Development of vesicular glutamate transnporter inhibitors

谷氨酸是哺乳动物中枢神经系统中一种主要的兴奋性神经递质，与记忆和学习等高级大脑功能有关。谷氨酸通过谷氨酸水疱转运蛋白（VGLUTs）集中到突触小泡中，并储存在神经末梢的小泡中。由于已知的抑制剂也显示谷氨酸受体和突触谷氨酸转运体（EAATs）的亲和力，因此需要选择性的VGLUTs抑制剂。抑制剂通过质膜来抑制神经元内的VGLUTs是很重要的。为了提高VGLUTs的选择性、效力和细胞膜通透性，我们合成了新的反式acpd衍生物。首先，我们合成了甲基类似物来研究取代基的位置和立体化学。其中，5r -甲基类似物抑制谷氨酸进入囊泡。因此，我们试图引入羟基类似物和醚类似物来研究取代基的影响。与甲基类似物的合成方案类似，用相应的酮制得的烷基烯卡宾用于苯氧基类似物的合成。然而，苯基位置反应得到一个醚环，而不是五元环环。我们研究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，将γ-羧基转化为甲酯（ACPD-OMe）。ACPD-OMe不抑制谷氨酸向离体囊泡的转运，但降低了谷氨酸通过突触体的转运。另一方面，当使用突触体时，反式acpd不影响谷氨酸的转运。ACPD-OMe通过膜后被酶转化为反式acpd，抑制VGLUTs。这些结果表明羧酸的修饰有助于提高细胞膜的通透性和开发新型的VGLUT抑制剂。

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.