「基孔肯雅病」是現今廣泛流行的傳染性疾病之一，且近年有從非洲和南亞蔓延到歐洲和日本的趨勢。目前世上還未有防治此症的疫苗或藥物，治療方式只能減輕該症狀。我們合成出具有抗「基孔肯雅病毒」的「香荳素共軛含氮鹼基化合物」，並成功的運用「聚乳酸–甘醇酸」（poly(lactic-co- glycolic acid)，即PLGA）包覆形成奈米粒子，作為藥物載體。
合成出的「香荳素共軛含氮鹼基化合物」抗「基孔肯雅病毒」之EC50最佳值為12.3 μM，本人使用24.4–40.6 KD、40.6–57.8 KD和49.67–69.58
KD三種不同分子量大小的PLGA做為包覆材料，利用「奈米沉澱法」（nanoprecipitation）製備包覆有「香荳素共軛化合物」PLGA奈米粒子大小，形成為200–450 nm之微胞，最高包覆效率（encapsulation efficiency，即EE）達55.3%。
本人測試發現被包覆之藥物濃度比「香荳素共軛化合物」在水中之濃度多達82–207倍，最多達兩個量級，此結果可以提高「香荳素共軛化合物」在作為口服藥的使用效率。

中文摘要 ………………………………………...………………......… i
英文摘要 ………………………………………...……………...…...… ii
謝誌 ………………………………………...……………...…...… iii
目錄 ………………………………………...………………......… iv
圖目錄 ………………………………………...……………...…...… viii
表目錄 ………………………………………...………………......… x
一、 緒論 ……………………………………...………………….. 1
二、 結果 ……………………………………...………………….. 8
2-1 合成「香荳素」化合物(12) ....................................... 8
2-2 合成「香荳素共軛嘌呤」(1)及「肌苷」(2)化合物於C-6號位 ......................................................................
9
2-3 合成「香荳素」共軛「嘌呤」(3)、「肌苷」(4)及具乙酸基修飾之「肌苷」(5)化合物於C-8號位 ............
9
2-4 合成「香荳素共軛尿嘧啶」(6)、「尿苷」(7)及具乙酸基修飾之「尿苷」(8)化合物於C-4號位 ..…..….....
11
2-5 合成新型「香荳素共軛化合物」(18) ....................... 12
2-6 製備包覆「香荳素共軛化合物」的PLGA奈米粒子 12
2-7 測量包覆「香荳素共軛化合物」的PLGA奈米粒子 14

2-8 生物型穿透式電子顯微鏡（Bio-TEM）下觀察包含有「香豆素共軛化合物」的PLGA奈米粒子 .............
20
2-9 提高「香荳素共軛化合物」用量的實驗 ................. 26
2-10 測試「香荳素共軛化合物」之水溶性和脂溶性 ..... 27
2-11 對包覆有「香荳素共軛化合物」的PLGA奈米粒子進行熱穩定性測試 .........................................................
30
2-12 藥物濃度測試實驗 ..................................................... 31
2-13 由「Bio-TEM」觀察包覆有藥物之PLGA奈米粒子在水中之降解 .................................................................
32
三、 討論 ……………………………………...………………….. 34
3-1 討論「香荳素共軛化合物」的水溶性和脂溶性差異原因 34
3-3 討論Yield、Drug Contnet和EE值的關係 .............. 35
3-4 討論包覆有「香荳素共軛化合物」的PLGA奈米粒子之藥物濃度 .................................................................
36
3-5 藥物濃度之比較 ......................................................... 39
3-5 改善合成路徑討論 ...................................................... 41
3-6 新型「香荳素共軛化合物」18的合成與產物鑑定 . 42
3-7 「脂質體」（liposome）包覆 .................................... 45
四、 結論 ……………………………………...………………….. 46
五、 實驗部分 ……………………………………...……………... 47
General Procedure ……………………….............…...…...….. 47
Preparation of Nucleobase–Coumarin Conjugates loaded PLGA
Nanoparticless ……..................................................…
48
Nanoparticle Recovery (%) ........................................................ 48
Quantification of Drug Loading in PLGA Nanoparticles ......... 49
Determination of Encapsulation Efficiency ............................... 49
Transmission Electron Microscopy（TEM） .......................... 49
Water Solubility ......................................................................... 49
Partition Coefficient Determinations ......................................... 50
6-[(6'-Chlorocoumarin-3'-yl)methylthio]purine (1) .................. 50
8-[(6'-Chlorocoumarin-3'-yl)methylthio]purine (3) ................... 51
4-[(6'-Chlorocoumarin-3'-yl)methylthio]uracil (6) .................... 52
Methyl 3-Hydroxy-2-methylene-3-(2-benzyloxy-5-chloropheny)
propanoate (11) ..............................................................
52
2,4-bis[(6'-Chlorocoumarin-3'-yl)methylthio]uracil (18) ......... 53
六、 參考文獻 ……………………………………...………...…... 55
七、 圖譜 ……………………………………...……………............ 62
1H NMR of 6-[(6'-Chlorocoumarin-3'-yl)methylthio]purine 1 . 63
1H NMR of 8-[(6'-Chlorocoumarin-3'-yl)methylthio]purine 3 . 63
1H NMR of 4-[(6'-Chlorocoumarin-3'-yl)methylthio]uracil 6 .. 64
1H NMR of Methyl 3-hydroxy-2-methylene-3-(2-benzyloxy-5-chloropheny) propanoate 12 ...................................................... 64
1H NMR of 2,4-Thiopyrimidine–coumarin conjugate 18 ......... 65
13C NMR of 2,4-Thiopyrimidine–coumarin conjugate 18 ........ 65
IR of 2,4-Thiopyrimidine–coumarin conjugate 18 ................. 66
HPLC chromatogram of 2,4-Thiopyrimidine–coumarin
conjugate 18 ..................................................................
66
UV of 6-Thiopurine–coumarin conjugate 1 ............................... 67
Calibration curve of 6-thiopurine–coumarin conjugate 1 .......... 67
UV of 8-Thiopurine–coumarin conjugate 3 .............................. 68
Calibration curve of 8-thiopuri coumarin conjugate 3 ............... 68
UV of purine ribofuranoside–coumarin conjugate 5 ................. 69
Calibration curve of purine ribofuranoside–coumarin conjugate 5 69
UV of 4-Thiopyrimidine–coumarin conjugate 6 ....................... 70
Calibration curve of 4-Thiopyrine–coumarin conjugate 6 ........ 70
UV of 4-Thiopyrimidine ribofuranoside–coumarin conjugate 8 71
Calibration curve of 4-Thiopyrimidine ribofuranoside–coumarin
conjugate 8 .....................................................................
71

(1) Rezza, G.; Nicoletti, L. Infection with Chikungunya Virus in Italy: an Outbreak in a
Temperate Region. Lancet 2007, 370, 1840–1846.
(2) Charrel, R. N. First Cases of Autochthonous Dengue Fever and Chikungunya Fever in
France: from Bad Dream to Reality! Clin. Microbiol. Infect. 2010, 10, 1702–1704.
(3) Strobel, M. Chikungunya, an Epidemic Arbovirosis. Lancet Infect. Dis. 2007, 7, 319–327.
(4) Marimoutou, C. Chikungunya Virus Infection. Curr. Infect. Dis. Rep. 2011, 13, 218–228.
(5) Voss, J. E.; Vaney, M.-C.; Duquerroy, S.; Vonrhein, C.; Girard-Blanc, C.; Crublet, E.;
Thompson, A.;Bricogne, G.; Rey, F. A. Glycoprotein Organization of Chikungunya
Virus Particles Revealed by X-ray Crystallography. Nature 2010, 468, 709–714.
(6) Mirkin, C. A. Multiple Thiol-Anchor Capped DNA-gold Nanoparticle Conjugates.
Nucleic Acids Res. 2002, 30, 1558–1562.
(7) Pingarrón, J. M. Enzyme-Controlled Sensing–Actuating NanomachineBased on Janus
Au–Mesoporous Silica Nanoparticles. Chem. Eur. J. 2013, 19, 7889–7894.
(8) Farokhzad, O. C. Formulation of Functionalized PLGA-PEG Nanoparticles for in Vivo
Targeted Drug Delivery. Biomaterials 2007, 28, 869–876.
(9) Hill, L. E.; Taylor, T. M.; Gomes, C. Antimicrobial Efficacy of Ppoly(DL-lactide-co-
glycolide) (PLGA) Nanoparticles with Entrapped Cinnamon Bark Extract Against
Listeria monocytogenes and Salmonella typhimurium. J. Food Sci. 2013, 78, 626–632.
(10) Keselowsky, B. G. Combinatorial Co-Encapsulation of Hydrophobic Molecules in
Poly(lactide-co-glycolide) Microparticles. Biomaterials 2013, 34, 3422–3430.
(11) Lowman, A. M. Novel Oral Insulin Delivery Systems Based on Complexation Polymer
Hydrogels: Single And Multiple Administration Studies in Type 1 and 2 Diabetic Tats.
Curr. Opin. Solid State Mater. Sci. 2002, 6, 319–327.
(12) Feng, S. S. Targeted Delivery of Paclitaxel Using Folate-Decorated Poly(lactide) -
Vitamin E TPGS Nanoparticles. Biomaterials 2008, 29, 2663–2672.
(13) Rossi, C. Leucinostatin-A Loaded Nanospheres: Characterization and in Vivo Toxicity
and Efficacy Evaluation. Int. J. Phytorem. 2004, 275, 61–72.
(14) Moffatt, S. Uptake Characteristics of NGR-Coupled Stealth PEI/pDNA Nanoparticles
Loaded with PLGA-PEG-PLGA tri-Block Copolymer for Targeted Delivery to Human
Monocyte-Derived Dendritic Cells. Int. J. Phytorem. 2006, 321, 143–154.
(15) Kissel, T. Morphological Characterization of Microspheres, Films and Implants
Prepared from Poly(lactide-co-glycolide) and ABA Triblock Copolymers: is the
Erosion Controlled by Degradation, Swelling or Diffusion? Eur. J. Pharm. Sci. 2001,
51, 171–178.
(16) Kunda, N. K. ;Somavarapu, S.; Gordon, S. B.; Hutcheon, G. A.; Saleem, I. Y.
Nanocarriers Targeting Dendritic Cells for Pulmonary Vaccine Delivery. Pharm. Res.
2013, 30, 325–324.
(17) Kumar, S. Aptamer Conjugated Paclitaxel and Magnetic Fluid Loaded Fluorescently
Tagged PLGA Nanoparticles for Targeted Cancer Therapy. J. Magn. Magn. Mater.
2013, 344, 116–123.
(18) Whitesides, G. M. The 'Right' Size in Nanobiotechnology. Nat. Biotechnol. 2003, 21,
1161–1165.
(19) Reyes-Ortega, F. Encapsulation of Low Molecular Weight Heparin (bemiparin) into
Polymeric Nanoparticles Obtained from Cationic Block Copolymers: Properties and
Cell Activity. J. Mater. Chem. B. 2013, 1, 850–860.
(20) Stride, E. Encapsulation of Superparamagnetic Iron Oxide Nanoparticles in Poly-
(lactide-co-glycolic acid) Microspheres for Biomedical Applications. Mater. Sci. Eng.
C. 2013, 33, 3129–3137.
(21) Shapiro, E. M. The Effect of Cryoprotection on the use of PLGA Encapsulated Iron
Oxide Nanoparticles for Magnetic Cell Labeling. J. Nanosci. Nanotechnol. 2013,
13, 3778–3783.
(22) Maleki, M.; Latifi, M.; Amani-Tehran, M. Mathur, S. Non-Invasive Delivery of
Nanoparticles to Hair Follicles: A Perspective for Transcutaneous Immunization.
Polym. Eng. Sci. 2013, 16, 1–10.
(23) Langer, R. Drug Delivery and Targeting. Nature 1998, 392, 5–10.
(24) Langer, R. New Methods of Drug Delivery. Science 1990, 249, 1527–1533.
(25) Dahan, A.; Hoffman, A. Rationalizing the Selection of Oral Lipid Based Drug Delivery
Systems by an in Vitro Dynamic Lipolysis Model for Improved Oral Bioavailability of
Poorly Water Soluble Drugs. J. Control. Release 2008, 129, 1–10. 
(26) Dhar, S.; Gu, F. X.; Langer, R.; Farokhzad, O. C.; Lippard, S. J. Targeted Delivery of
Cisplatin to Prostate Cancer Cells by Aptamer Functionalized Pt(IV) Prodrug-PLGA-
PEG Nanoparticles. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 17356–17361.
(27) Fujiyama, J.; Nakase, Y.; Osaki, K.; Sakakura, C.; Yamagishi, H.; Hagiwara, A.
Cisplatin Incorporated in Microspheres: Development and Fundamental Studies for Its
Clinical Application. J. Control. Release 2003, 89, 397–408.
(28) Prabaharan, M.; Grailer, J. J.; Pilla, S. Gold Nanoparticles with a Monolayer of
Doxorubicin-Conjugated Amphiphilic Block Copolymer for Tumor-Targeted Drug
Delivery. Biomaterials 2009, 30, 6065–6075.
(29) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.;
Zink, J. I. Multifunctional Inorganic Nanoparticles for Imaging, Targeting, and Drug
Delivery. ACS Nano 2008, 2, 889–896.
(30) Lammers, T.; Subr, V.; Ulbrich, K.; Hennink, W. E.; Storm, G.; Kiessling, F.
Polymeric Nanomedicines for Image-Guided Drug Delivery and Tumor-Targeted
Combination Therapy. Nano Today 2010, 5, 197–212.


(31) Kim, T. W.; Slowing, I. I.; Chung, P. W. Ordered Mesoporous Polymer-Silica Hybrid
Nanoparticles as Vehicles for the Intracellular Controlled Release of Macromolecules.
ACS Nano 2011, 5, 360–366.
(32) Khdair, A.; Handa, H.; Mao, G. Z. Nanoparticle-Mediated Combination Chemotherapy
and Photodynamic Therapy Overcomes Tumor Drug Resistance in Vitro. Eur. J. Pharm.
Biopharm. 2009, 71, 214–222.
(33) Hwu, J. R. Synthesis of New Benzimidazole-Coumarin Conjugates as Anti-Hepatitis C
Virus Agents. Antiviral Res. 2008, 77, 157–162.
(34) Bookser, B. C. Adenosine Kinase Inhibitors. 6. Synthesis, Water Solubility, and
Antinociceptive Activity of 5-Phenyl-7-(5-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-
d]pyrimidines Substituted at C4 with Glycinamides and Related Compounds..
J. Med. Chem. 2005, 48, 7808–7820.
(35) Labhasetwar, V. Biodegradable Nanoparticles for Drug and Gene Delivery to Cells and
Tissue. Adv. Drug Delivery Rev. 2012, 64, 61–67.
(36) Fessi, H.; Puisieux, F.; Devissaguet, J. P.; Ammoury, N.; Benita, S. Nanocapsule
Formation by Interfacial Polymer Deposition Following Solvent Displacement.
Int. J. Pharm. 1989, 55, R1–R4.
(37) Galindo-Rodriguez, S.; Allémann, E.; Fessi, H.; Doelker, E. Physicochemical
Parametersassociated with Nanoparticle Formation in the Salting-Out, Emulsification-
Diffusion, and Nanoprecipitation Methods. Pharm. Res. 2004, 21, 1428–1439.
(38) Lee, K. J. A New Synthesis of Methyl 7H-Dibenz[b,g]oxocin-6-carboxylates from
Morita-Baylis-Hillman Adducts of 2-Phenoxybenzaldehydes. Synthesis 2011, 3, 377–386.
(39) Sabliov, C. M. Size Control of Poly(D,L-lactide-co-glycolide) and Poly(D,L-lactide-co-
glycolide)-Magnetite Nanoparticles Synthesized by Emulsion Evaporation Technique.
Colloid Surj. A–Physicochem. Eng. Asp. 2007, 299, 209–216.

(40) Bechet, D. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents.
Trends Biotechnol. 2008, 26, 612–621.
(41) Giunchedi, P. Nasal administration of Carbamazepine using chitosan microspheres: In
Vitro/in Vivo Studies. Int. J. Phytorem. 2006, 307, 9–15.
(42) Kumar, V.; Prud'Homme, R. K. Thermodynamic Limits on Drug Loading in
Nanoparticle Cores. J. Pharm. Sci. 2008, 97, 4904–4914.
(43) Delie, F. Nanomedicines for Active Targeting: Physico-Chemical Characterization of
Paclitaxel-Loaded Anti-HER2 Immunonanoparticles and in Vitro Functional Studies on
Target cells. Eur. J. Pharm. Sci. 2009, 38, 230–237.
(44) Winey, K. I. Haloperidol-Loaded PLGA Nanoparticles: Systematic Study of Particle
Size and Drug Content. Int. J. Pharm. 2007, 336, 367–375.
(45) Fattal, E. Encapsulation of Dexamethasone into Biodegradable Polymeric
Nanoparticles. Int. J. Pharm. 2007, 331, 153–159.
(46) Simõesa, S. Paclitaxel-Loaded PLGA Nanoparticles: Preparation, Physicochemical
Characterization and in Vitro Anti-Tumoral Activity. J. Control Release 2002, 83, 274–287.
(47) Acharya, S.; Sahoo, S. K. PLGA Nanoparticles Containing Various Anticancer Agents
and Tumour Delivery by EPR Effect. Adv. Drug Deliv. Rev. 2011, 63, 170–183.
(48) Maeda, H. The Enhanced Permeability and Retention (EPR) Effect in Tumor
Vasculature: The Key Role of Tumor-Selective Macromolecular Drug Targeting. Adv.
Enzyme Regul. 2001, 41, 189–207.
(49) Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapeutics in
Cancer-Chemotherapy - Mechanism of Tumoritropic Accumulation of Proteins and the
Antitumor Agent Smancs. Cancer Res. 1986, 46, 6387–6392.
(50) Maeda, H.; Matsumura, Y. Tumoritropic and Lymphotropic Principles of
Macromolecular Drugs. Crit. Rev. Ther. Drug Carrier Syst. 1989, 6, 193–210.

(51) Torchilin, V. Tumor Delivery of Macromolecular Drugs Based on the EPR Effect. Adv.
Drug Deliv. Rev. 2011, 63, 131–135.
(52) Danhier, F.; Feron, O.; Preat, V. To Exploit the Tumor Microenvironment: Passive and
Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Control.
Release 2010, 148, 135–146.
(53) Dean, D. R. Fabrication and Characterization of Aligned Nanofibrous PLGA/Collagen
Blends as Bone Tissue Scaffolds. Polymer 2009, 50, 3778–3785.
(54) Islam, S. Lipophilic and Hydrophilic Drug Loaded Pla/Plga in Situ Implants Implants:
Studies on Thermal Behavior of Drug & Polymer and Observation of Parmeters
Influencing Drug Burst Release with Corresponding Effects on Loading Efficiency &
Morphology of Implants. Int. J. Pharm. Pharm. Sci. 2011, 3, 181–188.
(55) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Supuran, C. T. Carbonic Anhydrase
Inhibitors: a General Approach for the Preparation of Water-Soluble Sulfonamides
Incorporating Polyamino−Polycarboxylate Tails and of Their Metal Complexes
Possessing Long-Lasting, Topical Intraocular Pressure-Lowering Properties. J. Med.
Chem.2002, 45, 1466–1476.
(56) Kraszni, M.; Banyai, I.; Noszal, B. Determination of Conformer-Specific Partition
Coefficients in Octanol/Water Systems. J. Med. Chem. 2003, 46, 2241–2245.
(57) Shih, H. T. Surface Modification Characterization and Biocompatibility of
PHBV/PLGA Blend Membrances. 2008, Tatung University, Taiwan, ROC.
(58) Möller, M. Validation of a Novel Molecular Dynamics Simulation Approach for
Lipophilic Drug Incorporation into Polymer Micelles. J. Phys. Chem. B. 2012, 116,
4338–4345.
(59) Guzelian, P. S. 5 of 12 Forms of Vaccinia Virus-Expressed Human Hepatic Cytochrome-
P450 Metabolically Activate Aflatoxin-B1. Nati. Acad. Sci. USA 1990, 87, 4790–4793.
(60) Grimaudo, V.; Gueissaz, F.; Hauert, J.; Sarraj, A.; Kruithof, E. K. O.; Bachmann, F.
Necrosis of Skin Induced by Coumarin in a Patient Deficient in Protein-S. Brit. Med. J.
1989, 298, 233–234.
(61) Robins, R. K.; Hitching, G. H. Studies on Condensed Pyrimidine Systems. XIV. Some
Pyrido [3,2-d] pyrimidines. J. Am. Chem. Soc. 1956, 78, 973–976.
(62) Bangham, A. D.; Standish, M. M.; Watkins, J. C. Diffusion of Univalent Oons Across
Lamellae of Swollen Phospholipids. J. Mol. Biol. 1965, 13, 238–252.
(61) Mosrin, M.; Boudet, N.; Knochel, P. Regio- and Chemoselective Magnesiation of
Protected Uracils and Thiouracils Using TMPMgCl Center Dot LiCl and TMP2Mg
Center Dot 2LiCl. Org. Biomol. Chem. 2008, 6, 3237–3239.
(62) McKinnon, D. M.; Chauhan, M. C-13 Nuclear Magnetic-Resonance Spectra of N-
Methylated, O-Methylated, and S-Methylated Uracil and Thiouracil Derivatives. Can. I.
Chem. 1978, 56, 725–729.
(63) Lin, S. Y. New Nucleoside– and Nucleobase–Coumarin Conjugates as Anti-Hepatitis C
Virus Agents. 2010, National Tsing Hua University, Taiwan, ROC.
(64) Leserman, L. D.; Barbet, J.; Kourilsky, F.; Weinstein, J. N. Targeting to Cells of
Fluorescent Liposomes Covalently Coupled with Monoclonal Antibody or Protein-A.
Nature 1980, 288, 602–604.
(65) Heath, T. D.; Fraley, R. T.; Papahdjopoulos, D. Antibody Targeting of Liposomes - Cell
Specificity Obtained by Conjugation of F(Ab')2 to Vesicle Surface. Science 1980, 210,
539–541.
(66) Allen, T. M.; Chonn, A. Large Unilamellar Liposomes with Low Uptake into the
Reticuloendothelial System. FEBS Lett. 1987, 223, 42–46.
(67) Klibanov, A. L.; Maruyama, K.; Torchilin, V. P.; Huang, L. Amphipathic
Polyethyleneglycols Effectively Prolong the Circulation Time of Liposomes. FEBS Lett.
1990, 268, 235–237.