Michael English UC ID: MO1233850

PROBLEM:

Carcinogenesis is one of the most perplexing problems facing modern medicine. Unlike other

disorders, it is not caused by a specific disease. Since there is no bullet there is no ample understanding of

the wound. Cancer is caused by a failure of the cell cycle. In order for cancer to take hold in a cell, the

mechanism for both cell division and cell regulation must be damaged. The accelerator must be stuck and

the break must be broken. This can only occur if multiple mutations accumulate within the same cell.

Tissue cells accumulate mutations that allow them to more effectively compete for space, glucose and

oxygen.4 Cells that acquire mutations that enhances their fitness will produce more daughter cells that

share the genetic and epigenetic mutations of their parents.4 This somatic evolution allows mutated cells

to propagate until they become cancerous.4 This progression denies drug development an etiology to

target.7 Existing treatments such as chemotherapy and surgery lack effectiveness since somatic evolution

allows surviving cancer cells to become resistant and migrate to regrow tumors.

ABSTRACT:

This study has developed an amino acid analogue that is a promising

lead to more effective MCF-7 chemotherapeutic agent compared to current

FDA approved drugs. Chemotherapeutic agents that target DNA replication

in cancer cells have a narrow therapeutic window and are difficult to

administer. New advancements in cancer research has provided new targets

for novel chemotherapeutic agents. MTT cell cytotoxicity assays have revealed

that terminating moiety thymidine analogues are more selective and are more

effective chemotherapeutic agents compared to current FDA approved agents

such as fluorouracil (5-FU). Terminating moiety thymidine analogues can be

synthesized by first applying a silyl ether and a di-tert-butyloxycarbonyl

protecting group to thymidine. The 5’ pyrimidine methyl group was

brominated by an allylic bromination with NBS. A terminating moiety

auxiliary group was synthesized with an alcohol so a SN2 reaction could be

used to combine the two species together. The di-tert-butyloxycarbonyl amine

protecting groups and the silyl ether alcohol protecting groups were removed

with fluorides. Further research is needed to find suitable passive targeting

nanocarriers for these novel chemotherapeutic agents.5

INTRODUCTION: Hanahan and Weinberg’s innovative

paper “The Hallmarks of Cancer” revealed six

features that are present in all cancer cells. They

exhibit self-sufficiency in growth signals by

producing them themselves.4 This allows cancer

cells to propagate in the absence of external

feedback. They are insensitive to anti-growth

signals from their environment so inhibitors no

longer interrupt the cell cycle.4 They avoid

apoptosis by either losing the ability to monitor

themselves or the trigger is impaired.4 They

have limitless replicative potential by

maintaining their telomeres.4 They also sustain

themselves by producing new lymphatic vesicles

and can use them migrate to other tissues to

form secondary tumors.4 This indicates that

neoplastic tissue has a metabolome that differs

from that of healthy tissue.

The goal of this study is to advance the

development of nanocarrier transported

chemotherapeutic agents that can passively

target tumor cells. Hanahan and Weinberg’s

discovery of a tumor metabolism provides more

specific targets for anti-cancer agents.

Specificity will allow novel cancer agents to be

less toxic to healthy cells and less likely to cause

drug resistance in cancer cells. In this study, two

hallmarks will be targeted. Tumors have leaky

vascular systems.4 This will allow nanocarriers

of sufficient volume to selectively target cancer

cells by accumulating in tumors.5 Cancer cells

tend to divide rapidly thus inducing them to use

more nutrients than healthy cells. This makes

cancer cells more vulnerable to passive targeting

low molecular weight anti-tumor agents.5 This

study will forward the development of low

molecular weight DNA transcription

terminators.

DNA replication is highly coordinated

and must be done rapidly. DNA polymerase can

incorporate 2,000 base pairs per second.1 This is

done by highly processive polymerases that

catalyze many consecutive reactions with high

fidelity without releasing its substrate.1 Nucleic

acid analogues can be made to be incompatible

with the DNA polymerase republication fork

and terminate transcription prematurely.6 A

potential problem with this method is the cells

natural defenses against faulty nucleotides. An

effective nucleic acid analogue must be able to

bypass these defenses and terminate DNA

replication.

The eukaryotic genome is a formidable

target since it has a variety of defenses against

faulty nucleotides.1 Furthermore, faulty

nucleotides must be small enough to be

packaged into nanocarriers for passive targeting.

This allows them to be less toxic than other

chemotherapeutic drugs. A faulty nucleotide

could halt DNA replication by refusing to anneal

or force DNA polymerase from the template

entirely.1

In eukaryotic cells, DNA replication can

begin on multiple origins of replication.1 Human

eukaryotes requires around 30,000 origins of

replication.1 One chromosome typically has

several hundred.1 Each origin of replication is

loosely defined by AT-rich sequences that act as

assembly sites for origin of replication

complexes.1

First the origin of replication complex

assembles to initiate DNA replication.1 The

origin of replication complex consists of six

different proteins and acts as a foundation for

the assembly of the replication fork.1 Second,

licensing factors recruit a helicase to unwind the

DNA double helix.1 The single strands are

stabilized by the binding of replication proteins.1

Last, two distinct DNA polymerases are

recruited.1 Polymerase α, the initiator, binds to

the DNA template.1 This polymerase has a

primase subunit which adds a RNA primer and a

stretch of about 20 deoxynucleotides.1 This

polymerase is then replaced by DNA

polymerase δ which is the primary replicative

polymerase.1

The incorporation of numerous incorrect

or modified bases will result in cell

transformation or apoptosis by blockage of DNA

replication.1 Because of this, cells have evolved

a variety of DNA repair mechanisms that can

detect defects in the genome and restore

damaged DNA.1 Single strand DNA damage can

be repaired by using the sequence from the

uncompromised strand.1 In order to be effective,

a repair mechanism must be able to recognize

and remove the damaged base. The gap must

then be repaired with DNA polymerase and

sealed with DNA ligase.1

DNA repair is triggered when DNA

polymerase δ stalls over a faulty base.1 This

allows a proofreading exonuclease to repair the

faulty base.1 Nucleotide excision repair is

utilized when DNA damage is bulky and distorts

the shape of the double helix.1 Nucleotide

excision repair proteins assemble at the site and

a segment of DNA containing the distortion is

removed.1 The undamaged complementary

strand is used by DNA polymerase to fill the

lesion.1 DNA ligase seals the segment by

catalyzing the formation of phosphodiester

bonds.1 If replication stall completely, DNA

recombination can be used to allow replication

to continue.1 Recombination is used to repair

double strand breaks by using a homologous

chromosome as a template.1

Currently, drugs that can bypass the

cell’s DNA repair mechanisms by targeting

DNA or proteins that interact with DNA tend to

be very toxic and are reserved for life

threatening diseases.7 Toxicity tends to be more

severe in areas where rapidly dividing cells are

normal such as in the bone marrow, the

gastrointestinal tract, the mucosa and the hair.7

There is a need for a drug that can

selectively target cancer cells using known

“hallmarks” and disrupt a cancer cells ability to

divide. This will allow the drug to be

administered more easily and increase its

effectiveness.2

HYPOTHESIS:

A nucleic acid analogue that irreversibly

inhibits DNA polymerase would bypass a cells

heroic efforts to preserve its genome. Passive

targeting will allow drugs to accumulate in

neoplastic tissue and spare healthy tissue. This

will allow a wider therapeutic window thus

making these chemotherapeutics more effective

than current FDA approved drugs.

Approach:

Nucleotide analogues are modified

naturally occurring nucleotides, which gives

them new properties. Sanger DNA sequencing

relies on analogues that are radioactively or dye

labeled to allow them to be traced and had their

3’-hydroxyl group removed to prevent DNA

polymerase from adding another nucleotide

during polymerase chain reaction.5 When DNA

polymerase incorporates one of these analogues,

DNA replication is halted.5 Multiple fragments

of different lengths with labeled ends are

separated by gel electrophoresis, thus providing

the sequence.5 However, the 2’,3’-

dideoxynucleotide terminators, as well as 2’-

deoxy nucleotides bearing a blocking group at

3’-OH, are rather poor substrates for natural

DNA polymerases due to DNA repair systems,

which makes their therapeutic value low, as they

do not have the potential to compete with natural

nucleotides.6

According to a study conducted by

Litosh et al. modified nucleotides that have their

3’-hydroxyl groups intact have better DNA

incorporation compared sanger sequencing DNA

terminators, especially for pyrimidine analogs.6

The presence of a terminating moiety on the

nucleobase is more effective than termination

due to a missing 3’-hydroxyl group in living

cells.5 Incorporation of these nucleotide analogs

leads to obstruction of further DNA synthesis.5

Litosh et al. used terminating moiety

thymidine analogs to carry out cytotoxicity

studies in MCF-7 breast cancer cells5.

According to Litosh et al. the best auxiliary

group to add to the 5’ carbon of thymidine was a

benzene ring with electron withdrawing groups

in the ortho and para positions.5 This auxiliary

group must be bound to the 5’ carbon of

thymine. The 4’ carbon of the auxiliary benzene

ring should be linked by a triple bond linker

bridge to a large bulky group.

Figure 1: Thymidine with a 5’ methyl group auxiliary

DNA replication terminator.5

METHODS:

All of the reagents, solvents and

catalysts used were purchased from Sigma-

Aldrich Inc., TCl and Fischer Scientific.

Analytical thin glassed chromatography (TLC)

was carried out with glass back silica plates

(5x20 cm, 60 Å, 250 μm) and visualized with a

254nm UV lamp. Purified intermediates were

vacuum dried within a desiccator. 1H and 13C

HMR spectra were conducted with either a

Bruker Avance 400 MHz spectrometer or a

Bruker DPX 500 MHz spectrophotometer.

Samples were dissolved in chloroform,

methanol, acetonitrile, or water.

Introduce Thymidine to an alcohol

protecting group:

Figure 2: Alcohols on thymidine sugar were protected by

silyl ether protecting groups.

In order to prevent the alcohols on

thymidine from unwanted reactions during this

multi-step synthesis, tert-bytyldimethylsilyl

chloride (TBSCl) was added in order to form a

silyl ether protecting group on the 3’ and 5’

alcohol on the deoxyribose sugar.3 This was

done by first adding 1 equivalent of thymidine

powder to a 2.5ml round bottom flask under

argon gas. 6.4 equivalents of the amine base

imidazole and 3.2 equivalents of TBSCl to the

2.5ml round bottom flask. The contents of the

round bottom flask was dissolved in 5.50ml of

anhydrous dimethylformamide. The flask was

stirred for 24 hours at room temperature (24oC)

A TLC plate revealed the end of the

reaction (SiO2, hexane/ethyl acetate = 2:1 ⟶

dichloromethane/methanol = 10:1). The TLC

plate was then placed into an iodine chamber.

The iodine and imidazole formed an iodine

charge complex and became visible on the TLC

plate. The imidazole spot was distinct and was

separate from the spot that contained the

protected thymidine product. The reaction

mixture was then placed into a separatory funnel

and diluted with 50ml of water. The aqueous

layer was decanted. The organic layer was

washed with 2 aliquats of 15ml of diethyl ether.

The organic layer was decanted and the diethyl

ether was washed with 2 aliquats of 15ml of

water and then brine was added (NaCl saturated

water). 0.4g of solid Na2SO4 was added to the

diethyl ether. After 24 hours, the solution was

roto-evaporated to give an oil. This oil was

recrystallized with 60ml of boiling Et2O/Hexane

1:10.

The product of this reaction was purified

by column chromatography. The stationary

phase consisted of silica powder (SiO2,

hexane/ethyl acetate 4:1). A sample of

previously made precursor was used to spot a

TLC plate and this was used to determine which

fraction contained the product. This fraction was

further purified by column chromatography then

the solvent was removed with a roto-evaporator.

Introduce Thymidine to an amine

protecting group:

Figure 3: The pyrimidine 3’ amine was protected with a

tert-butyloxycarbonyl (BOC) protecting group.

To protect the pyrimidine 3’ amine, a di-

tert-butyloxycarbonyl (BOC) protecting group

must be added.3 1 equivalent of silyl ether

protected precursor synthesized in the last

reaction and 0.1 equivalents of 4-

dimethylaminopyridine was dissolved in 25ml

of anhydrous dimethylformamide in a 2.5ml

round bottom flask. 2.2 equivalents of

triethylamine was added to the flask and the

mixture was stirred for 15 minutes. Then 2.2

equivalents of di-tert-butyl dicarbonate was

added to the flask. The reaction mixture was

stirred for 24 hours. The reaction flask was then

diluted with 25ml of water and placed into a

separatory funnel. The product was extracted

with 4 aliquots of 25ml chloroform. The

combined extracts were washed with brine and

dried over sodium sulfate. The reaction mixture

was then roto-evaporated under reduced

pressure. A TLC of the reaction mixture

revealed the presence of the product. A

previously made precursor was used to identify

the product on the TLC plate. The residue within

the reaction flask was purified by column

chromatography with silica powder (SiO2,

hexane/ethyl acetate = 10:1⟶

dichloromethane/methanol = 10:1). Next the

methyl group on the 5’ carbon on thymidine’s

pyrimidine ring must be brominated.

Bromination of 5’ Thymidine

Pyrimidine Carbon.

Figure 4: The 5’ pyrimidine methyl group was brominated.

The methyl group on the 5’ pyrimidine

methyl group was brominated by an allylic

bromination with NBS.3 1 equivalent of the

precursor from the last reaction and 2.10

equivalents of NBS were placed in a 50 ml

round bottom flask. This was mixed with 25ml

of anhydrous carbon tetrachloride. 0.003

equivalents of benzoyl peroxide was added to

the flask and placed in a 100oC oil bath for 45

minutes. The reaction mixture turned to an

umber color. The color subsided at the end of

the reaction. A TLC plate revealed the end of the

reaction. A spot using a previously made

precursor identified the presence of the product.

The mixture was purified by silica powder

column chromatography (SiO2, hexane/ethyl

acetate = 2:1).

Synthesis of Alcohol Auxiliary

Group:

Figure 5: An auxiliary group was prepared. 1 alcohol must

be present so it can be added to thymidine’s pyrimidine

ring. A halide was needed to add a linker and bulky group

to the auxiliary group.

An auxiliary group that matches the

motif of the molecule in figure 1 must be

synthesized. This study synthesized a 3-

phenolethyne-nitrobenzene terminating moiety.

The auxiliary nitrobenzene ring was connected

to the brominated thymidine precursor

synthesized in the last reaction by an ortho ester

bridge. A methanol must be present on the

auxiliary benzene ring’s 2’ ortho carbon so a

SN2 reaction can establish this bridge with the

brominated thymidine precursor. First, 1

equivalent of 4-iodo-1-methylTBS-2-

nitrobenzene was dissolved in 20ml of

tetrahydrofuran in a 25ml round bottom flask

and was placed into a 0oC ice bath with stirring.

2.1 equivalents of tetra-n-butylammonium

fluoride (TBAF*3H2O) was added. The reaction

mixture stirred for 1 hour. A TLC of the reaction

mixture revealed the presence of the product. A

previously made precursor was used to identify

the product. The product was purified by silica

powder column chromatography (SiO2,

hexane/ethyl acetate = 2:1).

Synthesis of a Thymidine Analogue:

Figure 6: The auxiliary alcohol synthesized in the last

reaction was added to the brominated thymidine precursor.

1 equivalent of the brominated

thymidine precursor and 3.4 equivalents of the

auxiliary alcohol synthesized in the last reaction

were mixed in a clean 25ml round bottom flask.

A small amount of ethyl acetate and the turning

motion of a roto-evaporator was used to transfer

the reactants to the new flask. This yielded a

yellow viscous oil. This mixture was heated in

an oil bath at 105oC-113oC for 3 hours. A TLC

plate of the product revealed the presence of the

product. A previously made precursor was used

to identify the product. The product was purified

by silica powder column chromatography (SiO2,

hexane/ethyl acetate = 1:1 ⟶ 2:1 ⟶ 30:1 ⟶ 4:1 ⟶2:1 ⟶ dichloromethane/methanol =

20:1)

1 equivalent of the purified product was

added to a clean 25ml round bottom flask. 3.0

equivalents of phenylacetylene, 17.1 equivalents

of N,N-Diisopropylethylamine, 0.1 equivalents

of tetrakis(triphenylphosphine)palladium(0), 0.2

equivalents of copper(I) iodide and 0.2

equivalents of anhydrous DMF were added to

the flask. The mixture was stirred for 24 hours.

A TLC of the reaction mixture revealed the

presence of the product. A previously made

precursor was used to identify the product. The

product was purified by silica powder column

chromatography (SiO2,

dichloromethane/methanol = 20:1 ⟶ 10:1)

Figure 7: After adding the auxiliary alcohol to thymidine’s

pyrimidine ring, a linker and a bulky group was added to

the auxiliary moiety.

MTT Cell Cytotoxicity Assay

Terminating moiety thymidine

analogues were tested on MCF-7 breast cancer

cells.2 MCF-7 cells were grown in RPMI 1640

media supplemented with 10% bovine serum,

1% penicillin, 10nm estrogen and 1mM insulin.2

These cells were then tyrisinized and re-

suspended at a density of 2.2 x 104 cells per mL.

24 well plates were prepared with 500μl of this

suspension in each of them.2 These plates were

incubated at 37oC in a 5% CO2 atmosphere for

12 hours.2 The media was then changed and the

plates were dosed 3 times with media dissolved

in DMSO that did not exceed a concentration of

0.5%.2 Plates containing 100μM, 50μM, 25μM,

12.5μM, and 6.25μM of compound media were

prepared.2 A plate containing 5-Fluorouracil was

used as a positive control.2 These plates were

incubated for 65 hours after adding the MTT

solution.2 500μl of 193μg/mL MTT and media

solution was added to each well.2 The plates

were incubated for an additional 3 hours.2 Then

the MTT solution was decanted and 500μl of

DMSO was added to each well.2 The cells were

imaged using a GS 800 Bio Rad scanner using

Quality One Software or a BioTek plate reader

using Gene5 software.2 IC50 curves were

prepared by plotting viability vs compound

concentration.2 Kaleidagraph software was used

to calculate the R value for each logarithmic

curve fitting.2

RESULTS

Figure 8: 1H NMR spectrum of a purified sample of a thymidine analogue with a cyclohexyl phenyl terminating moiety.8

Figure 9: MCF-7 cell assays have revealed that thymidine nucleic acid analogues with terminating moieties (fourth row in blue

bracket) are more selective and more antagonistic to breast cancer cells than some FDA approved chemotherapeutic agents such

as fluorouracil (second row in red bracket).5

CONCLUSION

MCF-7 cell assays have revealed that

thymidine nucleic acid analogues with

terminating moieties have a much better chance

of bypassing a cells defenses and being

incorporated into a cell’s DNA during

replication.6 Crystal structure studies of DNA

polymerases suggests that high fidelity proof

reading polymerases have a ‘tight fit’ active site

to accommodate proper nucleic acids and stalls

on damaged nucleic acids due to steric

hindrance.6 This initiates the cell’s DNA repair

machinery. Low fidelity polymerases have

larger active sites to replicate DNA rapidly.6

DNA polymerase selectivity is due in part to the

flexibility of its active sites.6 The effectiveness

of nucleic acid analogues with terminating

moieties may be due to the size difference in

these two different types of DNA polymerases.6

Terminating moiety thymidine analogues are

promising cheomotheraputic agents due to their

higher selectivity and greater effectiveness at

halting cell division in MCF-7 breast cancer

cells. Further research is needed to develop a

nanocarrier to deliver these modified

nucleosides to MCF-7 cancer cells by passive

targeting.5

References

1 Berg, Jeremy M., Jogn L. Tymoczko and Lubert Stryer. Biochemistry. New York: W.H. Freeman and

Company, 2012.

2 Borland, Kayla M., et al. "Base-modified thymidines capable of terminating DNA synthesis are novel

bioactive compounds with activity in cancer cells." Bioorganic & Medicinal Chemistry (2015):

1869-1881.

3 Brown, William H., et al. Organic Chemistry. Boston: Brooks Cole, 2011.

4 Hanahan, D. and RA Weinberg. "The Hallmarks of Cancer." Cell (2000): 57-60.

5 Litosh, Vladislav A. Development of Novel Base-Modified Nucleosides as Anti-Cancer Agents and

Finding a Suitable Nanocarrier for Drug Delivery. Presentation. Cincinnati: University of

Cincinnati, 2015. Microsoft PowerPoint.

6 Litosh, Vladislav A., et al. "Improved nucleotide selectivity and termination of 3'-OH unblocked

reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates."

Nucleic Acids Research (2011): 1-13.

7 Silverman, Richard B. The Organic Chemistry of Drug Design and Drug Action. Burlington: Elsevier

Academic Press, 2004.

8 Summer, Wise, Courtney A. Stockman and Vladislav A. Litosh. Development Novel Cancer

Chemotherapeutic Agents. Research Paper. Cincinnati: University of Cincinnati, 2014. Microsoft

Word.