Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

Dissertation Proposal for Jeffrey Trautmann

Photodynamic Therapy (PDT) is a technique that destroys oncogenic (cancerous) cells. It has been used in several countries since the 1980’s. Molecules that contain chromophores can function as photosensitizers, and will absorb light at various wavelengths (that being dependent upon the extent of their conjugation). Certain functional groups, upon absorption of light, can be used to convert O2 to singlet oxygens which are destructive to the cellular environment. Once an in-vitro cell contains a high enough concentration of photosensitizer material and light is able to reach the photosensitizer material, PDT can be used effectively to destroy that cell.1-3

Folic acid (vitamin B9) 1 is an important material for cellular function. In particular, folate, a metabolite of folic acid, is necessary for DNA biosynthesis and repair, and ultimately for cell growth. When cells, (cancerous or healthy) are deprived of normal amounts of folic acid and then later introduced to a surplus, their rate of folic acid uptake becomes exaggerated. Under these conditions, cancerous cells, due to exhibiting unregulated proliferation, tend to exhibit folic acid uptake at a significantly greater rate than healthy cells.4

Dyes, porphyrins, and chlorophylls have been studied for use as photosensitizers in PDT. Porphyrins and many chlorophylls contain macrocyclic systems that coordinate with various metal cations (often heavy in molecular mass) through heteroatom ligand formation. Dyes are not inherently macrocyclic, and they tend to have a lower molecular mass, making them more applicable for oral bio-availability. Phenothiazine 2, a relatively small chromophore-containing dye, can be amide-linked to folic acid with the appropriate carbon tether to create a molecule that has a molecular mass less than 700 g/mol. Additionally, the nitrogen and the sulfur functionalities of phenothiazine offer opportunity for N-O and/or S-O oxide formation, enabling photo-induced generation of destructive oxygen radicals. The cells of skin-visible tumors are within close proximity to the skin surface, thus allowing skin-penetrating light to reach their cellular environment. By introducing phenothiazine-tethered folamides to the cells of a tumor, it is hoped that PDT will be able to effectively destroy the tumor.5-7

Concurrent studies have employed the use of phenothiazine derivative 3,7-dibromophenothiazine 3 as a precursor for preparing photosensitizers via Buchwald-Hartwig and/or Ullmann methodology. A model system was considered that would provide NMR spectra illustrating well-resolved peaks for the purpose of developing the methodologies of the project. Comparison of 4-bromoaniline 4 with 3 revealed that they both have a nitrogen atom with a pair of non-bonding electrons that can function as an electron donor to a benzene ring, and a bromine at the position para to the nitrogen.

Based on these comparisons, it was believed that 4 would make a suitable analog of 3 for use in a model system. A molecule was also needed to serve as an alternative to the short-chained linker (2-hydroxyethanamine) previously used to tether phenothiazine analogs to folic acid. 4 offered a nitrogen atom that could be joined to folic acid via amidification, and a bromine leaving group that could be used by the same Buchwald-Hartwig or Ullmann coupling methodology previously employed. Furthermore, the planarity of 4-bromoaniline offered reduced sterics, thereby facilitating both N-acylations and transition metal couplings. This led to the belief that 4-bromoaniline would also make a suitable linker for tethering phenothiazine analogs to folic acid.

A comparison between 2 and carbazole 6 derivatives is also underway. The use of a carbomethoxy ester benzyl protecting group allows for coupling of the selected destructive dye to folic acid. Previous studies by P. Low et al.8 demonstrated that polyethylene glycol (PEG) can be used as a spacer between fluorescent liposomes (spherical vessels capable of serving as a vehicle for nutrients and pharmaceuticals) and folate. It was found that tumor-specific delivery could be obtained by using short PEG chains (less than 2000 units in length) with small-size liposomes (less than 100 nm in diameter). Crocetin 12, a potential anti-cancer agent, can be installed as a tether in order to boost UV activity.9-10

Most recently, L. Donahue (Loyola University Chicago) has reported a PEG-folate conjugate that in-part resembles those prepared by Low, in that it consists of a 2000 ethylenedioxy unit linker. Donahue’s target, 16a, exhibits nearly quantitative death within HeLa (immortal-line oncogenic) cells at micromolar concentrations within 120 seconds of exposure to 660 nm light. Pure Chlorin-e6 was also shown by Donahue to kill cells, although to a lesser extent than the conjugate.

This research will focus on preparative synthesis and delivery of folate-linked photosensitizer precursors to folate receptors on cancer cells. Optimization of protection-deprotection sequences, metal-catalyzed oxidative additions, esterifications, and amidifications are of significance. Additional chlorin-e6-PEG-folate conjugates containing ethylenedioxy units approximating 3,400, 10,000, and 20,000 in number are to be prepared (16b-d, respectively). Preparation of certain carotenoids (crocetin) and dyes (phenothiazine-based and carbazole-based carbomethoxy esters) are also being explored as potential photosensitizer precursors. Methods of compound analysis are to include proton-NMR, TLC, and melting point. Exposure of these conjugates to near-IR light for 5.0 µM and 0.5 µM concentrations in phosphate buffer saline (PBS) are to be conducted at intervals of 1 min, 2 min, 4 min, and 6 min. Graphs summarizing in-vitro results will be generated.

Experimental

Methyl-4-(9H-phenothiazin-9-yl)methyl)benzoate (8). CAS 468066-07-1P This compound has been previously reported by Taguchi et al.11 The method for which its preparation is based upon has been previously reported by Bordwell et al.12 To an oven-dried 20 mL round bottom flask containing a spin bar was added potassium hydride slurry (30% KH/mineral spirits) (268 mg slurry, 2.01 mmol actual reagent). The mineral spirits were removed with petroleum ether (3 x 3 mL), and to the potassium hydride was immediately added N,N-dimethylformamide (DMF) (2 mL). 6 (268 mg, 1.60 mmol) was then added, followed by 7 (368 mg, 1.61 mmol). The flask was rinsed with DMF (2 mL), and the reaction was stirred under argon for 48 hours. The reaction was quenched with de-ionized water dropwise to initiate precipitate formation, and the quenched reaction was stirred at room temperature for 10 minutes. The precipitate was filtered through fritted glass, rinse with cold de-ionized water, and concentrated under vacuum to afford 272 mg (54.0%) of 8 as a white solid: TLC Rf 0.61 (50% Et2O/pet ether); 1H NMR (300 MHz, CDCl3)  8.18 (d, 2H),  7.94 (d, 2H), δ 7.43 (d, 2H), δ 7.32 (m, 4H), δ 7.19 (d, 2H),  5.59 (s, 2H),  3.88 (s, 3H).

References

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2. Wilson BC. Photodynamic therapy for cancer: principles. Canadian Journal of Gastroenterology 2002; 16(6):393–396.

3. Vrouenraets MB, Visser GW, Snow GB, van Dongen GA. Basic principles, applications in oncology and improved selectivity of photodynamic therapy. Anticancer Research 2003; 23(1B):505–522.

4. Weinstein SJ, Hartman TJ, Stolzenberg-Solomon R et al. (November 2003). "Null association between prostate cancer and serum folate, vitamin B(6), vitamin B(12), and homocysteine". Cancer Epidemiol. Biomarkers Prev. 12 (11 Pt 1): 1271–2.

5. Marker, A. F. H. (1972). "The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin". Freshwater Biology 2 (4): 361.

6. Jeffrey, S. W.; Shibata, Kazuo (February 1969). "Some Spectral Characteristics of Chlorophyll c from Tridacna crocea Zooxanthellae". Biological Bulletin (Marine Biological Laboratory) 136 (1): 54–62.

7. Gilpin, Linda (21 March 2001). "Methods for analysis of benthic photosynthetic pigment". School of Life Sciences, Napier University.

8. Low, P. S.; Antony, P. (2004). “Folate Receptor-Targeted Drugs for Cancer and Inflammatory

Diseases”. Advanced Drug Delivery Reviews 56: 1055-58.

9. Bodea, C.; Raileanu, M. Ann. Chem. 1960, 631, 194-198.

10. Gutheil, W.; Reed, G.; Ray, A.; Shrikant, A.; and Dhar, A., Current Pharmaceutical Biotechnology. 2012, 13(1), 173-179(7).

11. Taguchi, T. (2002). In Fuji Photo Film Co., Ltd., Japan (Ed.), Novel polymer and its use in luminescent device. Patent Application Country: Application: JP; JP; Priority Application Country: JP: Main IPC: C08F012-32.; Secondary IPC: C08F012-26; C08F026-12; C09K011-06; H05B033-14.

12. Bordwell, F., & Hughes, D. L. (1984). SN2 reactions of nitranions with benzyl chlorides. Journal of the American Chemical Society, 106(11), 3234-40. doi:10

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