合成α-GalCer之衍生物作為免疫刺激劑

Abstract

從海棉中萃取出的醣脂質化合物— α-Galactosylceramide (α-GalCer)，在in vivo與in vitro研究顯示其具有抗腫瘤活性。這種醣脂質與抗原呈現細胞上的CD1d蛋白結合後，形成CD1d/醣脂質/自然殺手T細胞複合體，而刺激自然殺手T細胞產生Th1 細胞激素和Th2細胞激素。在醯基鏈或醣基之C6”有芳香基取代之α-GalCer衍生物，有較佳之刺激Th1細胞激素分泌之選擇性。目前尚未有科學家研究這兩類化合物芳香基上之取代的結構與活性關係，因此，根據Topliss方法，我們設計苯基取代的癸醯基鏈類似物，使用A20CD1d 和mNK1.2系統測試其對IL-2分泌之刺激，結果顯示這類癸醯基鏈苯基取代基的化合物具有與α-GalCer相當之活性。我們在C6”位置上以苯基醯胺取代，發現碳鏈長度最短之苯基乙醯胺取代的87在87 - 89當中最有活性。因此，我們進一步合成C6”和醯基鏈雙苯基取代之化合物，其中在C6”之苯基有推電子取代之94, 98和100，相較於α-GalCer的免疫刺激活性略為增加。另外，我們合成醯基鏈有雙羥基之化合物，為增加化合物之水溶性，活性測試顯示其活性與α-GalCer相當。 目前最有活性之α-GalCer類似物C34，被選為進入前臨床及臨床試驗之抗癌藥物。為了藥物化學之研究，文獻中僅有小量之醣脂質合成方法，但大量的製程研究還未被報導過。為此，我們開發大量的C34製程，以phytosphingosine HCl為起始物，經由八個化學反應，總產率為14%。關鍵的醣化反應，我們選擇 thioglycoside做為供給者以Me2S2-Tf2O在溫和的條件下反應，較適合於工業上之應用。經過反應條件之最佳化，我們能夠在實驗室操作50克之批次。

α-Galactosylceramide (α-GalCer), a glycolipid derived from marine sponge, found to exhibit antitumor activity in vitro and in vivo. Upon binding with CD1d molecule on APC, this CD1d/glycolipid/NKT cell complex stimulates the production of both Th1 and Th2 cytokines by NKT cells. Aromatic acyl chain modifications and aromatic substituents on ceramide and galactose C6” of α-GalCer, respectively, were found to exhibit potent agonistic activity to Th1 cytokines secretions. The structure-activity relationship (SAR) study of the substitutions on both aromatic rings has not yet been reported. Base on Topliss rule, we designed various phenyl-substituted acyl chain analogs of α-GalCer and evaluated their IL-2 secretion in A20CD1d & mNK1.2 system. Compounds with different aromatic substitutions on undecanoyl chain were shown to have comparable activity to α-GalCer. The C6” phenyl acetamido substituted analog 87 was the most potent one among 87 - 89. Furthermore, the C6” and acyl chain bi-modified analogs were synthesized and evaluated, compounds with electron-donating property (94, 98, 100) at C6” phenyl acetamido group were found to slightly increase immunostimulating activity compared to α-GalCer. The most potent α-GalCer analog, C34, was chosen for preclinical and clinical application for anticancer therapy. Although milligram-scale syntheses of glycolipids have been developed for medicinal chemical research, a large scale synthesis and process development have not been reported. To this end, we developed a scale-up synthesis of C34 in an eight-step process commenced from phytosphingosine HCl in total 14% yield. The critical step of this process is glycosylation, thiolglycoside was selected as a glycosyl donor which was activated by Me2S2-Tf2O under mild condition. Such strategy is more applicable in industrial practice. After each step was fine tuned and optimized, we were able to operate up to 50 g scale in the laboratorial batch.