Discovery of Imidazopyridines as Potent Inhibitors of Indoleamine 2,3-Dioxygenase 1 for Cancer Immunotherapy

Indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as a target for small-molecule immunotherapy for the treatment of a variety of cancers including renal cell carcinoma and metastatic melanoma. This work focuses on the identification of IDO1 inhibitors containing replacements or isosteres for the amide found in BMS-986205, an amide-containing, IDO1-selective inhibitor currently in phase III clinical trials. Detailed subsequently are efforts to identify a structurally differentiated IDO1 inhibitor via the pursuit of a variety of heterocyclic isosteres, leading to the discovery of highly potent, imidazopyridine-containing IDO1 inhibitors.     

A selective plasmin inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), induces thymocyte apoptosis

The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.     

Regulation of anandamide tissue levels by N-arachidonylglycine

N-arachidonylglycine (NAGly), the carboxylic analog of the endocannabinoid anandamide, occurs in rat and bovine brain as well as in peripheral sites and shows activity against tonic, formalin-induced pain. It was also observed, using cell membrane preparations, that it inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide (N-arachidonylethanolamide). These data suggested that it may serve as an endogenous regulator of tissue anandamide concentrations. In this report, we show findings derived from mass spectrometric analyses, indicating that blood levels of anandamide in rats given 10 mg/kg p.o. of NAGly were increased significantly by more than 9-fold when compared with vehicle-treated controls. In vitro evidence in RAW 264.7 cells using a deuterium-labeled NAGly demonstrated that it was not a precursor or source of arachidonic acid for the observed 50% rise in anandamide levels, suggesting that the increase was due to some effect other than increased biosynthesis of anandamide. Moreover, the findings presented here suggest that NAGly can serve as a model for the design of agents to provide pharmacological control of tissue anandamide concentrations.     

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine6‐oxytocin and penicillamine6‐5‐tert‐butylproline7‐oxytocin analogs

Abstract: Six [Pen⁶]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa¹]oxytocin, [dPen¹]oxytocin, [5‐t‐BuPro⁷]oxytocin, [Mpa¹, 5‐t‐BuPro⁷]oxytocin and [dPen¹, 5‐t‐BuPro⁷]oxytocin. When tested in the uterotonic test in vitro[Pen⁶]oxytocin, [Pen⁶, 5‐t‐BuPro⁷]oxytocin, [Mpa¹, Pen⁶]oxytocin and [Mpa¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen¹, Pen⁶]Oxytocin and [dPen¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin were found to be pure antagonists with similarly high pA2 values of ≈ 8.2. Replacement of proline by 5‐tert‐butylproline increased binding affinity by a factor of two in [Pen⁶]oxytocin and had no influence on the binding affinity of [Mpa¹, Pen⁶]oxytocin and [dPen¹, Pen⁶]oxytocin. Assignment of the proton signals for prolyl amide cis‐ and trans‐isomers by NMR experiments in water indicated that the Pen⁶?5‐tert‐BuPro⁷ peptide bond cis‐isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5‐tert‐butylproline⁷ analogs of [Pen⁶]oxytocin, [Mpa¹, Pen⁶]oxytocin and [dPen¹, Pen⁶]oxytocin. This augmentation in cis‐isomer population was correlated with a 21‐fold reduction in the agonistic potency and 2‐fold augmentation in antagonistic potency for [Pen⁶, 5‐t‐BuPro⁷]oxytocin relative to [Pen⁶]oxytocin. Augmentation of cis‐isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa¹, Pen⁶]oxytocin to [Mpa¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin. In the potent oxytocin antagonist, [dPen¹, Pen⁶]oxytocin, substitution of 5‐tert‐butylproline for proline augmented the cis‐isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen⁶]oxytocin and [Pen⁶, 5‐t‐BuPro⁷]oxytocin analogs 1–6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis‐isomer population caused by 5‐tert‐butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.     

Oxotremorine antagonism by prolyl-leucyl-glycine-amide administered by different routes and with several anticholinergics

Prolyl-leucyl-glycine amide (PLG) was given by oral, intravenous, or subcutaneous routes of administration and shown to antagonize the central and peripheral effects of oxotremorine. The previously demonstrated action of PLG in this system when injected intraperitoneally in a single dose was found to be unchanged when administered by the same route for five consecutive days. Although three anticholinergic compounds were also effective in reducing the effects of oxotremorine, no interactions between PLG and trihexyphenidyl hydrochloride, benzotropin mesylate, or scopolamine were observed. The results confirm and extend the demonstration of the extra-endocrine effects of PLG.     

Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis--a difficult issue to handle

There is considerable debate at present as to whether the uptake of anandamide (AEA) into cells is by a facilitated transport process or by passive diffusion driven by fatty acid amide hydrolase (FAAH). The possibility that both processes occur, but to different extents depending upon the cell type used, has been difficult to investigate pharmacologically since available compounds show little selectivity between inhibition of AEA uptake and inhibition of FAAH. Recently, three compounds, UCM707 [N-(Fur-3-ylmethyl)arachidonamide], OMDM-1 and OMDM-2 [the 1'-(S)- and 1'-(R)-enantiomers of the 1'-4-hydroxybenzoyl analogue of oleoylethanolamide], selective for the uptake process, have been described and we have used these compounds, together with AM404 [(N-(4-hydroxyphenyl) arachidonoyl amide)] and VDM11 [(5Z,8Z,11Z,14Z)-N-(4-Hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide]), with the initial aim of determining which mechanism of uptake predominates in C6 glioma and RBL-2H3 cells. AM404 and VDM11 were both found to decrease the uptake of 2 microM AEA into cells (IC50 values 6-11 microM), but they also inhibited rat brain FAAH (IC50 values 1-6 microM). However, when using a different FAAH assay protocol, VDM11 was a much less potent FAAH inhibitor (IC50>50 microM) regardless of the cell type and animal species used. In contrast, we confirmed that UCM707, OMDM-1 and OMDM-2 were weak inhibitors of FAAH (IC50 values >50 microM) under all conditions used. However, their potency as inhibitors of AEA cellular accumulation appears to be largely dependent on the cell type and assay conditions used. In particular, the potency of UCM707 (IC50 value > or =25 microM) was considerably lower than the submicromolar potency previously reported for U937 cells. It is concluded that the cause/effect relationship between AEA uptake and hydrolysis cannot be investigated uniquely by using supposedly selective inhibitors of each process.     

人胎肾上腺线粒体细胞色素P-450体外脱甲基功能(英文)

目的:了解胎肾上腺线粒体代谢外源性化合物的能力和特征,方法:制备亚细胞组分,酶学检测脱甲基反应代谢产物—甲醛的含量,结果:在光谱分析和SDS-PAGE证实线粒体存在P-450的基础上,进一步证明线粒体P-450具有脱甲基功能,其脱甲基作用呈蛋白浓度(1-4 mg)和反应时间(10-30 min)依赖性增加,与底物浓度间有良好的量效关系,并与胎龄呈正比,线粒体中红霉素、苄非他明和氨基比林的脱甲基反应分别为微粒体中的89％,162％和62％。醋竹桃霉素增强肾上腺的红霉素脱甲基反应。结论:胎肾上腺线粒体有较强的脱甲基功能,提示胎儿肾上腺线粒体兼有药物代谢功能。     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere

Abstract: We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Pheψ[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)₂ where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)₂-Pheψ[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)₂ where (SMe)Pen(O)₂ stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the ψ of Phe(cep) is maintained about 130° and the φ angle of (cep)Gly prefers ± 150°[where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule (‘flap water’), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Acid and base catalyzed intramolecular cyclizations ofN-benzoylthiocarbamoyl-acetals

Acid and base catalyzed intramolecular cyclizations ofN-benzoylthioureidoacetal, containing four functional groups adjacent to thiourea such as benzocarbamoyl, acetal, thioure and amide, were investigated. The condensation reaction ofN-benzoyl thiocarbamoylglycine amide in the presence of 10% aqueous NaOH provided 1-(2,2-dimethoxy)ethyl-imidazolidine-2-thione exclusively. In the presence of pyridine, it was transformed to 2-thiohydantoin.N-Benzoyl thiocarbamoyl glycine amide was completely transformed to an iminothiazolidine exclusively in the presence of Lewis acid such as borontrifluoride etherate or trimethylsilyl iodide. 1-(2,2-Dimethoxy)ethyl-imidazolidine-2-thione was transformed to imidazole[2,1-b]thiazole and pyrazino[5,1-a]imidazole in the presence of BF₃.Et₂O and formic acid, respectively.