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Accessible cytotoxic and antiviral drug analogues: improved synthetic approaches to isoindolinones and bioisosteric difluoromethylated nucleotides, and the search for therapeutic organotelluranes

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Accessible cytotoxic and antiviral drug analogues: improved synthetic approaches to isoindolinones and bioisosteric difluoromethylated nucleotides, and the search for therapeutic organotelluranes

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Content ACCESSIBLE CYTOTOXIC AND ANTIVIRAL DRUG ANALOGUES:
IMPROVED SYNTHETIC APPROACHES TO ISOINDOLINONES AND
BIOISOSTERIC DIFLUOROMETHYLATED NUCLEOTIDES,
AND THE SEARCH FOR THERAPEUTIC ORGANOTELLURANES

by

Alexandra N. Aloia

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CHEMISTRY)

August 2019

Copyright Alexandra N. Aloia
2
DEDICATION

To those who did not believe in me, you pushed me to prove you wrong.
To those who did believe in me, you reminded me that I only need to accept myself.
This is not an end, but a beginning.

3
ACKNOWLEDGEMENTS

Thanks to my advisor, Dr. G. K. Surya Prakash, who gave me my first research experience
as an undergraduate, and who accepted me a second time as a graduate student. I have learned
many lessons in my time at USC that have come to shape who I am as a scientist and as a human
being. This experience would not have been possible without Dr. Prakash’s kind personality and
willingness to support his students even at the most difficult times.
Thanks to Dr. George Olah, whom I was fortunate to know in my time at USC. He filled
our group meetings with diverse topics and philosophical ideas; he reminded me always of a
genuine thirst for knowledge and my inspiration for becoming a chemist. He is dearly missed, as
are his memorable voice and humor.
Thanks to the staff and faculty of the Chemistry Department at USC for their tireless
support. Thanks to Michele Dea, without whom the department as we know it would crumble.
Thanks to Dr. Hanna Riesler and Dr. Jahan Dawlaty who always made themselves available to
hear my struggles and aspirations. Thanks to Magnolia Benitez, whose heartfelt support and
mutual love of fashion helped me through tough days. Thanks to Allan Kershaw, Dr. Travis
Williams, and Dr. Ralf Haiges for teaching me fundamental skills and caring for the instruments
which were essential to my research. Thanks also to Dr. Jennifer Moore, Dr. Thomas Bertolini,
and Dr. Rebecca Broyer for their guidance while I was a Teaching Assistant, and who allowed me
to contribute my own input. Thanks to my classmates who helped me survive homework, exams,
and teaching. Thanks to my labmates who exchanged ideas and suffered equally with me through
the challenges of research and of working in a large group environment. Thanks to the Chemistry
Department, the Loker Hydrocarbon Research Institute, and USC for its financial support, without
which I would not be able to pursue this goal.
4
Thanks to my teachers, my mentors, for influencing me, whether by direct encouragement
or through tough love, to pursue my best self and my passions. Thanks to my early inspirations,
Mrs. Pascquarelli, Mrs. Pancoast, Mr. Marine, and Mr. Matt, and to my undergraduate advisors
and mentors, Dr. Blessing, Dr. Chinni, Dr. Kremer, Prof. Schalk, Dr. Donnelly, Dr. Vigna, Dr.
Sholtz, Kevin Burns, Prof. Schwanger, Dr. Williams. You have all impacted my life more than
you could ever know and I will never forget you.
Last but not least, thanks to those closest to me who have been my foundation. Thank you
to my family, past and present, especially: my late grandmother, Dahlia, who will forever be my
best friend and who I wear on my arm like my heart on my sleeve; my late grandfather, Joseph,
who gave me my instruments and fueled my education; my cousin, R.J., and aunt, Linda, who have
served for years as voices of reason; my father, George, and aunt, Debi, who saw that I made it
through college; my aunt, “Dumbo” Debby, who reminds me the value of tradition; and my mother,
Rosanne, who, despite disagreements, shows me that we can begin again. Thank you to my near-
family, the Marchianos, Rachel Mummolo, and Carol Ann Berchtold Santo, for endless support
throughout my childhood and beyond. Thank you to my best friends from college who have
become my chosen family, Becky Chavan, Leah Della Croce, and Mallory Mautz. Thank you to
my friends from graduate school, especially Kavita Belligund who has become my best friend and
kindred spirit in surviving the trials of our degree program. Above all, thanks to my fiancé, Daniel
Kwasniewski, who has stayed by my side more than 10 years now, without whom I cannot imagine
living. He is my stability, my sanity, my adventure, my soulmate, an irreplaceable part of my
identity.
I hope dearly that I have made you all proud, because I would not be here without any one
of you. Thank you.

5
ABSTRACT
This work summarizes efforts in our laboratory to develop novel drug analogues with
cytotoxic and antiviral properties. Chapter 1 details the development and implementation of a
greener, more efficient and cost-effective synthetic method for substituted isoindolinones.
Isoindolinone scaffolds can be found in a number of naturally occurring alkaloids isolated from
plants and fungi. These natural products have demonstrated substantial biological activities,
including anticancer and antiviral functions. Synthetic methods to access isoindolinone derivates
are mostly multistep processes that utilize harmful organic media and expensive reagents.
Presented here is a tandem Cu catalyzed cross-coupling heterocyclization reaction under aqueous
conditions which utilizes convenient alkynylsilanes for the first time. The method is suitable for a
range of electron withdrawing and electron donating groups, is high yielding and selective. This
method was further developed by colleagues in our laboratory into a convenient one-pot synthesis.
This work should better enable the preparation of a wide variety of potential cytotoxic and antiviral
isodinolinone analogues.
Chapter 2 demonstrates improvements upon the synthesis and purification of novel
bioisosteric difluoromenthylphosphonate nucleotide analogues, which were previously
investigated in our laboratory. Nucleotide analogues have been utilized as successful drugs for the
treatment of cancers and viral infections. To circumvent problems with stability and
bioavailability, prodrug technology was developed in which bioisosteric groups mimic structural
aspects of nucleotides to improve lipophilicity, stability, and bioactivity. While a number of
monophosphate nucleotide prodrugs have been developed, the literature surprisingly lacks usage
of fluorine in phosphate modifications. Known for its powerful biological and chemical properties,
fluorine, specifically difluoromethylene, can be used to effectively mask the phosphate hydroxyl
group. A series of novel difluoromethylphosphonate nucleotide analogues have been proposed by
6
our laboratory, but the synthesis and purification of these compounds has involved many
challenges and has not produce products suitable for effective biological testing. This work
evaluates previous methods to determine the best approach. It has been demonstrated that the
current methods can be modified to obtain pure analogues of even the sensitive gemcitabine
substrate, but that success and reproducibility depend upon the complexity of the starting materials.
Alternative methods potentially more suitable have been proposed, though a few pure products are
reported here. These unique nucleotide analogues have great potential as more effective prodrugs,
and these improvements to their preparation make biological testing feasible in the near future.
Chapter 3 is my Gedankenexperiment. Still in pursuit of developments in drug discovery
for anticancer and antiviral agents, I started to investigate elements I had heard little about in the
literature, and I happened upon reviews for tellurium in biological applications, a field that is
grossly undervalued. Despite promising results in the literature and in historical reports for
tellurium compounds as anticancer therapeutics, irrational distrust from only a few reports of
toxicity has prevented much exploration in this area. Tellurium has demonstrated a unique
potential to function as a “sensor/effector” redox modulator which selectively targets cancer cells
with high levels of reactive oxygen species (ROS). Though studies have been performed for
ditellurides and inorganic telluranes, organotelluranes are almost wholly unexplored. The few
available examples illustrate the stability, potency, and safety of these compounds, but are too few
and lack comparable details to draw conclusions for intelligent drug design. It appears that a broad,
systematic structure activity relationship (SAR) study of organotelluranes is needed. Proposed here
is SAR study of selected organotelluranes to determine effects of structural differences on
behaviors as cysteine inhibitors and generators of ROS. It is expected that such a study could
hugely benefit the development of potential organotellurane cancer therapeutics.

7
Table of Contents
DEDICATION ....................................................................................................................... 2
ACKNOWLEDGEMENTS ..................................................................................................... 3
ABSTRACT ........................................................................................................................... 5
1 Green Synthesis of Substituted Isoindolin-1-one Analogues ............................................. 9
1.1 Introduction ........................................................................................................................... 9
1.1.1 Biological Importance of Isoindolinones .............................................................................................. 9
1.1.2 Methods of Preparation ....................................................................................................................... 10
1.2 Results and Discussion ......................................................................................................... 13
1.2.1 Optimization of Reaction Conditions .................................................................................................. 13
1.2.2 Scope of Synthetic Method ................................................................................................................. 15
1.3 Conclusions .......................................................................................................................... 20
1.4 Experimental ....................................................................................................................... 20
1.4.1 General ................................................................................................................................................ 20
1.4.2 Preparation of benzamide substrates ................................................................................................... 21
1.4.3 Preparation of trimethylsilylalkynes ................................................................................................... 23
1.4.4 NMR Spectroscopic data of trimethylsilylalkynes .............................................................................. 23
1.4.5 Preparation of isoindolin-1-ones from silylalkynes ............................................................................ 27
1.4.6 Preparation of isoindolin-1-ones 4 from terminal alkynes .................................................................. 28
1.4.7 NMR spectroscopic data of isoindolin-1-one products ....................................................................... 28
1.5 References ............................................................................................................................ 42
2 Improved Synthesis and Purification of Bioisosteric Difluoromethylated Nucleotides ..... 44
2.1 Introduction ......................................................................................................................... 44
2.1.1 Nucleotides and Enzymatic Phosphorylation ...................................................................................... 44
2.1.2 Fluorine and Bioisosteres: Improving Drug Delivery ......................................................................... 46
2.2 Results and Discussion ......................................................................................................... 49
2.2.1 Assessment of Previous Methods and Results .................................................................................... 49
2.2.2 Alternative Synthesis of Dichloride .................................................................................................... 51
2.2.3 Alternative Synthesis of Monophosphate Nucleotide Analogues ....................................................... 51
8
2.2.4 Identification of Pure Products ............................................................................................................ 52
2.2.5 Challenges in Isolation and Purification ............................................................................................. 54
2.3 Conclusions .......................................................................................................................... 56
2.4 Experimental ....................................................................................................................... 57
2.4.1 General Preparation of Difluoromethylphosphonate Analogues ......................................................... 57
2.5 Representative NMR and Mass Spectrometry data ............................................................ 57
2.5.1 2’deoxy-2’,2’-difluorocytidine-3’-difluoromethylphosphonate .......................................................... 57
2.5.2 2’Deoxy-2’,2’-difluorocytidine-5’-difluoromethylphosphonate ......................................................... 60
2.5.3 3’-deoxy-2’,3’-didehydro-thymidine-5’-difluoromethylphosphonate ................................................. 62
2.6 References ............................................................................................................................ 64
3 Proposed Organotelluranes for Cancer Therapy ............................................................. 67
3.1 Introduction ......................................................................................................................... 67
3.1.1 Tellurium’s Absurdly Toxic Reputation ............................................................................................. 68
3.1.2 The “Forgotten” Element .................................................................................................................... 69
3.1.3 New Investigations into Tellurium’s Biological Effects ..................................................................... 71
3.2 Discussion and Anticipated Results ..................................................................................... 75
3.2.1 Stage One: Diaryl Telluranes .............................................................................................................. 75
3.2.2 Stage Two: Exploring Other Structural Interests ................................................................................ 76
3.2.3 DFT Predicted Geometries and NMR Shifts ....................................................................................... 79
3.2.4 Model NMR study with l-Cysteine and Seleno-Cysteine ................................................................... 81
3.2.5 Assessing Inhibitory Activity, ROS Levels, and Toxicity .................................................................. 81
3.3 Conclusions .......................................................................................................................... 82
3.4 Experimental ....................................................................................................................... 82
3.4.1 Stage One Synthesis: ........................................................................................................................... 82
3.4.2 Stage Two Synthesis: .......................................................................................................................... 85
3.5 References ............................................................................................................................ 86

9
1 GREEN SYNTHESIS OF SUBSTITUTED ISOINDOLIN-1-ONE ANALOGUES
1.1 INTRODUCTION
1.1.1 BIOLOGICAL IMPORTANCE OF ISOINDOLINONES

Figure 1: a) isoindole and isoindolinone structural cores;
1
b) representative
natural structures;
2, 3
c) representative synthetic analogues.
1

The isoindolinone scaffold belongs to a larger family of biologically interesting compounds
containing the isoindole skeleton. Though known for more than a century, natural isoindoles were
first isolated in the early 1980s
1
and have since grown into a field of natural product total synthesis
as well as small molecule development for medicinal purposes. Insoindolinones in particular are
derived from naturally occurring alkaloids, which are identified as heterocycles containing at least
one basic nitrogen.
4
These alkaloids can be derived from both plants and fungi, including the
families of Berberis (Berberidaceae),
1, 2
Aspergillus,
5
Entonaema
3
and Meyerozyma
guilliermondii.
6
Isoindolinones have numerous biological activities and have been applied as
antipsychotics,
7-9
anesthetics,
10, 11
antiviral
12, 13
and anticancer agents.
14, 15

10
The ability to manipulate substitution and functional groups is highly desirable in drug
discovery. The position and type of functional group not only affect the conformation of the
molecule in relation to active sites, but control lipophilicity and permeability.
16
A strong example
of this is a study by Hardcastle et. al. of isoindolinone inhibitors of MDM2-p53; this gene has been
found to be amplified in about 11% of all tumors but up to 44% percent in hepatocellular
carcinoma,
17
the most common liver cancer and leading cancer-related cause of death in the
world.
18
The study systematically assesses substitutions at positions that directly correspond to
pockets within the active site. Selectivity is equally important, because often times only one
enantiomer or isomer demonstrates desirable effects.
19

Figure 2: Representation of isoindolinone within active site which
demonstrates the importance of functionalization in relation to
pockets of MDM2 based on NMR and docking studies performed
by Hardcastle et. al.
17

1.1.2 METHODS OF PREPARATION
There is a myriad of synthetic pathways in the literature to obtain substituted
isoindolinones,
20-23
but many are tedious, multistep procedures utilizing harmful organic media
and expensive reagents. Focusing on more recent developments, Scheme 1, Path A depicts a Pd
11
catalyzed elimination-cyclization-Suzuki coupling sequence, which is a multistep process that
utilizes reagents difficult to prepare.
24
Alternative methods explored the use of accessible
alkynylbenzamides through metal catalyzed cyclization and carbonylation sequences.
25-30
Owing
to their ease of access, alkynylbenzamides have been employed by many to access unsaturated
isoinolin-1-ones by treatment with strong bases ( Scheme 1, Path B).
31-35
Unfortunately, this
approach not only requires isolation of the alkynylbenzamide, but it lacks desirable regio- and
stereoselectivity.

Scheme 1: Selected synthetic procedures used to synthesize isoindolinones.
Multistep synthesis and isolation of intermediates are huge limitations within the literature.
More recent developments have succeeded in reducing steps and waste by use of tandem cross-
coupling cyclization sequences. Several Pd/Cu,
36, 37
Pd,
38, 39
and Cu
35, 40, 41
catalyzed protocols have
been developed from o-halobenzamides and terminal alkynes (Scheme 1, Path C). While the
utilization of inexpensive copper catalysts is attractive, these methods still require use of organic
media and long reaction times. A method developed by You et. al. explored the use of an
inexpensive Cu(II) salt under oxidative conditions for direct C-H bond functionalization (Scheme
1, Path D).
41
The scope of this method is limited, however, as it is restricted to benzamides with
12
the N-(quinolin-8-yl) directing group which is difficult to remove. The method also requires long
reaction times and large amounts of catalyst (3 equiv.)
While several tandem cross-coupling cyclization methods for 3-methylene-isoindolin-1-
ones have been reported,
36-42
none, to the best of our knowledge, utilize silylated (TMS)
derivatives. Organosilicon compounds (silanes and silanols) are known for their stability,
nontoxicity, and abundance, and have become popular since pioneering work by Hiyama
43, 44
and
Denmark.
45
These compounds have found particular application in metal catalyzed cross-coupling
reactions.
46-53
Alkynylsilanes can be easily accessed by Sonogashira
54
coupling of Aryl-X
55-60
with
trialkylsilylacetylene derivates as shown in Sheme 2.

Scheme 2: Metal catalyzed cross-coupling reactions using organosilicon alkynes
In the work presented here (Scheme 3), alkynylsilanes have been implemented in a triple
tandem desilylation cross-coupling heterocyclization under aqueous phase-transfer conditions to
obtain a wide variety of 3-methylene-isoindolin-1-ones. Alkynylsilanes are not only inexpensive
and convenient to access, but they simplify the procedure and expand the scope of the method
beyond the limited availability of terminal alkynes. With few predictable exceptions, the method
affords products with exclusive Z-configuration. This method also reflects elements of pot, atom,
and step economy (PASE)
61
in efforts to “green” the synthesis by reducing steps, waste and use of
organic solvents, and by efficient use of reagents to obtain products in increased yields.
13

Scheme 3: Our work demonstrates a triple tandem desilylation cross-coupling heterocyclization.

1.2 RESULTS AND DISCUSSION
1.2.1 OPTIMIZATION OF REACTION CONDITIONS
A series of reactions were performed to determine the most effective conditions, which are
summarized in Table 1. Though transmetalation to form Cu(I) acetylides has been shown to occur
even in the absence of an external activator,
58, 62, 63
phenylacetylene (3a) was used in lieu of the
silyl derivative (2a), which would afford 3a by in-situ deprotection in a protic solvent. To begin,
methanol was used as a basic, protic solvent expected to promote protodesilylation. Of the copper
salts, ligands, and bases available, 10 mol% of CuCl2, 20 mol% of salicylic acid, and 2
equivalences of Cs2CO3 were used, respectively. The initial result was a promising 60% yield, but
over 24 hours at 80°C which could be improved. To green the synthesis, water was next screened
as a solvent and gratifyingly resulted in 52% yield in only 30 minutes at 130°C without further
optimization. To ensure the reaction would take place under aqueous conditions, a concentration
of 0.75M n-tetrabutylammounium bromide (TBAB) was used as a phase-transfer agent.
14

Table 1: Optimization of the reaction conditions
a

a
Reaction conditions: 0.5 mmol of 1a, 1.5 equiv. of alkyne Cs2CO3 (2 equiv),
ligand and solvent (1mL, degassed) were placed in a crimp top vial, sealed and
heated at 130
o
C for the indicated time. Isolated Yields are shown. Average of
2 consecutive runs
b
Reaction was performed at 80
o
C.
c
n-Bu4NBr (0.75 M) was
added.
d
K2CO3 (2 equiv) was used.
e
K3PO4 was used (2 equiv) fn-Bu4NBr (0.5
M) was used
g
Reaction was performed at 100
o
C. h3 equiv. of Cs2CO3 were
used.

Screening of copper catalysts became the next focus and revealed that CuCl was the most
efficient option. Of the variety of N-N, N-O, and P ligands, PPh3 was determined to be the most
effective giving a yield of 65%, which improved to 70% after a slight increase in loading to 30
mol%. As a control, the reaction was performed without a ligand to demonstrate its importance.
15
As expected, yield significantly decreased. Though efforts were made to further optimize
conditions by evaluating the performance of different bases (Table 1, entries 14-15), variations
made no improvement. It was also determined that reductions in TBAB or temperature led to
incomplete conversion of starting materials (Table 1, entries 16-17). When implementing 2a, in-
situ desilylation occurred as expected followed by cross-coupling heterocyclization without a loss
in yield.
1.2.2 SCOPE OF SYNTHETIC METHOD
The optimized conditions were applied to a study of alkynylsilanes giving a broad scope
of 3-methylene-isoindolin-1-ones (Table 2). Isolated yields were good overall, ranging from 60-
91%. Electron-donating and electron-withdrawing groups were equally well tolerated, though
substrates with electron-donating groups typically resulted in better yields. The two highest
yielding derivatives, 4b and 4d, reflect the nature of the electron-donating functional groups, 4-
MeO and 4-NH2, respectively, but required slightly longer reaction times due to the lowered
electrophilicity of the o-alkynylbenzamide intermediates. Of particular interest are 4e, 4f, 4j, and
4l which have medicinally important functional groups (CF3, OCF3, and OCF2H), and which were
also obtained in good yields. Alkynylsilanes with ortho substituents were also readily
incorporated, even ortho-NH2 for which the competitive cyclization to form the corresponding
indole
32, 64
was completely suppressed. Products were confirmed by NOSY experiments to have
exclusive Z-configuration about the exocyclic double bond, though with specific exceptions owing
to their strongly electron-withdrawing substituents (4g, 4h, 4i, and 4k). The Z-configuration was
unambiguously confirmed by X-ray crystallography (4a). Exclusive Z-configuration is
advantageous in medicinal applications. Isomers can have significantly different effects in the
body and typically require separation, which is not a concern using this method.
16

Table 2: Triple tandem desilylation-cross-coupling-heterocyclization of
alkynylsilanes 2 with iodobenzamides 1a

a
Reaction conditions: 0.5 mmol of 1b, silylalkyne 4 (1.5 equiv), Cs2CO
3
(3 equiv), n-tetrabutylammonium
bromide (TBAB, 0.75 M), CuCl (10 mol%), PPh3 (30 mol%) in 1 mL of H2O (degassed) were heated at
130
o
C for 30 min in a crimp top vial under N2.
b
Reaction time = 40 min.
c
Z/E ratio = 2
d
Z/E ratio = 1.2
e
Z/E ratio = 0.2

17

Figure 3: X ray crystal structure of 4a confirmed the Z-configuration of the isoindaloninoes.

Table 3: Tandem cross-coupling, hydroamidation of 2-iodobenzamides 1 with terminal
alkynes 3 under aqueous phase-transfer conditions.
a

a
Isolated yields, average of two runs are shown. Reactions conditions: 0.5 mmol 2-Iodobenzamide derivatives 1, 1.5 equiv of terminal alkyne 3,
CuCl (10 mol%), PPh3 (30 mol%), Cs2CO3 (2 equiv), n-tetrabutylammonium bromide (TBAB, 0.75 M) in 1 mL of H2O (degassed) were heated at
130
o
C for 30 min.
b
Reaction time = 40 min.
c
3 equiv of alkyne were used.
d
Reaction time was 90 min.

18
The method was further applied to a study using terminal alkynes (Table 3). Yields
were comparable to those in Table 2, though the use of terminal alkynes requires fewer
equivalents of base. What is worthy to note is that amino-substituted isoindolin-1-ones were
obtained in good yield and without competitive C-N bond formation (Table 2, 4d, 4n, and
Table 3, 4s). Some drawbacks to be addressed concern the following functionalities: acetyl,
formyl, 4-cyano, 4-nitro, and aliphatics. Though competitive 1,2-addition was not observed
for the acetyl and formyl functionalities, there was a decrease in Z-selectivity (Z/E ratio =
2, Table 2, 4g and 4h). Similarly, the 4-cyano substituent resulted in decreased Z-selectivity
(Z/E = 1.2, Table 2, 4k). Reverted stereoselectivity was observed for the 4-nitro substituted
alkyne (Z/E ratio = 0.2, Table 2, 4i). The use of aliphatic alkynes led to products 4x and
4y successfully, though in lower yields and over longer reaction times (90 min).
Additionally, the X-ray crystal structure of 4y was also obtained.
Despite these small drawbacks, the method clearly demonstrates selectivity for Z-
configuration. The method also offers the ability to vary substrate functionalities on the
benzamide as well as the alkyne. While N-Me, N-Bn, and free N-H were shown to be well
tolerated, it is important to highlight that enantiomerically enriched isoindolinone 4z was
isolated in high yield and could lead to further asymmetric transformations valued in
medicinal chemistry. The use of halo-substituted alkynes also allows for further
functionalization by transition metal catalyzed coupling processes. The method was further
applied to alkynylsilanes with medicinally relevant heteroaromatic substituents. The resulting
isoindolin-1-ones were isolated in high yields and exclusive Z-configuration. Nitrogen
heterocycles (6e and 6f)) were successfully converted to the corresponding products without
competitive N-arylation,
65
further demonstrating the superior nature of this method for N-H
functionalities.
19

Table 4: Triple tandem desilylation, cross-coupling, hydroamidation of
heteroaromatic silyl-acetylenes with 1b

a
Reaction conditions: 0.5 mmol of 1b, heteroaryl silylalkyne 5 (1.5 equiv), Cs 2CO 3 (3 equiv), n-
tetrabutylammonium bromide (0.75 M), CuCl (10 mol%), PPh 3 (30 mol%) in 1 mL of H 2O
(degassed) were heated at 130
o
C for 30 min in a crimp top vial under N 2

Figure 4: X ray crystal structure of 4y
20
1.3 CONCLUSIONS
Presented here is a green, inexpensive and efficient CuCl/PPh 3 catalyzed method for the
synthesis of 3-methylene-isoindolin-1-ones from 2-iodobenzamides, terminal alkynes and
alkynylsilanes. Highlights include aqueous phase-transfer conditions, short reaction times, high
selectivity for Z-configuration, and tolerance for a wide variety of functional groups. Further
functionalization is made available by incorporation of Br-, Cl-, COMe-, CHO-, NH 2-, CN-, and
NO 2- substituents, as well as N-R substitutions on the benzamide. The advantages of this method
serve well the need for further systematic studies of isoindolinones as potential antiviral and
anticancer therapeutics. Efforts by collogues in our laboratory have further developed the method
as a greener, convenient one-pot process from aryl halides.
1.4 EXPERIMENTAL
1.4.1 GENERAL
Unless otherwise mentioned, all the chemicals were purchased from commercial sources and
used without further purification. Dry DMF was obtained by distillation over CaH2 and the
distillate was stored over molecular sieves in a Strauss flask under N2 atmosphere. Water was
degassed by several cycles (usually 3 cycles) of sonication- repressurization with N2 under static
vacuum. Flash column chromatography was performed to isolate products with suitable eluent as
determined by TLC.
1
H,
13
C, and
19
F spectra were recorded on 400 MHz or 500 MHz Varian NMR
spectrometers.
1
H NMR chemical shifts were determined relative to CDCl3 as the internal standard
at δ 7.26 ppm.
13
C NMR shifts were determined relative to CDCl3 at δ 77.16 ppm.
19
F NMR
chemical shifts were determined relative to CFCl3 at δ 0.00 ppm. Mass spectra were recorded on
a high-resolution mass spectrometer, only for new compositional matter, the results are reported
in the EI or in ESI mode.
21
1.4.2 PREPARATION OF BENZAMIDE SUBSTRATES
2-iodo-N-methylbenzamide, 1b

Benzoic acid (2.8 g, 11 mmol) were weighed into a 250 mL round bottom flask and 25 mL of dry
dichloromethane (DCM) was added under nitrogen. To this suspension, SOCl2 (1.57 g, 13.2 mmol)
and a few drops of dimethylformamide (DMF) was added by syringe and stirred at room
temperature overnight until the solution became complete clear which denoted the complete
consumption of the benzoic. This solution was concentrated to half of its volume under reduced
pressure and cooled down to 0
o
C with an ice/water bath. To this solution, a pre-cooled, 40%
aqueous solution of MeNH2 (5 mL, 64 mmol) was added slowly (exothermic reaction) and the
ensuing reaction was marked by the immediate formation of a white precipitate. Upon complete
addition of MeNH2, the mixture was allowed to warm up to room temperature and stirring was
continued for 1 h. The solid was recovered by filtration, wash with cold DCM (15 mL) and cold
water (15 mL). This solid was then dried under high vacuum overnight, affording an analytically
pure 1b as a white crystalline powder.
1
H NMR (400 MHz, CDCl3) δ 7.88 – 7.83 (m, 1H), 7.39 – 7.36 (m, 2H), 7.09 (ddd, J = 8.0, 6.3,
2.9 Hz, 1H), 5.78 (s, 1H), 3.02 (d, J = 5.0 Hz, 4H).
13
C NMR (100 MHz, CDCl3) δ 170.2, 142.4,
139.9, 131.1, 128.3, 128.2, 92.6, 26.8. These values are in agreement to those of the previously
reported authentic compound.

22
(S)-2-iodo-N-(2-(methoxymethyl)pyrrolidin-1-yl)benzamide, 1z

Benzoic acid (2 mmol, 496 mg) was weighed into a 50 mL round bottom flask and suspended in
dry DCM (6 mL). The flask was closed with a septum under nitrogen. Subsequently, DMF (1
drop), SOCl2 (1.2 eq, 2.4 mmol, 174 uL) were added via micro-syringe and the mixture was stirred
at room temperature until the solution became clear (usually 2 h), which indicated the full
consumption of benzoic acid. This solution of was then concentrated under reduced pressure to
remove all volatiles to obtain crude 2-iodobenzoyl chloride. Subsequently, a fresh portion of dry
DCM (6 mL) was added. In a separate vial, a solution of (S)-2-(methoxymethyl)pyrrolidin-1-
amine (SAMP, 2 equiv, 4.4 mmol, 622 L) in dry DCM (10 mL) was prepared and added dropwise
to the solution of 2-iodobenzoyl chloride at 0
o
C. The mixture was allowed to warm up to room
temperature and stirred for 30min.H2O (10 mL) was added and the product extracted with EtOAc
(10 mL, 3 times). The organic layer was then concentrated and purified by flash column
chromatography to obtain pure 1z in 68% yield.
Major rotamer
1
H NMR (400 MHz, CDCl3) δ 7.87 – 7.82 (m, 1H), 7.40 – 7.36 (m, 2H), 7.10 (ddd,
J = 7.9, 5.7, 3.5 Hz, 1H), 6.68 (s, 1H), 3.64 (dd, J = 9.6, 5.2 Hz, 1H), 3.52 (dd, J = 9.6, 5.5 Hz,
1H), 3.46 (ddd, J = 8.9, 6.9, 4.1 Hz, 1H), 3.38 (d, J = 0.4 Hz, 3H), 3.23 (ddd, J = 13.2, 7.9, 5.3 Hz,
1H), 3.00 (q, J = 8.5, 8.5, 8.5 Hz, 1H), 2.12 – 2.01 (m, 1H), 1.96 – 1.86 (m, 2H), 1.77 – 1.64 (m,
1H).
13
C NMR (100 MHz, CDCl3) δ 168.1 , 141.2 , 139.9 , 131.3 , 128.5 , 128.2 , 92.9 , 75.3 ,
64.4 , 59.4 , 55.3 , 26.7 , 21.5 . HRMS (ESI) Calcd. for C13H18N2O2I (M+H)+ = 361.0413, Found
= 361.0411.

23
1.4.3 PREPARATION OF TRIMETHYLSILYLALKYNES

Trimethysilylalkynes 2 and some heterosilylalkynes 5, were prepared from the corresponding
(hetero)aryl iodides 7 or bromides 8 and trimethylsilylacetylene (TMSA) by a procedure adapted
from a published report. 2a, 2e and 5a-5e are commercially available. A representative procedure
is as follows:
The aryl iodide 7 or bromide 8 (3 mmol), Pd(PPh3)4 (69.3 mg, 0.06 mmol), CuI (22.9 mg, 0.12
mmol), trimethylsilylacetylene(0.51 mL, 3.6 mmol), diethylamine (4 mL) and dimethylformamide
(1 mL) were mixed and stirred under nitrogen in a crimp-top vial at 120 °C until full consumption
of starting material as determined by GC-MS analysis. Usually, aryl iodides 7, required 5-10 min,
while aryl bromides 8, required from 15-30 min. Then, the reaction mixture was poured into 0.1
M aqueous HCl (5-10 mL) and extracted three times with diethyl ether (5-10 mL). The combined
organic layers were washed with concentrated aqueous NaHCO3 solution (5-10 mL) and water (5-
10 mL) and then concentrated under reduced pressure. The residue was purified by flash
chromatography using the appropriate eluent. The combined product fractions were concentrated
on a rotatory evaporator.
1.4.4 NMR SPECTROSCOPIC DATA OF TRIMETHYLSILYLALKYNES

((4-Methoxyphenyl)ethynyl)trimethylsilane, 2b

Obtained as yellow oil by the reaction of 4-Iodoanisole and TMSA in 86% isolated yield.
1
H
NMR (400 MHz, CDCl3) δ 7.43 – 7.38 (m, 2H), 6.84 – 6.79 (m, 2H), 3.80 (s, 3H), 0.24 (s, 9H).
24
13
C NMR (100 MHz, CDCl3) δ 159.9 , 133.6 , 115.4 , 113.9 , 105.3 , 92.6 , 55.4 , 0.2 . These
values are in agreement to those of the previously reported authentic compound.
((3,5-Dimethylphenyl)ethynyl)trimethylsilane, 2c

Obtained by the reaction of 1-iodo-3,5-dimethylbenzene and TMSA with 63% isolated yield as a
yellow oil.
1
H NMR (400 MHz, CDCl3) δ 7.10 (s, 2H), 6.95 (s, 1H), 2.27 (s, 6H), 0.24 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 137.9 , 130.5 , 129.8 , 122.8 , 105.7 , 93.4 , 21.2 , 0.2 .
4-((Trimethylsilyl)ethynyl)aniline, 2d

Obtained as a brown solid by the reaction of 4-iodoaniline and TMSA with 66% isolated yield.
1
H
NMR (400 MHz, CDCl3) δ 7.29 – 7.25 (m, 2H), 6.60 – 6.54 (m, 2H), 3.79 (br. s, 2H, NH2), 0.22
(s, 9H).
13
C NMR (100 MHz, CDCl3) δ 146.9 , 133.5 , 114.7 , 112.6 , 106.1 , 91.5 , 0.3 .

These
values are in agreement to those of the previously reported authentic compound.

((3,5-bis(trifluoromethyl)phenyl)ethynyl)trimethylsilane, 2f

Obtained by the reaction of 1-bromo-3,5-bis(trifluoromethyl)benzene and TMSA with a 86%
isolated yield as a colorless crystals after purification by sublimation as reported earlier.

These
values are in agreement to those of the previously reported authentic compound.
1
H NMR (400
MHz, CDCl3) δ 7.88 (s, 2H), 7.79 (s, 1H), 0.27 (d, J = 0.6 Hz, 9H).
13
C NMR (100 MHz, CDCl3)
δ 131.7 – 131.6 (m), 131.6 (q, J = 33.7, 33.7, 33.7 Hz), 125.3, 122.7 (q, J = 272.9, 272.8, 272.8
Hz), 121.6 (hept, J = 3.9, 3.9, 3.8, 3.8 Hz), 101.3, 98.6 , -0.5.
19
F NMR (376 MHz, CDCl3) δ -
63.7.

25
1-(4-((Trimethylsilyl)ethynyl)phenyl)ethanone, 2g

Obtained by the reaction of 1-(4-iodophenyl)ethanone and TMSA with a 83% isolated yield as a
brown oil.
1
H NMR (400 MHz, CDCl3) δ 7.91 – 7.86 (m, 2H), 7.56 – 7.51 (m, 2H), 2.60 (s, 3H),
0.26 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 197.5 , 136.5 , 132.2 , 128.3 , 128.1 , 104.1 , 98.3 ,
26.8 , 0.0 . These values are in agreement to those of the previously reported authentic compound.
4-((Trimethylsilyl)ethynyl)benzaldehyde, 2h

Obtained by the reaction of 4-iodobenzaldehyde and TMSA with a 53% isolated yield as a brown
solid.
1
H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 7.94 – 7.72 (m, 2H), 7.70 – 7.51 (m, 2H), 0.27
(s, 9H).
13
C NMR (100 MHz, CDCl3) δ 191.6 , 135.7 , 132.6 , 129.6 , 129.5 , 104.0 , 99.2 , -0.1 .
These values are in agreement to those of the previously reported authentic compound.
Trimethyl((4-nitrophenyl)ethynyl)silane, 2i

Obtained by the reaction of 1-iodo-4-nitrobenzene and TMSA with a 69% isolated yield as a
orange solid.
1
H NMR (400 MHz, CDCl3) δ 8.17 (m, 2H), 7.70 – 7.49 (m, 2H), 0.27 (s, 9H).
13
C
NMR (100 MHz, CDCl3) δ 147.3 , 132.8 , 130.1 , 123.6 , 102.8 , 100.8 , -0.1 . These values are in
agreement to those of the previously reported authentic compound.
Trimethyl((4-(trifluoromethoxy)phenyl)ethynyl)silane, 2j

Obtained by the reaction of 1-iodo-4-(trifluoromethoxy)benzene and TMSA with a 70% isolated
yield as a clear oil.
1
H NMR (400 MHz, CDCl3) δ 7.50 – 7.46 (m, 2H), 7.16 – 7.12 (m, 2H), 0.25
(s, 9H).
13
C NMR (100 MHz, CDCl3) δ 149.1 (q, J = 1.8 Hz), 133.6 , 122.1 , 120.8 , 120.5 (q, J =
26
257.8 Hz), 103.6 , 95.4 , 0.0 .
19
F NMR (376 MHz, CDCl3) δ -58.3 . HRMS (EI) Calcd for
C12H13OSiF3 (M
+
) = 258.06879, found = 258.06883.
4-((Trimethylsilyl)ethynyl)benzonitrile, 2k

Obtained by the reaction of 4-bromobenzonitrile and TMSA with a 60% isolated yield.
1
H NMR
(400 MHz, CDCl3) δ 7.63 – 7.55 (m, 2H), 7.57 – 7.50 (m, 2H), 0.26 (s, 9H).
13
C NMR (100 MHz,
CDCl3) δ 132.5 , 132.0 , 128.0 , 118.4 , 111.8 , 103.0 , 99.6 , -0.2 . These values are in agreement
to those of the previously reported authentic compound.
((3-(Difluoromethoxy)phenyl)ethynyl)trimethylsilane, 2l

Obtained as a clear oil by the reaction of 1-(difluoromethoxy)-3-iodobenzene and TMSA with a
71% isolated yield.
1
H NMR (400 MHz, CDCl3) δ 7.33 – 7.26 (m, 2H), 7.23 – 7.21 (m, 1H), 7.10
– 7.06 (m, 1H), 6.50 (t, J = 73.6 Hz, 1H), 0.25 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 151.0 ,
129.8 , 129.2 , 125.0 , 122.9 , 120.2 , 115.9 (t, J = 260.3 Hz), 103.7 , 95.7 , 0.0.
19
F NMR (376
MHz, CDCl3) δ -81.3 (d, J = 73.8 Hz). HRMS (EI) Calcd for C12H14OSiF2 (M
+
) = 240.07821,
found = 240.07788.
((4-Chloro-2-methoxyphenyl)ethynyl)trimethylsilane, 2m

Obtained by the reaction of 4-chloro-1-iodo-2-methoxybenzene and TMSA with a 61% isolated
yield.
1
H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 2.7 Hz, 1H), 7.22 (dd, J = 8.8, 2.7 Hz, 1H), 6.77
(d, J = 8.9 Hz, 1H), 3.86 (s, 3H), 0.26 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 159.1 , 133.7 , 129.8
, 125.2 , 114.0 , 111.9 , 100.1 , 99.8 , 56.3 , 0.1 . HRMS (EI) Calcd for C12H15OClSi (M
+
) =
238.05808, found = 238.05844

27
5-(Trifluoromethyl)-2-((trimethylsilyl)ethynyl)aniline, 2n

Obtained by the reaction of 2-iodo-5-(trifluoromethyl)aniline and TMSA with a 73% isolated
yield.
1
H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 2.5 Hz, 1H), 7.32 (dd, J = 8.6, 2.3 Hz, 1H), 6.71
(d, J = 8.6 Hz, 1H), 4.53 (br. s, 2H), 0.27 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 150.7 , 129.8 (q,
J = 3.7 Hz), 126.8 (q, J = 3.7 Hz), 124.4 (q, J = 270.7 Hz), 119.8 (q, J = 33.0 Hz), 113.7 , 107.5 ,
101.3 , 100.2 , 0.2 . These values are in agreement to those of the previously reported authentic
compound.
5-((Trimethylsilyl)ethynyl)-1H-indole, 5f

Obtained by the reaction of 5-iodo-1H-indole and TMSA with a 61% isolated yield.
1
H NMR (400
MHz, CDCl3) δ 8.17 (s, 1H), 7.81 (q, J = 1.0 Hz, 1H), 7.32 – 7.29 (m, 2H), 7.22 (dd, J = 3.3, 2.4
Hz, 1H), 6.52 (dd, J = 3.3, 2.0 Hz, 1H), 0.26 (s, 9H).
13
C NMR (100 MHz, CDCl3) δ 135.7 , 127.8
, 126.2 , 125.4 , 125.1 , 114.5 , 111.0 , 107.1 , 103.1 , 91.2 , 0.3 . These values are in agreement to
those of the previously reported authentic compound.
1.4.5 PREPARATION OF ISOINDOLIN-1-ONES FROM SILYLALKYNES

Inside an argon glovebox, the corresponding iodobenzamide 1 (0.5 mmol, 1 equiv), alkynylsilane
2 or heteroaryl alkynylsilane derivative 5 (0.75 mmol, 1.5 equiv), CuCl (5 mg, 0.05 mmol), PPh3
(39 mg, 0.15 mmol), n-tetrabutylammonium bromide (242 mg, 0.75 mmol) and Cs2CO3 (488 mg,
28
1.50 mmol) were placed in a crimp-top vial and sealed. Liquid derivatives 2 or 5, they were added
using a syringe outside the glovebox. Then, 1 ml of water (degassed) was also added by syringe
and the vial was place into a pre-heated oil bath at 130
o
C for 30-40 min. Subsequently, the reaction
mixture was allowed to cool down to room temperature, the vial was opened and the contents were
poured into 10 ml of a 0.1 M solution of NH4OH and extracted with EtOAc (3 times, 15 mL). The
organic layers were combined, washed with brine (10 mL), water (5 mL), dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by flash chromatography using the
appropriate eluent (usually Hex/EtOAc). The combined product fractions were concentrated on a
rotatory evaporator.
1.4.6 PREPARATION OF ISOINDOLIN-1-ONES 4 FROM TERMINAL ALKYNES
The preparation of 4 from terminal alkynes 3 is analogous to the one described above, but using
only 2 equiv of Cs2CO3 (325.8 mg, 1mmol).
1.4.7 NMR SPECTROSCOPIC DATA OF ISOINDOLIN-1-ONE PRODUCTS
The configuration around the double bond of all products reported herein was confirmed by
NOESY experiments (vide infra).
(Z)-3-Benzylideneisoindolin-1-one (4a)

1
H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.89 (dt, J = 7.5, 1.0 Hz, 1H), 7.80 (dt, J = 7.8, 0.9
Hz, 1H), 7.69 – 7.62 (m, 1H), 7.53 (td, J = 7.5, 1.0 Hz, 1H), 7.44 (d, J = 4.4 Hz, 4H), 7.36 – 7.29
(m, 1H), 6.57 (s, 1H).
13
C NMR (100 MHz, CDCl3) δ 169.4 , 138.4 , 135.0 , 133.1 , 132.3 , 129.3
, 129.2 , 128.8 , 128.7 , 127.8 , 123.6 , 119.9 , 106.2 . The chemical shift corresponding to the N–
H varies significantly with sample concentration. Data reported here corresponds to a
concentration of 10 mg/mL. This data corresponds to the previously reported structure.

29
(Z)-3-(4-Methoxybenzylidene)-2-methylisoindolin-1-one (4b)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.5, 1.0 Hz, 1H), 7.73 (dt, J = 7.8, 0.9 Hz, 1H), 7.58
(td, J = 7.5, 1.2 Hz, 1H), 7.48 (td, J = 7.5, 0.9 Hz, 1H), 7.31 – 7.24 (m, 2H), 6.95 – 6.91 (m, 2H),
6.74 (s, 1H), 3.85 (s, 3H), 3.07 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.1 , 159.2 , 138.3 ,
135.8 , 131.9 , 131.1 , 128.9 , 128.6 , 127.1 , 123.3 , 119.3 , 113.8 , 106.6 , 55.5 , 30.7 . HRMS
(ESI) Calcd. for C17H16NO2 (M+H)
+
= 266.1181, Found = 266.1181.
(Z)-3-(3,5-Dimethylbenzylidene)-2-methylisoindolin-1-one (4c)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.5, 0.9 Hz, 1H), 7.71 (dt, J = 7.7, 0.9 Hz, 1H), 7.57
(td, J = 7.7, 1.1 Hz, 1H), 7.47 (td, J = 7.4, 0.9 Hz, 1H), 6.97 – 6.94 (m, 3H), 6.73 (s, 1H), 3.05 (s,
3H), 2.34 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 138.2 , 137.7 , 136.0 , 134.7 , 131.9 ,
129.2 , 128.9 , 128.6 , 127.6 , 123.2 , 119.3 , 107.0 , 30.7 , 21.4 . HRMS (ESI) Calcd. for C18H18NO
(M+H)
+
= 264.1388, Found = 264.1389.
(Z)-3-(4-Aminobenzylidene)-2-methylisoindolin-1-one (4d)

1
H NMR (400 MHz, CDCl3) δ 7.84 (dt, J = 7.5, 0.9 Hz, 1H), 7.72 (dt, J = 7.7, 0.9 Hz, 1H), 7.57
(td, J = 7.7, 1.2 Hz, 1H), 7.46 (td, J = 7.5, 0.9 Hz, 1H), 7.16 – 7.12 (m, 2H), 6.72 – 6.68 (m, 3H),
3.78 (br. s, 2H), 3.12 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.2 , 146.0 , 138.4 , 135.1 , 131.8
30
, 131.1 , 128.7 , 124.7 , 123.2 , 119.2 , 115.2 , 114.7 , 107.5 , 30.8 . HRMS (ESI) Calcd. for
C16H15N2O (M+H)
+
= 251.1184, Found = 251.1186.
(Z)-2-Methyl-3-(4-(trifluoromethyl)benzylidene)isoindolin-1-one (4e)

1
H NMR (400 MHz, CDCl3) δ 7.87 (dd, J = 7.6, 1.0 Hz, 1H), 7.75 (dd, J = 7.7, 1.0 Hz, 1H), 7.68
– 7.59 (m, 3H), 7.52 (tt, J = 7.5, 7.5, 1.1 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 6.73 (s, 1H), 3.03 (s,
3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 138.8 , 137.9 , 137.6 , 132.9 (d, J = 218.2 Hz), 130.1
, 129.6 (q, J = 32.7 Hz), 129.6 , 128.6 , 125.2 (q, J = 3.8, 3.7, 3.7 Hz), 124.2 (q, J = 272.0 Hz),
123.5 , 119.5 , 30.8 .
19
F NMR (282 MHz, CDCl3) δ -63.1 . HRMS (ESI) Calcd. for C17H13NOF3
(M+H)
+
= 304.0949, Found = 304.0948.
(Z)-3-(3,5-Bis(trifluoromethyl)benzylidene)-2-methylisoindolin-1-one (4f)

1
H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.81 (s, 2H), 7.74 (dt, J =
7.7, 0.8 Hz, 1H), 7.65 (td, J = 7.6, 1.2 Hz, 1H), 7.55 (td, J = 7.5, 7.4, 0.9 Hz, 1H), 6.69 (s, 1H),
3.01 (d, J = 0.6 Hz, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 138.9 , 137.7 , 137.3 , 132.5 ,
131.7 (q, J = 33.7, 33.3, 32.9 Hz), 130.0, 129.9 – 129.6 (m), 128.5 , 123.7 , 123.3 (d, J = 272.8
Hz), 121.1 (d, J = 3.8 Hz), 119.6 , 102.1 , 31.0 .HRMS (ESI) Calcd. for C18H12NOF6 (M+H)
+
=
372.0823, Found = 372.0818.

31
(Z)-3-(4-Acetylbenzylidene)-2-methylisoindolin-1-one (Z-4g)

1
H NMR (400 MHz, CDCl3) δ 8.03 – 7.95 (m, 2H), 7.87 (dt, J = 7.5, 0.7 Hz, 1H), 7.75 (dt, J =
7.8, 1.2 Hz, 1H), 7.62 (td, J = 7.4, 1.2 Hz, 1H), 7.52 (td, J = 7.4, 1.4 Hz, 1H), 7.46 – 7.43 (m, 2H),
6.74 (s, 1H), 3.04 (s, 3H), 2.64 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 197.6 , 169.1 , 140.1 ,
138.0 , 137.6 , 136.0 , 132.3 , 130.1 , 129.6 , 128.5 , 128.2 , 123.5 , 119.5 , 105.0 , 30.9 , 26.8 .
HRMS (ESI) Calcd. for C18H16NO2 (M+H)
+
= 278.1181, Found = 278.1183.
(E)-3-(4-Acetylbenzylidene)-2-methylisoindolin-1-one (E-4g)

1
H NMR (400 MHz, CDCl3) δ 8.05 – 8.00 (m, 2H), 7.85 (dt, J = 7.5, 1.0, 1.0 Hz, 1H), 7.60 – 7.55
(m, 2H), 7.48 – 7.42 (m, 1H), 7.38 – 7.30 (m, 2H), 6.47 (s, 1H), 3.40 (s, 3H), 2.67 (s, 3H).
13
C
NMR (100 MHz, CDCl3) δ 197.7 , 166.7 , 140.6 , 138.6 , 136.3 , 134.7 , 131.8 , 130.8 , 130.0 ,
129.8 , 128.8 , 123.5 , 123.1 , 108.9 , 26.8 , 26.3 . HRMS (ESI) Calcd. for C18H16NO2 (M+H)
+
=
278.1181, Found = 278.1183.
(Z)-4-((2-Methyl-3-oxoisoindolin-1-ylidene)methyl)benzaldehyde (Z-4h)

1
H NMR (400 MHz, CDCl3) δ 10.04 (s, 1H), 7.93 – 7.89 (m, 2H), 7.86 (dt, J = 7.5, 1.1 Hz, 1H),
7.75 (dt, J = 7.8, 0.9 Hz, 1H), 7.62 (td, J = 7.7, 1.2 Hz, 1H), 7.55 – 7.49 (m, 3H), 6.73 (s, 1H), 3.04
(s, 3H).
13
C NMR (100 MHz, CDCl3) δ 191.7 , 169.1 , 141.6 , 137.9 , 137.9 , 135.3 , 132.3 , 130.5
32
, 129.7 , 129.6 , 128.5 , 123.5 , 119.6 , 104.7 , 30.9 . HRMS (ESI) Calcd. for C17H14NO2 (M+H)
+

= 264.1025, Found = 264.1023.
(E)-4-((2-Methyl-3-oxoisoindolin-1-ylidene)methyl)benzaldehyde (E-4h)

1
H NMR (400 MHz, CDCl3) δ 10.07 (s, 1H), 7.97 – 7.93 (m, 2H), 7.85 (dt, J = 7.5, 1.0, 1.0 Hz,
1H), 7.65 (dt, J = 8.1, 0.7, 0.7 Hz, 2H), 7.47 – 7.42 (m, 1H), 7.38 – 7.30 (m, 3H), 6.46 (s, 1H),
3.39 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 191.7 , 166.6 , 142.0 , 138.9 , 135.6 , 134.6 , 131.8 ,
130.8 , 130.4 , 130.1 , 129.9 , 123.5, 123.0, 108.6 , 26.3 . HRMS (ESI) Calcd. for C17H14NO2
(M+H)
+
= 264.1025, Found = 264.1023.
(E)-2-Methyl-3-(4-nitrobenzylidene)isoindolin-1-one (E-4i)

1
H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 8.8 Hz, 2H), 7.86 (dd, J = 7.6, 1.0 Hz, 1H), 7.65 (dd,
J = 9.0, 0.9 Hz, 2H), 7.50 – 7.45 (m, 1H), 7.39 – 7.31 (m, 2H), 6.43 (s, 1H), 3.40 (s, 3H).
13
C NMR
(100 MHz, CDCl3) δ 166.6 , 147.2 , 142.6 , 139.5 , 134.4 , 132.0 , 130.8 , 130.6 , 130.2 , 124.1 ,
123.7 , 122.9 , 107.3 , 26.4.

HRMS (ESI) Calcd. for C16H13N2O3 (M+H)
+
= 281.0926, Found =
281.0925.

33
(Z)-2-Methyl-3-(4-nitrobenzylidene)isoindolin-1-one (Z-4i)

1
H NMR (400 MHz, CDCl3) δ 8.26 (d, J = 8.7 Hz, 2H), 7.88 (dt, J = 7.5, 1.0, 1.0 Hz, 1H), 7.75
(dt, J = 7.7, 0.9, 0.9 Hz, 1H), 7.64 (td, J = 7.6, 7.5, 1.2 Hz, 1H), 7.55 (dd, J = 7.5, 1.0 Hz, 1H),
7.53 – 7.49 (m, 2H), 6.71 (s, 1H), 3.04 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 168.9 , 146.7 ,
142.0 , 138.5 , 137.6 , 132.3 , 130.5 , 129.8, 128.3 , 123.5 , 123.4 , 119.5 , 103.2 , 30.9 . HRMS
(ESI) Calcd. for C16H13N2O3 (M+H)
+
= 281.0926, Found = 281.0925.
(Z)-2-Methyl-3-(4-(trifluoromethoxy)benzylidene)isoindolin-1-one (4j)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.6, 1.0 Hz, 1H), 7.73 (dt, J = 7.8, 0.9 Hz, 1H), 7.60
(td, J = 7.5, 1.2 Hz, 1H), 7.50 (td, J = 7.5, 1.0 Hz, 1H), 7.39 – 7.34 (m, 2H), 7.26 – 7.22 (m, 2H),
6.70 (s, 1H), 3.03 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 148.5 , 137.9 , 137.0 , 133.6 ,
132.1 , 131.2 , 129.4 , 128.5 , 123.4 , 120.7 , 120.6 (q, J = 257.5 Hz), 119.4 , 104.7 , 30.7 .
19
F
NMR (376 MHz, CDCl3) δ -58.3. HRMS (ESI) Calcd. for C17H13NO2F3 (M+H)
+
= 320.0898,
Found = 320.0897.
(Z)-4-((2-Methyl-3-oxoisoindolin-1-ylidene)methyl)benzonitrile (Z-4k)

1
H NMR (400 MHz, CDCl3) δ 7.86 (dt, J = 7.5, 1.1 Hz, 1H), 7.74 (dt, J = 7.8, 0.9 Hz, 1H), 7.70
– 7.66 (m, 2H), 7.62 (td, J = 7.4, 1.2 Hz, 1H), 7.53 (td, J = 7.4, 1.0 Hz, 1H), 7.48 – 7.43 (m, 2H),
6.68 (s, 1H), 3.02 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.1 , 140.1 , 138.2 , 137.8 , 132.4 ,
34
132.0 , 130.5 , 129.8 , 128.5 , 123.6 , 119.6 , 118.8 , 111.1 , 103.8 , 31.0 . HRMS (ESI) Calcd. for
C17H13N2O (M+H)
+
= 261.1028, Found = 261.1027.
(E)-4-((2-Methyl-3-oxoisoindolin-1-ylidene)methyl)benzonitrile (E-4k)

1
H NMR (400 MHz, CDCl3) δ 7.86 (dt, J = 7.5, 1.0 Hz, 1H), 7.77 – 7.69 (m, 2H), 7.61 – 7.56 (m,
2H), 7.47 (td, J = 7.5, 1.1 Hz, 1H), 7.36 (td, J = 7.4, 1.2 Hz, 1H), 7.29 (dt, J = 7.9, 0.9 Hz, 1H),
6.41 (s, 1H), 3.38 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 166.6 , 140.5 , 139.2 , 134.4 , 132.6 ,
131.9 , 130.8 , 130.5 , 130.1 , 123.7 , 122.8 , 118.8 , 111.4 , 107.8 , 26.3 . HRMS (ESI) Calcd. for
C17H13N2O (M+H)
+
= 261.1028, Found = 261.1027.
(Z)-3-(3-(Difluoromethoxy)benzylidene)-2-methylisoindolin-1-one (4l)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.8, 0.9 Hz, 1H), 7.73 (dt, J = 7.7, 0.8 Hz, 1H), 7.60
(td, J = 7.6, 1.2 Hz, 1H), 7.50 (td, J = 7.5, 0.9 Hz, 1H), 7.38 (t, J = 7.9, 7.9 Hz, 1H), 7.22 – 7.17
(m, 1H), 7.12 (s, 1H), 7.11 – 7.06 (m, 1H), 6.70 (s, 1H), 6.56 (t, J = 73.6 Hz, 1H), 3.04 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 150.8 , 137.9 , 137.1 , 136.9 , 132.1 , 129.6 , 129.4 , 128.6
, 127.0 , 123.4 , 121.0 , 119.4 , 118.7 , 115.8 (t, J = 261.0 Hz), 104.9 , 30.7 .
19
F NMR (376 MHz,
CDCl3) δ -81.4 (d, J = 73.6 Hz). HRMS (ESI) Calcd. for C17H14NO2F2 (M+H)
+
= 302.0993, Found
= 302.0995.

35
(Z)-3-(4-Chloro-2-methoxybenzylidene)-2-methylisoindolin-1-one (4m)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.7, 1.0 Hz, 1H), 7.76 (dt, J = 7.6, 0.9 Hz, 1H), 7.59
(td, J = 7.6, 1.1 Hz, 1H), 7.49 (td, J = 7.5, 0.9 Hz, 1H), 7.27 (dd, J = 8.8, 2.5 Hz, 1H), 7.21 (dd, J
= 2.6, 0.9 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.58 (s, 1H), 3.85 (s, 3H), 3.06 (s, 3H).
13
C NMR
(100 MHz, CDCl3) δ 169.0 , 156.2 , 138.0 , 137.5 , 132.0 , 131.2 , 129.2 , 128.8 , 128.6 , 125.4 ,
125.2 , 123.3 , 119.7 , 111.6 , 101.2 , 55.9 , 30.2 . HRMS (ESI) Calcd. for C17H15NO2Cl (M+H)
+

= 300.0791, Found = 300.0791.
(Z)-3-(2-Amino-4-(trifluoromethyl)benzylidene)-2-methylisoindolin-1-one (4n)

1
H NMR (400 MHz, CDCl3) δ 7.87 (dt, J = 7.5, 1.0 Hz, 1H), 7.77 (dt, J = 7.7, 0.9 Hz, 1H), 7.62
(td, J = 7.6, 1.2 Hz, 1H), 7.53 (td, J = 7.5, 1.0 Hz, 1H), 7.42 – 7.37 (m, 2H), 6.77 (d, J = 8.3 Hz,
1H), 6.44 (s, 1H), 4.14 (s, 2H), 3.04 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ 168.6 , 147.8 , 138.9
, 137.3 , 132.2 , 129.6 , 128.7 , 128.2 (q, J = 3.8 Hz), 126.2 (q, J = 3.8 Hz), 124.7 (q, J = 270.8
Hz), 123.4 , 119.9 (q, J = 33.1 Hz), 119.6 , 119.4 , 114.4 , 100.2 , 29.2 .
19
F NMR (376 MHz,
CDCl3) δ -61.8 . HRMS (ESI) Calcd. for C17H14N2OF3 (M+H)
+
= 319.1058, Found = 319.1057.
(Z)-3-Benzylidene-2-methylisoindolin-1-one (4o)

36
1
H NMR (500 MHz, CDCl3) δ 7.86 (dt, J = 7.5, 1.0 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.60 (td, J
= 7.5, 1.2 Hz, 1H), 7.49 (td, J = 7.5, 0.9 Hz, 1H), 7.42 – 7.37 (m, 2H), 7.37 – 7.30 (m, 3H), 6.79
(s, 1H), 3.04 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.1 , 138.1 , 136.3 , 134.9 , 132.0 , 129.8
, 129.1 , 128.6 , 128.2 , 127.6 , 123.3 , 119.4 , 106.7 , 30.7. This data corresponds to the previously
reported structure.
(Z)-3-(4-Ethylbenzylidene)-2-methylisoindolin-1-one (4p)

1
H NMR (500 MHz, CDCl3) δ 7.85 (dt, J = 7.7, 0.9 Hz, 1H), 7.73 (dt, J = 7.7, 0.8 Hz, 1H), 7.58
(td, J = 7.6, 1.1 Hz, 1H), 7.48 (td, J = 7.5, 0.9 Hz, 1H), 7.29 – 7.19 (m, 4H), 6.76 (s, 1H), 3.06 (s,
3H), 2.69 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.1 ,
143.8 , 138.2 , 135.9 , 132.1 , 131.9 , 129.8 , 129.0 , 128.6 , 127.7 , 123.2 , 119.3 , 106.9 , 30.7 ,
28.8 , 15.6 . HRMS (ESI) Calcd. for C18H18NO (M+H)
+
= 264.1388 Found = 264.1398
(Z)-2-Methyl-3-(4-methylbenzylidene)isoindolin-1-one (4q)

1
H NMR (300 MHz, CDCl3) δ 7.84 (dt, J = 7.4, 1.0 Hz, 1H), 7.71 (dt, J = 7.6, 0.9 Hz, 1H), 7.57
(td, J = 7.7, 1.2 Hz, 1H), 7.46 (td, J = 7.4, 1.0 Hz, 1H), 7.25 – 7.14 (m, 4H), 6.74 (s, 1H), 3.05 (s,
3H), 2.38 (s, 3H).
13
C NMR (75 MHz, CDCl3) δ 168.9 , 138.1 , 137.3 , 135.8 , 133.9 , 133.7 ,
131.8 , 129.6 , 128.8 , 128.4 , 123.1 , 119.2 , 106.7 , 30.6 , 21.3 . HRMS (ESI) Calcd. for C17H16NO
(M+H)
+
= 250.1232, Found= 250.1232.

37
(Z)-3-(4-Methoxybenzylidene)isoindolin-1-one (4r)

1
H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.88 (dt, J = 7.6, 0.9 Hz, 1H), 7.77 (dt, J = 7.8, 0.8
Hz, 1H), 7.63 (td, J = 7.8, 1.1 Hz, 1H), 7.50 (td, J = 7.6, 1.0 Hz, 1H), 7.42 – 7.35 (m, 2H), 7.04 –
6.92 (m, 2H), 6.52 (s, 1H), 3.86 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.0 , 159.3 , 138.4 ,
132.3 , 131.8 , 130.8 , 129.9 , 129.0 , 127.6 , 123.7 , 119.8 , 114.9 , 106.0 , 55.6 . These values are
in agreement to those of the previously reported authentic compound.
(Z)-3-(3-Aminobenzylidene)-2-methylisoindolin-1-one (4s)

1
H NMR (400 MHz, DMSO-D6) δ 8.03 (d, J = 7.8 Hz, 1H), 7.74 (dt, J = 7.5, 0.7 Hz, 1H), 7.68
(td, J = 7.7, 1.2 Hz, 1H), 7.54 (td, J = 7.8, 0.7 Hz, 1H), 7.05 (t, J = 7.7 Hz, 1H), 6.94 (s, 1H), 6.60
– 6.57 (m, 1H), 6.56 – 6.52 (m, 2H), 5.16 (s, 2H), 3.01 (s, 3H).
13
C NMR (126 MHz, CDCl3) δ
169.0 , 146.2 , 138.2 , 136.1 , 135.9 , 131.9 , 129.2 , 129.0 , 128.6 , 123.2 , 120.3 , 119.3 , 116.1 ,
114.4 , 107.0 , 30.5 . HRMS (ESI) Calcd. for C16H15N2O (M+H)
+
= 251.1184, Found = 251.1186.
(Z)-3-(4-Fluorobenzylidene)-2-methylisoindolin-1-one (4t)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dt, J = 7.6, 0.6 Hz, 1H), 7.72 (dt, J = 7.7, 0.8 Hz, 1H), 7.59
(td, J = 7.8, 0.6 Hz, 1H), 7.49 (td, J = 7.4, 0.8 Hz, 1H), 7.34 – 7.28 (m, 2H), 7.12 – 7.05 (m, 2H),
6.71 (s, 1H), 3.02 (s, 1H).
19
F NMR (376 MHz, CDCl3) δ -114.4 – -114.6 (m).
13
C NMR (100
MHz, CDCl3) δ 169.0 , 162.2 (d, J = 247.6 Hz), 138.0 , 136.6 , 132.1 , 131.4 (d, J = 8.0 Hz), 130.8
38
(d, J = 3.5 Hz), 129.2 , 128.6 , 123.3 , 119.3 , 115.3 (d, J = 21.6 Hz), 105.4 , 30.7 . HRMS (ESI)
Calcd. for C16H13NOF (M+H)
+
= 254.0981, Found = 254.0977.
(Z)-3-(4-Bromobenzylidene)-2-methylisoindolin-1-one (4u)

1
H NMR (400 MHz, CDCl3) δ 7.85 (dd, J = 7.6, 1.1 Hz, 1H), 7.72 (dd, J = 7.7, 0.7 Hz, 1H), 7.60
(t, J = 7.6 Hz, 1H), 7.54 – 7.47 (m, 3H), 7.22 (d, J = 8.6 Hz, 2H), 6.65 (s, 1H), 3.03 (s, 4H).
13
C
NMR (100 MHz, CDCl3) δ 169.0 , 138.0 , 136.9 , 133.9 , 132.1 , 131.4 , 129.3 , 128.5 , 123.4 ,
121.7 , 119.4 , 105.0 , 30.8 (ipso carbon not seen). HRMS (ESI) Calcd. for C16H13NOBr (M+H)
+

= 314.0181, Found = 314.0176.
(Z)-3-(3,5-Difluorobenzylidene)-2-methylisoindolin-1-one (4v)

1
H NMR (400 MHz, CDCl3) δ 7.86 (dt, J = 7.5, 0.9, 1H), 7.71 (dt, J = 7.7, 0.9 Hz, 1H), 7.61 (td,
J = 7.5, 1.2 Hz, 1H), 7.52 (td, J = 7.4, 1.0 Hz, 1H), 6.91 – 6.83 (m, 2H), 6.78 (tt, J = 9.0, 2.3 Hz,
1H), 6.61 (s, 1H), 3.06 (s, 3H).
19
F NMR (376 MHz, CDCl3) δ -110.2 (ddd, J = 8.4, 6.4, 1.4 Hz).

13
C NMR (100 MHz, CDCl3) δ 169.0 , 162.7 (dd, J = 249.5, 13.2 Hz), 138.3 (t, J = 9.9 Hz), 137.8
, 137.7, 132.3 , 129.7 , 128.6 , 123.5 , 119.5 , 113.3 – 112.2 (m), 103.5 (t, J = 2.6 Hz), 103.1 (t, J
= 25.4, Hz), 30.6. HRMS (ESI) Calcd. for C16H12NOF2 (M+H)
+
= 272.0887, Found = 272.0889.
(Z)-2-Benzyl-3-benzylideneisoindolin-1-one (4w)

39
1
H NMR (400 MHz, CDCl3) δ 7.94 (dd, J = 7.5, 1.0 Hz, 1H), 7.75 (dd, J = 7.7, 1.0 Hz, 1H), 7.63
(td, J = 7.7, 1.1 Hz, 1H), 7.53 (td, J = 7.4, 1.0 Hz, 1H), 7.29 – 7.22 (m, 3H), 7.10 – 7.02 (m, 5H),
6.72 (s, 1H), 6.53 (dd, J = 7.0, 1.3 Hz, 2H), 4.94 (s, 2H).
13
C NMR (100 MHz, CDCl3) δ 169.2 ,
138.6 , 136.9 , 134.7 , 134.5 , 132.2 , 129.8 , 129.2 , 128.2 , 128.1 , 128.0 , 127.5 , 126.8 , 126.5 ,
123.7 , 119.6 , 107.7 , 45.0 . These values are in agreement to those of the previously reported
authentic compound.
(Z)-3-(Cyclohex-1-en-1-ylmethylene)-2-methylisoindolin-1-one (4x)

1
H NMR (400 MHz, CDCl3) δ 7.80 (dt, J = 7.4, 0.9, 0.9 Hz, 1H), 7.61 (dt, J = 7.4, 0.9, 0.9 Hz,
1H), 7.52 (td, J= 7.6, 7.5, 1.1 Hz, 1H), 7.42 (td, J = 7.4, 7.4, 1.0 Hz, 1H), 6.03 (s, 1H), 5.73 – 5.68
(m, 1H), 3.32 (s, 3H), 2.22 – 2.16 (m, 2H), 2.16 – 2.12 (m, 2H), 1.76 – 1.68 (m, 2H), 1.68 – 1.60
(m, 2H).
13
C NMR (100 MHz, CDCl3) δ 168.8 , 138.3 , 134.7 , 132.1 , 131.7 , 129.0 , 128.6 , 128.5
, 123.1 , 119.1 , 110.1 , 30.1 , 29.8 , 25.6 , 22.7 , 22.1 . HRMS (ESI) Calcd. for C16H18NO (M+H)
+

= 240.1388, Found = 240.1385.
(Z)-3-(Cyclopropylmethylene)-2-methylisoindolin-1-one (4y)

1
H NMR (400 MHz, CDCl3) δ 7.81 (dt, J = 7.5, 1.0 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.40 (td, J =
7.4, 1.5 Hz, 1H), 5.08 (d, J = 9.9 Hz, 1H), 3.63 (s, 3H), 2.12 (dtt, J = 9.7, 8.0, 4.7 Hz, 1H), 1.04 –
0.98 (m, 2H), 0.65 – 0.60 (m, 2H).
13
C NMR (100 MHz, CDCl3) δ 167.8 , 137.5 , 134.9 , 131.5 ,
128.1 , 128.0 , 123.1 , 118.5 , 113.6 , 29.5 , 9.6 , 9.2 . HRMS (ESI) Calcd. for C13H14NO (M+H)
+

= 200.1075, Found = 200.1078.

40
(S, Z)-3-Benzylidene-2-(2-(methoxymethyl)pyrrolidin-1-yl)isoindolin-1-one, 4z

1
H NMR (400 MHz, DMSO-d6) δ 8.05 (dd, J = 7.7, 1.0 Hz, 1H), 7.75 – 7.67 (m, 2H), 7.56 (td, J
= 7.5, 1.1 Hz, 1H), 7.49 – 7.44 (m, 2H), 7.34 (dd, J = 7.5, 1.6 Hz, 2H), 7.30 – 7.23 (m, 1H), 6.95
(s, 1H), 3.71 (p, J = 6.8 Hz, 1H), 3.59 (q, J = 8.0 Hz, 1H), 3.27 – 3.20 (m, 1H), 2.90 (s, 3H), 2.82
– 2.76 (m, 1H), 2.58 – 2.52 (m, 1H), 1.96 – 1.85 (m, 1H), 1.85 – 1.77 (m, 1H), 1.52 – 1.40 (m,
1H), 1.15 – 1.04 (m, 1H).
13
C NMR (100 MHz, DMSO-d6) δ 166.2 , 136.6 , 135.4 , 134.3 , 132.9
, 130.3 , 129.5 , 127.4 , 127.2 , 127.1 , 122.6 , 120.6 , 108.3 , 75.2 , 60.0 , 58.4 , 51.4 , 27.7 , 22.2
. HRMS (ESI) Calcd. for C21H23N2O2 (M+H)
+
= 335.1760, Found = 335.1754.
(Z)-2-Methyl-3-(pyridin-2-ylmethylene)isoindolin-1-one (5a)

1
H NMR (400 MHz, CDCl3) δ 8.67 (d, J = 4.8 Hz, 0H), 7.85 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 8.5
Hz, 1H), 7.71 (td, J = 7.7, 1.8 Hz, 1H), 7.60 (td, J = 7.7, 0.6 Hz, 1H), 7.51 (d, J = 7.4 Hz, 1H),
7.39 (d, J = 7.8 Hz, 1H), 7.23 – 7.16 (m, 1H), 6.72 (s, 1H), 3.23 (s, 3H).
13
C NMR (100 MHz,
CDCl3) δ 169.1 , 154.2 , 149.4 , 138.4 , 138.3 , 136.1 , 132.1 , 129.6 , 128.7 , 125.3 , 123.4 , 121.9
, 119.6 , 105.2 , 31.0 HRMS (ESI) Calcd. for C15H13N2O (M+H)
+
= 237.1028, Found = 237.1029.
(Z)-2-Methyl-3-(pyridin-3-ylmethylene)isoindolin-1-one (5b)

N
O
N
OMe
H
H
41
1
H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.57 (s, 1H), 7.86 (dt, J = 7.5, 1.0 Hz, 1H), 7.75 (dt,
J = 7.6, 0.8 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.62 (td, J = 7.7, 1.2 Hz, 1H), 7.52 (td, J = 7.5, 1.0
Hz, 1H), 7.34 (dd, J = 7.8, 4.7 Hz, 1H), 6.66 (s, 1H), 3.04 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ
168.9 , 150.5 , 148.6 , 138.0 , 137.8 , 136.8 , 132.3 , 131.1 , 129.6 , 128.6 , 123.5 , 123.1 , 119.5 ,
101.9 , 30.8 .HRMS (ESI) Calcd. for C15H13N2O (M+H)
+
= 237.1028, Found = 237.1028.
(Z)-2-Methyl-3-(thiophen-2-ylmethylene)isoindolin-1-one (5c)

1
H NMR (400 MHz, CDCl3) δ 7.8 (dt, J = 7.5, 1.1 Hz, 1H), 7.7 (dt, J = 7.8, 0.9 Hz, 1H), 7.6 (td,
J = 7.4, 1.1 Hz, 1H), 7.5 (td, J = 7.5, 1.0 Hz, 1H), 7.4 (dd, J = 5.1, 1.2 Hz, 1H), 7.1 – 7.0 (m, 1H),
7.0 (dt, J = 3.5, 1.3 Hz, 1H), 6.7 (s, 1H), 3.2 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 168.8 , 137.8
, 137.3 , 136.4 , 132.1 , 129.3 , 128.9, 128.4 , 127.3 , 126.6 , 123.4 , 119.4 , 98.6 , 30.2 . HRMS
(ESI) Calcd. for C14H12NOS (M+H)
+
= 242.0640, Found = 242.0639.
(Z)-3-(Furan-3-ylmethylene)-2-methylisoindolin-1-one (5d)

1
H NMR (400 MHz, CDCl3) δ 7.83 (dt, J = 7.8, 1.2 Hz, 1H), 7.69 (dt, J = 7.8, 0.9 Hz, 1H), 7.57
(td, J = 7.8, 1.2 Hz, 1H), 7.50 – 7.44 (m, 3H), 6.46 (dd, J = 1.3, 0.7 Hz, 1H), 6.41 (s, 1H), 3.25 (s,
3H).
13
C NMR (100 MHz, CDCl3) δ 168.7 , 143.2 , 138.0 , 136.8 , 131.9 , 129.0 , 128.4 , 123.3 ,
119.2 , 119.0 , 112.5 , 96.6 , 30.1. HRMS (ESI) Calcd. for C14H12NO2 (M+H)
+
= 226.0868, Found
= 226.0867.

42
(Z)-2-Methyl-3-((1-methyl-1H-imidazol-5-yl)methylene)isoindolin-1-one (5e)

1
H NMR (400 MHz, CDCl3) δ 7.77 (dt, J = 7.3, 1.1, 1.1 Hz, 1H), 7.69 (dt, J = 7.7, 0.9, 0.9 Hz,
1H), 7.54 (td, J = 7.7, 7.5, 1.2 Hz, 1H), 7.50 (s, 1H), 7.44 (td, J = 7.5, 7.4, 1.0 Hz, 1H), 7.02 (s,
1H), 6.21 (s, 1H), 3.61 (s, 3H), 3.11 (s, 3H).
13
C NMR (100 MHz, CDCl3) δ 168.5 , 138.6 , 137.3
, 132.0 , 130.9 , 129.5 , 128.3 , 125.8 , 123.3 , 122.6 , 119.3 , 91.4 , 32.0 , 29.5. HRMS (ESI)
Calcd. for C14H14N3O (M+H)
+
= 240.1137, Found = 240.1141.
(Z)-3-((1H-Indol-5-yl)methylene)-2-methylisoindolin-1-one (5f)

1
H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.86 (dd, J = 7.6, 0.9 Hz, 1H), 7.77 (dd, J = 7.8, 0.9
Hz, 1H), 7.62 – 7.56 (m, 2H), 7.47 (tt, J = 7.5, 7.5, 0.9, 0.9 Hz, 1H), 7.41 (dd, J = 8.3, 0.9 Hz, 1H),
7.28 – 7.25 (m, 1H), 7.19 – 7.14 (m, 1H), 6.97 (d, J = 0.9 Hz, 1H), 6.57 (td, J = 2.1, 2.1, 1.0 Hz,
1H), 3.07 (d, J = 0.7 Hz, 3H).
13
C NMR (100 MHz, CDCl3) δ 169.3 , 138.4 , 135.3 , 135.3 , 131.8
, 128.7 , 128.5 , 127.8 , 126.2 , 125.3 , 124.1 , 123.2 , 122.1 , 119.3 , 110.9 , 108.8 , 102.9 , 30.8 .
HRMS (ESI) Calcd. for C18H15N2O (M+H)
+
= 275.1184, Found = 275.1186.
1.5 REFERENCES
1. Speck, K.; Magauer, T., Beilstein J. Org. Chem. 2013, (9), 2048-2078.
2. Blasko, G.; Gula, D. J.; Shamma, M., J. Nat. Prod. 1982, (45), 105-122.
3. Choomuenwai, V.; Beattie, K. D.; Healy, P. C.; Andres, K. T.; Fechner, N.; Davis, R.
A., Phytochemistry 2015, (117), 10-16.
4. Ziegler, J.; Facchini, P. J., Annu. Rev. Plant Biol. 2008, (59), 735-769.
5. Scherlach, K.; Schuemann, J.; Dahse, H.-M.; Hertweck, C., J. Antibiot. 2010, (63), 375-
377.
6. Chen, S.; Liu, Z.; Liu, Y.; Lu, Y.; He, L.; She, Z., Beilstein J. Org. Chem. 2015, (11),
1187-1193.
7. Linden, L.; Hadler, D.; Hofmann, S., Psycopharmacol. 1997, (12), 445-452.
8. Zhuang, Z.-P.; Kung, M.-P.; Mu, M.; Kung, H. F., J. Med. Chem. 1998, (41), 157-166.
43
9. Norman, M. H.; Minick, D. J.; Rigdon, G. C., J. Med. Chem. 1998, (39), 149-157.
10. Chem. Abstr. 1984, (101), 54922.
11. Japanese Patent 5,946,268,1984.
12. Pendrak, I.; Barney, S.; Wittrock, R.; Lambert, D. M.; Kingsbury, W. D., J. Org. Chrm.
1994, (59), 2623.
13. De Clercq, E., J. Med. Chem. 1995, (38), 2491.
14. Taylor, E. C.; Zhou, P.; Jennings, L. D.; Mao, Z.; Hu, B.; Jun, J.-G., Tetrahedron Lett.
1997, (38), 521.
15. Riedinger, C.; Endicott, J. A.; Kemp, S. J.; Smyth, L. A.; Watson, A.; Valeur, E.;
Golding, B. T.; Griffin, R. J.; Hardcastle, I. R.; Noble, M. E.; McDonnell, J. M., J. Am. Chem.
Soc. 2008, (130), 16038.
16. Bohm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-Sander,
U.; Stahl, M., Fluorine in Medicinal Chemistry. ChemBioChem 2004, (5).
17. Hardcastle, I. R.; Liu, J.; Valeur, E.; Watson, A.; Ahmed, S. U.; Blackburn, T. J.;
Bennaceur, K.; Clegg, W.; Drummond, C.; Endicott, J. A.; Golding, B. T.; Griffin, R. J.; Gruber,
J.; Haggerty, K.; Harrington, R. W.; Hutton, C.; Kemp, S.; Lu, X.; McDonnell, J. M.; Newell,
D. R.; Noble, M. E. M.; Payne, S. L.; Revill, C. J.; Riedinger, C.; Xu, Q.; Lunec, J., J. Med.
Chem. 2011, (54), 1233-1243.
18. Balogh, J.; Victor III, D.; Asham, E. H.; Burroughs, S. G.; Boktour, M.; Saharia, A.;
Li, X.; Ghobrial, R. M.; Monsour, J., H. P., J. Hepatocell. Carcinoma 2016, (3), 41-53.
19. Chhabra, N.; Aseri, M. L.; Padmanabhan, D., Int. J. Appl. Basic Med. Res. 2013, (3), 16-
18.
20. Peter, H.; Flitsch, W., Tetrahedron Lett. 1969, (15), 1161-1162.
21. Wang, E.-C.; Chen, H.-F.; Feng, P.-K.; Lin, Y.-L.; Hsu, M.-K., Tetrahedron Lett. 2002,
(43), 9163-9165.
22. Kato, Y.; Takemoto, M.; Achiwa, K., Chem. Pharm. Bull. 1993, (41), 2003-2006.
23. Freccero, M.; Fasani, E.; Albini, A., J. Org. Chem. 1998, (58), 1740-1745.
24. Sun, C.; Xu, B., J. Org. Chem. 2008, (73), 7361-7364.
25. Kondo, Y.; Shinga, F.; Murata, N.; Sakamoto, T.; Tamanaka, H., Tetrahedron 1994,
(50), 11803-11812.
26. Marosvolgyi-Hasko, D.; Takacs, A.; Riedl, Z.; Kollar, L., Tetrahedron 2011, (67), 1036-
1040.
27. Fujioka, M.; Morimoto, T.; Tsumagari, T.; Tanimoto, H.; Nishiyama, Y.; Kakiuchi, K.,
J. Org. Chem. 2012, (77), 2911-2923.
28. Mancuso, R.; Ziccarelli, I.; Armentano, D.; Marino, N.; Giofre, S. V.; Gabriele, B., J.
Org. Chem. 2014, (79), 3506-3518.
29. Xu, Y.; Hu, w.; Tang, X.; Zhao, J.; Wu, W.; Jiang, H., Chem. Commun. (Camb). 2015,
(51), 6843-6846.
30. Cao, H.; Mcnamee, L.; Alper, H., Org. Lett. 2008, (10), 5281-5284.
31. Koseki, Y.; Kusano, S.; Nagasaka, T., Tetrahedron Lett. 1998, (39), 3517-3520.
32. Koradin, C.; Dohle, W.; Rodriquez, A. L.; Schmid, B.; Knochel, P., Tetrahedron 2003,
(59), 1571-1587.
33. Bianchi, G.; Chiarini, M.; Marinelli, F.; Rossi, L.; Arcadi, A., Adv. Synth. Catal. 2010,
(352), 136-142.
34. Hu, J.; Liu, L.; Wang, X.; Hu, Y.; Yang, S.; Liang, Y., Green Sustain. Chem. 2011, (01),
165-169.
35. Li, D. Y.; Shi, K. J.; Mao, X. F.; Le Zhao, Z.; Wu, X. Y.; Liu, P. N., Tetrahedron 2014,
(70), 7022-7031.
44
36. Khan, M. W.; Kundu, N. G., Synlett. 1997, 1435-1437.
37. Kundu, N. G.; Khan, M. W., Tetrahedron 2000, (56), 4777-4792.
38. Hellal, M.; Cuny, G. D., Tetrahedron Lett. 2011, (52), 5508-5511.
39. Sarkar, S.; Dutta, S.; Dey, R.; Naskar, S., Tetrahedron Lett. 2012, (53), 6789-6792.
40. Pan, J.; Xu, Z.; Zeng, R.; Zou, J., Chinese J. Chem. 2013, (31), 1022-1026.
41. Dong, I. J.; Wang, F.; You, J., Org. Lett. 2014, (16), 2884-2887.
42. Li, L.; Wang, M.; Zhang, X.; Jiang, Y.; Ma, D., Org. Lett. 2009, (11), 1309-1312.
43. Hatanaka, Y.; Hiyama, T., J. Org. Chem. 1988, (53), 918-920.
44. Hatanaka, Y.; Matsui, K.; Hiyama, T., Tetrahedron Lett. 1989, (30), 2403-2406.
45. Denmark, S. E.; Wehrli, D., Org. Lett. 2000, (2), 565-568.
46. Denmark, S. E.; Sweis, R. F., Acc. Chem. Res. 2002, (35), 835-846.
47. Denmark, S. E.; Tymonko, S. A., J. Org. Chem. 2003, (68), 9151-9154.
48. Denmark, S. E.; Neuville, L.; Christy, M. E. L.; a Tymonko, S., J. Org. Chem. 2006, (71),
8500-8509.
49. E., D. S.; Baird, J. D., Chem. Eur. J. 2006, (12), 4954-4963.
50. Chang, S.; Yang, S. H.; Lee, P. H., Tetrahedron Lett. 2001, (42), 4833-4835.
51. Alacid, E.; Najera, C., J. Org. Chem. 2008, (73), 2315-2322.
52. Hatanaka, Y.; Hiyama, T., Synlett. 1991, 845-853.
53. Nakao, Y.; Hiyama, T., Chem. Soc. Rev. 2011, (40), 4893-4901.
54. Plenio, H., Angew. Chem. Int. Ed. Engl. 2008, (47), 6954-6956.
55. Takahashi, S.; Kuroyama, Y.; Sonogashira, K.; Hagihara, N., Synthesis (Stuttg).
1980 627.
56. Erdelyi, M.; Gogoll, A., J. Org. Chem. 2001, (66), 4165-4169.
57. Mohamed Ahmed, M. S.; Sekiguchi, A.; Masui, K.; Mori, A., Bull. Chem. Soc. Jpn. 2005,
(78), 160-168.
58. Nishihara, Y.; Ikegashira, K.; Mori, A.; Hiyama, T., Chem. Lett. 1997, 1233-1234.
59. Chinchilla, R.; Najera, C., Chem. Rev. 2007, (107), 874-922.
60. Negishi, E.; Anastasia, L., Chem. Rev. 2003, (103), 1979-2017.
61. Clarke, P. A.; Santos, S.; Martin, W. H. C., Green Chem. 2007, (9), 438-440.
62. Nishihara, Y.; Ikegashira, K.; Mori, A.; Hiyama, T., Tetrahedron Lett. 1998, (39), 4075-
4078.
63. Nishihara, Y.; Ikegashira, K.; Hirabayashi, K.; Ando, J.; Mori, A.; Hiyama, T., J. Org.
Chem. 2000, (65), 1780-1787.
64. Kabalka, G. W.; Wang, L.; Pagni, R. M., Tetrahedron 2001, (57), 8017-8028.
65. Antilla, J. C.; Klapars, A.; Buchwald, S. L., J. Am. Chem. Soc. 2002, (124), 11684-11688.
2 IMPROVED SYNTHESIS AND PURIFICATION OF BIOISOSTERIC
DIFLUOROMETHYLATED NUCLEOTIDES
2.1 INTRODUCTION
2.1.1 NUCLEOTIDES AND ENZYMATIC PHOSPHORYLATION
Protein function is regulated by enzymatic phosphorylation moreso than any other
modification.
1, 2
The addition and removal of phosphate groups by protein kinases and
45
phosphatases, respectively, impact protein conformations and control virtually all types of cell
signaling.
3, 4
In DNA and RNA replication, phosphorylation is responsible for the assembly of the
sugar-phosphate backbone. Given that cancers and viruses arise from malfunctioning or
overexpressed protein kinases,
3
as well as uncontrollable growth (replication),
5
phosphorus
containing molecules have been given substantial attention as drug candidates.
Nucleotides, specifically, have been a leading family of small molecules exploited as drug
candidates. Composed of a sugar ring, one of four purine and pyrimidine bases, and a series of
phosphate groups, a nucleotide links to others in a chain to form nucleic acids.
6
Modifications to
the base, sugar ring, or phosphate group(s) lead to changes in charge and conformation (ring pucker
and torsion angles),
6
impacting interactions with enzymes and proteins.
7
Analogues inhibit
replication by directly interfering with the initiation or process of replication, by affecting
checkpoint responses accountable for repairing drug-induced damage.
8

Many nucleotide drug analogues are ineffective due to challenges in drug delivery.
9

Analogues’ instabilities leave them prone to dephosphorylation in the bloodstream and prevent
them from crossing membranes due to charges on the phosphate(s).
10, 11
These problems are
circumvented using prodrug technology, which refers to lipophilic, biologically inactive
precursors to drugs which are activated after administration by enzymatic or chemical processes.
12

Because triphosphate analogues have more greater instability and polarity to overcome,
monophosphate analogues have been superior attention. Monophosphate analogues “mask” one or
both charged oxygens to improve lipophilicity, and bypass the first phosphorylation process which
has been acknowledged as the limiting step (Figure 1).
13-16

46

Figure 2: depiction of the mechanism of nucleoside/nucleotide drugs versus
modified prodrugs.
ProTide technology, pioneered by Prof. Chris McGuigan,
10, 17
is the best-known approach
in the field currently. The approach masks both charged oxygens on a monophosphate nucleotide
(ProTides) using a phosphoramidate and a substituted phenol group, which are later metabolized
to give the active monophosphate.
10, 11
Successful nucleotide prodrugs have demonstrated
anticancer and antiviral activities.
18-20
Some examples of FDA-approved nucleotide prodrugs are
tenofovir DF and sofosbuvir.
13

2.1.2 FLUORINE AND BIOISOSTERES: IMPROVING DRUG DELIVERY
Despite advances in prodrug technology, the literature lacks the use of fluorine in
phosphate modifications. Fluorine, a small atom with a big attitude, is known to have significant
impact on physiochemical properties. The pharmaceutical industry has seen an explosion of
fluorine drug analogues (20-30% of all pharmaceuticals)
21
and synthetic methodology since the
first approved fluorinated drug in 1955.
22-27
Fluorine molecules uniquely behave as bioisosteres,
having comparable size, shape, and distribution of electrons to other molecules such that they
would be recognized similarly by biological systems.
28-31
Sterically, the van der Waals radius of
47
fluorine (1.35 A) is similar to that of hydrogen (1.2 A) and oxygen (1.4 A) (Figure 2). Substituting
fluorine for hydrogen causes insignificant steric impact on active site compatibility or enzymatic
processes. Of particular interest is the difluoromethyl (CF2H) group, which is an isosteric and
isopolar group to oxygen and hydroxyl group. The C-F bond length (1.39 A) is close to that of the
C-O bond length (1.43 A), and the two fluorine atoms mimic the positioning and electronegativity
of the oxygen lone pairs (Figure 2). Due to its high bond dissociation energy, the C-F bond poses
little threat in the formation of radical species during metabolism.

Figure 3: fluorine’s bioisosteric properties.
While the difluoromethyl group has been utilized as a bioisostere of bridging oxygens in
di- and tri-phosphates,
32, 33
to the best of our knowledge, it has not been employed as a neutral
48
mimetic of the hydroxyl group of the preferred monophosphate prodrug derivatives. Incorporation
of a difluoromethylphosphonate should serve to: 1) eliminate one charge, thus increasing
lipophilicity; 2) retain one charge to increase solubility in water (a constant challenge); 3) increase
hydrolytic stability; 4) stabilize adjacent functional groups;
25-27, 34, 35
and 5) potentially improve
cytotoxicity and antiviral activity. With these improvements, CF2H-modified analogues could
demonstrate enhanced the bioavailability, requiring lower dosage.
34, 35
The modification can be
extended to existing FDA-approved prodrugs
13, 36-38
such as gemcitabine and sofosbuvir, or to
other potential prodrug scaffolds, to determine changes in efficacy.

Figure 4: difluoromethyl mimetic of a phosphate group.
This work builds from previous efforts by colleagues in our laboratory to develop a library
of CF2H-modified monophosphate nucleotide prodrugs. Existing methods were adapted to
incorporate the CF2H moiety into a variety of nucleosides ranging from unaltered purine and
pyrimidine bases, to existing FDA-approved drugs such as gemcitabine. While previous efforts
made substantial progress, significant challenges arose in the purification and isolation of the
products. This work continues efforts to identify and resolve synthetic challenges to better isolate
pure products for biological testing.
49
2.2 RESULTS AND DISCUSSION
2.2.1 ASSESSMENT OF PREVIOUS METHODS AND RESULTS
To begin, gemcitabine was chosen as the substrate of interest due to its success as an FDA-
approved antiviral and anticancer drug.
36-38
The methods employed in previous work in our
laboratory were continued initially to reproduce results for further assessment.
39
To generate the
phosphorylating reagent (Scheme 1), difluoromethanephosphonyl dichloride,
diethyldifluoromethyl phosphonate was treated with TMS-Br for four days under an inert
atmosphere. After all volatiles were removed and without further purification, the silyl ester was
reacted with oxalyl chloride and catalytic amount of DMF for three days. Though a high yield was
expected, initial attempts at this synthesis was problematic due to the difficult removal of volatiles
between steps and due to high sensitivity to moisture. Subsequent attempts improved the yield to
38%, but no higher and without decent purity.

Scheme 1: synthesis of difluoromenthanephosphonyl dichloride.
To synthesize the nucleotide analogues, the prepared difluoromethanephosphonyl
dichloride was utilized as a phosphorylating reagent under reaction conditions adapted from the
preparation of phosphoramidates (Scheme 1).
10, 17, 40
Gemcitabine was treated with t-BuMgCl as
base and reacted with the dichloride under inert atmosphere. After stirring for 24 hours,
tetrabutylammonium hydroxide in MeOH was added to quench the reaction. The crude product
was purified using reverse phase flash chromatography with a 10% MeOH/H20 solvent system.
50
Initial results were not promising and suffered from poor separation, unreacted starting material,
and/or hydrolyzed product.

Scheme 2: preparation of difluoromethanephosphonyl dichloride.
After repeated reactions under these conditions, many issues were identified that
contributed to low yield and poor separation. While THF was freshly distilled, the potential for
unwanted moisture existed if the nucleoside was left untreated and if glassware was not adequately
flame dried. Excess base likely deprotonated both 3’ and 5’ hydroxyl groups of gemcitabine. The
purity and rate of addition of the difluoromethylphosphonyl dichloride at room temperature led to
unwanted side products and phosphorylation at both the 3’ and 5’ positions of the unprotected
nucleoside. An excess of tetrabutyl- or tributylammonium hydroxide created sticky residues which
caused separation and purification issues, though addition of too little hydroxide would fail to form
the salt. The slight variations in loading, solvent gradient, and collection during reverse phase
chromatogrphy dramatically impacted the abiity to separate crude mixtures.
After careful evaluation of
1
H,
19
F, and
31
P NMR spectra from previous efforts on this
project, it is evident that, though the product is present, there are signs of contamination from
unreacted nucleoside or hydrolyzed product, as well as byproducts formed during the reaction.
Due to poor separation and closely related peak signatures of components in the mixture, it is
difficult to make clear identifications. Alterations to the method were made in the expectation that
fewer complications during the reaction would lead to more successful separation.

51
2.2.2 ALTERNATIVE SYNTHESIS OF DICHLORIDE
As an alternative approach to the preparation of the difluoromethanephosphonyl
dichloride, thionyl chloride was added in excess to diethyldifluoromethyl phosphonate and
pyridine and left to reflux for three days (Scheme 2).
41
Excess thionyl chloride was removed by
fractional distillation resulting in 66% yield of the dichloride. Subsequent fractional distillations
can be performed to improve purity, but at the loss of yield.

Scheme 3: preparation of difluoromethanephosphonyl dichloride by reflux with thionyl chloride.
This method of preparation is more convenient as it has fewer steps and reagents, and it
reaches completion over the course of just three days as compared to seven. It is also easier to
ensure higher purity through fractional distillation and by observation of color and odor of the
dichloride should there be contamination from thionyl chloride. It is extremely difficult to detect
contamination from oxalyl chloride in the previous method, which increases the probability of the
formation of side products. The dichloride can be prepared in larger quantities, purified, and stored
in a Schlenk flask in a desiccator for use in subsequent reactions.
2.2.3 ALTERNATIVE SYNTHESIS OF MONOPHOSPHATE NUCLEOTIDE ANALOGUES
To address the abovementioned shortcomings of the given nucleotide synthesis, the
following modifications were made: 1) nucleoside starting materials were lyophilized to remove
the presence of moisture; 2) glassware was flame dried under vacuum or oven dried overnight; 3)
t-BuMgCl was titrated
42
and added dropwise to deprotonate the 5’ hydroxyl group only; 4) the
52
purified difluoromethanphosphonyl dichloride was added dropwise in a dry ice bath to control
phosphorylation at the 5’ position; 5) in amount directly related to equivalents of dichloride,
tributylammonium hydroxide in H2O/THF was added at 0°C to quench reaction to avoid
contamination and separation issues; 6) during reverse phase chromatography, solvent gradients
were made very gradual over time and fractions were collected in small test tubes to reduces
chances of contamination from neighboring or overlapping peaks.

Scheme 4: modified synthetic method for monophosphate nucleotide analogues.
These modifications resulted in vastly improved separations of five or more peaks, which
allowed for distinctions between nucleoside starting material, phosphate derivatives,
contamination from tributylammonium salts, and other byproducts. There were still samples
remaining with similar characteristics, so a determination was needed to firmly identify the
products.
2.2.4 IDENTIFICATION OF PURE PRODUCTS
Knowing that the present method can phosphorylate the nucleoside at two positions, it was
assumed that both could be present in the reaction mixture. The samples identified as pure product,
whether 3’ or 5’, lacked noticeable contamination and demonstrated proper proton integration. The
best way to identify 3’ and 5’ nucleotides is by multiplicity in
31
P NMR. The splitting patterns
differ by position (Figure 4): while the phosphorus atom is split at both positions by the
53
difluoromethyl moiety, it is split by two methylene hydrogens at the 5’ (giving a tdt) versus a
single hydrogen at the 3’ position (giving a tdd). This information is corroborated by HRMS. After
successful isolation of these products, comparison to previous data showed slight variations in
reported chemical shifts and multiplicity, as these were difficult to determine with contamination
of overlapping peaks. While the 5’ and 3’ gemcitabine analogues can be identified as pure, the
yields are grievously low, 1.8% and 2.5%, respectively. This is a drastic difference compared to
previous results, but it simply emphasizes the underlying problems in the synthetic and purification
methods.

Figure 5:
31
P NMR of 3' analogue (top);
31
P NMR of 5' analogue (bottom).
54
2.2.5 CHALLENGES IN ISOLATION AND PURIFICATION
Despite some improvement in separation, the complexity of the reaction mixture remains
problematic. The modifications made to the given method did not resolve issues of substantial side
products or loss of production on the column. Gemcitabine’s structural properties make it
considerably harder to implement in this method. Not only can the glyosidic bond sever, but it has
two vulnerable hydroxyl groups and.a powerful gem-difluoro moiety. The base can lead to other
side products in the reaction and, as previously mentioned, the unprotected 3’ hydroxyl group may
also be phosphorylated as opposed to the preferred 5’ position. The gem-difluoro moiety is very
interesting in that it affects polarity and ring pucker,
6, 43
thus the conformation of the molecule
throughout synthesis and purification. The 3’ position may be favored in this case because the ring
pucker makes it more accessible.

Scheme 5: preparation of 3'deoxy-2'3'-didehydrothymidine analogue.
To determine how much of an impact the choice of substrate has on the success of this
method, a nucleoside lacking fluorine substitution or hydroxyl group was chosen for the next
experiments. The 3’-deoxy-2’,3’-didehydrothymidine analogue was isolated in 15% yield without
any further optimization (Scheme 4). Given that each nucleoside has its own structural challenges,
this synthetic method will not be as effective for all substrates. With fewer structural variables,
there was less contamination from side products in the reaction, increasing yield and simplifying
purification. The reaction was repeated and successfully gave the more challenging
55
tetrabutylammonium salt in similar yield, though lower purity. These results indicate that the
current method could be applied to a number of simple nucleoside substrates.
For more structurally interesting nucleosides, other methods will have to be employed to
generate enough product for biological testing. Samples should be above 97% purity, though the
required amounts are on the scale of mere milligrams. The aim of this work is not, at this stage,
optimization of reaction conditions to increase product yields. The synthetic methods used simply
need to produce enough product, less than 10mg, to run biological tests for toxicity and potency.
While the current method produces about 5-20mg of product based on these substrates, this meager
amount leads to difficulties in reproducibility, especially by purification with flash
chromatography, and does not leave opportunity for
13
C NMR studies. Such small amounts of
product are also easy to lose through transfers, so it is more practical to pursue methods which will
produce modest yields.
In the interest of simplifying the synthesis of these compounds, methods for unprotected
nucleosides were first investigated. Based on the literature, a well-known successful method for
preferential phosphorylation at the 5’ position introduced by Yoshikawa and coworkers
44
may be
the best option. The reaction is a simple addition of POCl3 to the nucleoside in trimethyl phosphate
at 5°C. After two hours stirring at room temperature, the reaction is complete. This method has
been employed for a number of different nucleosides including gemcitabine,
45-47
and could be
utilized for the work presented here by substituting the difluoromethanephosphonyl dichloride for
POCl3. This method may be the most convenient direction for synthesis as it has only one step,
few reagents, and short reaction times. The use of trimethyl phosphate, however, can be
discouraging. It is postulated that the success of this method could be the superior solubility of
nucleosides in trimethyl phosphate, or a more active ionized formed of the phosphorylating reagent
due to interaction with trimethylphosphate.
48
Should this option fail, methods with protecting
56
groups will be necessary in the case of gemcitabine. For other nucleosides, different synthetic
methods may need to be applied for each unique case.
The last consideration for this work is the use of reverse phase flash chromatography. This
method is simply inadequate. In most cases, nucleotide monophosphates are purified by HPLC. In
addition to adapting synthetic methods to desired substrates, the use of HPLC for purification will
be implemented in future work, which should considerably resolve separation issues.
2.3 CONCLUSIONS
Presented here is an evaluation of synthetic and purification challenges facing the
development of potential difluoromethylphosphonate nucleotide prodrugs. After successive
attempts using existing methods, modifications were introduced to limit the production of side
products and promote conversion of product. These modifications successfully improved isolation
of pure gemcitabine and 3’-deoxy’2’3’-didehydrothymidine analogues identified by proton
integration, phosphorus multiplicity, and HRMS. The method was shown to be more suitable for
simpler nucleosides without competing hydroxyl groups or substitution on the sugar ring. While
the yields were extremely low (below 20%), for the purpose of this work the amount of product
produced (between 5-20mg) is adequate in the interest of biological testing. The purity and
reproducibility, however, are still not reliable with the current methods. It has been determined
that the implementation of trimenthylphosphate could significantly improve the synthesis of
unprotected nucleosides. Based on the literature and previous work in our laboratory, HPLC has
been proven to be a superior method of purification and will be likely be used over flash
chromatography for future experiments. The efforts presented here should enable the completion
of a small library of difluoromethylphosphonate nucleotide prodrugs with adequate purity for
biological testing to assess the expected improvements made by the difluoromethyl mimetic.
57
2.4 EXPERIMENTAL
2.4.1 GENERAL PREPARATION OF DIFLUOROMETHYLPHOSPHONATE ANALOGUES
Nucleosides were lyophilized overnight. Dried nucleoside (0.5mmol) was added to dried
round bottom flask under nitrogen or argon and secured by septum. Using syringe, 7.5mL freshly
distilled THF was added. Titrated t-BuMgCl (1.2 equiv.) was added dropwise and the reaction was
left to stir at room temperature for 30 minutes. Previously prepared difluoromethanephosphonyl
dichloride (2 equiv.) was added dropwise at -78°C over 30 minutes using syringe-pump. The
reaction was allowed to warm to room temperature and was left to stir overnight. Reaction was
quenched with tributylammonium hydroxide (2 equiv. in respect to dichloride) in H2O/MeOH at
0°C over 10 minutes. Crude product was purified by reverse phase flash chromatography using a
gradient of 20% MeOH/H20.
2.5 REPRESENTATIVE NMR AND MASS SPECTROMETRY DATA
2.5.1
2’DEOXY-2’,2’-DIFLUOROCYTIDINE-3’-DIFLUOROMETHYLPHOSPHONATE

1
H NMR (399 MHz, Methanol-d4) δ 7.67 (dd, J = 7.7, 1.1 Hz, 1H), 6.33 (t, J = 8.7 Hz, 1H), 5.98 (d, J = 7.7 Hz, 1H),
5.86 (td, J = 49.4, 24.0 Hz, 1H), 4.32 (ddd, J = 7.3, 4.5, 2.9 Hz, 1H), 4.07 (dd, J = 12.6, 2.9 Hz, 1H), 3.98 (dd, J =
12.6, 4.7 Hz, 1H), 3.16 – 3.07 (m, 5H), 1.76 – 1.60 (m, 5H), 1.42 (dq, J = 14.8, 7.4 Hz, 5H), 1.00 (t, J = 7.4 Hz, 7H).
19
F NMR (376 MHz, Methanol-d4) δ -115.32 – -118.23 (m), -135.36 (ddd, J = 81.0, 49.5, 6.4 Hz).
31
P NMR (162 MHz, Methanol-d4) δ 1.51 (tdd, J = 81.0, 23.9, 9.1 Hz).
58

59

60
2.5.2
2’DEOXY-2’,2’-DIFLUOROCYTIDINE-5’-DIFLUOROMETHYLPHOSPHONATE

1
H NMR (399 MHz, Methanol-d4) δ 7.89 (d, J = 7.6 Hz, 2H), 6.24 (d, J = 15.2 Hz, 1H), 5.98 (d, J = 6.0 Hz, 2H), 5.88
(td, J = 49.5, 23.8 Hz, 3H), 4.41 – 4.28 (m, 4H), 4.24 (ddd, J = 12.2, 5.9, 2.5 Hz, 2H), 3.99 (d, J = 8.4 Hz, 2H), 3.16
– 3.08 (m, 6H), 1.74 – 1.62 (m, 6H), 1.42 (h, J = 7.3 Hz, 6H), 1.00 (t, J = 7.4 Hz, 8H).
19
F NMR (376 MHz, Methanol-d4) δ -118.91 – -120.62 (m), -135.16 (dd, J = 78.9, 49.4 Hz).
31
P NMR (162 MHz, Methanol-d4) δ 2.27 (tdt, J = 78.5, 24.1, 5.0 Hz).
HRMS: ESI, Negative mode. Calculated as 376.03, found 376.0312 (100%).

61

62

2.5.3
3’-DEOXY-2’,3’-DIDEHYDRO-THYMIDINE-5’-DIFLUOROMETHYLPHOSPHONATE

1
H NMR (399 MHz, Methanol-d4) δ 7.65 (d, J = 1.3 Hz, 1H), 6.95 (dt, J = 3.4, 1.6 Hz, 1H), 6.43 (dt, J = 6.1, 1.7 Hz,
1H), 5.91 (ddd, J = 5.8, 2.4, 1.4 Hz, 1H), 5.80 (td, 1H), 4.96 (tt, J = 3.8, 1.5 Hz, 1H), 4.25 (ddd, J = 11.7, 6.6, 3.1 Hz,
1H), 4.12 (ddd, J = 11.8, 5.5, 2.9 Hz, 1H), 3.20 – 3.04 (m, 6H), 1.89 (d, J = 1.2 Hz, 4H), 1.77 – 1.59 (m, 7H), 1.42 (h,
J = 7.4 Hz, 7H), 1.00 (t, J = 7.4 Hz, 9H).
19
F NMR (376 MHz, Methanol-d4) δ -134.84 (ddd, J = 78.5, 49.4, 5.9 Hz).
31
P NMR (162 MHz, Methanol-d4) δ 1.83 (td, J = 78.6, 23.5 Hz).
HRMS: ESI, Negative mode. Calculated as 337.04, found 337.0387 (100%).
63

64

2.6 REFERENCES
1. Nestler, E. J.; Greengard, P., Protein phosphorylation is of fundamental importance in
biological regulation. In Basic Nurochemistry: Molecular, Cellular, and Medical Aspects, 6 ed.;
Siegle, G. J.; Agranoff, B. W.; al., e., Eds. Lippincott-Raven: Philadelphia, 1999.
2. Voet, D.; Voet, J. G., Biochemistry. 2 ed.; John Wiley and Sons: New York, 1995.
3. Ardito, F.; Giuliani, M.; Perrone, D.; Troiano, G.; Muzio, L. L., Int. J. Mol. Med. 2017,
(40), 271-280.
4. Gumina, G.; Chong, Y.; Choo, H.; Song, G.; Chu, C. K., Curr. Topics Med. Chem. 2002,
(2).
5. White, M. K.; Pagano, J. S.; Khalili, K., Clin. Microbio. Rev. 2014, (27), 463-481.
6. Roy, B.; Depaix, A.; Perigaud, C.; Peyrottes, S., Chem. Rev. 2016, (116), 7854-7897.
7. Jordheim, L. P.; Durantel, D.; Zoulin, F.; Dumontet, C., Nat. Rev. Drug. Disc. 2013,
(12), 447-464.
8. Pommier, Y.; Diasio, R. B., Pharmacological and therapeutics agents that target DNA
replication. In DNA Replication in Eukaryotic Cells, 2 ed.; DePamphilis, M. L., Ed. Cold spring
Harbor Press.
9. Jones, R. J.; Bischofberger, N., Antivir. Res. 1995, (27), 1-17.
10. Mehellou, Y.; Valente, R.; Mottram, H.; Walsby, E.; Mills, K. I.; Balzarini, J.;
McGuigan, C., Med. Chem. Lett. 2010, (18), 2439.
11. Mehellou, Y.; Rattan, H. S.; Balzarini, J., J. med. Chem. 2018, (61), 2211-2226.
12. Abu-jaish, A.; Jumaa, S.; Karaman, R., Prodrugs Overview. In Prodrugs Design - A New
Era, Karaman, R., Ed. Nova Publisher: USA, 2014.
65
13. Pradere, U.; Garnier-Amblard, E. C.; Coats, S. J.; Amblard, F.; Schinazi, R. F., Chem.
Rev. 2014, (114), 9154-9218.
14. Krise, J. P.; Stella, V. J., Adv. Drug Deliv. Rev. 1996, (19), 287.
15. Zemlicka, J., Biochim. Biophys. Acta-Mol. Basis Dis. 2002, (1587), 276.
16. de Schutter, C.; Ehteshami, M.; Hammond, E. T.; Amblard, F.; Schinazi, R. F., Curr.
Pharm. Des. 2017, (23), 6984-7002.
17. McGuigan, C.; Madela, K.; Aljarah, M.; Gilles, A.; Brancale, A.; Zonta, N.;
Chamberlain, S.; Vernachio, J.; Hutchins, J.; Hall, A.; Ames, B.; Gorovits, E.; Ganguly, B.;
Kolykhalov, A.; Wang, J.; Muhammad, J.; Patti, J. M.; Henson, G., Bioorg. Med. Chem. Lett.
2010, (20), 4850.
18. De Clercq, E.; Fields, H. J., Br. J. Pharmacol. 2006, (147), 1.
19. Orr, D. C.; Figueiredo, H. T.; Mo, C. L.; Penn, C. R.; Cameron, J. M., J. Biol. Chem.
1992, (267), 4177.
20. Anderson, K. S., Biochim. Biomys. Acta-Mol. Basis Dis. 2002, (1587), 296.
21. Taylor, E. C.; Zhou, P.; Jennings, L. D.; Mao, Z.; Hu, B.; Jun, J.-G., Tetrahedron Lett.
1997, (38), 521.
22. Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Acena, J. L.; Soloshonok, V. A.; Izawa,
K.; Liu, H., Chem. Rev. 2016, (116), 422-518.
23. Tozer, M. J.; Herpin, T. F., Tetrahedron 1996, (52), 8619.
24. Liu, P.; Sharon, A.; Chu, C. K., J. Fluor. Chem. 2008, (129), 743.
25. Welch, J. T., Tetrahedron 1987, (43), 3123.
26. Purrington, S. T.; Kagen, B. S.; Patrick, T. B., Chem. Rev. 1986, (86), 997.
27. Olah, G. A.; Chambers, R. D.; Prakash, G. K. S., Synthetic Fluorine Chemistry. John Wiley
and Sons: New York, 1992.
28. Burger, A., Prog Drug Res 1991, (37), 287-371.
29. Bohm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-Sander,
U.; Stahl, M., ChemBioChem 2004, (5).
30. Patani, G. A.; LaVoie, E. J., Chem. Rev. 1996, (96), 3147-3176.
31. Bondi, A., J. Phys. Chem. 1964, (68), 441.
32. Prakash, G. K. S.; Zibinsky, M.; Upton, T. G.; Kashemirov, B. A.; McKenna, C. E.;
Oertell, K.; Goodman, M. F.; Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Shock, D. D.; Wilson,
S. H.; Olah, G. A., PNAS 2010, (107), 15693-15698.
33. Shakhmin, A.; Jones, J.-P.; Bychinskaya, I.; Zibinsky, M.; Oertell, K.; Goodman, M.
F.; Prakash, G. K. S., J. Fluor. Chem. 2014, (167), 226-230.
34. Lin, G.-Q.; You, Q.-D.; Cheng, J.-F., Chiral Drugs: Chemistry and Biology Action. John
Wiley and Sons: 2011.
35. Ojima, I., Fluorine in Medicinal Chemistry and Chemical Biology. John Wiley and Sons:
2009.
36. Goodman; Gilman, The Pharmacological Basis of Therapeutics. 10 ed.; MaGraw Hill:
2001.
37. Clinical Oncology. 3 ed.; Elsevier: 2003.
38. Cancer Chemotherapy and Biotherapy, Principles and Practice. Lippincott and Wilkins:
2001.
39. Shakhmin, A. Synthesis of novel nucleotide analogues based on the traditional and
nontraditional bioisosteres. University of Southern California, California, 2014.
40. Zhang, H.-W.; Zhou, L.; Coats, S. J.; McBrayer, T. R.; Tharnish, P. M.; Bondada, L.;
Detorio, M.; Amichai, S. A.; Johns, M. D.; Whitaker, T.; Schianzi, R. F., Bioorg. Med. Chem.
Lett. 2011, (21), 6788.
66
41. Bergstrom, D. E.; Shum, P. W., J. Org. Chem. 1988, (53), 3953-3958.
42. Love, B. E.; Jones, E. G., J. Org. Chem. 1999, (64), 3755-3756.
43. Dolain, C.; Patwa, A.; Godeau, G.; Barthelemy, P., Appl. Sci. 2012, (2), 245-259.
44. Yoshikawa, M.; Kato, T.; Takenishi, T., Terahedron Lett. 1967, (50), 5065-5068.
45. Cappellacci, L.; Franchetti, P.; Vita, P.; Petrelli, R.; Grifantini, M., Nucleosides,
Nucleotides, and Nucleic Acids 2008, (27), 460-468.
46. Jansen, R. S.; Rosing, H.; Schellens, J. H. M.; Beijnen, J. H., Nucleosides, Nucleotides,
and Nucleic Acids 2010, (29), 14-26.
47. Risbood, P. A.; Kane Jr., C. T.; Hossain, M. T.; Vadapalli, S.; Chadda, S. K., Bioorg.
Med. Chem. Lett. 2008, (18), 2957-2958.
48. Yoshikawa, M.; Kato, T.; Takenishi, T., Bull. Chem. Soc. Japan 1969, (42), 3505-3508.

67
3 PROPOSED ORGANOTELLURANES FOR CANCER THERAPY
3.1 INTRODUCTION

In the interest of pursuing a new direction in the search for accessible cytotoxic and
antiviral drugs, the lesser known metalloid Tellurium became a focus for this work (Figure 1).
What was immediately obvious was an overwhelmingly negative reputation that makes
approaching Tellurium as anything other than a poison exceptionally challenging. Upon further
research, a few dedicated research laboratories have slowly investigated the biological effects of
tellurium-based drugs, which have been demonstrated to outperform some existing drugs.
1-6
A
recent revival in the exploration of tellurium’s biological effects motivated this work to focus on
the lesser known, but considerably more stable
8, 9
organotelluranes as possible cancer therapeutics.

Figure 6: tellurium.
68
3.1.1 TELLURIUM’S ABSURDLY TOXIC REPUTATION
Tellurium has long been regarded as a rare, toxic, non-essential trace element having no
role in biology. Since its discovery in 1782, it has historically been used in industry, mainly for
metallurgy and ore refinery, and has continued to gain popularity in materials science for
applications in electronic memory storage and semiconductors.
10, 11
Despite routine use of and
exposure to tellurium, literature on its toxicology is sparse and often contradictory. The majority
of toxicity claims reference specific reports of acute toxicity to the central nervous system
12
and
inactivation of human squalene monoogygenase.
13
Little discussion follows these citations
detailing the mechanism of toxicity.

Ironically, the popularity selenium gained in biological applications stemmed from the
same reputation. Tellurium comes from the same group in the periodic table as selenium and sulfur,
and all share similarities. Selenium has similar toxicity to tellurium, though not as potent.
14
The
same author who published on tellurium’s inhibition of squalene monooxygenase, published
similar results for selenium a year later.
15
Both selenium and tellurium have the ability to mimic
glutathione peroxidase, essential to cellular defense against oxidative damage; both can directly
affect sensitive thiol groups in cysteines, leading to inactivation of important proteins and
Figure 7: comparison of toxic Se and Te metabolites.
69
enzymes.
14
Comparison of the toxic selenium and tellurium compounds reveals structural
similarities, specifically of methylated metabolites and dichalcogenides (Figure 2). The toxicity of
these compounds relates to unstable bonds to the chalcogen, as well as hydrophobicity and size of
the compounds in relation to active site environments.
1
In short, there is no general toxicity of
tellurium. Chemical species, organism species, concentration, and route of exposure contribute to
highly variable toxicities of tellurium compounds.
1, 14
It is remarkable that this is an accepted fact
for selenium and other elements, but is ignored for tellurium. For instance, arsenic is famous for
its toxicity and use as a poison, but it is accepted as Trisenox, a commercial drug which treats
leukemia.
16, 17
Yet, tellurium’s absurd reputation continues to overshadow its potential in medicine.
3.1.2 THE “FORGOTTEN” ELEMENT
In stark comparison to the misplaced apprehension in recent literature, early 20
th
century
reports illustrate a fairer, more optimistic approach to tellurium. After tellurium’s discovery in
1782, tellurium was not recognized as an element until 16 years later and virtually nothing was
known about its toxicity. Following advances in the electro-refinement of ores, tellurium became
a much more prominent issue in industry as a natural impurity. Investigations into lead poisoning
revealed unrelated symptoms among workers, such as a strong garlic-like odor, dry skin and an
inability to sweat, which were soon attributed to tellurium poisoning.
18
This is of no surprise,
owing to shared traits with selenium and sulfur, which are also known to produce garlic-like
odors.
19
The most common routes of exposure are reported as inhalation and absorption through
the skin.
The 1920 report notes that most symptoms disappear within a few days, except for the garlic-
like odor which can last up to a few months. Though laboratory experiments resulted in severe
tellurium poisoning, the authors emphasize that there had been no severe cases in the workforce.
The authors insist that tellurium may have therapeutic benefits, but that the intolerable garlic odor
70
and metallic taste would not merit use. The authors also include an interesting side note: early
experiments at the time showed evidence that tellurium effectively targets malignant growths by
destroying tumor nuclei.
18

Another 1920 report acknowledges that tellurium and selenium had not been given enough
attention, considering their similarities to biologically important sulfur. This report also mentions
tellurium’s potential to treat cancer and other diseases, but that data was limited and questionable.
The greatest deterrence, again, is the strong garlic odor tied to methyl telluride, which the author
insists should be overlooked for potential applications.
20
A Chemical Education article in 1931
mentions tellurium’s potential as a drug for long-term, treatment-resistant illness, and that its
success hinged on the tolerance for, or attraction to, a strong garlic odor.
21
These reports are only
a few representative samples reflecting the attitude toward tellurium at the time.
With such historical accounts in mind, it is no wonder that tellurium has been labeled as
“forgotten” in modern research.
1
Selenium was also considered toxic and displayed the same
symptoms of poisoning as tellurium, but greater efforts were made during the early 20
th
century to
understand its toxicity.
22
When selenium’s importance as a nutrient was discovered in the 1950s,
its wider role in biology was extensively explored. Tellurium lacked any role in biology and was
not met with the same interest, despite the optimism of early reports. Thankfully, the last 30 years
have seen a healthy growth in the development of biologically active tellurium compounds. The
best-known success is compound AS-101, an inorganic tellurane experimental drug.
23
First
developed in the 1980s, AS-101 has excelled in phase I and II clinical trials as an antitumoral
agent.
7
It has since been extensively explored for other applications, especially for inflammatory
diseases.
23
Inorganic telluranes are often given greater consideration because they are ionic and do
not pose the same risk as organotelluranes in the aforementioned toxicity reports. Realistically,
organotelluranes with more stable ligands are preferable because they are less likely to be
71
metabolized into biologically available compounds. Inorganic telluranes are more likely to
hydrolyze, form toxic metabolites, and may also cross the blood-brain barrier.
3.1.3 NEW INVESTIGATIONS INTO TELLURIUM’S BIOLOGICAL EFFECTS
Recently, Tellurium’s role in biology has been marked as an emerging research area by the
Royal Society of Chemistry.
1
Tellurium compounds have new applications as biomarkers,
antioxidants, antibiotics, and anticancer agents, among others.
2, 3, 6, 7, 11, 23, 24
In nearly all cases,
tellurium compounds exceed their selenium counterparts in effectiveness and often have lower
toxicity. In cancer therapy applications, tellurium compounds are redox modulators which
demonstrate a powerful “sensor/effector”
5
ability for naturally high levels of reactive oxygen
species (ROS) in cancer cells. In this sense, tellurium compounds have chemical selectivity and
heightened reactivity toward ROS, resulting in selective toxicity toward cancer cells (Figure 3).

Figure 8: concept of "sensor/effector” drug design.
72

Tellurium also exhibits strong reactivity with thiol groups, which are abundantly present
in cell membranes and biologically important enzymes and proteins.
1, 6, 9
Tellurium compounds
with labile ligands undergo ligand exchange with thiol groups, most commonly cysteine thiols,
that form stable complexes or that result in oxidation of the thiol, leading to programmed cell
death. Though it is suggested that tellurium inhibitors may have a higher affinity for seleno-
cysteine,
1
no study has been conducted to explore this possibility (Scheme 1). With these
properties, tellurium compounds can be utilized as more effective redox modulators and inhibitors
than existing selenium counterparts.
There is a consensus in the literature that tellurium compounds have great potential as
anticancer agents, but there is little research available yet on their biological behaviors. There are
two prevalent mechanistic proposals (Figure 4): Te(II) compounds behave as antioxidants,
contributing to cell death through a catalytic cycle that mimics the natural glutathione process; in
other cases, organotelluranes lead to programmed cell death by inhibiting key components of
enzymes and proteins, which cause changes in cell signaling.
1
Though tellurium (II) compounds
are decently reported and studied,
1, 9
far less is known about organotelluranes. A rational approach
to the design of novel organotelluranes is largely absent in the field, and biological assays vary
across existing studies. There is not enough comparable data in the literature to draw conclusions
Scheme: 1 mechanism of inorganic tellurane inhibitors on cystienes and seleno-cysteins.
1, 7

73
or to determine how best to advance the potential of new organotelluranes for cancer therapy.
Proposed is a SAR study aimed to: (1) provide data on basic structural correlations to Te
(IV) reactivity in colon cancer cells; (2) evaluate whether Te (IV) can act in one or more capacities;
(3) determine relative toxicity. A core selection of nine Te (IV) compounds will be used, first with
variations in leaving groups and second with variations in p-substituted aryl ligands (Table 1).

Evaluation will be two-fold: model NMR studies with two cysteines, as well as the lesser
acknowledged seleno-cysteine, will determine the inhibitory capacity of the compounds; a series
of biological assays will be pursued through collaborations to determine inhibitory activity and
inactivation kinetics, impacts on ROS levels, and relative toxicity. The use of DFT calculations
will assist in predicting ground state geometries, lability of ligands, and NMR shifts. In addition
to the core selection of compounds, a small series of compounds with other structural aspects of
interest will be explored for future expansion of this study (Table 2).

Figure 9: comparison of natural glutathione process to Te activity.
74

Table 1: core organotelluranes for SAR study.

Table 2: additional scaffolds of interest for SAR study.

75
3.2 DISCUSSION AND ANTICIPATED RESULTS
3.2.1 STAGE ONE: DIARYL TELLURANES
The core nine organotelluranes were designed with existing tellurium (II) studies in mind, as
well as two limited organotellurane studies and the dependence of toxicity on structural stability.
6,
9
Weak Te-Te and Te-C were avoided, and the number of reactive Te-O and Te-Hal bonds limited.
The more stable Te-Ar bonds, most common in tellurium (II) literature, were the focus of the non-
labile ligands. The sp
2
carbons of aryl ligands have greater s character and form stronger, polarized
bonds to tellurium.
25
Using structurally similar compounds to existing tellurium (II) and tellurium
(IV) literature will provide comparable data to draw conclusions for future studies.
While the two existing organotellurane SAR studies emphasize the necessity of the leaving
groups for inhibitory effects, only Br, Cl, OH, and OC(O)CH3 groups were included. The studies
draw the main conclusions: 1) the labile ligands of tellurium (IV) compounds are necessary for
inhibitory effects compared to ineffective tellurium (II);
6
2) of the leaving groups mentioned, Br
is the most effective;
9
3) achiral compounds performed better than chiral compounds.
9
Only one
study investigated in vitro and in vivo toxicity, which indicate the most inhibitory organotellurane
was selectively toxic in cancer cells and non-toxic to the host.
6

It is of interest to compare Br to I and oxide as leaving groups. Iodine is closer to tellurium in
size, but is less electronegative and more ionizable than Br. Tellurium oxides have been given
more attention in the literature since an aqueous solution study on AS-101 revealed that the drug
immediately hydrolyzes once it enters the body,
7
[cite] but tellurium oxides have not been studied
in comparison to traditional leaving groups. It is worth mentioning that tellurium, unlike selenium
and sulfur, does not readily form pi bonds, which are significantly less stable than single Te-O
bonds.
11
Tellurium also tends to form more highly coordinated complexes in trigonal bipyramidal
76
or octahedral geometries, which indicates that tellurium (IV) moieties may be more stable, less
toxic, and more effective drug candidates than better known tellurides.
Varying p-substituents on the aryl ligands is expected to affect the strength of the Te-C bond
and electrophilicity of the tellurium atom. The aryl ligands are typically treated as non-labile
ligands, but there is indication that these could also participate in ligand exchange.
6
Similar studies
claim that the number of labile ligands corresponds to the number of cysteines consumed, but
experimental procedures only asses for two equivalents of cysteine.
6, 7
No study includes
additional equivalents of cysteine to address the lability of ligands other than the leaving groups.
Selenocysteine, potentially more sensitive to tellurium inhibitors,
1
has not been assessed at all. A
recent aqueous solutions study of organotelluranes highlights the stability of organotelluranes
against hydrolysis.
26
The results demonstrate that the electronics of the ligands and intramolecular
interactions contribute to greater stability, in both basic and acidic conditions, as well as at high
temperatures. This suggests that organotelluranes are not easily hydrolyzed to unknown reactive
species, and that controlled design of organotelluranes for more potent inhibitors and antioxidants
is possible. The solutions study had a limited number of examples with little structural variation
and calls for additional, more detailed SAR studies as the one proposed here.
3.2.2 STAGE TWO: EXPLORING OTHER STRUCTURAL INTERESTS

The systematic approach described in Stage One will provide much needed data for the
rational design of new organotellurane drugs. It is still valuable, however, to include representative
samples with other key structural variants to determine the best pathways for future SAR studies.
Stage Two of this proposed study will look at some of the most notable structural aspects in
tellurium literature.
77
Aromatic tellurium heterocycles are considered similarly stable to diaryl tellurium
compounds, but the literature is almost devoid of examples.
1
Compounds 7, 8 and 11 incorporate
aromatic tellurium heterocycles in response. Aromatic tellurium (II) compounds may prove more
successful, because the addition of halogens to tellurium’s p orbitals alters geometry and disrupts
conjugation.
27

Intramolecular interactions are known to stabilize tellurium compounds and are expected
to lower toxicity. Stabilization occurs when an ortho-substituted nitrogen or oxygen group
contributes an electron pair to a non-bonding σ* orbital, most often from a Te-X bond, where
stability increases as the electronegativity of the halogen increases.
11, 28, 29
The non-bonding
interaction also leads to elongated Te-X bonds, facilitating nucleophilic attack on the tellurium
atom.
11, 28
Compounds 7, 9, and 11 incorporate such intramolecular interactions, but in varied
capacities. Though the oxygen in 7 is in the ortho position, its likelihood to participate is limited
by its position in the aromatic ring. Both nitrogens in 9 have the potential to participate in the non-
bonding orbitals of the two halogens. In compound 11, the carbonyls may contribute to the non-
bonding orbitals of the Te-C, but due to steric hindrance and carbon’s relatively low
electronegativity, the effect may be negligent. Minor stabilization may also occur due to
electrostatic effects or d-orbital participation.
29
It would be of interest to compare these compounds
with varied leaving groups to clearly demonstrate a trend.
As stated previously, tellurium tends not to form stable pi bonds, which makes telluroxides
particularly reactive. Telluroketones are similarly unstable, but scarcer, though some stable
derivatives have been made.
11
Compound 11 is a hypervalent telluroketone which may be
stabilized from some contribution from the aromatic ring and sp
2
carbons, as well as the lone pairs
on the neighboring carbonyls. Though it has been used as an intermediate step in organic
synthesis,
28
this compound has not been utilized for biological applications. It is worth comparing
78
the reactivity of the telluroketone to the telluroxides. If the bonds are stable, it would be interesting
if this compound does not at all participate as an inhibitor and instead only contributes to affecting
ROS levels directly.
In the literature, nitrogen is often discussed as having pronounced effects on tellurium
through intramolecular interactions, as previously stated. Its effect through direct bonds to
tellurium is not as general a topic, despite the common use of this structural aspect in the
literature.
11, 28
A study on glutathione peroxidase mimetics revealed that the strength of the Se-N
bonds of Ebselen derivatives was significant and dramatically lowered antioxidant activity.
30
[cite]
The authors emphasize that the direct Se-N gave the lowest antioxidant activity and highest
cytotoxicity. Tellurium is a larger atom and may have a longer, weaker bond to nitrogen, but
tellurium is also less electronegative than selenium and may have a more polarized bond with
nitrogen. It is of interest to see if the trend observed for reported selenium compounds is
comparable to tellurium compounds. Compounds 8 and 12 incorporate direct Te-N bonds, with
sp
2
and sp
3
hybridizations, respectively.
Compound 10 is a derivate of an existing Te (II) compound that has been attributed to
having significant redox acitivity.
4
The quinone group is given more credit for contributing to the
generation of radicals than the tellurium atom in most cases. The tellurium is supposed to
simultaneously participate in a redox cycle which affects cysteines, resulting in a twice-deadly
anti-cancer drug design. By studying the Te (IV) derivative, the results may be compared to the
reactivity of the Te (IV) compounds lacking a quinone group, as well as to the Te (II) parent. It is
expected that the Te (IV) derivative will demonstrate greater inhibitory properties, while
maintaining its potent radical production from the quinone. The proximity of the carbonyl may
also enhance stability and reactivity.
79
The most interesting of this series is compound 12. The structure is derived from a
successful colon cancer drug, Oxaliplatin. This drug operates by way of affecting DNA in the cells,
which also makes them considerably toxic to patients.
31
Oxaliplatin’s structure if applied to
tellurium chemistry could make for a potentially effective cysteine inhibitor. The oxalate would
act as a good labile ligand, while the amine ligand provides stabilization. Interestingly, neither the
Te (II) or Te (IV) derivatives have been made. The Te (II) compound could potentially have greater
inhibitory effects than traditional Te (II) compounds. The Te (IV) derivative would be interesting
because it could have up to four potential labile ligands, but remain a more stable tellurium
compound because of the stability from the amine ligand. It may be, because the sp
3
nitrogens are
more loosely coordinated to the tellurium, rather than participating in a strong direct bond, that all
ligands are potentially labile in this compound. What could be concerning about this compound is
its geometry. Because organotelluranes tend to adopt specific geometries, this compound would
most likely form a trigonal bipyramidal structure, weakening the bonds to both the oxalate and
amine ligands. Preparing an alternative to 12 is beneficial to investigate for comparison. In place
of the sp
3
amines, more rigid and electronegative sp
2
amines will be used to strengthen the bonds
to tellurium as well as to affect the overall geometry of the molecule.
3.2.3 DFT PREDICTED GEOMETRIES AND NMR SHIFTS
There are several advantages to studying tellurium compounds. Tellurium has a natural
abundance of 7%, more favorable than 1% abundance of carbon, and has wide chemical shifts
associated with its chemical environment.
1
Organic tellurides fall between 0 and -1000 ppm, while
organic telluranes fall between 0 and +1000; inorganic telluranes, such as AS-101, are further
downfield (Figure 5).
32

80

Figure 5: Typical NMR shifts of Te compounds.
32

Figure 6: DFT calculated ground state geometries of 12.
81
Many of the compounds in this study have not been synthesized or biologically tested.
Hartre-Fock and DFT calculations may be used to predict NMR shifts and optimized geometries
(Figure 6). This is particularly useful for discerning between Te (II) and Te (IV) compounds during
different stages of synthesis and during NMR model studies with cysteines. Other DFT studies
may reveal the potential lability of ligands based on bond length and orbital overlap.
3.2.4 MODEL NMR STUDY WITH L-CYSTEINE AND SELENO-CYSTEINE
The proposed NMR study is based on those performed by Silberman et al. in an effort to
debunk the reactivity and likely mechanism of organotelluranes as inhibitors.
6
This study is
particularly useful in analyzing the impact of ligands on the role of organotelluranes as inhibitors.
Multiple equivalents of l-cysteine, a common cysteine in the body and one that is
commercially available, will be added one equivalent at a time to the organotelluranes proposed
in this study. Because interaction with cysteines occurs by ligand exchange, experiments could
result in stable complexes, irreversibly oxidized cysteines, or no reaction based on the lability and
number of available ligands.
NMR studies using seleno-cysteine have not been performed previously, yet promises
interesting results based on indications in the literature that seleno-cysteine is more sensitive to
oxidation than other cysteines and may have specific affinity for these potential drugs.
1

3.2.5 ASSESSING INHIBITORY ACTIVITY, ROS LEVELS, AND TOXICITY
For these purposes, collaborators would be invited to investigate the proposed
organotelluranes through biological testing. Based on the literature, there are three areas of interest,
inhibitory activity, effects on ROS levels, and general toxicity. While the NMR studies give great
insight into expected behaviors, direct testing is necessary to determine effects on cells.
82
3.3 CONCLUSIONS
In contrast to known telluride and selenium compounds, organotelluranes have not been
given the attention of a systematic study to understand their biological trends for intelligent drug
design. Proposed here is a SAR study of organotelluranes as potential cancer therapeutics. Based
on results demonstrated in the literature, organotelluranes are expected to be more potent than their
selenium counterparts and other comparable drugs, but more selectively toxic to cancer cells than
to healthy cells. These organotelluranes are not anticipated to cross the blood-brain barrier or have
affects related to the often-cited examples of acutely toxic tellurium compounds. This is due to
their design which avoids weak, hydrolysable bonds, limits ligand lability and utilizes two stable
complexes, (II) and (IV). Several of the proposed organotelluranes have been prepared previously
and utilized for other applications, while others have never before been made.
Utilizing DFT calculations for ground state geometries and predicted NMR shifts will give
early insight into these compounds during preparation. NMR studies with l-cysteine and seleno-
cysteine will effectively demonstrate the impact of labile ligands and the stability of the overall
structures to give insight into the expected behaviors in biological testing. Working with
collaborators will enable biological testing to assess the effectiveness of inhibitory activities,
effects on ROS levels, and general toxicity. Future work can be directed based on results and by
expanding the library of organotelluranes with interesting structural aspects such as aromatic rings.
3.4 EXPERIMENTAL
3.4.1 STAGE ONE SYNTHESIS:
Diaryl telluranes 1a-c and 1-3 will be synthesized using a modified version of a recent
solventless method under microwave irradiation (Scheme 2).
26
Starting materials will be purchased
83
from Sigma Aldrich. Alternatively, TeCl4 can be prepared with elemental tellurium and excess
SO2Cl2 under microwave irradiation at 65°C for 4 h. The reported method for organotellurane
synthesis requires a 1:1 ratio of activated arene to TeCl4, giving the aryltellurium trichloride
product. For this study, 2 equivalents arene will be used to one equivalent TeCl4 to give the diaryl
tellurane. Reported conditions call for 50°C for 3 min, but either one or both conditions will have
to be increased for this study, because the second aromatic substitution occurs more slowly.

Scheme: 3 alternative method for amino substitution.

Scheme: 2 general preparation of diaryl telluranes 1a-c and 1-3.
84

The conventional method may also be used, which requires two equivalents of activated
arene to one equivalent TeCl4 refluxed at 160°C for 6 h. The resulting diaryltellurium dichlorides
can be converted to the dibromides and diiodides by ionic exchange of KBr and KI in dry
methanol.
33
The products may also be converted to the telluroxides by hydrolysis with aqueous
NaOH.
25
Aryltellurium dihalides and telluroxides are odorless solids stable to air and moisture. In
the event that the p-NH2 substituted arene does not form the desired product and instead forms a
species with HCl present in the reaction,
34
an alternative synthesis will be used (Scheme 3).
35

The synthetic method for the first six compounds of this series is only appropriate for
activated arenes. Non-activated arenes such as toluene require high reaction temperatures, and
weakly deactivating substituents, Cl and Br, require addition of Lewis acid.
36
Therefore a different
method is necessary for deactivated arenes. Diaryl ditellurides are thermolabile and will eliminate
a tellurium atom under high temperature, or by reflux in either toluene or dioxane in the presence
of electrolytic copper (Scheme 4).
25
The diaryl telluride can afterward undergo halogen addition
to give the desired diaryl tellurane. The starting materials are not commercially available but may
be prepared with existing methods.
34, 37, 38

Scheme: 4: alternative method for deactivated arenes.
85

Scheme: 5: Synthesis of 12 based off of method for Oxaliplatin.

Scheme: 6 Potentially more stable alternative to 12.

3.4.2 STAGE TWO SYNTHESIS:

Compounds 7, 8, 9, and 10 may be prepared using existing methods for the parent tellurides
followed by halogen addition to give the telluranes.
25, 27
Compound 11 is a known telluroketone
used in organic synthesis and may be prepared according to an existing method.
27
Compound 12
will be prepared for the first time by adapting the synthetic method for the parent platinum (II)
drug, oxaliplatin,
39
followed by halogen addition (Scheme 5). The geometry of 12 may weaken
the already labile sp
3
amines, so an alternative compound with more rigid, more electronegative
sp
2
amines will be used for comparison. This alternative compound will be synthesized using an
86
existing method to attach the amine ligand,
27
follow by the remaining steps for 12 (Scheme 6). It
is of interest to test both the Te (II) derivates and Te (IV) derivatives in this study, though the focus
of this study is Te (IV), as neither have been made.
3.5 REFERENCES
1. Ba, L. A.; Doring, M.; Jamier, V.; Jacob, C., Org. Biomol. Chem. 2010, 8, 4203-4216.
2. Du, P.; Saidu, N. E. B.; Intemann, J.; Jacob, C.; Montenarh, M., Ciochimicia et
Biophysica Acta 2014, 1840, 1808-1816.
3. Du, P.; Viswanathan, U. M.; Khairan, K.; Buric, T.; Saidu, N. E. B.; Xu, Z.; Hanf, B.;
Bazukyan, I.; Trchounian, A.; Hannemann, F.; Bernhardt, I.; Burkholz, T.; Diesel, B.; Keimer,
A. K.; Schafer, K. H.; Montenarh, M.; Kirsch, G.; Jacob, C., Med. Chem. Commun. 2014, 5, 25-
31.
4. Fry, F. H.; Holme, A. L.; Giles, N. M.; Giles, G. I.; Collins, C.; Holt, K.; Pariagh, S.;
Gelbrich, T.; Hursthouse, M. B.; Gutowski, N. J.; Jacob, C., Org. Biomol. Chem. 2005, 3, 2579-
2587.
5. Jamier, V.; Ba, L. A.; Jacob, C., Chem. Eur. J. 2010, (16), 10920-10928.
6. Silberman, A.; Kalechman, Y.; Hirsch, S.; Erlich, Z.; Sredni, B.; Albeck, A.,
ChemBioChem 2016, 17, 918-927.
7. Silberman, A.; Albeck, M.; Sredni, B.; Albeck, A., Inorg. Chem. 2016, 55, 10847-10850.
8. Princival, C. R.; Archilha, M. V. L. R.; Dos Santos, A. A.; Franco, M. P.; Braga, A. A.
C.; Rodrigues-Oliveira, A. F.; Correra, T. C.; Cunha, R. L. O. R.; Comasseto, J. V., ACS Omega
2017, 2, 4431-4439.
9. Piovan, L.; Alves, M. F. M.; Juliano, L.; Dromme, D.; Cunha, R. L. O. R., Bioorg. Med.
Chem. 2011, 19, 2009-2014.
10. Goldfarb, R., Tellurium - The bright Future of Solar Energy. USGS science for a changing
world 2015.
11. Chivers, T.; Laitinen, R. S., Chem. Soc. Rev. 2015, 44, 1725-1739.
12. Jackson, K. F.; Hammang, J. P.; Worth, S. F.; Duncan, I. DActa Neuropathol. 1989, 78
(3), 301-309.
13. Laden, B. P.; Porter, T. DJ. Lipid Res. 2001, 42, 235-240.
14. Nogueira, C. W.; Zeni, G.; Rocha, J. B. T., Chem. Rev. 2004, 6255-6285.
15. Gupta, N.; Porter, T. D., J. Biochem. Mol. Toxicol. 2002, 16, 18-23.
16. List, A.; Beran, M.; DiPersio, J.; Slack, J.; Vey, N.; Rosenfeld, C. S.; Greenberg, P.,
Leukemia 2003, (17), 1499-1507.
17. Sekhon, B. S., Res Pharm Sci 2013, 8 (3), 145-158.
18. Shie, M. D.; Deeds, F. E., Public Health Reports (1896-1970) 1920, 35 (16), 939-954.
19. Boudreaux, K. A. Group 6A - The Chalcogens.
https://www.angelo.edu/faculty/kboudrea/periodic/periodic_main6.htm.
20. Lenher, V., Selenium and Tellurium. The Journal of Industrial and Engineering Chemistry
1920, 12 (1), 597-598.
21. Service, S., J. Chem. Educ. 1931, 8 (1), 142.
22. Kurokawa, S.; Berry, M. J., Seleium. Met. Ions. Life. Sci. 2015, 13, 499-534.
23. Halpert, G.; Eitan, T.; Voronov, E.; Apte, R. N.; Rath-Wolfson, L.; Albeck, M.;
Kalechman, Y.; Sredni, B., J. Biol. Chem. 2014, 289, 17215-17227.
87
24. Sredni, B.; Caspi, R. R.; Klein, A.; Kalechman, Y.; Danziger, Y.; Benyaakov, M.;
Tamari, T.; Shalit, F.; Albeck, M., Nature 1987, 330, 173-176.
25. Petragnani, N.; Stefani, H. A., Tellurium in Organic Synthesis. Academic Press: California,
2007.
26. Princival, C.; Dos Santos, A. A.; Comasseto, J. V., J. Braz. Chem. Soc. 2015, 26 (5), 832-
836.
27. Detty, M. R.; O'Regan, M. B., Tellurium-Containing Heterocycles. John Wiley and Sons,
Inc.: New York, 1994; Vol. 53.
28. Kandasamy, K.; Kumar, S.; Singh, H. B.; Wolmershauser, G., Organometallics 2003, 22,
5069-5078.
29. Minyaev, R. M.; Minkin, V. I., Can. J. Chem. 1998, 76, 776-788.
30. Pacula, A. J.; Kaczor, K. B.; Wojtowicz, A.; Antosiewicz, J.; Janecka, A.; Dlugosz, A.;
Janecki, T.; Scianowski, J., Bioorg. Med. Chem. 2016, 25, 126-131.
31. Alcindor, T.; Beauger, N., Curr. Oncol. 2011, (18), 18-25.
32. Tellurium NMR. http://chem.ch.huji.ac.il/nmr/techniques/1d/row5/te.html#te125prop
(accessed April).
33. Narwal, J. K.; Chhabra, S.; Malik, R. K.; Garg, S.; Verma, K. K., Orient. J. Chem. 2013,
29 (4), 1339-1349.
34. Engman, L.; Persson, J., J. Organomet. Chem. 1990, 388 (1-2), 71-74.
35. Zhang, S.; Karra, K.; Koe, A.; Jin, J., Tetrahedron Lett. 2013, 54, 2452-2454.
36. Bergman, J., Tellurium in organic chemistry. Tetrahedron 1972, 28, 3323-3334
37. Haller, W. S.; Irgolic, K. J., J. Organomet. Chem. 1972, 38 (1), 97-103.
38. Botteselle, G. V.; Godoi, M.; Galetto, F. Z.; Bettanin, L.; Singh, D.; Rodriques, O. E.
D.; Braga, A. L., J. Mol. Catal. A: Chem. 2012, 635, 186-193.
39. Ermondi, G.; Caron, G.; Ravera, M.; Gabano, E.; Bianco, S.; Platts, J. A.; Osella, D.,
Dalton Tans. 2013, (42), 3482-3489.

Abstract (if available)

Abstract This work summarizes efforts in our laboratory to develop novel drug analogues with cytotoxic and antiviral properties. Chapter 1 details the development and implementation of a greener, more efficient and cost-effective synthetic method for substituted isoindolinones. Isoindolinone scaffolds can be found in a number of naturally occurring alkaloids isolated from plants and fungi. These natural products have demonstrated substantial biological activities, including anticancer and antiviral functions. Synthetic methods to access isoindolinone derivates are mostly multistep processes that utilize harmful organic media and expensive reagents. Presented here is a tandem Cu catalyzed cross-coupling heterocyclization reaction under aqueous conditions which utilizes convenient alkynylsilanes for the first time. The method is suitable for a range of electron withdrawing and electron donating groups, is high yielding and selective. This method was further developed by colleagues in our laboratory into a convenient one-pot synthesis. This work should better enable the preparation of a wide variety of potential cytotoxic and antiviral isodinolinone analogues. ❧ Chapter 2 demonstrates improvements upon the synthesis and purification of novel bioisosteric difluoromenthylphosphonate nucleotide analogues, which were previously investigated in our laboratory. Nucleotide analogues have been utilized as successful drugs for the treatment of cancers and viral infections. To circumvent problems with stability and bioavailability, prodrug technology was developed in which bioisosteric groups mimic structural aspects of nucleotides to improve lipophilicity, stability, and bioactivity. While a number of monophosphate nucleotide prodrugs have been developed, the literature surprisingly lacks usage of fluorine in phosphate modifications. Known for its powerful biological and chemical properties, fluorine, specifically difluoromethylene, can be used to effectively mask the phosphate hydroxyl group. A series of novel difluoromethylphosphonate nucleotide analogues have been proposed by our laboratory, but the synthesis and purification of these compounds has involved many challenges and has not produce products suitable for effective biological testing. This work evaluates previous methods to determine the best approach. It has been demonstrated that the current methods can be modified to obtain pure analogues of even the sensitive gemcitabine substrate, but that success and reproducibility depend upon the complexity of the starting materials. Alternative methods potentially more suitable have been proposed, though a few pure products are reported here. These unique nucleotide analogues have great potential as more effective prodrugs, and these improvements to their preparation make biological testing feasible in the near future. ❧ Chapter 3 is my Gedankenexperiment. Still in pursuit of developments in drug discovery for anticancer and antiviral agents, I started to investigate elements I had heard little about in the literature, and I happened upon reviews for tellurium in biological applications, a field that is grossly undervalued. Despite promising results in the literature and in historical reports for tellurium compounds as anticancer therapeutics, irrational distrust from only a few reports of toxicity has prevented much exploration in this area. Tellurium has demonstrated a unique potential to function as a “sensor/effector” redox modulator which selectively targets cancer cells with high levels of reactive oxygen species (ROS). Though studies have been performed for ditellurides and inorganic telluranes, organotelluranes are almost wholly unexplored. The few available examples illustrate the stability, potency, and safety of these compounds, but are too few and lack comparable details to draw conclusions for intelligent drug design. It appears that a broad, systematic structure activity relationship (SAR) study of organotelluranes is needed. Proposed here is SAR study of selected organotelluranes to determine effects of structural differences on behaviors as cysteine inhibitors and generators of ROS. It is expected that such a study could hugely benefit the development of potential organotellurane cancer therapeutics.

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Asset Metadata

Creator Aloia, Alexandra N. (author)

Core Title Accessible cytotoxic and antiviral drug analogues: improved synthetic approaches to isoindolinones and bioisosteric difluoromethylated nucleotides, and the search for therapeutic organotelluranes

School College of Letters, Arts and Sciences

Degree Master of Science

Degree Program Chemistry

Publication Date 08/07/2019

Defense Date 05/20/2019

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

Tag biochemistry,bioisostere,chemistry,drug analogue,fluorine,isoindolinone,medicinal chemistry,nucleotide,OAI-PMH Harvest,organic chemistry,organotellurane,prodrug,synthesis,tellurium

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Advisor Prakash, Surya (committee chair), Shing, Katherine (committee member), Thompson, Barry (committee member)

Creator Email aloia.alexandra@gmail.com,aloia@usc.edu

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