Synthesis of O-phenyl-N-(9-acridinyl)-hydroxylamine

Alexys McGuire
Adolph Coors Foundation Scholar
Frontiers of Science Institute

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INTRODUCTION
Cancer, the leading cause of death worldwide after heart disease, has been around for

generations and can often be difficult to treat. It has been proven that about half of men and onethird of women in the United States will develop cancer in their lifetime.1 Unfortunately, cancer
can take multiple forms, and because of this, it has become vital to create different forms of
treatment in order to attack the various types of cancer that are out there. These treatments
include chemotherapy, radiation, hormone therapy, and anti-tumor drugs. However, out of these
different treatments, anti-tumor drugs have more potential to target patients cancer cells, leading
to a greater need to develop novel anti-tumor drugs. As you can imagine, this has the potential to
lead to more effective treatment options for cancer patients worldwide.
Novel substituted 9-aminoacridine derivatives have been proven to inhibit cell
proliferation of pancreatic cancer cell lines by inducing apoptosis.2 In addition, Oppeguard,
Mugolkov, and Luchini show that these same derivatives were able to intercalate into DNA and
inhibit the catalytic activity of human topoisomerase II, leading to the conclusion that these
acridine-based compounds were able to act as catalytic inhibitors of human topoisomerase II.
Although it is possible for these compounds to target healthy cells as well as cancerous cells,
cancer cells tend to divide more rapidly, making it more likely for 9-aminoacridine based
compounds to target cancerous cells rather than healthy cells. Based on these results, it has been
hypothesized that a novel anti-tumor drug, O-phenyl-N-(9-acridinyl)-hydroxylamine, can be
created that will produce the same results. Although these drugs do have the potential to harm
healthy cells, the compounds are more likely to target cancer cells due to the nature of these
cells. Cancerous cells have been shown to divide very rapidly, meaning that they have to
replicate their DNA often. Because of this, the hope is that this new novel anti-tumor drug will

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show the same results as existing 9-aminoacridine based compounds, with the intercalation

evoked by these compounds eventually arresting the cell cycle, leading to apoptosis, slowing the
proliferation of the cancerous cells.
Many different forms of 9-aminoacridine based compounds and their effects have already
been tested. The results of these tests have shown that many DNA intercalating drugs are
susceptible to undergoing hydrolysis reactions in vivo.2 However, there are no known studies
done for O-phenyl-N-(9-acridinyl)-hydroxylamine, a derivative of 9-aminoacridine. This new
compound should be less susceptible to hydrolysis, which should lead to more effective cancer
treatments. This, in turn, leads scientists to wonder: Can this new anti-tumor drug be synthesized
in higher yields, thus leading to a higher anti-tumor efficiency?

REVIEW OF LITERATURE
There has been a multitude of research done on 9-aminoacridine based compounds over
the past few years.2,3,4,5 The results obtained from this research have shown, among other things,
the effects of these compounds on DNA and how this relates to cancer research in general. For
example, it has been proven that 9-aminoacridines can interact with DNA in one of two ways: it
can either act as a DNA topoisomerase poison, or it can act as a DNA topoisomerase catalytic
inhibitor. Although 9-aminoacridines have the potential to act as a DNA topoisomerase poison,
these compounds have shown to be more effective acting as catalytic inhibitors of DNA.
Previous studies have shown how 9-aminoacridines interact with DNA, thus illustrating
the process of exactly how these compounds act as catalytic inhibitors.2,3 One such study details
this same process, explaining how 9-aminoacridine based compounds were observed
intercalating into the DNA of pancreatic cell cancer lines, creating constrained negative

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supercoils, inhibiting relaxation of the DNA.4 As a result, the plasmid remains supercoiled in the
presence of an intercalator, creating a catalytic inhibitor. Credibility for these results was further
solidified in another study,4 in which is demonstrated the positive effects of 9-aminoacridine
based compounds in mice, and their effects on the mices DNA in terms of catalytic inhibitors.
Although mice and humans are different in multiple ways, their basic cell structure is still the
same, which leads to the conclusion that these same results will hold true for humans. Based on
the results on the plethora of experiments presented, there is much evidence to support the role of
9-aminoacridine based compounds acting as catalytic inhibitors of DNA.
9-aminoacridines have also been proven to be toxic to tumor cells displaying
simultaneous inhibition of NF-kB and induction of p53, which plays a major role in cancer cell
proliferation. One study explains how quinacrine, a derivative of 9-aminoacridine, was observed
having similar effects on the NF-kB and p53 signaling pathways as 9-aminoacridines. Therefore,
derivatives of this compound have been proven to have the potential to fight cancerous tumors.
A multitude of scholarly articles have been presented about how to best approach the
issue of synthesizing 9-aminoacridine based compounds, showing the different steps that were
used for each experiment. One article discusses some methods for the synthesis of
diaryliodonium triflate salts.6 These methods include using aryl iodides, iodines, and arenes to
synthesize diaryliodonium triflate salts. Both of these procedures have proven to be not only high
yielding, but also to be easily applicable with relatively short reaction times, as compared to
other protocols.
Unfortunately, in this experiment, there are some steps that must be substituted for others.
For instance, Albrecht, Defoin, and Tarnus7 portray their process, saying that a direct method was
used to transform the alcohol, R-OH, into O-substituted hydroxylamines, R-ONH2.7 The method

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begins with an O-alkylation of the tert-butyl N-hydroxycarbamate with the alcohol mesylates,
which is then followed by acidic N-deprotection. Mesylation of the alcohols occurs in the next
step, followed by the reaction of the crude mesylates with the hydroxyamic acid. The end result
yields O-hydroxamates. This study repeated the same procedure with the exception of R-OH
being substituted for cyclo-hexyl mesylate in the second reaction. Another study describes the
usefulness of using amino ethers as intermediates in organic synthesis, explaining their use in the
work of the anhydrous copper acetate mediated reaction O-arylation and O-styrylation of amino
alcohols for a new series of aminoethers synthesis.8
Although anti-tumor drugs have proven to be effective in killing cancerous cells and
inducing apoptosis, as with any treatment, there are unwanted side effects.1 Anti-tumor drugs
function by targeting rapidly dividing cells and trying to stimulate apoptosis in them. However,
sometimes healthy cells are targeted as well, leading to the destruction of these healthy cells.
Although there is no way for this to be avoided, there are still ways to minimize these side
effects. For example, many 9-aminoacridines undergo hydrolysis reactions in vivo, creating a
need for higher doses to be administered to patients. Because of this, the novel compound, Ophenyl-N-(9-acridinyl)-hydroxylamine, is to be created in the hopes that this compound will be
less susceptible to undergoing hydrolysis reactions, therefore allowing smaller doses to be
administered to patients, creating a more effective drug overall.
The need to create novel anti-tumor drugs cannot be understated. 9-aminoacrdine based
compounds have been deemed extremely effective in this area, binding to DNA, catalytically
inhibiting the function of topoisomerase II. The only downfall to this specific treatment, that has
been observed thus far, is the fact that this compound tends to undergo hydrolysis reactions in
vivo. This creates a need for higher dosages to be administered to patients. A series of steps has

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been obtained from a variety of sources that detail certain methods about how to best synthesize

9-aminoacridine based compounds.6,7,8,9,10 All of these steps could be used as alternative methods
in this experiment. Our goal in creating the novel anti-tumor drug, O-phenyl-N-(9-acridinyl)hydroxylamine, is to create a drug that has a stronger bond to DNA, allowing the drug to be
administered in smaller dosages to patients. This should, in turn, lead to a more effective antitumor drug overall.

METHODS AND MATERIALS

Main Reaction: Synthesis of O-phenyl-N-(9-acridinyl)-hydroxylamine
Overview
The synthesis described below of O-phenyl-N-(9-acridinyl)-hydroxylamine was
performed in a multi-step process that began with the synthesis of diaryliodonium triflate salt by
reacting iodobenzene with benzene. Next, Step 2 illustrated how to synthesize Nphenyloxyphthalimide by mixing the diaryliodonium triflate salt with N-hydroxyphthalimide,
thus causing a reaction to occur. This was followed by Step 3, which detailed the hydrolysis of
N-phenyloxyphthalimide, which produced the compound O-phenylhydroxylamine. Finally, in
Step 4, O-phenyl-N-(9-acridinyl)-hydroxylamine was synthesized by mixing Ophenylhydroxylamine with 9-chloroacridine, thus causing a reaction, producing O-phenyl-N-(9acridinyl)-hydroxylamine.

Step 1: Synthesis of Diaryliodonium Triflate

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Figure 1: Synthesis of Diaryliodonium Triflate

Step 2: Synthesis of N-phenyloxyphthalimide

Figure 2: Synthesis of N-phenyloxyphthalimide

Step 3: Hydrolysis of N-phenyloxyphthalimide

Figure 3: Hydrolysis of N-phenyloxyphthalimide

Step 4: Synthesis of O-phenyl-N-(9-acridinyl)-hydroxylamine

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Figure 4: Synthesis of O-phenyl-N-(9-acridinyl)-hydroxylamine

Step 1: Synthesis of Diaryliodonium Triflate

Based off of the procedure done by Marcin Bielawski6, 26 L (0.23 mmol) of
iodobenzene and 58 mg (0.26 mmol, 1.1 equivalents) of m-chloroperbenzoic acid (mCPBA) was
dissolved in 1 mL of dichloromethane under Argon gas in a 10 mL round bottom flask. Next, 23
L (0.26 mmol) of benzene was injected via needle to the same flask. This is illustrated in Figure
1. Then, 61 L (0.69 mmol, 3 equivalents) of trifluoromethanesulfonic acid (TfOH) was added
dropwise as well and the solution was left to stir for 10 minutes at room temperature and was
then concentrated using the rotary evaporator. Next, 1 mL of diethyl ether (Et2O) was added and
was left to be stirred in the mixture for 10 minutes and evaporated with the rotary evaporator,
after which time the solution was put in the fridge for 30 or more minutes. This allowed enough
time for a white solid to precipitate. After this, the white solid was washed with Et2O and was
vacuum dried in order to obtain the diaryliodonium triflate salt crystals, which had, at that time,
formed.

Step 2: Synthesis of N-phenyloxyphthalimide

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Based off of the methods done by Raju Ghosh10, to a dry 10 mL round bottom flask, 21
mg (0.55 mmol) of Potassium t-butoxide (t-BuOK) was dissolved in 2 mL of anhydrous
dimethylformamide (DMF). Next, 27 mg (0.50 mmol) of N-hydroxyphthalimide was added. This
is shown in Figure 2. Then, the flask was stoppered and Argon gas was injected in order to
perform the procedure under inert atmosphere conditions. The reaction was then stirred for 10
minutes at room temperature. Afterwards, the diaryliodonium triflate salt created in the previous
step was added to the solution and the reaction was heated to 60 Celsius and was left to stir for
one hour. Next, the reaction flask was cooled to room temperature and the organic phase was
washed with 20 mL of ethyl acetate (EtOAc) and 20 mL of deionized water in a separatory
funnel. The aqueous layer was then removed and the organic phase was washed with 20 mL of
concentrated brine solution. Afterwards, the aqueous layer was again removed and the remaining
organic phase was dried with anhydrous magnesium sulfate (MgSO4). Finally, the mixture was
filtered in order to remove the MgSO4 and was then concentrated using the rotary evaporator.
Thin Layer Chromatography (TLC) analysis using 20:80 hexane:EtOAc as the eluent and 1HNMR spectrum analysis were performed to verify the presence of N-phenyloxyphthalimide.

Step 3: Hydrolysis of N-phenyloxyphthalimide

Again, based on the procedure detailed by Raju Ghosh10, to the compound synthesized in
the previous step, N-phenyloxyphthalimide, 2 mL of chloroform (CHCl3) was added and the
solution was then stoppered in a round bottom flask. This is described in Figure 3. The reaction
was then injected with Argon gas in order to perform the procedure under inert atmospheric
conditions. 1 mL of 7N ammonia (NH3) was then added to the solution dropwise via needle and
was stirred for 15 hours at room temperature. After this, the mixture was absorbed to silica and

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was washed with 100 mL of 20:80 Et2O:pentane. Next, the solution was concentrated using a
rotary evaporator, which produced an oily, yellow substance, O-phenylhodroxylamine.

Step 4: Synthesis of O-phenyl-N-(9-acridinyl)-hydroxylamine

The last step of the experiment was based off of Carlsons9 reaction, in which a mixture
of 75 mol molten phenol (PhOH): 1 mol 9-chloroacridine was prepared. This is illustrated in
Figure 4. Then, the mixture was stirred at a range of 80-100 Celsius and was stirred for 6 hours.
Afterwards, the mixture was cooled to room temperature, and 50 mL of CH2Cl2 was then added.
Then, 0.1 M of sodium hydroxide was added to the solution, which was then subsequently added
to a separatory funnel in order to remove excess phenol from the mixture. Next, the organic
matter that was created was dried over anhydrous MgSO4 and then gravity filtered in order to
remove the drying agent. Finally, the solution was concentrated using a rotary evaporator and, if
needed, was purified.

Product Analysis
Methods such as proton (1H-NMR) and carbon (13C-NMR) nuclear magnetic resonance
analysis, Infrared (IR) spectroscopy, and mass spectrometry were used for spectral analysis of
the product. Additional methods may be used as well in order to identify the synthesized product.

Side Reaction: Synthesis of O-(4-methyl)-phenyl-N-(9-acridinyl)-hydroxylamine

Overview

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The synthesis described below was performed in a multi-step process which begins in
Step 1 with the synthesis of diaryliodonium triflate salt by mixing toluene and 4-iodotoluine in
order to create a reaction. This was followed by Step 2, in which the diaryliodonium triflate salt
reacted with N-hydroxyphthalimide to create N-phenyloxyphthalimide.

Step 1: Synthesis of Diaryliodonium Triflate

Figure 5: Synthesis of Diaryliodonium Triflate

Step 2: Synthesis of N-(4-methyl)-phenyloxyphthalimide

Figure 6: Synthesis of N-(4-methyl)-phenyloxyphthalimide

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Step 3: Hydrolysis of N-(4-methyl)-phenyloxyphthalimide

Figure 7: Hydrolysis of N-(4-methyl)-phenyloxyphthalimide

Step 4: Synthesis of O-(4-methyl)-phenyl-N-(9-acridinyl)-hydroxylamine

Figure 8: Synthesis of O-(4-methyl)-phenyl-N-(9-acridnyl)-hydroxylamine

Step 1: Synthesis of Diaryliodonium Triflate

Based off of the procedure done by Marcin Bielawski6, 120 mg of m-chloroperbenzoic
acid (mCPBA) was dissolved in 2 mL of dichloromethane under Argon gas in a 10 mL round

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bottom flask. Next, 100 mg of 4-iodotoluine and 55 L of toluene were added dropwise to the
same flask. Then, 80 L of trifluoromethanesulfonic acid (TfOH) was added dropwise as well
and the solution was left to stir for 10 minutes at room temperature. This reaction is detailed in
Figure 5. Then, 2 mL of ethyl acetate (EtOAc) was added and was left to be stirred in the mixture
for 10 minutes and evaporated with the rotary evaporator, after which time the solution was put
in the fridge overnight. This allowed enough time for a white solid to precipitate. After this, the
white solid was washed with EtOAc and was vacuum dried in order to obtain the diaryliodonium
triflate salt crystals, which had, at that time, formed.

Step 2: Synthesis of N-(4-methyl)-phenyloxyphthalimide

Based off of the methods done by Raju Ghosh10, to a dry 10 mL round bottom flask, 64
mg of Potassium t-butoxide (t-BuOK) was dissolved in 4 mL of anhydrous dimethylformamide
(DMF). Next, 82 mg of N-hydroxyphthalimide was added. Then, the flask was stoppered and
Argon gas was injected in order to perform the procedure under inert atmosphere conditions. The
reaction was then stirred for 10 minutes at room temperature. This reaction is detailed in Figure
6. Afterwards, the diaryliodonium triflate salt created in the previous step was added to the
solution and the reaction was heated to 60 Celsius and was left to stir for two hours. Next, the
reaction flask was cooled to room temperature and the organic phase that was created was
washed with 20 mL of ethyl acetate (EtOAc) and 20 mL of deionized water in a separatory
funnel. Then, the aqueous layer was removed and the solution was mixed with 20 mL of
concentrated brine solution. The organic phase was then transferred to an Erlenmeyer flask and
put in the hood overnight to evaporate the EtOAc.

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Step 3: Hydrolysis of N-(4-methyl)-phenyloxyphthalimide

Again, based on the procedure detailed by Raju Ghosh10, to the compound synthesized in
the previous step, N-phenyloxyphthalimide, 2 mL of chloroform (CHCl3) was added and the
solution was then stoppered in a round bottom flask. The reaction was then injected with Argon
gas in order to perform the procedure under inert atmospheric conditions. 1 mL of 7N ammonia
(NH3) was then added to the solution dropwise via needle and was stirred for 15 hours at room
temperature. This process is detailed in Figure 7. After this, the mixture was absorbed to silica
and was washed with 100 mL of 20:80 Et2O:pentane. Next, the solution was concentrated using a
rotary evaporator, which produced an oily, yellow substance, O-phenylhodroxylamine.

Step 4: Synthesis of O-(4-methyl)-phenyl-N-(9-acridinyl)-hydroxylamine

The last step of the experiment was based off of Carlsons9 reaction, in which a mixture
of 75 mol molten phenol (PhOH): 1 mol 9-chloroacridine was prepared. Then, the mixture was
stirred at a range of 80-100 Celsius and was stirred for 6 hours. Afterwards, the mixture was
cooled to room temperature, and 50 mL of CH2Cl2 was then added. This process is illustrated in
Figure 8. Then, 0.1 M of sodium hydroxide was added to the solution, which was then
subsequently added to a separatory funnel in order to remove excess phenol from the mixture.
Next, the organic matter that was created was dried over anhydrous MgSO4 and then gravity
filtered in order to remove the drying agent. Finally, the solution was concentrated using a rotary
evaporator and, if needed, was purified.

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Product Analysis

Methods such as proton (1H-NMR) and carbon (13C-NMR) nuclear magnetic resonance
analysis, Infrared (IR) spectroscopy, and mass spectrometry were used for spectral analysis of
the product. Additional methods may be used as well in order to identify the synthesized product.
RESULTS AND DISCUSSION
Results
A series of N-phenyloxyphthalimides was synthesized using methods proposed by
Bielawski6, Ghosh10, and Carlson9 in order to create the compounds O-phenyl-N-(9acridinyl)hydroxylamine and O-(4-methyl)-phenyl-N-(9acridinyl)-hydroxylamine. After running through
both of these reactions, the results showed that the compound O-phenyl-N-(9acridinyl)hydroxylamine (116-029) had a 25% yield, and the compound O-(4-methyl)-phenyl-N(9acridinyl)-hydroxylamine (116-031) had a 175% yield.

Discussion
Based on the 25% yield of reaction 116-029, it can be concluded that, during the reaction
process, as the product was transferred from containers, a fairly large amount of air was allowed
into the reaction flasks as well. As a result, the percent yield of the product was significantly
lower than what was hoped for. However, based off of the 175% yield of reaction 116-031, it can
be determined that the solvent that was used, ethyl acetate (EtOAc), was not allowed enough
time to evaporate when the reaction was placed in the hood. Because of this, more product was

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in the reaction flask then what was originally supposed to be there, allowing for a yield higher
than 100% to occur.
For future research, measurements should be more precise. During the course of the
project, not all of the measurements used were exact, leading to some problems in our results.
For instance, if we had been more exact in reaction 116-031, then the amount of EtOAc would
not have been overestimated. This would have led to a more precise percent yield than the 175%
yield that was obtained. It has also been hypothesized that substituting benzene for nitrobenzene
will be beneficial. Nitrobenzene has one less hydrogen, but also has an extra nitrite, which may
prove to be more successful in terms of binding with the DNA. Additional research will be
conducted at the University of Northern Colorado, located in Greeley, CO, in order to obtain
further results.

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WORKS CITED
1. Sudhakar, A. (2009, December 1). History of Cancer, Ancient and Modern Treatment
Methods. Retrieved June 28, 2016, from file:///E:/FSI/Research/Scholarly Article.pdf
2. Oppegard, L. M.; Ougolkov, A. V.; Luchini, D. N. et al. (2009). Novel acridine-based
compounds that exhibit an anti-pancreatic cancer activity are catalytic inhibitors of
human topoisomerase II. Retrieved June 25, 2016. Eur. J. Pharmacol, 602, 223-229.

3. Kumar, P.; Kumar, R.; Prasad, D. (2013). Synthesis and anticancer study of 9-aminoacridine
derivatives. Retrieved June 25, 2016. Arabian Journal of Chemistry, 6, 79-85.
4. Glvez-Peralta, M.; Hackbarth, J. S.; Flatten, K. S. et al. (2009). On the role of topoisomerase
I in mediating the cytotoxicity of 9-aminoacridine-based anticancer agents. Retrieved
June 25, 2016. Bioorg. Med. Chem. Lett, 19, 4459-4462.
5. Guo, C.; Gasparian, A.; Zhuang, Z. et al. (2009). 9-Aminoacridine-based anticancer drugs
target the PI3K AKT mTOR, NF-B and p53 pathways. Retrieved June 25, 2016.
Oncogene, 28, 1151-1161.
6. Bielawski, M.; Zhu, M.; Olofsson, B. (2007). Efficient and General One-Pot Synthesis of
Diaryliodonium Triflates: Optimization, Scope and Limitations. Retrieved June 25, 2016.
Advanced Synthetic Catalysis, 349, 2610-2618.
7. Albrecht, S.; Defoin, A.; Tarnus, C. (2007). Simple Preparation of O-Substituted
Hydroxylamines from Alcohols. Retrieved June 25, 2016. Synthesis, 10, 1635-1638.

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8. Al-Masum, M., Quinones, L., & Cain, L. T. (2016, June). Microwave Application and
Anhydrous Cu(OAc)2 Mediated O-Arylation of Aliphatic Amino Alcohols. Retrieved
June 27, 2016, from file:///E:/FSI/Research/Scholarly Article Source #11.pdf
9. Carlson, A. et al. (2015). O-benzyl-N-(9-acridinyl)-hydroxylamines as Potential Antitumor
Agents(Unpublished masters thesis). Master of Science Thesis, University of Northern
Colorado, Greeley, CO. Retrieved June 27, 2016.
10. Ghosh, R.; Oloffson, B. (2014). Metal-Free Synthesis of N-Aryloxyimides and
Aryloxyamines. Organic Letters. Retrieved June 27, 2016.