SYNTHESIS OF p38α MAP KINASE INHIBITORS

by

Vita Koren

A Thesis Submitted in Partial Fulfillment

of the Requirements for the Degree of

Bachelor of Science

In

The Faculty of Science

Biological Sciences

University of Ontario Institute of Technology

April 2016

© Vita Koren, 2016

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Abstract

Dibenzo[b,e]oxepin-11(6H)-one derivatives have shown to be able to fit in the binding pocket of p38α MAP kinase and to act as inhibitors. The formation of 3-phenylbenzo[c]oxepin-5(1H)-one would be biologically important in improving the inhibitory capabilities while being able to remain in the binding pocket. A significant progress toward the preparation of 3-phenylbenzo[c]oxepin-5(1H)-one has been achieved. A diol was monoprotected as a silyl ether. Subsequently, an alcohol functionality was oxidized to afford an aldehyde, which was further coupled with phenylacetylene. An oxidation of the resulting alcohol afforded propargyl benzyl ketone functionality. The next step would be to cleave the protecting group from the compound and to protonate it back into an alcohol. Furthermore, the scaffold may be achieved by the successful closing of the cycloheptane ring.

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Acknowledgements

I would like to thank my supervisor Dr. Yuri Bolshan for all of his guidance, advice and support. I am sincerely grateful for the confidence he had in me and for helping me strive for my highest potential. I would not have maintained such a positive attitude without his reinforcement.

Thank you to Kayla Fisher for being a mentor, lab companion and new friend over the past 8 months. I am very lucky to have had her by my side throughout this project with the knowledge that somebody else has been through this before. I would also like to thank Ms. Fisher for all her help in the laboratory and her ability to have an explanation for any question.

I would like to thank Ifedi Orizu and Stefan Ramkissoon for working alongside with pleasure and mutual collaboration.

Thank you to the members of Dr. Jean-Paul Desaulnier’s laboratory as well as Olena Zenkina’s laboratory for their cooperation and support.

I would like to thank Kevin Coultier, Genevieve Barnes and Darcy Burns for their assistance in the operation and maintenance of instrumentation throughout the project. As well, Dr. Coultier for sharing his knowledge on the characterization of compounds.

A thank you to Edmond Courville for his knowledge and assistance in obtaining laboratory supplies.

Thank you to the University of Ontario Institute of Technology for providing the facilities and funding necessary to make this project possible.

I would also like to thank my family for their continued love and support throughout all my studies at UOIT.

Lastly, I would like to thank the rest of the laboratory personnel, my advisory committee and everyone that has provided a positive impact during my undergraduate studies at UOIT.

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Table of Contents

Abstract……………………………………………………………………………………………2

Acknowledgements………………………………………………………………………………..3

Table of Contents………………………………………………………………………………….4

List of Tables……………………………………………………………………………………...6

List of Figures……………………………………………………………………………………..6

List of Schemes……………………………………………………………………………………6

List of Abbreviations……………………………………………………………………………...7

1. Introduction……………………………………………………………………………………..9

1.1 General Background…………………………………………………………………...9

1.2 p38α in the MAP kinase Pathway……………………………………………………...9

1.3 Enzyme Inhibitors……………………………………………………………………10

1.4 Effects of Inhibitors on p38α in the MAP kinase Pathway………………………..…..11

1.5 Steps of the New Methodology………………………………………………………13

2. Materials and Methods………………………………………………………………………...15

2.1 General Experimental Considerations………………………………………………..15

2.2 Use of methyl 2-formylbeonzate………………………………………………….….15

2.3 Use of 1,2-benzenedimethanol……………………………………………………….16

2.3.1 Adding a protective group to an alcohol……………………………….…...16

2.3.2 Converting an alcohol group into an aldehyde……………………….…….16

2.4 Use of phenylacetylene……………………………………………………….………17

2.4.1 Oxidizing a secondary alcohol into a ketone………………………………18

3. Results and Discussion………………………………………………………………………..19

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3.1 Summary of Reaction Procedure…………………………………………………….19

3.2 Attempted Reductions of 2-carboxybenzaldehyde…………………………....……..20

3.3 Attempted Reductions of methyl 2-formylbenzoate……………………….……...…21

3.3.1 Proposed Mechanism………………………………………………………22

3.4 Attempted use of Phthalide as Starting Material…………………………...…….….22

3.5 Successful use of 1,2-benzenedimethanol………………………………….………...23

3.5.1 Attempted conversion of a primary alcohol into a carboxylic acid………....24

3.5.2 Successful conversion of a primary alcohol into an aldehyde………..……..25

3.5.3 Attempted conversion of an aldehyde into a carboxylic acid………..……...26

3.6 Successful addition of Phenylacetylene…………………………………….………...27

3.6.1 Propose Mechanism………………………………………………………..27

3.6.2 Successful conversion of a secondary alcohol into a ketone………………28

3.7 Removal of the TBDMSCl protecting group………………………….……………...29

4. Conclusions and Future Directions………………………………………………….…………30

5. Appendices…………………………………………………………………………………….31

Appendix I…………………………………………………………………………….….31

Appendix II………………………………………………………………………………34

6. References……………………………………………………………………………………..39

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List of Tables

Table 1: Reagent data table for the attempted reduction of 2-carboxybenzaldehyde…………..…20

Table 2: Reducing agents for the attempted synthesis of methyl 2-(hydroxymethyl)benzoate…...21

Table 3: Conditions for the addition of TBDMSCl protecting group to an alcohol group……...…23

Table 4: Oxidizing agents for the attempted synthesis of a carboxylic acid from an alcohol……...24

Table 5: Oxidizing reaction conditions for the synthesis of an aldehyde from an alcohol…...……25

Table 6: Oxidizing agents for the attempted synthesis of a carboxylic acid from an aldehyde...….26

Table 7: Addition of phenylacetylene to an unprotected aldehyde……………………………..…27

Table 8: Oxidizing reaction conditions for the synthesis of a ketone from an alcohol……….….28

Table 9: Cleavage of the TBDMSCl protecting group to reform an alcohol…………………….29

List of Figures

Figure 1: Signal cascade of the MAP kinase pathway when activated……………………..………9

Figure 2: Enzyme-Substrate Interactions of various Inhibition Types…………………….….…..10

Figure 3: Inhibitors of p38α MAP kinase previously studied……………………………..………12

Figure 4: Proposed binding mode of p38α MAP kinase inhibitor…………………….….……….13

List of Schemes

Scheme 1: Proposed steps for the novel synthesis of an inhibitory scaffold for p38α………...…..14

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Scheme 2: Proposed mechanistic pathway for the synthesis of phthalide……………………..….22

Scheme 3: Reaction conditions that did not result in the desired ester products……………..…....23

Scheme 4: Proposed mechanistic pathway for the synthesis of a propargylic alcohol………..…..28

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List of Abbreviations

MAPK

ATP

RAF

MEK

RTK

RAS

ERK

TNF-α

IL-1β

Gly110

Met109

TMS

MeI

K2CO3

DMF

DCM

MgSO4

H2O

HCl

TBDMSCl

MeOH

MeCN

Ca(OCl)2

PCC

NABH4

Na(CH₃COO)₃BH

NaBH3CN

Mitogen activated protein kinase

Adenosine triphosphate

Mitogen activated protein kinase kinase kinase

Mitogen activated protein kinase kinase

Receptor tyrosine kinase

Ras protein

Extracellular signal-regulated kinase

Necrosis factor-alpha

Interleukin-1beta

Glycine110

Methionine109

Trimethylsilyl group

Methyl iodide

Potassium carbonate

Dimethylformamide

Dichloromethane

Magnesium sulfate

Water

Hydrochloric acid

Tert-butyldimethylsilyl chloride

Methanol

Acetonitrile

Calcium hypochlorite

Pyridinium chlorochromate

Sodium borohydride

Sodium triacetoxyborohydride

Sodium cyanoborohydride

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C12H28BNa

NaOCH3

NaOH

Et3N

MnSO4*H2O

C10H11IO4

MnO2

TBAF

Cs2CO3

HF

N - Selectride

Sodium methoxide

Sodium hydroxide

Triethylamine

Manganese(II) sulfate monohydrate

(Diacetoxyiodo)benzene

Manganese dioxide

Tetrabutylammonium fluoride

Cesium carbonate

Hydrofluoric acid

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1. Introduction1.1 General Background

In regards to the health and wellness of the human population, biology used to always seem as the area of expertise where a solution could be found. Nowadays, there are many technological advances and it has been determined that other forces such as physics and chemistry come into play, in the human body. It is now possible to synthesize organic compounds in laboratories that may then be used as treatments for diseases, syndromes and many other symptoms that may be effecting the human population negatively.

The focus of this project has been the development of a multi-step methodology for the formation of a novel inhibitor specifically at the p38α region of the MAP kinase pathway in the human cell. The new inhibitor will hopefully have a higher success rate than its previous counterparts. This idea has been approached from several angles and starting points.

1.2 p38α in the MAP kinase Pathway

The MAPK pathways are evolutionarily conserved enzymes that catalyzes the transfer of a phosphate group from ATP to a specified molecule. This process is vital for central process such as growth, proliferation, differentiation, migration and apoptosis. The MAPK pathway consists of three parts in which MAPK is activated once it is phosphorylated by RAF and then it is activated once more by MEK [7]. In the case of cancer cell invasion, there would be a loss of control in regards to proliferation, apoptosis, growth, replication and invasion [7]. Therefore, it is important that drug resistance be obtained in order to prevent such damage.

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Figure 1: Signal cascade of the MAP kinase pathway when activated

In overview, the MAPK pathway begins with already having phosphorylated RTK on the extracellular membrane with growth factors. An adaptor protein is then able to recognize the activated state and bind to RTK as well. The adaptor protein proceeds to activate the G protein-coupled receptor RAS; its phosphorylated state becomes energized [10]. The energized signal is then able to be transmitted to the next molecule, the RAF protein. RAF (MAP KKK) now possess’ the activation and continues the cycle by passing it on to MEK (MAP KK) who then passes the activity to ERK (MAP K). MAPK is then able to enter the nucleus and phosphorylate transcription factors for growth, proliferation etc [10].

This p38α MAP kinase component is activated during the secondary phosphorylation MAP KK stage of MEF to ERK [Roux]. Accordingly, p38α MAPK’s are able to phosphorylate a variety of targets downstream (200 to 300 substrates) which are activated via phosphorylation as mentioned previously. Most importantly, it is able to activate partners such as RPS6KA5/MSK1 and RPS6KA4/MSK2 which play big roles in stress response by inducing chromatin remodeling of genes or by recruiting the transcription machinery [7].

In the case of physical or chemical stress such as oxidative stress, UV irradiation or hypoxia p38α MAP kinase component of the MAPK pathway have a very important role in regulation of cytokine production [3]. That is, p38α MAP kinase helps several substances prepared by the immune system to protect cells from harm. Therefore, p38α MAPK has induced phosphorylation, of membrane-associated metalloproteases which mediates the extracellular portion of cytokines such as TNF- α to shed; activating the next part of the MAPK signaling pathway (MEF) and allowing for cell proliferation and growth to occur [3]. However, TNF-α along with IL-1β among others, promote the beginning of inflammatory diseases such as Rheumatoid Arthritis and Crohn’s Disease as well as autoimmune diseases such as Multiple Sclerosis, Type 1 Diabetes and Cancer.

1.3 Enzyme Inhibitors

Enzyme inhibitors are substances that modify the catalytic activity of an enzyme to obtain an altered consequence of the catalysis. Usually, enzyme inhibitors reduce the rate of activity and this may be a permanent or temporary solution [20]. The well-known inhibitors are the competitive type and the non-competitive type.

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Figure 2: Enzyme-Substrate Interactions of various Inhibition Types

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both in contact with the unaltered enzyme [19]. This follows the “lock and key” theory in that the enzyme has a shape with a strong affinity to fit with the complementary shape substrate. The enzyme is considered the lock and the substrate is the key, which can then be inserted in order to unlock the enzyme and fulfill its role [19]. If an inhibitor with the same shape is able to bind to the active site first, the enzyme will accept it as a substrate, but the enzyme will not unlock thus slowing down its activity. Competitive inhibition is usually temporary and the inhibitor will leave. Therefore, the level of inhibition depends on the relative concentrations of substrate and inhibitor competing [19].

On the other hand, non-competitive inhibitors alter the enzyme rendering the enzyme unable to bind to its regular substrate and not obtaining a product [19]. As substrates will continue to pile up, with none being able to activate with the enzyme, the reaction velocity will start to decrease. This occurs at an allosteric site, meaning not on the enzyme because there is a large amount of competition to enter the active site of the enzymes surface. This blocks the site and prevents any molecule from occupying them. The reaction rate drops due to the enzyme not being used [19].

1.4 Effects of Inhibitors on p38α in the MAP kinase Pathway

More recently, the idea of using pharmacological inhibitors of MEK and ERK in multiple cellular functions such as cellular proliferation, inflammatory responses and cell survival as a few examples has emerged. Most research has centered on the amino-flavone MEK inhibitor PD98059. This inhibitor has been found to stop the activation of MEK by RAF at low micromolar concentrations of 2 to 7 µm [2]. A study by Sebolt-Leopold et al. (1999) discovered that analogous inhibitor PD184352 inhibits MAPK and blocks the growth of colon carcinoma cells in mice [24]. A set of structurally unrelated compounds have also been found to act as inhibitors of the ATP binding site [2]. Overall, the balance of pro and anti-survival signaling of p38α MAPK may be a positive for the needed lethal behavior of chemotherapeutic agents in vivo [24].

The inhibition of p38α MAPK is regarded as a promising therapeutic technique for controlling inflammatory diseases. In recent studies it has been reported that most inhibitors target the ATP binding site, which contains a conserved phenylalanine amino acid residue of the Asp-Phe-Gly (DFG) motif buried in a hydrophobic pocket between the two lobes of the p38α MAP kinase [8]. Another set of inhibitors occupies the ATP binding site as well as the allosteric pocket (which is only available when the Phe side chain of the DFG motif moves out from the hydrophobic pocket) [8].

Due to the fact that p38α MAPK regulates the productions of TNF-α and IL-1β, p38 inhibitors are projected to impede the production of pro-inflammatory cytokines, as well as their actions. This will result in interfering the negative cycle that often occurs in inflammatory and immune-responsive diseases [8]. The goal is to produce drugs that will be able to disrupt the molecular foundations of these responses.

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Figure 3: Inhibitors of p38α MAP kinase previously studied

The fluorophenyl ring of these previously studied inhibitors bind in such a way that only residues that are the size of the threonine amino acid or smaller can accommodate the fluorophenyl ring [8]. p38α, which has a threonine, are sensitive to these inhibitors with submicromolar IC50 values. Additional chemistry has refined the potency and specificity of these p38 inhibitors relative to Raf-1. Therefore, the inhibitors were generated to act at subnanomolar concentrations with 5000-fold selectivity for p38 over Raf-1 [8]. The research obtained from structural studies and sequence databases has helped the development of selective p38 inhibitors.

Another group of inhibitors of p38 MAPK belong to the class of pyridinylimidazoles, the most common one being known as SB203580 [3]. After conducting animal studies, these inhibitors showed high toxicity due to the interaction of the imidazole heterocycle with enzymes as well as having poor selectivity [3]. More recently, a new version of p38α inhibitors was established with scaffolds of dibenzo[b,e]oxepin-11(6H)-one and 5H-dibenzo[a,d][7]annulen-5-one. These scaffolds demonstrated positive biological behavior with IC50 values of 38 nM. However, when exposed to human whole blood TNF-α assays, values grew to 22 µM [3].

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Figure 4: Proposed binding mode of p38α MAP kinase inhibitor

In an improved study of the above, 2-(2-amino)amino-substituted dibenzosuberone was used as the starting material and introduced the concept of hydrophilic residues [3]. The resulting compound was a complex consisting of Skepinone-L with p38α MAP kinase, with an induced peptide flip of Gly110 rotating 180º. Additionally, the peptide flip resulted in two hydrogen bonds between the central carbonyl oxygen of Skepinone- and the NH of Met109 and Gly110 to form [3].

Due to these newly formed hydrogen bonds, 2,3-dufluorophenyl residue was able to occupy the hydrophobic region I (selectivity pocket) of the p38α MAP kinase [2]. Meanwhile, the hydrophobic region II remained unoccupied, leaving it as an open opportunity for investigation. Overall, hydrophobicity results in more bioavailability.

1.5 Steps of the New Methodology

Section 1.4 highlighted the use of dibenzo[b,e]oxepin-11(6H)-one as the primary scaffold for a p38α MAP kinase inhibitor. It has been determined to be a weak solution due to low yield of product as well as low activity count.

The goal now was to develop a straightforward, multi-step procedure for the preparation of a derivative of dibenzo[b,e]oxepin-11(6H)-one, known as 3-phenylbenzo[c]oxepin-5(1H)-one. As with traditional novel experimentation, the approach was constant and consistent trial and error of attacking starting material, hoping to obtain the desired product. In this case, only tools and materials available to the undergraduates was used.

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Scheme 1: Proposed steps for the novel synthesis of an inhibitory scaffold for p38α

The overall objective of this research is to explore the formation of a modified new p38α MAP kinase inhibitor using alcohol starting materials. Specifically, the goal of this project is to develop and optimize a synthetic route for the preparation of a scaffold which can then be applied towards drug development. This compound would be considered more flexible due to it having a carbon bond between the cycloheptane ring and one of the aromatic rings. The novel flexibility may allow for more functionality and substituent options.

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2 Experimental Methods

2.1 General Experimental Considerations

Unless otherwise noted, all reagents and materials were obtained from commercial suppliers and used without further purification. Reactions were performed under air conditions unless written otherwise. NMR characterization data was obtained at 25ºC on an Oxford AS400 NMR spectrometer as solutions in deuterated solvents (CDCl3 and Methanol obtained from Cambridge Isotope Laboratories, Inc.). 1H NMR spectra were recorded at 400 MHz and referenced to the residual solvent peak at 0.00 ppm for TMS in relevance to CDCl3 and 3.30 ppm for Methanol. Flash column chromatography on silica gel (60 Å, low acidity, obtained from EMD Chemicals Inc.) was performed using reagent grade solvents unless otherwise specified.

2.2 Use of methyl 2-formylbenzoate

2-carboxybenzaldehyde was converted into methyl 2-formylbenzoate and then exposed to various reducing agents under numerous conditions to attempt generating methyl 2-(hydroxymethyl)benzoate.

General Procedure for the successful formation and unsuccessful reduction of methyl 2-formylbenzoate:

A solution of 2-carboxybenzaldehyde (2 g, 1.0 equiv.) was mixed with MeI (1.53 mL, 1.86 equiv.) and K2CO3 (0.995, equiv.) in DMF and acetone. The solution was stirred at ambient temperature overnight according to a procedure modified from Yuan et al (2015). The reactions were quenched with H2O (30 mL) and extracted with DCM (30 mL x 3). The combined DCM layers were washed with brine (30 mL x 1), dried over MgSO4, filtered and concentrated. The solvent was evaporated in vacuo and the resulting products were dried under vacuum for 1 hr. The product was purified using column chromatography with ethyl acetate and hexane (1:6) to produce methyl 2-formylbenzoate. Methyl 2-formylbenzoate was then dissolved in methanol and reducing agents were added (Table 2). The reactions were quenched with HCl (1 M) and extracted with ethyl acetate (30 mL x 3). The organic layer was then washed with brine (30 mL x 1) and dried over MgSO4. The solvent was removed in vacuo and the resulting products were placed under vacuum for 1 hr. The products were purified using column chromatography with ethyl acetate and hexane (1:6).

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2.3 Use of 1,2-benzenedimethanol

2.3.1 Adding a protective group to an alcohol

1,2-benzenedimethanol was initially substituted at one alcohol position a protective group in order to produce (2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol according to a procedure modified from Huff et al. (2008).

General procedure for the successful preparation of

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol:

1,2-benzenedimethanol (273.96 mg, 1.0 equiv.) and TBDMSCl (268.97 mg, 0.9 equiv.) were dissolved in DCM (6.96 mL) and cooled to 0ºC. To the solution trimethylamine (1.24 mL, 4.5 equiv.) in DCM (7.89 mL). The mixture was stirred at room temperature for 1 hr and the solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate and washed with brine (30 mL x 3). The organic layer was dried over MgSO4 and solvent was removed once again by rotary evaporation. The resulting product was then purified via column chromatography in DCM and MeOH (10:1) to be a light yellow oil.

.2.3.2 Converting an alcohol group into an aldehyde

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol was then attempted to be converted into an aldehyde via several different procedures. Procedures were followed and modified according to Nwaukwa et al. (1982) and Corey et. al (1975).

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General procedure for the successful use of calcium hypochlorite [13]:

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol ( 21.6 mL, 1.0 equiv.) was initially dissolved in MeCN and acetic acid (3:2). Over the course of 10 minutes, the stirred solution was added dropwise to Ca(OCl)2 (8.2 mg, 0.67 equiv.) in H2O at 0ºC. Stirring continued for 1 hr and then the reaction was quenched with H2O. The solution was extracted with DCM (30 mL x 3) and the organic layer was washed with 10% sodium bicarbonate. Lastly, the solution was exposed to an aqueous wash, dried with MgSO4, solvent was evaporated and the resulting product was placed under vacuum for 1 hr.

General procedure for the successful use of pyridinium chlorochromate [6]:

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol (270.5 mg, 1.0 equiv.) was combined with PCC (352.28 mg, 1.5 equiv.) and dissolved in DCM (0.2M) at room temperature. The reaction was quenched with H2O (30 mL) and extracted with ethyl acetate (30 mL x 3). The organic layer was dried over MgSO4 and then washed with brine. The organic layer was once again dried with MgSO4 and solvent was evaporated. The resulting product was placed under vacuum overnight. The product was isolated in a DCM and MeOH (40:1) column.

2.4 Use of Phenylacetylene

1,2-diphenylprop-2-yn-1-ol was attempted to be prepared from (2-((benzyloxy)methyl)benzaldehyde according to a procedure modified from Yuan et. al (2014).

General procedure for the successful use of zinc iodide [25]:

Zinc iodide (645.98 mg, 2.5 equiv.) was placed in a flask under argon conditions. Anhydrous toluene (0.25M) was then added and the solution was stirred for 5 minutes at room temperature. In the following order, phenylacetylene (115.57 mL, 1.3 equiv.), trimethylamine (282 mL, 2.5 equiv.) and (2-((benzyloxy)methyl)benzaldehyde (202.70 mL, 1.0 equiv.) were then added. The reaction was steadily heated to 80ºC (25 minutes) and was then stirred for 30 minutes at a constant 80ºC. The reaction was quenched with 0.2 M HCl and extracted with ethyl acetate (30 mL x 1). The organic layer was washed with HCl (30 mL x 3) and with brine (30 mL

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x 1). The organic layer was then dried with MgSO4 and solvent was evaporated. The product was left on the vacuum overnight and was then purified with an ethyl acetate and hexane (2:10) column chromatography.

2.4.1 Oxidizing a secondary alcohol into a ketone

1-(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-3-phenylprop-2-yn-1-one prepared from its alcohol derivative according to a procedure modified from Corey et. al (1975).

General procedure for the successful use of pyridinium chlorochromate [6]:

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol (98.30 mg, 1.0 equiv.) was combined with PCC (90.16 mg, 1.5 equiv.) and dissolved in DCM (0.2M) at room temperature. The reaction was quenched with H2O (30 mL) and extracted with ethyl acetate (30 mL x 3). The organic layer was washed with brine and then dried with MgSO4 and solvent was evaporated. The resulting product was placed under vacuum overnight. The product was isolated in an ethyl acetate and hexane column.

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3. Results and Discussion

3.1 Summary of Reaction Procedure

Initially, we focused on the preparation of 2-hydroxymethyl benzoic acid from 2-carboxybenzaldehyde and sodium borohydride. In order to find suitable reaction conditions, various loadings of NaBH4 were attempted (Table 1). Sodium borohydride has been known to be a fast acting reducing agent. Unfortunately, this reducing agent was too reactive and did not promote the formation of product. An ester group is known to be more stable than a carboxylic acid, therefore 2-carboxybenzaldehyde was able to be converted.

Upon running various reduction reactions with different equivalences, temperatures and time frames (Table 2), it was determined that even with the newly formed ester, the desired product was not able to form and instead phthalide (scheme 2) was consistently produced. Therefore, attempts were made using commercially available phthalide. Still, the desired product was not able to be generated.

Next, 1,2-benzenedimethanol was attempted as a starting material. First, an alcohol functionality was protected to avoid interference with the reaction (Table 3). Then, the protected di-alcohol was subjected to numerous attempts at modifying the monoprotected alcohol group (Table 4-6). The yields were modest but nevertheless successfully produced an aldehyde, allowing to move on to the next step.

Then, phenylacetylene was attempted to be bonded to the carbonyl carbon of the aldehyde functionality. First, n-butyllithium was used as the attacking agent as it had been found to be very successful in the past by Taylor (2014). However, throughout this experiment, the results were ineffective and a new method had to be attempted. The introduction of zinc iodide and trimethylamine was able to generate the needed alcohol with two aromatic rings and a triple bond. Then, oxidation of the alcohol into a ketone was accomplished using the same methodology as the one that was able to produce an aldehyde from an alcohol. The procedure was found to work the same for a secondary alcohol; producing a ketone (Table 8).

Lastly, the removal of the protecting group was attempted. After attempting basic, acidic and fluoride conditions, the removal of TBDMSCl was unsuccessful.

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3.2 Attempted Reductions of 2-carboxybenzaldehyde

Table 1. Reagent data table for the attempted reduction of 2-carboxybenzaldehyde

Reaction NaBH4 (equiv.) Time Yield (%)1 2.0 10 minutes 02 1.0 15 minutes 03 0.5 45 minutes 04 0.5 10 minutes 0

Reactions were run with 1 equiv. 2-carboxybenzaldehyde. The reactions were done in dried glassware under argon.

Conducting the reaction under excessive load conditions, resulted in a low yield. Although sodium borohydride is known to selectively reduce aldehydes and not carboxylic acids [5], the load may have been too high, thus over-reducing the desired product and not yielding the desired monoalcohol as indicated by a 1H and 13C NMR spectrum. Lowering the load of sodium borohydride to be equal to that of the starting material did not yield the desired product either, therefore, it was thought that sodium borohydride was reacting too quickly and thus continuing to over-reduce. The load was lowered further and then, the time of the reaction was lengthened. The yield obtained was still low indicating that timing was not the concern. Returning the timing back to its original length and maintaining the lowered load did not yield the desired product. Therefore, it was concluded that sodium borohydride was not able to reduce an aldehyde when there was a carboxylic group present nearby.

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Desired Product

3.3 Attempted Reduction of methyl 2-formylbenzoate

Table 2. Reducing agents for the attempted synthesis of methyl 2-(hydroxymethyl)benzoate

Reducing Agent Equivalence Temperature Time Yield (%)

NaBH4 1.0 0ºC 10 minutes 0Na(CH₃COO)₃BH [3] 0.7 21ºC 15 minutes 0

1.0 90ºC 1 hour 03.0 75ºC Overnight 80

NaBH3CN [2] 1.5 21ºC 1 hour 01.0 25ºC 2 hours, pH= 4 80

C12H28BNa 1.2 0ºC 2 hours 0Reactions were run with 1 equiv. methyl 2-formylbenzoate. The reaction was done in dried glassware under air.

In order to continue the attempt of obtaining the desired alcohol, 2-carboxybenzaldehyde was turned into methyl 2-formylbenzoate and then attacked with several reducing agents under varying conditions.

Commercially available 2-carboxybenzaldehyde was converted into methyl 2-formylbenzoate with a yield of 75%. The modification of the starting material was performed because borohydride reducing agents are able to chemoselectively reduce aldehydes in the presence of esters [16]. Esters are reduced at a much lower rate due to the functional group being less electrophilic. It was thought that adding a less reactive group than a carboxylic acid would help yielding the desired product by having the reducing agents focus on the more desirable aldehyde for reduction.

Regrettably, the NMR spectrum determined unsuccessful transformation of the aldehyde into an alcohol was continued. Sodium borohydride was the first reducing agent attempted, and concluded that even with replacing the carboxylic acid into an ester, an alcohol was not produced and required further investigation. Next, sodium triacetoxyborohydride was thought to be a solution mainly because it has only one hydride. Usually, sodium triacetoxyborohydride is suited

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Desired Product

Actual Product

for reductive animations because the rate of reduction is much faster than that of aldehydes thus a slower acting agent is necessary [1]. After attempting several variations of loads, temperatures and time frames, it was determined that this milder reducing agent was not the solution. However, at the highest used load and time frame, an 80% yield of phthalide was formed. Next, sodium cyanoborohydride was used as a reducing agent. Under neutral conditions the reduction of an aldehyde functionality since only traces of the final product were observed. Under acidic conditions the rate of reduction was sufficiently rapid to synthesize an 80% yield. Sodium cyanoborohydride worked better under acidic conditions because its hydrolysis was acid-catalyzed. Its rate of hydrolysis was 10-8 that of sodium borohydride because it has one less hydrogen. This allowed for more neuclophilicity to be allocated to the remaining hydrides [17]. Once again, the product obtained was phthalide. Lastly, N-selectride did not yield a product and this was most likely due to the fact that it is a bulkier hydride reagent and thus would not be able to attack a sterically hindered functional group [23].

3.3.1 Proposed Mechanism

Scheme 2 illustrates the proposed mechanistic pathway for the phthalide formation reaction. Interaction of the aldehyde species (1) with borohydride reducing agents results in the formation of a primary alcohol (2). The primary alcohol is now able to act as a strong nucleophile and attack the carbonyl carbon (3). A proton transfer to the ester (4) forms an intermediate (5) by transfer of a hydrogen from the alcohol. The phthalide (6) is obtained once the newly formed methanol has left.

Scheme 2: Proposed mechanistic pathway for the synthesis of phthalide

3.4 Attempted use of Phthalide as starting material

Although phthalide was generated, using it as a starting material did not result in successful reactions (Scheme 3).

23

Scheme 3: Reaction conditions that did not result in the desired ester products

In order to break open the oxygen ring, phthalide was treated with selected bases. It was thought that the nucleophilic OH group would attack the ring at the least substituted position and result in an inversion of stereochemistry. This would use the SN2 mechanism and then transfer a proton to the alkoxide. Unfortunately, no yield was obtained for either base used. In the case of sodium methoxide, a 0% yield may have occurred due to the fact that solvent had evaporated throughout the reaction. Without a sufficient amount of solvent, mostly starting material was left because there was no equilibrium established between sodium methoxide and its conjugate acids [14]. Meanwhile, the flaw with using sodium hydroxide may have been that the reaction was quenched with HCl. A weakly acidic solution such as H2O may have made it easier to add a proton to the alkoxide to obtain a neutral alcohol molecule [14].

3.5 Successful use of 1,2-benzenedimethanol

Table 3. Conditions for the addition of TBMSCl protecting group to an alcohol group

Reaction TBDSCl (equiv.) Et3N (equiv.) Yield (%)1 1.0 4.0 n.a2 1.0 4.0 433 1.0 4.0 04 0.9 4.5 55

Reactions were run with 1 equiv. 1,2-dibenzenedimethanol for 1h at room temperature. Reactions 1-2 & 4 were done in dried glassware under air while reaction 3 was under argon.

A protective group was selectively added to one of the alcohol groups in order to proceed to further reactions with monoalcohol conditions. After performing the addition of the protective

24

Desired Product

group, it was found that ethyl acetate/ hexane ratio on the column were not polar enough to push the purified product through the column. Recovery of product was attempted by passing methanol through the column but an insufficient amount was retrieved. TBDMSCl was able to attach and yield 43% on a MeOH/ DCM column which was more polar. For curiosity, TBDMSCl was added to the di-alcohol under argon conditions. The desired product was not formed and this may have been because of the lack of moisture. TBDMSCl was stable when it was in the presence of aqueous base but may have never attacked the alcohol without the presence of water in the air [11]. When modifying the loads, the yield was improved to 55%. A larger amount of amine base and a lower amount of TBDMSCl allowed for more stability in the reaction.

3.5.1 Attempted conversion of a primary alcohol into a carboxylic acid

Table 4. Oxidizing agents for the attempted synthesis of a carboxylic acid from an alcohol

Reaction Reagents (equiv.) Solvent Temperature Time Yield (%)

1 Oxone (2.5), MnSO4*H2O (1.5)

H2O 90ºC 45 mins n.a

2 Iodine (1.0), C10H11IO4 (1.1)

MeCN r.t 2 hrs 0

Reactions were run with 1 equiv. TBDMS protected 1,2-dibenzenedimethanol. Reactions were done in dried glassware under air.

The newly formed monoprotected alcohol was not successfully converted into a carboxylic acid. As shown in Table 4, the first attempted reaction yielded no product, meaning starting material was still present (indicated by NMR spectrum). No reaction may have occurred due to the combination of temperature, reflux and solvent selected. The boiling point of water is 100ºC, therefore the fact that the reaction took place at 90ºC indicated that water already had bubbles from extreme heating and was moving quickly into the condenser. It was thought that water vapor was not able to cool fast enough, leaving the flask with no solvent for a significant amount of time. In the case of reaction 2, before purification, TLC spots showed that a carboxylic acid had potentially formed and the TBDMSCl protecting group was present as well. However, after column purification, an NMR spectrum determined that C10H11IO4 may have been too reactive because the protecting group was no longer present. It was found that in combination with a catalyst, such as iodine, the hypervalent iodine reagent became even more reactive under mild conditions and was able to remove the protective group [18].

25

Desired Product

3.5.2 Successful conversion of a primary alcohol into an aldehyde

Table 5. Oxidizing reaction conditions for the synthesis of an aldehyde from an alcohol

Reaction Reagents (equiv.) Solvent Temperatur

e Time Yield (%)

1 Ca(ClO)2 (0.67) MeCNAcetic acid

H2O

0ºC 1 hr 20

2 MnO2 (1.1) Hexane -10 ºC 3 hrs 0

3 PCC (1.5) DCM r.t 2 hrs 60

Reactions were run with 1 equiv. TBDMS protected 1,2-dibenzenedimethanol. Reactions were done in dried glassware under air.

Due to the unsuccessful conversions of an alcohol to a carboxylic acid, it was thought to take a step back and to first try forming an aldehyde from an alcohol. Reaction 1 utilized calcium hypochlorite which is known to be stable because it is in solid form and is able to synthesize aldehydes from primary and secondary alcohols [12]. Unfortunately, the yield was 20% which was not a sufficient amount to proceed with the experiment. The reaction may have not gone to completion because usually, calcium hypochlorite is combined with a phase-transfer catalyst to transport the hypochlorite ion from solid to liquid phase [12].

Reaction 2 did not yield the product because no alcohol or any other substituent group was observed at the benzylic alcohol carbon. Manganese dioxide only oxidizes activated alcohols and proceeds via the hydrated aldehyde [22]. However, MnO2 required very acidic conditions which were not established, therefore, oxidation was not possible.

The successful reaction of PCC with the TBDMSCl protected benzylic alcohol generated the desired product in 60% yield. While performing the reaction workup, the solution was mistakenly washed with MgSO4 before the workup procedure had been completed. Although the experiment did not follow standard procedure, the NMR spectrum was the cleanest compared to other experiments thus far. It was previously found that PCC often formed side products that complicated product isolation [6]. The addition of MgSO4 throughout the workup steps helped reduce chromium salts and other reagent-derived byproducts by the deposition on the solids and removal by filtration [6].

26

Desired Product

3.5.3 Attempted conversion of an aldehyde into a carboxylic acid

Table 6. Oxidizing agents for the attempted synthesis of a carboxylic acid from an aldehyde

Reaction Reagents (equiv.) Solvent Temperature Time Yield (%)

1 Oxone (1.0) DMF r.t 3 hrs 0

2 Periodic acid (1.1), PCC (0.2)

MeCN r.t 1.5 hrs 0

Reactions were run with 1 equiv. TBDMS protected benzaldehyde. Reactions were done in dried glassware under air.

In the challenge of converting the newly formed aldehyde into a carboxylic acid, both attempted reactions were not able to generate the desired product. In both cases, the NMR spectrum indicated the possibility that the protecting group had been removed. It was recently studied that when trying to use oxone alone as the attacking group, it allowed for deprotection of phenolic and benzylic TBDMSCl under mild conditions [4]. Novel procedures have been established such as combining a TEMPO catalyst with Oxone to improve functional group tolerance avoiding over-oxidation [4]. Additionally, quarternary ammonium salts have been used to obtain high yields of oxidation without bothering the protecting group [4]. In regards to reaction 2, it was already shown that PCC was a strong oxidizing agent. Therefore, using PCC as a catalyst for periodic acid may have also caused a deprotection of TBDMSCl.

27

Desired Product

3.6 Successful addition of Phenylacetylene

Table 7. Addition of Phenylacetylene to an unprotected aldehyde

Reaction Reagents (equiv.) Solvent Temperature Time Yield (%)

1 Phenylacetylene (5.0)n-butyllithium (5.0)

THF -78ºCrt 2 hrs 0

2 Phenylacetylene (1.3) Zinc iodide (2.5)

Triethylamine (2.5)

Toluene 80ºC 30 mins 44

Reactions were run with 1 equiv. TBDMS protected benzaldehyde. Reactions were done in dried glassware under argon.

A new plan was established, attaching a phenylacetylene molecule to the aldehyde in order to obtain an alcohol as well as a triple bond for further reactions later on. The first attempt at achieving this new compound was by referring to the previous work of Taylor (2014) which had been successful. Unfortunately, the reaction was not successful and the reason is not entirely clear. It was possible that due to the compound having a bulky substituent, n-butyllithium was interacting with unwanted areas of the compound. Even the compound obtained after the purification was not identified because the aldehyde peak was no longer present in 1H NMR, while new peaks were observed. Next, zinc iodide was attempted because zinc (II) acts as a Lewis acid and bimetallic zinc compounds have been found to promote C-C bond formation [22]. Therefore, the addition of a terminal alkyne to the aldehyde was sufficiently successful by obtaining a yield of 44%.

3.6.1 Proposed Mechanism

Scheme 4 illustrates the proposed mechanistic pathway for the promotion of a C-C bond formation between a terminal alkyne and an aldehyde. Zinc is known to easily replace hydrogens on acetylenes (1). Hydrogen iodide is then generated and neutralized by a base. The acetylene (2) is then more reactive and can attack the carbonyl carbon. A benzylic carbocation (3) will be formed which can easily lose a proton to a base such as trimethylamine. The propargylic alcohol (4) is obtained once the compound is protonated.

28

Desired Product

Scheme 4: Proposed mechanistic pathway for the synthesis of a propargylic alcohol

3.6.2 Successful conversion of a secondary alcohol into a ketone

Table 8. Oxidizing reaction conditions for the synthesis of a ketone from an alcohol

Reaction Reagents (equiv.) Solvent Temperature Time Yield (%)1 PCC (1.5) DCM r.t 2 hrs 65

Reactions were run with 1 equiv. of starting material. Reactions were done in dried glassware under air.

The oxidation of propargylic alcohol with PCC afforded the product in 65%. The procedure was followed similarly to that of the benzylic primary alcohol.

29

Desired Product

3.7 Removal of the TBDMSCl protecting group

Table 9. Cleavage of the TBDMSCl protecting group to reform an alcohol

Reaction Reagents (equiv.) Solvent Temperature Time Yield (%)1 Cs2CO3 (1.0) DMF

H2O100ºC 1 hr 0

2 TBAF (1.5) DMF r.t 18 hrs 0

3 TBAF (1.5) DMF r.t 15 mins 0

4 TBAF (1.1) DMF r.t 15 mins 0

5 TBAF (2.5) DMF r.t 3 hrs 0

6 HF (1.9) DMF r.t 1 hr 0

7 TBAF (1.1) DMF -10ºC 1 hr 0

8 HF-pyridine (1.5) DMF r.t 40 mins 0

Reactions were run with 1 equiv. TBDMS protected benzaldehyde. Reaction 1-4 was done in dried glassware under argon. Reaction 5-8 was performed in dried glassware under air.

Silyl ethers are usually used as protecting groups for alcohols in organic synthesis. Reaction with acids or fluorides remove the silyl group when protection is no longer needed. Under basic conditions, TBDMSCl is fairly stable [9], therefore Cs2CO3 was not able to cleave the C-Si bond. Next, TBAF was attempted because fluorides have a strong deprotection ability due to its formation of S-F bonds which is stronger than Si-O bonds [9]. However, the propargyl ketone being a larger substituent may have increased resistance to hydrolysis and therefore fluoride conditions were not sufficient. Then, HF was used because it possess’ acidic and fluoride properties. Unfortunately, HF did not succeed either and it was possible that all the attempted conditions were too harsh. Subsequently, TBAF was attempted at lower temperatures but did not have an impact. Finally, a HF-pyridine complex was attempted because it was found to be less volatile [15] but after the workup, starting material had been regenerated.

30

Desired Product

4. Conclusions and Future Directions

A synthetic route for the preparation of a novel scaffold for an inhibitor of the p38α MAP kinase from 1,2-benzenedimethanol has been developed. Operational fluidity is a key feature of this method. This approach boasts unique reagent conditions, illustrated by the successful preparation of 3-phenylbenzo[c]oxepin-5(1H)-one.

In order to obtain the applicable scaffold for the preparation of p38α MAP kinase inhibitors, many other starting materials had been attempted to make the process faster. Compared to benzylic alcohols, other commercially available compounds were unsuccessful. This left not many synthetic opportunities given that over reduction had been the biggest issue with aldehyde and carboxylic acids.

In the presence of the TBDMSCl protecting group, the benzylic alcohol was able to be oxidized into an aldehyde. As a result, this method allows for the hydrogen to be a good leaving group. Phenylacetylene was able to be added to the newly formed benzaldehyde to produce a propargylic alcohol and the methodology was further employed toward the production of the scaffold.

From the propargylic alcohol, a ketone was the fastest and easiest step to derive by following the same oxidative procedure as that of producing the aldehyde. The removal of the TBDMSCl protecting group was then able to become the focus. This was found to be more difficult than anticipated.

The future steps will consist of continuing the attempt at removing the protecting group as well as closing the cycloheptane ring. Once the novel scaffold has been established, future directions of this project involve the preparation of a large amount of the aldehyde step. Then, various phenylacetylene derivatives will attempt to attack the aldehyde. This procedure will allow for the preparation of novel 3-phenylbenzo[c]oxepin-5(1H)-one derivatives, opening up the possibility for further transformations to synthesize novel inhibitory pharmaceuticals.

31

5. Appendices

Appendix I: Compound Characterization Data

Phthalide: Derived from methyl 2-formylbenzoate. Phthalide was obtained in 80% yield as a yellow oil. 1H NMR (400MHz, MeOD) δ 7.94 (m, 1H), 7.69 (m, 1H), 7.51 (m, 1H), 7.51 (m, 1H), 5.33 (s, 2H); 13C {1H] NMR (MeOD, 100MHz) δ 146.50, 133.98, 129.02, 125.78, 122.06, 77.33, 77.00, 76.68, 69.63

(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol: Derived from 1,2-benzenedimethanol. (2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)methanol was obtained in 55% yield as a yellow oil. 1H NMR (400MHz, CDCl3) δ 7.31 (s, 1H), 7.29 (s, 1H), 4.80 (s, 2H), 4.68 (s, 2H), 0.92 (s, 9H), 0.13 (s, 3H); 13C {1H] NMR (CDCl3, 100MHz) δ 139.83, 129.52, 128.76, 128.28, 127.96, 64.70, 63.92, 25.86, 18.25, -5.25

32

2-(((tert-butyldimethylsilyl)oxy)methyl)benzaldehyde: Derived from (2-(((tert- butyldimethylsilyl)oxy)methyl)phenyl)methanol. 2-(((tert-butyldimethylsilyl)oxy)methyl)benzaldehyde was obtained in 60% yield as a yellow oil. 1H NMR (400MHz, CDCl3) δ 10.16 (s, 1H), 7.80 (m, 1H), 7.62 (m, 1H), 7.45 (m, 1H), 5.15 (s, 2H), 0.93 (s, 9H), 0.21 (s, 3H); 13C {1H] NMR (CDCl3, 100MHz) δ 144.28, 122.90, 133.31, 132.60, 126.97, 126.62, 62.88, 25.92, 18.36, -5.35

1,3-diphenylprop-2-yn-1-ol: Derived from 2-(((tert-butyldimethylsilyl)oxy)methyl)benzaldehyde. 1,3-diphenylprop-2-yn-1-ol was obtained in 44% yield as a yellow oil. 1H NMR (400MHz, CDCl3) δ 7.74 (d, 1H), 7.47 (d, 2H), 7.33 (m, 3H), 7.32 (m, 4H), 5.86 (d, 1H), 5.11 (s, 1H), 4.90 (s, 1H), 0.93 (s, 9H), 0.15 (s, 6H); 13C {1H] NMR (CDCl3, 100MHz) δ 139.64, 137.88, 131.69, 129.42, 128.39, 128.25, 123.77, 122.73, 88.41, 86.41, 64.72, 64.00, 63.00, 25.86, 18.26

33

1-(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-3-phenylprop-2-yn-1-one: Derived from 1,3-diphenyl-2-yn-1-ol. 1-(2-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-3-phenylprop-2-yn-1-one was obtained in 65% yield as a yellow oil. 1H NMR (400MHz, CDCl3) δ 8.37 (d, 1H), 7.68 (m, 1H), 7.44 (m, 3H), 7.42 (m, 3H), 5.18 (s, 2H), 0.98 (s, 9H), 0.14 (s, 6H); 13C {1H] NMR (CDCl3, 100MHz) δ 179.41, 145.16, 132.93, 128.63, 126.31, 120.27, 91.97, 87.78, 63.45, 26.01. 18.42, -5.34

34

Appendix II: NMR Spectra for Synthesized Compounds

35

O

O

O

O

phthalide

36

37

38

39

40

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