臺灣博碩士論文加值系統

紫杉醇是一種由太平洋紫杉的樹皮中萃取出來的抗癌藥物,已經被應用於臨床治療。然而,由於紫杉醇缺乏對癌細胞的專一性,在治療癌症的同時也會傷害到其他的正常組織。這項缺點限制了紫杉醇在癌症治療上的廣泛運用。具有專一傳輸性質的前驅藥物將能解決這項缺點。前驅藥物以低毒性的形態在體內傳送,在到達目的地後,它的毒性才會被釋放出來,而達到專一消滅癌細胞的效果。
在這篇論文中,我們設計合成了四種分別以葡萄糖和葡萄糖醛酸做為傳輸載體的前驅藥物。這四種藥物的設計概念是基於葡萄糖傳輸孔道與葡萄糖醛酸水解酶在癌細胞上的過度表現。在這四種前驅藥物中,以prodrug 1對人類癌症細胞株(NCI-H838, Hep-3B, A498, MES-SA, HCT-116, NPC-TW01,和MKN-45)具有最高的毒殺性質,僅較紫杉醇低二至十六倍。除此之外,我們也測試了這些藥物對正常細胞株(HUV-EC-C和CHO-K1)的細胞毒性。除了prodrug 4之外,我們的醣化前驅藥物對癌細胞都展現了高度的選擇性。
在本研究中,我們也證明了prodrug 1確實可以被癌細胞所攝取。為了追蹤藥物,我們在紫杉醇的C7上加上了一個螢光基團—間位胺基苯甲酸(m-aminobenzoyl group)。經過五小時的藥物處理後,該藥物出現於細胞質中。這表示癌細胞可以在短時間內將該藥物吸收。若將該藥物施予癌症病患,將可能降低藥物被正常細胞吸收的機率,以減緩化學治療所帶來的副作用。
當細胞處理藥物二十四小時後,我們觀察到微質管(microtubule)在細胞內累積聚集的現象。我們利用可移除式的連接鍊(linker)暫時抑止這些前驅藥物的毒性。我們假設在醣基被切除後,連接鍊尾端的官能基將攻擊連接紫杉醇活性區的羧酸基(carboxylic group),並釋放出紫杉醇以執行毒殺癌細胞的功能。由雷射掃描共軛焦顯微鏡的影像顯示,無論細胞被處理紫杉醇或醣化前驅藥物,都有微質管累積的情形。這項結果支持了我們先前所提出的藥物釋放假說。
我們所設計的前驅藥物也具有與紫杉醇相似的另一項生物活性—促使細胞凋亡(apoptosis)。相對於壞死(necrosis),細胞凋亡是一種較為溫和的細胞死亡方式。為了驗證醣化前驅藥物是否將引發細胞凋亡,我們利用核染色的方式觀察染色體的型態。當細胞凋亡時,它的染色體會開始濃縮、聚集。當細胞處理我們的前驅藥物後也出現這個現象。這顯示前驅藥物是藉由引發細胞凋亡的路徑造成細胞死亡。
總結而論,我們已設計合成出四種醣化紫杉醇前驅藥物並測試其生物活性。這些藥物造成細胞內的微質管累積聚集並引發細胞凋亡以達到殺死癌細胞的藥理效果。我們也利用螢光標定prodrug 1的方式證明了該藥物確實可以在短時間內被癌細胞所攝取。在這四種藥物中,以prodrug 1的毒性與癌細胞專一性最佳,未來將做更深入的研究與探討。

Paclitaxel (Taxol), a natural diterpene compound, is a promising anticancer drug that was shown substantial clinical efficacy to solid tumor. However, like other cytotoxic agent, taxol lacks of selectivity and contributes to serious side effects. The conversion of taxol into prodrugs that can be specifically delivered to cancer cells may prevent this shortcoming.
In this study, we designed and synthesized four glycan-based paclitaxel prodrugs. Compound 1 and 2 are glucose-based prodrugs; and 3 and 4 are glucuronic acid-based ones. Given the high level expressions of glucose transporters and -glucuronidase in carcinomas, there is rationale to develop glycan-targeting prodrugs. Within these four compounds, prodrug 1 has the best cytotoxicity against seven human tumor cell lines (NCI-H838, Hep-3B, A498, MES-SA, HCT-116, NPC-Two1, and MKN-45). The cytotoxicity of prodrug 1 is only 2 to 16-fold lower than taxol. In addition, we also tested the toxicity of these drugs to normal cell lines (HUV-EC-C and CHO-K1). Except prodrug 4, the IC50 of other compounds are more than 100 μM. Our prodrugs showed good cytotoxicity and selectivity to cancer cells.
We also confirmed the internalization of prodrugs 1 into cancer cells. In order to trace this drug, we added a chromophore, m-aminobenzoyl group, in C7 position of taxol skeleton. After five hours treatment, the drug was seen in the plasma regions of NPC-TW01 cells. This result suggested that glucose-based prodrug 1 can be taken up by cancer cells within short time treatment. By means of rapid internalization by cancer cells, our prodrug may ease the side effects when administrating to patients.
Microtubule aggregation was observed in NPC-TW01 cells after stimulation with these prodrugs for 24 hours. In our prodrugs, the biological properties of taxol are masked temporarily by blocking its active site with a self-immolative linker. We except that paclitaxel will be liberated after glycan removal and spacer autocleavage.
In confocoal images, microtubules in cells treated with taxol or prodrugs were observed to lose silk structures. This result supported the hypothesis that our prodrugs can be converted into taxol and interference the dynamics of microtubule.
Like taxol, our prodrugs can cause cell death through apoptosis. In contrast to necrosis, apoptosis is a milder cell death pathway. In order to avoid inflammation caused by necrosis, we must demonstrate that these drugs kill cells in apoptosis pathway. Chromosome condensation is a distinct character of programmed cell death; and we found this phenomenon in cells treated with these four compound. This result indicated that apoptosis is the major death mechanism triggered by our prodrugs.
In summary, we have synthesized four glycan-based paclitaxel prodrugs and analyzed their biological characters. These prodrugs showed antitumor effect on promoting microtubule assembly and inducing apoptosis. Within these four drugs, prodrug 1 has the best cytotoxicity and selectivity to cancer cells according to the result of cytotoxicity assay. Furthermore, prodrug 1 was also proved to internalize into cells after five-hour treatment. This compound is a potential candidate for further investigation.

Acknowledge i
Abstract (Chinese) ii
Abstract (English) iv


1. Introduction 1
2. Result
2.1 Chemistry
2.1.1. Synthesis of prodrug 1 and 2 5
2.1.2. Synthesis of prodrug 3 and 4 6
2.1.3. Synthesis of fluorescence-labeled taxol (21) 8

2.2 Biology
2.2.1. Cytotoxicity of prodrugs 1, 2, 3, and 4 to cancer and normal
cell lines 9
2.2.2. Internalize of fluorescence-label drugs in NPC-TW01 cell
lines 11
2.2.3. Confocoal microscopy analysis of NPC-TW01 cells which
were treated with prodrugs 1-4 11

3. Experimental
3.1. Materials 13
3.2. Chemistry
3.2.1. Succinic acid mono-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano
[3,2-d][1,3]dioxin-7-yl) ester(5) 14
3.2.2. C15 (6) 15
3.2.3. Prodrug 1 (1) 16
3.2.4. Benzoic acid 4-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d]
[1,3]dioxin-7-yloxy)-butyl ester (7) 18
3.2.5. 4-(8-Benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d]
[1,3]dioxin-7-yloxy)-butan-1-ol (8) 19
3.2.6. 4-(8-Benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d]
[1,3]dioxin-7-yloxy)-butyric acid (9) 20
3.2.7. Taxol-ether-C14 (10) 21
3.2.8. Prodrug (2) 23
3.2.9. Benzoic acid 8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d]
[1,3]dioxin-7-yl ester (11) 25
3.2.10. Benzoic acid 4,5-bis-benzyloxy-6-hydroxymethyl-2-methoxy-tetrahydro-
pyran-3-yl ester (12) 26
3.2.11. 3,4-Bis-benzyloxy-5-hydroxy-6-methoxy-tetrahydro-pyran-
2-carboxylic acid benzyl ester (13) 27
3.2.12. Succinic acid mono-(4,5-bis-benzyloxy-6-benzyloxycarbonyl-2-methoxy-
tetrahydro-pyran-3-yl) ester (14) 28
3.2.13. Glucronic-ester-taxol (15) 29
3.2.14. Prodrug 3 (3) 31
3.2.15. Benzoic acid 4-(4,5-bis-benzyloxy-6-hydroxymethyl-2-methoxy-tetrahydro-
pyran-3-yloxy)-butyl ester (16) 33
3.2.16. 3,4-Bis-benzyloxy-5-(3-carboxy-propoxy)-6-methoxy-
tetrahydro-pyran-2-carboxylic acid (17) 34
3.2.17. Glucronic-ether-taxol (18) 35
3.2.18. Prodrug 4 (4) 37
3.2.19. Taxol-2’TES (19) 39
3.2.20. Taxol-2’TES-7F (20) 40
3.2.21. Taxol-2’OH-7F synthesis (21) 41
3.2.22. Fluoresce-labeled prodrug 1 (22) 43
3.3 Biology
3.3.1. Cell proliferation assay 44
3.3.2. Fluorescence microscopy 45
3.3.3. Confocoal microscopy 46
4. Discussion 47
5. References 52
6. Schemes 55
7. Figures 62
8. Tables 70
9. Appendix 71

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