CN114369060B - 吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 - Google Patents

吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用 Download PDF

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CN114369060B
CN114369060B CN202011100808.9A CN202011100808A CN114369060B CN 114369060 B CN114369060 B CN 114369060B CN 202011100808 A CN202011100808 A CN 202011100808A CN 114369060 B CN114369060 B CN 114369060B
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nicotinic acid
ido1
tumor
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CN114369060A (zh
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谭瀛轩
向道凤
颜道静
谭相宝
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Hangzhou Xing'ao Biological Technology Co ltd
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Abstract

本发明属于生物医药技术领域，发明了一类吲哚胺2，3‑双加氧酶IDO1的抑制剂，即烟酸衍生物(3‑吡啶丙酸的衍生物)。该类3‑吡啶丙酸的衍生物能有效抑制吲哚胺2，3‑双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性低。这些烟酸衍生物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂联合使用/或与免疫检查点抑制剂联合使用，抑制肿瘤生长药效更佳，安全性也很好。因此，这类烟酸衍生物在制备免疫抗肿瘤药物中具有广阔的应用前景。

Description

吲哚胺2，3-双加氧酶抑制剂及其在制备抗肿瘤药物中的应用
技术领域
本发明属于生物医药技术领域具体涉及一类烟酸衍生物对吲哚胺2，3-双加氧酶IDO1的抑制剂其在制备免疫抗肿瘤药物中的应用
背景技术
肿瘤是一类严重危害人类生命健康的重大疾病之一，表现为细胞过度增殖和分化异常。据WHO统计，全球范围内每年癌症病例可能增加到2400万。与此同时，癌症给全球经济造成了巨大的负担。肿瘤免疫治疗是继手术、放疗、化疗后的新兴肿瘤治疗方式。它是一种利用刺激或增强人体自身免疫***的力量来预防、控制和消灭癌症的癌症治疗方法。与传统的肿瘤治疗方法相比，免疫治疗具有自身特有的优势，可以提高传统治疗的疗效，减轻传统治疗带来的不良反应等。先天免疫***激动剂、免疫检测点阻断剂和细胞免疫治疗是目前免疫治疗的主要研究领域。基于代谢调控的肿瘤免疫治疗策略，可以提高免疫治疗的有效性，让更多的患者受益，肿瘤代谢的调控为改善肿瘤免疫治疗开辟了新方向。
人体的免疫***负责识别自身与非自身，从而保护人体免受外源性和内源性疾病的侵害。人体免疫***由白细胞和淋巴器官以及淋巴组织构成，包括胸腺、脾脏、扁桃体、***、***和骨髓，免疫***通过识别并消除多种威胁来保持机体的持续稳态。癌症免疫疗法是指通过引入疫苗、细胞因子、抗体或转移的免疫细胞本身来刺激针对癌细胞的免疫***。免疫疗法在肿瘤治疗上的成功，例如免疫检查点抑制剂和CAR-T细胞疗法，已经确立了它在癌症治疗中的作用。癌症是使用多种机制来选择宿主-肿瘤免疫相互作用，从而导致免疫逃逸。在过去的几年中，我们对宿主-肿瘤相互作用的研究不断有新的进展，产生了各种有前景的免疫治疗方法。
吲哚胺2，3-双加氧酶(IDO1)是一种单体血红素蛋白，分子量为45KD。IDO1能对具有吲哚环的分子进行催化，因此被称为吲哚胺2，3-双加氧酶。IDO 1是色氨酸代谢途径中的关键限速代谢酶。大量研究表明IDO1在多种类型的人类癌症中高度表达，随后导致色氨酸代谢产物的积累，造成机体对肿瘤抗原的免疫耐受，最终导致肿瘤的免疫逃逸。许多临床前和临床试验研究表明IDO1抑制剂是对抗多种癌症的有效工具。目前，IDO1抑制剂***的临床研究正在进行，它们可以有效激活肿瘤浸润性T细胞和/或减少肿瘤驻留性免疫抑制性调节性T细胞，从而提高人体免疫，达到抗肿瘤的目的。基于IDO1在肿瘤免疫治疗中的重要作用且目前没有上市用于肿瘤治疗的IDO1抑制剂，因此，在新的IDO1抑制剂的筛选、及其基于IDO1酶抑制剂的抗肿瘤药物研究具有重要的意义。
环二核苷酸合成酶(cGAS)是先天性免疫通路中重要的细胞质DNA感受器。cGAS催化体内ATP和GTP合成cGAMP,cGAMP作为二级信使分子通过内质网膜上的STING蛋白通路诱导干扰素IFN-β和其他细胞因子的产生，调节下游蛋白质表达，诱导细胞生长停滞和凋亡，产生抗病毒效应。STING通路可以调节免疫原性肿瘤的先天免疫识别，促进干扰素的抗肿瘤作用。IFN-γ在体内通过TRAIL(tumor necrosis factor-related apoptosis-inducingligand)发挥抗肿瘤作用，促进肿瘤细胞凋亡。cGAMP是先天免疫反应的关键刺激物，是STING的内源性激活剂，具有免疫抗肿瘤作用。
本研究发明了一类创新的吲哚胺2，3-双加氧酶IDO1的抑制剂，即烟酸衍生物(3-吡啶丙酸的衍生物)。该类3-吡啶丙酸的衍生物能有效抑制吲哚胺2，3-双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性较低。这些烟酸类似物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂联合使用/或与免疫检查点抑制剂联和使用/或与抗肿瘤化药联合使用，抑制肿瘤生长药效更佳，安全性也很好。因此，这类烟酸衍生物在制备免疫抗肿瘤药物中具有广阔的应用前景。
发明内容
本发明提供了一种创新的吲哚胺2，3-双加氧酶IDO1的抑制剂，即烟酸衍生物(3-吡啶丙酸的衍生物)。该类3-吡啶丙酸的衍生物能有效抑制吲哚胺2，3-双加氧酶IDO1催化色氨酸代谢，在人源癌细胞中能显著抑制高表达的IDO1酶活性，而且细胞毒性较低。这些烟酸衍生物作为IDO1酶的抑制剂单独使用能显著抑制小鼠移植肿瘤生长，其与先天免疫通路STING激动剂或免疫检查点抑制剂联合使用，抑制肿瘤生长药效更佳，安全性也很好。因此，这类烟酸衍生物在制备免疫抗肿瘤药物中具有广阔的应用前景。
具体发明内容特征为：
1、烟酸衍生物(3-吡啶丙酸的衍生物),是新型吲哚胺2，3-双加氧酶IDO1的抑制剂。烟酸衍生物的具体特征为：以烟酸为结构母核，利用羧酸等排体的方式进行引入S，N，O，F等易于形成氢键的原子，增强这些抑制剂与IDO1酶蛋白分子的结合力，稳定抑制剂与IDO1蛋白分子形成的复合物，增加抑制IDO1酶活性效果。具体烟酸衍生物举例见附图1(烟酸衍生物(1-10)结构式及其名称)。
2、烟酸衍生物(3-吡啶丙酸的衍生物)在制备抗肿瘤药物中的应用。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤，包括但不于结直肠癌、乳腺癌、睾丸癌、卵巢癌、***癌、肺癌、鼻咽癌、食道癌、恶性淋巴瘤、头颈部癌、甲状腺癌、成骨肉瘤、膀胱癌、子***、和生殖细胞瘤等。
3、烟酸衍生物(3-吡啶丙酸的衍生物)与免疫通路STING激动剂联合(包括复合物)在制备抗肿瘤药物中的应用。STING激动剂包括但不限于cGAMP及其衍生物。所述的抗肿瘤药物，应用于治疗各种类型的肿瘤。
4、烟酸衍生物(3-吡啶丙酸的衍生物)与免疫检查点抑制剂联合用药在制备抗肿瘤药物中的应用。免疫检查点抑制剂包括但不限于抗PD1/PD-L1、抗CD-47、抗VEGF、抗CTLA-4等单克隆抗体及其单克隆抗体的纳米抗体；所述的抗肿瘤药物，应用于治疗各种类型的肿瘤。
5、烟酸衍生物(3-吡啶丙酸的衍生物)与化疗抗肿瘤药物联合用药在制备抗肿瘤药物中的应用。化疗药物包括但不限于铂类金属药物、5-氟尿嘧啶、紫山醇等抗肿瘤药物，应用于治疗各种类型的肿瘤。
6、烟酸衍生物(3-吡啶丙酸的衍生物)在制备抗肿瘤药物中的应用，包含不同规格的单位制剂及药学上可接受的药物制剂，包括但不限于静脉注射、肌肉注射、皮下注射、静脉滴注、鼻腔滴注、口服等中的一种或多种，进行上述相关肿瘤的治疗。
附图说明
图1为烟酸衍生物(1-10)结构式及其名称。
具体实施方式
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。
实施例1：烟酸衍生物对IDO1金属酶的抑制作用研究
所有生物化学试剂均为分析纯，购于Sigma-Aldrach。烟酸衍生物1-10的结构式及其英文名称见附图1.
1、吲哚胺2，3-双加氧酶1(IDO1)按照文献方法制备
选择pGEX-6P-1作为构建人源IDO1表达质粒的载体，带有GST融合标签的IDO1蛋白在大肠杆菌中可容表达，IDO1融合蛋白使用GST亲和柱进行吸附纯化且蛋白纯度极高，采用重组HRV 3C蛋白酶进行酶切GST标签蛋白，在16℃的条件下酶过夜，酶切后的IDO1蛋白经二次使用Glutathione Sepharose亲和柱纯化，得到电泳纯蛋白，经SDS-Page和质谱鉴定。SDS-PAGE显示IDO1蛋白纯度高于95％，MALDI-TOF质谱进一步确认了IDO1的分子量为45KD，这与理论分子量相吻合；紫外可见光谱表征显示特征吸收峰为404nm，电化学还原电位为-0.37V。实验结果证明，我们成功的获得了高纯度的人源IDO1蛋白，为后续IDO1抑制剂筛选及酶活性相关实验的顺利进行奠定了基础。
2、烟酸衍生物抑制IDO1酶活性的IC50值测定
实验方法：
用KH2PO4(100mM，pH 6.5)缓冲溶液，根据IDO1的摩尔消光系数计算1μM蛋白在404nm紫外吸收值来准备蛋白样品。准备1.5mL的EP管，做好化合物各浓度梯度的标记；然后取1mL、1μM蛋白于EP管中，作为反应体系的体积；随后分别向其中加入亚甲基蓝、过氧化氢酶、L-抗坏血酸(用NaOH调pH至6.5)，试剂终浓度分别为10μM、200μg/mL和10mM；再向每个管中加入对应浓度梯度的化合物并设置不加化合物空白孔，上下颠倒混合均匀后在37℃水浴锅中孵育10min；随后在动力学测试前加入100μM底物L-色氨酸，然后立即监测320nm处反应产物N'-甲酰犬尿氨酸的吸收值，反应时间为60s。实验选择100μM色氨酸浓度下的抑制率计算抑制剂的IC50值。
数据处理：
数据处理上，首先对各个反应的动力学实验数据进行线性拟合，求出初始速率V；然后根据公式计算出不同化合物浓度下对应的抑制率，以抑制率对烟酸衍生物抑制剂浓度的Log10值进行作图，根据IC50公式进行数据拟合，即可得到各化合物的IC50值。
实验结果：IC50值测定表明10个烟酸衍生物对IDO1均有抑制作用。化合物6、7、8、9、10上，它们的IC50值均在10μM左右。8个化合物均表现出比阳性对照化合物4-PI(IC50:49.03±6.67μM)更高的IDO1抑制效果，其中化合物9和10的效果最好，它们在最大浓度200μM的抑制率最高，而化合物9的抑制率可以达到90％。
表1.烟酸衍生物抑制IDO1金属酶的IC50和Ki值
3、烟酸衍生物抑制IDO1抑制常数Ki测定
实验方法与数据处理：
对同一种烟酸衍生物抑制剂分别在50μM、100μM和150μM三个底物浓度下进行平行的抑制活性动力学实验。首先对各个反应的动力学实验数据进行线性拟合，求出初始速率V，然后采用Cornish-Bowden作图法，在同一个数轴中以抑制剂浓度为横坐标，底物浓度与初始速率V的比值(S/V)为纵坐标，最后对三组数据进行线性拟合，拟合直线交点的绝对值即为抑制常数Ki，而通过交点Ki所处坐标轴的位置来判定抑制剂的抑制类型。导出的实验数据用Origin 7.5进行作图分析。
实验结果：
Ki值测定选择在100μM的浓度下抑制率在60％以上的化合物进行，分别是化合物2，4，5，8，9，10。实验分别用不同抑制剂浓度在50μM、100μM、150μM三种烟酸衍生物底物浓度下进行平行实验，以S/V对烟酸衍生物抑制剂浓度作图，三组数据线性拟合的直线交点的绝对值即为抑制常数Ki。
如表1所示，三种强IDO1烟酸衍生物抑制剂8(15.56μM)，9(8.68μM)和10(9.93μM)的Ki值在15μM范围内。Ki值越低，则抑制效果越强，因此，烟酸衍生物抑制剂9对IDO1具有最强的抑制效果。以上的结果表明，两种含氟化合物9和10作为以3-吡啶丙酸为母核的化合物对IDO1抑制活性最强。两者的最低IC50和Ki约为10μM。抑制剂9的抑制效果效果最为显著主要由两方面原因促成，一方面，与其他抑制剂相比，只有9具有氧和三个氟原子，F的引入可以增强IDO1和抑制剂之间的氢键结合力。特别是，9含有三氟乙基，它可以显著提高抑制剂的结合能力。另一方面，9中的羧酸被氟取代，这增加了抑制剂的疏水性，并且有利于9占据IDO1的疏水腔。总之，含有容易形成氢键的元素，特别是含有三氟乙基的化合物，它们更具有抑制IDO1的潜在优势。
实施例2：烟酸衍生物对癌细胞IDO1酶抑制作用研究
1、实验试剂耗材
0.25×胰酶-EDTA、双抗(青霉素/链霉素)购自Gibco公司；1640培养基、FBS胎牛血清购自维森特生物技术(南京)有限公司；异丙醇、DMSO、冰醋酸、无水乙醇、硫酸铜购自国药集团化学试剂有限公司；Nalgene程序降温盒购自北京索莱宝科技有限公司；无菌PBS缓冲液、无菌0.22μm水相滤膜购自生工生物工程(上海)股份有限公司；CCK-8试剂盒购自碧云天生物技术有限公司；60mm和100mm细胞培养皿、细胞冻存管购自Corning公司；96孔板购自NEST公司；人***细胞(Hela)、人肝癌细胞(HepG-2)、人乳腺癌(MCF-7)、人正常肝细胞(L02)购自上海中科院细胞库；IFNγ购自Sigma-Aldrich公司；对-二甲氨基苯甲醛，三氯乙酸，4-苯基咪唑均购自阿拉丁试剂(上海)有限公司。
2、实验仪器
-80℃冰箱(安徽中科都菱商用电器股份有限公司)；电热恒温水浴锅(上海科恒实业发展有限公司)；立式压力蒸汽灭菌锅(上海博迅实业有限公司医疗设备厂)；双人单面垂直送风洁净工作台(上海天恒医疗器械有限公司)；二氧化碳培养箱(型号：BPN-50CH，上海一恒科学仪器有限公司)；显微镜(上海光学仪器一厂)；立式电冰箱(合肥美菱股份有限公司)；离心机(Sorvall Legend Micro 17R/21R,Thermo Fisher Scientific)；酶标仪(BioTek CytationTM 3)。
3、实验内容及实验方法
本实验采用IFNγ诱导癌细胞产生IDO1，从而进行活细胞水平上的IDO1抑制剂的活性检测。
实验第一天：将用100mm培养至一定细胞密度的HeLa、HepG-2和MCF-7癌细胞用胰蛋白酶消化，显微镜下观察，细胞形态变圆后用完全培养基中止消化，将细胞用移液器轻轻吹落，离心去除胰酶。以5×104个/mL的细胞数目接种到96孔板中并设置空白对照。将其放在37℃、5％CO2的二氧化碳培养箱中培养12小时。
实验第二天：将96孔板中的细胞换液并加入200μL培养基，随后在每个培养空分别加入终浓度位10ng/mL的IFNγ、100μM的色氨酸和不同浓度梯度的抑制剂，共同培养48h。抑制剂的终浓度分别为0μM、0.5μM、1μM、5μM、10μM、20μM、40μM、60μM、80μM、100μM、150μM、200μM。阳性对照药4-PI的终浓度分别为0μM、10μM、20μM、40μM、6μM、100μM、150μM、200μM。每个样品浓度复孔3个。
实验第四天：细胞加药培养48h后，每个培养孔取140μL上清至1.5mL离心管中，加入10μL 30％(w/v)的三氯乙酸溶液，在65℃水浴条件下孵育15min终止IDO1对色氨酸的催化反应，并使N′-甲酰犬尿氨酸转化为犬尿氨酸；随后10000rpm离心10min，再取100μL的上清溶液至新的96孔板中，加入相同体积的2％(w/v)对-二甲氨基苯甲醛的乙酸溶液混合，进行显示反应；最后采用酶标仪检测492nm处的吸收值，根据产物的量来计算IDO1酶的反应速率。数据导出为excel格式，然后用GraphPad 7.00作图。细胞实验进行两次技术重复。IDO1活细胞上的抑制率计算公式：Inhibition％＝[1-(A/B)]×100％(1)(A表示含有抑制剂时的吸收值，B表示不含抑制剂时的吸收值。)
4、实验结果
为了评估烟酸衍生物对活细胞中IDO1酶的抑制活性，我们对实施例1酶抑制实验筛选的抑制率大于60％的六种抑制剂进行细胞实验分析，选用三种人源癌细胞系。首先测定了20μM相同抑制剂浓度下药物对三种癌细胞中IDO1的抑制率，实验结果如表2，在Hela细胞系中，9和10对IDO1的抑制率最高，分别为14.70％和20.06％，在HepG-2细胞系中，9和10同样产生了最好的抑制效果，抑制率分别为45.61％和29.52％，而在抑制剂在MCF-7细胞系中产生的抑制作用较弱，抑制剂9略微显著，为13.54％，其他5种抑制剂均在10％左右。我们用9和10抑制剂确定它们对HepG-2细胞的IDO1的EC50。结果表明，9和10的EC50值分别问11.04μM，14.06μM。其他抑制剂的EC50值均超过100μM(表2)。
表2、烟酸衍生物抑制人源癌细胞中IDO1抑制活性参数
实施例3：采用荷瘤鼠模型检测烟酸衍生物的抗肿瘤作用
动物
BALB/C普通小鼠，雄性，体重20-22g，7-8周龄，SPF级，购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005]。
饲养条件
所有小鼠均自由觅食和饮水，在室温(25±2)℃下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。
剂量设置
(1)烟酸衍生物(化合物9)设置1个剂量组20mg/kg；
(2)烟酸衍生物与cGAMP联合用药：烟酸衍生物20mg/kg+cGAMP20
mg/kg；
(3)烟酸衍生物与抗PD-L1联合用药：烟酸衍生物20mg/kg/天+抗PD-L1单抗，200μg/次，每周一次；
试验对照
阴性对照：生理盐水溶液
阳性对照：
(1)cGAMP，20mg/kg；
(2)抗PD-L1单抗，200μg/次，每周一次；
给药方法
给药途径：尾静脉注射
给药体积：100微升/只；给药次数：连续21天给药，每隔两天给药1次。
每组动物数：10只
小鼠结直肠癌细胞株CT26，小鼠乳腺癌细胞株4T1均购自中国科学院细胞库。
试验主要步骤
肿瘤模型鼠的建立与干预
细胞培养，传代，在细胞对数期收集细胞，做成浓度为每毫升(1.0×107)的细胞悬液，小鼠右前肢腋下注射0.2ml细胞悬液(细胞数目为2.0×106个/只)，8天左右致瘤成功。按照两种小鼠肿瘤模型实验类别，分别随机均分6组，包括模型对照组、cGAMP阳性药对照组、抗-PD-L1阳性药对照组、烟酸衍生物9、烟酸衍生物9与cGAMP联合用药组、烟酸衍生物9与抗PD-L1单抗联合用药组。21天后，处死小鼠并称瘤体重量，计算抑瘤率。
分别制备小鼠结直肠癌细胞株CT26，移植到BalB/C普通小鼠，小鼠乳腺癌细胞株4T1，移植到BalB/C普通小鼠，观察不同药物抗肿瘤效果。
统计分析
数据用x±s表示，利用SPSS10.0软件进行处理，采用单因素方差分析(one-wayANOVA)检验比较各组瘤重差异的显著性，显著性水平a＝0.05。
实验结果
小鼠皮下接种肿瘤细胞后制备成功皮下移植瘤模型，烟酸衍生物(化合物9)可明显抑制肿瘤生长，给药21天后的瘤重显著低于阴性对照模型组(P<0.05，P<0.01)；烟酸衍生物(化合物9)联合cGAMP、烟酸衍生物(化合物9)联合抗PD-L1单抗联合用药组，均表现出比各自单一成分单独用药显著提高的抑制肿瘤药效作用。具体结果见表2-3。
表2.烟酸衍生物及其联合用药对小鼠结直肠癌细胞CT26皮下移植瘤的抑制作用
表3.烟酸衍生物及其联合用药对小鼠乳腺癌4T1皮下移植瘤的抑制作用
实施例4.烟酸衍生物小鼠急性毒性研究
实验方法：
ICR小鼠20只(购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005])，雌雄各半，体重18～22g，动物以颗粒饲料喂养，自由摄食和饮水。
烟酸衍生物(化合物9)用生理盐水配制成浓度为200mg/mL的溶液。
ICR小鼠按体重单次腹腔注射2g/kg的新型复合物给药，观察给药后小鼠14天内的毒性反应及死亡情况。结果发现，小鼠单次腹腔注射给药后，小鼠活动正常。给药后14天内，小鼠未出现死亡，第15天，全部小鼠处死，解剖，肉眼检查各脏器，均未见明显病变。
实验结果：
上述急性毒性实验结果表明，腹腔给药最大耐受量MTD不低于2g/Kg，说明烟酸衍生物9的急性毒性低。
附图1:烟酸衍生物(1-10)结构式及其名称。

Claims (3)

1.烟酸衍生物作为抗肿瘤有效成分在制备抗肿瘤药物中的应用，所述的抗肿瘤药物应用于治疗结直肠癌、乳腺癌；所述烟酸衍生物的结构式为：
2.烟酸衍生物作为抗肿瘤有效成分与免疫通路STING激动剂联合在制备抗肿瘤药物中的应用，所述免疫通路STING激动剂为cGAMP；所述的抗肿瘤药物应用于治疗结直肠癌、乳腺癌，所述烟酸衍生物的结构为:
3.根据权利要求1所述的烟酸衍生物作为抗肿瘤有效成分在制备抗肿瘤药物中的应用，其特征在于，所述药物为药学上可接受的药物制剂。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1623987A1 (de) * 2004-08-02 2006-02-08 Curacyte Discovery GmbH Anellierte Pyrido[3',2':4,5]thieno[3,2-d]pyrimidine und Pyrido[3',2':4,5]furo[3,2-d]pyrimidine
CN101611018A (zh) * 2006-11-10 2009-12-23 阿斯利康(瑞典)有限公司 具有Edg-I拮抗活性的杂环磺酰胺
WO2011081968A1 (en) * 2009-12-31 2011-07-07 Corning Incorporated Pi3k modulators, rho kinase modulators and methods of identifying and using same
CN103819393A (zh) * 2010-03-01 2014-05-28 瑞科西有限公司 化合物及其治疗应用
WO2014081856A2 (en) * 2012-11-20 2014-05-30 Azzazy Hassan Ligands that target hcv-e2 binding sites on cd81 and therapeutic methods using them
WO2019005559A1 (en) * 2017-06-28 2019-01-03 Genentech, Inc. INHIBITORS OF TDO2 AND IDO1

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10980807B2 (en) * 2016-02-09 2021-04-20 Inventisbio Llc Inhibitor of indoleamine-2,3-dioxygenase (IDO)

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1623987A1 (de) * 2004-08-02 2006-02-08 Curacyte Discovery GmbH Anellierte Pyrido[3',2':4,5]thieno[3,2-d]pyrimidine und Pyrido[3',2':4,5]furo[3,2-d]pyrimidine
CN101611018A (zh) * 2006-11-10 2009-12-23 阿斯利康(瑞典)有限公司 具有Edg-I拮抗活性的杂环磺酰胺
WO2011081968A1 (en) * 2009-12-31 2011-07-07 Corning Incorporated Pi3k modulators, rho kinase modulators and methods of identifying and using same
CN103819393A (zh) * 2010-03-01 2014-05-28 瑞科西有限公司 化合物及其治疗应用
WO2014081856A2 (en) * 2012-11-20 2014-05-30 Azzazy Hassan Ligands that target hcv-e2 binding sites on cd81 and therapeutic methods using them
WO2019005559A1 (en) * 2017-06-28 2019-01-03 Genentech, Inc. INHIBITORS OF TDO2 AND IDO1

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
An SAR study of hydroxy-trifluoromethylpyrazolines as inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader assay;Stevenson, Ralph J.，等;Bioorganic & Medicinal Chemistry;第26卷(第12期);第3406-3413页 *
Hauck, Stefanie，等.Pyrazolo[1,5α]pyrimidines as a new class of FUSE binding protein 1 (FUBP1) inhibitors.Bioorganic & Medicinal Chemistry.2016,第24卷(第22期), 第5717-5729页. *
Najlaa S. Al-Radad，等.Artichoke (Cynara scolymus L.,) mediated rapid analysis of silver nanoparticles and their utilisation on the cancer cell treatments.Journal of Computational and Theoretical Nanoscience.2018,第15卷(第06期),第1818-1829页. *
Selected pharmacotherapy agents as antiproliferative and anti-inflammatory compounds;Mthanna AlKhalil，等;Drug Development Research;第81卷(第04期);第470-490页 *
尼克酸治疗恶性黑素瘤的相关机制研究;陈龙，等;中国医疗美容;第09卷(第02期);第88-91页 *

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