Development of vesicular glutamate transnporter inhibitors

谷氨酸是哺乳动物中枢神经系统中的主要兴奋性神经递质，与大脑的高级功能如记忆和学习有关。谷氨酸通过囊泡谷氨酸转运蛋白（VGLUT）浓缩到突触囊泡中，并储存在神经末梢的囊泡中。由于已知的抑制剂还显示出对谷氨酸受体和突触谷氨酸转运体（EAAT）的亲和力，因此需要VGLUT的选择性抑制剂。重要的是抑制剂渗透质膜以抑制神经元内的VGLUT。为了提高对VGLUT的选择性、效力和细胞膜通透性，我们合成了新型反式ACPD衍生物。首先，我们合成了甲基类似物，以检查取代基的位置和立体化学。其中，5R-甲基类似物表现出抑制谷氨酸摄取到囊泡。因此，我们尝试引入羟基类似物和醚类似物来检查取代基的影响。类似于甲基类似物的合成方案，由相应的酮制备的亚烷基卡宾被应用于苄氧基类似物。然而，苄基位置反应得到醚环，而不是五元环环化。我们研究了羟基上的其他保护基团来克服这个问题。此外，为了提高细胞膜的通透性，γ-羧基被转化为甲酯（ACPD-OMe）。ACPD-OMe不抑制谷氨酸向囊泡的转运，但可抑制突触体的谷氨酸转运。另一方面，trans-ACPD对突触体的谷氨酸转运无影响。ACPD-OMe经膜渗透后被酶转化为反式ACPD，从而抑制VGLUTs。这些结果表明，羧酸的修饰对于改善细胞膜通透性和开发新型VGLUT抑制剂是有用的。

Glutamate is a major excitatory neurotransmitter in mammalian central nervous systems and is linked to higher brain functions such as memory and learning. Glutamate is concentrated into synaptic vesicles through Vesicular Glutamate Transporters (VGLUTs) and is stocked in vesicles in nerve endings. As known inhibitors also show affinity for glutamate receptors and synaptic glutamate transporters (EAATs), selective inhibitors for VGLUTs have been required. It would be important that inhibitors permeate plasma membrane to inhibit VGLUTs inside neurons. In order to improve selectivity for VGLUTs, potency, and cell membrane permeability, we synthesized novel trans-ACPD derivatives. First, we synthesized methyl analogs to examine the position and stereochemistry of a substituent. Among them, 5R-methyl analog showed inhibition of glutamate uptake into vesicles. Thus, we tried to introduce a hydroxyl analog and ether analogs to examine the effects of a substituent. Similar to the synthetic scheme of methyl analogs, alkylidenecarbene prepared from the corresponding ketone was applied for the benzyloxy analog. However, benzyl position reacted to give an ether ring instead of the five membered-ring cyclization. We examine other protective groups on the hydroxyl group to overcome this problem. Furthermore, to improve the cell membrane permeability, γ-carboxyl group was converted into methyl ester (ACPD-OMe). ACPD-OMe didn't inhibit the transport of glutamate into the isolated vesicles, while it decreased the glutamate transport using synaptosomes. On the other hand, trans-ACPD didn't affect the glutamate transport when synaptosomes were used. ACPD-OMe would be converted to trans-ACPD by enzyme after membrane permeation and, then, inhibit VGLUTs. These results demonstrate that the modification of carboxylic acid is useful for the improvement of cell membrane permeability and development of novel type of VGLUT inhibitors.