O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine
1
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine
Eva R. Key
2015 Noble Energy Scholar
Frontiers of Science Institute, Mathematics and Science Teaching Institute,
University of Northern Colorado, Greeley CO
Mentored by Alysa Carlson Department of Chemistry and Biochemistry
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 2
Acknowledgments
I would like to acknowledge the financial assistance of Noble Energy. I would also like to thank
Frontiers of Science Institution , The University of Northern Colorado, Alyssa Carlson, and Sean
Hawskins. Without the help of them this would not be possible. Thank you.
Table Of Contents
Introduction…………………………………………………………………………………………… 3
Literature Review……………………………………………………………………………………. 4
Materials……………………………………………………………………………………………… 6
Methodology………………………………………………………………………………………...... 6
Results and Discussion……………………………………………………………………………...9
Conclusion…………………………………………………………………………………………….16
References …………………………………………………………………………………………...17
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 3
Introduction
Cancer is a disease that kills an average of 7.6 million people a year (UICC, 2015). It is an
epidemic that needs to be put to a stop. The funding for cancer research has averaged to $4.9 billion over
the past six years (Brill, 2014). Even when considering the money, time, and research spent on cancer
treatments, there are still many problems encountered when developing treatments. Cancer is easily one
of the worst diseases out there. The genes change how the cells grow and divide. (NIH, 2015) The cells
affected by these genes are called cancer cells. Cancer cells grow out of control and become invasive in
the body. They grow without limit unlike regular healthy cells, which are very specific. (NIH, 2015)
Normal healthy cells have particular functions for example neurons. They are used to transport messages
from the body to the brain. Cancerous cells don’t have these particular functions. They grow in masses
and don’t contribute to the functioning body.
Cancer can be treated in many different ways such as radiation and chemotherapy. Radiation is
when an oncologist uses x-ray treatment or other wavelengths to kill cancer cells. Chemotherapy is when
an oncologist prescribes medication, which have chemical compounds that are meant to destroy the cells.
The problem with these treatments is that they don’t only target cancer cells. They can target and effect
regular cells as well. (Lundberg, 2015) This effect is what causes the severe symptoms of cancer patients
who undergoing treatment.
One of the compounds that has been used in chemotherapy and observed as effective, mamsacrine, undergoes nucleophilic attack under physiological conditions. It has a short half-life, which
means that the patient must be treated with a high dosage for it to be effective. Increasing the dosage
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 4
amount of a therapeutic compound taken may also increases the negative effects it has on the body.
Researchers have considered using varying substitutions within the cancer drugs in order to optimize
effectiveness. Using varying substituent groups on the benzene ring of O-benzyl-N-(9’acridinyl)hydroxylamine, the resulting binding affinity of the compound for genomic DNA will be
evaluated, thus identifying the potential of the medication to interact with DNA.
Lit Review
For decades scientists and doctors have been working to find a cure to cancer. Although lots of
medical advancements have taken place there is still no perfect answer.
Before the use of chemotherapy doctors used surgery to remove tumors. Surgically removing
tumors wouldn’t work if the tumor was too big or if it had started to spread. Sometimes cancer cells were
left behind in the body, and they would continue to multiply. Later on radiation became a common way to
treat cancer. Even with its severe effects on the body it was still more effective than surgery on its own.
Radiation could help with a lot more types of cancer than surgery. Often time’s oncologists would
prescribe both for a patient. The most recent and most common cancer treatment is chemotherapy.
Chemotherapy uses chemical compounds that interact with the DNA and stop the cancerous cells growth.
Paul Ehrlich, a famous German chemist, is known for his work in medication. He was one of the
first people to use animal models to show how the medication would affect the body. Ehrlich was the first
one to come up with the term chemotherapy. He used the term to describe the usage of chemicals to treat
diseases. Before Ehrlich, the main treatments of cancer were radiotherapy and surgery. (Devita, 2015)
The survival rate increased significantly with the introduction the chemotherapy.
George Clowes presented another example of a great medical achievement. He figured out how to
place a tumor into a rodent. By being able to do this we could see the direct results of tumor treatment
with therapeutic compounds. Later that century, during WW2, it was found that mustard gas could be
used to treat lymphoma. (ACS 2014) There were certain chemicals in the gas that were good for attacking
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 5
and destroying cells. The first times that chemo was used to successfully treat cancer was recorded in the
1960s. Chemo has ever since been a popular treatment for cancer.
Figure 1. Mustard gas chemical composition.
Lerman concluded that altering a intercalating agents could benefit the DNA by bettering the
interactions they had together. Intercalation is where a acridine binds to the DNA between the alternating
base pairs and is inserted into the DNA phosphate backbone. He suggested that the changes would
include an increased healthy DNA, the coiling of DNA. Lerman believed that we could note these
changes by measuring the viscosity in the DNA. (Lerman 1960)
Cain and colleagues believed that groups of chemicals in the acridine might increase how the
medication bonds with DNA and therefore keep it from multiplying. (Cain, Seelye, & Atwell, 1974).
They developed m-amsacrine.. Although m-amsacrine was commonly used for cancers such as acute
leukemia and Hodgkin’s and non-Hodgkin’s lymphomas, it was observed to have some toxic effects as
well. Patients experienced harmful outcomes such as cardiac issues.
Figure 2. Chemical structure of m-amsacrine.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 6
Later, Baugley and co-workers made some anti-tumor compounds containing an acridine base.
They substituted on varying positions on the 9-aminoacridine . The purpose of this was to observe the
impact of the substitutions on the compounds ability to interact with DNA. They found that some of
substitutions did affect the interaction with DNA by preventing replication. (Baugley, Denny, Atwell, &
Cain, 1981).
Figure 3. An example of an acridine base.
Denny, Twigden, and Baugley looked more into m-amsacrine and of o-amsacrine and their
abilities to interact with DNA. m-Amasacrine was found to be the most effective. Under physiological
conditions m-amsacrine goes under nucleophilic attack, which would be very harmful to the human body.
The substitutions took part in different places of the molecule and lowered its ability to bind to DNA.
Hydroxylamines were studied and like amino’s they have become useful in treating illnesses. (Benson,
1956)
Work continues to be completed on substitution in common cancer drugs. With new technology
and information it is desired to find a compound with a greater affinity for DNA and lesser susceptibility
for nucleophilic attack. Doctors have found ways to lessen the side affects until then using other drugs
such as anti nausea and pain medications.
Materials
The following glassware was used; funnels, beakers, round bottoms, separatory funnels,
condensers, an Ostwald viscometer, and pipettes. The following chemicals were used; anhydrous,
Na2SO4, HCl, N-hydroxphthalimide, Bu4NHSO4, NaHCO3, CH2Cl2, NHSO4, 3 -chlorobenzyl chloride,
and deionized H2O. Other then that lab spatulas, weighing paper, heating plates, variacs, Tygon tubes,
heating nests, stirring bars, a vacuum creator, and a rotary evaporator (rotovap) were used.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 7
Methodology
First 62 milligrams of NaHCO3 was weighed.10 mL of deionized water was measured into a 20
mL graduated cylinder. 10 mL of CH2Cl2 (dichloromethane) was added into a graduated cylinder using a
pipette. A piece of weighing paper was put on a scale and the scale was zeroed. Bu4NHSO4 was added in
small increments using a small lab spatula, until it reached the mass of 12.5 milligrams. 6.5mL of 3 chlorobenzyl chloride was put into a graduated cylinder using a different pipette. 62 mg of NaHCO3,
10mL of deionized water, 10mL of CH2Cl2, 12.5 mg of Bu4NHSO4, and 6.5Ml 3 -Chlorobenzyl Chloride
was thoroughly mixed into one appropriate sized beaker.
A reflux was set up to heat up the mixture it so the reaction can occur. The reflux was needed so
that the mixture does not evaporate but does heat up. To set up a reflux, pour the mixture into a round
bottom flask. The round bottom flask that contains the mixture was put into a heating mantle. The
reaction flask was put onto a stirring plate and clamped so the round bottom so it would not move. A
stirring bar was put into the round bottom. A heating plate was hooked up to a variac. A condenser was
connected onto the round bottom flask. The condenser has two holes. Tygon tubes were attached to both
the holes. The bottom tube goes to the green water faucet. The top tube goes to the drain. The water was
turned on. Using a piece of weighing paper, a lab spatula, and a scale 600 mg of N-hHydroxphthalimide
was weighed. The N-hydroxphthalimide was put into a small flask. After the mixture started to boil Nhydroxphthalimide was added in small portions every 10-20 minutes until there was none left.
After performing the reflux, an extraction was set up using a separatory funnel. Using a graduated
cylinder, 20 mL of CH2Cl2 was measured. Two extractions were performed using CH2Cl2. To extract, a
funnel was needed. The aqueous solution was added to the separatory funnel followed by Ch2Cl2.
The solutions were shook and then the aqueous solutions were drained. The similar steps were
repeated with deionized H2O, NaHCO3, and 5% HCL (diluted with water). Anhydrous Na2SO4, was
added to the combined organic solution to dry it. The powder was added until it looked feathery and then
gravity filtered into another flask. The mixture was put into a round bottom flask. The solution was
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 8
evaporated to dryness using a rotary evaporator. The solvent was removed until the solution had provided
a white powdery substance. The substance was put into a flask and labeled.
Next an acid reflux was set up again. The obtained crude N-(3-chlorobenzyl) phthalimide was
mixed with 10 mL of acetic acid in a beaker. Acetic acid was measured using a graduated cylinder and a
pipette. Using a separate graduated cylinder and pipette 5 mL of HCl acid was measured.
HCl acid was added to the N-b(3-chloroenzyloxy)phthalimide and acetic acid mixture. The
mixture was put into a round bottom flask and put it into a reflux. HCl was added every 5-10 minutes for
an hour with a pipette. Following reflux, the solution was placed on a rotary evaporator and the remaining
acid solution was removed yielding an off-white solid.
Water was then added to the solid to produce a suspension. 10% NaOH was added the mixture
with a pipette until it became basic. This was tested using litmus paper. The mixture was put into a
separatory funnel. An extraction was performed three times using 30 mL of CH2Cl2. After the aqueous
layer was drained we put the organic layer into another flask. The mixture was dried using anhydrous
Na2SO4. The solution was then isolated through gravity filtration. The mixture was put into a round
bottom flask and attached to a rotatory evaporator to condense it for 5-10mins. After it was condensed it
was put it in a flask and ran it through a process called “Bubbling HCl”. Bubbling HCl makes the
negative and the positive combine to make a solid using HCl.
A vacuum filtration was then set up. The purpose of this particular filtration was to isolate the
produced solid. The dry product was then put into a vial and weighed using the electronic scale. The
product weighed 0.139 grams. The yield of the reaction was measured and percent yield was determined.
A DNA solution was then created using genomic calf thymus DNA. 10 mL of the solution was
measured out using a volumetric flask. Placed in a 10 mL volumetric flask was .38 milliliters of
synthesized compound. In the same flask as the anticancer drug medication .01M phosphate buffer PH 7
with 5% DMSO was put.
The two 10 mL measures were then combined and mixed into one flask. The viscosity was then
measured 5 times using an Ostwald viscometer. Between each testing the Ostwald viscometer was put
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 9
into a hot water bath for 10 minutes. The average efflux time for the viscosity was determined. Thermal
denaturation plots were provided.
Figure 4. Synthetic route for the preparation of meta-substituted O-benzyl-N-(9’acridinyl)hydroxylamines.
Results and Discussion
N-(3-chlorobenzyl)oxyphthalimide was synthesized using a procedure made by Bonaccorsi and
Giorgi. Benzyl chloride was substituted with chloride at the meta-position. Using the N-(3chlorobenzyl)oxyphthalimide, O-(3-chlorobenzyl)hydroxylamine hydrochloride, a salt, was made. An
extraction to isolate the salt was then performed using dichloromethane. The purpose of this was to
increase the bonding of the medication with the DNA. This will stop the DNA from multiplying and
spreading. The higher the viscosity is the better the medication bonded to the DNA. The medication is
made to be potentially used for chemo. It’s when an oncologist prescribes a medication that will destroy
the cells in the body. Amsacrine, a chemo medication, was prescribed to cancer patients, particularly
diagnosed with leukemia.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 10
Figure 5. Structure of O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine.
To test the bonding of the DNA with proposed therapeutic compound viscosity and thermal
denaturation data were collected. The synthesis to produce the O-(3-chlorobenzyl)hydroxylamine
hydrochloride produced a 30% yield. The concentration of the DNA solution was determined using Beer's
law, A=𝜖bc. The concentration came out to be 1.0316x10^-4 mols/liter. The concentration of the DNA
solution determined the desired concentration of the acridine solution, which was made using a stock
solution of known concentration. Using my previous calculations I found the volume by using the
formula M1V1=M2V2. The volume was 343.4 microleters. We measured the viscosity using an Ostwald
viscometer. The viscosity was measured five times and the average efflux time was determined. The
average efflux time was used to determine the kinematic viscosity of the solution. The longer the times
are the higher the viscosity is. The viscosity was compared to the viscosity of the untreated DNA solution
and there is a clear increase in the viscosity meaning that is binding to the DNA was strong.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 11
Table 1. Efflux Time
Efflux time for untreated
DNA
185.44s
187.25s
187.24s
186.52s
N/A
Table 2. Efflux Time
Efflux Time (In seconds)
for O-(3-chlorobenzyl)-N(9’acridinyl)hydroxylamine
401.21s
406.305s
411.49s
417.89s
407.78s
Average Efflux
408.934s
Standard Deviation
6.21838
Kinetic Viscosity
2.06 mm/s2
Proton Nuclear Magnetic Resonance (1H NMR) were taken throughout this process. The point of
the NMR was to make sure the desired product was formed. The first NMR collected was that of N-(3chlorobenzyl)oxyphthalimide. The 1H NMR represented 15 hydrogen’s, which was excessive for those
within the desired compound. There were 8 hydrogen’s were from the phthalimide. The others were from
N-Hydroxysuccinimide, a reactant that was put into the reflux but didn’t fully dissolve. The second NMR
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 12
represents O-(3-chlorobenzyl)hydroxylamine hydrochloride. It identifies that even with the present
impurities in the N-(3-chlorobenzyl)oxyphthalimidethe corresponding salt still came out fine.
Figure 7. 1H NMR of phthalimide using 3-chlorobenzyl chloride
Figure 8. 1H NMR of salt using 3-chlorobenzyl chloride
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 13
Thermal Denaturation is another way to test the compounds effectiveness through its melting
point. The higher the melting point of the DNA when treated with the compound, the more effective the
compound interacts with DNA. As seen on the diagrams below the O-(3-chlorobenzyl)-N-(9’acridinyl)hydroxylamine was very effective. The line is constant and then starts to curve upwards. The
diagram shows that the chloro has a higher melting point than nitro, which means that it is more effective
in its ability to bond with DNA. The high melting point also indicates it will last and not precipitate in an
aqueous environment, such as a human body.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 14
Figure 9. Original thermal denaturation plot
Figure 10. Thermal denaturation plot of DNA treated with O-(3-chlorobenzyl)-N-(9’acridinyl)hydroxylamine.
A comparison of the collected data with the known Hammett sigma values provides for the
development of quality structure activity relationship (QSAR) plot. These plots represent trends relating
how well the compound binds to DNA to the electronic characteristics of the substituents.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 15
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 16
As you can see in the graphs above there's a noticeable parabolic trend. Chlorobenzyl chloride is
on the top indicating its high melting point. The nitrobenzyl is lower on the graph than the chlorobenzyl
but both are higher than the standard hydrogen. Both of the graphs show a similar trend indicating that
results are correct.
Conclusion
After synthesizing two O-benzyl-N-(9’-acridinyl)hydroxylamines each with a different
substituents on the meta-position of the benzene ring, it was determined that the 3-chloro substituent was
more effective at interacting with DNA than the 3-nitro substituent. The ability to bind with the DNA is
important because it can stop replication and therefore cell growth will be limited and more controlled. It
has a higher melting point and therefore is more likely to have a strong bonding. The O-(3-chlorobenzyl)N-(9’-acridinyl)hydroxylamine has a higher kinematic viscosity and efflux time, which indicates its
ability and effectiveness to bond to DNA is greater. Although the 3-benzyl substituent was found to better
enhance binding ability than the 3-nitro substituent, both compounds were found to be more effective than
the standard hydrogen substituent. Electron withdrawing groups, such as chloro- and nitro- are better
substituents than hydrogen, but a chloro- substituent is better than a nitro- substituent at the meta-position
on the benzene ring.
O-(3-chlorobenzyl)-N-(9’-acridinyl)hydroxylamine 17
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