The
reaction mixture was diluted in EtOA and washed with 10% Na2CO3. The organic layer was
washed with brine, dried over NaSO4, and concentrated by rotary evaporation to yield 1-c.4
(45 mg, 88%) as a dark brown solid, which was used in the next reaction without further
purification.

1
H NMR (400 MHz, Chloroform-d) δ 8.56 (s, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.16 (t, J = 8.1
Hz, 1H), 7.11 – 7.03 (m, 2H), 6.76 (d, J = 2.6 Hz, 1H), 6.62 (dd, J = 8.2, 2.5 Hz, 1H), 6.50
– 6.29 (m, 5H), 4.65 (d, J = 2.4 Hz, 2H), 2.51 (t, J = 2.4 Hz, 1H).

2-chloro-N-(4-((3-(prop-2-yn-1-yloxy)phenyl)amino)quinolin-6-yl)acetamide (1-c)

1-c.4 (45 mg, 0.156 mmol) was dissolved in 2 mL anhydrous DCM/THF (1:4) under nitrogen
gas. DIPEA (67.8 uL, 0.39 mmol) was added via syringe. Chloroacetyl chloride (15.8 uL,
59
0.20 mmol) was added via syringe slowly dropwise. The reaction was stirred under nitrogen
gas at room temperature for 5 hours. Once completed, the reaction was diluted with EtOAc
and washed with brine. The organic layer was dried with Na2SO4, and concentrated. The
crude product was purified via flash chromatography (Hex/EtOAc) to produce 1-c as a dark
yellow solid (11.2 mg, 20% yield).

1
H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 9.03 (s, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.42
(d, J = 5.4 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.76 (dd, J = 9.0, 2.3 Hz, 1H), 7.29 (t, J = 8.2
Hz, 1H), 7.04 (d, J = 5.3 Hz, 1H), 7.00 – 6.93 (m, 2H), 6.70 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H),
4.78 (d, J = 2.3 Hz, 2H), 4.33 (s, 2H), 3.60 (t, J = 2.3 Hz, 1H).

13
C NMR (151 MHz, DMSO-d6) δ 164.83, 158.01, 147.59, 142.27, 135.07, 133.71, 130.10,
129.06, 123.82, 120.48, 117.45, 114.17, 111.59, 109.67, 107.67, 103.41, 79.27, 78.34, 55.36,
43.53.
HRMS (ESI) m/z: [M+H]
+
calcd for C20H16ClN3O2 365.0931; Found 366.1005.

60

Methyl (E)-2-(2-(dimethylamino)vinyl)-5-nitrobenzoate (1-d.1)
2

A mixture of 1.8g of methyl 2-methyl-5-nitrobenzoate (9.22 mmol) and 4.8 g of DMA (40
mmol) in 12 mL of DMF was heated to 100
o
C and allowed to reflux for 3 hr. The mixture
was poured into ice-water and the resulting precipitating solid was filtered and washed with
water to give 1-d.1 as a yellow solid (1g, 44% yield).
1
H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 2.6 Hz, 1H), 7.96 (dd, J = 9.2, 2.7 Hz, 1H),
7.77 – 7.65 (m, 2H), 6.19 (d, J = 13.3 Hz, 1H), 3.81 (s, 3H), 2.96 (s, 6H).

2-(2,4-dimethoxybenzyl)-7-nitroisoquinolin-1(2H)-one (1-d.2)
1g (4 mmol) 1-d.1 and 2,4-dimethoxylbenxylamine (0.840 mL, 5.6 mmol) were dissolved
in 20 mL of toluene. The mixture was stirred at 125
o
C until the reaction was completed (2.5
hr). The mixture was cooled to room temperature and triturated with hexanes/ethyl acetate
(2:1). The mixture was left at room temperature over night and the precipitate was collected
by filtration to give 1-d.2 (940 mg, 69% yield) as a white solid.
61

1
H NMR (400 MHz, DMSO-d6) δ 8.91 (d, J = 2.6 Hz, 1H), 8.44 (dd, J = 8.8, 2.2 Hz, 1H),
7.88 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 6.79 (d, J =8.7, 1H), 6.77 – 6.45 (m, 3H),
5.05 (s, 2H), 3.74 (s, 3H), 3.72 (s, 3H).

7-nitroisoquinolin-1(2H)-one (1-d.3)
1-d.2 (940 mg, 2.76 mmol) was dissolved in 15 mL of CF3COOH at 85
o
C for 5 hr. The
mixture was cooled to room temperature and concentrated by rotary evaporation. The residue
was re-suspended in EtOAc and stirred for 30 min. The solid was filtered and washed with
EtOAc to afford 1-d.3 as a beige solid (240 mg, 46% yield).
1
H NMR (400 MHz, DMSO-d6) δ 11.74 (s, 1H), 8.88 (d, J = 2.5 Hz, 1H), 8.43 (dd, J = 8.8,
2.5 Hz, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.44 (dd, J = 7.1, 5.9 Hz, 1H), 6.72 (d, J = 7.1 Hz,
1H).

1-chloro-7-nitroisoquinoline (1-d.4)

The intermediate 1-d.3 (200 mg, 1.05 mmol) was suspended in phosphoryl trichloride (3
mL) and heated to 100
o
C until 1-d.3 was completely consumed. The mixture was then
poured into ice-water and extracted with EtOAc (3x). The combined organic layer was
washed with sat. NaHCO3 followed by brine, dried over Na2SO4, and concentrated to afford
a yellow solid after flash chromatography to give 1-d.4 (150 mg, 68% yield).

62
1
H NMR (400 MHz, DMSO-d6) δ 9.03 (dt, J = 2.3, 0.8 Hz, 1H), 8.59 (dd, J = 9.0, 2.3 Hz,
1H), 8.54 (d, J = 5.7 Hz, 1H), 8.36 (d, J = 9.0 Hz, 1H), 8.11 (dd, J = 5.7, 1.0 Hz, 1H).

7-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)isoquinolin-1-amine (1-d.5)

104 mg (0.5 mmol) of 1-d.4 and 101 mg of 1a (0.55 mmol) dissolved in 3 mL of n-butanol.
77 uL (0.55 mmol) of TEA was added. The mixture was refluxed for 2 hours. After cooling,
EtOAc was added. The organic layer was washed with sat. Na2CO3, separated, dried with
63
Na2SO4, and concentrated. The residue was purified via flash chromatography
(Hexane/EtOAc) to give 1-d.5as a bright red solid (100 mg, 63% yield).

1
H NMR (400 MHz, Chloroform-d) δ 8.95 (d, J = 2.1 Hz, 1H), 8.42 (dd, J = 9.0, 2.1 Hz,
1H), 8.27 (d, J = 5.7 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.58 (t, J = 2.3 Hz, 1H), 7.37 – 7.28
(m, 2H), 7.20 (dd, J = 6.0, 1.0 Hz, 1H), 6.77 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 6.44 – 6.25 (m,
1H), 4.75 (d, J = 2.4 Hz, 2H), 2.54 (t, J = 2.4 Hz, 1H).

N
1
-(3-(prop-2-yn-1-yloxy)phenyl)isoquinoline-1,7-diamine (1-d.6)

100 mg of 1-d.5 (0.313 mmol) and 87 mg Fe (1.57 mmol) were dissolved in 4 mL
EtOH/AcOH/H2O (2:2:1). The reaction was allowed to stir for 3.5 hours. After the reaction
was completed, the mixture was diluted with EtOAc and washed with sat. Na2CO3 until the
aqueous layer was very basic. The organic layer was washed with brine, separated, dried
with Na2SO4, and concentrated to yield 1-d.6 as a dark green solid (89 mg, 90% yield) which
was used without further purification.
1
H NMR (400 MHz, Chloroform-d) δ 7.85 (d, J = 5.7 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H),
7.40 (t, J = 2.3 Hz, 1H), 7.10 – 6.92 (m, 4H), 6.75 (br s, 1H), 6.58 (ddd, J = 8.2, 2.5, 0.9 Hz,
1H), 6.36 – 6.20 (m, 1H), 4.65 (d, J = 2.4 Hz, 2H), 3.91 (br s, 2H), 2.45 (t, J = 2.4 Hz, 1H).

64
2-chloro-N-(1-((3-(prop-2-yn-1-yloxy)phenyl)amino)isoquinolin-7-yl)acetamide (1-d)

1-d.6 (89 mg, 0.308 mmol) was dissolved in 2.5mL anhydrous DCM/THF (1:4) under
nitrogen gas. DIPEA (133.9 uL, 0.77 mmol) was added via syringe. Chloroacetyl chloride
(31.2 uL, 0.392 mmol) was added via syringe slowly dropwise. The reaction was stirred
under nitrogen gas at room temperature for 5 hours. Once completed, the reaction was
diluted with EtOAc and washed with 10% Na2CO3. The organic layer was washed with
brine, separated, dried with Na2SO4, and concentrated. The crude product was purified via
flash chromatography (Hex/1% TEA in EtOAc) to produce 1-d as a dark brown oil (60 mg,
53% yield).

1
H NMR (600 MHz, Chloroform-d) δ 8.71 (s, 1H), 8.44 (s, 1H), 7.97 (d, J = 5.8 Hz, 1H),
7.65 (d, J = 8.7 Hz, 1H), 7.59 (dd, J = 8.7, 2.0 Hz, 1H), 7.45 (t, J = 2.2 Hz, 1H), 7.20 (t, J =
8.1 Hz, 1H), 7.16 – 7.12 (m, 1H), 7.03 (dd, J = 5.9, 0.9 Hz, 1H), 6.64 (ddd, J = 8.0, 2.5, 0.9
Hz, 1H), 4.66 (d, J = 2.4 Hz, 2H), 4.21 (s, 2H), 2.51 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, Chloroform-d) δ 164.44, 158.19, 151.89, 141.30, 139.62, 135.12,
134.75, 129.62, 128.32, 123.56, 118.99, 113.88, 113.00, 111.56, 109.13, 107.53, 78.72,
75.44, 55.86, 42.99.
HRMS (ESI) m/z: [M+H]
+
calcd for C20H16ClN3O2; 365.0931; Found 366.1035

65

2.9.2 Materials and methods for biological characterization

Cell culture
Frozen HEK293 cell stocks were obtained from ATCC and ThermoFisher Scientific. Cells
were grown in DMEM (Corning) containing 4.5 g/L glucose and glutamine, supplemented
with 10% FBS and in a 37 C humidified incubator with 5% CO2.

Probe treatment in situ and in-gel fluorescence imaging
Cells were seeded in 6-well plates at 5x10
5
per well, in DMEM supplemented with 10%
FBS. The next day, media was replaced with fresh media containing 1 µL of (1000x) desired
concentration of the probe. Following the probe treatment, cells were washed twice with ice
cold DPBS prior to harvesting them. Harvested cells were pelleted down using
centrifugation at 2500 g for 5 minutes at 4 C. The supernatants were removed, and cells
were lysed using 1%NP40 lysis buffer (1%NP40, 50 mM HEPES (pH 7.4) and 150 mM
NaCl). Soluble cellular fractions were separated from cell debris, by centrifugation at
18000g for 10 minutes at 4 C. Cell lysate concentrations were measured using BCA assay
and CuACC reactions were set up at in 100 L of cell lysates at 1 mg/mL protein
concentrations with, 25µΜ TAMRA-azide (Click Chemistry Tools), 1 mM Tris(2-
carboxyethyl)phosphine(TCEP, Thermo-Scientific), 100 µΜ Tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl]amine(TBTA, TCI), and 1 mM CuSO4 (Sigma-Aldrich). Click
chemistry reaction mixtures were incubated at room temperature for 1 hr in the dark,
66
followed by reaction quenching by adding 33 L of 4x Laemmli sample buffer (Bio-Rad)
and boiling for 5 min. A volume of 30 µL of the samples were loaded and resolved by SDS-
PAGE (18%, 4-30% or 7.5% depending on the purpose of experiment) before visualization
at 532 nm for excitation and 610 nm for emission on a Typhoon 9400 Variable Mode Imager
(GE Healthcare). Fluorescence images are displayed as gray scale. For the cells transfected
with 3×FLAG tagged GMPS, after fluorescence scanning, proteins were transferred to
PVDF membranes for immunoblotting with FLAG and GMPS antibodies to monitor the
transfection level.

Biotin streptavidin enrichment and protein identification
HEK293 cells were seeded in 15 cm plates with 10x10
6
per plate, with 20 mL of DMEM
media supplemented with 10% FBS. The next day, media was replaced with fresh media
containing either mock (DMSO) or 100 nM of probe 3. Three 15 cm plates were used for
each condition. Following 30-min incubation, media was removed and cells were washed
2x with ice cold DPBS, before cells were scraped using minimum volume of cold DPBS.
Harvested cells were pelleted down using centrifugation at 2,000 g and 4 C for 2min.
Supernatant was carefully aspirated and proceeded to cell lysis and biotin-azide conjugation
procedure as previously reported
1
.

Plasmid construction and transfection
Standard cloning procedures were followed to obtain all the constructs. Full length human
GMPS sequence was amplified using PCR and cloned into p3xFLAG-CMV 10 vector in
order to be used for transient transfection. Full-length human GMPS were amplified using
67
PCR and cloned into pET28a vector in order to be used for recombinant protein expression.
Mutants were generated using QuikChange
TM
site-directed mutagenesis (Agilent). Detailed
cloning information and sequences will be provided upon request.
Cells were seeded at 5x10
5
cells per well in 6-well plates and transfected with GMPS
constructs (p3XFLAG-CMV 10) the next day using Lipofectamine 2000 (Life
Technologies). Cells were used for downstream experiments following 24hrs of transfection.

Western Blotting
Following SDS-PAGE gel, proteins were transferred in to PVDF membranes using TRANS-
Blot Turbo Transfer System (Bio-Rad). Membrane containing transferred proteins were
incubated with respective antibody following recommended rations by the manufacturer and
developed with ECL substrate (Bio-Rad) prior to visualization with ChemiDoc XRS+
molecular imager (Bio-Rad). Antibodies used: anti-FLAG tag (Sigma, cat# F3165, 1:1000)
and anti-GMPS D-5 (Santa Cruze, sc-374225, 1:1000)

Recombinant GMPS expression and purification
GMPS constructs (pET28a) were transformed into E. coli BL21 cells and grown under
Kanamycin resistance. Individual colonies were inoculated in 5 mL of LB broth overnight,
followed by large culture growth in total of 1L of LB. Previously reported standard bacterial
protein purification protocols were followed to obtain purified recombinant GMPS protein
2,
3
.
Purified protein variants were utilized for in vitro labeling experiments as well as in vitro
activity assay experiments.
68

In vitro probe treatment and in-gel visualization
Purified recombinant GMPS protein solutions were prepared at 100 nM concentration.
Protein solutions were reacted with 1 µL of desired concentrations of the probes (20x) in a
20 µL of total volume at 37 C for the desired time. Following probe treatment click reactions
were performed using 1.05 µL of the click reagent containing, 25 µΜ TAMRA-azide (Click
Chemistry Tools), 1 mM Tris(2-carboxyethyl)phosphine(TCEP, Thermo-Scientific), 100
µΜ Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine(TBTA, TCI), and 1 mM CuSO4
(Sigma-Aldrich). After one hour, reactions were quenched using 7 µL of 4X LB containing
10% BME. Samples were boiled and subjected to SDS-PAGE analysis following in-gel
fluorescence visualization to identify labeling.

In vitro activity assay for GMPS
The reaction buffer was prepared containing EPPS (pH 7.8), EDTA (100 M), MgCl2 (10
mM), ATP (2 mM), XMP (200 M), Glutamine (5 mM). The blank buffer was prepared
with the same ingredients at equal concentrations except for the absence of XMP. Protein
solutions were prepared in EPPS (pH 7.8) at varying concentrations (ranging from 0.1 M
to 100 M). UV-vis was set to kinetics mode and the absorbance wavelength was set to 290
nM. Following auto zeroing with the blank buffer, 495 µL of the reaction buffer was
incubated with 5 µL of each protein solutions and the decrease of absorbance at 290 nM was
observed. The reaction rates were calculated using the change in absorbance over the time
course of 30 minutes.
69
For inhibition assays 0.5 µL of 1000x of the desired concentration of the drug was added to
the reaction mixture and the decrease in absorbance over 30 minutes was observed. IC50
values for probes and competitor molecules were calculated using non-linear regression
“Dose Response-Inhibition” model on Prism 7 software (GraphPad Software, La Jolla, CA).

MS analysis of cellular metabolites upon treatment with probe 3
Cells were seeded in 6-well plates at 5x10
5
per well, in DMEM supplemented with 10%
FBS. The next day, media was replaced with fresh media containing 1 μL of 100 μM of
probe 3. Following the probe treatment, cells were washed with ice cold ammonium acetate
(pH 7.3) using 1 mL per well. Following washing 1 mL of cold Methanol (cooled in -80 C
freezer couple of hours prior to the experiment) was added to each well and cells were
scraped off. Scraped cell suspensions were transferred to Eppendorf lo-bind tubes (Catalog
No. 0030122348) and centrifuged at 5000 g at 4 C. The supernatant in each tube was
transferred to a clean Eppendorf tube and was kept on ice. The pellet was re-suspended in
200 µL of 80% cold methanol and re-centrifuged at 5000g and each supernatant was
transferred to the same tube containing previous extracts. The supernatants were lyophilized
without heating and stored at -80C until ready for mass spectrometry analysis.

For the time-course experiment, cells were treated with 300 nM of compound 1 and
harvested at different time points. Metabolite extraction was performed 24 h after adding
labeled media. For extraction of intracellular metabolites, cells were washed on ice with 1
ml ice-cold 150 mM ammonium acetate (NH4AcO, pH 7.3). 1 ml of -80 °C cold 80% MeOH
was added to the wells, samples were incubated at -80 °C for 20 mins, then cells were scraped
70
off and supernatants were transferred into microfuge tubes. Samples were pelleted at 4°C
for 5 min at 15k rpm. The supernatants were transferred into LoBind Eppendorf microfuge
tube, the cell pellets were re-extracted with 200 µl ice-cold 80% MeOH, spun down and the
supernatants were combined. Metabolites were dried at room temperature under vacuum and
re-suspended in water for LC-MS run.
Samples were randomized and analyzed on a Q Exactive Plus hybrid quadrupole-Orbitrap
mass spectrometer coupled to an UltiMate 3000 UHPLC system (Thermo Scientific). The
mass spectrometer was run in polarity switching mode (+3.00 kV/-2.25 kV) with an m/z
window ranging from 65 to 975. Mobile phase A was 5 mM NH4AcO, pH 9.9, and mobile
phase B was acetonitrile. Metabolites were separated on a Luna 3 µm NH 2 100 Å (150 ×
2.0 mm) column (Phenomenex). The flowrate was 300 µl/min, and the gradient was from
15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for
7 min. All samples were run in biological triplicate. Metabolites were detected and quantified
as area under the curve based on retention time and accurate mass (≤ 5 ppm) using
TraceFinder 3.3 (Thermo Scientific) software. Metabolites data was normalized to cell
number at the time of extraction.

71

Spectra:

72

73

74

75

2.10 References

1. Wang, Q. et al. Inhibition of guanosine monophosphate synthetase (GMPS) blocks
glutamine metabolism and prostate cancer growth. The Journal of Pathology 254,
135–146 (2021).

2. Welin, M. et al. Substrate Specificity and Oligomerization of Human GMP
Synthetase. Journal of Molecular Biology 425, 4323–4333 (2013).

76

Chapter 3. Novel covalent compounds with anti-COVID activities

Coronaviruses (CoV) comprise of a family of positive-sense RNA viruses
responsible for inducing severe respiratory disease in humans and animals. Several CoV
exist, with SARS-CoV and MERS-CoV being the most notable due to their history of large
outbreaks over the last two decades.
1,2
The replication and transcription mechanisms behind
these CoV are both sophisticated and complex, and this coupled with our limited
understating of the CoV RNA synthesis and processing, hinders drug development.

CoV contain a positive-sense RNA genome of ~30 kb, with approximately two-thirds
encoding for non-structural proteins (nsps).
3
Individual coronavirus particles are comprised
of four main structural proteins: spike (S), membrane (M), envelope (E), and nucleo-capsid
(N). The S protein contains an N-terminal signal sequence allowing it to gain access to the
endoplasmic reticulum. Homotrimers of the S protein form the distinctive spike-structure on
the surface of the virus, and are responsible for mediating attachment to the host receptor.
3

The M protein, believed to exist as a dimer, is the most abundant structural protein in the
virion and is thought to be responsible for maintaining the virion’s shape.
3
The E protein is
the least abundant protein in the virion, and is responsible for facilitating the assembly and
release of the virus.
3
In addition to the main structural proteins, the 16 non-structural proteins
contained in the individual virions also work together as an enzyme complex during
replication.
4

77
Nearly two-thirds of the SARS-CoV-2 genome is comprised of ORF1a/b which is translated
into polyproteins pp1a and pp1ab, with pp1a consisting of Nsp1-Nsp11 and pp1ab consisting
of Nsp1-Nsp16.
5
These NSPs, namely Nsp1 to Nsp10 and Nsp12 to Nsp16, along with nine
accessory proteins (ORF3a, 3d, 6, 7a, 7b, 8, 9b, 14, and 10), play a crucial role in viral
replication.
6
Due to their significant contributions to SARS-CoV-2 viral replication,
proteins may prove promising drug targets to combat pathogenesis.

3.1 Generation of open-ring GMPS analogs 2-d and 2-c

Following the discovery of the GMPS covalent inhibitor 1, we aimed to utilize some
of the compounds developed for that project to pursue a new study. We were very intrigued
by the results we saw from the labeling experiments of 1-c and 1-d (Figure 17), from which
we learned that only the isoquinoline analog of 1 was capable of labeling GMPS. With only
that particular nitrogen necessary for protein labeling, we inquired into whether the
remainder of the core, which was rather rigid, was necessary for protein labeling as well.

78

Figure 29. Generation of open-ring analogs. 2-d and 2-c closely resemble 1-d and 1-c,
with greater flexibility.

To investigate this, we designed two new analogs, 2-d and 2-c based off of 1-d and 1-c
respectively (Figure 29). The difference between these two molecules has to do with the
order of the carbonyl and amide. In 2-d, the carbonyl is directly attached to the phenyl group
containing the chloroacetamide electrophile. This design resembles 1-d, in which an extra
carbon exists between the nitrogen and the phenyl of the isoquinoline ring. In 2-c on the
other hand, the amide and carbonyl groups are reversed such that the amide is directly
attached to the phenyl containing the electrophile. This order resembles 1-c, in which the
nitrogen of the quinoline ring is adject to the phenyl.
We tested these new compounds in HEK293 cells using our standard protocol
described previously followed by a “click” reaction to Tamra-N3 (Figure 30).

79

Figure 30. Treatment of 2-d and 2-c in cells and purified recombinant protein. Only 2-d
was able to label p75 (GMPS).

In HEK293 cells, we observed labeling of a protein band ~75 kDa, presumably GMPS, with
2-d treatment. Given that 2-d was based of off 1-d proven to label GMPS in cells, this is
data was consistent. 2-c showed no labeling of the 75 kDa band. As described above, this
compound was designed to resemble 1-c which was shown unable to label GMPS in cells.
To further assess these compounds, we tested them on purified recombinant GMPS. At 100
nM concentration of protein, only 2-d was able to label GMPS. Compared to compound 1,
our main probe from Chapter 2, the labeling efficiency of 2-d was not as good. Despite this,
the results of these two experiments were both interesting and promising. They supported
our hypothesis that removing unnecessary nitrogens and carbons from the original
quinazoline ring would still allow for labeling of GMPS. Both 1-d and 2-d were able to label
GMPS in cells and purified protein, supporting the notion that the isoquinoline nitrogen of
the original scaffold was essential for protein labeling. We found this new open-ring scaffold
80
to be interesting, both in its simplicity and opportunity for modifications.

Figure 31. Design of competitor version of 2-d. The propargyloxy moiety was replaced with
meta-methoxy.
We decided to synthesize a competitor version of 2-d, 2-d-comp, similar to what we did
with 1 and 4. 2-d-comp consisted of a meta-methoxy in place of the propargyloxy, and
would serve useful in future experiments.

Around this time, we were faced with the Covid-19 pandemic. We sought out to
investigate the potential of this new scaffold using SARS-CoV-2 as our model, and aimed
to assess the antiviral capabilities of a new a set of inhibitors based on 2-d and 2-d-comp.

81
3.1.1 2-d-comp can increase IFN signaling activation

To first assess whether these new compounds could have potential as anti-viral
agents, we wanted to test them using a Sendai Virus (SeV) IFN-B mRNA expression assay
(Figure 32). The Feng Lab at USC School of Dentistry assisted us with this project, and
generated all viral models talked about in this chapter. NSP8 overexpressed A549 lung
cancer cells infected with SeV were treated with 1, 2-d, 2-c, and 2-d-comp. The IFN-B
mRNA expression, the immune response induced by each compound, was measured. Out of
the 4 compounds tested, 2-d-comp performed the best, inducing the strongest interferon
response.

Figure 32. IFN-B mRNA expression assay of 1, 2-d, 2-c, and 2-d-comp. 2-d-comp
generated the strongest interferon response in A549 cells infected with SeV.
82
The data suggested that these open-ring analogs, namely 2-d-comp, could have potential
antiviral activity, and that optimization of the compounds through SAR studies was worth
pursuing.

3.2 Design and synthesis of 1
st
generation panel

Using 2-d and 2-d-comp as the parental molecules for our SAR studies, we designed
and synthesized 8 new small molecules. We made modifications to investigate various
structural effects of different areas of the parental compounds (Figure 33). The aniline was
notably a prime area for optimization. 2-d-comp possess a meta-methoxy substituent on the
aniline. We thought it would prove interesting to include para-methoxy (C4) and an ortho-
methoxy (C5) variants in our first-generation panel. We also made, C1 which has a
cyclohexane as opposed to an phenyl ring, to assess the effects of removing aromaticity in
that substituent. In C2, we methylated the central amide bond to see whether that nitrogen
nitrogen, a hydrogen donor, was necessary for the observed IFN response.

Figure 33. First generation panel. Molecules based off of 2-d and 2-d-comp.
83

For C6, we incorporated an imidazole ring in place of the phenyl to investigate how a
heterocyclic ring in that position would perform. C9 was unique in that we used a 5-carbon
chain in place of a closed ring, making that part of the molecule longer and more flexible.
Lastly, C7 and C8 saw changes to the center ring by converting to a thiophene.

3.2.1 IFN reporter assay of 1
st
generation panel
We tested the panel using the same IFN reporter assay discussed earlier to check
whether any of these compounds induced an immune response in virus-infected mammalian
cells. We also included the parental 2-d-comp for comparison (Figure 34). Of the 8
compounds, 5 were shown to increase IFN activity. C4 and C5 induced notable responses at
1 uM of compound, whereas C7, C8, and C9 induced similar responses at only 0.5 uM of
compound. 2-d-comp performed well as expected, however at 0.5 uM, both C8 and C9
performed better.
84

Figure 34. IFN reporter assay of 1
st
generation panel. C4 and C5 induced an increase in
reporter activity at 1 uM. C7, C8, and C9 induced an increase in reporter activity at 0.5 uM.

We took these top 5 performing compounds and repeated the experiment using lower
concentrations of each drug (Figure 35). All 5 showed an increase in IFN reporter activity,
mostly at 1 uM of drug. C9 stood out to us for a couple of reasons. The first being that in the
reduced concentration experiment, C9 showed a clear dose-dependent IFN activity response
(boxed in figure). This dose-response was the most prominent of the 5 compounds. Second,
in looking at the structure of C9, it is distinctly different from the others in that it contains a
5-carbon chain in place of a closed ring. Because of this, we took note of C9’s promise. C7
and C8 were also notable as both contained a thiophene ring in place of the center phenyl.
85

Figure 35. IFN reporter assay repeated for best-performing drugs at lower concentrations.

Given these were the only two compounds in the panel to possess a thiophene, we postulated
whether this feature was advantageous. The performances of C4 and C5 were somewhat
unexpected. 2-d-comp contains a meta-methoxy substituent, and C4 and C5 contain para-
and ortho- methoxy. The modification is rather small, and so these two performing similar
to that of 2-d-comp is consistent with what we would expect.

3.2.2 1
st
Gen treatment in SARS-CoV-2 infected Caco2 cells

Taking these 5 compounds, we next tested them in Caco2 cells infected with SARS-
CoV-2. Caco2 cells were treated with 2 uM of each drug for 2 hours and then were infected
with SARS-CoV-2. The medium was changed after infection and replaced with new medium
containing another 2 uM of each drug. Fresh drugs were added every 24 hours for 72 hours.
86
The supernatant was harvested for a plaque assay and cells were harvested for qPCR at 72 h
post-viral infection.

Figure 36. SARS-CoV-2 infected Caco2 cells treatment. Cells were trearted with 2 uM of
each drug for 2 hours and then were infected with SARS-CoV-2. The medium was changed
after infection and replaced with new medium containing another 2 uM of each drug. Fresh
drugs were added every 24 hours for 72 hours. The supernatant was harvested for a plaque
assay and cells were harvested for qPCR at 72 h post-viral infection

The first experiment shown on the left side, measured the cellular viral titers after post drug
treatment. C9 significantly performed the best, followed by C5 and C7 (note Log units). The
data to the right hand side measured the relative gene expression of two SARS-CoV-2
proteins N and E. In this experiment too, C9 performed that best at reducing the gene
expression of these viral proteins, followed by C5 and C7. Importantly, all performed better
87
in these experiments compared to the parental 2-d-comp, indicating that beneficial structural
modifications had been made with our 1
st
generation panel. We were very interested again
by how well C9 performed, and flagged this compound as one to move on for additional
SAR studies. C5 was also quite interesting considering it performed better compared to its
C4 counterpart. From this data it appears the ortho-methoxy substituent is more beneficial
than the para- and meta- versions. C7, with a thiophene ring also caught our attention. While
C8 had a thiophene ring as well, the particular orientation of the ring seen in C7 proved to
be a better performer in these experiments. With this SAR information, we moved on to
designing a 2
nd
Generation Panel based off of C5, C7, and C9.

88
3.3 Design and synthesis of 2
nd
generation panel

Following the SAR we acquired from the 1
st
generation panel and the subsequent
experiments, we designed a set of 8 new compounds based on C5, C7, and C9 (Figure 37)
which were the best performing compounds in both the IFN activity assay and SARS-CoV-
2 viral assays. For the ones based on C5, we chose to explore how different substituents at
the ortho-position of the aniline ring would affect the drugs’ performance.

Figure 37. 2
nd
Generation panel. Compounds based on best performing compounds from
previous generation (C5,C7,C9).

For B5, we incorporated a simple methyl in place of the methoxy to see how a smaller and
less polar substituent would perform. In B6 we replaced the methoxy with a chlorine and in
B8 we incorporated an ethyl group, essentially removing the oxygen from the original
89
methoxy substituent. For compounds based on C7, we wanted to keep the central thiophene
ring, they key feature of C7, while altering some of the other substituents on the structure.
B2 and B7 explored the combination of a thiophene central ring with orth- and meta-
methoxy substituents on the aniline, similar to that of C5 and 2-d-comp. B1 combined the
two main features of C7 and C9, the thiophene center and the 5-carbon chain, and thus
became of particular interest to us. We were curious as to whether putting together the two
seemingly best performing features of the 1
st
generation into one compound would produce
an even better small-molecule. Lastly, the two new compounds we designed based on C9
were B3, which included an oxygen in its 5-carbon chain to see what the effects of a more
polar chain would do, and B4, with a cyclopentane meant to serve as a closed-ring of the 5-
carbon chain.

3.3.1 2
nd
Gen treatment in SARS-CoV-2 infected Caco2 cells

Like with the 1
st
generation panel, we treated the 2
nd
generation molecules into Caco2
cells infected with SARS-CoV-2 using the same workflow as described previously (Figure
38). We first measured the RNA abundance of the two viral proteins N and E after 2 uM
pretreatment of drugs. We included C9 for comparison. B1, B2, B5, and B7 all demonstrated
a prominent decrease in viral protein RNA abundance, performing better even than C9. In
the viral titers assay, these same four performed best at inducing the greatest reduction. It
was noted however, that cells treated with B2 and B7 exhibited a phenotypic appearance
consistent with toxicity. As a result, the anti-viral effects of B2 and B7 observed in the
previous data was taken to be false and simply the result of toxicity. That left B1 and B5 as
90
being the best performing from this set of compounds. In looking at the structures, B1 is
noteworthy. It incorporates two features from the previous generation that were shown to
have better anti-viral abilities, the thiophene center ring and the 5-carbon flexible chain. B5,
with its ortho methyl substituent was a bit surprising, and suggests that a smaller substituent
at the ortho position may be beneficial. Though C9 did not perform as well as B1 and B5 in
these experiments, the fact that C9, with its 5-carbon chain, was the best performing of the
first generation, and now B1 with the same moiety proved superior in the second generation,
indicated that the 5-carbon chain is an important feature responsible for the observed anti-
viral activity of these drugs.

91

Figure 38. SARS-CoV-2 infected Caco2 cells treatment of 2
nd
gen panel.

As a result, in addition to B1 and B5 being selected for optimization for the 3
rd
generation
panel we planned to design, we also continue to include C9 as being one of the top
performing compounds we had made and tested. With these three, we moved onto designing
a new panel of drugs, taking inspiration from all of the SAR we had acquired thus far.

92
3.4 Design and synthesis of 3
rd
generation panel

For our third and final generation panel, we designed and synthesized 6 small
molecules (Figure 39). The first A1, contained a thiophene ring in the center with a
cyclopentane amine. Given that the 5-carbon chain appeared to be an important substituent
in the previous panels, we wanted to see the effects of using a substituent that mimicked a
closed-ring of this. Similarly, A4 and A5 possess a substituted cyclopentane (2-methyl)
representative of an “ortho” substitution which was noted to be an important modification
in earlier studies, with the difference being that A5 has a thiophene center.

Figure 39. 3
rd
generation panel. Molecules designed to explore various “close- ring”
variants of the 5-carbon chain seen on C9.

For A3 we used a thiophene center with a cyclohexane amine, meant to also mimic a closed
carbon chain. A7 was given a substituted (2-methyl) cyclohexane, also mimicking an ortho-
93
substitution. Lastly A6 can be seen with a 2,6-dimethyl-aniline, allowing us to explore
whether a double ortho- substitution would produce a better performing drug.

3.4.1 3
rd
Gen treatment in SARS-CoV-2 infected Caco2 cells

We tested our 3
rd
and final panel using the same experiment as the last two panels in
which we measured the RNA abundance of two SARS-CoV-2 viral proteins (Figure 40).
Using C9 and B1 as comparisons, we were disappointed to see that none of the new
molecules performed well in this assay. C9 was the clear winner, and we did not note any
interesting results from any of the other molecules.

Figure 40. SARS-CoV-2 infected Caco2 cells treatment of 3
rd
gen panel

Though this panel was not successful in improving upon the SARS-CoV-2 anti-viral
activity, we felt we had obtained several promising candidates in C9, B1, and B5, from
previous generations, and decided to continue with additional studies using these drugs.
94
3.5 Best Performing Compounds in SARS-CoV-2 Infected Caco2 Cells and Generation of
C9 probe

C9, B1, and B5, proved to be the best performing compounds of our 3 generations.
We decided to repeat the SARS-CoV-2 viral protein RNA assay using these 3 compounds
and various increasing concentrations to see whether a noticeable dose-response could be
seen (Figure 41). We tested the 3 compounds at 0.25 uM, 0.5 uM, 1 uM, and 2 uM. We
were very pleased to see C9 significantly reduced (note Log units) RNA abundance of N and
E in a very prominent dose-dependent manner. At 0.25 uM of drug, C9 reduces N levels
nearly 3 fold times more than B1 and B5.

Figure 41. SARS-CoV-2 infected Caco2 cells treatment of best performing compounds from
all three panels (C9, B1, B5). C9 shows prominent dose-response in reducing cellular
abundance of N and E, and is selected as most promising compound. C9-probe is
subsequently synthesized for additional studies.
95
Given this data and all data acquired thus far, we identified C9 to be our most promising
compound for possessing anti-viral activity against SARS-CoV-2. To further study C9 and
identify potential protein targets, we generated C9-probe, with a terminal alkyne. This
alkyne, as discussed in previous chapters, allows us to conduct pull-down and labeling
studies to further our investigation.

3.6 Target engagement of 2-d & C9-probe with SARS-CoV-2 viral proteins

After researching the possible SARS-CoV-2 protein targets C9 could be interacting
with, we narrowed our hypothesis down to it being one or more of the many non-structural
proteins (NSPs) or CTPS1. CTPS1 or CTP Synthase 1 is responsible for the catalytic
conversion of UTP to CTP. It does this through the hydrolysis of glutamine to glutamate and
generating a free amine which it will use to amidate UTP. The reaction is similar to that of
GMPS discusses in Chapter 2. In fact, both UTP and GMPS belong to the same the same
class I glutamine amidotransferase-like domain family and possess a cysteine, histidine,
glutamic acid (glutamate) catalytic triad. Given that our parental compounds, 2-d and 2-d-
comp were shown to bind GMPS, we speculated that C9 could be interacting with and
inhibiting viral CTPS1. For the NSPs, we knew that the protein or proteins of interest played
must be essential for SARS-CoV-2 survival, which C9 is able to somehow perturb. The
various SARS-CoV-2 NSPs are essential for survival, with all serve unique roles in
maintaining the replication process.

96
To check the target engagement of C9-probe with potential SARS-CoV-2 viral
proteins, we first transfected a handful of NSPs, Nsp5, the Nsp 7+8+12 complex, Nsp13,
and Nsp15, as well as CTPS1 into HEK293 cells. After confirming transfection, we treated
each set of transfected cells with 2 uM of either 2-d (original parental compound) or C9-
probe (Figure 42).

97

Figure 42. Treatment of 2-d and C9-probe on HEK293 cells transfected with various
SARS-CoV-2 proteins. Both probes labeled CTPS1 and the Nsp 7+8+12 RNA polymerase
complex.

After click with N3-azide, we were able to visualize the targets C9-probe was binding to.
Both 2-d and C9-probe were shown to label CTPS1 and the Nsp 7+8+12 RNA polymerase
complex.

98
3.7 CTPS1 labeling
To assess the labeling of CTPS1, we performed a simple experiment involving
immunoprecipitated FLAG-CTPS1 generated by our collaborators in the Feng Lab. We
treated the IP FLAG-CTPS1 with increasing concentration of 2-d followed by click for
visualization (Figure 43).

Figure 43. 2-d Labeling of IP FLAG-CTPS1.

We were able to observe dose-dependent but weak labeling of CTPS1. We then
moved on to investigating occupancy of this probe in competition of the molecules generated
from our 3 panels. We knew that several of these compounds exhibited strong antiviral
activity. Presumably, we were observe high occupancy of these drugs to their protein of
interest. To test this, we performed a competition labeling experiment with 2-d at a fixed
concentration of 2 uM and the 22 compounds, from the 3 panels shown earlier in this chapter,
at a fixed concentration of 10 uM.

99

Figure 44. Competition labeling experiment between 2-d (2uM) and the 22 compounds
from the 3 generations of panels. No significant inhibition of the signal was observed.

The 5-fold increase in concentration for the competitors should outcompete the probe signal
should CTPS1 be the primary binding target. What we observed instead was no significant
inhibition of signal from any of the 22 inhibitors (Figure 44). This suggested that while
labeled, CTPS1 is not the primary target of these compounds and is not responsible for the
anti-viral activity observed in previous experiments.

3.8 Target engagement of 2-d and C9-probe on Nsp12

With CTPS1 being ruled out as the primary target of the C9, we moved onto
investigating whether Nsp12 could be the target of interest. We transfected Nsp12 into
HEK293 cells and after confirming transfection, performed a couple competition
100
experiments (Figure 45). The first was between C9-probe at 10 uM and inhibitors C9, B5,
B2, B1, and B8 at 10 uM. Due to the transfection quality, visualizing the competition of the
signal was a bit unclear, so we included normalized fluorescence values.

Figure 45. Competition labeling experiment on cells transfected with Nsp12 and Nsp15
(negative control). C9-probe and 2-d were treated at 10 uM and 2 uM against C9, B5, B2,
B1, and B8 at 10 uM. Inhibition of signal seen with all inhibitors in the Nsp12 cells while
no competing of the signal was observed in the Nsp15 cells.

Both C9-probe and 2-d were shown to be outcompeted by all inhibitors included in
the experiment, notably B1 which performed the best at competing off the signal against
both probes. As a negative control, we also performed this experiment using 2-d on cells
transfected with Nsp15 which was not identified to be a target of these probes (Figure 43).
No competing of the signal was observed in the competition for the Nsp15 transfected cells.
The results from these experiments strongly suggest that Nsp12 could be the primary target
of C9.
101

We further tested this by performing another competition experiment using cells
transfected with Nsp12. Using B1, because of how it performed in the previous experiment,
and C9, we treated these compounds into the Nsp12 cells at increasing concentrations from
0.1-30 uM. For the probe we used 2-d at a fixed concentration of 2 uM.

Figure 46. Competition labeling experiment on cells transfected with Nsp12. 2-d was treated
at 2 uM against B1 and C9 at 0.1, 0.3, 1, 3, 10 and 30 uM. Both B1 and C9 exhibited dose-
dependent inhibition of the signal.

We were able to actually visualize the competing off of the signal, which was confirmed
using normalized fluorescence values. Both B1 and C9 exhibited dose-dependent inhibition
of the signal (Figure 46). If one recalls, both B1 and C9 were identified to be the most
promising compounds of the three generations of panels that were tested. Given that B1 and
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C9 both exhibited strong anti-viral activity in cells, and both were shown to compete off the
fluorescence signal when treated in Nsp12 transfected cells, we believe Nsp12 is the protein
of interest both B1 and C9 are binding to.

3.9 Site identification of C9-probe on Nsp12

To determine the potential sites of modification of our compounds on Nsp12, we sent
out our lead compounds, B1 and C9, along with our probes 2-d and C9-probe for mass spec
analysis. Several cysteine sites were identified as being covalently labeled by both 2-d and
C9-probe: C396, C623, C800, and C814. To confirm the selectivity of our probes against
these identified cysteines we generated alanine mutants C396A, C623A, C800A, and C814A
in order for us to perform live-cell labeling experiments. The mutants were transfected into
HEK293T cells and the cells were treated at 3 concentration points, 0 uM, 1 uM, 2 uM, of
both 2-d and C9-probe. After 30 minutes of incubation with the probes, cells were lysed
and lysates were using the StrepTactin enrichment method (See methods). On-resin, Cu-
catalyzed tamra-azide click reactions subsequently followed, and protein concentrations
were standardized and run via gel electrophoresis.

103

Figure 47. Treatment and flourecence visualization of 2-d against panel of alanine Nsp12
mutants.

The fluorescence bands were normalized in order to accurately interpret the effects of each
alanine mutant on labeling. Compared to labeling of WT Nsp12, a reduction in labeling can
be observed with the C396A, C564A, and C800A mutants, with C564A showing the largest
reduction at both 1uM and 2uM of 2-d. In the C9-probe treated groups, a similar trend is
observed, with C564A showing the most inhibition of signal compared to WT. In both cases,
the signal reduction is between 40-60%.

104

Figure 48. Treatment and flourecence visualization of C9-probe against panel of alanine
Nsp12 mutants.

While a complete elimination of signal is not observed, it is not entirely surprising. Mass
spec analysis reveal both 2-d and C9-probe bind several cysteines within Nsp12, so it is
very unlikely that any one mutation would completely eliminate the signal. A double or even
triple mutant would likely necessary in order to observe a more significant reduction of
signal. What does seem to be apparent from both data sets is that C564A appears to be the
largest contributor to signal reduction, suggesting that the primary site of modification of
these probes in C564 within Nsp12.

3.10 Biochemical data
105
3.11 C9 demonstrates anti-viral activity in vivo

To investigate whether our compounds exhibit antiviral activity given the role
of Nsp12 in SARS-CoV-2, we sought the help of valued collaborators who developed a
robust method for preparing SARS-CoV-2-infected AAV-hACE2 mice for a series of animal
studies.

Figure 49. Workflow schematic to generate SARS-CoV-2-infected AAV-hACE2 mice for
animal studies

Though C9 has consistently proven to be our best performing compound in cellular
experiments as discussed previously, we wanted to compare C9’s performance in SARS-
CoV-2 infected mice compared its parental compared 2-d-comp. Both compounds were
treated intravenously(35 mg/kg) in AAV-hACE2 C57BL/6J mice daily over a course of 15
days before they were euthanized and their lungs harvested for qRT-PCR analysis. The
relative mRNA abundance of several interferons (IFNs), the signaling proteins released by
host cells in response the viral infection, were measured. Only C9 was found to have induced
an increase in interferon response indicating that the drug caused an immune response in
these SARS-CoV-2 infected mice.

106

Figure 50. Relative mRNA abundance of IFNs in lungs harvested from SARS-CoV-2-
infected AAV-hACE2 mice treated with 2-d-comp and C9

Further experiments supported these results. The relative mRNA abundance of the SARS-
CoV-2 viral structural protein E, the RNA-binding protein critical for viral genome
packaging, N, and the non-structural protein 1 (Nsp1), were next measured. C9 and not 2-d-
comp was shown to significantly reduce the abundance of viral proteins in treated mice by
1.4-1.9 log units. Finally, lung viral titers of the two groups of mice were measured. While
2-d-comp did reduce viral titers, the clear performer was C9 with a nearly 100x reduction
in viral titer compared to the control and a 10x reduction compared to 2-d-comp.

107

Figure 51. Relative RNA abundance of SARS-CoV-2 viral proteins in lungs harvested from
mice treated with 2-d-comp and C9, and lung viral titer measurements.

These initial animal experiments of 2-d-comp, one of our parental compounds, and C9, our
lead compound thus far, revealed that while structurally similar, the SAR optimization we
performed to develop C9 has lead to the compound possessing significantly stronger anti-
108
viral activity in SARS-CoV-2 infected mice, which compliments the prior cellular data we
have collected.

Figure 52. C9 (35mg/kg) increased IFN-B and ISGs expression in the lungs of SARS-Co-
V-2 infected AAV-hACE2 delivered mice (10 mice sample size).

The experiments were repeated using just C9 with 10 transgenic mice instead of 5, and we
pleased to see that C9 showed consistent antiviral activity in vivo. Once again, C9 was
shown to increase interferon-B and interferon stimulating gene expression in the lungs of
SARS-CoV-2-infected AAV-hACE2 delivered mice (Figure 52).
109

Figure 53. C9 (35mg/kg) inhibits SARS-CoV-2 gene expression and viral titers in lungs of
AAV-hACE2 delivered mice (10 mice sample size).

C9 was also shown to inhibit SARS-CoV-2 gene expression by reducing the expression of
the N and E viral proteins as well as the non-structural protein Nsp1. Finally, the larger
sample of group mice given C9 were shown to exhibit decreased viral titers in the lungs,
consistent with the previously acquired data set.
We then moved on to investigating the effects of C9 on body weight and percent
survival. C9 was introduced to 9 mice by intraperitoneal injection (IP) at dose of 35 mg/kg
(0.6 mg per mouse) 2 hours before viral infection. The mice were infected with SARS-CoV-
2 (1 x 10
3
PFU) via intranasal route and given drug daily for 7 days. The beneficial difference
between C9 and control is marginal, however a notable decrease in body weight of the
control group can be seen at day 3 with only 2 mice in this group showing body weight above
90% at the end of 6 days. On the other hand, mice given C9 did not begin to show a notable
decrease in body weight until day 4. After day 4 and even up to day 6, 4 of the mice in the
C9 treatment group only showed a decrease in body weight of about 5-10%.
110

Figure 54. C9 helps reduce loss of body % weight and increases percent survival compared
to control

In looking at percent survival, mice treated with C9 showed a 40% better chance of survival
at 5 days post-injection compared to the control and a 20% better chance of survival 6 dpi.
Although after 6 days, no difference in percent survival between C9 and control groups are
observed, we were still pleased to have see that C9 had a positive effect on increasing
survival, even marginally.

111

Figure 55. C9 reduces lung tissue inflammation in SARS-CoV-2 infected mice

Lastly, we wanted to acquire a visual representation of the anti-viral activity C9 is able to
induce in SARS-CoV-2 infected mice. Stained lung tissues of mice that received in C9 can
be seen in Figure 55 compared control. Inflammation of the lungs represented by the darker
tissue stain can be seen in the vehicle sample. Comparing the vehicle sample to the C9
sample, a clear reduction in inflammation can be observed. Comparing the C9 sample to the
mock, coloring is similar with perhaps slightly higher inflammation in the C9 sample. Still,
notable difference in inflammation between vehicle and C9, indicating that C9 was able to
reduce lung inflammation in infected mice, and supporting the notion that C9 shows
demonstratable anti-viral activity in vivo.
Through this work we have identified a promising drug-candidate with anti-
SARS-CoV-2 activity in vitro and in vivo. We designed and synthesized three series of
112
analogs, performing numerous rounds of structural activity relationship studies, and
identified the tops hits of each generation. Our best performing compound was identified to
be C9, as it consistently showed strong anti-viral activity in cellular and animal models. To
further study our lead compound, we developed a probe version, C9-probe, giving us the
ability to perform labeling experiments using live cells, and allowing to determine its
potential binding partner. Through a series of pull-downs and labeling experiments, we
determined Nsp12 was the primary target of C9, and believe through mass spec analysis and
mutagenesis experiments that C9 is covalently binding to C564, though it is very possible
multiple cysteines on Nsp12 are being labeled and contributing to the observed anti-viral
activity. We demonstrated that C9 is able to reduce the loss of body weight in mice infected
with SARS-CoV-2, and increase the % chance of survival over a period of days. Lastly, we
provided visual evidence of C9 actively reducing inflammation in lung samples of infected
mice. Overall, we were able to provide a comprehensive study of our lead compound
showing antiviral activity against the most detrimental virus of our generation.

3.13 Experimental details

3.13.1 Chemical synthesis

Reagents and solvents were obtained from commercial suppliers and used without further
purification, unless otherwise stated. Flash column chromatography was carried out using
an automated system (Teledyne Isco CombiFlash). All anhydrous reactions were carried out
under nitrogen atmosphere. NMR spectra were obtained on Varian VNMRS-500, VNMRS-
113
600, or Mercury-400. The following abbreviations were used to explain NMR peak
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, b =
broad.

Preparation of probes and competitors for labeling experiments

3-(prop-2-yn-1-yloxy)aniline (1a)

NaOH pellets (1.49 g, 37.3 mmol) were weighed out and added to methanol (69.4 mL). 3-
aminophenol (3.82 g, 35 mmol) was then added and the solution was kept in an ultrasonic
bath for about 15 minutes until everything was completely dissolved. The volatiles were
evaporated by rotary evaporation and the residual water was removed by four successive co-
distillations with abs EtOH (27.8 mL, 4x). The obtained dried residue was dissolved in
CH3CN (69.4 mL) and propargyl bromide (80 wt. % solution in toluene, 4.62 mL, 41.56
mmol) was added in three portions over a 1hr period. The reaction mixture was allowed to
H
2
N OH
Br
+
1. NaOH, MeOH, rt
2. in-situ HCl, MeOH, rt
ClH
3
N O
1a
114
stir overnight. It was concentrated under rotary evaporation and the residue was partitioned
between aq NaOH (0.1 M, 69.4 mL) and diethyl ether (69.4 mL). The layers were separated
and the aqueous layer was extracted with diethyl ether (2x69.4 mL). The combined layers
were washed brine and dried over Na2SO4. Evaporation of the solvent gave a brownish oily
residue which was re-dissolved in MeOH (20.83 mL) and combined with in-situ generated
HCl (prepared from 104.2 mL of MeOH and 5.3 mL (74.76 mmol) of acetyl chloride). The
volatiles were evaporated resulting in a brownish solid which was resuspended in boiling
EtOAc (34.7 mL). After cooling the suspension to room temperature, white crystals were
collected by filtration and dried to afford compound 1a. (3.14 g, 49%).

1
H NMR (400 MHz, DMSO-d6) δ 9.73 (br s, 3H), 7.40 – 7.29 (m, 1H), 6.94 – 6.81 (m, 3H),
4.80 (d, J = 2.4 Hz, 2H), 3.60 (t, J = 2.4 Hz, 1H).

13
C NMR (151 MHz, DMSO-d6) δ 158.26, 134.14, 131.06, 116.19, 114.01, 110.46, 79.22,
79.13, 56.17.

115

N-(3-methoxyphenyl)-3-nitrobenzamide (2-d-comp.1)

3-nitrobenzoic acid (1 g, 5.98 mmol) and m-anisidine (803.9 uL, 7.18 mmol) were dissolved
in 10 mL of DCM/DMF (4:1). HBTU (9.07 g, 23.92 mmol) and triethylamine (3.33 mL,
23.92 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give 2-d-comp.1 (1.6 g, 99%) as a light orange
solid.
1
H NMR (600 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.76 (t, J = 1.8 Hz, 1H), 8.41 (ddd, J = 8.2,
2.3, 1.0 Hz, 1H), 8.37 (ddd, J = 7.8, 1.8, 1.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.44 (t, J =
2.2 Hz, 1H), 7.36 (ddd, J = 8.1, 2.0, 0.9 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 6.70 (ddd, J = 8.3,
2.6, 0.9 Hz, 1H), 3.74 (s, 3H).
13
C NMR (151 MHz,DMSO-d6) δ 163.78, 159.90, 148.18, 140.32, 136.72, 134.60, 130.69,
130.53, 126.62, 122.91, 113.30, 110.13, 106.60, 55.56.

116
3-amino-N-(3-methoxyphenyl)benzamide (2-d-comp.2)

2-d-comp.1 (250 mg, 0.92 mmol) was dissolved in 4 mL MeOH. Zn (300.19 mg, 4.59 mmol)
and NH4Cl (245.52 mg 4.59 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 follwoed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield 2-d-comp.2 (183 mg, 82%) as a beige solid, which was used
in the next reaction without further purification.
1
H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H), 7.44 (t, J = 2.3 Hz, 1H), 7.34 (ddd, J = 8.1,
2.0, 0.9 Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 2.0 Hz, 1H),
7.03 (ddd, J = 7.6, 1.8, 1.1 Hz, 1H), 6.73 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 6.63 (ddd, J = 8.2,
2.5, 0.9 Hz, 1H), 5.28 (s, 2H), 3.72 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 166.86, 159.85, 149.20, 141.03, 136.41, 129.74, 129.20,
117.22, 115.16, 113.40, 112.86, 109.27, 106.36, 55.42.

3-(2-chloroacetamido)-N-(3-methoxyphenyl)benzamide (2-d-comp)

2-d-comp.2 (150 mg, 0.62 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (238.53 uL, 1.36 mmol) was added via syringe. Chloroacetyl chloride
(79 uL, 0.87 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
117
chromatography (EtOAc/Hexane, 0-50%) to yield 2-d-comp as a white solid (100 mg,
51%).
1
H NMR (600 MHz, DMSO-d6) δ 10.48 (s, 1H), 10.22 (s, 1H), 8.07 (t, J = 2.0 Hz, 1H), 7.80
(ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.64 (ddd, J = 7.8, 1.8, 1.0 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H),
7.43 (t, J = 2.3 Hz, 1H), 7.34 (ddd, J = 8.1, 1.9, 0.9 Hz, 1H), 7.23 (t, J = 8.1 Hz, 1H), 6.66
(ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.26 (s, 2H), 3.73 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 165.86, 165.35, 159.87, 140.74, 139.06, 136.29, 129.94,
129.47, 123.32, 122.83, 119.36, 113.09, 109.67, 106.61, 55.53, 43.98.
HRMS (ESI) m/z: [M+H]
-
calcd for C16H15ClN2O3 318.0771; Found 317.0701.

3-nitro-N-(3-(prop-2-yn-1-yloxy)phenyl)benzamide (2-d.1)

3-nitrobenzoic acid (250 mg, 1.5 mmol) and 1a (330.55 mg, 1.80 mmol) were dissolved in
5 mL of DCM/DMF (4:1). HBTU (2.28 g, 6.0 mmol) and triethylamine (836.6 uL, 6 mmol)
were added and the solution was allowed to stir at room temperature for 16 hours. After 16
hours, the reaction mixture was diluted with EtOAc and washed with 10% Na2CO3 followed
by brine 3 times. The organic layer was dried with Na2SO4 and concentrated by rotary
118
evaporation. The residue was purified via flash column chromatography (EtOAc/Hex = 0-
50%) to give 2-d.1 (268 mg, 60%) as a pink solid.
1
H NMR (600 MHz, DMSO-d6) δ 10.56 (s, 1H), 8.78 – 8.72 (m, 1H), 8.43 – 8.40 (m, 1H),
8.39 – 8.36 (m, 1H), 7.86 – 7.76 (m, 1H), 7.49 (t, J = 2.2 Hz, 1H), 7.40 (ddd, J = 8.2, 2.0,
0.9 Hz, 1H), 7.29 (t, J = 8.2 Hz, 1H), 6.77 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 4.77 (d, J = 2.4
Hz, 2H), 3.56 (t, J = 2.4 Hz, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 163.85, 157.89, 148.20, 140.31, 136.70, 134.64,
130.64, 129.94, 126.64, 122.87, 113.95, 110.79, 107.83, 79.67, 78.72, 55.90.

3-amino-N-(3-(prop-2-yn-1-yloxy)phenyl)benzamide (2-d.2)

2-d.1 (250 mg, 0.84 mmol) was dissolved in 4 mL MeOH. Zn (274.68 mg, 4.2 mmol) and
NH4Cl (224.66 mg 4.2 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 follwoed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield 2-d.2 (164 mg, 73%) as a red oil, which was used in the next
reaction without further purification.
1
H NMR (600 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.45 (t, J = 2.2 Hz, 1H), 7.28 – 7.18
(m, 3H), 7.19 – 7.11 (m, 4H), 6.81 (ddd, J = 7.9, 2.4, 1.0 Hz, 1H), 6.75 (ddd, J = 8.2, 2.5,
0.9 Hz, 1H), 4.69 (d, J = 2.4 Hz, 2H), 2.52 (t, J = 2.4 Hz, 1H).
13
C NMR (151 MHz, CDCl3) δ 166.03, 158.13, 146.89, 139.25, 136.09, 129.76, 129.62,
118.33, 116.41, 113.83, 113.21, 111.16, 106.84, 75.58, 55.90, 38.59.
119
3-(2-chloroacetamido)-N-(3-(prop-2-yn-1-yloxy)phenyl)benzamide (2d)

2-d.2 (164 mg, 0.62 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (237.53 uL, 1.36 mmol) was added via syringe. Chloroacetyl chloride
(79 uL, 0.87 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield 2-d as a white solid (90 mg, 43%).
1
H NMR (600 MHz, DMSO-d6) δ 10.49 (s, 1H), 10.27 (s, 1H), 8.07 (s, 0H), 7.81 (d, J = 8.1
Hz, 1H), 7.65 (d, J = 7.8 Hz, 0H), 7.51 – 7.45 (m, 2H), 7.39 (d, J = 8.1 Hz, 1H), 7.25 (t, J =
8.1 Hz, 1H), 6.73 (dd, J = 8.2, 2.5 Hz, 1H), 4.76 (d, J = 2.4 Hz, 2H), 4.27 (s, 2H), 3.55 (t, J
= 2.3 Hz, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 165.90, 165.35, 157.87, 140.75, 139.07, 136.26, 129.84,
129.38, 123.24, 122.77, 119.33, 113.74, 110.28, 107.62, 79.71, 78.66, 55.86, 43.98.
HRMS (ESI) m/z: [M+H]
-
calcd for C18H15ClN2O3 342.0771; Found 341.0703.

120

N-cyclohexyl-3-nitrobenzamide (C1-s1)

3-nitrobenzoic acid (400 mg, 2.39 mmol) and cyclohexamine (284.4 mg, 2.87 mmol) were
dissolved in 5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and triethylamine (1.33
mL, 9.56 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C1-s2 (378 mg, 64%) as a white solid.
1
H NMR (600 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.58 (d, J = 7.8 Hz, 1H), 8.34 (d, J = 7.7 Hz,
1H), 8.26 (d, J = 7.8 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 3.76 (tdt, J = 11.2, 7.6, 3.9 Hz, 1H),
1.85 – 1.77 (m, 2H), 1.72 (dd, J = 9.4, 3.8 Hz, 2H), 1.62 – 1.55 (m, 1H), 1.36 – 1.23 (m,
4H), 1.16 – 1.07 (m, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 163.57, 148.14, 136.64, 134.26, 130.48, 126.07,
122.45, 49.23, 49.09, 32.74, 25.34.

121
3-amino-N-cyclohexylbenzamide (C1-s2)

C1-s1 (300 mg, 1.21 mmol) was dissolved in 4 mL MeOH. Zn (395.02 mg, 6.04 mmol) and
NH4Cl (323.08 mg 6.04 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C1-s2 (225 mg, 85%) as a white solid, which was used in the
next reaction without further purification.
1
H NMR (600 MHz, DMSO-d6) δ 7.91 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.7 Hz, 1H), 6.98 (t,
J = 2.0 Hz, 1H), 6.91 (dt, J = 7.6, 1.3 Hz, 1H), 6.64 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 5.15 (s,
2H), 3.74 – 3.64 (m, 1H), 1.80 – 1.71 (m, 2H), 1.73 – 1.65 (m, 2H), 1.61 – 1.53 (m, 1H),
1.31 – 1.21 (m, 4H), 1.14 – 1.03 (m, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 166.62, 148.97, 136.39, 128.92, 116.64, 114.89,
113.40, 48.64, 48.50, 32.90, 25.42.

3-(2-chloroacetamido)-N-cyclohexylbenzamide (C1)

C1-s2 (150 mg, 0.69 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (264.63 uL, 1.51 mmol) was added via syringe. Chloroacetyl chloride
(87.62 uL, 0.96 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C1 as a white solid (47.2 mg, 23%).
122
1
H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.17 (d, J = 7.9 Hz, 1H), 7.91 (t, J = 2.0
Hz, 1H), 7.74 (dt, J = 7.9, 1.1 Hz, 1H), 7.51 (dt, J = 7.7, 1.4 Hz, 1H), 7.37 (t, J = 7.9 Hz,
1H), 4.23 (s, 2H), 3.74 – 3.64 (m, 1H), 1.80 – 1.74 (m, 2H), 1.73 – 1.67 (m, 2H), 1.61 – 1.54
(m, 1H), 1.35 – 1.18 (m, 4H), 1.13 – 1.08 (m, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 165.63, 165.22, 138.84, 136.25, 129.18, 122.74,
122.28, 119.18, 48.88, 48.74, 43.96, 32.83, 25.38.
HRMS (ESI) m/z: [M+H]
+
calcd for C15H19ClN2O2 294.1135; Found 295.1198.

N-(3-methoxyphenyl)-3-nitrobenzamide (C2-s1)

3-nitrobenzoic acid (1 g, 5.98 mmol) and m-anisidine (803.9 uL, 7.18 mmol) were dissolved
in 10 mL of DCM/DMF (4:1). HBTU (9.07 g, 23.92 mmol) and triethylamine (3.33 mL,
23.92 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
123
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C2-s2 (1.6 g, 99%) as a light orange solid.
1
H NMR (600 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.76 (t, J = 1.8 Hz, 1H), 8.41 (ddd, J = 8.2,
2.3, 1.0 Hz, 1H), 8.37 (ddd, J = 7.8, 1.8, 1.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.44 (t, J =
2.2 Hz, 1H), 7.36 (ddd, J = 8.1, 2.0, 0.9 Hz, 1H), 7.26 (t, J = 8.1 Hz, 1H), 6.70 (ddd, J = 8.3,
2.6, 0.9 Hz, 1H), 3.74 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 163.78, 159.90, 148.18, 140.32, 136.72, 134.60, 130.69,
130.53, 126.62, 122.91, 113.30, 110.13, 106.60, 55.56.

N-(3-methoxyphenyl)-N-methyl-3-nitrobenzamide (C2-s2)

C2-s1 (500 mg, 1.84 mmol) was dissolved in dry THF and cooled to 0
o
C. NaH (60% in oil)
(112.60 mg, 2.76 mmol) was added portion wise to the stirring solution. Methyl iodide
(125.75 uL, 2.02 mmol) was added drop wise. The reaction mixture was allowed to warm to
room temperature. The round bottom was transferred to oil bath and refluxed at 75
o
C for 2
hrs. The reaction mixture was poured in ice water and was extracted with EtOAc 3 times.
The organic layer was washed with brine 2 times, dried with Na2SO4, and concentrated by
rotary evaporation. The residue was purified via flash column chromatography to yield C2-
s2 (90 mg, 17%) as a yellow solid.
1
H NMR (500 MHz, Methanol-d4) δ 8.15 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.8
Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.13 (t, J = 8.1 Hz, 1H), 6.79 – 6.66 (m, 3H), 3.65 (s, 3H),
3.45 (s, 3H).
124
13
C NMR (126 MHz, Methanol-d4) δ 194.40, 186.25, 173.20, 170.51, 163.31, 159.56,
155.59, 154.64, 149.62, 148.65, 144.85, 138.62, 138.51, 62.96, 54.96.

3-amino-N-(3-methoxyphenyl)-N-methylbenzamide (C2-s3)

C2-s2 (90 mg, 0.34 mmol) was dissolved in 4 mL MeOH. Zn (102.7 mg, 1.57 mmol) and
NH4Cl (83.98 mg 1.57 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 follwoed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C2-s3 (78 mg, 97%) as a yellow oil, which was used in the
next reaction without further purification.
1
H NMR (600 MHz, Chloroform-d) δ 7.07 (t, J = 8.1 Hz, 1H), 6.85 (t, J = 7.8 Hz, 1H), 6.70
– 6.67 (m, 1H), 6.64 (ddd, J = 8.4, 2.5, 0.9 Hz, 1H), 6.59 (ddd, J = 7.8, 2.1, 0.9 Hz, 1H),
6.57 – 6.52 (m, 2H), 6.49 (ddd, J = 8.0, 2.4, 1.0 Hz, 1H), 3.63 (s, 3H), 3.52 (bs, 2H), 3.40 (s,
3H).
13
C NMR (151 MHz, Chloroform-d) δ 170.90, 159.92, 146.15, 145.95, 136.96, 129.64,
128.48, 119.01, 118.59, 116.32, 115.07, 112.61, 111.96, 55.28, 38.31.

3-(2-chloroacetamido)-N-(3-methoxyphenyl)-N-methylbenzamide (C2)

C2-s3 (78 mg, 0.30 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (117.17 uL, 0.67 mmol) was added via syringe. Chloroacetyl chloride
(38.8 uL, 0.43 mmol) was added via syringe slowly dropwise. The reaction mixture was
125
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C2 as a yellow solid (54 mg, 53%).
1
H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 7.64 (s, 1H), 7.43 (ddd, J = 8.1, 2.3, 1.0
Hz, 1H), 7.11 (t, J = 8.0 Hz, 2H), 6.86 (dt, J = 7.7, 1.4 Hz, 1H), 6.74 (t, J = 2.2 Hz, 1H), 6.70
(ddd, J = 8.3, 2.5, 0.9 Hz, 1H), 6.65 (ddd, J = 7.8, 2.0, 0.9 Hz, 1H), 4.19 (s, 2H), 3.62 (s,
3H), 3.32 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 169.61, 165.13, 160.00, 145.94, 138.58, 137.55, 130.25,
128.68, 123.76, 120.65, 119.50, 119.28, 113.23, 112.54, 55.69, 43.96, 38.30.
HRMS (ESI) m/z: [M+H]
+
calcd for C17H17ClN2O3 332.0928; Found 333.0988.

N-(4-methoxyphenyl)-3-nitrobenzamide (C4-s1)

3-nitrobenzoic acid (400 mg, 2.39 mmol) and p-anisidine (353.4 mg, 2.87 mmol) were
dissolved in 5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and triethylamine (1.33
mL, 9.56 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
126
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C4-s2 (657 mg, 99%) as a yellow solid.
1
H NMR (600 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.76 (s, 1H), 8.44 – 8.31 (m, 2H), 7.81 (t,
J = 8.0 Hz, 1H), 7.66 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 3.73 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 163.31, 156.33, 148.21, 136.82, 134.49, 132.13,
130.60, 126.44, 122.74, 122.65, 114.27, 55.64.

3-amino-N-(4-methoxyphenyl)benzamide (C4-s2)
C4-s1 (450 mg, 1.65 mmol) was dissolved in 4 mL MeOH. Zn (539.60 mg, 8.25 mmol) and
NH4Cl (441.30 mg 8.25 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C4-s2 (154 mg, 39%) as a white solid, which was used in the
next reaction without further purification.
1
H NMR (600 MHz, cd3od) δ 5.99 – 5.96 (m, 2H), 5.70 – 5.57 (m, 3H), 5.40 – 5.29 (m,
3H), 3.33 (s, 3H), 2.23 (s, 3H).
13
C NMR (151 MHz, cd3od) δ 166.48, 155.25, 146.44, 134.24, 129.81, 127.32, 121.17,
116.48, 114.65, 112.03, 111.99, 52.93.

127
3-(2-chloroacetamido)-N-(4-methoxyphenyl)benzamide (C4)

C4-s2 (142 mg, 0.59 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (225.92 uL, 1.29 mmol) was added via syringe. Chloroacetyl chloride
(55.91 uL, 0.70 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C4 as a white solid (22.8 mg, 12%).
1
H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.97 (s, 1H), 7.88 (d, J = 4.0 Hz, 1H), 7.59
(dd, J = 8.0, 2.6 Hz, 1H), 7.46 – 7.42 (m, 3H), 7.28 (t, J = 7.9 Hz, 1H), 6.75 – 6.70 (m, 2H),
4.07 (s, 2H), 3.54 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 165.41, 156.07, 138.94, 136.30, 132.45, 129.38,
123.17, 122.52, 119.30, 114.20, 55.62, 49.03, 48.90, 43.91.
HRMS (ESI) m/z: [M+H]
+
calcd for C16H15ClN2O3 318.0771; Found 319.0844.

128

N-(2-methoxyphenyl)-3-nitrobenzamide (C5-s1)

3-nitrobenzoic acid (400 mg, 2.39 mmol) and o-anisidine (353.4 mg, 2.87 mmol) were
dissolved in 5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and triethylamine (1.33
mL, 9.56 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C5-s2 (525 mg, 81%) as a light yellow solid.
1
H NMR (600 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.74 (s, 1H), 8.41 (d, J = 8.2 Hz, 1H), 8.37
(d, J = 7.7 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.64 (dd, J = 7.8, 1.6 Hz, 1H), 7.21 (t, J = 8.7
Hz, 1H), 7.10 (d, J = 8.3 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 3.81 (s, 3H).
13
C NMR (151 MHz, DMSO-d6) δ 163.66, 152.69, 148.24, 136.44, 134.41, 130.65, 126.98,
126.65, 126.54, 125.93, 122.91, 120.63, 112.08, 56.13.

129
3-amino-N-(2-methoxyphenyl)benzamide (C5-s2)

C5-s1 (450 mg, 1.65 mmol) was dissolved in 4 mL MeOH. Zn (539.60 mg, 8.25 mmol) and
NH4Cl (441.30 mg 8.25 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C5-s2 (190 mg, 46%) as a yellow oil, which was used in the
next reaction without further purification.
1
H NMR (600 MHz, Chloroform-d) δ 8.48 (bs, 1H), 8.46 (dd, J = 7.9, 1.6 Hz, 1H), 7.20 –
7.09 (m, 5H), 7.04 – 6.98 (m, 1H), 6.94 (td, J = 7.7, 1.4 Hz, 1H), 6.84 (dd, J = 8.1, 1.4 Hz,
1H), 6.75 (ddd, J = 7.8, 2.5, 1.3 Hz, 1H), 3.81 (s, 3H).
13
C NMR (151 MHz, cdcl3) δ 165.70, 165.61, 148.27, 147.30, 136.28, 129.54, 127.75,
123.88, 120.99, 119.84, 118.19, 116.20, 113.63, 55.75.

3-(2-chloroacetamido)-N-(2-methoxyphenyl)benzamide (C5)

C5-s2 (190 mg, 0.78 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (300.53 uL, 1.72 mmol) was added via syringe. Chloroacetyl chloride
(74.76 uL, 0.94 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C5 as a yellow solid (157 mg, 84%).
130
1
H NMR (600 MHz, Chloroform-d) δ 8.53 (s, 2H), 8.46 (dd, J = 8.0, 1.6 Hz, 1H), 8.01 (t, J
= 2.0 Hz, 1H), 7.88 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.62 (ddd, J = 7.7, 1.8, 1.0 Hz, 1H), 7.46
(t, J = 7.9 Hz, 1H), 7.08 (ddd, J = 8.1, 7.6, 1.6 Hz, 1H), 7.00 (td, J = 7.8, 1.4 Hz, 1H), 6.91
(dd, J = 8.1, 1.4 Hz, 1H), 4.18 (s, 2H), 3.91 (s, 3H).
13
C NMR (151 MHz, Chloroform-d) δ 164.63, 164.23, 148.31, 137.52, 136.20, 129.60,
127.47, 124.20, 123.22, 123.15, 121.14, 120.04, 118.89, 110.02, 55.84, 42.90.
HRMS (ESI) m/z: [M+H]
+
calcd for C16H15ClN2O3 318.0771; Found 319.0668.

N-(1H-imidazol-2-yl)-3-nitrobenzamide (C6-s1)

3-nitrobenzoic acid (400 mg, 2.39 mmol) and bis(1h-imidazol-2-amine;sulfuric acid (504.96
mg, 1.91 mmol) were dissolved in 5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and
triethylamine (2.0 mL, 14.4 mmol) were added and the solution was allowed to stir at room
temperature for 16 hours. After 16 hours, the reaction mixture was diluted with EtOAc and
washed with 10% Na2CO3 followed by brine 3 times. The organic layer was dried with
Na2SO4 and concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C6-s2 (253 mg, 46%) as a beige solid.
131
1
H NMR (400 MHz, DMSO-d6) δ 11.96 (bs, 1H), 8.86 (s, 1H), 8.45 (dt, J = 7.7, 1.4 Hz,
1H), 8.31 (ddd, J = 8.2, 2.5, 1.0 Hz, 1H), 7.92 (s, 1H), 7.72 (t, J = 8.0 Hz, 1H), 6.87 (s,
2H).

3-amino-N-(1H-imidazol-2-yl)benzamide (C6-s2)

C6-s1 (241 mg, 1.21 mmol) was dissolved in 4 mL MeOH. Zn (340.08 mg, 5.2 mmol) and
NH4Cl (278.15 mg 5.2 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C6-s2 (122 mg, 58%) as a white solid, which was used
immediately in the next reaction without further purification.

3-(2-chloroacetamido)-N-(1H-imidazol-2-yl)benzamide (C6)

C6-s2 (114 mg, 0.56 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (217.17 uL, 1.24 mmol) was added via syringe. Chloroacetyl chloride
(72 uL, 0.79 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (DCM/MeOH, 0-10%) to yield C6 as an orange solid (6.9 mg, 4%).
132
1
H NMR (400 MHz, DMSO-d6) δ 11.72 (bs, 2H), 10.44 (s, 1H), 8.20 (s, 1H), 7.80 – 7.70
(m, 2H), 7.40 (t, J = 7.9 Hz, 1H), 6.79 (s, 2H), 4.25 (s, 2H).
13
C NMR (151 MHz, DMSO-d6) δ 206.95, 165.29, 138.98, 129.26, 123.67, 122.99,
119.73, 44.00, 42.24, 29.45, 18.52, 17.18.
HRMS (ESI) m/z: [M+H]
+
calcd for C12H11ClN4O2 278.0571; Found 279.0634.

5-nitrothiophene-2-carboxylic acid (C7-s1) & 4-nitrothiophene-2-carboxylic acid (C8-
s1)

3.0 mL of sulfuric acid was added to 2.0 mL of nitric acid at 0
o
C. 2-Thiophenecarboxylic
acid (2.8 g, 21.87 mmol) was added slowly to the mixture over a 15 minute period. The
mixture was stirred at 0
o
C for 1 hr. After 1 hr, the mixture was poured in ice cold water and
stirred for 30 min. The precipitate was filtered out and the filtrate was collected. The filtrate
133
was washed with water and brine, was dried with Na2SO4, and concentrated by rotary
evaporation. Hexane was added to the residue and stirred for 20 min. The solid was filtered
and allowed to dry to give a mixture of C7-s1 and C8-s1 as a light yellow solid (observed
by
1
H NMR) (1.55 g).
Mixture of two products:
1
H NMR (400 MHz, DMSO-d6) δ 9.01 (d, J = 1.7 Hz, 1H), 8.11 (d, J = 4.3 Hz, 1H), 8.10
(d, J = 1.7 Hz, 1H), 7.73 (d, J = 4.3 Hz, 1H).

5-nitro-N-phenylthiophene-2-carboxamide (C7-s2) & 4-nitro-N-phenylthiophene-2-
carboxamide (C8-s2)

The mixture containing C7-s1 and C8-s1 (1 g, 5.78 mmol) and aniline (632.82 uL, 6.93
mmol) were dissolved in 10 mL of DCM/DMF (4:1). HBTU (8.76 g, 23.10 mmol) and
triethylamine (3.22 mL, 23.10 mmol) were added and the solution was allowed to stir at
room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with EtOAc
and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was dried with
Na2SO4 and concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C7-s2 (208 mg) and C8-s2 (313 mg), both
orange solids, as separated products.

134
5-nitro-N-phenylthiophene-2-carboxamide (C7-s2)

1
H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.18 (d, J = 4.4 Hz, 1H), 8.03 (d, J = 4.4
Hz, 1H), 7.69 (d, J = 7.6 Hz, 2H), 7.41 – 7.31 (m, 2H), 7.18 – 7.07 (m, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 158.66, 153.79, 146.83, 138.35, 130.55, 129.29,
128.78, 125.06, 121.10.
4-nitro-N-phenylthiophene-2-carboxamide (C8-s2)
1
H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.99 (d, J = 1.5 Hz, 1H), 8.68 (d, J = 1.6
Hz, 1H), 7.74 – 7.66 (m, 2H), 7.40 – 7.30 (m, 2H), 7.16 – 7.07 (m, 1H).

5-amino-N-phenylthiophene-2-carboxamide (C7-s3)

C7-s2 (200 mg, 0.81 mmol) was dissolved in 4 mL MeOH. Zn (264.87 mg, 4.0 mmol) and
NH4Cl (216.63 mg 4.0 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C7-s3 (160 mg, 96%) as a red solid, which was used in the
next reaction without further purification.
1
H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H), 7.79 – 7.60 (m, 2H), 7.56 (d, J = 4.1 Hz,
1H), 7.32 – 7.20 (m, 2H), 7.03 – 6.92 (m, 1H), 6.40 (s, 1H), 6.14 (s, 1H), 5.85 (d, J = 4.1
Hz, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 170.84, 161.59, 139.95, 131.12, 129.24, 123.13,
120.16, 104.49, 60.22.
135
5-(2-chloroacetamido)-N-phenylthiophene-2-carboxamide (C7)

C7-s3 (160 mg, 0.73 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (281.97 uL, 1.61 mmol) was added via syringe. Chloroacetyl chloride
(93.81 uL, 1.03 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C7 as a peach solid (47 mg, 22%).
1
H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 10.02 (s, 1H), 7.80 (d, J = 4.2 Hz, 1H),
7.67 (d, J = 7.4 Hz, 2H), 7.34 – 7.26 (m, 2H), 7.04 (ddt, J = 8.5, 7.2, 1.2 Hz, 1H), 6.77 (d, J
= 4.2 Hz, 1H), 4.33 (s, 2H).
13
C NMR (151 MHz, DMSO-d6) δ 164.13, 160.95, 144.61, 139.38, 130.34, 129.16,
127.66, 123.92, 120.75, 112.92, 42.85.
HRMS (ESI) m/z: [M+H]
+
calcd for C13H11ClN2O2S 294.0203; Found 295.0288.

4-amino-N-phenylthiophene-2-carboxamide (C8-s3)

C8-s2 (140 mg, 0.56 mmol) was dissolved in 4 mL MeOH. Zn (184.43 mg, 2.8 mmol) and
NH4Cl (150.84 mg 2.82 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
136
by rotary evaporation to yield C8-s3 (134 mg, 98%) as a red solid, which was used in the
next reaction without further purification.
1
H NMR (600 MHz, DMSO-d6) δ 10.06 (s, 1H), 7.72 – 7.66 (m, 2H), 7.43 (s, 1H), 7.34 –
7.28 (m, 2H), 7.11 – 7.03 (m, 1H), 6.28 (s, 1H), 5.17 (s, 1H), 4.97 (s, 1H).
13
C NMR (151 MHz, DMSO-d6) δ 160.57, 148.24, 139.39, 129.03, 123.93, 122.76,
120.67, 103.23, 60.19.

4-(2-chloroacetamido)-N-phenylthiophene-2-carboxamide (C8)

C8-s3 (120 mg, 0.55 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (211.91 uL, 1.21 mmol) was added via syringe. Chloroacetyl chloride
(70.13 uL, 0.77 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C8 as a yellow solid (23 mg, 14%).
1
H NMR (400 MHz, cdcl3) δ 8.46 (s, 1H), 7.74 (d, J = 1.6 Hz, 1H), 7.68 (s, 1H), 7.56 (d, J
= 1.5 Hz, 1H), 7.55 – 7.50 (m, 2H), 7.34 – 7.21 (m, 2H), 7.13 – 7.04 (m, 1H), 4.14 (s, 2H).
HRMS (ESI) m/z: [M+H]
+
calcd for C13H11ClN2O2S 294.0203; Found 295.0288.

137

3-nitro-N-pentylbenzamide (C9-s1)

3-nitrobenzoic acid (400 mg, 2.39 mmol) and amylamine (332.45 uL, 2.87 mmol) were
dissolved in 5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and triethylamine (2.0
mL, 14.4 mmol) were added and the solution was allowed to stir at room temperature for 16
hours. After 16 hours, the reaction mixture was diluted with EtOAc and washed with 10%
Na2CO3 followed by brine 3 times. The organic layer was dried with Na2SO4 and
concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give C9-s2 (276 mg, 49%) as a white solid.
1
H NMR (400 MHz, DMSO-d6) δ 8.81 – 8.77 (m, 1H), 8.64 (t, J = 1.8 Hz, 1H), 8.34 (ddd,
J = 8.2, 2.4, 1.0 Hz, 1H), 8.25 (ddd, J = 7.7, 1.7, 1.0 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 3.25
(td, J = 7.1, 5.6 Hz, 2H), 1.51 (p, J = 7.2 Hz, 2H), 1.36 – 1.20 (m, 4H), 0.91 – 0.79 (m,
3H).
13
C NMR (151 MHz, DMSO-d6δ 164.33, 148.20, 136.48, 134.03, 130.49, 126.10, 122.29,
39.86, 29.12, 29.04, 22.29, 14.32.

138
3-amino-N-pentylbenzamide (C9-s2)
C9-s1 (150 mg, 0.64 mmol) was dissolved in 4 mL MeOH. Zn (207.65 mg, 3.18 mmol) and
NH4Cl (169.83 mg 3.18 mmol) were added, and the mixture was allowed to stir at room
temperature for 16 hr. The reaction mixture was dissolved in EtOA and washed with 10%
Na2CO3 follwoed by brine 3 times. The organic layer was dried over NaSO4 and concentrated
by rotary evaporation to yield C9-s2 (122 mg, 99%) as a yellow solid, which was used
immediately in the next reaction without further purification.

3-(2-chloroacetamido)-N-pentylbenzamide (C9)

C9-s2 (112 mg, 0.54 mmol) was dissolved in anhydrous DCM/THF (5 mL, 1:4) under
nitrogen gas. DIPEA (208.06 uL, 1.19 mmol) was added via syringe. Chloroacetyl chloride
(68.8 uL, 0.76 mmol) was added via syringe slowly dropwise. The reaction mixture was
allowed to stir overnight. The mixture was diluted with EtOAc and washed with 10%
Na2CO3. The organic layer was dried over NaSO4 and the residue was purified via flash
chromatography (EtOAc/Hexane, 0-50%) to yield C9 as a white solid (22.8 mg, 15%).
1
H NMR (400 MHz, Chloroform-d) δ 8.45 (s, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.74 (d, J =
5.7 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 6.25 (s, 1H), 4.18 (s, 2H),
1.70 (s, 2H), 1.64 – 1.54 (m, 2H), 1.39 – 1.30 (m, 4H), 0.93 – 0.85 (m, 3H).
13
C NMR (151 MHz, Chloroform-d) δ 166.76, 164.12, 137.10, 135.84, 129.45, 123.37,
122.75, 118.52, 42.85, 40.19, 29.30, 29.11, 22.36, 13.96.
HRMS (ESI) m/z: [M+H]
+
calcd for C14H19ClN2O2 282.1135; Found 283.1195.

139

3-(2-chloroacetamido)-N-(pent-4-yn-1-yl)benzamide (C9-probe)

BE-2-12 (200 mg) and pent-4-yn-1-amine (1.2 eq) were dissolved in 5 mL of DCM/DMF
(4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir
at room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with
EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was
dried with Na2SO4 and concentrated by rotary evaporation. The residue was purified via
flash column chromatography (EtOAc/Hex = 0-50%) to give C9-probe (56%)as a white
solid.
1
H NMR (600 MHz, dmso) δ 10.40 (s, 1H), 8.46 (t, J = 5.6 Hz, 1H), 7.98 (s, 1H), 7.73 (d, J
= 7.0 Hz, 1H), 7.52 (d, J = 9.1 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 4.24 (s, 2H), 3.30 – 3.26
(m, 2H), 2.77 (q, J = 2.9 Hz, 1H), 2.20 (td, J = 7.1, 2.7 Hz, 2H), 1.68 (p, J = 7.2 Hz, 2H).
13
C NMR (151 MHz, dmso) δ 166.52, 165.24, 138.93, 135.91, 129.20, 122.71, 122.33,
119.14, 84.56, 71.81, 43.97, 38.92, 28.49, 15.97.
HRMS (ESI) m/z: calcd for C
14
H
15
ClN
2
O
2
278.08; Found 278.09

140

5-nitro-N-pentylthiophene-2-carboxamide (B1-s1)

5-nitrothiophene-2-carboxylic acid (200 mg) and pentan-1-amine (1.2 eq) were dissolved in
5 mL of DCM/DMF (4:1). HBTU (3.6 g, 9.56 mmol) and triethylamine (2.0 mL, 14.4 mmol)
were added and the solution was allowed to stir at room temperature for 16 hours. After 16
hours, the reaction mixture was diluted with EtOAc and washed with 10% Na2CO3 followed
by brine 3 times. The organic layer was dried with Na2SO4 and concentrated by rotary
evaporation. The residue was purified via flash column chromatography (EtOAc/Hex = 0-
50%) to give B1-s1 (76%)as a white solid.
1
H NMR (600 MHz, dmso) δ 8.91 (t, J = 5.7 Hz, 1H), 8.10 (d, J = 4.4 Hz, 1H), 7.76 (d, J =
4.2 Hz, 1H), 1.50 (p, J = 7.2 Hz, 3H), 1.27 (dqd, J = 14.0, 8.2, 2.7 Hz, 5H), 0.85 (t, J = 6.9
Hz, 3H).
13
C NMR (151 MHz, dmso) δ 159.05, 153.68, 152.71, 130.38, 128.68, 127.82, 125.34,
120.75, 112.12, 56.08.

141
5-amino-N-pentylthiophene-2-carboxamide (B1-s2)

B1-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried over NaSO4 and concentrated by rotary evaporation to yield B1-s2 (99%) as
a yellow solid, which was used immediately in the next reaction without further purification.
1
H NMR (600 MHz, dmso) δ 8.74 (s, 1H), 7.75 (dd, J = 7.9, 1.6 Hz, 1H), 7.49 (d, J = 4.1
Hz, 1H), 7.06 (ddd, J = 8.9, 7.3, 1.6 Hz, 2H), 6.89 (td, J = 7.6, 1.5 Hz, 1H), 6.41 (s, 2H),
5.87 (d, J = 4.1 Hz, 1H), 3.81 (s, 3H), 2.66 (d, J = 1.3 Hz, 4H).
13
C NMR (151 MHz, dmso) δ 161.44, 160.59, 150.85, 130.99, 120.64, 119.80, 111.53,
104.60, 60.20, 56.12.

5-(2-chloroacetamido)-N-pentylthiophene-2-carboxamide (B1)

B1-s2 100 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas.
DIPEA (2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe
slowly dropwise. The reaction mixture was allowed to stir overnight. The mixture was
diluted with EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4
and the residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield B1
as a white solid (20%).
142
1
H NMR (400 MHz, dmso) δ 11.70 (s, 1H), 8.25 (t, J = 5.7 Hz, 1H), 7.50 (d, J = 4.1 Hz,
1H), 6.70 (d, J = 4.1 Hz, 1H), 4.30 (s, 2H), 3.16 (q, J = 6.7 Hz, 2H), 1.47 (p, J = 7.3 Hz, 2H),
1.27 (td, J = 8.6, 4.9 Hz, 4H), 0.89 – 0.81 (m, 3H).
13
C NMR (151 MHz, dmso) δ 163.88, 162.02, 143.32, 130.77, 126.10, 112.68, 42.85, 39.38,
29.41, 29.14, 22.32, 14.37.
HRMS (ESI) m/z: calcd for C
12
H
17
ClN
2
O
2
S 288.07; Found 288.06

N-(2-methoxyphenyl)-5-nitrothiophene-2-carboxamide (B2-s1)

5-nitrothiophene-2-carboxylic acid (200 mg) and o-anisidine (1.2 eq.) were dissolved in 5
mL of DCM/DMF (4:1). HBTU (5 eq) and triethylamine (6 eq.) were added and the solution
was allowed to stir at room temperature for 16 hours. After 16 hours, the reaction mixture
was diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The
organic layer was dried with Na2SO4 and concentrated by rotary evaporation. The residue
was purified via flash column chromatography (EtOAc/Hex = 0-50%) to give B2-s1 (80%)
as a white solid.
1
H NMR (600 MHz, dmso) δ 10.10 (s, 1H), 8.16 (d, J = 4.4 Hz, 1H), 8.03 (d, J = 4.4 Hz,
1H), 7.53 (d, J = 7.6 Hz, 1H), 7.24 (t, J = 7.9 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.96 (t, J =
7.6 Hz, 1H), 3.81 (s, 3H).
13
C NMR (151 MHz, dmso) δ 158.77, 153.70, 152.99, 146.64, 130.60, 128.69, 127.66,
126.52, 125.60, 120.68, 112.20, 56.11.

143

5-amino-N-(2-methoxyphenyl)thiophene-2-carboxamide (B2-s2)

B2-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried over NaSO4 and concentrated by rotary evaporation to yield B2-s2 (89%) as
a yellow solid, which was used immediately in the next reaction without further purification.

5-(2-chloroacetamido)-N-(2-methoxyphenyl)thiophene-2-carboxamide (B2)

B2-s2 150 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas.
DIPEA (2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe
slowly dropwise. The reaction mixture was allowed to stir overnight. The mixture was
diluted with EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4
and the residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield B2
as a white solid (43%).
1
H NMR (600 MHz, dmso) δ 11.82 (s, 1H), 9.27 (s, 1H), 7.77 (d, J = 4.2 Hz, 1H), 7.65 (dd,
J = 7.8, 1.7 Hz, 1H), 7.17 – 7.11 (m, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.98 – 6.90 (m, 1H), 6.77
(d, J = 4.1 Hz, 1H), 4.33 (s, 2H), 3.81 (s, 3H).
13
C NMR (151 MHz, dmso) δ 164.10, 160.76, 151.94, 144.41, 130.10, 127.61, 126.92,
126.08, 125.07, 120.63, 112.95, 111.83, 56.13, 42.85.
HRMS (ESI) m/z: calcd for C
14
H
13
ClN
2
O
3
S 324.03 Found; 324.03
144

Methyl 3-(2-chloroacetamido)benzoate (BE-2-11)

Methyl 3-aminobenzoate 1g was dissolved in anhydrous DCM/THF (1:4) under nitrogen
gas. DIPEA (2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via
syringe slowly dropwise. The reaction mixture was allowed to stir overnight. The mixture
was diluted with EtOAc and washed with 10% Na2CO3. The organic layer was dried over
NaSO4 and the residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to
yield BE-2-11 as a white solid (75%).
1
H NMR (600 MHz, dmso) δ 10.50 (s, 1H), 8.24 (t, J = 1.9 Hz, 1H), 7.81 (ddd, J = 8.2, 2.3,
1.1 Hz, 1H), 7.69 – 7.63 (m, 1H), 7.46 (t, J = 7.9 Hz, 1H), 4.25 (s, 2H), 3.83 (s, 3H).
13
C NMR (151 MHz, dmso) δ 166.40, 165.42, 139.30, 130.66, 129.82, 124.85, 124.26,
120.27, 52.67, 43.95.

BE-2-11
1
H NMR (600 MHz, dmso) δ 12.97 (s, 1H), 10.46 (s, 1H), 8.20 (t, J = 1.9 Hz, 1H), 7.79
(ddd, J = 8.1, 2.3, 1.1 Hz, 1H), 7.64 (dt, J = 7.8, 1.4 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 4.25
(s, 1H).
13
C NMR (151 MHz, dmso) δ 167.59, 165.34, 139.12, 131.67, 129.62, 125.06, 124.36,
121.01, 43.97.

145

3-(2-chloroacetamido)-N-(2-ethoxyethyl)benzamide (B3)

BE-2-12 (200 mg) and 2-ethoxyethan-1-amine (1.2 eq) were dissolved in 5 mL of
DCM/DMF (4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was
allowed to stir at room temperature for 16 hours. After 16 hours, the reaction mixture was
diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic
146
layer was dried with Na2SO4 and concentrated by rotary evaporation. The residue was
purified via flash column chromatography (EtOAc/Hex = 0-50%) to give B3 (11.5%)as a
white solid.
1
H NMR (600 MHz, dmso) δ 10.41 (s, 1H), 8.47 (t, J = 5.6 Hz, 1H), 8.00 (s, 1H), 7.93 (s,
1H), 7.73 (dd, J = 7.9, 2.1 Hz, 1H), 7.53 (ddt, J = 7.7, 1.9, 1.0 Hz, 1H), 7.39 (t, J = 7.9 Hz,
2H), 4.24 (s, 2H), 3.38 (q, J = 6.0 Hz, 3H), 2.87 (s, 3H), 2.71 (d, J = 0.7 Hz, 1H).
13
C NMR (151 MHz, dmso) δ 166.51, 165.24, 162.75, 138.95, 135.76, 129.22, 122.69,
122.39, 119.16, 68.71, 65.82, 43.96, 15.56.
HRMS (ESI) m/z: calcd for C
13
H
17
ClN
2
O
3
284. 09; Found 284.09

3-(2-chloroacetamido)-N-cyclopentylbenzamide (B4)

BE-2-12 (200 mg) and cyclopentanamine (1.2 eq) were dissolved in 5 mL of DCM/DMF
(4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir
at room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with
EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was
dried with Na2SO4 and concentrated by rotary evaporation. The residue was purified via
flash column chromatography (EtOAc/Hex = 0-50%) to give B4 (5%)as a white solid.
1
H NMR (600 MHz, dmso) δ 10.39 (s, 1H), 8.26 (d, J = 7.3 Hz, 1H), 7.93 (s, 1H), 7.73 (d,
J = 8.9 Hz, 1H), 7.52 (d, J = 10.6 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 4.24 (s, 2H), 4.19 (q, J
= 7.1 Hz, 1H), 1.91 – 1.73 (m, 2H), 1.69 – 1.65 (m, 2H), 1.53 – 1.46 (m, 4H).
147
13
C NMR (151 MHz, dmso) δ 166.23, 165.23, 165.05, 138.81, 136.20, 129.08, 122.90,
122.20, 119.23, 51.39, 43.95, 38.69, 32.53, 24.06.
HRMS (ESI) m/z: calcd for C
14
H
17
ClN
2
O
2
280.12 Found; 280.09

3-(2-chloroacetamido)-N-(o-tolyl)benzamide (B5)

BE-2-12 (200 mg) and o-toluidine (1.2 eq) were dissolved in 5 mL of DCM/DMF (4:1).
HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir at
room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with EtOAc
and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was dried with
Na2SO4 and concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give B5 (22%)as a white solid.

1
H NMR (600 MHz, dmso) δ 10.48 (s, 1H), 9.88 (s, 1H), 8.12 (s, 1H), 7.81 (d, J = 9.6 Hz,
1H), 7.70 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.26 (d, J =
7.4 Hz, 1H), 7.18 (dt, J = 29.1, 7.3 Hz, 2H), 4.27 (s, 2H), 2.22 (s, 3H).
13
C NMR (151 MHz, dmso) δ 165.59, 165.33, 139.10, 136.82, 135.88, 134.11, 130.75,
129.40, 126.99, 126.46, 123.14, 122.70, 119.46, 43.98, 40.39, 18.33.
HRMS (ESI) m/z: calcd for C
16
H
15
ClN
2
O
2
302.08 Found; 302.08

148
3-(2-chloroacetamido)-N-(2-chlorophenyl)benzamide (B6)

BE-2-12 (200 mg) and 2-chloroaniline (1.2 eq) were dissolved in 5 mL of DCM/DMF (4:1).
HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir at
room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with EtOAc
and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was dried with
Na2SO4 and concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give B6 (18%)as a white solid.
1
H NMR (600 MHz, dmso) δ 13.57 (s, 1H), 12.99 (s, 1H), 8.21 (t, J = 2.0 Hz, 1H), 7.96 (t,
J = 8.0 Hz, 1H), 7.79 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.65 (d, J =
7.8 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 4.30
(s, 2H).
13
C NMR (151 MHz, dmso) δ 167.46, 165.34, 143.24, 139.89, 139.14, 131.86, 130.76,
129.81, 129.61, 125.06, 123.90, 120.52, 120.35, 109.95, 43.97.
HRMS (ESI) m/z: [M+H]
+
calcd for C
15
H
12
Cl
2
N
2
O
2
322.03 Found; 322.18

3-(2-chloroacetamido)-N-(2-ethylphenyl)benzamide (B8)

BE-2-12 (200 mg) and 2-ethylaniline (1.2 eq) were dissolved in 5 mL of DCM/DMF (4:1).
HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir at
room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with EtOAc
and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was dried with
149
Na2SO4 and concentrated by rotary evaporation. The residue was purified via flash column
chromatography (EtOAc/Hex = 0-50%) to give B8 (27%)as a white solid.
1
H NMR (600 MHz, dmso) δ 10.47 (s, 1H), 9.88 (s, 1H), 8.11 (s, 1H), 7.81 (d, J = 9.1 Hz,
1H), 7.69 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.28 (dt, J = 5.7, 3.1 Hz, 2H), 7.21
(dd, J = 5.8, 3.5 Hz, 2H), 4.26 (s, 2H), 2.60 (q, J = 7.6 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H).
13
C NMR (151 MHz, dmso) δ 165.98, 165.32, 140.24, 139.11, 136.18, 135.90, 129.41,
128.96, 127.95, 126.95, 126.47, 123.07, 122.68, 119.44, 43.99, 24.43, 14.59.
HRMS (ESI) m/z: calcd for C
17
H
17
ClN
2
O
2
316.10 Found; 316.09

N-(2-methoxyphenyl)-5-nitrothiophene-2-carboxamide (B7-s1)

5-nitrothiophene-2-carboxylic acid (200 mg) and m-anisidine (1.2 eq.) were dissolved in 5
mL of DCM/DMF (4:1). HBTU (5 eq) and triethylamine (6 eq.) were added and the solution
was allowed to stir at room temperature for 16 hours. After 16 hours, the reaction mixture
was diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The
organic layer was dried with Na2SO4 and concentrated by rotary evaporation. The residue
was purified via flash column chromatography (EtOAc/Hex = 0-50%) to give B7-s1 (70%)
as a yellow solid.
1
H NMR (600 MHz, dmso) δ 10.56 (s, 1H), 8.17 (d, J = 4.4 Hz, 1H), 8.03 (d, J = 4.4 Hz,
1H), 7.37 (s, 1H), 7.31 – 7.23 (m, 2H), 6.72 (d, J = 11.4 Hz, 1H), 3.74 (s, 3H).
13
C NMR (151 MHz, dmso) δ 159.94, 158.68, 153.81, 146.77, 139.51, 130.51, 130.09,
128.76, 113.23, 110.60, 106.73, 55.52.

150
5-amino-N-(3-methoxyphenyl)thiophene-2-carboxamide (B7-s2)

B7-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried over NaSO4 and concentrated by rotary evaporation to yield B7-s2 (77%) as
a yellow solid, which was used immediately in the next reaction without further purification.

5-(2-chloroacetamido)-N-(3-methoxyphenyl)thiophene-2-carboxamide (B7)

B7-s2 80 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas. DIPEA
(2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe slowly
dropwise. The reaction mixture was allowed to stir overnight. The mixture was diluted with
EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4 and the
residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield B7 as a
yellow solid (6%).
1
H NMR (600 MHz, dmso) δ 11.84 (s, 1H), 10.00 (s, 1H), 7.81 (d, J = 4.2 Hz, 1H), 7.37 (t,
J = 2.2 Hz, 1H), 7.27 (ddd, J = 8.1, 1.9, 0.9 Hz, 1H), 7.21 (t, J = 8.1 Hz, 1H), 6.78 (d, J =
4.2 Hz, 1H), 6.63 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 4.33 (s, 2H), 3.73 (s, 3H).
13
C NMR (151 MHz, dmso) δ 164.14, 160.97, 159.88, 144.70, 140.61, 130.32, 129.82,
127.79, 112.90, 112.82, 109.40, 106.27, 55.44, 42.85.
HRMS (ESI) m/z:

calcd for C
14
H
13
ClN
2
O
3
S 324.03 Found; 324.03
151

N-cyclopentyl-5-nitrothiophene-2-carboxamide (A1-s1)

5-nitrothiophene-2-carboxylic acid (200mg) and cyclopentanamine (1.2 eq.) were dissolved
in 5 mL of DCM/DMF (4:1). HBTU (5 eq) and triethylamine (6 eq.) were added and the
solution was allowed to stir at room temperature for 16 hours. After 16 hours, the reaction
mixture was diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times.
The organic layer was dried with Na2SO4 and concentrated by rotary evaporation. The
residue was purified via flash column chromatography (EtOAc/Hex = 0-50%) to give A1-s1
(63%) as a white solid.
1
H NMR (600 MHz, dmso) δ 8.75 (d, J = 7.2 Hz, 1H), 8.09 (d, J = 4.4 Hz, 1H), 7.81 (d, J =
4.4 Hz, 1H), 4.15 (h, J = 7.2 Hz, 1H), 1.92 – 1.81 (m, 2H), 1.72 – 1.62 (m, 2H), 1.58 – 1.46
(m, 4H).
13
C NMR (151 MHz, dmso) δ 159.44, 153.14, 147.28, 130.53, 129.87, 128.00, 127.66,
51.81, 32.41, 24.08.

5-amino-N-cyclopentylthiophene-2-carboxamide (A1-s2)

A1-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
152
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried over NaSO4 and concentrated by rotary evaporation to yield A1-s2 (62%) as
a white solid, which was used immediately in the next reaction without further purification.

5-(2-chloroacetamido)-N-cyclopentylthiophene-2-carboxamide (A1)

A1-s2 150 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas.
DIPEA (2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe
slowly dropwise. The reaction mixture was allowed to stir overnight. The mixture was
diluted with EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4
and the residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield A1
as a white solid (21%).
1
H NMR (600 MHz, dmso) δ 11.68 (s, 1H), 8.08 (d, J = 7.1 Hz, 1H), 7.55 (d, J = 4.1 Hz,
1H), 6.69 (d, J = 4.1 Hz, 1H), 4.30 (s, 2H), 4.14 (dq, J = 14.1, 6.9 Hz, 1H), 1.88 – 1.79 (m,
2H), 1.70 – 1.60 (m, 2H), 1.56 – 1.42 (m, 4H).
13
C NMR (151 MHz, dmso) δ 163.87, 161.58, 143.37, 130.89, 128.21, 126.79, 126.26,
112.62, 51.18 (d, J = 2.6 Hz), 42.86, 32.63 (d, J = 11.0 Hz), 24.04.
HRMS (ESI) m/z: calcd for C
12
H
15
ClN
2
O
2
S 286.05 Found; 286.05

153
N-cyclohexyl-5-nitrothiophene-2-carboxamide (A3-s1)

5-nitrothiophene-2-carboxylic acid (200mg) and cyclohexanamine (1.2 eq.) were dissolved
in 5 mL of DCM/DMF (4:1). HBTU (5 eq) and triethylamine (6 eq.) were added and the
solution was allowed to stir at room temperature for 16 hours. After 16 hours, the reaction
mixture was diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times.
The organic layer was dried with Na2SO4 and concentrated by rotary evaporation. The
residue was purified via flash column chromatography (EtOAc/Hex = 0-50%) to give A3-s1
(82%) as a white solid.
1
H NMR (400 MHz, dmso) δ 8.74 (d, J = 7.2 Hz, 1H), 8.10 (d, J = 4.4 Hz, 1H), 7.83 (d, J =
4.4 Hz, 1H), 4.16 (h, J = 7.0 Hz, 1H), 1.93 – 1.81 (m, 2H), 1.75 – 1.57 (m, 3H), 1.59 – 1.44
(m, 5H).

5-amino-N-cyclohexylthiophene-2-carboxamide (A3-s2)

A3-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried over NaSO4 and concentrated by rotary evaporation to yield A3-s2 (63%) as
a white solid, which was used immediately in the next reaction without further purification.

154
5-(2-chloroacetamido)-N-cyclohexylthiophene-2-carboxamide (A3)

A3-s2 70 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas. DIPEA
(2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe slowly
dropwise. The reaction mixture was allowed to stir overnight. The mixture was diluted with
EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4 and the
residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield A3 as a
white solid (3%).
1
H NMR (400 MHz, dmso) δ 11.69 (s, 1H), 8.09 (d, J = 7.3 Hz, 1H), 7.55 (d, J = 4.1 Hz,
1H), 6.69 (d, J = 4.1 Hz, 1H), 4.31 (d, J = 2.6 Hz, 2H), 3.45 (q, J = 7.2 Hz, 1H), 1.91 – 1.79
(m, 2H), 1.66 (s, 3H), 1.47 (dd, J = 15.3, 4.7 Hz, 5H).
HRMS (ESI) m/z: calcd for C
13
H
17
ClN
2
O
2
S 300.07 Found; 300.07

155

3-(2-chloroacetamido)-N-(2-methylcyclopentyl)benzamide (A4)

BE-2-12 (200 mg) and 2-methylcyclopentan-1-amine (1.2 eq) were dissolved in 5 mL of
DCM/DMF (4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was
allowed to stir at room temperature for 16 hours. After 16 hours, the reaction mixture was
diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried with Na2SO4 and concentrated by rotary evaporation. The residue was
purified via flash column chromatography (EtOAc/Hex = 0-50%) to give A4 (4%)as a white
solid.
1
H NMR (600 MHz, dmso) δ 10.55 (s, 1H), 8.20 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.92 (d,
J = 8.4 Hz, 1H), 7.77 (d, J = 11.4 Hz, 1H), 7.67 – 7.59 (m, 2H), 7.48 – 7.42 (m, 2H), 5.31
(s, 2H), 3.82 (s, 3H), 3.30 (s, 4H), 2.06 (s, 1H).
13
C NMR (151 MHz, dmso) δ 166.36, 165.21, 138.88, 130.67, 129.87, 128.91, 127.62,
125.51, 124.98, 124.39, 120.35, 119.98, 110.65, 78.02, 52.69.
HRMS (ESI) m/z: calcd for C
15
H
19
ClN
2
O
2
294.11 Found; 294.18

156

N-(2-methylcyclopentyl)-5-nitrothiophene-2-carboxamide (A5-s1)

5-nitrothiophene-2-carboxylic acid (200mg) and 2-methylcyclopentan-1-amine (1.2 eq.)
were dissolved in 5 mL of DCM/DMF (4:1). HBTU (5 eq) and triethylamine (6 eq.) were
added and the solution was allowed to stir at room temperature for 16 hours. After 16 hours,
the reaction mixture was diluted with EtOAc and washed with 10% Na2CO3 followed by
brine 3 times. The organic layer was dried with Na2SO4 and concentrated by rotary
evaporation. The residue was purified via flash column chromatography (EtOAc/Hex = 0-
50%) to give A5-s1 (23%) as a white solid.
1
H NMR (600 MHz, dmso) δ 9.24 (s, 1H), 8.11 (d, J = 4.4 Hz, 1H), 7.83 (d, J = 4.5 Hz, 1H),
7.31 (d, J = 4.3 Hz, 4H), 7.27 – 7.21 (m, 1H), 4.91 (s, 5H), 4.46 (d, J = 6.0 Hz, 1H).
13
C NMR (151 MHz, dmso) δ 164.71, 164.55, 141.85, 136.76, 132.92, 129.56, 128.87 (d, J
= 3.1 Hz), 127.86, 127.72, 44.21, 43.30.

5-amino-N-(2-methylcyclopentyl)thiophene-2-carboxamide (A5-s2)

A5-s1 (1 eq) was dissolved in 4 mL MeOH. Zn (5 eq) and NH4Cl (5 eq) were added, and the
mixture was allowed to stir at room temperature for 16 hr. The reaction mixture was
dissolved in EtOA and washed with 10% Na2CO3 followed by brine 3 times. The organic
157
layer was dried over NaSO4 and concentrated by rotary evaporation to yield A5-s2 (87%) as
a yellow solid, which was used immediately in the next reaction without further purification.

5-(2-chloroacetamido)-N-(2-methylcyclopentyl)thiophene-2-carboxamide (A5)

A5-s2 70 mg was dissolved in anhydrous DCM/THF (5 mL, 1:4) under nitrogen gas. DIPEA
(2.2 eq.) was added via syringe. Chloroacetyl chloride (1.4 eq) was added via syringe slowly
dropwise. The reaction mixture was allowed to stir overnight. The mixture was diluted with
EtOAc and washed with 10% Na2CO3. The organic layer was dried over NaSO4 and the
residue was purified via flash chromatography (EtOAc/Hexane, 0-50%) to yield A5 as a
white solid (9%).
1
H NMR (600 MHz, dmso) δ 11.73 (s, 1H), 8.85 (t, J = 6.0 Hz, 1H), 7.57 (d, J = 4.1 Hz,
1H), 7.41 – 7.08 (m, 8H), 6.72 (d, J = 4.1 Hz, 1H), 4.40 (d, J = 6.0 Hz, 2H), 4.31 (s, 3H).
13
C NMR (151 MHz, dmso) δ 163.95, 162.20, 143.68, 140.16, 130.28, 128.73, 127.70,
127.21, 126.57, 112.79, 42.85, 42.80, 40.40.
HRMS (ESI) m/z: calcd for C
13
H
17
ClN
2
O
2
S 300.07 Found; 300.06

158

3-(2-chloroacetamido)-N-(2,6-dimethylphenyl)benzamide (A6)

BE-2-12 (200 mg) and 2,6-dimethylaniline (1.2 eq) were dissolved in 5 mL of DCM/DMF
(4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was allowed to stir
at room temperature for 16 hours. After 16 hours, the reaction mixture was diluted with
EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic layer was
dried with Na2SO4 and concentrated by rotary evaporation. The residue was purified via
flash column chromatography (EtOAc/Hex = 0-50%) to give A6 (45%)as a white solid.
1
H NMR (600 MHz, dmso) δ 10.51 (s, 1H), 9.74 (s, 1H), 8.07 (s, 1H), 8.03 (d, J = 6.6 Hz,
1H), 7.93 (d, J = 10.4 Hz, 1H), 7.86 (d, J = 10.2 Hz, 1H), 7.78 (d, J = 10.3 Hz, 1H), 7.71 (d,
J = 6.6 Hz, 1H), 7.65 – 7.59 (m, 1H), 7.09 (s, 6H), 3.37 (s, 2H).
13
C NMR (151 MHz, dmso) δ 165.49, 165.06, 143.18, 139.09, 136.04, 129.43, 128.90,
128.00, 127.64, 125.51, 119.97, 119.50, 110.68, 78.06, 72.12, 59.13, 18.48.
HRMS (ESI) m/z: calcd for C
17
H
17
ClN
2
O
2
316.1 Found; 316.09

159

BE-2-12 (200 mg) and 2-methylcyclohexan-1-amine (1.2 eq) were dissolved in 5 mL of
DCM/DMF (4:1). HBTU (5 eq) and triethylamine (5 eq) were added and the solution was
allowed to stir at room temperature for 16 hours. After 16 hours, the reaction mixture was
diluted with EtOAc and washed with 10% Na2CO3 followed by brine 3 times. The organic
layer was dried with Na2SO4 and concentrated by rotary evaporation. The residue was
purified via flash column chromatography (EtOAc/Hex = 0-50%) to give A7 (8%)as a white
solid.
1
H NMR (600 MHz, dmso) δ 10.40 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 9.4 Hz,
1H), 7.52 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 4.24 (s, 2H), 3.50 – 3.41 (m, 1H),
3.30 (s, 4H), 1.92 – 0.88 (m, 9H).
13
C NMR (151 MHz, dmso) δ 165.99, 165.22, 138.75, 136.43, 129.10, 122.75, 122.15,
119.25 (d, J = 10.2 Hz), 54.48, 43.96, 37.35, 34.59, 33.27, 26.03, 25.80, 19.68.
HRMS (ESI) m/z: calcd for C
16
H
21
ClN
2
O
2
308.13 Found; 308.13

160
3.13.2 Methods and materials for biological characterization

Materials and methods for the biological characterization Nsp12 cysteine sites

Plasmid transfection
HEK293T cells were seeded at 7.5x10
5
cells per well in 6-well plates 16 hours before
transfection with Nsp12 or CTPS1 constructs (pLVX-EF1α-2xStrep-IRES) using
Lipofectamine 2000 (Life Technologies).

Inhibitor treatment in situ
The cells were treated with various concentrations of compounds for various times at 37°C.
After compound treatment, the media was removed, and cells were washed twice with ice-
cold D-PBS. The cells were harvested, and the pellet was resuspended in 50 µL of NP40
lysis buffer (50 mM HEPES, pH 7.4, 1% NP-40, 150 mM NaCl), supplemented with 5
mg/mL protease inhibitor cocktail (Roche). The lysate was incubated on ice for 20 min and
fractionated by centrifugation at 18,000 x g for 10 min at 4°C. The protein concentrations
were measured from the supernatant by BCA assay (Pierce).

Twin-Strep-tag® : StrepTactin® enrichment
The lysate was adjusted to 2 mg/mL and 100 µg total protein was incubated with
StrepTactin® sepharose beads (Cytiva), pre-washed twice with 1% NP40, for 20 min at 4°C.
Following protein binding, the beads were triply washed with 5x column volume of 1%
NP40.

161
On-resin Cu-catalyzed azide-alkyne cycloaddition (CuAAC)
Beads were resuspended in PBS and subjected to on-resin CuAAC at a final concentration
of 25 µM TAMRA-azide (Click Chemistry Tools), 1 mM tris(2-carboxyethyl)phosphine
(TCEP, Thermo-Scientific), 100 µM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(TBTA, TCI), and 1 mM CuSO4 (Sigma-Aldrich) in a total volume of 15 µL. The reaction
was performed at RT for 1 hr in the dark before termination by addition of 15 µL of 2X
Laemmli sample buffer (Bio-Rad) and boiled for 15 min. The entire protein sample was
resolved by 4-20% SDS-PAGE (Bio-Rad) and visualized at 532 nm excitation/610 nm
emission by ChemiDoc MP imaging (Bio-Rad).

Silver Stain
Following visualization of fluorescence, protein gels were stained by Silver Stain
(ThermoFisher Scientific). Briefly, gels were first washed twice for 5 minutes in ultrapure
water. They were then incubated in a fixing solution (30% ethanol:10% acetic acid) twice
for 15 min at RT before washes with twice 10% ethanol and twice ultrapure water for 5
minutes each. Gels were incubated in Sensitizer Working Solution (0.2% Sensitizer in
ultrapure water) for 1 minute, followed by 2 brief washes with ultrapure water. Gel was
stained in Stain Working Solution (2% Enhancer in Silver Stain Solution) at RT for 20 min.
After two 20 second washes with ultrapure water, gel was developed in Developer Working
Solution (2% Enhancer in Developer Solution) until bands were visible. Staining was
terminated by addition of 5% acetic acid and protein bands were visualized by ChemiDoc
MP imaging (Bio-Rad).

162
Materials and methods for the animal experiments

Cells and Viruses
HEK293T (ATCC, cat. no. ACS-4500), HCT116 (ATCC, cat. no. CCL-247), mouse
embryonic fibroblasts (MEFs) (isolated from the fetuses of different mouse strains) and Vero
E6-hACE2 (a kind gift from Dr. Jae Jung, Cleveland Clinic Foundation, Cleveland, OH)
were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone) supplemented
with 10% fetal bovine serum (FBS; Gibco), penicillin (100 U/mL) and streptomycin (100
μg/mL). Caco-2 (ATCC, cat. no. HTB-37) and Calu-3 (ATCC, cat. no. HTB-55) were
cultured in Minimum Essential Medium supplemented with 10% FBS and antibiotics.
Normal human bronchial epithelial (NHBE) cells (ATCC, cat. no. PCS-300-010) were
cultured in Airway Epithelial Cell Basal Medium (ATCC) supplemented with Bronchial
Epithelial Cell Growth factors (ATCC) and antibiotics. Sendai Virus (Cantell strain) was
purchased from Charles River (cat. no. 10100774).

Mice

K18-hACE2 C57BL/6 mice were purchased from the Jackson Laboratory. All strains were
confirmed by genotyping

qRT-PCR

qRT-PCR was performed as previously described (3, 4). Total RNA was extracted using
TRIzol reagent (Invitrogen). Complementary DNA (cDNA) was synthesized from total
RNA using reverse transcriptase (Invitrogen). cDNA was diluted and analyzed by qRT-PCR
163
using SYBR Green Master Mix (Applied Biosystems) with CFX Connect PCR instrument
(Bio-Rad Lab.). Relative mRNA expression for each target gene was calculated by the 2-
ΔΔCt method using β-actin or Gapdh as an internal control.
Mouse Infection and Treatment with Compounds

Mouse Infection and Treatment with Compounds

AAV9 encoding hACE2 was purchased from Vector Biolabs (AAV-CMV-hACE2). Mice
(regular C57BL/6 mice were anesthetized with 2.5% isoflurane in O2 (1 L/min). A volume
of 40 μL AAV (1011 GC) was delivered to mice intranasally. At 14 days post-infection,
mice were intranasally infected with SARS-CoV-2 (5 x 105 PFU) in a volume of 30 μL of
DMEM. All mice were monitored every day and euthanized at 4 dpi to collect lung tissues
to determine viral titer by plaque assay and extract RNA for gene expression profiling.

Histology and Immunofluorescence Analysis

Fresh lung pieces were fixed with formalin solution for 24 h. The post-caval lobes of left
lung were sent to histology lab in USC of School of Pharmacy Core Facilities for processing,
and hematoxylin and eosin (H&E) staining. In brief, tissues were dehydrated and embedded
in paraffin. Paraffin-embedded tissue blocks were sectioned with 5 μm thickness. Sections
were processed by H&E staining and imaged by ZEISS Axio Scope.A1 microscope (Zeiss)
and analyzed by ZEISS ZEN Blue software. Pathological scoring on blinded H&E-stained
sections was implemented via a semiquantitative, 5-point grading scheme (0 - within normal
limits, 1 - mild, 2 - moderate, 3 - marked, 4 severe) that considered four different
histopathological parameters: 1) perivascular inflammation, 2) bronchial or bronchiolar
epithelial degeneration or necrosis, 3) bronchial or bronchiolar inflammation, and 4) alveolar
164
inflammation (6). Immunofluorescence staining was performed on the middle lobes of left
lung fixed with formalin solution. Tissues were embedded in optimal cutting temperature
(OCT) compound and frozen immediately at -80°C. Subsequently, the frozen tissues were
cut into 8 μm sections using a cryostat-microtome. Sections were blocked with 10 % normal
goat serum diluted in PBST for 1 h, and incubated with primary antibodies diluted in 10%
normal goat serum overnight at 4°C. After washing with PBST, sections were incubated with
species-matched secondary antibodies for 30 min at room temperature. Then tissue sections
were washed with PBST, mounted with Mounting Medium (Vector Laboratories), and
analyzed with a confocal microscope (Nikon).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism software to perform Student’s t-
test or analysis of variance (ANOVA) on at least three independent replicates. P values of
˂0.05 were considered statistically significant for each test.

165
3.14 References

1. Kirchdoerfer, R. N. & Ward, A. B. Structure of the SARS-CoV nsp12 polymerase bound
to nsp7 and nsp8 co-factors. Nature Communications 2019 10:1 10, 1–9 (2019).

2. Lau, S. K. P. et al. Coronavirus HKU1 and other coronavirus infections in Hong Kong.
Journal of Clinical Microbiology 44, 2063–2071 (2006).

3. Fehr, A. R. & Perlman, S. Coronaviruses: An Overview of Their Replication and
Pathogenesis. Coronaviruses 1282, 1 (2015).

4. Snijder, E. J., Decroly, E. & Ziebuhr, J. The Nonstructural Proteins Directing Coronavirus
RNA Synthesis and Processing. Advances in Virus Research 96, 59–126 (2016).

5. Irwin, J. J. & Shoichet, B. K. Docking Screens for Novel Ligands Conferring New
Biology. Journal of Medicinal Chemistry 59, 4103–4120 (2016).

6. Yadav, R. et al. Role of Structural and Non-Structural Proteins and Therapeutic Targets of
SARS-CoV-2 for COVID-19. Cells 10, (2021).

Abstract (if available)

Abstract One of the most important aims of drug discovery is to develop ligands that are both potent and selective for target proteins. Conventional medical chemistry methods rely on having adequate prior knowledge of a target of interest, identifying a potential scaffold, and modifying the scaffold to instigate selective inhibition. Although these traditional methods have proven powerful in delivering potent ligands in many cases, they alone often do not produce strong drug candidates in a timely or efficient manner. To meet this challenge, our lab has developed a robust chemical proteomic method which allows us to develop potent and selective covalent small molecules that can be used to rapidly identify and modulate diverse protein targets. In this dissertation, I will discuss three projects in which this approach was utilized to identify unique protein targets, and the design, synthesis, and biological characterization of the electrophilic small molecule probes that were responsible for their identification.

Chapter 1 details our efforts to investigate the dependence of electrophile position and electrophile type on the ability of a pharmacophore to covalently bind to different proteins. A panel of electrophilic probes was designed and developed in such a way that we were able to study how minor changes in electrophilic position and type can dramatically alter the range of possible targets of a conserved scaffold. We identified several unique protein targets that were shown to be covalently labeled by our probes through a series of Cu-catalyzed click-chemistry experiments that allowed for target visualization or enrichment.
Chapter 2 focuses on one of the targets identified in the first chapter, Guanosine Monophosphate Synthetase (GMPS). Structural activity relationship studies were performed for optimization and a series of in vitro labeling experiments allowed for the visualization of the covalent interaction of our hit compound with GMPS. We identify the site-of-modification to be a single cysteine residue within the glutamine amido transferase domain of the protein, and prove that mutagenesis of this residue abolishes labeling. The biochemical effects of our compound against GMPS are demonstrated through mass spec analysis and reveals that binding of our compound leads to an accumulation of substrate and subsequent reduction of GMP production. Lastly, in vivo experimentation reveals both the anti-cancer and anti-viral activity of our hit compound and provides compelling data for our comprehensive study.
Chapter 3 looks at the design and synthesis of a covalent small molecule that was inspired by our lead compound in Chapter 2. We utilize in vitro data of a SARS-CoV-2 cellular model to inform our design and synthesis of three generations of analogs. Of the compounds identified to exhibit the strongest anti-viral activity in vitro, we synthesized analogous probe versions, giving us the ability to perform visualization labeling experiments. Through a series of pull-downs and enrichments, we identified nonstructural protein 12 (Nsp12) to be the protein target being labeled, and believe the binding of our compounds to this target is responsible for the observed anti-covid activity. Mass spec analysis revealed several potential cysteine sites-of-modification, and site-directed mutagenesis of these cysteines helped to reveal to be what we believe is the primary cysteine being labeled on Nsp12. Lastly, through collaboration efforts, we have demonstrated that our lead compound exhibits anti-Covid activity in both cellular and animal models.

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Asset Metadata

Creator Espinosa, Bianca Ashley (author)

Core Title Development of selective covalent probes to identify and modulate protein targets

Contributor Electronically uploaded by the author (provenance)

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 2022-12

Publication Date 08/31/2022

Defense Date 08/18/2022

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

Tag chemical biology,chemical genetics,chemical probe,covalent probe,cysteine,GMPS,inhibitor,NSP12,OAI-PMH Harvest,SARS-CoV-2

Format application/pdf (imt)

Language English

Advisor Zhang, Chao (committee chair), Feng, Pinghui (committee member), Prakash, Surya (committee member)

Creator Email baespino@usc.edu,biancaespin5@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111384903

Unique identifier UC111384903

Legacy Identifier etd-EspinosaBi-11166

Document Type Dissertation

Format application/pdf (imt)

Rights Espinosa, Bianca Ashley

Type texts

Source 20220901-usctheses-batch-976 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

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