Synthesis and in vitro and in vivo screening of fused and tethered heterocyclic peptidomimetics for the discovery of new analgesics with decreased side effects

我们推测，外周限制的混合活性阿片受体激动剂的开发将 产生强有力的镇痛作用，同时表现出减少的副作用，包括缺乏呼吸抑制或 增强性能在初步研究中，体外筛选新的双咪唑烷和稠合 杂环先导化合物BIM-22和FDC-14鉴定了混合阿片受体激动剂活性。后 外周（i. p.）给药小鼠，两种化合物表现出与吗啡相当的抗伤害感受作用 在55 ℃温水断尾（甩尾）试验中，FDC-14的效力是吗啡的25倍 在醋酸扭体试验中，镇痛效力比为72.7，提示外周- 限制活动。为此，LC-MS/MS研究在收获的小鼠血液中检测到BIM-22和FDC-14， 而不是脑。该建议涉及两个特别感兴趣的研究领域， FOA：i）开发新的和创新的受体分子探针和新的策略， 创新的肽模拟物设计，和ii）鉴定结构多样的、口服活性的、代谢上 稳定的外周限制性阿片激动剂。我们提出了六个相互作用的目标：在目标1中，我们提出 杂环肽模拟物的计算指导合成：双咪唑烷-2-亚胺、哌嗪、双- 哌嗪、双咪唑酮和稠合杂环文库。在目标2中，我们将筛选 竞争性放射性配体结合试验，用于测定μ-（莫尔）、δ-（DOR）和κ-（KOR）阿片样物质的亲和力 受体，并在功能测定中鉴定双重δ/κ或双重δ/μ激动剂。在目标3中，选择了十种化合物 将在体外评价药代动力学特性，并使用 小鼠甩尾测定。从这一目标中确定的铅化合物将指导第二代SAR研究， 增强外周选择性活性。在目标4中：我们将进行完整的体内抗伤害性表征， 先导激动剂化合物。在目的2+3中，不穿过BBB的最稳定、活性最高的4种激动剂将是 在腹膜内给药后，用小鼠甩尾和醋酸扭体试验评价其功效、持续时间、 作用和阿片受体选择性。在目标5中：我们将表征所选的4种化合物在小鼠中的生物利用度。 在口服给药后，这些化合物被证实不能穿透血脑屏障。目标6： 将检查目的1-5中鉴定的两种最有效的生物可利用的新型激动剂的责任， 特别是抗伤害耐受性，在CLAMS生理和呼吸和过度运动的影响， 行为评估系统，在旋转杆测定中镇静和协调运动活动的破坏， 以及在条件性位置偏爱（CPP）测定中评价奖励或厌恶效应。影响 还将评价GI转运，以评估对便秘的影响。总而言之，我们希望产生新的 作为探针和镇痛剂的外周限制性混合活性拟肽类阿片受体激动剂 副作用减少，从而显著影响镇痛药的开发。

We hypothesize that the development of peripherally-restricted, mixed-activity opioid receptor agonists will produce robust analgesia while exhibiting reduced side effects, including a lack of respiratory depression or reinforcing properties. In preliminary studies, the in vitro screening of novel bis-imidazolidines and fused heterocyclic lead compounds BIM-22 and FDC-14 identified mixed-opioid receptor agonist activity. After peripheral (i.p.) administration to mice, both compounds demonstrated antinociception equivalent to morphine in the 55oC warm-water tail withdrawal (tail-flick) assay, but FDC-14 was 25 times more potent than morphine in the acetic acid writhing test, with an antinociceptive potency ratio of 72.7, suggestive of peripherally- restricted activity. Confirming this, LC-MS/MS studies detected BIM-22 and FDC-14 in harvested mouse blood, but not brain, after i.v. administration. This proposal addresses two research areas of particular interest for this FOA: i) The development of new and innovative molecular probes for receptors and new strategies for innovative peptidomimetic design, and ii) The identification of structurally diverse, orally active, metabolically stable peripherally-restricted opioid agonists. We propose six interacting aims: In Aim 1, we propose the computationally guided synthesis of heterocyclic peptidomimetics: bis-imidazolidin-2-imines, piperazines, bis- piperazine, bis-imidazolone and fused heterocyclic libraries. In Aim 2, we will screen all compounds in competition radioligand binding assays to determine affinity for μ- (MOR), δ- (DOR), and κ- (KOR) opioid receptors, and in functional assays to identify dual δ/κ or dual δ/µ agonists. In Aim 3, ten selected compounds will be evaluated in vitro for pharmacokinetic properties and screened in vivo for antinociception using the mouse tail-flick assay. Lead compounds identified from this aim will guide 2nd generation SAR studies to enhance peripherally-selective activity. In Aim 4: we will perform full in vivo antinociceptive characterization of lead agonist compounds. The most stable, active 4 agonists not crossing the BBB in Aims 2+3 will be evaluated after i.p. administration with mouse tail-flick and acetic-acid writhing assays for efficacy, duration of action, and opioid receptor selectivity. In Aim 5: we will characterize the bioavailability in mice of the selected 4 agonists following oral administration and confirm their inability to penetrate the blood brain barrier. In Aim 6: The two most potent bioavailable novel agonists identified in Aims 1-5 will be examined for liabilities, specifically antinociceptive tolerance, respiratory and hyperlocomotor effects in the CLAMS physiological and behavioral assessment system, sedation and disruption of coordinated locomotor activity in the rotorod assay, and assessment for rewarding or aversive effects in the conditioned place preference (CPP) assay. Effects on GI transit will also be evaluated to assess effects on constipation. In summary, we expect to generate novel peripherally-restricted, mixed-activity peptidomimetic opioid receptor agonists as both probes and analgesics with reduced side effects, thereby significantly impacting analgesic development.