【正文】 human fibroblasts were seeded in 96well plates at subconfluent density (1000 cells/well) or confluent density (10000 cells/well). After 24 h, fresh medium containing 10 μM MOMIPP or an equal volume of vehicle (DMSO) was added. MTT viability assays were performed at a 48 h end point. Values are the means (177。 SD), with the results expressed as percent of the mean from parallel control dishes treated with an equivalent volume of vehicle alone (DMSO). (C) Parental and doxorubicinresistant (DoxR) MCF7 breast cancer cells(56) were treated with 10 μM MOMIPP for 24 h and then examined by phasecontrast microscopy. (D) Longterm viability of parental or DoxR MCF7 cells was assessed by colonyforming assay after a 48 h of treatment with the indicated concentrations of MOMIPP. The results are the mean 177。MOMIPP對(duì)亞融合與融合的成纖維細(xì)胞的影響差別提供了一個(gè)可能的解釋?zhuān)@意味著，以空泡為典型的內(nèi)體販運(yùn)的中斷可能在不分裂的細(xì)胞中比在正常增殖的細(xì)胞中具有相對(duì)較好的耐受性。然而，當(dāng)纖維母細(xì)胞接種在一個(gè)較高的初始密度，使他們?cè)贛OMIPP加入時(shí)以形成固定相，那么細(xì)胞活性基本上不受影響（圖5H），即使細(xì)胞發(fā)生空泡（未顯示）。在所有其他細(xì)胞測(cè)試實(shí)驗(yàn)中，到24小時(shí)10μMMOMIPP明顯誘導(dǎo)成纖維細(xì)胞空泡化（圖5G）。到48小時(shí)，細(xì)胞活力減少約40％，而在類(lèi)似的細(xì)胞密度下同期處理的MCF7乳腺癌細(xì)胞減少了70％（圖5F）。為了確定對(duì)腫瘤細(xì)胞造成最大限度毒性作用的濃度的MOMIPP是否也會(huì)對(duì)正常細(xì)胞產(chǎn)生毒性，我們用10μMMOMIPP處理人類(lèi)乳腺上皮細(xì)胞（HMEC）。MOMIPP通過(guò)methuosis形式殺死有抗藥性的腫瘤細(xì)胞的能力并不僅僅局限于膠質(zhì)母細(xì)胞瘤。 SD from three separate cultures.Figure 3. Comparison of the abilities of MOMIPP and MIPP to induce the morphological hallmarks of methuosis. One day after plating, U251 GBM cells were treated with MOMIPP or MIPP at final concentrations of (A) or 10 μM (B). Controls received an equivalent volume of vehicle (DMSO). Cells were observed by phase contrast microscopy on three sequential days after addition of the pounds, without changing the medium or replenishing the pounds. Methuosis is characterized by extensive accumulation of phaselucent cytoplasmic vacuoles, with eventual cell rounding and detachment from the substratum as viability Figure 4. Comparison of the abilities of MOMIPP and MIPP to inhibit survival of U251 GBM cells in colonyforming assays. (A) Cells were plated for colonyforming assays as described in the Experimental Section. One day after plating the cells, MOMIPP (●) or MIPP (■) was added to the medium at the indicated concentrations, and cells were maintained in the presence of the pounds for 48 h. Thereafter, the pounds were removed, and colonies 50 cells were counted after 2 weeks. Each point represents the mean (177。Figure 2. Comparison of the effects of MOMIPP (pound 19) and MIPP (pound 2) on growth and viability of U251 GBM cells. (A). One day after plating the cells in 96well dishes, MOMIPP (●) or MIPP (■) was added at the indicated concentrations. Controls consisted of cells in parallel wells treated with an equivalent volume of vehicle (DMSO). Medium containing fresh pound was added after 24 h, and MTT assays were performed after a total 48 h of treatment. Each point represents the mean (177。當(dāng)細(xì)胞處理2天時(shí)，MOMIPP顯??然在減少細(xì)胞集落形成方面比MIPP有效（圖4A）。這些研究表明，單獨(dú)作用時(shí)，MOMIPP比MIPP對(duì)細(xì)胞形態(tài)和細(xì)胞活力的影響更持久。在用MOMIPP處理的組中細(xì)胞數(shù)量的減少以大規(guī)模早期細(xì)胞空泡的出現(xiàn)和底層非活性細(xì)胞的損傷的形式表現(xiàn)。與可行性研究不同的是，在這些實(shí)驗(yàn)中，該化合物在研究開(kāi)始時(shí)被添加，并沒(méi)有持續(xù)的補(bǔ)充。分別添加每個(gè)化合物到指定的濃度，保持2天，并且在第一天后要補(bǔ)充原料。 MOMIPP與化合物2（MIPP）高生物活性在研究U251膠質(zhì)瘤細(xì)胞中被證實(shí)，研究中我們采用MTT法的可行性分析，細(xì)胞生長(zhǎng)實(shí)驗(yàn)，形態(tài)評(píng)估，和集落形成實(shí)驗(yàn)來(lái)比較MOMIPP和MIPP。 (2) 5 N NaOH, 99%. Compound 26: 4acetylpyridine, piperidine, MeOH, reflux, 34%. Compound 27: 4acetylpyridine, piperidine, MeOH, reflux, 21%.化合物19（MOMIPP）與化合物2（MIPP）生物活性比較上述研究確定化合物19是最有力誘導(dǎo)methuosis的化合物。 (2) NaHCO3, 90%. Compound 25: (1) POCl3, DMF, 0 176。C, 93%. Compound 23: methyl4(bromomethyl)benzoate, NaOH, TBAB, CH2Cl2, RT, 63%. Compound 24: (1) POCl3, DMF, 0 176。然而，在神經(jīng)母細(xì)胞瘤的實(shí)驗(yàn)測(cè)試發(fā)現(xiàn)，既不是26也不是27誘導(dǎo)methuosis（見(jiàn)表1）。相關(guān)反應(yīng)在溫和條件基礎(chǔ)下（碳酸氫鈉）生產(chǎn)了酯24，而在5 N的氫氧化鈉條件下產(chǎn)生酸25。 4 甲基（溴甲基）苯甲酸烷基化產(chǎn)物在相轉(zhuǎn)移條件下用于合成單 O被烷基化產(chǎn)品23。因此，一個(gè)新的路線即在引入吡啶基團(tuán)（計(jì)劃2）前官能化吡