sgonzalez honors thesis

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Synthesis and Biological Testing of Novel Pharmacological Agents: Dihydropyrimidinone Derivatives

By Santiago Gonzalez

Presented to The Honors Collegein partial fulfillment of the requirements for Honors Senior Thesis

Arkansas State University

_________________ __________________________________

Dr. John Hershberger, Advisor

_________________ _________________________________

Dr. Allyn Ontko

_________________ ________________________________

Dr. Mohammad Alam

_________________ __________________________________

Ms. Rebecca Oliver, Director of The

Honors College

May 2016

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TABLE OF CONTENTS

TITLE PAGE…………………………………………………………………………………… .1

TABLE OF CONTENTS……………………………………………………………….………. 2

LIST OF FIGURES……………………………………………………………………………... 3

ACKNOWLEDGEMENTS………………………………………………………….…………. .4

ABSTRACT…………………………………………………………………….………………...5

INTRODUCTION/BACKGROUNG…………………………………………………………….6

DIHYDROPYRIMIDINONE BIOACTIVITY..............................................................................6

REASONS FOR DIHYDROPYRIMIDINONE ACTIVITY…………………………….……....7

DIHYDROPYRIMIDINONE SYNTHESIS…………………………………………………......8

PHENETHYLAMINE BIOACTIVITY………………………………………………………….9

APPLICATION OF THE RESEARCH…………………………………………………………10

SYNTHETIC PATHWAY OUTLINE..........................................................................................11

DIHYDROPYRIMIDINONE WITH DIFFERENT FUNCTIONAL GROUPS……………......12

BROMINATION OF THE ALLYLIC SITES IN DIHYDROPYRIMIDINONES………….….14

NBS VS. TRIMETHYLAMMONIUM TRIBROMIDE………………………………...............14

CYCLIZATION METHODOLOGY AND MECHANISM…………………………………. ...15

SUBSTITUTED PHENETHYLAMINES USED IN THE PROJECT.........................................16

PHENETHYLAMINE...................................................................................................................16

INCREASED TEMPERATURES…………………………………………….……………........18

DECREASED STERIC HINDRANCE…………….…………………………………………....19

SYNTHESIZED STRUCTURES..................................................................................................19

EXPERIMENTAL SECTION...................................................................................................... 21

EXPERIMENTAL DATA FROM SYNTHESIZED PRODUCTS..............................................21

ANTIBIOTIC TESTING...............................................................................................................31

ANTIBIOTIC TESTING RESULTS.............................................................................................31

CONCLUSIONS ……………………………………………………………………….……….32

REFERENCES………………………………………………………………….……………….34

APPENDICES (1-10) ...................................................................................................................37

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List of Figures FIGURE 1: DIHYDROPYRIMIDINONES IN PHARMACOLOGICAL AGENTS

FIGURE 2: DNA INTERCALATION

FIGURE 3: BIGINELLI REACTION

FIGURE 4: BASIC PHENETHYLAMINE SCAFFOLD

FIGURE 5: NOREPINEPHRINE

FIGURE 6: DOPAMINE

FIGURE 7: ALDEHYDE AND PHENETHYLAMINE VARIANTS

FIGURE 8: SYNTHETIC PATHWAY OUTLINE

FIGURE 9: DIHYDROPYRIMIDINONE SCAFFOLDS

FIGURE 10: BROMINATION REACTION

FIGURE 11: CYCLIZATION

FIGURE 12: CYCLIZED PRODUCTS MADE WITH REGULAR PHENETHYLAMINE

FIGURE 13: INTERMEDIATE PRODUCTS

FIGURE 14: CYCLIZED PRODUCTS AT 100° CELSIUS

FIGURE 15: ETHYL ESTERS VS. METHYL ESTERS

FIGURE 16: SYNTHESIZED PRODUCTS

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Acknowledgements This thesis could not have been completed without the support of my mentor and committee

members. I would like to thank Dr. John Hershberger for his mentorship for the past two years in

the process of gathering data, presenting, and writing of the thesis. I am also grateful with Dr.

Hershberger for his guidance in the process of making career decisions, applying to medical

schools, and pursuing various academic opportunities. My experience working in your lab and

having you as a mentor have made me grow as a person and as a student of science, and for all of

this, I am deeply grateful. I would like to thank my committee members Dr. Allyn Ontko, and

Dr. Mohammad Abrar Alam for their input throughout the research process. I would like to

thank Dr. David Gilmore for his assistance in the antibiotic testing of the synthesized

compounds. Lastly, I would like to thank my parents, grandmother, and sister for their trust and

support throughout my entire college career. I could not have done it if it were not for your

unconditional love, for your bold decision of sacrificing everything to seek a better future for all

of us, and for the fact that you all are living role models of the importance of education and the

pursuit of one’s dreams.

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Abstract The goal of this project is to synthesize novel pharmacological agents by combining two

privileged structures; dihydropyrimidinones and phenethylamines. Dihydropyrimidinones are

significant building blocks and versatile synthons that are frequently seen in medicinal chemistry

due to their many pharmacological properties, including calcium channel blocking, bacterial

growth inhibiting, HIV inhibiting, and antitumor activity. Phenethylamines form the scaffold for

catecholamine adrenergic hormones such as norepinephrine and dopamine. By combining these

two privileged bioactive scaffolds, we predict that the resulting structure will also be bioactive.

After various experimental trials, we have developed a reaction pathway that encompasses three

straightforward steps to synthesize the desired products. We have characterized and tested the

synthesized products for bioactivity using the Kirby-Bauer disk diffusion assay.

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Introduction/Background Dihydropyrimidinone bioactivity

Dihydropyrimidinones are significant building blocks and versatile synthons that are frequently

seen in medicinal chemistry and are used in pharmacological agents as calcium channel blockers,

HIV inhibitors, antitumor agents, HIV gp-120-CD4 inhibitors and neuropeptide Y antagonists

(1). One of the most widely used cancer drugs is a dihydropyrimidinone derivative named

Monastrol, which is an inhibitor of human kinesin Eg5 that leads to mitotic arrest and apoptotic

cell death (Figure 1) (2). Another example of a bioactive dihydropyrimidinone is Nitractin,

which is a highly effective antiviral agent. (Figure 1) (3).

Figure 1: Dihydropyrimidinones in Pharmacological agents; Monastrol and Nitractin

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Reasons for Dihydropyrimidinone Bioactivity

Dihydropyrimidinones have the ability to interact with DNA. A planar polycyclic aromatic

portion of the drug is inserted between adjacent stacked base pairs of a double stranded DNA in

a process named intercalation, which results in helix extension and unwinding of the DNA

(Figure 2) (4). The insertion of an intercalating molecule occurs in the minor groove of DNA and

is held together by hydrogen bonds, charge transfer bonds, and van der Waals forces (5). The

most effective compounds are ligands capable of structure and sequence selective binding to

DNA, since they are able to unwind a specific target sequence that causes the inhibition of a

particular gene or protein that produces a disease (5). The unwinding of the DNA molecule

prevents transcription, which blocks the replication process of the cell containing the intercalated

DNA (6). There is not a consensus on how the unwinding disrupts the transcription process, but

one hypothesis is that it inhibits topoisomerases, which are necessary for uncoiling a DNA

molecule prior to replication (6). Inhibiting cell replication leads to cell death, which can cause a

decrease in tumor size in cancer patients (6). By being able to stop transcription, and thus

causing death of specific cells, dihydropyrimidinones are able to act as a powerful cancer

treatment.

Figure 2: DNA Intercalation

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Dihydropyrimidinone Synthesis

The first dihydropyrimidinone was first synthesized by Pietro Biginelli in 1893 by an acid-

catalyzed cyclocondensation reaction of ethyl acetoacetate, urea, and benzaldehyde (Figure 3)

(7). The reaction was performed by heating a mixture of the three components dissolved in

ethanol with hydrochloric acid as a catalyst (7). The product of this synthesis was identified by

Pietro Biginelli as 3, 4- dihydropyrimidine- 2 (1H) – one (Figure 3) (7). Although this reaction is

simple, it has poor yield. Many catalytic processes have been discovered (8). A method to

catalyze the cyclization of the Biginelli product is to use a Bronsted or Lewis acid with methods

based on metal salts with non-nucleophilic anions (8). The most effective catalysts use reagents

which have dehydrating properties as well as protic or Lewis acidic behavior. Some of these

compounds are: ethyl polyphosphate, acetic anhydride, and trimethylsilyl chloride (TMSCl) (8).

Synthetic studies also show that triethylorthoformate (TEOF), associated with citric acid or

oxalic acid acts as a system that highly increases yields of dihydropyrimidinones, especially

when weak acids are used (8).

Figure 3: Biginelli Reaction. Cyclocondensation of ethyl acetoacetate, benzaldehyde, and urea

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Phenethylamine Bioactivity

Phenethylamines (Figure 4) are a basic scaffold for a series of compounds that have high

biological activity. Some of these compounds are neurotransmitters such as norepinephrine,

sympathomimetics such as ephedrine and amphetamine, and hallucinogens such as mescaline

(9). Many phenethylamines are also ubiquitous in hallucinogenic designer drugs such as alpha-

methyltryptamine (AMT), dimethyltryptamine (DMT), and psilocybin (10).

Figure 4: Phenethylamine

The reason for the neurologic activity of phenethylamine scaffolds has to do with the structural

similarity between the tryptamines and serotonin, and phenethylamines and dopamine, which

makes these compounds act as 5 hydroxytryptamine (5HT) receptor agonists in the central

nervous system (10). Norepinephrine is an example of one of the most significant

phenethylamines, which can be seen by the many crucial effects that it has on the body (Figure

5) (11). Norepinephrine controls the flight-or fight response by acting as an alpha-adrenergic

receptor activator, which increases heart rate, dilates bronchioles, increases gluconeogenesis and

sugar breakdown, and it increases blood flow to muscles (11).

Figure 5: Norepinephrine

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Another important example of a bioactive phenethylamine is dopamine (Figure 6) (12).

Dopamine regulates hormonal, cardiovascular, retinal, renal, and immune system functions

among others (12). Proper release and functioning of dopamine and its receptors is so crucial in

the body, that malfunctions in these systems have been linked to Parkinson’s disease, ADHD,

and Tourette’s syndrome (12).

Figure 6: Dopamine

As it can be seen by the information above, phenethylamines and its derivatives have lots of

bioactive properties in the body, which is expected to also occur in the compounds that were

synthesized in this project.

Applications to the Research

The main idea of the project is to combine two privileged structures; dihydropyrimidinones and

phenethylamines in order to form a highly bioactive chimeric compound. The strategy of

combining two or more bioactive structures with the goal of enhancing pharmacological benefits,

and decreasing side effects has been seen in literature. The name of this strategy is the synthesis

of polyfunctional drugs (13). These polyfunctional drugs are made by combining two or more

bioactive agents in order to produce a single compound with multiple biological activities (13).

The advantages of using polyfunctional drugs include the possibility of synergistic drug effects,

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and more predictable pharmacokinetics and pharmacodynamics than drug cocktails which use

various drugs to achieve the same effect (13). As a result of combining dihydropyrimidinones

and phenethylamines, it is expected to synthesize a chimeric molecule with unique

pharmacological properties, which will be locally tested for antibiotic activity with the use of the

Kirby-Bauer disk diffusion assay. Pharmacological Screening will also be performed by sending

the synthesized products to the High Throughput Screening (HTS) services of the National

Institutes of Health (NIH) (14).

Synthetic pathway outline

The synthesis of the target molecules is split into three separate steps. The first step is to

synthesize the Biginelli products with various functional groups. Different functional groups are

added to the molecule by changing the aldehyde at the beginning of the reaction, which end up

carrying over to the final synthesized products (Figure 7).

Figure 7: Aldehyde and Phenethylamine

The second step of the synthetic pathway is to brominate the allylic site in the Biginelli product.

The third step is to perform a novel cyclization of a lactam ring by using a phenethylamine as a

nucleophile. Various functional groups can be added to the target product by changing the

functional groups in the phenethylamines used for cyclization (Figure 7). The various

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modifications to the reaction conditions in each of these steps will be explored in depth

throughout the remainder of the thesis. The outline can be seen below (Figure 8):

Figure 8: Synthetic Pathway Outline. Biginelli Synthesis, Bromination, and Cyclization

Synthesis of dihydropyrimidinone scaffolds with various functional groups

In this section of the project, Nozomi Arai and Kei Ohgo helped with the synthesis of some

dihydropyrimidinones. As mentioned earlier in the paper, the synthesis of dihydropyrimidinones

is done through the standard Biginelli reaction. The reaction is done by an acid-catalyzed

cyclocondensation reaction of ethyl acetoacetate, urea, and benzaldehyde (2). The way in which

we added functional groups to the dihydropyrimidinones is by using different commercially

available aldehydes at the beginning of the cyclocondensation reaction. The aldehydes used in

the project include: benzaldehyde, 1-napthaldehyde, 2- naphthaldehyde, 4-nitrobenzaldehyde, 4-

trifluoromethylbenzaldehyde, 2-nitrobenzaldehyde, 3-chloro benzaldehyde, 2-

bromobenzaldehyde, 3-bromobenzaldehyde, 2-furaldehyde, 3-furaldehyde, and 4-

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trifluoromethoxy benzaldehyde. The raw synthesized products were recrystallized with ethanol

in order to increase purity. The synthesized dihydropyrimidinone scaffolds can be seen below

(Figure 9).

Figure 9: Synthesized Dihydropyrimidinone Scaffolds

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Bromination of the allylic sites in dihydropyrimidinones

The next step in the project was to brominate the allylic site in the synthesized

dihydropyrimidinone structures (Figure 10). The two brominating agents we used for this step in

the process are N-bromosuccinimide (NBS) and phenyltrimethylammonium tribromide. The

NBS reacts with the allylic site through a radical reaction that involves the homolytic cleavage of

the Br2 with light, followed by the extraction of the allylic H and the reaction of this radical with

another equivalent of Br2 to give the desired product (15). The preferred method due to

experimental observations is the bromination with phenyltrimethylammonium tribromide due to

its tendency to cause only a single bromination (16).

Figure 10: Bromination Reaction

N-bromosuccinimide (NBS) vs. Phenyltrimethylammonium Tribromide

The procedure for the Bromination reaction with NBS involved 1.1 NBS equivalent, and 1

equivalent of the Biginelli product dissolved in dichloromethane (DCM), and reacted at room

temperature. As predicted by literature, NBS was a strong brominating agent (15), and the NMR

spectra showed dibrominations at the allylic site. Phenyltrimethylammonium tribromide (PMTB)

was used due to its behavior as a milder brominating agent (16). The methodology for the

Bromination reaction with PMTB involved 1 PMTB equivalent, and 1 equivalent of the Biginelli

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product dissolved in DCM, and reacted at room temperature. The 1H Nuclear Magnetic

Resonance (NMR) Spectra showed a single Bromination at the allylic site. In order to increase

the purity of the brominated Biginelli product, recrystallization with ethanol was performed. The

desired goal of recrystallizing the molecule was not reached however, as the NMR spectra after

the recrystallization showed the dissociation of the bromine from the molecule. Due to the

repeated debromination after various recrystallization procedures, we decided to perform a one

pot reaction mechanism. The one pot reaction mechanism involves the evaporation of DCM from

the reaction flask by rotavaping, followed by the addition of methanol in order to re-dissolve the

brominated Biginelli product. Lastly, the phenethylamine is added to perform the cyclization.

Cyclization Methodology and Mechanism The third step in the synthesis of the desired product is the addition of the primary

phenylethylamine group to perform a second order nucleophilic substitution (SN2) intermolecular

cyclization. The cyclization reaction was done by adding the 5 equivalents of the phenethylamine

to the round bottom flask with the brominated Biginelli dissolved in methanol. The reaction was

stirred at room temperature overnight. The hypothesized mechanism involves the nucleophilic

attack from the lone pair on the nitrogen of the primary amine on the electrophilic carbon

attached to the bromine. The bromine will be knocked off the molecule as a leaving group. The

amino group from the extra equivalents of the phenethylamine will pull a proton off of the

nitrogen, and the remaining lone pair in the nitrogen will then nucleophilically attack the

electrophilic carbonyl carbon in the ester group, the O-R group will act as a leaving group, and

an intra-molecular cyclization will occur as a result (Figure 11). A similar mechanism is seen in

literature in a reaction called the Pictet-Spengler cyclization in which the primary amine acts as a

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nucleophile by attacking an electrophilic carbonyl group that subsequently undergoes an

intramolecular cyclization (17).

Figure 11: SN2 reaction followed by an intramolecular cyclization

Substituted Phenethylamines used in the project

In order to add variety to the functional groups present in the final cyclized products, pre-

manufactured phenethylamines with different functional groups were used to perform the

cyclization. The phenethylamines that were used include: phenethylamine, tyramine, 2-

methoxyphenethylamine, 3-methoxyphenethylamine, 4-methoxyphenethylamine, 3,4-

dimethoxyphenethylamine, 2-(3-chlorophenyl) ethylamine, 4-trifluoromethylphenethylamine, 2-

(3-trifluoromethylphenyl) ethylamine, 3-fluorophenethylamine, and 4-fluorophenethylamine.

Phenethylamine

The phenethylamine that worked the best for the cyclization step is the unsubstituted

phenethylamine with no groups attached to it. As soon as the phenethylamine was added to the

reaction, there was a visible color change from light yellow to clear, and soon thereafter a white

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precipitate was formed. The product of this reaction was then easily filtered, and recrystallized

with ethanol. The following products were formed with phenethylamine (Figure 12).

Figure 12: Cyclized products made with phenethylamine

Not all of the phenethylamine variants precipitated in the cyclization step. We assume most

phenethylamines worked to perform the first SN2 reaction in which the lone pair on the

phenethylamine’s nitrogen nucleophilically attacks the brominated site on the molecule. Most of

the phenethylamine variants ended up forming an intermediate in which the cyclization did not

yet occur. The intermediate product of the SN2 prior to the cyclization, which was precipitated

from methanol can be seen below (Figure 13).

Figure 13: Intermediate product from the SN2 reaction

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In order to increase the chances for cyclization, various changes to the reaction conditions were

performed. The modifications made to the conditions were focused on increasing the entropy of

the system by raising the temperature in order to increase chances for favorable molecular

collisions, and a decrease in the steric hindrance in areas of the molecule that are crucial for

proper cyclization.

Increased Temperature The first attempt at increasing the entropy of the system was done by increasing the temperature

of the reaction from room temperature to 60° Celsius with methanol as a solvent. As indicated by

NMR spectra, there were no intramolecular cyclizations that occurred at this temperature. In

order to further increase the entropy of the system the solvent was changed to propanol, and the

temperature was brought up to 100° Celsius. Favorable results were encountered at this

temperature, and two molecules that were unable to cyclize prior to the increase in temperature

showed peaks on the NMR that indicated full cyclization. The structures that were synthesized

after the increase in temperature are seen below (Figure 14).

Figure 14: Cyclized structures at 100° Celsius

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Decreased steric hindrance In order to decrease the steric hindrance at the site of cyclization, a different acetoacetate was

used in the cyclocondensation reaction of the starting dihydropyrimidinone (Biginelli) product.

The acetoacetate used at the beginning of the project was ethyl acetoacetate. In order to decrease

the hindrance of the molecule, methyl acetoacetate was used instead. By taking away a carbon

from the R group in the ester group, steric hindrance was decreased. The difference in the

structures made with the ethyl acetoacetate vs. the methyl acetoacetate are seen below (Figure

15). By decreasing the hindrance, there is an increased chance of a favorable collision between

the nitrogen and the carbonyl carbon from the ester group in the molecule.

Figure 15: Structures made with ethyl acetoacetate vs. methyl acetoacetate Synthesized Structures After the methyl acetoacetate modification was done to decrease hindrance, the success in the

making of the cyclized final products increased dramatically. Multiple phenethylamines were

able to trigger a full inner molecular cyclization at room temperature. The synthesized structures

are seen below (Figure 16).

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Figure 16: Synthesized Structures

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Experimental Section The reagents were used as received and no precautions were taken to exclude air or moisture.

Experimental Data from Synthesized Products

Product 2 (APPENDIX 1,2): Dihydropyrimidinone 1A (2 mmol) was dissolved in 40 ml of

DCM, and mixed with Phenyltrimethylammonium tribromide (2 mmol) at room temperature and

the reaction was stirred for 2 hours. The DCM was rotavaped, and 40 ml methanol was added as

a solvent. Five equivalents of Phenethylamine (10 mmol) was added to the reaction flask, and it

was stirred overnight. The precipitate was filtered, and later recrystallized with ethanol. A yield

of 356.8 mg was obtained, with a percent yield of 52.5%. 1H NMR (300 MHz, DMSO-d6) δ 2.74

(t, 2H, J= 7.3 Hz), 3.44 (m, 7H), 3.84 (s, 2H), 5.14 (s, 1H), 7.16-7.35 (m, 10H), 7.54 (d, 7H, J=

2.1 Hz), 9.52 (s, 2H); 13C NMR 126.7, 126.8, 128.0, 128.8(2), 129.0, 139.4, 143.7, 150.2, 152.5,

168.7.

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Product 3: Dihydropyrimidinone 1K (4.1 mmol) was dissolved in 40 ml of DCM, and mixed

with Phenyltrimethylammonium tribromide (4.1mmol) at room temperature and the reaction was

stirred for 2 hours. The DCM was rotavaped, and 30 ml methanol was added as a solvent. Five

equivalents of Phenethylamine (20.5 mmol) was added to the reaction flask, and it was stirred

overnight. The precipitate was filtered, and later recrystallized with ethanol. A yield of 31.0 mg

was obtained, with a 2.7% yield.

Product 4: Dihydropyrimidinone 1G (1.7 mmol) was dissolved in 40 ml of DCM, and mixed

with Phenyltrimethylammonium tribromide (1.7 mmol) at room temperature and the reaction

was stirred for 2 hours. The DCM was rotavaped, and 30 ml methanol was added as a solvent.

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Five equivalents of Phenethylamine (8.5 mmol) was added to the reaction flask, and it was

stirred overnight. The precipitate was filtered, and later recrystallized with ethanol. The yield

was small, and it was used in its entirety for 1H NMR analysis.

Product 5 (APPENDIX 3): Dihydropyrimidinone 1A (.9605 mmol) was dissolved in 10 ml of

DCM, and mixed with Phenyltrimethylammonium tribromide (1.1mmol) at room temperature

and the reaction was stirred for 2 hours. The DCM was rotavaped, and 10 ml propanol was added

as a solvent. Five equivalents of 4-fluorophenethylamine (4.8 mmol) was added to the reaction

flask, and it was stirred for 2 hours at room temperature, followed by 4 hours at 100° Celsius.

The precipitate was filtered, and later recrystallized with ethanol. The yield was small, and it was

used in its entirety for 1H NMR analysis.

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Product 6 (APPENDIX 4): Dihydropyrimidinone 1A (.9605 mmol) was dissolved in 10 ml of

DCM, and mixed with Phenyltrimethylammonium tribromide (1.1mmol) at room temperature

and the reaction was stirred for 2 hours. The DCM was rotavaped, and 10 ml propanol was added

as a solvent. Five equivalents of 4-fluorophenethylamine (4.8 mmol) was added to the reaction

flask, and it was stirred for 2 hours at room temperature, followed by 4 hours at 100° Celsius.

The precipitate was filtered, and later recrystallized with ethanol. The yield was small, and it was

used in its entirety for 1H NMR analysis.

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Product 7: Dihydropyrimidinone 1D (1.7 mmol) was dissolved in 10 ml of DCM, and mixed



Five equivalents of phenethylamine (8.5 mmol) was added to the reaction flask, and it was

stirred overnight. The precipitate was filtered, and later recrystallized with ethanol. A yield of

85.1 mg was obtained, with a percent yield of 13.5%.

Product 8: Dihydropyrimidinone 1E (1.2 mmol) was dissolved in 10 ml of DCM, and mixed


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Five equivalents of phenethylamine (6 mmol) was added to the reaction flask, and it was stirred

overnight. The precipitate was filtered, and later recrystallized with ethanol. A yield of 224.4 mg

was obtained, with a percent yield of 35.4%.

Product 9 (APPENDIX 5): Dihydropyrimidinone 1L (.314 mmol) was dissolved in 10 ml of

DCM, and mixed with Phenyltrimethylammonium tribromide (.314 mmol) at room temperature

and the reaction was stirred for 2 hours. The DCM was rotavaped, and 10 ml methanol was

added as a solvent. Five equivalents of phenethylamine (1.57 mmol) was added to the reaction

flask, and it was stirred overnight. The precipitate was filtered, and later recrystallized with

ethanol. A yield of 26.4 mg was obtained, with a percent yield of 14.7%.

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Product 10: Dihydropyrimidinone 1C (1.7 mmol) was dissolved in 10 ml of DCM, and mixed




stirred overnight. The precipitate was filtered, and later recrystallized with ethanol. The yield

was small, and it was used in its entirety for 1H NMR analysis.

Product 11: Dihydropyrimidinone 1B (1.7 mmol) was dissolved in 10 ml of DCM, and mixed


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stirred overnight. The precipitate was filtered, and later recrystallized with ethanol. A yield of

69.3 mg was obtained, with a percent yield of 10.6%.

Product 12 (APPENDIX 6, 7): Dihydropyrimidinone 1A (1 mmol) was dissolved in 10 ml of



a solvent. Five equivalents of 4 trifluoromethylphenylethylamine (5 mmol) was added to the

reaction flask, and it was stirred overnight. The precipitate was filtered, and later recrystallized

with ethanol. A yield of 135.4 mg was obtained, with a percent yield of 35.6%.

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Product 13 (APPENDIX 8): Dihydropyrimidinone 1A (1 mmol) was dissolved in 10 ml of



a solvent. Five equivalents of 2-(3-chlorophenylthylamine) (5 mmol) was added to the reaction






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a solvent. Five equivalents of 2-methoxyphenethylamine (5 mmol) was added to the reaction






a solvent. Five equivalents of 3-methoxyphenethylamine (5 mmol) was added to the reaction



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Antibiotic testing Local antibiotic testing was done with the use of the Kirby Bauer Disk Diffusion Assay. The

synthesized compounds were dissolved in dimethylsulfoxide (DMSO) to a concentration of

0.1M, and were tested against a strain of gram positive bacteria (Staphylococcus aureus), and a

strain of gram negative bacteria (Enterobacter aerogenes). The extent of the antibiotic activity is

qualitatively seen by the diameter of the zone of inhibition produced by the compound (18). The

larger the zone of inhibition, the higher the antibacterial activity of the compound (18). The

positive control substance was chloramphenicol, which is always expected to have a relatively

large zone of inhibition with both gram positive and gram negative bacterial strains.

Antibiotic testing results

The final cyclized scaffolds, the intermediate uncycled products, and some of the starting

dihydropyrimidinone scaffolds were tested. The results of the experiment indicate that none of

the compounds synthesized thus far possess antibacterial properties as indicated by the lack of a

zone of inhibition.

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Conclusion

The goal of the experiment was to synthesize novel pharmacological agents by combining two

privileged structures; dihydropyrimidinones and phenethylamines. The project was separated

into four parts; making dihydropyrimidinone scaffolds with different functional groups,

brominating the allylic site in the dihydropyrimidinone, performing a cyclization with various

phenethylamines, and testing for bioactivity of the compounds.

The first part of the project involved the synthesis of dihydropyrimidinones with various

functional groups by using pre-manufactured aldehydes. Twelve dihydropyrimidinone scaffolds

were synthesized, and their purity was increased through recrystallization with ethanol as a

solvent. The second part of the project involved the bromination of the allylic site in the

dihydropyrimidinone scaffolds. It was found that phenytrimethylammonium tribromide was the

preferred brominating agent. Due to the dissociation of the bromine group during the

recrystallization, a one pot reaction was chosen. The goal of the third part of the project was to

perform an intermolecular cyclization which combined pre-manufactured phenethylamines with

different functional groups with the brominated dihydropyrimidinone scaffolds. The temperature

was raised to 60° Celsius, and 100° Celsius respectively, which led to improved results. The

reaction was also maximized by decreasing the steric hindrance of the molecule by altering the

ester group from an ethyl ester to a methyl ester. The increased temperature, and the decreased

hindrance led to the successful synthesis of fourteen cyclized compounds. The fourth part of the

project involved the antibacterial testing of the synthesized compounds using the Kirby-Bauer

disk diffusion assay.

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Although the compounds did not show any antibacterial activity, it is a possibility that they

possess other pharmacological properties of dihydropyrimidinones such as calcium channel

blockers, anti-cancer drugs, and anti-inflammatory agents. These pharmacological properties will

be tested for by the Developmental Therapeutics Program (DTP) of the NIH. This NIH program

is designed to help the general research community to screen bioactive compounds with the goal

of discovering new chemical leads and biological mechanisms. Another program that we will

send our compounds to the Lilly Open Innovation Drug Discovery (OIDD) Program for

bioactivity screenings (19). Once hits are discovered, we will move to more conventional SAR

studies to determine how to improve the absorption and bioavailability of these compounds in

both in vitro and in vivo.

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APPENDIX 1: 1H NMR from Product 2

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APPENDIX 2: 13C NMR from Product 2

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APPENDIX 7: 19F NMR from Product 12

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sgonzalez honors thesis

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