TW202241888A - Deuterated colony stimulating factor-1 receptor (csf-1r) inhibitors - Google Patents
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本文公開了下式的氘化化合物，所述氘化化合物可用作群落刺激因子1受體抑制劑（“CSF-1R抑制劑”）。Disclosed herein are deuterated compounds of the formula that are useful as
本申請要求2020年12月23日提交的美國臨時申請號63/129,939和2021年7月28日提交的美國臨時申請號63/226,549的優先權權益，將所述臨時申請出於任何目的通過引用以其整體併入本文。This application claims the benefit of priority to U.S. Provisional Application No. 63/129,939, filed December 23, 2020, and U.S. Provisional Application No. 63/226,549, filed July 28, 2021, which are incorporated by reference for any purpose incorporated herein in its entirety.
藥物通常可能具有差的吸收、分佈、代謝和/或排泄（ADME）特性，從而阻礙其更廣泛的用途或限制其在某些適應症中的用途。差的ADME特性也可能是臨床試驗中藥物候選物失敗的主要原因。儘管在一些情況下可以採用配製品和前驅藥策略以改善某些ADME特性，但是這些方法通常無法解決許多藥物和藥物候選物存在的根本ADME問題。Drugs often may have poor absorption, distribution, metabolism and/or excretion (ADME) properties, preventing their wider use or limiting their use in certain indications. Poor ADME properties may also be a major reason for failure of drug candidates in clinical trials. Although formulation and prodrug strategies can be employed in some cases to improve certain ADME properties, these approaches often fail to address the underlying ADME problems present with many drugs and drug candidates.
一個此類問題是快速代謝，其引起許多藥物從身體中快速清除 - 否則所述藥物將在治療疾病方面非常有效。快速藥物清除的可能解決方案是頻繁投藥或高劑量投藥以獲得足夠高的血漿藥物水準。然而，這種方法具有潛在的缺點，包括患者對投藥方案的依從性較差、劑量越大副作用越嚴重以及治療成本增加。快速代謝的藥物也可能將患者暴露於不希望的毒性代謝物或反應性代謝物。One such problem is rapid metabolism, which causes many drugs to be cleared from the body quickly - drugs that would otherwise be very effective in treating disease. A possible solution to rapid drug clearance is frequent dosing or high dose dosing to achieve sufficiently high plasma drug levels. However, this approach has potential disadvantages, including poor patient compliance with the dosing regimen, higher doses with more severe side effects, and increased treatment costs. Rapidly metabolizing drugs may also expose patients to undesired toxic or reactive metabolites.
毒性代謝物或生物反應性代謝物的降解也可能是一個問題，導致一些接受藥物的患者經歷毒性，或者對安全投藥的限制，使得患者接受次優量的活性藥劑。有時，改變投藥間隔或配製方法可以有助於降低臨床不良反應，但是通常此類不希望的代謝物的形成是化合物代謝固有的。Degradation of toxic or bioreactive metabolites may also be a problem, causing some patients receiving the drug to experience toxicity, or limitations on safe dosing, such that patients receive suboptimal amounts of the active agent. Sometimes, changes in dosing intervals or formulation methods can help reduce clinical adverse effects, but usually the formation of such undesired metabolites is inherent to the metabolism of the compound.
活體中的酶（諸如醛氧化酶）可以導致不想要的代謝降解。醛氧化酶（AO）是含胞質鉬的酶，涉及許多藥物的生物轉化。由AO介導的代謝代表的挑戰是由幾種重疊因素驅動的，包括酶的複雜生物學以及作為AO受質的結構基序（例如，氮雜雜環化合物和醯胺）的廣泛使用。 參見例如，Manevski, N.等人, Metabolism by Aldehyde Oxidase: Drug Design and Complementary Approaches to Challenges in Drug Discovery, J. Med.Chem.2019, 62, 10955-10994。此外，AO介導的代謝的差異（不僅在物種之間，而且還在個體之間）也有助於暴露的可變性並且使人劑量選擇複雜化。 Enzymes in living organisms such as aldehyde oxidase can cause unwanted metabolic degradation. Aldehyde oxidase (AO) is a cytosolic molybdenum-containing enzyme involved in the biotransformation of many drugs. The challenges posed by AO-mediated metabolism are driven by several overlapping factors, including the complex biology of the enzyme and the widespread use of structural motifs (e.g., azaheterocycles and amides) as AO substrates. See , eg, Manevski, N. et al., Metabolism by Aldehyde Oxidase: Drug Design and Complementary Approaches to Challenges in Drug Discovery, J. Med. Chem. 2019, 62, 10955-10994. In addition, differences in AO-mediated metabolism (not only between species, but also between individuals) also contribute to variability in exposure and complicate human dose selection.
雖然簡單地避免易受AO代謝影響的受質的策略可能似乎很有吸引力，但是這將不切實際地消除了廣大潛在的藥核（pharmacore）。因此，已經提出了各種策略來調節藥物化合物的潛在AO代謝。這些策略包括試圖阻止AO反應（例如，與AO抑制劑結合投予化合物）；試圖降低AO反應的速率；以及使用AO代謝物作為新型支架或前驅藥的靈感。 參見例如，Manevski等人。此外，為了停止或減輕藥核的AO代謝，必須確定AO與藥核之間的反應位點。Manevski等人提供了減輕AO代謝的建議策略的表，諸如阻斷AO反應的位點、將碳用雜原子替代、將氮用碳替代、去除芳香性、降低環大小、動力學氘化同位素效應（“KDIE”）和減少logD；但是在每個實例中AO降解位點的知識是至關重要的。參見Manevski等人的表4。這些策略均包括基於實驗室測試來預測人體清除率的補充措施。換言之，對於一般技術者來說，沒有可預測的方法來瞭解任何一種建議的策略是否將能夠開發出針對特定靶標的藥物，出於其預期目的維持藥物的預期效果（例如，高功效、靶結合或生物利用度），同時還減輕AO降解，而無需使用適當的生物樣品進行大量測試。 While a strategy of simply avoiding substrates susceptible to AO metabolism might seem attractive, this would impractically eliminate a vast potential pharmacocore. Therefore, various strategies have been proposed to modulate the potential AO metabolism of pharmaceutical compounds. These strategies include attempts to block the AO response (eg, by administering compounds in combination with AO inhibitors); attempts to reduce the rate of the AO response; and the use of AO metabolites as inspiration for novel scaffolds or prodrugs. See , eg, Manevski et al. In addition, in order to stop or alleviate the AO metabolism of drug cores, the reaction sites between AO and drug cores must be identified. Manevski et al. provide a table of suggested strategies to mitigate AO metabolism, such as blocking sites for AO reactions, replacing carbons with heteroatoms, replacing nitrogens with carbons, removing aromaticity, reducing ring size, kinetic deuterated isotope effects ("KDIE") and reduce logD; but knowledge of the site of AO degradation is critical in each instance. See Table 4 of Manevski et al. Each of these strategies includes additional measures to predict human clearance based on laboratory testing. In other words, there is no predictable way for one of ordinary skill to know whether any one proposed strategy will enable the development of a drug against a specific target that maintains the desired effect of the drug for its intended purpose (e.g., high efficacy, target binding or bioavailability) while also mitigating AO degradation without extensive testing using appropriate biological samples.
令人驚訝地發現，用氘取代的如WO 2017/015267中所示的CSF-1R抑制劑化合物可以改善ADME特性。在本揭露的一些方面，在特定位置處用氘取代的CSF-1R抑制劑化合物具有改善的ADME特性，特別是對AO降解的顯著抗性，因此潛在地改善藥物功效和體內藥物的暴露。本文公開了對體內酶促降解具有抗性的氘化群落刺激因子-1受體抑制劑（“CSF-1R抑制劑”）。本揭露的CSF-1R抑制劑是能夠穿透血腦屏障以到達中樞神經系統（CNS）的小分子化合物。因為這些化合物能夠有利地穿透血腦屏障（在神經適應症中高度希望的特性），所以所述化合物需要能夠展現出足夠的吸收、代謝、分佈和排泄（ADME）特性，以確保適當的投藥。代謝問題可以包括快速代謝以及代謝降解，這二者均可以導致活性藥劑的毒性和/或次優投藥。It was surprisingly found that substitution of CSF-1R inhibitor compounds as shown in WO 2017/015267 with deuterium can improve ADME properties. In some aspects of the present disclosure, CSF-1R inhibitor compounds substituted with deuterium at specific positions have improved ADME properties, in particular significant resistance to AO degradation, thus potentially improving drug efficacy and drug exposure in vivo. Disclosed herein are deuterated colony stimulating factor-1 receptor inhibitors ("CSF-1R inhibitors") that are resistant to enzymatic degradation in vivo. The CSF-1R inhibitors of the present disclosure are small molecule compounds capable of penetrating the blood-brain barrier to reach the central nervous system (CNS). Because these compounds are able to advantageously penetrate the blood-brain barrier (a highly desirable property in neurological indications), the compounds need to be able to exhibit sufficient absorption, metabolism, distribution and excretion (ADME) properties to ensure proper dosing . Metabolic problems can include rapid metabolism and metabolic degradation, both of which can lead to toxic and/or suboptimal dosing of the active agent.
本揭露涉及氘化CSF-1R抑制劑並且涉及氘化CSF-1R抑制劑和包含CSF-1R抑制劑的醫藥組合物治療疾病的用途，其具有令人驚訝地降低的AO降解和高的功效。The present disclosure relates to deuterated CSF-1R inhibitors and to the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors to treat diseases with surprisingly reduced AO degradation and high efficacy.
此類化合物包括式 (I) 的化合物：
在至少一態樣，本揭露涉及式 (I) 的化合物：
在至少一態樣，本揭露涉及式 (I') 的化合物：
本揭露還涉及包含氘化CSF-1R抑制劑的醫藥配製品並且涉及氘化CSF-1R抑制劑和包含CSF-1R抑制劑的醫藥組合物用於治療疾病的用途。本文進一步公開了氘化CSF-1R抑制劑和包含氘化CSF-1R抑制劑的醫藥組合物用於治療免疫介導的疾病和神經疾病的用途，所述抑制劑具有醛氧化酶降解抗性，所述免疫介導的疾病包括多發性硬化症、狼瘡性腎炎和類風濕性關節炎，所述神經疾病包括肌萎縮側索硬化症（ALS）、多系統萎縮症（MSA）、進行性核上性麻痺（PSP）和亨廷頓病。The present disclosure also relates to pharmaceutical formulations comprising deuterated CSF-1R inhibitors and to the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors for the treatment of diseases. Further disclosed herein is the use of deuterated CSF-1R inhibitors and pharmaceutical compositions comprising deuterated CSF-1R inhibitors for the treatment of immune-mediated and neurological diseases, said inhibitors being resistant to degradation by aldehyde oxidase, The immune-mediated diseases include multiple sclerosis, lupus nephritis, and rheumatoid arthritis, and the neurological diseases include amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), progressive supranuclear sexual paralysis (PSP) and Huntington's disease.
關於氘修飾，人們試圖通過用氘原子替代一個或多個氫原子來減緩藥物的CYP介導的代謝或減少不希望的代謝物的形成。氘是氫的安全、穩定的、非放射性同位素。與氫相比，氘與碳形成較強的鍵。在所選情況下，氘賦予的鍵強度增加可以積極地影響藥物的ADME特性，產生改善的藥物功效、安全性和/或耐受性的潛力。同時，由於氘的尺寸和形狀與氫的尺寸和形狀基本上相同，因此與僅含有氫的原始化學實體相比，氘替代氫預期將不會實質性地影響藥物的生物化學效力和選擇性。With respect to deuterium modification, attempts have been made to slow down the CYP-mediated metabolism of drugs or reduce the formation of undesired metabolites by replacing one or more hydrogen atoms with deuterium atoms. Deuterium is a safe, stable, non-radioactive isotope of hydrogen. Deuterium forms stronger bonds with carbon than hydrogen. In selected cases, increased bond strength conferred by deuterium can positively affect the ADME profile of a drug, leading to the potential for improved drug efficacy, safety and/or tolerability. Also, since deuterium is substantially the same size and shape as hydrogen, substitution of deuterium for hydrogen is not expected to substantially affect the biochemical potency and selectivity of the drug compared to the original chemical entity containing only hydrogen.
應當注意，已經報導了對於非常小百分比的批准藥物，氘取代對代謝速率的影響（參見例如，Blake, MI 等人, J Pharm Sci, 1975, 64:367-91；Foster, AB, Adv Drug Res 1985, 14:1-40（“Foster”）；Kushner, DJ等人, Can J Physiol Pharmacol 1999, 79-88；Fisher, MB等人, Curr Opin Drug Discov Devel, 2006, 9:101-09（“Fisher”））。然而，結果是可變的且不可預測的。對於一些化合物，氘化引起體內的代謝清除率降低。對於其他化合物，代謝沒有變化。仍有其他化合物表現出增加的代謝清除率。氘效應的可變性也使專家質疑或不考慮氘修飾作為抑制不良代謝的可行藥物設計策略（參見Foster的第35頁和Fisher的第101頁）。It should be noted that the effect of deuterium substitution on metabolic rate has been reported for a very small percentage of approved drugs (see e.g., Blake, MI et al., J Pharm Sci, 1975, 64:367-91; Foster, AB, Adv Drug Res. 1985, 14:1-40 (“Foster”); Kushner, DJ et al., Can J Physiol Pharmacol 1999, 79-88; Fisher, MB et al., Curr Opin Drug Discov Devel, 2006, 9:101-09 (“ Fisher")). However, results are variable and unpredictable. For some compounds, deuteration causes decreased metabolic clearance from the body. For the other compounds, there was no change in metabolism. Still other compounds exhibit increased metabolic clearance. The variability in deuterium effects has also led experts to question or not consider deuterium modification as a viable drug design strategy to suppress undesirable metabolism (see p. 35 in Foster and p. 101 in Fisher).
本揭露的化合物是用氘取代的如WO 2017/015267中所示的CSF-1R抑制劑化合物，並且具有改善的ADME特性並且特別是對AO降解的高抗性，因此潛在地改善藥物功效和體內藥物的暴露。鑒於如Manevski等人所描述的克服AO介導的代謝（例如，需要評估和平衡多種矛盾因素（諸如化合物結構基序））、實驗確定體外和體內特性（例如，在肝微粒體或肝細胞中）的相互聯鎖且矛盾的挑戰；和以上指出的用氘取代獲得改善的ADME特性、特別是AO降解的降低的不確定性，這種結果非常令人驚訝和出乎意料。The compounds of the present disclosure are CSF-1R inhibitor compounds substituted with deuterium as shown in WO 2017/015267 and have improved ADME properties and especially high resistance to AO degradation, thus potentially improving drug efficacy and in vivo drug exposure. Given that overcoming AO-mediated metabolism as described by Manevski et al. (e.g., requires assessment and balancing of multiple conflicting factors such as compound structural motifs), experimentally determined in vitro and in vivo properties (e.g., in liver microsomes or hepatocytes ) and the above noted substitution with deuterium to obtain improved ADME properties, especially the reduced uncertainty of AO degradation, this result is very surprising and unexpected.
在一個實施例中，本揭露涉及一種治療需要此類治療的個體的由群落刺激因子-1受體（CSF-1R）介導的疾病或障礙或其中牽涉CSF-1R的疾病或障礙的方法，包括向所述個體投予有效量的根據式(I)或式(I')的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在另一個實施例中，所述疾病或障礙是需要此類治療的個體的神經疾病和免疫介導的疾病，包括多發性硬化症、ALS、MSA、PSP、亨廷頓病、狼瘡、狼瘡性腎炎和類風濕性關節炎，此類治療包括向所述個體投予有效量的根據式(I)或式(I')的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。In one embodiment, the present disclosure relates to a method of treating a colony stimulating factor-1 receptor (CSF-1R) mediated disease or disorder, or a disease or disorder in which CSF-1R is implicated, in an individual in need of such treatment, comprising administering to said individual an effective amount of a compound according to formula (I) or formula (I′) and/or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and/or or a pharmaceutically acceptable salt. In another embodiment, the disease or disorder is a neurological and immune-mediated disease in an individual in need of such treatment, including multiple sclerosis, ALS, MSA, PSP, Huntington's disease, lupus, lupus nephritis, and Rheumatoid arthritis, such treatment comprising administering to said individual an effective amount of a compound according to formula (I) or formula (I') and/or its stereoisomers, optical isomers, racemic and Diastereomeric mixtures and/or pharmaceutically acceptable salts.
本揭露還涉及包含根據式 (I) 或式 (I') 的化合物的醫藥組合物。The present disclosure also relates to pharmaceutical compositions comprising compounds according to formula (I) or formula (I').
本揭露涉及群落刺激因子-1受體抑制劑（“CSF-1R抑制劑”），所述抑制劑是能夠穿透血腦屏障以到達中樞神經系統（CNS）的小分子。本揭露還涉及包含CSF-1R抑制劑的醫藥配製品並且涉及CSF-1R抑制劑和包含CSF-1R抑制劑的醫藥組合物治療疾病的用途。此類疾病包括免疫介導的疾病（包括多發性硬化症、狼瘡性腎炎、類風濕性關節炎）和神經疾病（包括肌萎縮側索硬化症（ALS）和亨廷頓病。The present disclosure relates to colony stimulating factor-1 receptor inhibitors ("CSF-1R inhibitors"), which are small molecules capable of penetrating the blood-brain barrier to reach the central nervous system (CNS). The present disclosure also relates to pharmaceutical formulations comprising CSF-1R inhibitors and to the use of CSF-1R inhibitors and pharmaceutical compositions comprising CSF-1R inhibitors to treat diseases. Such diseases include immune-mediated diseases (including multiple sclerosis, lupus nephritis, rheumatoid arthritis) and neurological diseases (including amyotrophic lateral sclerosis (ALS) and Huntington's disease.
多發性硬化症是引起間歇性復發和進行性神經系統惡化的CNS的慢性、發炎、脫髓鞘疾病。在多發性硬化症中，活化的小神經膠質細胞和巨噬細胞有助於CNS損傷，並且在疾病進展和神經變性中起顯著作用。這些活化的先天免疫細胞可以參與抗原呈遞，並且產生對神經元和少突膠質細胞具有破壞性的發炎和神經毒性介質。CSF-1R是在巨噬細胞、單核細胞和小神經膠質細胞上表達的受體-酪胺酸激酶，並且代表效應子功能的治療性調節的潛在靶標。Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the CNS causing intermittent relapses and progressive neurological deterioration. In multiple sclerosis, activated microglia and macrophages contribute to CNS injury and play a prominent role in disease progression and neurodegeneration. These activated innate immune cells can participate in antigen presentation and produce inflammatory and neurotoxic mediators that are destructive to neurons and oligodendrocytes. CSF-IR is a receptor-tyrosine kinase expressed on macrophages, monocytes and microglia, and represents a potential target for therapeutic modulation of effector functions.
本文所述的CSF-1R抑制劑在治療多發性硬化症中特別有用，並且在臨床前體外和體內研究中證明了以下內容：發炎細胞介素/趨化介素的減少、巨噬細胞/小神經膠質細胞的擴增和活化的抑制而沒有對其吞噬活性產生負面影響、在多個體內疾病模型中CNS浸潤的抑制和在小鼠疾病模型中的治療益處。這些資料表明，通過降低發炎、脫髓鞘和軸突損失，通過CSF-1R拮抗作用抑制CNS巨噬細胞/小神經膠質細胞效應子功能在多發性硬化症中提供神經保護作用。還發現CSF-1R信號傳導在ALS中上調，並且它也可能在例如PSP和MSA中上調，並且已經在文獻中注意到CSF-1R抑制似乎在ALS、MSA和PSP的臨床前模型中是有效的。參見例如，Gowing, G.等人, Macrophage colony stimulating factor (M-CSF) exacerbates ALS disease in a mouse model through altered responses of microglia expressing mutant superoxide dismutase,
Exp Neurol. 2009年12月; 220(2):267-75；Martínez-Muriana, A.等人, CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves,
Sci Rep. 2016年5月13日; 6:25663；Neal, M.L.等人, Pharmacological inhibition of CSF1R by GW2580 reduces microglial proliferation and is protective against neuroinflammation and dopaminergic neurodegeneration.
FASEB J. 2020年1月; 34(1):1679-1694；Oh, S.J.等人, Evaluation of the Neuroprotective Effect of Microglial Depletion by CSF-1R Inhibition in a Parkinson's Animal Model. Mol Imaging Biol. 2020年8月; 22(4):1031-1042；Mancuso, R.等人, CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice. Brain. 2019年10月1日; 142(10):3243-3264；Lodder, C.等人, CSF1R inhibition rescues tau pathology and neurodegeneration in an A/T/N model with combined AD pathologies, while preserving plaque associated microglia.
Acta Neuropathol Commun. 2021年6月8日; 9(1):108。
The CSF-1R inhibitors described herein are particularly useful in the treatment of multiple sclerosis and have demonstrated the following in preclinical in vitro and in vivo studies: reduction of inflammatory cytokines/chemokines, reduction of macrophages/small Inhibition of expansion and activation of glial cells without negatively affecting their phagocytic activity, inhibition of CNS infiltration in multiple in vivo disease models, and therapeutic benefit in mouse disease models. These data suggest that inhibition of CNS macrophage/microglia effector function through CSF-1R antagonism provides neuroprotection in multiple sclerosis by reducing inflammation, demyelination, and axonal loss. CSF-1R signaling was also found to be upregulated in ALS, and it may also be upregulated in e.g. PSP and MSA, and it has been noted in the literature that CSF-1R inhibition appears to be effective in preclinical models of ALS, MSA and PSP . See eg, Gowing, G. et al., Macrophage colony stimulating factor (M-CSF) exacerbates ALS disease in a mouse model through altered responses of microglia expressing mutant superoxide dismutase, Exp Neurol . 2009 Dec;220(2):267 -75; Martínez-Muriana, A. et al., CSF1R blockade slows the progression of amyotrophic lateral sclerosis by reducing microgliosis and invasion of macrophages into peripheral nerves, Sci Rep . 2016 May 13; 6:25663; Neal, ML, et al Human, Pharmacological inhibition of CSF1R by GW2580 reduces microglial proliferation and is protective against neuroinflammation and dopaminergic neurodegeneration. FASEB J . 2020 Jan; 34(1):1679-1694; Ohial, SJ et al., Evaluation of the Neuroprotective Microprotect Depletion by CSF-1R Inhibition in a Parkinson's Animal Model. Mol Imaging Biol. 2020 Aug;22(4):1031-1042; Mancuso, R. et al., CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice . Brain. 2019
在一個實施例中，本揭露涉及式 (I) 的化合物：
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 1是N。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 2是N。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 3是CR 7。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 4是CR 7。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 5是CR 7。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 6是N。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 7是CR 7。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 8是C。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 9是C。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得X 1是N；x 2是N；X 3是CR 7；X 4是CR 7；X 5是CR 7；X 6是N；X 7是CR 7；X 8是C；並且X 9是C。 In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 1 is N. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 2 is N. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 3 is CR 7 . In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 4 is CR 7 . In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 5 is CR 7 . In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 6 is N. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 7 is CR 7 . In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X 8 is C. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that X is C. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that X 1 is N; X 2 is N; X 3 is CR 7 ; X 4 is CR 7 ; X 5 is CR 7 ; X 6 is N ; X 7 is CR 7 ; X 8 is C; and X 9 is C.
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得T 1是CR 10。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得T 2是CR 10。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得T 3是CR 10。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得T 1、T 2和T 3中的至少兩個各自獨立地是CR 10。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得T 1、T 2和T 3各自獨立地是CR 10。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that T 1 is CR 10 . In at least one embodiment of the present disclosure, the compound according to formula (I) is such that T 2 is CR 10 . In at least one embodiment of the present disclosure, the compound according to formula (I) is such that T 3 is CR 10 . In at least one embodiment of the present disclosure, the compound according to formula (I) is such that at least two of T 1 , T 2 and T 3 are each independently CR 10 . In at least one embodiment of the present disclosure, the compound according to formula (I) is such that T 1 , T 2 and T 3 are each independently CR 10 .
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得每個R 10獨立地選自H、(C 1-C 10)烷基、(C 3-C 10)環烷基、(C 1-C 10)烷氧基和鹵基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得每個R 10獨立地選自H、(C 1-C 10)烷基和鹵基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得每個R 10獨立地選自H、(C 1-C 10)烷基、(C 1-C 10)烷氧基和鹵基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得每個R 10獨立地選自H、(C 1-C 10)烷基、(C 3-C 10)環烷基和鹵基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得每個R 10獨立地選自H和鹵基。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that each R 10 is independently selected from H, (C 1 -C 10 )alkyl, (C 3 -C 10 )cycloalkyl, ( C 1 -C 10 )alkoxy and halo. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that each R 10 is independently selected from H, (C 1 -C 10 )alkyl, and halo. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that each R 10 is independently selected from H, (C 1 -C 10 )alkyl, (C 1 -C 10 )alkoxy and halo base. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that each R 10 is independently selected from H, (C 1 -C 10 )alkyl, (C 3 -C 10 )cycloalkyl, and halo base. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that each R 10 is independently selected from H and halo.
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Y 1是O。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Y 2是O。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Y1和Y2各自是O。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that Y 1 is O. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that Y 2 is O. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that Y1 and Y2 are each O.
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Z 1選自H、鹵基和(C 1-C 10)烷基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Z 1是(C 1-C 10)烷基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Z 1是鹵基。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得Z 1是H。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that Z 1 is selected from H, halo and (C 1 -C 10 )alkyl. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that Z 1 is (C 1 -C 10 )alkyl. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that Z 1 is halo. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that Z 1 is H.
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 1和R 2各自獨立地選自H和D。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 1和R 2均是H。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 1和R 2均是D。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 1和R 2中的一個是H並且另一個是D。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that R 1 and R 2 are each independently selected from H and D. In at least one embodiment of the present disclosure, compounds according to formula (I) are such that R 1 and R 2 are both H. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that R 1 and R 2 are both D. In at least one embodiment of the present disclosure, the compound according to formula (I) is such that one of R 1 and R 2 is H and the other is D.
在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 6選自(C 3-C 10)環烷基、(C 2-C 9)雜芳基、R 14-(C 6-C 14)芳基、R 14-(C 2-C 9)雜芳基和R 14-(C 1-C 10)烷基胺；其中R 14各自獨立地選自H、(C 1-C 10)烷基、(C 1-C 10)烷基胺、(C 1-C 10)烷氧基-、HO-、F 2HC-O-、F 3C-C(O)-、F 3C-和F 2HC-；並且其中每個(C 3-C 10)環烷基或(C 2-C 9)雜環烷基進一步任選地被一至四個選自以下的基團取代：(C 1-C 10)烷基、HO-、鹵基或H 2N-。在本揭露的至少一個實施例中，根據式 (I) 的化合物使得R 6選自(C 3-C 10)環烷基和(C 2-C 9)雜芳基；並且其中每個(C 3-C 10)環烷基或(C 2-C 9)雜環烷基進一步任選地被選自以下的一至兩個基團取代：(C 1-C 10)烷基、HO-、鹵基或H 2N-。 In at least one embodiment of the present disclosure, the compound according to formula (I) is such that R 6 is selected from (C 3 -C 10 )cycloalkyl, (C 2 -C 9 )heteroaryl, R 14 -(C 6 -C 14 )aryl, R 14 -(C 2 -C 9 )heteroaryl and R 14 -(C 1 -C 10 )alkylamine; wherein each R 14 is independently selected from H, (C 1 -C 10 ) Alkyl, (C 1 -C 10 ) Alkylamine, (C 1 -C 10 ) Alkoxy-, HO-, F 2 HC-O-, F 3 CC(O)-, F 3 C- and F 2 HC-; and wherein each (C 3 -C 10 )cycloalkyl or (C 2 -C 9 )heterocycloalkyl is further optionally substituted by one to four groups selected from: (C 1 -C 10 ) alkyl, HO-, halo or H 2 N-. In at least one embodiment of the present disclosure, according to the compound of formula (I), R 6 is selected from (C 3 -C 10 ) cycloalkyl and (C 2 -C 9 ) heteroaryl; and wherein each (C 3 -C 10 )cycloalkyl or (C 2 -C 9 )heterocycloalkyl is further optionally substituted by one to two groups selected from: (C 1 -C 10 )alkyl, HO-, halo group or H 2 N-.
在另一態樣，本揭露涉及式 (I') 的化合物：
在本揭露的至少一個實施例中，根據式 (I') 的化合物使得R 1和R 2各自獨立地選自H和D。在本揭露的至少一個實施例中，根據式 (I') 的化合物使得R 6選自(C 3-C 10)環烷基、(C 2-C 9)雜芳基、R 14-(C 6-C 14)芳基、R 14-(C 2-C 9)雜芳基和R 14-(C 1-C 10)烷基胺；其中R 14各自獨立地選自H、(C 1-C 10)烷基、(C 1-C 10)烷基胺、(C 1-C 10)烷氧基-、HO-、F 2HC-O-、F 3C-C(O)-、F 3C-和F 2HC-；並且其中每個(C 1-C 10)烷基、(C 6-C 14)芳基、(C 2-C 9)雜芳基、(C 3-C 10)環烷基或(C 2-C 9)雜環烷基進一步任選地被一至四個選自以下的基團取代：(C 1-C 10)烷基、HO-、鹵基或H 2N-。 In at least one embodiment of the present disclosure, according to the compound of formula (I′), R 1 and R 2 are each independently selected from H and D. In at least one embodiment of the present disclosure, the compound according to formula (I′) is such that R 6 is selected from (C 3 -C 10 )cycloalkyl, (C 2 -C 9 )heteroaryl, R 14 -(C 6 -C 14 ) aryl, R 14 -(C 2 -C 9 ) heteroaryl and R 14 -(C 1 -C 10 ) alkylamine; wherein each R 14 is independently selected from H, (C 1 - C 10 )alkyl, (C 1 -C 10 )alkylamine, (C 1 -C 10 )alkoxy-, HO-, F 2 HC-O-, F 3 CC(O)-, F 3 C - and F 2 HC-; and wherein each (C 1 -C 10 ) alkyl, (C 6 -C 14 ) aryl, (C 2 -C 9 ) heteroaryl, (C 3 -C 10 ) ring Alkyl or (C 2 -C 9 )heterocycloalkyl is further optionally substituted by one to four groups selected from (C 1 -C 10 )alkyl, HO-, halo or H 2 N- .
在另一態樣，本揭露涉及表A的化合物，和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽：
表 A 
在本揭露的至少一個實施例中，所述化合物選自3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的至少一個實施例中，所述化合物是3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d。 In at least one embodiment of the present disclosure, the compound is selected from 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3- Methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d and/ Or stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In at least one embodiment of the present disclosure, the compound is 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methoxy base-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d .
本揭露的另一態樣是一種醫藥組合物，所述醫藥組合物包含醫藥上可接受的賦形劑和式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述醫藥組合物包含式 (I) 的化合物和/或其醫藥上可接受的鹽。Another aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of formula (I) and/or its stereoisomers, optical isomers, racemic and diastereomer mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of formula (I) and/or a pharmaceutically acceptable salt thereof.
本揭露的另一態樣是一種醫藥組合物，所述醫藥組合物包含醫藥上可接受的賦形劑和至少一種式 (I') 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述醫藥組合物包含式 (I') 的化合物和/或其醫藥上可接受的鹽。Another aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and at least one compound of formula (I') and/or its stereoisomers and optical isomers , racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of formula (I') and/or a pharmaceutically acceptable salt thereof.
本揭露的另一態樣是一種醫藥組合物，所述醫藥組合物包含醫藥上可接受的賦形劑和表A的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述醫藥組合物包含表A的化合物和/或其醫藥上可接受的鹽。在本揭露的一態樣，所述醫藥組合物包含3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述醫藥組合物包含3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d形式A。 Another aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a compound of Table A and/or its stereoisomers, optical isomers, racemic and Diastereomeric mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, the pharmaceutical composition comprises a compound of Table A and/or a pharmaceutically acceptable salt thereof. In one aspect of the present disclosure, the pharmaceutical composition comprises 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methoxy base-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d and/or Stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the pharmaceutical composition comprises 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methoxy yl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d Form A.
本揭露的另一態樣是一種治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的方法，所述方法包括投予治療有效量的如本文所述的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述方法包括投予治療有效量的如本文所述的式 (I') 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述方法包括投予治療有效量的如本文所述的表A的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述醫藥組合物包含3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述醫藥組合物包含3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d形式A。 Another aspect of the present disclosure is a method of treating a disease or disorder (such as neurological diseases and immune-mediated diseases) in an individual in need thereof, the method comprising administering a therapeutically effective amount of a formula (I ) and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In at least one embodiment, the method comprises administering a therapeutically effective amount of a compound of formula (I') as described herein and/or stereoisomers, optical isomers, racemic and diastereoisomers thereof Isomer mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a therapeutically effective amount of a compound of Table A as described herein and/or stereoisomers, optical isomers, racemates and diastereomers thereof Mixtures and/or pharmaceutically acceptable salts. In one aspect of the present disclosure, the pharmaceutical composition comprises 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methoxy base-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d and/or Stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the pharmaceutical composition comprises 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methoxy yl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d Form A.
本揭露的另一態樣是一種治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的方法，所述方法包括投予一種醫藥組合物，所述醫藥組合物包含治療有效量的如本文所述的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述方法包括投予一種醫藥組合物，所述醫藥組合物包含治療有效量的如本文所述的式 (I') 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，所述方法包括投予治療有效量的醫藥組合物，所述醫藥組合物包含如本文所述的表A的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述方法包括投予治療有效量的醫藥組合物，所述醫藥組合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽和/或其醫藥上可接受的鹽。在本揭露的一態樣，所述方法包括投予治療有效量的醫藥組合物，所述醫藥組合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d形式A和/或其醫藥上可接受的鹽。 Another aspect of the disclosure is a method of treating a disease or disorder, such as neurological and immune-mediated diseases, in an individual in need thereof, the method comprising administering a pharmaceutical composition comprising a therapeutic An effective amount of a compound of formula (I) as described herein and/or its stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In at least one embodiment, the method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I') as described herein and/or its stereoisomer, optical Isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of Table A as described herein and/or its stereoisomers, optical isomers, Racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In one aspect of the present disclosure, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a 3-(((2 S ,3 S )-8-methoxy -2-(6-Methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl)-3 H -imidazo[4,5- b ]pyridine-2- d and/or its stereoisomers, optical isomers, racemic and diastereoisomer mixtures and/or pharmaceutically acceptable salts thereof and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a 3-(((2 S ,3 S )-8-methoxy -2-(6-Methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl)-3 H -imidazo[4,5- b ]pyridine-2- d Form A and/or a pharmaceutically acceptable salt thereof.
在另一態樣，本揭露提供了如本文所述用作藥劑的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用作藥劑的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是如本文所述的式 (I') 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用作藥劑的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是如本文所述的表A的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用作藥劑的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是選自以下的化合物：3-(((2S,3S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[b][1,4]二噁英-6-基)甲基)-3H-咪唑並[4,5-b]吡啶-2-d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述化合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽和/或其醫藥上可接受的鹽。在本揭露的一態樣，所述化合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d形式A和/或其醫藥上可接受的鹽。 In another aspect, the present disclosure provides a compound of formula (I) and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof for use as a medicament as described herein and /or a pharmaceutically acceptable salt. In at least one embodiment, a compound of formula (I) and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof as described herein for use as a medicament and/or a pharmaceutical Acceptable salts are compounds of formula (I') as described herein and/or stereoisomers, optical isomers, racemic and diastereoisomer mixtures and/or pharmaceutically acceptable of salt. In at least one embodiment, a compound of formula (I) and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof as described herein for use as a medicament and/or a pharmaceutical Above acceptable salts are compounds of Table A as described herein and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In at least one embodiment, a compound of formula (I) and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof as described herein for use as a medicament and/or a pharmaceutical Acceptable salts above are compounds selected from the group consisting of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2, 3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine-2-d and/or its stereoisomers , optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In one aspect of the present disclosure, the compound comprises 3-((( 2S , 3S )-8-methoxy-2-(6-methoxypyridin-3-yl)- as described herein 3-Methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof and/or pharmaceutically acceptable salts thereof and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the compound comprises 3-((( 2S , 3S )-8-methoxy-2-(6-methoxypyridin-3-yl)- as described herein 3-Methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d Form A and/or a pharmaceutically acceptable salt thereof.
在另一態樣，本揭露提供了如本文所述用於治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用於治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是如本文所述的式 (I') 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用於治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是如本文所述的表A的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在至少一個實施例中，如本文所述用於治療有需要的個體的疾病或障礙（諸如神經疾病和免疫介導的疾病）的式 (I) 的化合物和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽是選自以下的化合物：3-(((2S,3S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[b][1,4]二噁英-6-基)甲基)-3H-咪唑並[4,5-b]吡啶-2-d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽。在本揭露的一態樣，所述化合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d和/或其立體異構物、光學異構物、外消旋和非對映異構物混合物和/或醫藥上可接受的鹽和/或其醫藥上可接受的鹽。在本揭露的一態樣，所述化合物包含如本文所述的3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-2- d形式A和/或其醫藥上可接受的鹽。 In another aspect, the present disclosure provides a compound of formula (I) and/or stereoisomers thereof as described herein for use in the treatment of a disease or disorder in an individual in need thereof, such as neurological diseases and immune-mediated diseases compounds, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, a compound of formula (I) and/or stereoisomers thereof, optical Isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts are compounds of formula (I') as described herein and/or stereoisomers, optical isomers thereof , racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, a compound of formula (I) and/or stereoisomers thereof, optical Isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts are compounds of Table A as described herein and/or stereoisomers, optical isomers, racemic isomers thereof and diastereomer mixtures and/or pharmaceutically acceptable salts. In at least one embodiment, a compound of formula (I) and/or stereoisomers thereof, optical Isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts are compounds selected from the group consisting of 3-(((2S,3S)-8-methoxy-2-( 6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-3H-imidazo[ 4,5-b]pyridine-2-d and/or its stereoisomers, optical isomers, racemic and diastereomeric mixtures and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the compound comprises 3-((( 2S , 3S )-8-methoxy-2-(6-methoxypyridin-3-yl)- as described herein 3-Methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d and/or stereoisomers, optical isomers, racemic and diastereomeric mixtures thereof and/or pharmaceutically acceptable salts thereof and/or pharmaceutically acceptable salts thereof. In one aspect of the present disclosure, the compound comprises 3-((( 2S , 3S )-8-methoxy-2-(6-methoxypyridin-3-yl)- as described herein 3-Methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-2- d Form A and/or a pharmaceutically acceptable salt thereof.
在本揭露的一些方面，所述神經疾病和免疫介導的疾病包括多發性硬化症、ALS、MSA、PSP、亨廷頓病、狼瘡、狼瘡性腎炎和類風濕性關節炎。In some aspects of the present disclosure, the neurological and immune-mediated diseases include multiple sclerosis, ALS, MSA, PSP, Huntington's disease, lupus, lupus nephritis, and rheumatoid arthritis.
實例中描述了氘化CSF-1R抑制劑的體外和體內作用、其耐受代謝降解的能力、以及製備本揭露的選擇氘化CSF-1R抑制劑的方法。The in vitro and in vivo effects of deuterated CSF-1R inhibitors, their ability to withstand metabolic degradation, and methods of preparing selected deuterated CSF-1R inhibitors of the present disclosure are described in the Examples.
儘管本揭露的特定的實施例現在將參考製備和方案進行描述，但是應當理解，此類實施例僅通過舉例的方式，並且僅對可以表示本揭露的原理的應用的許多可能的特定的實施例中的少量進行說明。考慮到本揭露的益處，多種變化和修飾對於業內熟習此項技術者來說將是顯而易見的，並且如在所附申請專利範圍中進一步定義的，所述多種變化和修飾被認為是在本揭露的精神和範圍內。While specific embodiments of the disclosure will now be described with reference to preparations and protocols, it should be understood that such embodiments are by way of example only, and are merely representative of the many possible specific embodiments that may be indicative of applications of the principles of the disclosure. A small amount of them will be explained. Various changes and modifications will be apparent to those skilled in the art in view of the benefits of this disclosure, and as further defined in the appended claims, said various changes and modifications are considered to be within the scope of this disclosure. spirit and scope.
除非另有定義，否則本文使用的所有技術和科學術語具有與本揭露所屬的領域中的一般技術者通常理解的相同含義。儘管在實踐或測試中可以使用其他化合物或方法，但是現在在以下製備和方案的上下文中描述了某些較佳的方法。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. While other compounds or methods may be used in practice or testing, certain preferred methods are now described in the context of the following Preparations and Schemes.
如本文所用，術語“ 胺基”意指具有氮原子和1至2個氫原子的官能團。“胺基”通常可以在本文中用於描述一級胺、二級胺、或三級胺，並且在本揭露中使用此術語的上下文的情況下業內熟習此項技術者將能夠容易地確定所述胺基的身份。術語“ 胺”或“ 胺基（amine group）”或“ 氨基（ammonia group）”意指含有來源於氨（NH 3）的氮原子的官能團。胺基較佳是一級胺，這意味著氮鍵合至兩個氫原子和一個取代基，所述取代基包含經取代或未經取代的烷基或芳基或脂族基團或芳族基團。胺基可以是二級胺，這意味著氮鍵合至一個氫原子和兩個取代基，所述取代基包含經取代或未經取代的烷基或芳基或脂族基團或芳族基團，如下文所定義的。胺基可以是三級胺，這意味著氮鍵合至三個取代基，所述取代基包含經取代或未經取代的烷基或芳基或脂族基團或芳族基團。胺基也可以是四級胺，這意味著指定的胺基鍵合至第四基團，這導致帶正電荷的銨基。 As used herein, the term " amine group " means a functional group having a nitrogen atom and 1 to 2 hydrogen atoms. "Amino" may be used herein generally to describe primary, secondary, or tertiary amines, and those skilled in the art will be able to readily determine the The identity of the amine group. The term " amine " or " amine group" or " ammonia group" means a functional group containing a nitrogen atom derived from ammonia (NH 3 ). The amine group is preferably a primary amine, meaning that the nitrogen is bonded to two hydrogen atoms and a substituent comprising a substituted or unsubstituted alkyl or aryl or aliphatic or aromatic group group. The amine group may be a secondary amine, meaning the nitrogen is bonded to a hydrogen atom and two substituents comprising substituted or unsubstituted alkyl or aryl or aliphatic or aromatic groups group, as defined below. The amine group may be a tertiary amine, meaning that the nitrogen is bonded to three substituents comprising substituted or unsubstituted alkyl or aryl or aliphatic or aromatic groups. An amine group can also be a quaternary amine, meaning that the specified amine group is bonded to a fourth group, which results in a positively charged ammonium group.
應當理解，本揭露中的任何或所有的胺可以呈游離胺形式（即，作為一級胺的-NH 2），或呈具有醫藥上可接受的陰離子的質子化形式（即，作為一級胺的-NH 3 +Y -，其中Y -是醫藥上可接受的陰離子）。 It should be understood that any or all of the amines in this disclosure may be in the free amine form (i.e., -NH2 as the primary amine), or in protonated form with a pharmaceutically acceptable anion (i.e., -NH2 as the primary amine). NH 3 + Y - , where Y - is a pharmaceutically acceptable anion).
如本文所用，術語“ 醯胺基”意指包含與氮連接的羰基的官能團。 As used herein, the term " amido " means a functional group comprising a carbonyl group attached to nitrogen.
如本文所用，“ 羰基”意指包含由（C=O）表示的雙鍵鍵合至氧原子的碳原子的官能團。 As used herein, " carbonyl " means a functional group including a carbon atom represented by (C=O) double bonded to an oxygen atom.
如本文所用，術語“ 烷烴”意指通過單鍵鍵合的飽和烴。烷烴可以是直鏈的或支鏈的。“ 環烷烴”是通過單鍵鍵合的飽和烴環。 As used herein, the term " alkane " means a saturated hydrocarbon bonded by a single bond. Alkanes can be linear or branched. A " cycloalkane " is a saturated hydrocarbon ring bonded by a single bond.
如本文所用，術語 “(C 1-C 10) 烷基 ”意指基本上由1至10個碳原子和相應數量的氫原子組成的飽和直鏈或支鏈或環狀烴。典型地，直鏈或支鏈基團具有從一至十個碳或更典型的一至五個碳。例示性(C 1-C 10)烷基包括甲基（由-CH 3表示）、乙基（由-CH 2-CH 3表示）、正丙基、異丙基、正丁基、異丁基等。鑒於本揭露的益處，其他(C 1-C- 10)烷基對業內熟習此項技術者來說將是顯而易見的。 As used herein, the term "(C 1 -C 10 ) alkyl " means a saturated linear or branched or cyclic hydrocarbon consisting essentially of 1 to 10 carbon atoms and a corresponding number of hydrogen atoms. Typically, straight or branched chain groups have from one to ten carbons or more typically one to five carbons. Exemplary (C 1 -C 10 )alkyl groups include methyl (represented by -CH 3 ), ethyl (represented by -CH 2 -CH 3 ), n-propyl, isopropyl, n-butyl, isobutyl Wait. Other (C 1 -C- 10 )alkyl groups will be apparent to those skilled in the art given the benefit of this disclosure.
如本文所用，術語“ (C 2-C 9) 雜烷基”意指基本上由2至10個原子組成的飽和直鏈或支鏈或環狀烴，其中2至9個原子是碳並且剩餘的一個或多個原子選自氮、硫和氧。鑒於本揭露的益處，例示性(C 2-C 9)雜烷基對業內熟習此項技術者來說將是顯而易見的。 As used herein, the term " (C 2 -C 9 ) heteroalkyl " means a saturated linear or branched or cyclic hydrocarbon consisting essentially of 2 to 10 atoms, of which 2 to 9 atoms are carbon and the remaining One or more atoms of are selected from nitrogen, sulfur and oxygen. Exemplary (C 2 -C 9 )heteroalkyl groups will be apparent to those skilled in the art, given the benefit of this disclosure.
如本文所用，術語“ (C 3-C 10) 環烷基”意指非芳族飽和烴基，形成至少一個基本上由3至10個碳原子和相應數量的氫原子組成的環。(C 3-C 10)環烷基可以是單環的或多環的。除了共價鍵取代之外，多環的環烷基的單個環可以具有不同的連線性，例如，稠合、橋接、螺等。例示性(C 3-C 10)環烷基包括環丙基、環丁基、環戊基、環己基、降莰烷基(norbornanyl)、雙環-辛基、八氫并環戊二烯基、螺-癸基、被環丁基取代的環丙基、被環戊基取代的環丁基、被環丙基取代的環己基等。鑒於本揭露的益處，其他(C 3-C 10)環烷基對業內熟習此項技術者來說將是顯而易見的。 As used herein, the term " (C 3 -C 10 ) cycloalkyl " means a non-aromatic saturated hydrocarbon group forming at least one ring consisting essentially of 3 to 10 carbon atoms and a corresponding number of hydrogen atoms. A (C 3 -C 10 )cycloalkyl group can be monocyclic or polycyclic. In addition to covalent bond substitution, the individual rings of polycyclic cycloalkyls can have different linkages, eg, fused, bridged, spiro, and the like. Exemplary (C 3 -C 10 )cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornanyl, bicyclo-octyl, octahydropentalenyl, Spiro-decyl, cyclopropyl substituted by cyclobutyl, cyclobutyl substituted by cyclopentyl, cyclohexyl substituted by cyclopropyl, and the like. Other (C 3 -C 10 )cycloalkyl groups will be apparent to those skilled in the art given the benefit of this disclosure.
如本文所用，術語“ (C 2-C 9) 雜環烷基”意指具有形成至少一個環的3至10個原子的非芳族基團，其中2至9個環原子是碳並且剩餘的一個或多個環原子選自氮、硫和氧。(C 2-C 9)雜環烷基可以是單環的或多環的。除了共價鍵取代之外，此類多環的雜環烷基的單個環可以具有不同的連線性，例如，稠合、橋接、螺等。例示性(C 2-C 9)雜環烷基包括吡咯烷基、四氫呋喃基、二氫呋喃基、四氫吡喃基、吡喃基、硫代吡喃基、氮丙啶基、氮雜環丁烷基、環氧乙基、亞甲二氧基、色烯基、巴比妥基（barbituryl）、異噁唑啶基、1,3-噁唑啶-3-基、異噻唑啶基、1,3-噻唑啶-3-基、1,2-吡唑啶-2-基、1,3-吡唑啶-1-基、哌啶基、硫代嗎啉基、1,2-四氫噻嗪-2-基、1,3-四氫噻嗪-3-基、四氫噻二嗪基、嗎啉基、1,2-四氫二嗪-2-基、1,3-四氫二嗪-1-基、四氫吖庚因基、哌嗪基、哌嗪-2-酮基、哌嗪-3-酮基、色滿基、2-吡咯啉基、3-吡咯啉基、咪唑啶基、2-咪唑啶基、1,4-二噁烷基、8-氮雜雙環[3.2.1]辛基、3-氮雜雙環[3.2.1]辛基、3,8-二氮雜雙環[3.2.1]辛基、2,5-二氮雜雙環[2.2.1]庚基、2,5-二氮雜雙環[2.2.2]辛基、八氫-2H-吡啶並[1,2-a]吡嗪基、3-氮雜雙環[4.1.0]庚基、3-氮雜雙環[3.1.0]己基、2-氮雜螺[4.4]壬基、7-氧雜-1-氮雜-螺[4.4]壬基、7-氮雜雙環[2.2.2]庚基、八氫-1H-吲哚基等。所述(C 2-C 9)雜環烷基典型地經由碳原子或氮原子與主要結構附接。鑒於本揭露的益處，其他(C 2-C 9)雜環烷基對業內熟習此項技術者來說將是顯而易見的。 As used herein, the term " (C 2 -C 9 ) heterocycloalkyl " means a non-aromatic group having 3 to 10 atoms forming at least one ring, of which 2 to 9 ring atoms are carbon and the remaining One or more ring atoms are selected from nitrogen, sulfur and oxygen. A (C 2 -C 9 )heterocycloalkyl group can be monocyclic or polycyclic. In addition to covalent bond substitution, the individual rings of such polycyclic heterocycloalkyls can have different linkages, eg, fused, bridged, spiro, and the like. Exemplary (C 2 -C 9 )heterocycloalkyl groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydropyranyl, pyranyl, thiopyranyl, aziridinyl, azacyclic Butyl, oxirane, methylenedioxy, chromenyl, barbituryl, isoxazolidinyl, 1,3-oxazolidine-3-yl, isothiazolidine, 1,3-thiazolidin-3-yl, 1,2-pyrazolidine-2-yl, 1,3-pyrazolidine-1-yl, piperidinyl, thiomorpholinyl, 1,2-tetra Hydrothiazin-2-yl, 1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, morpholinyl, 1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrothiazinyl Hydrodiazin-1-yl, tetrahydroazepinyl, piperazinyl, piperazin-2-onyl, piperazin-3-onyl, chromanyl, 2-pyrrolinyl, 3-pyrrolinyl , imidazolidinyl, 2-imidazolidinyl, 1,4-dioxanyl, 8-azabicyclo[3.2.1]octyl, 3-azabicyclo[3.2.1]octyl, 3,8- Diazabicyclo[3.2.1]octyl, 2,5-diazabicyclo[2.2.1]heptyl, 2,5-diazabicyclo[2.2.2]octyl, octahydro-2H-pyridine And[1,2-a]pyrazinyl, 3-azabicyclo[4.1.0]heptyl, 3-azabicyclo[3.1.0]hexyl, 2-azaspiro[4.4]nonyl, 7- Oxa-1-aza-spiro[4.4]nonyl, 7-azabicyclo[2.2.2]heptyl, octahydro-1H-indolyl, etc. The (C 2 -C 9 )heterocycloalkyl is typically attached to the main structure via a carbon atom or a nitrogen atom. Other (C 2 -C 9 )heterocycloalkyl groups will be apparent to those skilled in the art given the benefit of this disclosure.
術語“ 脂族基團”或“ 脂族”意指由碳和氫組成的非芳族基團，並且可以任選地包括一個或多個雙鍵和/或三鍵。換言之，脂族基團是由含有無芳族官能團的碳和氫組成的任何基團。脂族基團可以是直鏈的、支鏈的或環狀的，並且典型地含有約1與約24個之間的碳原子。 The term " aliphatic group " or " aliphatic " means a non-aromatic group consisting of carbon and hydrogen, and may optionally include one or more double and/or triple bonds. In other words, an aliphatic group is any group consisting of carbon and hydrogen that contains no aromatic functionality. Aliphatic groups can be straight-chain, branched or cyclic, and typically contain between about 1 and about 24 carbon atoms.
術語“ 芳基（ aryl group）”可以與“ 芳基（ aryl）”、“ 芳環（ aryl ring）”、“ 芳族（ aromatic）”、“ 芳族基團（ aromatic group）”和“ 芳族環（ aromatic ring）”互換使用。芳基包括碳環芳族基團，典型地具有六至十四個環碳原子。芳基還包括雜芳基，其典型地具有五至十四個環原子，具有一個或多個選自氮、氧和硫的雜原子。 The term " aryl group " is interchangeable with " aryl ", " aryl ring " , " aromatic " , " aromatic group " and " aromatic Ring ( aromatic ring )" used interchangeably. Aryl includes carbocyclic aromatic groups, typically having six to fourteen ring carbon atoms. Aryl also includes heteroaryl, which typically has five to fourteen ring atoms, with one or more heteroatoms selected from nitrogen, oxygen and sulfur.
如本文所用，術語“ (C 6-C 14) 芳基”意指具有形成至少一個環的6至14個碳原子的芳族官能團。 As used herein, the term " (C 6 -C 14 ) aryl " means an aromatic functional group having 6 to 14 carbon atoms forming at least one ring.
如本文所用，術語“ (C 2-C 9) 雜芳基”意指具有形成至少一個環的5至10個原子的芳族官能團，其中2至9個環原子是碳並且剩餘的一個或多個環原子選自氮、硫和氧。(C 2-C 9)雜芳基可以是單環的或多環的。除了共價鍵取代之外，此類多環的雜芳基的單個環可以具有不同的連結，例如稠合等。例示性(C 2-C 9)雜芳基包括呋喃基、噻吩基、噻唑基、吡唑基、異噻唑基、噁唑基、異噁唑基、吡咯基、三唑基、四唑基、咪唑基、1,3,5-噁二唑基、1,2,4-噁二唑基、1,2,3-噁二唑基、1,3,5-噻二唑基、1,2,3-噻二唑基、1,2,4-噻二唑基、吡啶基、嘧啶基、吡嗪基、噠嗪基、1,2,4-三嗪基、1,2,3-三嗪基、1,3,5-三嗪基、吡唑並[3,4-b]吡啶基、噌啉基、喋啶基、嘌呤基、6,7-二氫-5H-[1]吡啶基、苯並[b]苯硫基、5,6,7,8-四氫-喹啉-3-基、苯並噁唑基、苯並噻唑基、苯並異噻唑基、苯並異噁唑基、苯並咪唑基、硫茚基、異硫茚基、苯並呋喃基、異苯並呋喃基、異吲哚基、吲哚基、吲哚嗪基、吲唑基、異喹啉基、喹啉基、呔嗪基、喹噁啉基、喹唑啉基和苯並噁嗪基等。(C 2-C 9)雜芳基典型地經由碳原子與主要結構附接，然而，業內熟習此項技術者意識到某些其他原子（例如，雜環原子）可以在何時與主要結構附接。鑒於本揭露的益處，其他(C 2-C 9)雜芳基對業內熟習此項技術者來說將是顯而易見的。 As used herein, the term " (C 2 -C 9 ) heteroaryl " means an aromatic functional group having 5 to 10 atoms forming at least one ring, of which 2 to 9 ring atoms are carbon and the remaining one or more ring atoms selected from nitrogen, sulfur and oxygen. A (C 2 -C 9 )heteroaryl group can be monocyclic or polycyclic. In addition to covalent bond substitution, the individual rings of such polycyclic heteroaryl groups may have different linkages, eg, fused, etc. Exemplary (C 2 -C 9 )heteroaryl groups include furyl, thienyl, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolyl, triazolyl, tetrazolyl, imidazolyl, 1,3,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-oxadiazolyl, 1,3,5-thiadiazolyl, 1,2 ,3-thiadiazolyl, 1,2,4-thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, 1,2,4-triazinyl, 1,2,3-tri Azinyl, 1,3,5-triazinyl, pyrazolo[3,4-b]pyridinyl, cinnolinyl, pteridinyl, purinyl, 6,7-dihydro-5H-[1]pyridine Benzyl, benzo[b]phenylthio, 5,6,7,8-tetrahydro-quinolin-3-yl, benzoxazolyl, benzothiazolyl, benzisothiazolyl, benzoisox Azolyl, benzimidazolyl, thioindenyl, isothioindenyl, benzofuryl, isobenzofuryl, isoindolyl, indolyl, indorazinyl, indazolyl, isoquinolyl , quinolinyl, oxazinyl, quinoxalinyl, quinazolinyl and benzoxazinyl, etc. (C 2 -C 9 )heteroaryl is typically attached to the primary structure via a carbon atom, however, those skilled in the art recognize when certain other atoms (eg, hetero ring atoms) may be attached to the primary structure . Other (C 2 -C 9 )heteroaryl groups will be apparent to those skilled in the art given the benefit of this disclosure.
術語“ 炔基”意指含有三鍵合碳的官能團，由(C 2-C 10)炔基-表示。 The term " alkynyl " means a functional group containing a triple bonded carbon, represented by (C 2 -C 10 )alkynyl-.
如本文所用，術語“ 烷基胺”意指含有代替一個氫原子的一級胺、二級胺或三級胺基團的(C 1-C 10)烷基，由(C 1-C 10)烷基胺和((C 1-C 10)烷基) 2胺表示。 As used herein, the term " alkylamine " means a (C 1 -C 10 )alkyl group containing a primary, secondary or tertiary amine group in place of a hydrogen atom, derived from (C 1 -C 10 )alkane Baseamine and ((C 1 -C 10 )alkyl) 2amine .
術語“ 炔基胺”意指含有三鍵合碳和胺基團的(C 2-C 10)基團，由(C 2-C 10)炔基胺表示。 The term " alkynylamine " means a (C 2 -C 10 ) group containing a triple bonded carbon and an amine group, represented by (C 2 -C 10 )alkynylamine.
術語“ 烷氧基”意指與氧鍵合的(C 1-C 10)烷基，由(C 1-C 10)烷基-O-或(C 1-C 10)烷氧基-表示。術語“ 烷氧基烷基”意指與另一個(C 1-C 10)烷基鍵合的氧鍵合的(C 1-C 10)烷基，由(C 1-C 10)烷基-O-(C 1-C 10)烷基-或(C 1-C 10)烷氧基-(C 1-C 10)烷基-表示。 The term " alkoxy " means a (C 1 -C 10 )alkyl group bonded to oxygen, represented by (C 1 -C 10 )alkyl-O- or (C 1 -C 10 )alkoxy-. The term " alkoxyalkyl " means an oxygen-bonded (C 1 -C 10 )alkyl group bonded to another (C 1 -C 10 )alkyl group consisting of (C 1 -C 10 )alkyl- O-(C 1 -C 10 )alkyl- or (C 1 -C 10 )alkoxy-(C 1 -C 10 )alkyl- represents.
術語“ 烷基酯”意指含有代替一個氫原子的酯基團的(C 1-C 10)烷基，由-O(O)C-(C 1-C 10)烷基表示。 The term " alkyl ester " means a (C 1 -C 10 )alkyl group containing an ester group in place of one hydrogen atom, represented by -O(O)C-(C 1 -C 10 )alkyl.
術語“ 烷基酸”意指含有代替一個氫原子的羧酸基團的(C 1-C 10)烷基，由(C 1-C 10)烷基-COOH表示。 The term " alkyl acid " means a (C 1 -C 10 )alkyl group containing a carboxylic acid group in place of one hydrogen atom, represented by (C 1 -C 10 )alkyl-COOH.
術語“ 脂族酸”意指非芳族烴的酸，由(C 1-C 10)烷基-COOH和(C 3-C 10)環烷基-COOH表示。 The term " aliphatic acid " means a non-aromatic hydrocarbon acid, represented by (C 1 -C 10 )alkyl-COOH and (C 3 -C 10 )cycloalkyl-COOH.
如本文所用，“ D”和“ d”二者均是指氘。 As used herein, both " D " and " d " refer to deuterium.
術語“ 二羰基”是指含有兩個或更多個相鄰羰基的有機分子。由C=O表示的羰基可以是例如醛、酮和具有與碳原子雙鍵鍵合的氧原子的其他基團。例子包括乙二醛、甲基乙二醛、二甲基乙二醛和3-去氧葡萄糖酮醛。 The term " dicarbonyl " refers to an organic molecule containing two or more adjacent carbonyl groups. The carbonyl group represented by C═O may be, for example, aldehydes, ketones, and other groups having an oxygen atom double-bonded to a carbon atom. Examples include glyoxal, methylglyoxal, dimethylglyoxal and 3-deoxyglucosone aldehyde.
術語“ 鹵基”或“ Hal”意指氟（F）、氯（Cl）、溴（Br）、碘（I）或砈（At）離子。 The term " halo " or " Hal " means a fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or astatium (At) ion.
如本文所用，“ i -”是指異。 As used herein , " i- " means different.
術語“ 甲氧基”意指含有替代一個氫原子的氧的(C 1)烷基，由-(O)CH 3表示。 The term " methoxy " means a (C 1 )alkyl group containing oxygen replacing one hydrogen atom, represented by -(O)CH 3 .
如本文所用，“ n- ”是指正。 As used herein, " n- " means positive.
術語“ 多元醇”意指含有多個羥基（-OH）基團的醇。 The term " polyol " means an alcohol containing multiple hydroxyl (-OH) groups.
如本文所用，“ Sec ”或“ s-”各自是指二級。 As used herein, " Sec " or " s- " each refer to secondary.
如本文所用，術語“ 立體異構物”是指對映異構物和非對映異構物二者。 As used herein, the term " stereoisomer " refers to both enantiomers and diastereomers.
“ 經取代的 ”意指烷基、雜環基團或芳基中的碳用一個或多個非碳取代基取代。非碳取代基選自氮、氧和硫。 " Substituted " means that a carbon in an alkyl, heterocyclic group or aryl group is replaced with one or more non-carbon substituents. Non-carbon substituents are selected from nitrogen, oxygen and sulfur.
如本文所用，“ Tert”和“ t-”各自是指三級。 As used herein, " Tert " and " t- " each refer to tertiary.
“ 未經取代的”意指僅由氫和碳組成的基團。 " Unsubstituted " means a group consisting only of hydrogen and carbon.
3至10元環指封閉環；3至10元環可以是無環的、芳族環的或雜環的。A 3- to 10-membered ring refers to a closed ring; a 3- to 10-membered ring may be acyclic, aromatic or heterocyclic.
術語“ 醫藥上可接受的陰離子”意指適用於藥學用途的陰離子。醫藥上可接受的陰離子包括鹵離子、碳酸根、碳酸氫根、硫酸根、硫酸氫根、氫氧根、硝酸根、過硫酸根、磷酸根、亞硫酸根、乙酸根、抗壞血酸根、苯甲酸根、檸檬酸根、檸檬酸二氫根、檸檬酸氫根、草酸根、琥珀酸根、酒石酸根、牛磺膽酸根、甘氨膽酸根和膽酸根。 The term " pharmaceutically acceptable anion" means an anion suitable for pharmaceutical use. Pharmaceutically acceptable anions include halides, carbonate, bicarbonate, sulfate, bisulfate, hydroxide, nitrate, persulfate, phosphate, sulfite, acetate, ascorbate, benzoic acid Root, citrate, dihydrogen citrate, hydrogen citrate, oxalate, succinate, tartrate, taurocholate, glycocholate and cholate.
“ 被氘取代”是指用相應數目的氘原子替代一或多個氫原子。 " Substituted by deuterium " refers to the replacement of one or more hydrogen atoms by a corresponding number of deuterium atoms.
本發明公開的化合物的所有醫藥上可接受的鹽、前驅藥、互變異構物、水合物和溶劑化物也在本揭露的範圍內。All pharmaceutically acceptable salts, prodrugs, tautomers, hydrates and solvates of the compounds disclosed herein are also within the scope of the present disclosure.
本發明公開了本質上是鹼性的化合物，通常能夠形成具有各種無機和/或有機酸的各種不同的鹽。雖然此類鹽對於投予動物和人通常是醫藥上可接受的，實踐中通常希望首先將化合物從反應混合物中作為醫藥上不可接受的鹽分離，並且然後將後者通過用鹼性試劑處理簡單轉化回游離鹼化合物，並且隨後將所述游離鹼轉化為醫藥上可接受的酸加成鹽。鹼性化合物的酸加成鹽可以使用常規技術容易地製備，例如，通過將鹼性化合物在水性溶劑介質中或在合適的有機溶劑（例如像，甲醇或乙醇）中用基本上等量的所選的無機酸或有機酸處理。謹慎蒸發溶劑後，獲得所希望的固體鹽。The present invention discloses that compounds that are basic in nature are generally capable of forming a wide variety of salts with various inorganic and/or organic acids. While such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt, and then convert the latter simply by treatment with a basic reagent. back to the free base compound, and then convert the free base into a pharmaceutically acceptable acid addition salt. Acid addition salts of basic compounds can be readily prepared using conventional techniques, for example, by dissolving the basic compound in an aqueous solvent medium or in a suitable organic solvent (such as, for example, methanol or ethanol) with a substantially equivalent amount of all Selected inorganic acid or organic acid treatment. After careful evaporation of the solvent, the desired salt was obtained as a solid.
可用於製備鹼性化合物的醫藥上可接受的酸加成鹽的酸是可以形成無毒酸加成鹽的那些，所述無毒酸加成鹽即含有藥理學上可接受的陰離子的鹽，諸如氯化物、溴化物、碘化物、硝酸鹽、硫酸鹽或硫酸氫鹽、磷酸鹽或酸性磷酸鹽、乙酸鹽、乳酸鹽、檸檬酸鹽或酸性檸檬酸鹽、酒石酸鹽或酒石氫酸鹽、琥珀酸鹽、馬來酸鹽、富馬酸鹽、葡萄糖酸鹽、糖酸鹽、苯甲酸鹽、甲磺酸鹽和雙羥萘酸鹽 [即，1,1'-亞甲基-雙-(2-羥基-3-萘甲酸鹽)] 鹽。Acids useful in the preparation of pharmaceutically acceptable acid addition salts of basic compounds are those which form non-toxic acid addition salts, i.e., salts containing a pharmacologically acceptable anion, such as chloride Amide, Bromide, Iodide, Nitrate, Sulfate or Bisulfate, Phosphate or Acid Phosphate, Acetate, Lactate, Citrate or Acid Citrate, Tartrate or Bitartrate, Succinate salt, maleate, fumarate, gluconate, saccharate, benzoate, mesylate and pamoate [i.e., 1,1'-methylene-bis- (2-Hydroxy-3-naphthoate)] salt.
本發明公開了本質上是酸性（例如，含有COOH或四唑部分）的化合物，通常能夠形成具有各種無機鹼和/或有機鹼的各種不同的鹽。雖然此類鹽對於投予動物和人通常是醫藥上可接受的，實踐中通常希望首先將化合物從反應混合物中作為醫藥上不可接受的鹽分離，並且然後將後者通過用酸性試劑處理簡單轉化回游離酸化合物，並且隨後將所述游離酸轉化為醫藥上可接受的鹼加成鹽。這些鹼加成鹽可以使用常規技術容易地製備，例如，通過將相應的酸性化合物用含有所希望的藥理學上可接受的陽離子的水溶液處理，並且然後將所得溶液蒸發至乾（較佳在減壓下）。可替代地，它們還可以通過以下方式製備：將酸性化合物的低級烷醇溶液和所希望的鹼金屬醇鹽混合在一起，並且然後以與以前相同的方式將所得溶液蒸發至乾。在任一種情況下，較佳採用化學計量量的試劑以確保反應的完整性和所希望的固體鹽的最大產物產率。The present invention discloses that compounds that are acidic in nature (eg, containing COOH or tetrazole moieties) are generally capable of forming a wide variety of salts with various inorganic and/or organic bases. While such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to first isolate the compound from the reaction mixture as a pharmaceutically unacceptable salt, and then convert the latter simply back to free acid compound, and subsequently converting said free acid into a pharmaceutically acceptable base addition salt. These base addition salts can be readily prepared using conventional techniques, for example, by treating the corresponding acidic compound with an aqueous solution containing the desired pharmacologically acceptable cation, and then evaporating the resulting solution to dryness (preferably under reduced pressure). depress). Alternatively, they can also be prepared by mixing together a lower alkanol solution of an acidic compound and the desired alkali metal alkoxide, and then evaporating the resulting solution to dryness in the same manner as before. In either case, stoichiometric amounts of reagents are preferably employed to ensure reaction integrity and maximum product yield of the desired solid salt.
可用於製備鹼性化合物的醫藥上可接受的鹼加成鹽的鹼是可以形成無毒鹼加成鹽的那些，所述無毒鹼加成鹽即含有藥理學上可接受的陽離子的鹽，諸如鹼金屬陽離子（例如，鉀和鈉）、鹼土金屬陽離子（例如，鈣和鎂）、銨或其他水溶性胺加成鹽諸如N-甲基葡糖胺（葡甲胺）、低級烷醇銨和其他有機胺的此類鹼。Bases useful in the preparation of pharmaceutically acceptable base addition salts of basic compounds are those which form non-toxic base addition salts, i.e., salts containing a pharmacologically acceptable cation, such as base Metal cations (for example, potassium and sodium), alkaline earth metal cations (for example, calcium and magnesium), ammonium or other water-soluble amine addition salts such as N-methylglucamine (meglumine), lower alkanolammonium and others Such bases of organic amines.
本發明公開的化合物的立體異構物（例如，順式和反式異構物）和所有光學異構物（例如，R和S對映異構物），以及外消旋異構物、非對映異構物和此類異構物的其他混合物在本揭露的範圍內。Stereoisomers (e.g., cis and trans isomers) and all optical isomers (e.g., R and S enantiomers) of the compounds disclosed herein, as well as racemic isomers, non- Enantiomers and other mixtures of such isomers are within the scope of the present disclosure.
本發明公開的化合物、鹽、前驅藥、水合物和溶劑化物可以以幾種互變異構的形式（包括烯醇與亞胺形式和酮與烯胺形式）和幾何異構物及其混合物存在。互變異構物以在溶液中互變異構物集的混合物存在。在固體形式中，通常是一種互變異構物占主導地位。即使可以描述一種互變異構物，所有互變異構物也在本揭露的範圍內。The compounds, salts, prodrugs, hydrates and solvates disclosed herein may exist in several tautomeric forms (including enol and imine forms and ketone and enamine forms) and geometric isomers and mixtures thereof. Tautomers exist as mixtures of sets of tautomers in solution. In solid form, usually one tautomer predominates. Even if one tautomer can be described, all tautomers are within the scope of the disclosure.
構型異構物也在本揭露的範圍內。構型異構物是指可以分離成旋轉限制的異構物的化合物。Configuration isomers are also within the scope of the present disclosure. Configurational isomers refer to compounds that can be separated into rotationally restricted isomers.
本揭露還提供了醫藥組合物，所述醫藥組合物包含至少一種本發明公開的化合物和至少一種醫藥上可接受的載劑。醫藥上可接受的載劑可以是業內已知的任何此類載劑，包括描述於例如Remington's Pharmaceutical Sciences, Mack Publishing Co., (A. R. Gennaro, 編輯 1985) 中的那些。本發明公開的化合物的醫藥組合物可以通過業內已知的常規方法（包括例如將至少一種本發明公開的化合物與醫藥上可接受的載劑混合）製備。The disclosure also provides pharmaceutical compositions comprising at least one compound disclosed herein and at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any such carrier known in the art, including those described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A. R. Gennaro, ed. 1985). Pharmaceutical compositions of the compounds disclosed in the present invention can be prepared by conventional methods known in the art (including, for example, mixing at least one compound disclosed in the present invention with a pharmaceutically acceptable carrier).
本發明公開的醫藥組合物可以用於動物或人。因此，本發明公開的化合物可以被配製成用於口服、口腔、腸胃外（例如，靜脈內、肌內或皮下）、局部、直腸或鼻內投予或呈適於通過吸入或吹入投予的形式的醫藥組合物。The pharmaceutical composition disclosed in the present invention can be used for animals or humans. Accordingly, the compounds disclosed herein may be formulated for oral, buccal, parenteral (e.g., intravenous, intramuscular, or subcutaneous), topical, rectal, or intranasal administration or in a form suitable for administration by inhalation or insufflation. Pharmaceutical compositions in the form of administration.
根據業內一般技術者熟知的方法，本發明公開的化合物也可以被配製用於持續遞送。此類配製品的例子可以在美國專利3,119,742；3,492,397；3,538,214；4,060,598；和4,173,626中找到。The compounds disclosed herein can also be formulated for sustained delivery according to methods well known to those of ordinary skill in the art. Examples of such formulations can be found in US Patent Nos. 3,119,742; 3,492,397; 3,538,214; 4,060,598; and 4,173,626.
對於口服投予，所述醫藥組合物可以採取例如通過常規手段用一種或多種諸如以下的醫藥上可接受的賦形劑製備的片劑或膠囊的形式：粘合劑（例如，預糊化的玉蜀黍澱粉、聚乙烯吡咯烷酮或羥丙基甲基纖維素）；填充劑（例如，乳糖、微晶纖維素或磷酸鈣）；潤滑劑（例如，硬脂酸鎂、滑石或二氧化矽）；崩解劑（例如，馬鈴薯澱粉或羥基乙酸澱粉鈉）；和/或潤濕劑（例如，十二烷基硫酸鈉）。可以通過業內熟知的方法將片劑包衣。用於口服投予的液體製劑可以採用例如溶液、糖漿或混懸劑的形式，或者它們可以呈現為乾燥產品，用於在使用前用水或其他合適的媒劑重構。此類液體製劑可以通過常規手段用一種或多種諸如以下的醫藥上可接受的添加劑製備：助懸劑（例如，山梨糖醇糖漿、甲基纖維素或氫化食用脂肪）；乳化劑（例如，卵磷脂或阿拉伯膠）；非水性媒劑（例如，扁桃仁油、油性酯或乙醇）；和/或防腐劑（例如，對羥基苯甲酸甲酯或對羥基苯甲酸丙酯或山梨酸）。For oral administration, the pharmaceutical compositions may take the form of tablets or capsules prepared, for example, by conventional means with one or more pharmaceutically acceptable excipients such as: a binder (for example, pregelatinized cornstarch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (for example, lactose, microcrystalline cellulose, or calcium phosphate); lubricants (for example, magnesium stearate, talc, or silicon dioxide); disintegrants disintegrants (for example, potato starch or sodium starch glycolate); and/or wetting agents (for example, sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with one or more pharmaceutically acceptable additives such as: suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., egg phospholipids or acacia); non-aqueous vehicles (for example, almond oil, oily esters, or ethanol); and/or preservatives (for example, methyl or propylparaben or sorbic acid).
用於口服、腸胃外或經頰投予一般成年人以治療或預防CSF-1R相關疾病狀態的本發明公開的化合物的建議劑量是約0.1 mg至約2000 mg。在某些實施例中，建議劑量是每單位劑量從約0.1 mg至約200 mg活性成分。無論建議劑量的量如何，所述化合物的投予都可以發生，例如1至4次/天。Suggested dosages of the compounds disclosed herein for oral, parenteral or buccal administration to the average adult human for the treatment or prevention of CSF-1R-associated disease states are about 0.1 mg to about 2000 mg. In certain embodiments, suggested dosages are from about 0.1 mg to about 200 mg of active ingredient per unit dose. Administration of the compound can occur regardless of the amount of the suggested dosage, for example 1 to 4 times per day.
醫藥組合物和包括投予至少一種本發明公開的化合物的前驅藥的治療或預防方法也在本揭露的範圍內。Pharmaceutical compositions and methods of treatment or prophylaxis comprising the administration of at least one prodrug of a compound disclosed herein are also within the scope of the present disclosure.
根據式(I)和式(I')的合適的CSF-1R抑制劑的非限制性例子呈現在以下實例中。應當理解，在根據式(I)和式(I')的抑制劑中呈現的結構的任何或所有胺在以下實例中呈現，可以呈具有游離胺的形式或呈具有醫藥上可接受的陰離子的質子化形式。較佳的醫藥上可接受的陰離子包括鹵離子、碳酸根、碳酸氫根、硫酸根、硫酸氫根、氫氧根、硝酸根、過硫酸根、磷酸根、亞硫酸根、乙酸根、抗壞血酸根、苯甲酸根、檸檬酸根、檸檬酸二氫根、檸檬酸氫根、草酸根、琥珀酸根、酒石酸根、牛磺膽酸根、甘氨膽酸根和膽酸根。最佳的醫藥上可接受的陰離子包括氯離子、碳酸根和碳酸氫根。還應理解，根據式(I)和式(I')的任何或所有CSF-1R抑制劑可以是外消旋體或外消旋體的對映異構物。 實例 實例 1 ：合成方法 Non-limiting examples of suitable CSF-1R inhibitors according to formula (I) and formula (I') are presented in the Examples below. It should be understood that any or all of the amines of the structures presented in the inhibitors according to formula (I) and formula (I') presented in the following examples may be in the form of a free amine or in the form of a pharmaceutically acceptable anion protonated form. Preferred pharmaceutically acceptable anions include halides, carbonates, bicarbonates, sulfates, bisulfates, hydroxides, nitrates, persulfates, phosphates, sulfites, acetates, ascorbates , benzoate, citrate, dihydrogen citrate, hydrogen citrate, oxalate, succinate, tartrate, taurocholate, glycocholate and cholate. Preferred pharmaceutically acceptable anions include chloride, carbonate and bicarbonate. It is also to be understood that any or all of the CSF-1R inhibitors according to formula (I) and formula (I') may be a racemate or an enantiomer of a racemate. Example Example 1 : synthetic method
通過參考本文參考和公開的製備、方案和實例，業內的合成化學技術者可以容易地實現式I的化合物的合成。在WO 2017/015267的通用方案和合成實例中公開了與用於製備式I的化合物及其中間體的製備、方案和程序類似的相關製備、方案和程序。參考以下呈現的合成製備和方案描述本揭露的特定實施例；應當理解，此類實施例僅作為舉例，並且僅對可以表示本揭露的原理的應用的許多可能特定實施例中的少數進行說明。鑒於本揭露的益處，對製備、方案和實例的各種變化和修飾對於業內熟習此項技術者來說將是顯而易見的。 合成實例（實例 1-9 ） Syntheses of compounds of Formula I can be readily accomplished by a skilled synthetic chemist by referring to the Preparations, Schemes and Examples referenced and disclosed herein. Related preparations, schemes and procedures similar to those used to prepare compounds of formula I and intermediates thereof are disclosed in the general schemes and synthetic examples of WO 2017/015267. Specific examples of the disclosure are described with reference to the synthetic preparations and schemes presented below; it is to be understood that such examples are by way of example only, and illustrative of only a few of the many possible specific examples that may illustrate applications of the principles of the disclosure. Various changes and modifications to the Preparations, Schemes, and Examples will become apparent to those skilled in the art having the benefit of this disclosure. Synthetic Examples (Examples 1-9 )
實例 1 ： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -5-
d （化合物 1 ）的合成
實例 1-1 ： (+/-)-( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 甲酸甲酯的製備 
向4-羥基-3-((1-羥基-1-(6-甲氧基吡啶-3-基)丙-2-基)氧基)-5-甲氧基苯甲酸甲酯（1.41 g，3.88 mmol，關於製備參見 WO 2017015267）、三苯基膦（1.23 g，4.66 mmol）和 N, N-二異丙基乙基胺（1.0 mL，5.82 mmol）在乙腈（30 mL）中的攪拌溶液中添加四氯化碳（1.9 mL，19.40 mmol）。將所得的無色溶液加熱至回流，並且在惰性氣氛下攪拌。45 min後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕褐色固體。層析純化（CombiFlash，80 g SiO 2gold柱，10%-30%乙酸乙酯/庚烷溶析）得到呈白色固體的 (+/-)-( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 甲酸甲酯（0.75 g，2.18 mmol，56%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.19 (d, J= 2.4 Hz, 1H), 7.59 (dd, J= 8.6, 2.4 Hz, 1H), 7.34 (d, J= 1.9 Hz, 1H), 7.23 (d, J= 1.9 Hz, 1H), 6.80 (d, J= 8.6 Hz, 1H), 4.70 (d, J= 7.8 Hz, 1H), 4.15 (dq, J= 7.8, 6.4 Hz, 1H), 3.96 (s, 3H), 3.90 (s, 6H), 1.22 (d, J= 6.4 Hz, 3H) ppm; (M+1) = 346。 4-Hydroxy-3-((1-hydroxy-1-(6-methoxypyridin-3-yl)prop-2-yl)oxy)-5-methoxybenzoic acid methyl ester (1.41 g, 3.88 mmol, see WO 2017015267 for preparation), a stirred solution of triphenylphosphine (1.23 g, 4.66 mmol) and N , N -diisopropylethylamine (1.0 mL, 5.82 mmol) in acetonitrile (30 mL) Carbon tetrachloride (1.9 mL, 19.40 mmol) was added. The resulting colorless solution was heated to reflux and stirred under an inert atmosphere. After 45 min, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a tan solid. Chromatographic purification (CombiFlash, 80 g SiO 2 gold column, 10%-30% ethyl acetate/heptane elution) gave (+/-)-( trans )-8- methoxy- 2 as a white solid -(6 -Methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- carboxylic acid methyl ester (0.75 g, 2.18 mmol , 56% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.19 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.6, 2.4 Hz, 1H), 7.34 (d, J = 1.9 Hz, 1H), 7.23 (d, J = 1.9 Hz, 1H), 6.80 (d, J = 8.6 Hz, 1H), 4.70 (d, J = 7.8 Hz, 1H), 4.15 (dq, J = 7.8, 6.4 Hz, 1H), 3.96 (s, 3H), 3.90 (s, 6H), 1.22 (d, J = 6.4 Hz, 3H) ppm; (M+1) = 346.
實例 1-2 ： (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲醇的製備 
向(+/-)-反式-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-甲酸甲酯（0.75 g，2.18 mmol）在四氫呋喃（30 ml）中攪拌的0ºC溶液中一次性添加氫化鋁鋰（0.12 g，3.27 mmol）（注意到少量氣體放出）。將所得的灰色混合物在惰性氣氛下在0ºC下攪拌。10 min後，LC/MS分析顯示反應完成。將混合物通過添加水（0.12 mL）、1 N氫氧化鈉溶液（0.12 mL）和水（0.38 mL）淬滅。將所得的混合物在0ºC下攪拌10分鐘，並且然後添加硫酸鎂（約5 g）。將混合物通過矽藻土過濾，並且將濾餅用乙酸乙酯（50 mL）洗滌。將濾液濃縮以提供呈粘性白色泡沫的 (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲醇（0.69 g，2.18 mmol，100%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.26 (d, J= 2.3 Hz, 1H), 7.77 (dd, J= 8.5, 2.3 Hz, 1H), 6.89 (d, J= 8.5 Hz, 1H), 6.55 (d, J= 1.9 Hz, 1H), 6.49 (d, J= 1.9 Hz, 1H), 5.09 (br s, 1H), 4.74 (d, J= 7.7 Hz, 1H), 4.38 (s, 2H), 4.31 (dq, J= 7.7, 6.3 Hz, 1H), 3.88 (s, 3H), 3.72 (s, 3H), 1.09 (d, J= 6.3 Hz, 3H); (M+1) = 318。 To (+/-)-trans-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][1, 4] To a stirred 0ºC solution of dioxin-6-carboxylate (0.75 g, 2.18 mmol) in tetrahydrofuran (30 ml) was added lithium aluminum hydride (0.12 g, 3.27 mmol) in one portion (note a small amount of gas evolution) . The resulting gray mixture was stirred at 0 ºC under an inert atmosphere. After 10 min, LC/MS analysis showed the reaction was complete. The mixture was quenched by adding water (0.12 mL), 1 N sodium hydroxide solution (0.12 mL) and water (0.38 mL). The resulting mixture was stirred at 0°C for 10 minutes, and then magnesium sulfate (about 5 g) was added. The mixture was filtered through celite, and the filter cake was washed with ethyl acetate (50 mL). The filtrate was concentrated to afford (+/-)-(( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl- 2 as a viscous white foam, 3 -Dihydrobenzo [ b ][1,4] dioxin -6- yl ) methanol (0.69 g, 2.18 mmol, 100% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.26 (d, J = 2.3 Hz, 1H), 7.77 (dd, J = 8.5, 2.3 Hz, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 1.9 Hz, 1H), 6.49 (d, J = 1.9 Hz, 1H), 5.09 (br s, 1H), 4.74 (d, J = 7.7 Hz, 1H), 4.38 (s, 2H), 4.31 (dq, J = 7.7, 6.3 Hz, 1H ), 3.88 (s, 3H), 3.72 (s, 3H), 1.09 (d, J = 6.3 Hz, 3H); (M+1) = 318.
實例 1-3 ： (+/-)-5- 溴 -3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶的製備 
向5-溴-1
H-咪唑並[4,5-
b]吡啶（0.21 g，0.99 mmol）和(+/-)-((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[
b][1,4]二噁英-6-基)甲醇（0.35 g，1.10 mmol）在甲苯（10 ml）中的攪拌溶液中添加(三丁基亞正膦基)乙腈（0.43 g，1.74 mmol）。將所得的混合物在密封容器中加熱至75ºC並且允許攪拌。18 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO
2gold柱，20%-60% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈棕褐色固體的
(+/-)-5- 溴 -3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶（0.33 g，0.67 mmol，67%產率）：
1H NMR (400 MHz, DMSO-
d6) δ 8.61 (s, 1H), 8.23 (d,
J= 2.3 Hz, 1H), 8.08 (d,
J= 8.3 Hz, 1H), 7.73 (dd,
J= 8.6, 2.3 Hz, 1H), 7.49 (d,
J= 8.3 Hz, 1H), 6.88 (d,
J= 8.6 Hz, 1H), 6.79 (d,
J= 1.9 Hz, 1H), 6.46 (d,
J= 1.9 Hz, 1H), 5.36 (s, 2H), 4.73 (d,
J= 7.8 Hz, 1H), 4.29 (dq,
J= 7.8, 6.3 Hz, 1H), 3.87 (s, 3H), 3.73 (s, 3H), 1.05 (d,
J= 6.3 Hz, 3H) ppm; (M+1) = 497。區域化學的確認：
1H-
13C HSQC NMR資料分別在8.61和145.8 ppm處鑑定了咪唑C-2質子和碳。接下來，
1H-
13C HMBC NMR資料示出了在8.61 ppm處的此質子與134.1和146.4 ppm的四級環碳之間的多鍵相關性，其中146.4 ppm處的碳與吡啶氮相鄰。最後，通過在HMBC NMR資料中證明的在5.36 ppm處的相鄰的亞甲基質子與145.8 ppm和146.4 ppm處的四級碳之間的
1H-
13C多鍵相關性來確認連接。
實例 1-4： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -5- d 的製備 Example 1-4 : (+/-)-3-((( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3- Preparation of Dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl ) -3H- imidazo [4,5- b ] pyridine -5- d
向(+/-)-5-溴-反式-3-((-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（0.53 mg，1.06 mmol）在2-丙醇- d8（5 mL）中的攪拌溶液（注意：在20 mL烘箱乾燥的微波反應容器中進行）中添加三(二亞苄基丙酮)二鈀(0)（0.19 g，0.21 mmol）、2-二環己基膦-2',6'-二甲氧基聯苯（0.27 g，0.63 mmol）和碳酸鉀（0.29 g，2.12 mmol）。將容器密封，並且將內容物在真空/用N 2回填下脫氣（x 3）。將混合物加熱至100ºC並且允許攪拌。2 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，20%-70% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈棕褐色固體的 (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -5- d （0.30 g，0.72 mmol，68%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.60 (s, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.10 (d, J= 8.1 Hz, 1H), 7.73 (dd, J= 8.6, 2.3 Hz, 1H), 7.30 (d, J= 8.1 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.76 (d, J= 1.9 Hz, 1H), 6.49 (d, J= 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.27 (dq, J= 7.8, 6.4 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J= 6.4 Hz, 3H) ppm; (M+1) = 420。 To (+/-)-5-bromo-trans-3-((-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-di Hydrobenzo [ b ][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5- b ]pyridine (0.53 mg, 1.06 mmol) in 2-propanol- d To a stirred solution of 8 (5 mL) (Caution: performed in a 20 mL oven-dried microwave reaction vessel) was added tris(dibenzylideneacetone)dipalladium(0) (0.19 g, 0.21 mmol), 2-di Cyclohexylphosphine-2',6'-dimethoxybiphenyl (0.27 g, 0.63 mmol) and potassium carbonate (0.29 g, 2.12 mmol). The vessel was sealed and the contents were degassed (x 3) under vacuum/backfill with N2 . The mixture was heated to 100°C and allowed to stir. After 2 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 20%-70% 3:1 ethyl acetate:ethanol/heptane elution) afforded (+/-)-3-(((( trans Formula ) -8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin - 6 -yl ) methyl ) -3H - imidazo [4,5- b ] pyridine -5- d (0.30 g, 0.72 mmol, 68% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.60 (s, 1H), 8.22 (d, J = 2.3 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.73 (dd, J = 8.6, 2.3 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 1.9 Hz, 1H), 6.49 (d, J = 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.27 (dq, J = 7.8, 6.4 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J = 6.4 Hz, 3H ) ppm; (M+1) = 420.
實例 2 ： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -2,5-
d2
（化合物 2 ）的合成
實例 2-1 ： 2- 溴 -3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -5-
d 的製備 
向2-溴-3-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-5- d（0.25 g，0.59 mmol）在 N, N-二甲基甲醯胺（3 ml）的攪拌溶液中添加四溴化碳（0.32 g，0.96 mmol）和三級丁醇鈉（0.23 g，2.35 mmol）。將所得的深棕色混合物在室溫下攪拌。30 min後，LC/MS分析顯示形成新產物並且仍然存在起始材料。將另外部分的四溴化碳（0.32 g，0.96 mmol）和三級丁醇鈉（0.23 g，2.35 mmol）添加到混合物中。1 h後，LC/MS分析顯示反應仍未完成。將混合物淬滅至飽和氯化銨溶液（50 mL）中。將混合物用乙酸乙酯（40 mL）萃取。將有機相用鹽水（30 mL）洗滌，經無水硫酸鎂乾燥，過濾並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，10%-50% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈灰白色固體的 (+/-)-2- 溴 -3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -5- d （0.089 g，0.18 mmol，31%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.22 (d, J = 2.3 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.6, 2.3 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 1.9 Hz, 1H), 6.27 (d, J = 1.9 Hz, 1H), 5.41 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.28 (dq, J = 7.8, 6.3 Hz, 1H), 3.87 (s, 3H), 3.69 (s, 3H), 1.02 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 498。 To 2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-5- d (0.25 g, 0.59 mmol) in N , N -dimethyl To a stirred solution of formamide (3 mL) was added carbon tetrabromide (0.32 g, 0.96 mmol) and sodium tert-butoxide (0.23 g, 2.35 mmol). The resulting dark brown mixture was stirred at room temperature. After 30 min, LC/MS analysis showed formation of new product and starting material still present. Additional portions of carbon tetrabromide (0.32 g, 0.96 mmol) and sodium tert-butoxide (0.23 g, 2.35 mmol) were added to the mixture. After 1 h, LC/MS analysis showed that the reaction was still not complete. The mixture was quenched into saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 10%-50% 3:1 ethyl acetate:ethanol/heptane elution) gave (+/-)-2- bromo - 3-( (( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin In- 6- yl ) methyl ) -3H- imidazo [4,5- b ] pyridine -5- d (0.089 g, 0.18 mmol, 31% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.22 (d, J = 2.3 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.6, 2.3 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 1.9 Hz, 1H), 6.27 (d, J = 1.9 Hz, 1H), 5.41 (s, 2H), 4.72 (d, J = 7.8Hz, 1H), 4.28 (dq, J = 7.8, 6.3Hz, 1H), 3.87 (s, 3H), 3.69 (s, 3H), 1.02 (d, J = 6.3Hz, 3H) ppm; ( M+1) = 498.
實例 2-2： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2,5- d2 的製備向(+/-)-2-溴-3-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶-5- d（0.085 mg，0.17 mmol）在2-丙醇- d8（3 mL）中的攪拌溶液（注意：在20 mL烘箱乾燥的微波反應容器中進行）中添加三(二亞苄基丙酮)二鈀(0)（0.031 g，0.034 mmol）、2-二環己基膦-2',6'-二甲氧基聯苯（0.043 g，0.10 mmol）和碳酸鉀（0.047 g，0.34 mmol）。將容器密封，並且將內容物在真空/用N 2回填下脫氣（x 3）。將混合物加熱至100ºC並且攪拌。2 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，24 g SiO 2gold柱，20%-70% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈淺黃色固體的 (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2,5- d2 （0.047 g，0.11 mmol，66%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.22 (d, J= 2.3 Hz, 1H), 8.10 (d, J= 8.0 Hz, 1H), 7.73 (dd, J= 8.7, 2.3 Hz, 1H), 7.30 (d, J= 8.0 Hz, 1H), 6.88 (d, J= 8.7 Hz, 1H), 6.76 (d, J= 2.0 Hz, 1H), 6.49 (d, J= 2.0 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.27 (dq, J= 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 421。 Example 2-2 : (+/-)-3-((( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3- Preparation of dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl ) -3H- imidazo [4,5- b ] pyridine -2,5- d2 to (+/ -)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo [ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine-5- d (0.085 mg, 0.17 mmol) in 2-propanol- To a stirred solution in d 8 (3 mL) (Caution: performed in a 20 mL oven-dried microwave reaction vessel) was added tris(dibenzylideneacetone)dipalladium(0) (0.031 g, 0.034 mmol), 2- Dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.043 g, 0.10 mmol) and potassium carbonate (0.047 g, 0.34 mmol). The vessel was sealed and the contents were degassed (x 3) under vacuum/backfill with N2 . The mixture was heated to 100°C and stirred. After 2 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Purification by chromatography (CombiFlash, 24 g SiO 2 gold column, 20%-70% 3:1 ethyl acetate:ethanol/heptane elution) gave (+/-)-3-(((( trans Formula ) -8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin - 6 -yl ) methyl ) -3H - imidazo [4,5- b ] pyridine -2,5- d2 (0.047 g, 0.11 mmol, 66% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.22 (d, J = 2.3 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.7, 2.3 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.49 (d, J = 2.0 Hz, 1H), 5.39 (s, 2H), 4.72 (d, ( _ _ M+1) = 421.
實例 3 ： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 -
d2)-3
H-
咪唑並 [4,5-
b]
吡啶（化合物 3 ）的合成
實例 3-1 ： (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲 -
d2-
醇的製備 
向(+/-)-(反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-甲酸甲酯（2.08 g，6.02 mmol）在四氫呋喃（60 mL）中的0ºC攪拌溶液中一次性添加氘化鋁鋰（0.34 g，8.13 mmol）（注意到少量氣體放出）。將所得的灰色混合物在0ºC下攪拌。15 min後，LC/MS分析顯示反應完成。將混合物通過添加水（0.50 mL）、1 N氫氧化鈉溶液（0.50 mL）和水（1.5 mL）淬滅。將所得的混合物在0ºC下攪拌15 min，並且然後添加硫酸鎂（約10 g）。將混合物通過矽藻土過濾，並且將濾餅用乙酸乙酯（100 mL）洗滌。將濾液濃縮以提供呈粘性白色泡沫的 (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲 - d2- 醇（1.92 g，6.01 mmol，100%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.15 (d, J= 2.5 Hz, 1H), 7.59 (dd, J= 8.6, 2.5 Hz, 1H), 6.79 (d, J= 8.6 Hz, 1H), 6.59 (d, J= 1.9 Hz, 1H), 6.57 (d, J= 1.9 Hz, 1H), 4.63 (d, J= 7.8 Hz, 1H), 4.14 (dq, J= 7.8, 6.4 Hz, 1H), 3.95 (s, 3H), 3.86 (s, 3H), 1.19 (d, J= 6.4 Hz, 3H); (M+1) = 320。 To (+/-)-(trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][ 1,4] To a stirred solution of methyl dioxin-6-carboxylate (2.08 g, 6.02 mmol) in tetrahydrofuran (60 mL) at 0ºC was added lithium aluminum deuteride (0.34 g, 8.13 mmol) in one portion (note that a small gas evolution). The resulting gray mixture was stirred at 0°C. After 15 min, LC/MS analysis showed the reaction was complete. The mixture was quenched by adding water (0.50 mL), 1 N sodium hydroxide solution (0.50 mL) and water (1.5 mL). The resulting mixture was stirred at 0°C for 15 min, and then magnesium sulfate (ca. 10 g) was added. The mixture was filtered through celite, and the filter cake was washed with ethyl acetate (100 mL). The filtrate was concentrated to afford (+/-)-(( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl- 2 as a viscous white foam, 3 -Dihydrobenzo [ b ][1,4] dioxin -6- yl ) meth - d2 - ol (1.92 g, 6.01 mmol, 100% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.15 (d, J = 2.5 Hz, 1H), 7.59 (dd, J = 8.6, 2.5 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 6.59 (d, J = 1.9 Hz, 1H ), 6.57 (d, J = 1.9 Hz, 1H), 4.63 (d, J = 7.8 Hz, 1H), 4.14 (dq, J = 7.8, 6.4 Hz, 1H), 3.95 (s, 3H), 3.86 (s , 3H), 1.19 (d, J = 6.4 Hz, 3H); (M+1) = 320.
實例 3-2 ： (+/-)-5-(( 反式 )-6-( 疊氮基甲基 -
d2)-8-
甲氧基 -3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -2- 基 )-2- 甲氧基吡啶的製備 
向(+/-)-((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲- d2-醇（1.92 g，6.01 mmol）和二苯基磷醯基疊氮化合物（2.07 mL，9.62 mmol）在四氫呋喃（50 mL）中的攪拌溶液中添加1,8-二氮雜雙環[5.4.0]十一-7-烯（1.4 mL，9.62 mmol）。將所得的混合物加熱至回流，並且在惰性氣氛下攪拌。1 h後，LC/MS分析顯示反應完成。將無色溶液冷卻至室溫並且濃縮以提供黃色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，10%-30%乙酸乙酯/庚烷溶析）得到呈白色固體的 (+/-)-5-(( 反式 )-6-( 疊氮基甲基 - d2)-8- 甲氧基 -3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -2- 基 )-2- 甲氧基吡啶（1.78 g，5.17 mmol，86%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.19 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.7, 2.4 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H), 4.65 (d, J = 7.9 Hz, 1H), 4.15 (dq, J = 7.9, 6.4 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 1.20 (d, J = 6.4 Hz, 3H) ppm; (M+1) = 345。 To (+/-)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ] [1,4]dioxin-6-yl)meth- d -2-ol (1.92 g, 6.01 mmol) and diphenylphosphoryl azide (2.07 mL, 9.62 mmol) in tetrahydrofuran (50 mL) To the stirred solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (1.4 mL, 9.62 mmol) was added. The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 1 h, LC/MS analysis showed the reaction was complete. The colorless solution was cooled to room temperature and concentrated to afford a yellow oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 10%-30% ethyl acetate/heptane elution) afforded (+/-)-5-(( trans )-6-( alkene ) as a white solid Nitromethyl- d2 )-8- methoxy- 3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -2- yl )-2- methoxy Pyridine (1.78 g, 5.17 mmol, 86% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.19 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 8.7, 2.4 Hz, 1H) , 6.80 (d, J = 8.7 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H), 4.65 (d, J = 7.9 Hz, 1H), 4.15 (dq, J = 7.9, 6.4 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 1.20 (d, J = 6.4 Hz, 3H) ppm; (M+1) = 345.
實例 3-3 ： (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲 -
d2-
胺的製備 
向(+/-)-5-((反式)-6-(疊氮基甲基- d2)-8-甲氧基-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-2-基)-2-甲氧基吡啶（1.78 g，5.17 mmol）在四氫呋喃（50 mL）和水（5 mL）的攪拌溶液中添加聚合物鍵合的三苯基膦（3.50 g，約10.50 mmol）。將橙色懸浮液加熱至回流並且在惰性氣氛下攪拌。2 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫，並且借助乙酸乙酯（50 mL）通過矽藻土過濾。將濾液經硫酸鎂乾燥，過濾並且濃縮以提供呈無色油狀物的 (+/-)-(( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲 - d2- 胺（1.61 g，5.06 mmol，98%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.17 (d, J= 2.4 Hz, 1H), 7.59 (dd, J= 8.6, 2.4 Hz, 1H), 6.78 (d, J= 8.6 Hz, 1H), 6.54 (d, J= 1.9 Hz, 1H), 6.50 (d, J= 1.9 Hz, 1H), 4.63 (d, J= 7.8 Hz, 1H), 4.13 (dq, J= 7.8, 6.4 Hz, 1H), 3.95 (s, 3H), 3.85 (s, 3H), 2.04 (s, 2H), 1.19 (d, J= 6.4 Hz, 3H) ppm; (M-16) = 302。 To (+/-)-5-((trans)-6-(azidomethyl- d2 )-8-methoxy-3-methyl-2,3-dihydrobenzo[ b ] To a stirred solution of [1,4]dioxin-2-yl)-2-methoxypyridine (1.78 g, 5.17 mmol) in tetrahydrofuran (50 mL) and water (5 mL) was added the polymer-bonded tris Phenylphosphine (3.50 g, about 10.50 mmol). The orange suspension was heated to reflux and stirred under an inert atmosphere. After 2 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and filtered through celite with ethyl acetate (50 mL). The filtrate was dried over magnesium sulfate, filtered and concentrated to afford (+/-)-(( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl ) as a colorless oil -3 -Methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) meth - d2 - amine (1.61 g, 5.06 mmol, 98% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.17 (d, J = 2.4 Hz, 1H), 7.59 (dd, J = 8.6, 2.4 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.54 ( d, J = 1.9 Hz, 1H), 6.50 (d, J = 1.9 Hz, 1H), 4.63 (d, J = 7.8 Hz, 1H), 4.13 (dq, J = 7.8, 6.4 Hz, 1H), 3.95 ( s, 3H), 3.85 (s, 3H), 2.04 (s, 2H), 1.19 (d, J = 6.4 Hz, 3H) ppm; (M-16) = 302.
實例 3-4 ： (+/-)-
N-(((
反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 -
d2)-3-
硝基吡啶 -2- 胺的製備 
向(+/-)-((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲- d2-胺（1.61 g，5.06 mmol）和 N, N-二異丙基乙基胺（1.3 mL，7.59 mmol）在乙腈（30 mL）中的攪拌溶液中添加2-氯-3-硝基吡啶（0.84 g，5.31 mmol）。將所得的混合物加熱至回流，並且在惰性氣氛下攪拌。16 h後，黃色混合物的LC/MS分析顯示反應完成。將混合物冷卻至室溫並且用水（50 mL）稀釋。將所得混合物用乙酸乙酯（2 x 50 mL）萃取。將合併的有機相經硫酸鎂乾燥，過濾並且濃縮以提供呈黃色固體的 (+/-)- N-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 - d2)-3- 硝基吡啶 -2- 胺（2.05 g，4.65 mmol，92%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.51 – 8.40 (m, 3H), 8.17 (d, J = 2.4 Hz, 1H), 7.58 (dd, J = 8.6, 2.4 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 6.70 – 6.66 (m, 1H), 6.61 (d, J = 1.9 Hz, 1H), 6.55 (d, J = 1.9 Hz, 1H), 4.63 (d, J = 7.8 Hz, 1H), 4.18 – 4.09 (m, 1H), 3.95 (s, 3H), 3.84 (s, 3H), 1.19 (d, J = 6.4 Hz, 3H) ppm; (M+1) = 441。 To (+/-)-((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ] [1,4]dioxin-6-yl)meth- d 2-amine (1.61 g, 5.06 mmol) and N , N -diisopropylethylamine (1.3 mL, 7.59 mmol) in acetonitrile (30 mL ) was added 2-chloro-3-nitropyridine (0.84 g, 5.31 mmol). The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 16 h, LC/MS analysis of the yellow mixture showed the reaction was complete. The mixture was cooled to room temperature and diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic phases were dried over magnesium sulfate, filtered and concentrated to afford (+/-)- N -((( trans )-8- methoxy- 2-(6 - methoxypyridine- 3- yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl - d2 )-3 -nitropyridin -2- amine (2.05 g, 4.65 mmol, 92% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.51 – 8.40 (m, 3H), 8.17 (d, J = 2.4 Hz, 1H), 7.58 (dd, J = 8.6, 2.4 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 6.70 – 6.66 (m, 1H), 6.61 (d, J = 1.9 Hz, 1H), 6.55 (d, J = 1.9 Hz , 1H), 4.63 (d, J = 7.8 Hz, 1H), 4.18 – 4.09 (m, 1H), 3.95 (s, 3H), 3.84 (s, 3H), 1.19 (d, J = 6.4 Hz, 3H) ppm; (M+1) = 441.
實例 3-5 ： (+/-)-
N 2-(((
反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 -
d2)
吡啶 -2,3- 二胺的製備 
向(+/-)- N-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基- d2)-3-硝基吡啶-2-胺（2.05 g，4.65 mmol）和氯化銨（1.99 g，37.23 mmol）在四氫呋喃（50 mL）/甲醇（20 mL）/水（10 mL）的混合物中的攪拌溶液中添加鋅粉（2.43 g，37.23 mmol）。將所得的混合物在室溫下攪拌。45 min後，LC/MS分析顯示反應完成。將灰色懸浮液通過矽藻土過濾，並且將濾餅用乙酸乙酯（75 mL）洗滌。將濾液用5 N氫氧化銨溶液（50 mL）洗滌。將有機相經硫酸鎂乾燥，過濾並且濃縮以提供呈深棕色固體的 (+/-)- N 2-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 - d2) 吡啶 -2,3- 二胺（1.72 g，4.19 mmol，90%產率）： 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 5.1, 1.5 Hz, 1H), 7.58 (dd, J = 8.6, 2.4 Hz, 1H), 6.88 (dd, J = 7.4, 1.5 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.65 (d, J = 1.9 Hz, 1H), 6.59 (d, J = 1.9 Hz, 1H), 6.56 (dd, J = 7.4, 5.1 Hz, 1H), 4.63 (d, J = 7.7 Hz, 1H), 4.40 (br s, 1H), 4.18 – 4.09 (m, 1H), 3.95 (s, 3H), 3.83 (s, 3H), 3.22 (br s, 2H), 1.19 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 411。 To (+/-)- N -(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo [ b ][1,4]dioxin-6-yl)methyl- d2 )-3-nitropyridin-2-amine (2.05 g, 4.65 mmol) and ammonium chloride (1.99 g, 37.23 mmol) To a stirred solution in a mixture of tetrahydrofuran (50 mL)/methanol (20 mL)/water (10 mL) was added zinc powder (2.43 g, 37.23 mmol). The resulting mixture was stirred at room temperature. After 45 min, LC/MS analysis showed the reaction was complete. The gray suspension was filtered through celite, and the filter cake was washed with ethyl acetate (75 mL). The filtrate was washed with 5 N ammonium hydroxide solution (50 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated to afford (+/-)- N2 - ((( trans )-8- methoxy- 2-(6 - methoxypyridine- 3- yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl - d 2) pyridine -2,3- diamine (1.72 g, 4.19 mmol, 90% yield): 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 2.4 Hz, 1H), 7.78 (dd, J = 5.1, 1.5 Hz, 1H), 7.58 (dd , J = 8.6, 2.4 Hz, 1H), 6.88 (dd, J = 7.4, 1.5 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.65 (d, J = 1.9 Hz, 1H), 6.59 (d, J = 1.9 Hz, 1H), 6.56 (dd, J = 7.4, 5.1 Hz, 1H), 4.63 (d, J = 7.7 Hz, 1H), 4.40 (br s, 1H), 4.18 – 4.09 (m , 1H), 3.95 (s, 3H), 3.83 (s, 3H), 3.22 (br s, 2H), 1.19 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 411.
實例example 3-63-6 ：: (+/-)-3-((((+/-)-3-((( 反式trans )-8-)-8- 甲氧基Methoxy -2-(6--2-(6- 甲氧基吡啶Methoxypyridine -3--3- 基base )-3-)-3- 甲基methyl -2,3--2,3- 二氫苯並Dihydrobenzo [ b][1,4] [ b ][1,4] 二噁英Dioxins -6--6- 基base )) 甲基methyl - d2)-3 H- - d 2)-3 H - 咪唑並imidazo [4,5- b] [4,5- b ] 吡啶的製備Preparation of pyridine
向(+/-)- N 2-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基- d2)吡啶-2,3-二胺（1.72 g，4.19 mmol）和原甲酸三乙酯（2.0 mL，11.78 mmol）在乙醇（75 mL）中的攪拌懸浮液中添加對甲苯磺酸一水合物（約0.050 g）。將所得的混合物加熱至回流，並且在惰性氣氛下攪拌。16 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，120 g SiO 2gold柱，20%-50% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）提供淺棕色固體。將固體懸浮在甲基三級丁基醚（12 mL）/乙酸乙酯（0.50 mL）的混合物中。將混合物加熱至55ºC。3 h後，將溫熱的混合物過濾，並且將濾餅用甲基三級丁基醚（10 mL）洗滌並且乾燥以得到呈棕褐色固體的 (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 - d2)-3 H- 咪唑並 [4,5- b] 吡啶（0.99 g，2.37 mmol，57%產率）： 1H NMR (400 MHz, CDCl 3) δ 8.45 (dd, J= 4.8, 1.4 Hz, 1H), 8.16 (d, J= 2.4 Hz, 1H), 8.10 (dd, J= 8.0, 1.4 Hz, 1H), 8.06 (s, 1H), 7.56 (dd, J= 8.6, 2.4 Hz, 1H), 7.27 (dd, J= 8.0, 4.8 Hz, 1H), 6.78 (d, J= 8.6, 1H), 6.54 (s, 2H), 4.62 (d, J= 7.8 Hz, 1H), 4.12 (dq, J= 7.8, 6.3 Hz, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 1.17 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 421。 To (+/-)- N 2 -(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzene and[ b ][1,4]dioxin-6-yl)methyl- d2 )pyridine-2,3-diamine (1.72 g, 4.19 mmol) and triethyl orthoformate (2.0 mL, 11.78 mmol ) to a stirred suspension in ethanol (75 mL) was added p-toluenesulfonic acid monohydrate (ca. 0.050 g). The resulting mixture was heated to reflux and stirred under an inert atmosphere. After 16 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 120 g SiO 2 gold column, 20%-50% 3:1 ethyl acetate:ethanol/heptane elution) provided a light brown solid. Suspend the solid in a mixture of methyl tert-butyl ether (12 mL)/ethyl acetate (0.50 mL). Heat the mixture to 55ºC. After 3 h, the warm mixture was filtered, and the filter cake was washed with methyl tert-butyl ether (10 mL) and dried to give (+/-)-3-((( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl - d2 )-3H - imidazo [4,5- b ] pyridine (0.99 g, 2.37 mmol, 57% yield): 1 H NMR (400 MHz, CDCl 3 ) δ 8.45 (dd, J = 4.8, 1.4 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.0, 1.4 Hz, 1H), 8.06 (s, 1H), 7.56 (dd, J = 8.6 , 2.4 Hz, 1H), 7.27 (dd, J = 8.0, 4.8 Hz, 1H), 6.78 (d, J = 8.6, 1H), 6.54 (s, 2H), 4.62 (d, J = 7.8 Hz, 1H) , 4.12 (dq, J = 7.8, 6.3 Hz, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 1.17 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 421.
實例 4 ： (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 -
d2)-3H-
咪唑並 [4,5-
b]
吡啶 -2-
d （化合物 4 ）的合成
實例example 4-14-1 ：: (+/-)-2-(+/-)-2- 溴bromine -3-(((-3-((( 反式trans )-8-)-8- 甲氧基Methoxy -2-(6--2-(6- 甲氧基吡啶Methoxypyridine -3--3- 基base )-3-)-3- 甲基methyl -2,3--2,3- 二氫苯並Dihydrobenzo [ b][1,4] [ b ][1,4] 二噁英Dioxins -6--6- 基base )) 甲基methyl - d2)-3H- -d 2)-3H- 咪唑並imidazo [4,5- b] [4,5- b ] 吡啶的合成Synthesis of pyridine
向(+/-)3-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基- d2)-3H-咪唑並[4,5- b]吡啶（0.45 g，1.08 mmol）在 N, N-二甲基甲醯胺（5 ml）中的攪拌溶液中添加四溴化碳（0.54 g，1.62 mmol）和三級丁醇鈉（0.41 g，4.31 mmol），導致深棕色混合物的形成。將混合物在室溫下攪拌。20 min後，LC/MS分析顯示形成新產物並且仍然存在起始材料（約1 : 1）。將另外部分的四溴化碳（0.54 g，1.62 mmol）和三級丁醇鈉（0.41 g，4.31 mmol）添加到混合物中（在t = 40 min、t = 60 min和t = 80 min時重複）。總計100 min後，LC/MS分析顯示反應接近完成。將混合物淬滅至飽和氯化銨溶液（50 mL）中。將混合物用乙酸乙酯（40 mL）萃取。將有機相用鹽水（30 mL）洗滌，經無水硫酸鎂乾燥，過濾並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，10%-50% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈灰白色固體的 (+/-)-2- 溴 -3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 - d2)-3H- 咪唑並 [4,5- b] 吡啶（0.39 g，0.79 mmol，73%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.42 (dd, J= 4.9, 1.5 Hz, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.10 (dd, J= 8.1, 1.5 Hz, 1H), 7.73 (dd, J= 8.6, 2.3 Hz, 1H), 7.35 (dd, J= 8.1, 4.9 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.70 (d, J= 1.9 Hz, 1H), 6.28 (d, J= 1.9 Hz, 1H), 4.72 (d, J= 7.9 Hz, 1H), 4.28 (dq, J= 7.9, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.03 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 499。 To (+/-)3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl- d2 )-3H-imidazo[4,5- b ]pyridine (0.45 g, 1.08 mmol) in N , N -dimethylformaldehyde To a stirred solution in amide (5 ml) was added carbon tetrabromide (0.54 g, 1.62 mmol) and sodium tert-butoxide (0.41 g, 4.31 mmol), resulting in the formation of a dark brown mixture. The mixture was stirred at room temperature. After 20 min, LC/MS analysis showed the formation of new product and the starting material was still present (approximately 1:1). Additional portions of carbon tetrabromide (0.54 g, 1.62 mmol) and sodium tert-butoxide (0.41 g, 4.31 mmol) were added to the mixture (repeated at t = 40 min, t = 60 min and t = 80 min ). After a total of 100 min, LC/MS analysis indicated that the reaction was nearly complete. The mixture was quenched into saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to afford a brown oil. Purification by chromatography (CombiFlash, 40 g SiO 2 gold column, 10%-50% 3:1 ethyl acetate:ethanol/heptane elution) afforded (+/-)-2- bromo - 3-( (( trans )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin In- 6- yl ) methyl - d2 )-3H- imidazo [4,5- b ] pyridine (0.39 g, 0.79 mmol, 73% yield): 1 H NMR (400 MHz, DMSO- d6 ) δ 8.42 (dd, J = 4.9, 1.5 Hz, 1H), 8.22 (d, J = 2.3 Hz, 1H), 8.10 (dd, J = 8.1, 1.5 Hz, 1H), 7.73 (dd, J = 8.6, 2.3 Hz, 1H), 7.35 (dd, J = 8.1, 4.9 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.28 (d, J = 1.9 Hz, 1H), 4.72 (d, J = 7.9 Hz, 1H), 4.28 (dq, J = 7.9, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 499.
實例example 4-24-2 ：: (+/-)-3-((((+/-)-3-((( 反式trans )-8-)-8- 甲氧基Methoxy -2-(6--2-(6- 甲氧基吡啶Methoxypyridine -3--3- 基base )-3-)-3- 甲基methyl -2,3--2,3- 二氫苯並Dihydrobenzo [ b][1,4] [ b ][1,4] 二噁英Dioxins -6--6- 基base )) 甲基methyl - d2)-3 H- - d 2)-3 H - 咪唑並imidazo [4,5- b] [4,5- b ] 吡啶pyridine -2- d -2- d 的製備preparation of
向(+/-)-2-溴-3-(((反式)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基- d2)-3 H-咪唑並[4,5- b]吡啶（0.32 g，0.65 mmol）在丙醇- d8（3 mL）中的攪拌溶液（注意：在20 mL烘箱乾燥的微波反應容器中進行反應）中添加三(二亞苄基丙酮)二鈀(0)（0.12 g，0.13 mmol）、2-二環己基膦-2',6'-二甲氧基聯苯（0.16 g，039 mmol）和碳酸鉀（0.18 g，1.29 mmol）。將容器密封，並且將內容物在真空/用N 2回填下脫氣（x 3）。將混合物在加熱塊中加熱至100ºC。2 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，80 g SiO 2gold柱，20%-70% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到不純的棕色油狀物。製備型HPLC純化（Interchim柱：F0040 - 51 g - 51.0 g（20巴））柱，20% 乙腈/水/0.1%甲酸至100%乙腈/0.1%甲酸溶析）提供兩個純流分。將流分合併並且用飽和碳酸氫鈉溶液（30 mL）稀釋。將混合物用乙酸乙酯（30 mL）萃取。將有機相經硫酸鎂乾燥，過濾並且濃縮以提供呈白色固體的 (+/-)-3-((( 反式 )-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 - d2)-3 H- 咪唑並 [4,5- b] 吡啶 -2- d （0.16 g，0.37 mmol，57%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.40 (dd, J= 4.8, 1.5 Hz, 1H), 8.22 (d, J= 2.5 Hz, 1H), 8.10 (dd, J= 8.0, 1.5 Hz, 1H), 7.73 (dd, J= 8.6, 2.5 Hz, 1H), 7.30 (dd, J= 8.0, 4.8 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.76 (d, J= 2.0 Hz, 1H), 6.50 (d, J= 2.0 Hz, 1H), 4.72 (d, J= 7.8 Hz, 1H), 4.27 (dq, J= 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 421。 To (+/-)-2-bromo-3-(((trans)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3- Dihydrobenzo[ b ][1,4]dioxin-6-yl)methyl- d2 ) -3H -imidazo[4,5- b ]pyridine (0.32 g, 0.65 mmol) in propanol - To a stirred solution in d 8 (3 mL) (Note: the reaction was performed in a 20 mL oven-dried microwave reaction vessel) was added tris(dibenzylideneacetone)dipalladium(0) (0.12 g, 0.13 mmol), 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl (0.16 g, 039 mmol) and potassium carbonate (0.18 g, 1.29 mmol). The vessel was sealed and the contents were degassed (x 3) under vacuum/backfill with N2 . Heat the mixture to 100 ºC in a heating block. After 2 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 80 g SiO 2 gold column, 20%-70% 3:1 ethyl acetate:ethanol/heptane eluent) gave an impure brown oil. Preparative HPLC purification (Interchim column: F0040 - 51 g - 51.0 g (20 bar)) column, 20% acetonitrile/water/0.1% formic acid to 100% acetonitrile/0.1% formic acid eluting) provided two pure fractions. Fractions were combined and diluted with saturated sodium bicarbonate solution (30 mL). The mixture was extracted with ethyl acetate (30 mL). The organic phase was dried over magnesium sulfate, filtered and concentrated to afford (+/-)-3-((( trans )-8- methoxy- 2-(6 -methoxypyridine- 3- yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl - d 2) -3H - imidazo [4,5- b ] pyridine -2- d (0.16 g, 0.37 mmol, 57% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.40 (dd, J = 4.8, 1.5 Hz, 1H), 8.22 (d, J = 2.5 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.73 (dd, J = 8.6, 2.5 Hz, 1H), 7.30 (dd, J = 8.0, 4.8 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 4.72 (d, J = 7.8 Hz, 1H), 4.27 ( dq, J = 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J = 6.3 Hz, 3H) ppm; (M+1) = 421.
實例 5 ： 3-(((2
R,3
R)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -2-
d （化合物 5 ）的合成
實例 5-1 ： 3-(((2
R,3
R)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶和 3-(((2
S,3
S)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶的製備和分離 
實例 5-2 ： 2- 溴 -3-(((2
R,3
R)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶的製備 
向3-(((2 R,3 R)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（0.44 g，1.05 mmol）在 N, N-二甲基甲醯胺（5 ml）中的攪拌溶液中添加四溴化碳（0.38 g，1.16 mmol）和三級丁醇鈉（0.40 g，4.21 mmol）。將所得的深棕色混合物在室溫下攪拌。30 min後，LC/MS分析顯示形成新產物並且仍然存在起始材料（約1 : 1）。將混合物淬滅至飽和氯化銨溶液（50 mL）中。將混合物用乙酸乙酯（40 mL）萃取。將有機相用鹽水（30 mL）洗滌，經無水硫酸鎂乾燥，過濾並且濃縮以提供棕色油狀物。層析純化（CombiFlash，24 g SiO 2gold柱，10%-50% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈棕褐色固體的 2- 溴 -3-(((2 R,3 R)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶（0.18 g，0.37 mmol，35%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.42 (dd, J= 4.9, 1.5 Hz, 1H), 8.22 (d, J= 2.4 Hz, 1H), 8.11 (dd, J= 8.1, 1.5 Hz, 1H), 7.73 (dd, J= 8.6, 2.4 Hz, 1H), 7.36 (dd, J= 8.1, 4.9 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.69 (d, J= 1.9 Hz, 1H), 6.27 (d, J= 1.9 Hz, 1H), 5.41 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.28 (dq, J= 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.02 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 497。 To 3-(((2 R ,3 R )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ] [1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine (0.44 g, 1.05 mmol) in N , N -dimethylformamide (5 ml) was added carbon tetrabromide (0.38 g, 1.16 mmol) and sodium tertiary butoxide (0.40 g, 4.21 mmol). The resulting dark brown mixture was stirred at room temperature. After 30 min, LC/MS analysis showed the formation of new product and the starting material was still present (approximately 1:1). The mixture was quenched into saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to afford a brown oil. Purification by chromatography (CombiFlash, 24 g SiO 2 gold column, 10%-50% 3:1 ethyl acetate:ethanol/heptane elution) afforded 2- bromo -3-((((2 R , 3 R )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 - dihydrobenzo [ b ][1,4] dioxin- 6- yl ) methyl )-3H - imidazo [4,5- b ] pyridine (0.18 g, 0.37 mmol, 35% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.42 (dd , J = 4.9, 1.5 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 8.1, 1.5 Hz, 1H), 7.73 (dd, J = 8.6, 2.4 Hz, 1H) , 7.36 (dd, J = 8.1, 4.9 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 1.9 Hz, 1H), 6.27 (d, J = 1.9 Hz, 1H) , 5.41 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.28 (dq, J = 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.02 ( d, J = 6.3 Hz, 3H) ppm; (M+1) = 497.
實例 5-3： 3-(((2 R,3 R)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2- d 的製備 Example 5-3 : 3-(((2 R ,3 R )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzene Preparation of [ b ][1,4] dioxin -6- yl ) methyl ) -3H- imidazo [4,5- b ] pyridine -2- d
向2-溴-3-(((2 R,3 R)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（0.16 g，0.32 mmol）在2-丙醇- d8（5 mL）中的攪拌溶液（注意：在20 mL烘箱乾燥的微波反應容器中進行反應）中添加三(二亞苄基丙酮)二鈀(0)（0.059 g，0.064 mol）、2-二環己基膦-2',6'-二甲氧基聯苯（0.080 g，0.19 mmol）和碳酸鉀（0.089 g，0.64 mmol）。將容器密封，並且將內容物在真空/用N 2回填下脫氣（x 3）。將混合物在加熱塊中加熱至100ºC。2 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，20%-70% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈黃色固體的 3-(((2 R,3 R)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2- d （0.075 g，0.18 mmol，56%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.40 (dd, J= 4.7, 1.5 Hz, 1H), 8.22 (d, J= 2.4 Hz, 1H), 8.10 (dd, J= 8.0, 1.5 Hz, 1H), 7.73 (dd, J= 8.6, 2.4 Hz, 1H), 7.30 (dd, J= 8.0, 4.7 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.76 (d, J= 1.9 Hz, 1H), 6.49 (d, J= 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.28 (dq, J= 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 420。 To 2-bromo-3-(((2 R ,3 R )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzene and[ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine (0.16 g, 0.32 mmol) in 2-propanol- d 8 ( 5 mL) (Note: the reaction was performed in a 20 mL oven-dried microwave reaction vessel) was added tris(dibenzylideneacetone)dipalladium(0) (0.059 g, 0.064 mol), 2-bicyclo Hexylphosphine-2',6'-dimethoxybiphenyl (0.080 g, 0.19 mmol) and potassium carbonate (0.089 g, 0.64 mmol). The vessel was sealed and the contents were degassed (x 3) under vacuum/backfill with N2 . Heat the mixture to 100 ºC in a heating block. After 2 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 20%-70% 3:1 ethyl acetate:ethanol/heptane elution) afforded 3-((((2 R ,3 R )-8 -Methoxy- 2- (6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methoxy base )-3 H - imidazo [4,5- b ] pyridine -2- d (0.075 g, 0.18 mmol, 56% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.40 (dd, J = 4.7, 1.5 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.73 (dd, J = 8.6, 2.4 Hz, 1H), 7.30 (dd, J = 8.0, 4.7 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 1.9 Hz, 1H), 6.49 (d, J = 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.28 (dq, J = 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d , J = 6.3 Hz, 3H) ppm; (M+1) = 420.
實例 6 ： 3-(((2
S,3
S)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -2-
d （化合物 6 ）的合成 方法 A ：
實例 6-1 ： 2- 溴 -3-(((2
S,3
S)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶的合成 
向3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（0.51 g，1.22 mmol）在 N, N-二甲基甲醯胺（5 ml）中的攪拌溶液中添加四溴化碳（0.53 g，1.58 mmol）和三級丁醇鈉（0.50 g，5.22 mmol）。將所得的深棕色混合物在室溫下攪拌。1 h後，LC/MS分析顯示形成新產物並且仍然存在起始材料（約1 : 1）。將混合物淬滅至飽和氯化銨溶液（50 mL）中。將混合物用乙酸乙酯（40 mL）萃取。將有機相用鹽水（30 mL）洗滌，經無水硫酸鎂乾燥，過濾並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，10%-60% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈灰白色固體的 2- 溴 -3-(((2 S,3 S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶（0.20 mg，0.39 mmol，32%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.42 (dd, J= 4.8, 1.5 Hz, 1H), 8.22 (d, J= 2.4 Hz, 1H), 8.11 (dd, J= 8.1, 1.5 Hz, 1H), 7.73 (dd, J= 8.6, 2.4 Hz, 1H), 7.36 (dd, J= 8.1, 4.8 Hz, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.69 (d, J= 2.0 Hz, 1H), 6.27 (d, J= 2.0 Hz, 1H), 5.41 (s, 2H), 4.72 (d, J= 7.9 Hz, 1H), 4.28 (dq, J= 7.9, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.02 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 497。 To 3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ] [1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine (0.51 g, 1.22 mmol) in N , N -dimethylformamide (5 ml) was added carbon tetrabromide (0.53 g, 1.58 mmol) and sodium tertiary butoxide (0.50 g, 5.22 mmol). The resulting dark brown mixture was stirred at room temperature. After 1 h, LC/MS analysis showed the formation of new product and the starting material was still present (approximately 1:1). The mixture was quenched into saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (40 mL). The organic phase was washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 10%-60% 3:1 ethyl acetate:ethanol/heptane elution) gave 2- bromo -3-((((2 S ,3 S )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin - 6 -yl ) methyl )-3H - imidazo [ 4,5- b ] pyridine (0.20 mg, 0.39 mmol, 32% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.42 (dd, J = 4.8, 1.5 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 8.11 (dd, J = 8.1, 1.5 Hz, 1H), 7.73 (dd, J = 8.6, 2.4 Hz, 1H), 7.36 (dd, J = 8.1, 4.8 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 2.0 Hz, 1H), 5.41 (s, 2H), 4.72 (d, J = 7.9 Hz, 1H), 4.28 (dq, J = 7.9, 6.3 Hz, 1H), 3.86 (s, 3H), 3.69 (s, 3H), 1.02 (d , J = 6.3 Hz, 3H) ppm; (M+1) = 497.
實例 6-2： 3-(((2 S,3 S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2- d 的製備 Example 6-2 : 3-(((2 S ,3 S )-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzene Preparation of [ b ][1,4] dioxin -6- yl ) methyl ) -3H- imidazo [4,5- b ] pyridine -2- d
向2-溴-3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（0.15 mg，0.30 mmol）在2-丙醇- d8（5 mL）中的攪拌溶液（注意：在20 mL烘箱乾燥的微波反應容器中進行反應）中添加三(二亞苄基丙酮)二鈀(0)（0.054 g，0.059 mmol）、2-二環己基膦-2',6'-二甲氧基聯苯（0.074 g，0.18 mmol）和碳酸鉀（82.52 mg，591.13 µmol）。將容器密封，並且將內容物在真空/用N 2回填下脫氣（x 3）。將混合物在加熱塊中加熱至100ºC。1 h後，LC/MS分析顯示反應完成。將混合物冷卻至室溫並且濃縮以提供棕色油狀物。層析純化（CombiFlash，40 g SiO 2gold柱，20%-70% 3 : 1乙酸乙酯 : 乙醇/庚烷溶析）得到呈黃色固體的 3-(((2 S,3 S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2- d （0.088 g，0.21 mmol，71%產率）： 1H NMR (400 MHz, DMSO- d6) δ 8.40 (dd, J= 4.8, 1.5 Hz, 1H), 8.22 (d, J= 2.4 Hz, 1H), 8.10 (dd, J= 8.0, 1.5 Hz, 1H), 7.73 (dd, J= 8.7, 2.4 Hz, 1H), 7.30 (dd, J= 8.0, 4.8 Hz, 1H), 6.88 (d, J= 8.7 Hz, 1H), 6.76 (d, J= 1.9 Hz, 1H), 6.49 (d, J= 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.28 (dq, J= 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d, J= 6.3 Hz, 3H) ppm; (M+1) = 420。 To 2-bromo-3-(((2 S ,3 S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzene [ b ][1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine (0.15 mg, 0.30 mmol) in 2-propanol- d8 (5 mL) in a stirred solution (Note: the reaction was performed in a 20 mL oven-dried microwave reaction vessel) was added tris(dibenzylideneacetone)dipalladium(0) (0.054 g, 0.059 mmol), 2-dicyclohexyl Phosphine-2',6'-dimethoxybiphenyl (0.074 g, 0.18 mmol) and potassium carbonate (82.52 mg, 591.13 µmol). The vessel was sealed and the contents were degassed (x 3) under vacuum/backfill with N2 . Heat the mixture to 100 ºC in a heating block. After 1 h, LC/MS analysis showed the reaction was complete. The mixture was cooled to room temperature and concentrated to afford a brown oil. Chromatographic purification (CombiFlash, 40 g SiO 2 gold column, 20%-70% 3:1 ethyl acetate:ethanol/heptane elution) afforded 3-((( 2S , 3S )-8 as a yellow solid -Methoxy- 2- (6 -methoxypyridin- 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methoxy base )-3 H - imidazo [4,5- b ] pyridine -2- d (0.088 g, 0.21 mmol, 71% yield): 1 H NMR (400 MHz, DMSO- d 6) δ 8.40 (dd, J = 4.8, 1.5 Hz, 1H), 8.22 (d, J = 2.4 Hz, 1H), 8.10 (dd, J = 8.0, 1.5 Hz, 1H), 7.73 (dd, J = 8.7, 2.4 Hz, 1H), 7.30 (dd, J = 8.0, 4.8 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 6.76 (d, J = 1.9 Hz, 1H), 6.49 (d, J = 1.9 Hz, 1H), 5.39 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.28 (dq, J = 7.8, 6.3 Hz, 1H), 3.86 (s, 3H), 3.70 (s, 3H), 1.04 (d , J = 6.3 Hz, 3H) ppm; (M+1) = 420.
實例example 77 ：: 3-(((2 S,3 S)-8- 3-(((2 S ,3 S )-8- 甲氧基Methoxy -2-(6--2-(6- 甲氧基吡啶Methoxypyridine -3--3- 基base )-3-)-3- 甲基methyl -2,3--2,3- 二氫苯並Dihydrobenzo [ b][1,4] [ b ][1,4] 二噁英Dioxins -6--6- 基base )) 甲基methyl )-3 H- )-3 H- 咪唑並imidazo [4,5- b] [4,5- b ] 吡啶pyridine -2- d -2- d （化合物(compound 66 ）的合成)Synthesis 方法method BB ：:
將3-(((2 S,3 S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[ b][1,4]二噁英-6-基)甲基)-3 H-咪唑並[4,5- b]吡啶（8.00 g，19.12 mmol）在加熱下溶解於2-甲基四氫呋喃（220 mL）中。將溶液蒸餾以去除20 mL的溶劑以使混合物乾燥。冷卻至室溫後，將混合物用三級丁醇鉀（3.2 g，28.7 mmol）和甲醇- d1（24.00 ml，646 mmol）處理。將溶液加熱至58ºC-61ºC。4 h後，將溶液冷卻至室溫並且用10% w/w氯化銨水溶液（150 mL）洗滌。將有機層用鹽水洗滌兩次，經硫酸鎂乾燥，過濾並且濃縮以提供7.60 g固體。將此材料通過加熱至60ºC溶解於2-甲基四氫呋喃（76 mL）中。將溶液接種並且在45ºC下攪拌1 h。將混合物在室溫下攪拌1 h，並且然後在0ºC-5ºC下攪拌1 h。將所得的固體過濾，用少量2-甲基四氫呋喃洗滌並且在真空下乾燥以得到呈灰白色結晶固體的 3-(((2 S,3 S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [ b][1,4] 二噁英 -6- 基 ) 甲基 )-3 H- 咪唑並 [4,5- b] 吡啶 -2- d （6.3 g，79%產率，LCMS：94.5% D，1H NMR：94% D）。 3-((( 2S , 3S )-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[ b ] [1,4]dioxin-6-yl)methyl) -3H -imidazo[4,5- b ]pyridine (8.00 g, 19.12 mmol) was dissolved in 2-methyltetrahydrofuran (220 mL )middle. The solution was distilled to remove 20 mL of solvent to dry the mixture. After cooling to room temperature, the mixture was treated with potassium tert-butoxide (3.2 g, 28.7 mmol) and methanol- d 1 (24.00 mL, 646 mmol). Heat the solution to 58ºC-61ºC. After 4 h, the solution was cooled to room temperature and washed with 10% w/w aqueous ammonium chloride (150 mL). The organic layer was washed twice with brine, dried over magnesium sulfate, filtered and concentrated to afford 7.60 g of solid. This material was dissolved in 2-methyltetrahydrofuran (76 mL) by heating to 60°C. The solution was inoculated and stirred at 45 ºC for 1 h. The mixture was stirred at room temperature for 1 h, and then at 0°C-5°C for 1 h. The resulting solid was filtered, washed with a small amount of 2-methyltetrahydrofuran and dried under vacuum to give 3-((( 2S , 3S )-8- methoxy- 2-(6- methanol ) as an off-white crystalline solid Oxypyridin - 3 -yl )-3 -methyl -2,3 -dihydrobenzo [ b ][1,4] dioxin -6- yl ) methyl ) -3H- imidazo [4, 5- b ] pyridine -2- d (6.3 g, 79% yield, LCMS: 94.5% D, 1H NMR: 94% D).
實例example 88 ：: 3-(((2 S,3 S)-8- 3-(((2 S ,3 S )-8- 甲氧基Methoxy -2-(6--2-(6- 甲氧基吡啶Methoxypyridine -3--3- 基base )-3-)-3- 甲基methyl -2,3--2,3- 二氫苯並Dihydrobenzo [ b][1,4] [ b ][1,4] 二噁英Dioxins -6--6- 基base )) 甲基methyl )-3 H- )-3 H- 咪唑並imidazo [4,5- b] [4,5- b ] 吡啶pyridine -2- d -2- d （化合物(compound 66 ）的大規模合成) for the large-scale synthesis of
將50 g的3-(((2S,3S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[b][1,4]二噁英-6-基)甲基)-3H-咪唑並[4,5-b]吡啶裝入500 mL的甲苯中。將所得的漿料加熱至120ºC-130ºC並且回流2-3 h，用Dean stark去除水。去除大部分溶劑後，將混合物在低於60ºC下用庚烷（2 × 250 mL）追加至3.0至4.0體積。將所得的漿料在低於60ºC下用2-甲基THF（2 × 250 mL）溶劑交換至3.0至4.0體積。在裝入800 mL（16體積）的2-甲基THF後，將混合物溫熱至60ºC至65ºC並且在60ºC-65ºC下裝入50 mL的MeOD和67 mL（1.0當量）的在THF中的20%三級丁醇鉀溶液。將反應混合物在60ºC-65ºC下維持3 h。將反應冷卻至20ºC-30ºC，並且用1000 mL（20體積）的10%氯化銨水溶液淬滅。將有機層用乙酸乙酯稀釋，並且用水（3 × 250 mL）和25%鹽水溶液（250 mL）洗滌。將有機層在真空下在60ºC下蒸餾至3.0至4.0體積。將混合物在低於60ºC下用甲苯（2 × 250 mL）追加至3.0至4.0體積，在低於60ºC下用庚烷（2 × 250 mL）追加至3.0至4.0體積。將所得的漿料在低於60ºC下用2-甲基THF（2 × 250 mL）溶劑交換至3.0至4.0體積。向混合物裝入1050 mL（21體積）的2-甲基THF並且將反應物溫熱至高達60ºC-65ºC以獲得澄清溶液。通過1H NMR，獲得的淺淡黃色澄清溶液含有80%-85% D化合物6。在60ºC-65ºC下向溶液裝入100 mL的MeOD和13.4 mL（0.2當量）的在THF中的20%三級丁醇鉀溶液。將反應在60ºC-65ºC下維持3 h。將反應冷卻至20ºC-30ºC，並且用500 mL（10體積）的10%氯化銨溶液淬滅。將有機層用水（3 × 250 mL）進一步洗滌。將有機層在真空下在60ºC下蒸餾至7.5至8.0體積。將所得的漿料在65ºC-70ºC下回流以得到澄清溶液。將混合物經20 min的時間段冷卻至60ºC-65ºC並且接種
3-(((2
S,3
S)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -2-
d （0.025 g）。將混合物經2-3 h的時間段緩慢冷卻至25ºC至30ºC並且經2-3 h的時間段進一步冷卻至0ºC-5ºC並且攪拌1-2 h。將固體過濾並且用50 mL（1.0體積）的預冷的2-甲基THF洗滌。將濕材料（39.5 g）在45ºC-50ºC下在高真空下放置16 h以獲得
3-(((2
S,3
S)-8-
甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [
b][1,4]
二噁英 -6- 基 ) 甲基 )-3
H-
咪唑並 [4,5-
b]
吡啶 -2-
d （39.2 g；78%產率；通過LCMS 95% D）。
50 g of 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b ][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine was charged into 500 mL of toluene. The resulting slurry was heated to 120ºC-130ºC and refluxed for 2-3 h, using a Dean stark to remove water. After most of the solvent was removed, the mixture was chased to 3.0 to 4.0 volumes with heptane (2 x 250 mL) below 60 ºC. The resulting slurry was solvent exchanged with 2-methyl THF (2 x 250 mL) to 3.0 to 4.0 volumes below 60 ºC. After charging 800 mL (16 volumes) of 2-methyl THF, the mixture was warmed to 60ºC to 65ºC and charged with 50 mL of MeOD and 67 mL (1.0 equiv) of 20 in THF at 60ºC-65ºC. % potassium tertiary butoxide solution. The reaction mixture was maintained at 60ºC-65ºC for 3 h. The reaction was cooled to 20°C-30°C and quenched with 1000 mL (20 vol) of 10% aqueous ammonium chloride. The organic layer was diluted with ethyl acetate and washed with water (3 x 250 mL) and 25% brine solution (250 mL). The organic layer was distilled under vacuum at 60°C to 3.0 to 4.0 volumes. The mixture was chased to 3.0 to 4.0 volumes with toluene (2 x 250 mL) below 60ºC and to 3.0 to 4.0 volumes with heptane (2 x 250 mL) below 60ºC. The resulting slurry was solvent exchanged with 2-methyl THF (2 x 250 mL) to 3.0 to 4.0 volumes below 60 ºC. The mixture was charged with 1050 mL (21 vol) of 2-methyl THF and the reaction was warmed up to 60ºC-65ºC to obtain a clear solution. The obtained pale yellow clear solution contained 80%-85
實例example
99
：:
另外的對化合物additional pairs of
實例 9-1 ： 2-(5-((3H- 咪唑 並 [4,5-b] 吡啶 -3- 基 ) 甲基 )-2-( 苄基氧基 )-3- 甲氧基苯氧基 )-1-(6- 甲氧基吡啶 -3- 基 ) 丙 -1- 酮的製備 
將2-溴-1-(6-甲氧基吡啶-3-基)丙-1-酮（21.2 g，87 mmol，1當量，CAS 1391089-35-2）、5-((3H-咪唑並[4,5-b]吡啶-3-基)甲基)-2-(苄基氧基)-3-甲氧基苯酚（32.9 g，91.3 mmol，1.05當量）（WO 2017015267實例1-193）和碳酸鉀（30 g，218 mmol，2.5當量）在乙腈（330 mL）中的混合物在室溫下攪拌4 h。HPLC分析顯示2-溴-1-(6-甲氧基吡啶-3-基)丙-1-酮的完全消耗。將甲基三級丁基醚（330 mL）添加到漿料中並且將混合物過濾並且將固體用甲基三級丁基醚洗滌。將濾液用稀釋的氫氧化鈉溶液（350 mL）和飽和氯化鈉溶液（300 mL）洗滌。將溶劑用甲醇交換。將甲醇溶液在室溫下與種晶（20 mg）一起攪拌。在室溫下攪拌16 h後，將結晶的產物通過過濾分離，用甲醇洗滌並且乾燥以得到呈灰白色結晶固體的 2-(5-((3H- 咪唑 並 [4,5-b] 吡啶 -3- 基 ) 甲基 )-2-( 苄基氧基 )-3- 甲氧基苯氧基 )-1-(6- 甲氧基吡啶 -3- 基 ) 丙 -1- 酮（35.9 g，85%產率），熔點72ºC； 1H NMR (400 MHz, CDCl 3) δ 8.83 (dd, J= 2.4, 0.7 Hz, 1H), 8.39 (dd, J= 4.8, 1.4 Hz, 1H), 8.13 – 8.03 (m, 2H), 7.96 (s, 1H), 7.48 – 7.41 (m, 2H), 7.36 – 7.21 (m, 4H), 6.69 (dd, J= 8.8, 0.8 Hz, 1H), 6.55 (d, J= 1.9 Hz, 1H), 6.44 (d, J= 2.0 Hz, 1H), 5.32 (s, 2H), 5.29 (q, J= 6.8, 1H), 4.99 (s, 2H), 3.98 (s, 3H), 3.74 (s, 3H), 1.60 (d, J= 6.8 Hz, 3H)ppm; (M+1) = 525。 2-Bromo-1-(6-methoxypyridin-3-yl)propan-1-one (21.2 g, 87 mmol, 1 equivalent, CAS 1391089-35-2), 5-((3H-imidazo [4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenol (32.9 g, 91.3 mmol, 1.05 equiv) (WO 2017015267 Example 1-193) and potassium carbonate (30 g, 218 mmol, 2.5 equiv) in acetonitrile (330 mL) was stirred at room temperature for 4 h. HPLC analysis showed complete consumption of 2-bromo-1-(6-methoxypyridin-3-yl)propan-1-one. Methyl tert-butyl ether (330 mL) was added to the slurry and the mixture was filtered and the solids were washed with methyl tert-butyl ether. The filtrate was washed with dilute sodium hydroxide solution (350 mL) and saturated sodium chloride solution (300 mL). The solvent was exchanged with methanol. The methanol solution was stirred with seed crystals (20 mg) at room temperature. After stirring at room temperature for 16 h, the crystallized product was isolated by filtration, washed with methanol and dried to give 2-(5-((3H- imidazo [4,5-b] pyridine -3 as an off-white crystalline solid -yl ) methyl )-2-( benzyloxy )-3 - methoxyphenoxy )-1-(6 -methoxypyridin- 3 -yl ) propan- 1 -one (35.9 g, 85 % yield), melting point 72ºC; 1 H NMR (400 MHz, CDCl 3 ) δ 8.83 (dd, J = 2.4, 0.7 Hz, 1H), 8.39 (dd, J = 4.8, 1.4 Hz, 1H), 8.13 – 8.03 (m, 2H), 7.96 (s, 1H), 7.48 – 7.41 (m, 2H), 7.36 – 7.21 (m, 4H), 6.69 (dd, J = 8.8, 0.8 Hz, 1H), 6.55 (d, J = 1.9 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H), 5.29 (q, J= 6.8, 1H), 4.99 (s, 2H), 3.98 (s, 3H) , 3.74 (s, 3H), 1.60 (d, J = 6.8 Hz, 3H)ppm; (M+1) = 525.
實例 9-2：
4-((3H- 咪唑 並 [4,5-b] 吡啶 -3- 基 ) 甲基 )-2-(((1S,2S)-1- 羥基 -1-(6- 甲氧基吡啶 -3- 基 ) 丙 -2- 基 ) 氧基 )-6- 甲氧基苯酚的製備 
將2-(5-((3H-咪唑並[4,5-b]吡啶-3-基)甲基)-2-(苄基氧基)-3-甲氧基苯氧基)-1-(6-甲氧基吡啶-3-基)丙-1-酮（14 g，25.2 mmol）、三級丁醇鉀（1.35 g，12.01 mmol，0.48當量）和RuCl 2[(S)-(DM-BINAP)][(S)-DAIPEN]（CAS 220114-01-2，0.33 g，0.27 mmol，0.01當量）溶解於異丙醇（230 mL）中並且裝入氫化反應器中。將反應器用氮氣吹掃並且裝入氫氣至70 psi。在22ºC下在70 psi氫氣壓力下攪拌5 h後，HPLC分析顯示起始材料完全消耗。通過將Pd/C（4.8 g，34 wt%，5%活性炭載Pd，50%濕度）裝入反應器中進行氫解。將Parr反應器用氮氣吹掃並且裝入氫氣至70 psi。在22ºC下在70 psi氫氣壓力下攪拌48 h後，HPLC分析顯示反應基本完成。將反應混合物通過矽藻土墊過濾，用異丙醇和甲醇洗滌。將濾液濃縮至澄清黃色油狀物。將油狀物溶解於乙酸乙酯（250 mL）中並且用氯化銨水溶液（130 mL）洗滌。將水層用乙酸乙酯（30 mL）萃取。將合併的有機層用飽和氯化鈉溶液洗滌，用硫酸鈉乾燥，過濾並且濃縮以獲得呈淺黃色硬泡沫的 4-((3H- 咪唑 並 [4,5-b] 吡啶 -3- 基 ) 甲基 )-2-(((1S,2S)-1- 羥基 -1-(6- 甲氧基吡啶 -3- 基 ) 丙 -2- 基 ) 氧基 )-6- 甲氧基苯酚（10.1 g，23.1 mmol，92%產率）。產物是大約84 : 16比率的1S,2S與1R,2S非對映異構物（通過 1H NMR）；＞ 98% ee（通過掌性HPLC） 1H NMR (400 MHz, CDCl 3) δ 8.43 (d, J= 4.6 Hz, 1H), 8.13–8.05 (m, 2H), 8.02 (d, J= 1.7 Hz, 1H), 7.68和7.61 (2 br d, J= 8.7Hz, 1H), 7.31–7.23 (m, 2H), 6.77–6.67 (m, 2H), 6.66 (d, J= 2.7 Hz, 1H), 5.36 (s, 2H), 4.82 和4.71 (br s和d, J= 8.3 Hz, 1H), 4.13 (m, 1H), 3.94 (br s, 3H), 3.83 (br s, 3H), 1.18–1.07 (d, J= 6.4Hz, 3H)ppm; (M+1) = 437。 2-(5-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(benzyloxy)-3-methoxyphenoxy)-1- (6-Methoxypyridin-3-yl)propan-1-one (14 g, 25.2 mmol), potassium tertiary butoxide (1.35 g, 12.01 mmol, 0.48 equivalents) and RuCl 2 [(S)-(DM -BINAP)][(S)-DAIPEN] (CAS 220114-01-2, 0.33 g, 0.27 mmol, 0.01 equiv) was dissolved in isopropanol (230 mL) and charged to the hydrogenation reactor. The reactor was purged with nitrogen and charged with hydrogen to 70 psi. After stirring for 5 h at 22 ºC under 70 psi hydrogen pressure, HPLC analysis showed complete consumption of the starting material. Hydrogenolysis was performed by charging Pd/C (4.8 g, 34 wt%, 5% Pd on activated carbon, 50% humidity) into the reactor. The Parr reactor was purged with nitrogen and charged with hydrogen to 70 psi. After stirring for 48 h at 22 ºC under 70 psi hydrogen pressure, HPLC analysis showed that the reaction was essentially complete. The reaction mixture was filtered through a pad of celite, washing with isopropanol and methanol. The filtrate was concentrated to a clear yellow oil. The oil was dissolved in ethyl acetate (250 mL) and washed with aqueous ammonium chloride (130 mL). The aqueous layer was extracted with ethyl acetate (30 mL). The combined organic layers were washed with saturated sodium chloride solution, dried over sodium sulfate, filtered and concentrated to obtain 4-((3H- imidazo [4,5-b] pyridin - 3 -yl ) as a light yellow hard foam Methyl )-2-(((1S,2S)-1 -hydroxyl- 1-(6 -methoxypyridin- 3 -yl ) prop -2- yl ) oxy )-6 -methoxyphenol (10.1 g, 23.1 mmol, 92% yield). Product is 1S,2S and 1R,2S diastereoisomers in approximately 84:16 ratio (by 1 H NMR); >98% ee (by chiral HPLC) 1 H NMR (400 MHz, CDCl 3 ) δ 8.43 (d, J = 4.6 Hz, 1H), 8.13–8.05 (m, 2H), 8.02 (d, J = 1.7 Hz, 1H), 7.68 and 7.61 (2 br d, J = 8.7Hz, 1H), 7.31– 7.23 (m, 2H), 6.77–6.67 (m, 2H), 6.66 (d, J = 2.7 Hz, 1H), 5.36 (s, 2H), 4.82 and 4.71 (br s and d, J = 8.3 Hz, 1H ), 4.13 (m, 1H), 3.94 (br s, 3H), 3.83 (br s, 3H), 1.18–1.07 (d, J = 6.4Hz, 3H)ppm; (M+1) = 437.
實例 9-3：
3-(((2S,3S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [b][1,4] 二噁英 -6- 基 ) 甲基 )-3H- 咪唑並 [4,5-b] 吡啶的製備 
將4-((3H-咪唑並[4,5-b]吡啶-3-基)甲基)-2-(((1S,2S)-1-羥基-1-(6-甲氧基吡啶-3-基)丙-2-基)氧基)-6-甲氧基苯酚（10.10 g；23.14 mmol；非對映異構物的84 : 16混合物；1.00當量）在乙酸乙酯（90 mL）中的溶液與二異丙基乙基胺（16.02 ml；92.56 mmol；4.00當量）和CCl 4（5.58 ml；57.85 mmol；2.50當量）在45ºC-50ºC下攪拌。經10 min逐滴添加三正丁基膦（11.99 ml；48.60 mmol；2.10當量），伴有輕微放熱。將所得的棕色溶液在45ºC-50ºC下攪拌1.5 h。向反應中添加氫氧化鈉溶液（15 wt%，40 mL，6.5當量）並且將混合物在45ºC下攪拌0.5-1 h。將反應冷卻至室溫。將各層分離。將水層用乙酸乙酯（40 mL）萃取。將合併的有機層用飽和氯化鈉溶液（50 mL）洗滌，經硫酸鈉乾燥，過濾並且濃縮至濕固體。將固體在甲基三級丁基醚（60 mL）中攪拌2 h，過濾並且在真空下乾燥。將灰白色固體在升高的溫度下溶解於乙醇（55 mL）中。將溶液在室溫下與種晶一起攪拌並且冷卻至0ºC-5ºC。將所得的固體過濾並且乾燥以得到呈灰白色固體粉末的 3-(((2S,3S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [b][1,4] 二噁英 -6- 基 ) 甲基 )-3H- 咪唑並 [4,5-b] 吡啶（6.16 g，63.6%）99 A%（通過HPLC），98% ee，Pd：1 ppm；Ru：225 ppm；形式A。熔點164.9ºC。 1H NMR (400 MHz, CDCl3) δ 8.45 (dd, J= 4.8, 1.5 Hz, 1H), 8.16 (d, J= 2.4 Hz, 1H), 8.13–8.03 (m, 2H), 7.56 (dd, J= 8.6, 2.5 Hz, 1H), 7.27 (dd, J = 8.0, 4.8 Hz, 1H), 6.78 (d, J= 8.6 Hz, 1H), 6.53 (br s, 2H), 5.38 (s, 2H), 4.62 (d, J= 7.8 Hz, 1H), 4.13 (m, 1H) 3.94 (s, 3H), 3.79 (s, 3H), 1.17 (d, J= 6.4 Hz, 3H)ppm。(M+1) = 419。 4-((3H-imidazo[4,5-b]pyridin-3-yl)methyl)-2-(((1S,2S)-1-hydroxyl-1-(6-methoxypyridine- 3-yl)propan-2-yl)oxy)-6-methoxyphenol (10.10 g; 23.14 mmol; 84:16 mixture of diastereomers; 1.00 equiv) in ethyl acetate (90 mL) The solution in ® was stirred with diisopropylethylamine (16.02 ml; 92.56 mmol; 4.00 equiv) and CCl 4 (5.58 ml; 57.85 mmol; 2.50 equiv) at 45°C-50°C. Tri-n-butylphosphine (11.99 ml; 48.60 mmol; 2.10 equiv) was added dropwise over 10 min with a slight exotherm. The resulting brown solution was stirred at 45ºC-50ºC for 1.5 h. Sodium hydroxide solution (15 wt%, 40 mL, 6.5 equiv) was added to the reaction and the mixture was stirred at 45°C for 0.5-1 h. The reaction was cooled to room temperature. The layers were separated. The aqueous layer was extracted with ethyl acetate (40 mL). The combined organic layers were washed with saturated sodium chloride solution (50 mL), dried over sodium sulfate, filtered and concentrated to a wet solid. The solid was stirred in methyl tert-butyl ether (60 mL) for 2 h, filtered and dried under vacuum. The off-white solid was dissolved in ethanol (55 mL) at elevated temperature. The solution was stirred with the seed crystals at room temperature and cooled to 0ºC-5ºC. The resulting solid was filtered and dried to give 3-(((2S,3S)-8- methoxy- 2-(6 -methoxypyridin- 3 -yl )-3 - methyl- 2,3 -Dihydrobenzo [b][1,4] dioxin -6- yl ) methyl )-3H- imidazo [4,5-b] pyridine (6.16 g, 63.6%) 99 A% (by HPLC), 98% ee, Pd: 1 ppm; Ru: 225 ppm; Form A. Melting point 164.9ºC. 1 H NMR (400 MHz, CDCl3) δ 8.45 (dd, J = 4.8, 1.5 Hz, 1H), 8.16 (d, J = 2.4 Hz, 1H), 8.13–8.03 (m, 2H), 7.56 (dd, J = 8.6, 2.5 Hz, 1H), 7.27 (dd, J = 8.0, 4.8 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.53 (br s, 2H), 5.38 (s, 2H), 4.62 (d, J = 7.8 Hz, 1H), 4.13 (m, 1H) 3.94 (s, 3H), 3.79 (s, 3H), 1.17 (d, J = 6.4 Hz, 3H) ppm. (M+1) = 419.
實例 9-4：
3-(((2S,3S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [b][1,4] 二噁英 -6- 基 ) 甲基 )-3H- 咪唑並 [4,5-b] 吡啶 -2-
d 的製備 
在60ºC下將3-(((2S,3S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[b][1,4]二噁英-6-基)甲基)-3H-咪唑並[4,5-b]吡啶（20 g；47.80 mmol）溶解於2-甲基四氫呋喃（400 mL）中。添加甲醇-d（20 mL；1 V，99% D），然後添加固體三級丁醇鉀（5.36 g；47.80 mmol；1當量）。將溶液加熱2 h。LCMS顯示87% D。將反應溶液冷卻至22ºC。將懸浮液用10% w/w氯化銨水溶液（400 mL）洗滌。將有機層分離，用乙酸乙酯（200 mL）稀釋並且用水（3 x 100 mL）洗滌，然後用1/2飽和氯化鈉溶液（100 mL）洗滌。將有機層乾燥（硫酸鈉），過濾並且濃縮為固體。將固體通過與甲苯（2 x 100 mL）共沸乾燥。在60ºC下將所得的棕褐色固體溶解於2-甲基四氫呋喃（500 mL）中並且添加甲醇-d（40 mL；2V），然後添加固體三級丁醇鉀（1.1 g；9.80 mmol；0.2當量）。將溶液在60ºC下加熱3 h。3 h後LCMS顯示96%-97% D。將反應溶液冷卻至室溫，用10% w/w氯化銨水溶液（200 mL；10 V）洗滌。將有機層分離並且用水洗滌3次（每次200 mL）。將有機溶液過濾，濃縮並且與甲苯共沸乾燥。在80ºC下將固體溶解於2-甲基四氫呋喃（560 mL）中。將反應溶液冷卻至75ºC，用形式A（200 mg）接種。將混合物攪拌同時將溫度冷卻至22ºC並且保持1 h。將混合物在0ºC-5ºC下攪拌1 h。將所得的固體過濾，用冷2-甲基四氫呋喃洗滌並且在真空烘箱中乾燥以獲得呈灰白色粉末的 3-(((2S,3S)-8- 甲氧基 -2-(6- 甲氧基吡啶 -3- 基 )-3- 甲基 -2,3- 二氫苯並 [b][1,4] 二噁英 -6- 基 ) 甲基 )-3H- 咪唑並 [4,5-b] 吡啶 -2-d 形式 A（100 A%（通過HPLC），掌性純度：99.5%；96.1% D（通過LCMS）；Pd 1 ppm；Ru 20 ppm，84%產率） 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 剩餘未氘化的, 0.02H), 8.40 (dd, J= 4.8, 1.5 Hz, 1H), 8.22 (d, J= 2.3 Hz, 1H), 8.10 (dd, J= 8.0,1.5, 1H), 7.72 (dd, J= 8.6, 2.4 Hz, 1H), 7.36 (dd, J= 8.0, 4.8, 1H), 6.88 (d, J= 8.6 Hz, 1H), 6.76 (d, J= 1.9, 1H), 6.50 (d, J= 1.9, 1H), 5.40 (s, 2H), 4.72 (d, J= 7.8 Hz, 1H), 4.26-4.29 (m, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 1.03 (d, J= 6.3 Hz, 3H)ppm。(M+1) = 420 3-(((2S,3S)-8-methoxy-2-(6-methoxypyridin-3-yl)-3-methyl-2,3-dihydrobenzo[b ][1,4]dioxin-6-yl)methyl)-3H-imidazo[4,5-b]pyridine (20 g; 47.80 mmol) was dissolved in 2-methyltetrahydrofuran (400 mL). Methanol-d (20 mL; 1 V, 99% D) was added, followed by solid potassium tert-butoxide (5.36 g; 47.80 mmol; 1 equiv). The solution was heated for 2 h. LCMS showed 87% D. The reaction solution was cooled to 22ºC. The suspension was washed with 10% w/w aqueous ammonium chloride (400 mL). The organic layer was separated, diluted with ethyl acetate (200 mL) and washed with water (3 x 100 mL), then with 1/2 saturated sodium chloride solution (100 mL). The organic layer was dried (sodium sulfate), filtered and concentrated to a solid. The solid was dried by azeotroping with toluene (2 x 100 mL). The resulting tan solid was dissolved in 2-methyltetrahydrofuran (500 mL) at 60°C and methanol-d (40 mL; 2V) was added followed by solid potassium ter-butoxide (1.1 g; 9.80 mmol; 0.2 eq. ). The solution was heated at 60 ºC for 3 h. LCMS showed 96%-97% D after 3 h. The reaction solution was cooled to room temperature, washed with 10% w/w aqueous ammonium chloride (200 mL; 10 V). The organic layer was separated and washed 3 times with water (200 mL each). The organic solution was filtered, concentrated and dried azeotropically with toluene. The solid was dissolved in 2-methyltetrahydrofuran (560 mL) at 80 ºC. The reaction solution was cooled to 75ºC and inoculated with Form A (200 mg). The mixture was stirred while cooling the temperature to 22 ºC for 1 h. The mixture was stirred at 0ºC-5ºC for 1 h. The resulting solid was filtered, washed with cold 2-methyltetrahydrofuran and dried in a vacuum oven to obtain 3-(((2S,3S)-8- methoxy- 2-(6 -methoxy Pyridin - 3 -yl )-3 -methyl -2,3 -dihydrobenzo [b][1,4] dioxin -6- yl ) methyl )-3H- imidazo [4,5-b ] Pyridine -2-d Form A (100 A% (by HPLC), chiral purity: 99.5%; 96.1% D (by LCMS); Pd 1 ppm; Ru 20 ppm, 84% yield) 1 H NMR (400 MHz, DMSO-d6) δ 8.57 (s, remaining undeuterated, 0.02H), 8.40 (dd, J = 4.8, 1.5 Hz, 1H), 8.22 (d, J = 2.3 Hz, 1H), 8.10 (dd , J = 8.0,1.5, 1H), 7.72 (dd, J = 8.6, 2.4 Hz, 1H), 7.36 (dd, J = 8.0, 4.8, 1H), 6.88 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 1.9, 1H), 6.50 (d, J = 1.9, 1H), 5.40 (s, 2H), 4.72 (d, J = 7.8 Hz, 1H), 4.26-4.29 (m, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H) ppm. (M+1) = 420
將3-(((2S,3S)-8-甲氧基-2-(6-甲氧基吡啶-3-基)-3-甲基-2,3-二氫苯並[b][1,4]二噁英-6-基)甲基)-3H-咪唑並[4,5-b]吡啶-2-d形式A通過XRPD、PLM、DSC、TGA和HPLC進一步表徵。結果總結於表2-1中，指示起始材料是結晶無水合物。
分析方法：Analytical method:
Xx 射線粉末衍射（X-ray powder diffraction ( XRPDXRPD ）)
儀器：Panalytical Empyrean粉末衍射儀Instrument: Panalytical Empyrean powder diffractometer
參數：X射線管Cu（Kα）；管電壓45 kV；管電流40 mAParameters: X-ray tube Cu (Kα); tube voltage 45 kV; tube current 40 mA
從2至40度2-θ掃描；0.013度/步；掃描速率6度/min2-theta scan from 2 to 40 degrees; 0.013 degrees/step; scan
熱重分析Thermogravimetry （( TGATGA ）)
儀器：TA Instruments Discovery TGA Q5500Instrument: TA Instruments Discovery TGA Q5500
參數：斜升10ºC/分鐘，環境溫度至250ºC/300ºC，50 mL/min N 2沖掃 Parameters: ramp up 10ºC/min, ambient temperature to 250ºC/300ºC, 50 mL/min N2 flush
差示掃描量熱Differential Scanning Calorimetry 法（Law( DSCDSC ）)
儀器：TA Instruments Discovery DSCInstrument: TA Instruments Discovery DSC
參數：斜升10ºC/分鐘，從環境溫度至250ºC/300ºC，50 mL/min N 2沖掃 Parameters: Ramp 10ºC/min from ambient to 250ºC/300ºC, 50 mL/min N2 purge
偏振光顯微術（polarized light microscopy ( PLMPLM ）)
儀器：Nikon Eclipse Ci PolInstrument: Nikon Eclipse Ci Pol
相機：NikonCamera: Nikon
軟體：NIS-Elements圖像軟體Software: NIS-Elements image software
將樣品以漿料形式分散在顯微鏡載玻片上，或者如果是乾燥的話，將樣品用矽油分散，並且在透射的偏振光下檢查。 磷酸化 cFMS 活性 Samples were dispersed as slurries on microscope slides, or in silicone oil if dry, and examined under transmitted polarized light. Phosphorylated cFMS activity
試劑和耗材購自Sigma Aldrich、Gibco LifeTechnologies、BD Biosciences、Perkin Elmer、R&D Systems、Cell Signaling、Thermo Scientific（Pierce）和Santa Cruz Biotechnology。將過表達人cFMS的HEK293細胞（HEK293/hFMS）在T225燒瓶中的RPMI培養基中培養，並且每週分割兩次。對於實驗，將細胞胰蛋白酶化，計數並且用無血清Megacell培養基（Sigma目錄號M3817）稀釋至600,000個細胞/ml（30,000個細胞/孔）。通過Echo 555（LABCYTE）使用Echo LDV板（目錄號LP-0200）製備連續稀釋的測試化合物；並且將500 nl的每種化合物濃度添加到96孔BD Biocoat聚-d-賴氨酸板（BD目錄號356640）中的DMSO中（0.5%最終）。然後添加50 μL/孔MegaCell無血清培養基以覆蓋化合物，然後以50 μL/孔細胞（30,000/孔）添加細胞。將板在1000 rpm下減速旋轉1分鐘，並且然後在臺上培育15-30分鐘；將板移至在37ºC下的CO
2培養箱中過夜培育。將白色96孔Perkin Elmer OptiPlates（目錄號6005509）用50 ng/孔（100 μL/孔）在PBS中的抗cFMS/CSF-1R（C-20）（Santa Cruz目錄號sc-692）預包被，用箔密封物密封，在1000 rpm下減速旋轉一分鐘並且在4ºC下培育過夜。
在第二天，在室溫下將預包被的OptiPlates板用200 ul/孔的在1x PBST中的1% BSA（具有0.1% Triton-X的PBS）封閉2-3小時。平行地，將100 μL/孔的2x hCSF1（最終150 ng/ml）（R&D Systems，目錄號216-MC-025/CF）（或培養基，作為陰性對照）添加到與化合物一起培育過夜的HEK293/hFMS細胞（BD培養板）中。在每個板上，將100%反應（用CSF1處理）和0%反應（沒有CSF1）對照柱用於計算測試化合物的抑制百分比和Z`prime值。將板在37ºC下培育10分鐘。將培養基/hCSF1吸去，並且將細胞用100 ul/孔的預冷的裂解緩衝液裂解，所述裂解緩衝液由裂解緩衝液（Cell Signaling目錄號9803）、蛋白酶/磷酸酶抑制劑（Pierce目錄號78444）和PMSF（Sigma目錄號93482）製成。將板振搖60秒；然後，在4ºC下在3200 rpm下旋轉5分鐘，並且保持在冰上。將90 ul的裂解物轉移至預包被/封閉的OptiPlates中。然後將板在1000 rpm下旋轉60秒並且在4ºC密封下培育過夜。
第二天，將裂解物從板中取出；並且將板使用Biotek洗滌機用300 μL/孔的1xPBS洗滌6次。將板上剩餘的PBS拍出。將90 μL/孔的在PBST中1% BSA中的1 : 10,000抗磷酸化Eu（Tyr 100）（Perkin Elmer目錄號AD0159）添加到板中；並且將板在室溫密封下培育一小時。一小時後，去除抗體，並且將板使用Biotek洗滌機用300 μL/孔的PBST洗滌6次。接下來添加90 μL/孔的增強溶液（Perkin Elmer目錄號4001-0010），並且將板密封並且振搖5分鐘。在Perkin Elmer Envision上立即讀取信號，以得到在320 nm處激發和615 nm處發射的時間分辨螢光。
將資料通過Pipeline Pilot分析以計算IC
50值；對於選定的CSF-1R抑制劑，磷光體c-FMS的IC
50值提供在下表B中。
表 B 
為了比較在CSF-1刺激後，CSF-1R抑制性化合物和本揭露的氘化CSF-1R抑制性化合物對細胞介素/趨化介素產生的影響，在BV2鼠小神經膠質細胞中進行以下實驗。In order to compare the effects of CSF-1R inhibitory compounds and deuterated CSF-1R inhibitory compounds of the present disclosure on the production of cytokines/chemokines following CSF-1 stimulation, the following was performed in BV2 murine microglial cells experiment.
將兩種不同代次的BV2小鼠小神經膠質細胞在不同的96孔板中鋪板以提供生物學四重複。
化合物
24根據WO 2017/015267的實例1-92中概述的程序製備。
將兩種測試化合物在稀釋的儲備溶液（10 mM）中用培養基製備以得到100 µM工作溶液，並且在3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM、200 nM或400 nM下處理。 方法 BV2 小神經膠質細胞的處理和刺激 Two test compounds were prepared in culture medium in diluted stock solutions (10 mM) to give 100 µM working solutions and were prepared at 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM or 400 nM Next process. Methods BV2 Microglia Treatment and Stimulation
將BV2小鼠小神經膠質細胞以5 x 10
5個/mL的濃度懸浮，並且將100 µL的此細胞懸浮液添加到96孔板的每個孔中。允許在37ºC、5% CO
2下將小神經膠質細胞靜置過夜。第二天，去除培養基並且在37ºC、5% CO
2下將細胞用二甲基亞碸（DMSO）、化合物
24RA10947016或化合物
6RA16100017處理30分鐘。然後將細胞用100 ng/mL重組小鼠M-CSF刺激24小時。刺激後，將培養上清液從每個孔中去除，並且等分至兩種不同的96孔板用於後續ELISA測定。
小鼠 MCP-1 ELISA BV2 mouse microglial cells were suspended at a concentration of 5 x 105 cells/mL, and 100 µL of this cell suspension was added to each well of a 96-well plate. Allow the microglia to rest overnight at 37 ºC, 5% CO . The next day, the medium was removed and the cells were treated with dimethylsulfoxide (DMSO),
將培養上清液用來自R&D Systems的Quantikine小鼠MCP-1 ELISA套組測定。將樣品用校準品稀釋液1 : 10稀釋。首先將50微升的測定稀釋液添加到每個孔中。然後將50 µL的標準品、測定對照和稀釋的樣品添加到孔中。將板通過輕輕敲擊框架混合，並且用膠帶密封。將板在室溫下培育2小時。培育後，將板使用噴射瓶用大約400 µL的洗滌緩衝液洗滌5次。在最後一次洗滌之後，將板在紙巾上輕輕敲擊以去除過量水分。將100 µLOne hundred microliters 的小鼠MCP-1接合物添加到每個孔中，用新的膠帶覆蓋，並且在室溫下培育2小時。培育後，如上所述將板洗滌。然後將受質溶液添加到每個孔中，並且在黑暗中在室溫下培育30分鐘。培育後，將酸性終止溶液添加到每個孔中，並且在450 nm下在ELISA板讀取器上讀取板。 結果 Culture supernatants were assayed with the Quantikine mouse MCP-1 ELISA kit from R&D Systems. Samples were diluted 1:10 with calibrator diluent. First add 50 microliters of assay dilution to each well. Then add 50 µL of standards, assay controls, and diluted samples to the wells. The plate was mixed by tapping the frame lightly and sealed with tape. Plates were incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 µL of wash buffer using a squirt bottle. After the last wash, the plates were tapped on paper towels to remove excess water. Add 100 µL of One hundred microliters of mouse MCP-1 conjugate to each well, cover with fresh tape, and incubate at room temperature for 2 hours. After incubation, plates were washed as described above. The substrate solution was then added to each well and incubated for 30 minutes at room temperature in the dark. After incubation, acidic stop solution was added to each well and the plate was read on an ELISA plate reader at 450 nm. result
將BV2鼠小神經膠質細胞以50,000個細胞/孔鋪板並且靜置過夜。將細胞用DMSO、化合物
24或化合物
6RA10947016預處理 RA1610001730分鐘，並且然後經受CSF-1刺激。將來自此實驗的細胞培養上清液在MCP1 ELISA中加工，以確定刺激/處理是否影響趨化介素產生。如
圖 1A- 圖 1B和
圖 2A- 圖 2B所示，CSF-1刺激誘導MCP-1（CCL2-趨化介素）的釋放顯著增加，並且兩種小分子CSF-1R抑制劑以濃度依賴性方式顯著降低MCP-1產生。基於未刺激的對照和刺激的對照計算抑制百分比並且生成IC
50曲線。如
圖 3A- 圖 3B 和圖 4A- 圖 4B所示，對於此測定，兩種化合物展現出在28.8 nM-36.5 nM之間的相似IC
50值。
BV2 murine microglia were plated at 50,000 cells/well and left overnight. Cells were pre-treated RA1610001730 min with DMSO,
圖形柱代表平均值和標準差。用具有多重比較的單因子變異數分析確定統計學顯著性，並且p值表示為*p ＜ 0.05，**p ＜ 0.01，***p ＜ 0.001和****p ＜ 0.0001。 實例 11 Graph bars represent mean and standard deviation. Statistical significance was determined with one-way ANOVA with multiple comparisons and p-values are expressed as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Example 11
為了比較在CSF-1刺激後，兩種CSF-1R抑制性化合物和本揭露的一種氘化CSF-1R抑制性化合物對小神經膠質細胞細胞介素/趨化介素產生的作用，在初代鼠小神經膠質細胞中進行以下實驗。
初代小鼠小神經膠質細胞 –obtained from Nellwyn Hagan (ELN# 20200702-082).
化合物 49根據WO 2017/015267的實例1-5中概述的程序製備。 Compound 49 was prepared according to the procedure outlined in Examples 1-5 of WO 2017/015267.
將所有測試化合物在稀釋的儲備溶液（10 mM）中用培養基製備以得到100 µM工作溶液，並且在3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM、200 nM或400 nM下處理。 方法 初代小神經膠質細胞的處理和刺激 All test compounds were prepared in diluted stock solutions (10 mM) in medium to give 100 µM working solutions and were prepared at 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM or 400 nM deal with. Methods Treatment and stimulation of primary microglial cells
將初代小鼠小神經膠質細胞以5 x 10
5個細胞/mL的濃度懸浮並且將100 µL的此細胞懸浮液添加到三個96孔板的內部60個孔中。允許在37ºC、5% CO
2下將小神經膠質細胞靜置過夜。第二天，去除培養基，並且在37ºC、5% CO
2下將細胞用二甲基亞碸（DMSO）、化合物
49RA03546849、PLX3397RA11275777或化合物
6RA16100017處理30分鐘。然後將細胞用100 ng/mL重組小鼠CSF-1刺激24小時。刺激後，將培養上清液從每個孔中去除，並且等分至兩種不同的96孔板用於後續ELISA測定。將板用4% PFA固定用於未來的免疫細胞化學分析。
小鼠 MCP-1 ELISA Primary mouse microglial cells were suspended at a concentration of 5 x 105 cells/mL and 100 µL of this cell suspension was added to the inner 60 wells of three 96-well plates. Allow the microglia to rest overnight at 37 ºC, 5% CO . The next day, the medium was removed and the cells were treated with dimethylsulfoxide (DMSO), compound 49 RA03546849, PLX3397RA11275777 or
將培養上清液用Quantikine小鼠MCP-1 ELISA套組（R&D Systems，目錄號SMJE00B）測定。將樣品用校準品稀釋液1 : 10稀釋。首先將50微升的測定稀釋液添加到每個孔中。然後將50微升的標準品、測定對照和稀釋的樣品添加到孔中。將板通過輕輕敲擊框架混合，並且用膠帶密封。將板在室溫下培育2小時。培育後，將板使用噴射瓶用大約400 µL的洗滌緩衝液洗滌5次。在最後一次洗滌之後，將板在紙巾上輕輕敲擊以去除過量水分。將100 µLOne hundred microliters 的小鼠MCP-1接合物添加到每個孔中，用新的膠帶覆蓋，並且在室溫下培育2小時。培育後，如上所述將板洗滌。然後將受質溶液添加到每個孔中，並且在黑暗中在室溫下培育30分鐘。培育後，將酸終止溶液添加到每個孔中，並且在450 nm下在具有SoftMax Pro軟體的FlexStation3多模式微板讀取器（Molecular Devices，目錄號Flex3）上讀取板。 初代小神經膠質細胞的免疫染色 The culture supernatant was assayed with the Quantikine mouse MCP-1 ELISA kit (R&D Systems, catalog number SMJE00B). Samples were diluted 1:10 with calibrator diluent. First add 50 microliters of assay dilution to each well. Then 50 microliters of standards, assay controls and diluted samples were added to the wells. The plate was mixed by tapping the frame lightly and sealed with tape. Plates were incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 µL of wash buffer using a squirt bottle. After the last wash, the plates were tapped on paper towels to remove excess water. Add 100 µL of One hundred microliters of mouse MCP-1 conjugate to each well, cover with fresh tape, and incubate at room temperature for 2 hours. After incubation, plates were washed as described above. The substrate solution was then added to each well and incubated for 30 minutes at room temperature in the dark. After incubation, acid stop solution was added to each well, and the plate was read at 450 nm on a FlexStation3 multimode microplate reader (Molecular Devices, cat# Flex3) with SoftMax Pro software. Immunostaining of primary microglia
在刺激後，在室溫下將細胞用4% PFA固定20分鐘。然後在室溫下將細胞在PBS中沖洗，在0.2% PBT（在PBS中的0.2% Triton X-100）中洗滌3 x 5分鐘，並且用10%驢血清/0.2% PBT封閉1小時。然後在4ºC下將細胞在10%驢血清/0.2% PBT中稀釋的一抗（兔抗Iba1，1 : 500；Wako，目錄號019-19741或兔抗Ki67，1 : 500；Abcam，目錄號ab15580）中培育過夜。第二天，將細胞在0.2% PBT中洗滌3 x 5分鐘並且在室溫下在1%驢血清/0.2% PBT中稀釋的二抗（驢抗兔Alexa Fluoro 488，1 : 500；Life Technologies，目錄號A21206）中培育1小時。然後將細胞在0.2% PBT中洗滌3 x 5分鐘，在室溫下在DAPI（在PBS中1 : 10,000）中培育5分鐘並且在PBS中沖洗。 顯微術 After stimulation, cells were fixed with 4% PFA for 20 min at room temperature. Cells were then rinsed in PBS, washed 3 x 5 minutes in 0.2% PBT (0.2% Triton X-100 in PBS) at room temperature, and blocked with 10% donkey serum/0.2% PBT for 1 hour. Cells were then incubated with primary antibody (rabbit anti-Iba1, 1:500; Wako, cat. no. 019-19741 or rabbit anti-Ki67, 1:500; Abcam, cat. no. ab15580) diluted in 10% donkey serum/0.2% PBT at 4ºC. ) overnight. The next day, cells were washed 3 x 5 minutes in 0.2% PBT and secondary antibody diluted in 1% donkey serum/0.2% PBT (donkey anti-rabbit Alexa Fluoro 488, 1:500; Life Technologies, Cat. No. A21206) for 1 hour. Cells were then washed 3 x 5 minutes in 0.2% PBT, incubated in DAPI (1 : 10,000 in PBS) for 5 minutes at room temperature and rinsed in PBS. Microscopy
染色後，將板在IN細胞分析儀2200上成像，每孔獲得9個視野。在IN細胞顯影分析軟體上進行定量，計算9個視野的IBA1染色的面積總和（以µm 2計）或Ki67+細胞的數量。針對每個技術三重複計算每個孔中每個視野的平均值（由於染色偽影（artifact）而排除某些視野，典型地每個孔6-9個視野），並且將其相對於DMSO對照孔的平均值進行歸一化。使用單因子變異數分析來確定樣品之間差異的統計學顯著性。用Prism 6（GraphPad軟體）進行統計學分析，並且p值指示為* ≤ 0.05，** ≤ 0.01，*** ≤ 0.001和**** ≤ 0.0001。 結果 After staining, plates were imaged on an IN Cytometer 2200, acquiring 9 fields per well. Quantification was carried out on the IN cell imaging analysis software, and the sum of the area of IBA1 staining (in µm 2 ) or the number of Ki67+ cells in 9 fields of view was calculated. The average of each field of view in each well was calculated for triplicate per technique (some fields were excluded due to staining artifacts, typically 6-9 fields per well) and compared to the DMSO control Wells were normalized to the mean. Statistical significance of differences between samples was determined using one-way analysis of variance. Statistical analysis was performed with Prism 6 (GraphPad software), and p-values are indicated as * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001 and **** ≤ 0.0001. result
將初代鼠小神經膠質細胞以50,000個細胞/孔鋪板並且靜置過夜。將細胞用DMSO、化合物
49RA03546849、RA11275777 (PLX3397)或化合物
6RA16100017預處理30分鐘，並且然後經受CSF-1刺激。將來自此實驗的細胞培養上清液在MCP1 ELISA中加工，以確定刺激/處理是否影響趨化介素產生。如
圖 5A- 圖 5C所示，CSF-1刺激誘導MCP-1（CCL2 - 趨化介素）的釋放顯著增加。CSF-1R抑制劑, RA11275777PLX3397和化合物
6RA16100017以濃度依賴性方式顯著降低了MCP-1產生（普通單因子變異數分析，p ＜ 0.0001）。計算PLX3397（IC
50= 17.4 nM）和化合物6（IC
50= 23.2 nM）二者的IC
50值。CSF-1誘導的MCP-1產生在用化合物
49RA03546849處理的板中不穩健（
圖 5B），因此不能生成此化合物的IC
50值。利用R&D MCP-1 Elisa套組，24小時後評估MCP-1分泌。每個數據點代表單個孔，而圖形柱代表六個孔的平均值和標準差。
Primary mouse microglia were plated at 50,000 cells/well and left overnight. Cells were pretreated with DMSO, compound 49 RA03546849, RA11275777 (PLX3397) or
在小神經膠質細胞刺激之後，用Iba1、Ki67和DAPI完成免疫細胞化學以確定小神經膠質細胞形態、增殖狀態和數量。使用InCell成像顯微鏡和分析軟體來定量培養物內的Iba1
+面積和DAPI
+細胞核的數量。Ki67無法定量，因為在ICC期間的凝聚將Iba1抗體交叉轉移至Ki67孔中。定量結果（
圖 6A- 圖 6C 和 7A- 圖 7C）分別證明了CSF-1刺激對Iba1
+面積和DAPI
+細胞數量的顯著影響。可以看到CSF-1R抑制以劑量依賴性方式阻斷這些CSF-1誘導的細胞變化。計算PLX3397RA11275777（Iba1的IC
50= 50.43 nM，並且DAPI的為68.2 nM）和化合物
6RA16100017（Iba1的IC
50= 84.6 nM，並且DAPI的為248 nM）的IC
50值。
Following microglial stimulation, immunocytochemistry was done with Ibal, Ki67 and DAPI to determine microglial morphology, proliferation status and number. The Iba1 + area and the number of DAPI + nuclei within the cultures were quantified using an InCell imaging microscope and analysis software. Ki67 could not be quantified because aggregation during ICC cross-transfers Iba1 antibody into Ki67 wells. Quantitative results ( Figure 6A -Figure 6C and 7A- Figure 7C ) demonstrated a significant effect of CSF-1 stimulation on Iba1 + area and DAPI + cell number, respectively. It can be seen that CSF-1R inhibition blocks these CSF-1-induced cellular changes in a dose-dependent manner. IC 50 values were calculated for PLX3397RA11275777 (IC 50 = 50.43 nM for Iba1 and 68.2 nM for DAPI) and
在 圖 6A- 圖 6C中，在小神經膠質細胞刺激測定後定量Iba1 +面積。CSF-1刺激顯著增加了Iba1 +面積並且用CSF-1R抑制劑的處理以濃度依賴性方式顯著消除了這種作用。從每種條件的三種不同孔中拍攝的九個圖像來定量小神經膠質細胞面積。資料點代表每個孔的平均Iba1 +面積，並且誤差條代表標準差（n = 3）。通過單因子變異數分析確定統計學顯著性，並且p值指示為*p ＜ 0.05，**p ＜ 0.01，***p ＜ 0.001和****p ＜ 0.0001。 In Figure 6A - 6C , Iba1 + area was quantified after the microglia stimulation assay. CSF-1 stimulation significantly increased Iba1 + area and treatment with CSF-1R inhibitors significantly abolished this effect in a concentration-dependent manner. Microglial area was quantified from nine images taken in three different wells for each condition. Data points represent mean Iba1 + area per well and error bars represent standard deviation (n = 3). Statistical significance was determined by one-way ANOVA and p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
在 圖 7A- 圖 7C中，用以下小神經膠質細胞刺激測來定量DAPI +標記的細胞核。CSF-1刺激增加了培養物內的細胞數量，並且CSF-1R抑制劑以濃度依賴性方式減少此數量。從每種條件的三種不同孔中拍攝的九個圖像來定量DAPI +細胞核。資料點代表每個孔的平均Iba1 +面積（來自9個圖像），並且誤差條代表標準差（n = 3）。通過單因子變異數分析確定統計學顯著性，並且p值指示為*p ＜ 0.05，**p ＜ 0.01，***p ＜ 0.001和****p ＜ 0.0001。 實例 12 In Figure 7A- 7C , the following microglia stimulation assay was used to quantify DAPI + labeled nuclei. CSF-1 stimulation increased the number of cells in culture, and CSF-1R inhibitors decreased this number in a concentration-dependent manner. DAPI + nuclei were quantified from nine images taken in three different wells for each condition. Data points represent mean Iba1 + area per well (from 9 images) and error bars represent standard deviation (n = 3). Statistical significance was determined by one-way ANOVA and p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Example 12
在以下實驗中檢查了在野生型初代小神經膠質細胞與SOD1突變的初代小神經膠質細胞中，在CSF-1或LPS刺激後本揭露的氘化CSF-1R抑制劑對細胞介素/趨化介素產生的影響。
初代小鼠小神經膠質細胞 -在ELN 20200624-062中分離
將初代小鼠小神經膠質細胞以5 x 10 5個/mL的濃度懸浮，並且將100 µL的此細胞懸浮液添加到96孔板的每個孔中。允許在37ºC、5% CO 2下將小神經膠質細胞靜置過夜。第二天，去除培養基，並且在37ºC、5% CO 2下，將細胞用二甲基亞碸（DMSO）或化合物 6RA16100017處理30分鐘或24小時。然後將細胞用100 ng/mL重組小鼠M-CSF刺激30分鐘或用10 ng/mL脂多醣刺激24小時。刺激後，將培養上清液從每個孔中去除，並且等分至兩種不同的96孔板用於後續ELISA測定。 CellTiter Glo 2.0 生存力測定 Primary mouse microglial cells were suspended at a concentration of 5 x 10 5 cells/mL, and 100 µL of this cell suspension was added to each well of a 96-well plate. Allow the microglia to rest overnight at 37 ºC, 5% CO . The next day, the medium was removed and the cells were treated with dimethylsulfoxide (DMSO) or compound 6RA16100017 for 30 minutes or 24 hours at 37ºC, 5% CO 2 . Cells were then stimulated with 100 ng/mL recombinant mouse M-CSF for 30 minutes or with 10 ng/mL lipopolysaccharide for 24 hours. After stimulation, the culture supernatant was removed from each well and aliquoted into two different 96-well plates for subsequent ELISA assays. CellTiter Glo 2.0 Viability Assay
使用Promega的Cell Titer Glo發光細胞生存力測定確定細胞生存力。首先允許測定試劑平衡至室溫30分鐘。去除培養上清液後，將100 µL新鮮室溫培養基添加到每個孔中。隨後，將100 µL的測定試劑添加到每個孔中。然後將測定板振搖兩分鐘，並且然後靜置10分鐘。將100 µL從每個孔轉移至白板中，並且立即在FlexStation3板讀取器上讀取發光。
小鼠 MCP-1 ELISA Cell viability was determined using Promega's Cell Titer Glo Luminescent Cell Viability Assay. The assay reagents were first allowed to equilibrate to room temperature for 30 minutes. After removing the culture supernatant, add 100 µL of fresh room temperature medium to each well. Subsequently, 100 µL of assay reagent was added to each well. The assay plate was then shaken for two minutes and then left to stand for 10 minutes.
將培養上清液用來自R&D Systems的Quantikine小鼠MCP-1 ELISA套組（目錄號SMJE00B）測定。將樣品用校準品稀釋液1 : 10稀釋。首先將50微升的測定稀釋液添加到每個孔中。然後將50微升的標準品、測定對照和稀釋的樣品添加到孔中。將板通過輕輕敲擊框架混合，然後用膠帶密封。將板在室溫下培育2小時。培育後，將板使用噴射瓶用大約400 uL的洗滌緩衝液洗滌5次。在最後一次洗滌之後，將板在紙巾上輕輕敲擊以去除過量水分。將100微升的小鼠MCP-1接合物添加到每個孔中，用新的膠帶覆蓋，並且在室溫下培育2小時。培育後，如上所述將板洗滌。然後將受質溶液添加到每個孔中，並且在黑暗中在室溫下培育30分鐘。培育後，將酸性終止溶液添加到每個孔中，並且在450 nm下在ELISA板讀取器上讀取板。 小鼠 IL-12p40 ELISA Culture supernatants were assayed with the Quantikine Mouse MCP-1 ELISA Kit from R&D Systems (Catalog No. SMJE00B). Samples were diluted 1:10 with calibrator diluent. First add 50 microliters of assay dilution to each well. Then 50 microliters of standards, assay controls and diluted samples were added to the wells. The plate was mixed by tapping the frame lightly, then sealed with tape. Plates were incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 uL of wash buffer using a spray bottle. After the last wash, the plates were tapped on paper towels to remove excess water. 100 microliters of mouse MCP-1 conjugate was added to each well, covered with fresh tape, and incubated for 2 hours at room temperature. After incubation, plates were washed as described above. The substrate solution was then added to each well and incubated for 30 minutes at room temperature in the dark. After incubation, acidic stop solution was added to each well and the plate was read on an ELISA plate reader at 450 nm. Mouse IL-12p40 ELISA
將細胞培養上清液用來自R&D Systems的Quantikine小鼠IL-12p40 ELISA套組（目錄號MP400）測定。將樣品用校準品稀釋液1 : 10稀釋。首先將50微升的測定稀釋液添加到每個孔中。然後將50微升的標準品、測定對照和稀釋的樣品以單個試樣添加到孔中。將板通過輕輕敲擊框架混合，然後用膠帶密封。將板在室溫下培育2小時。培育後，將板使用噴射瓶用大約400 uL的洗滌緩衝液洗滌5次。在最後一次洗滌之後，將板在紙巾上輕輕敲擊以去除過量水分。將100微升的小鼠IL-12p40接合物添加到每個孔中，用新的膠帶覆蓋，並且在室溫下培育2小時。培育後，如上所述將板洗滌。然後將受質溶液添加到每個孔中，並且在黑暗中在室溫下培育30分鐘。培育後，將酸性終止溶液添加到每個孔中，並且在450 nm下在ELISA板讀取器上讀取板。 結果 Cell culture supernatants were assayed with the Quantikine Mouse IL-12p40 ELISA Kit from R&D Systems (Catalog # MP400). Samples were diluted 1:10 with calibrator diluent. First add 50 microliters of assay dilution to each well. 50 microliters of standards, assay controls and diluted samples were then added to the wells as individual samples. The plate was mixed by tapping the frame lightly, then sealed with tape. Plates were incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 uL of wash buffer using a spray bottle. After the last wash, the plates were tapped on paper towels to remove excess water. 100 microliters of mouse IL-12p40 conjugate was added to each well, covered with fresh tape, and incubated for 2 hours at room temperature. After incubation, plates were washed as described above. The substrate solution was then added to each well and incubated for 30 minutes at room temperature in the dark. After incubation, acidic stop solution was added to each well and the plate was read on an ELISA plate reader at 450 nm. result
將初代鼠小神經膠質細胞以50,000個細胞/孔鋪板並且靜置過夜。將細胞用DMSO或化合物
6RA16100017預處理30分鐘或24小時，並且然後分別經受CSF-1或LPS刺激。24小時後，利用Promega的Cell Titer Glo測定套組評估細胞生存力。相比於未刺激的細胞，CSF-1刺激和LPS刺激二者均誘導細胞生存力讀數略微增加（
圖 8A- 圖 8B和
圖 9A- 圖 9B）。
Primary mouse microglia were plated at 50,000 cells/well and left overnight. Cells were pretreated with DMSO or
如
圖 8A和
圖 8B所示，CSF-1R抑制劑處理在所評估的濃度下對小神經膠質細胞沒有毒性作用。氘化CSF-1R抑制劑，化合物
6RA16100017，略微降低了CSF-1誘導的細胞生存力增加。圖形柱代表六個孔的平均值和標準差。如
圖 9A- 圖 9B所示，CSF-1R抑制對細胞生存力沒有有害作用。圖形柱代表六個孔的平均值和標準差。在野生型小神經膠質細胞與SOD1小神經膠質細胞細胞生存力中對於CSF-1或LPS刺激之反應沒有觀察到顯著差異。
As shown in Figure 8A and Figure 8B , CSF-1R inhibitor treatment had no toxic effect on microglia at the concentrations evaluated. A deuterated CSF-1R inhibitor,
將來自此實驗的細胞培養上清液在兩個單獨的ELISA（MCP-1和IL12p40）中處理，以確定刺激/處理是否影響趨化介素/細胞介素產生。Cell culture supernatants from this experiment were processed in two separate ELISAs (MCP-1 and IL12p40) to determine whether stimulation/treatment affected chemokines/cytokines production.
如
圖 10A- 圖 10B所示，CSF-1刺激誘導MCP-1（CCL2 - 趨化介素）的釋放顯著增加，並且化合物
6RA16100017以濃度依賴性方式顯著降低MCP-1產生。圖形柱代表六個孔的平均值和標準差。進行普通單因子變異數分析以確定各組之間的統計學差異性，並且p值表示為*** p ＜ 0.001和**** p ＜0.0001。
As shown in Figure 10A - 10B , CSF-1 stimulation induced a significant increase in the release of MCP-1 (CCL2 - a chemoattractant), and
如
圖 11A- 圖 11B所示，在鼠小神經膠質細胞培養物中，LPS刺激誘導IL12-p40產生的顯著增加。化合物
6RA16100017的CSF-1R抑制作用以濃度依賴性方式顯著降低了IL12-p40產生。圖形柱代表六個孔的平均值和標準差。進行普通單因子變異數分析以確定各組之間的統計學差異性，並且p值表示為** p ＜ 0.01，*** p ＜ 0.001和**** p ＜ 0.0001。
As shown in Figures 11A- 11B , LPS stimulation induced a significant increase in IL12-p40 production in murine microglia cultures. CSF-1R inhibition by
實例 13In Vitro Compounds:
Caco-2Caco-2
滲透性和流出測定以比較化合物Permeability and efflux assays to compare
使用Caco-2/TC7細胞在基於細胞的滲透性模型中進行Caco-2滲透性和流出測定。對於滲透性測定，將Caco-2/TC7細胞接種在Millipore Millicell 96上，並且對於流出測定，使用24孔板。具有細胞的板準備在培養21-25天之間使用。使用TECAN自動化液體處理平臺進行滲透性測定和流出測定二者。對於滲透性測定，在含有0.5% BSA的滲透性測定緩衝液（在HBSS緩衝液中的10 mM HEPES，pH 6.5）中以20 µM測試濃度製備測試化合物。底側滲透性緩衝液（pH 7.4）含有5% BSA。對於流出測定，在含有0.5% BSA的滲透性測定緩衝液（在HBSS緩衝液中的10 mM HEPES，pH 7.4）中以1 µM測試濃度製備測試化合物。對於流出測定，底側滲透性緩衝液（pH 7.4）含有0.5% BSA。通過將20 µM測試溶液添加到含有Caco‑2/TC7細胞的板的頂側來開始滲透性測定。在流出測定中，將1 µM測試化合物添加到頂端區室中用於頂端至底側（A至B）滲透性確定。對於底側至頂端（B至A）滲透性確定，將測試化合物添加到底側。將板在37ºC下在恒定振搖下培育90 min。在培育期結束時，將取得的樣品使用高壓液相層析法與串聯質譜法進行分析。對於每個測定，從質譜資料計算表觀滲透性（P app）和回收率。對於滲透性測定，將P app值報告為數字 x 10 -07cm/s。對於流出測定，除了回收率值之外，使用P app（基底側至頂端）與P app（頂端至底側）計算流出率。 Caco-2 permeability and efflux assays were performed in a cell-based permeability model using Caco-2/TC7 cells. For permeability assays, Caco-2/TC7 cells were seeded on Millipore Millicell 96, and for efflux assays, 24-well plates were used. Plates with cells were ready for use between days 21-25 in culture. Both permeability and efflux assays were performed using the TECAN automated liquid handling platform. For permeability assays, test compounds were prepared at 20 µM test concentrations in permeability assay buffer (10 mM HEPES in HBSS buffer, pH 6.5) containing 0.5% BSA. The bottom side permeabilization buffer (pH 7.4) contains 5% BSA. For efflux assays, test compounds were prepared at 1 µM test concentrations in permeability assay buffer (10 mM HEPES in HBSS buffer, pH 7.4) containing 0.5% BSA. For the efflux assay, the bottom side permeabilization buffer (pH 7.4) contains 0.5% BSA. Start the permeability assay by adding 20 µM test solution to the top side of the plate containing Caco‑2/TC7 cells. In the efflux assay, 1 µM of the test compound is added to the apical compartment for apical-to-basal (A to B) permeability determination. For bottom side to top (B to A) permeability determination, the test compound is added to the bottom side. Plates were incubated at 37ºC for 90 min with constant shaking. At the end of the incubation period, the samples taken were analyzed using high pressure liquid chromatography with tandem mass spectrometry. For each assay, the apparent permeability (P app ) and recovery were calculated from the mass spectral data. For permeability assays, P app values are reported as numbers x 10 −07 cm/s. For efflux assays, the efflux rates were calculated using P app (basal side to apical side) and P app (apical side to basal side) in addition to recovery values.
滲透性數據：
流出數據：
此測定方法的目的是使用人類肝微粒體（HLM）確定測試物品體外對特異性細胞色素P450（CYP）酶的抑制潛力。從純DMSO儲液稀釋測試化合物至在0.5% DMSO溶液中10 µM-0.07 µM的最終測試濃度範圍。在37ºC下將化合物與0.22 mg/mL人類肝微粒體（HLM）、50 mM磷酸鹽緩衝液、1.33 mM NADPH、3.33 mM 6-磷酸葡萄糖、3.33 mM六水鎂、0.4單位/mL 6-磷酸葡萄糖脫氫酶和適當濃度的單個化學探針一起共培育10-30分鐘。培育後，提取樣品，並且蛋白質在含0.1%甲酸的乙腈中沉澱。將樣品離心以去除過量的蛋白質，並且通過LDTD/MS/MS分析以確定IC
50值。受質的關鍵濃度如下：CYP2D6受質 - 10 µM的右美沙芬。CYP3A4受質 - 60 µM的睾酮和2 µM的咪達唑侖。將右美沙芬和睾酮各自與測試化合物一起培育30分鐘。將咪達唑侖與測試化合物一起培育10分鐘。如以下資料表所示，化合物6和24二者在高達10 μM下均未顯示出CYP抑制。
The purpose of this assay is to determine the inhibitory potential of a test article against specific cytochrome P450 (CYP) enzymes in vitro using human liver microsomes (HLM). Test compounds were diluted from neat DMSO stock solutions to a final test concentration range of 10 µM-0.07 µM in 0.5% DMSO solution. Compounds were mixed with 0.22 mg/mL human liver microsomes (HLM), 50 mM phosphate buffer, 1.33 mM NADPH, 3.33 mM glucose-6-phosphate, 3.33 mM magnesium hexahydrate, 0.4 units/mL glucose-6-phosphate at 37ºC Dehydrogenases are co-incubated with appropriate concentrations of individual chemical probes for 10-30 minutes. After incubation, samples were extracted and proteins were precipitated in acetonitrile containing 0.1% formic acid. Samples were centrifuged to remove excess protein and analyzed by LDTD/MS/MS to determine IC50 values. Critical concentrations of substrates are as follows: CYP2D6 substrate - 10 µM dextromethorphan. CYP3A4 Substrate -
數據：
以二重複將1 μM濃度的測試化合物與大鼠、人、犬、猴或小鼠肝細胞一起以50萬個肝細胞/mL進行培育，並且在測定時間點從培育取出樣品、然後進行液相層析與串聯質譜分析後，確定測試化合物耗盡的濃度-時間過程。使用有機溶劑終止培育。
以二重複將1 μM濃度的測試化合物在1 mg/mL人肝胞質溶膠（具有高醛氧化酶（AO）/黃嘌呤氧化酶（XO）活性）或2.5 mg/mL人肝S-9部分（具有高活性AO/XO）中進行培育，並且在測定時間點從培育中取出樣品、然後進行液相層析串聯質譜分析後，確定測試化合物耗盡的濃度-時間過程。使用有機溶劑終止培育。 醛 氧化酶受質的校準方法 Test compounds at 1 μM concentration in duplicate in 1 mg/mL human liver cytosol (with high aldehyde oxidase (AO)/xanthine oxidase (XO) activity) or 2.5 mg/mL human liver S-9 fraction (with highly active AO/XO), and after samples were removed from the incubation at assay time points followed by liquid chromatography tandem mass spectrometry analysis, the concentration-time course of test compound depletion was determined. Use an organic solvent to terminate the incubation. Aldehyde Oxidase Substrate Calibration Method
Zientek M, Jiang Y, Youdim K, Obach RS.In vitro-in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. Drug Metab Dispos.2010;38(8):1322-1327. doi:10.1124/dmd.110.033555描述了用於AO受質的基本校準方法。 Zientek M, Jiang Y, Youdim K, Obach RS. In vitro-in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. Drug Metab Dispos .2010;38(8):1322-1327. doi:10.1124/dmd. 110.033555 describes the basic calibration method for AO substrates.
此方法提供了一種使用已知被AO代謝的市售藥物的固有清除率的體外-體內相關性的基準工具。This method provides a benchmarking tool using the in vitro-in vivo correlation of intrinsic clearance of marketed drugs known to be metabolized by AO.
已知由於所述酶的人AOX1同種型的差異性表達，臨床前物種（小鼠、大鼠和犬）不能準確地預測AO代謝。小鼠和大鼠含有活性的四種同種型（AOX1、AOX2、AOX3和AOX4），犬缺乏活性AOX1酶，並且只有猴含有活性AOX1同種型。由於缺乏可用於準確預測AO受質的臨床前物種，預測人藥動學的傳統類比方法是困難的。因此，使用臨床中具有人藥動學的已知AO受質進行體外-體內校準方法。由於這些藥物中的幾種的PK特性較差，它們在臨床中失敗。通過使用具有可接受的人藥動學特性的紮來普隆（較低清除率AO受質）作為基準化合物，可以開發一種排序校準方法。Preclinical species (mouse, rat and dog) are known not to accurately predict AO metabolism due to differential expression of the human AOX1 isoform of the enzyme. Mice and rats contain active four isoforms (AOX1, AOX2, AOX3, and AOX4), dogs lack active AOX1 enzyme, and only monkeys contain active AOX1 isoforms. Traditional analog methods for predicting human pharmacokinetics are difficult due to the lack of preclinical species that can be used to accurately predict AO substrates. Therefore, an in vitro-in vivo calibration method was performed using known AO substrates with human pharmacokinetics in the clinic. Several of these drugs failed in the clinic due to their poor PK profile. A ranking calibration method could be developed by using zaleplon (a lower clearance AO substrate) with acceptable human pharmacokinetic properties as a reference compound.
使用三種體外系統（合併的人類肝胞質溶膠、肝臟S-9部分和分離自用HTK培養基灌注的肝的人肝細胞），分析了與測試化合物new chemical entities (SAR444024 and deuterated analogs)一起的這些可用的已知AO受質。計算測試化合物/新化學實體的放大的未結合固有清除率，並且與已知AO受質的體內未結合固有清除率進行比較。預測AO介導的體外放大的未結合固有清除率小於紮來普隆(zaleplon)的化合物具有可接受的AO體內清除率。Using three in vitro systems (pooled human liver cytosol, liver S-9 fraction, and human hepatocytes isolated from livers perfused with HTK medium), these available chemical entities (SAR444024 and deuterated analogs) together with test compounds were analyzed. known AO substrates. The amplified unbound intrinsic clearance of the test compound/NCE is calculated and compared to the in vivo unbound intrinsic clearance of known AO substrates. Compounds with AO-mediated in vitro amplification of unconjugated intrinsic clearance less than zaleplon are predicted to have acceptable AO clearance in vivo.
原始資料source material
放大的數據magnified data
實例example 1414 ：: 在不存在和存在醛氧化酶抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中化合物Compounds in human cryopreserved hepatocytes in the absence and presence of the aldehyde oxidase inhibitor hydralazine 24twenty four 的代謝特徵The metabolic characteristics of
在不存在和存在醛氧化酶（AO）抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中體外研究化合物
24的代謝特徵。將化合物
24在人類冷凍保存的肝細胞中培育2小時後，通過LC-MS鑑定並且定量總共九種代謝物。
The metabolic profile of
在人類冷凍保存的肝細胞中培育2小時後，剩餘72.2%的未改變的親本化合物，此數值是基於化合物
24及其鑑定的代謝物的總積分MS峰面積計算的。H10是檢測到的最豐富的代謝物，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的20.3%。代謝物H4a占化合物
24及其鑑定的代謝物的總積分MS峰面積的4.5%。所鑑定的每種其他代謝物均為化合物
24及其鑑定的代謝物的總積分MS峰面積的 ＜ 2%。
After 2 hours of incubation in human cryopreserved hepatocytes, 72.2% of the unchanged parental compound remained, calculated based on the total integrated MS peak areas of
在存在肼苯噠嗪的情況下在人類冷凍保存的肝細胞中培育2小時後，未改變的化合物
24占化合物
24及其鑑定的代謝物的總積分MS峰面積的90.9%。羥基化代謝物H10的形成被顯著抑制，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的1.6%。H4a是主要代謝物，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的5.4%。所鑑定的每種其他代謝物均為化合物
24及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
After incubation for 2 h in human cryopreserved hepatocytes in the presence of hydralazine,
提出了H10來源於在化合物
24的3H-咪唑[4,5-b]吡啶部分的2C位置處的羥基化。提出了H4a來源於在化合物
24的3H-咪唑[4,5-b]吡啶部分處的水合以及在3H-咪唑[4,5-b]吡啶部分的2C位置處的葡糖苷酸。提出了H11a來源於H10的3H-咪唑[4,5-b]吡啶部分的葡糖苷酸化。提出了H7來源於化合物
24的2-甲氧基
-吡啶部分的
O-去甲基化。提出了H6是化合物
24的葡糖苷酸接合物。
H10 was proposed to be derived from hydroxylation at the 2C position of the 3H-imidazo[4,5-b]pyridine moiety of
所提出的主要代謝途徑包括AO介導的羥基化，然後是葡糖苷酸化以及水合與葡糖苷酸化的組合。其他觀察到的代謝途徑包括非AO介導的羥基化然後葡糖苷酸化、直接葡糖苷酸化、去甲基化然後是葡糖苷酸化、以及氧化脫胺基化然後氧化的組合。The proposed major metabolic pathways include AO-mediated hydroxylation followed by glucuronidation and a combination of hydration and glucuronidation. Other observed metabolic pathways include non-AO mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation.
化合物
24根據WO 2017/015267的實例1-92中概述的程序製備。
培育條件
以下示出了含有肼苯噠嗪的通用實驗設計：
在研究取樣後，將來自三重複培育的剩餘樣品合併並且加工用於代謝物鑑定研究。 樣品製備 After study sampling, remaining samples from triplicate incubations were pooled and processed for metabolite identification studies. Sample Preparation
向每個樣品中添加等量的冰冷的乙腈（ v/v），並且然後將樣品渦旋混合。在大約13,000 rpm下離心10分鐘後，將上清液在35ºC下在氮氣流下濃縮直至剩餘大約0.1-0.2 mL的提取物。在分析前，將剩餘的提取物在大約13,000 rpm下離心15分鐘。將上清液注入LC/UV/MS用於分析。 儀器條件 An equal amount of ice-cold acetonitrile ( v/v ) was added to each sample, and the samples were then vortex mixed. After centrifugation at approximately 13,000 rpm for 10 min, the supernatant was concentrated at 35 ºC under nitrogen flow until approximately 0.1-0.2 mL of extract remained. The remaining extract was centrifuged at approximately 13,000 rpm for 15 minutes prior to analysis. The supernatant was injected into LC/UV/MS for analysis. Instrument conditions
代謝物鑑定在與UV（Thermo Vanquish）和質譜（MS）檢測（Thermo Orbitrap ID-X）偶聯的UPLC（Thermo Vanquish）上進行。
由於低樣品濃度，將質量峰面積用於代謝物特徵分析。假設等效摩爾濃度的代謝物或親本化合物的質譜反應相等，基於化合物
24及其鑑定的代謝物的總積分MS峰面積，計算代謝物或未改變的親本化合物的百分比。下表1中報告了峰面積占總積分MS峰面積的等於或大於0.1%的代謝物。
Due to low sample concentrations, mass peak areas were used for metabolite profiling. Based on the total integrated MS peak areas of
基於其準確的質量（容差 ≤ 5 ppm）、質量碎片模式和與其他體外研究的比較表徵代謝物。Metabolites were characterized based on their accurate mass (tolerance ≤ 5 ppm), mass fragmentation pattern, and comparison to other in vitro studies.
表surface
11
：在不存在或存在肼苯噠嗪的情況下，化合物: In the absence or presence of hydralazine, the compound
24twenty four
在人類冷凍的肝細胞中培育Growing in Human Frozen Hepatocytes
22
小時後的代謝物特徵Metabolite profile after hours
在不存在和存在AO抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中體外研究化合物
24的代謝特徵。將化合物
24在人類冷凍保存的肝細胞中培育2小時後，通過LC-MS鑑定並且定量總共九種代謝物。（參見
圖 12A- 圖 12B3）。
The metabolic profile of
在人類冷凍保存的肝細胞中培育2小時後，剩餘72.2%的未改變的親本化合物，此數值是基於化合物
24及其鑑定的代謝物的總積分MS峰面積計算的。H10是檢測到的最豐富的代謝物，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的20.3%。代謝物H4a占化合物
24及其鑑定的代謝物的總積分MS峰面積的4.5%。所鑑定的每種其他代謝物均為化合物
24及其鑑定的代謝物的總積分MS峰面積的 ＜ 2%。
After 2 hours of incubation in human cryopreserved hepatocytes, 72.2% of the unchanged parental compound remained, calculated based on the total integrated MS peak areas of
在存在AO抑制劑肼苯噠嗪的情況下在人類冷凍保存的肝細胞中培育2小時後，未改變的化合物
24占化合物
24及其鑑定的代謝物的總積分MS峰面積的90.9%。羥基化代謝物H10的形成被顯著抑制，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的1.6%。H4a是主要代謝物，並且占化合物
24及其鑑定的代謝物的總積分MS峰面積的5.4%。所鑑定的每種其他代謝物均為化合物
24及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
After incubation for 2 h in human cryopreserved hepatocytes in the presence of the AO inhibitor hydralazine,
提出了H10來源於在化合物
24的3H-咪唑[4,5-b]吡啶部分的2C位置處的羥基化。提出了H4a來源於在化合物
24的3H-咪唑[4,5-b]吡啶部分處的水合以及在3H-咪唑[4,5-b]吡啶部分的2C位置處的葡糖苷酸。提出了H11a來源於H10的3H-咪唑[4,5-b]吡啶部分的葡糖苷酸化。提出了H7來源於化合物
24的2-甲氧基
-吡啶部分的
O-去甲基化。提出了H6是化合物
24的葡糖苷酸接合物。
H10 was proposed to be derived from hydroxylation at the 2C position of the 3H-imidazo[4,5-b]pyridine moiety of
所提出的主要代謝途徑（ 圖 13）包括AO介導的羥基化，然後是葡糖苷酸化以及水合與葡糖苷酸化的組合。其他觀察到的代謝途徑包括非AO介導的羥基化然後葡糖苷酸化、直接葡糖苷酸化、去甲基化然後是葡糖苷酸化、以及氧化脫胺基化然後氧化的組合。 The proposed major metabolic pathway ( Fig. 13 ) involves AO-mediated hydroxylation followed by glucuronidation and a combination of hydration and glucuronidation. Other observed metabolic pathways include non-AO mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation.
實例example
1515
：:
在不存在和存在醛氧化酶抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中化合物Compounds in human cryopreserved hepatocytes in the absence and presence of the aldehyde
在不存在和存在醛氧化酶（AO）抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中體外研究化合物
6的代謝特徵。將化合物
6在人類冷凍保存的肝細胞中培育2小時後，通過LC-MS鑑定並且定量總共九種代謝物。
The metabolic profile of
在人冷凍保存的肝細胞中培育2小時後，剩餘87.1%的未改變的親本化合物，此數值是基於化合物
6及其鑑定的代謝物的總積分MS峰面積計算的。H10和H4a是檢測到的主要代謝物，並且分別占化合物
6及其鑑定的代謝物的總積分MS峰面積的6.6%和4.6%。所鑑定的每種其他代謝物均為化合物
6及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
After 2 hours of incubation in human cryopreserved hepatocytes, 87.1% of the unchanged parental compound remained, calculated based on the total integrated MS peak areas of
在存在AO抑制劑肼苯噠嗪的情況下在人類冷凍保存的肝細胞中培育2小時後，未改變的化合物
6占化合物
6及其鑑定的代謝物的總積分MS峰面積的92.0%。羥基化代謝物H10的形成被顯著抑制，並且占化合物
6及其鑑定的代謝物的總積分MS峰面積的0.9%。H4a是主要代謝物，並且占化合物
6及其鑑定的代謝物的總積分MS峰面積的5.7%。所鑑定的每種其他代謝物均為化合物
6及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
提出了H10來源於在化合物
6的3H-咪唑[4,5-b]吡啶部分的2C位置處的羥基化。提出了H4a來源於在化合物
6的3H-咪唑[4,5-b]吡啶部分處的水合以及在3H-咪唑[4,5-b]吡啶部分的2C位置處的葡糖苷酸。提出了H11a來源於H10的3H-咪唑[4,5-b]吡啶部分的葡糖苷酸化。提出了H7來源於化合物
6的2-甲氧基
-吡啶部分的
O-去甲基化。提出了H6是化合物
6的葡糖苷酸接合物。
It is proposed that H10 originates from hydroxylation at the 2C position of the 3H-imidazo[4,5-b]pyridine moiety of
所提出的主要代謝途徑包括AO介導的羥基化，然後是葡糖苷酸化以及水合與葡糖苷酸化的組合。其他觀察到的代謝途徑包括非AO介導的羥基化然後葡糖苷酸化、直接葡糖苷酸化、去甲基化然後是葡糖苷酸化、以及氧化脫胺基化然後氧化的組合。 培育條件 The proposed major metabolic pathways include AO-mediated hydroxylation followed by glucuronidation and a combination of hydration and glucuronidation. Other observed metabolic pathways include non-AO mediated hydroxylation followed by glucuronidation, direct glucuronidation, demethylation followed by glucuronidation, and combinations of oxidative deamination followed by oxidation. Breeding conditions
以下示出了含有肼苯噠嗪的通用DM20-03 實驗設計：
在研究DM20-03 取樣後，將來自三重複培育的剩餘樣品合併並且加工用於代謝物鑑定研究。 樣品製備 After study DM20-03 sampling, remaining samples from triplicate cultivations were pooled and processed for metabolite identification studies. Sample Preparation
向每個樣品中添加等量的冰冷的乙腈（ v/v），並且將樣品渦旋混合。在大約13,000 rpm下離心10分鐘後，將上清液在35ºC下在氮氣流下濃縮直至剩餘大約0.1-0.2 mL的提取物。在分析前，將剩餘的提取物在大約13,000 rpm下離心15分鐘。將上清液注入LC/UV/MS用於分析。 儀器條件 An equal volume of ice-cold acetonitrile ( v/v ) was added to each sample, and the samples were vortex mixed. After centrifugation at approximately 13,000 rpm for 10 min, the supernatant was concentrated at 35 ºC under nitrogen flow until approximately 0.1-0.2 mL of extract remained. The remaining extract was centrifuged at approximately 13,000 rpm for 15 minutes prior to analysis. The supernatant was injected into LC/UV/MS for analysis. Instrument conditions
代謝物鑑定在與UV（Thermo Vanquish）和質譜（MS）檢測（Thermo Orbitrap ID-X）偶聯的UPLC（Thermo Vanquish）上進行。
由於低樣品濃度，將質量峰面積用於代謝物特徵分析。假設等效莫耳濃度的代謝物或親本化合物的質譜反應相等，基於化合物
6及其鑑定的代謝物的總積分MS峰面積，計算代謝物或未改變的親本化合物的百分比。下表2中報告了峰面積占總積分MS峰面積的等於或大於0.1%的代謝物。
Due to low sample concentrations, mass peak areas were used for metabolite profiling. Based on the total integrated MS peak areas of
基於其準確的質量（容差 ≤ 5 ppm）、質量碎片模式和與其他體外研究的比較表徵代謝物 EID17-008 [ Error! Reference source not found.], EID17-009 [ Error! Reference source not found.], EID20-006 [ Error! Reference source not found.] and EID20-008 [ Error! Reference source not found.], and in vivostudies EID17-025 [ Error! Reference source not found.], EID17-026 [ Error! Reference source not found.], EID17-029 [ Error! Reference source not found.], EID19-035 [ Error! Reference source not found.], EID19-042 [ Error! Reference source not found.], EID19-046 [ Error! Reference source not found.], and EID19-051 [ Error! Reference source not found.]。 結果 Characterization of metabolites EID17-008 [ Error! Reference source not found. ], EID17-009 [ Error! Reference source not found. ], EID20-006 [ Error! Reference source not found. ] and EID20-008 [ Error! Reference source not found. ], and in vivo studies EID17-025 [ Error! Reference source not found. ], EID17-026 [ Error ! Reference source not found. ], EID17-029 [ Error! Reference source not found. ], EID19-035 [ Error! Reference source not found. ], EID19-042 [ Error! Reference source not found. ], EID19-046 [ Error! Reference source not found. ], and EID19-051 [ Error! Reference source not found. ]. result
表surface
22
：在不存在或存在肼苯噠嗪的情況下，化合物: In the absence or presence of hydralazine, the compound
66
在人類冷凍的肝細胞中培育Growing in Human Frozen Hepatocytes
22
小時後的代謝物特徵Metabolite profile after hours
在不存在和存在AO抑制劑肼苯噠嗪的情況下，在人類冷凍保存的肝細胞中體外研究化合物
6的代謝特徵。將化合物
6在人冷凍保存的肝細胞中培育2小時後，通過LC-MS鑑定並且定量總共九種代謝物。（參見
圖 14A-14B5）。
The metabolic profile of
在人類冷凍保存的肝細胞中培育2小時後，剩餘87.1%的未改變的親本化合物，此數值是基於化合物
6及其鑑定的代謝物的總積分MS峰面積計算的。H10和H4a是檢測到的主要代謝物，並且分別占化合物
6及其鑑定的代謝物的總積分MS峰面積的6.6%和4.6%。所鑑定的每種其他代謝物均為化合物
6及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
After 2 hours of incubation in human cryopreserved hepatocytes, 87.1% of the unchanged parental compound remained, calculated based on the total integrated MS peak areas of
在存在AO抑制劑肼苯噠嗪的情況下在人類冷凍保存的肝細胞中培育2小時後，未改變的化合物
6占化合物
6及其鑑定的代謝物的總積分MS峰面積的92.0%。羥基化代謝物H10的形成被顯著抑制，並且占化合物
6及其鑑定的代謝物的總積分MS峰面積的0.9%。H4a是主要代謝物，並且占化合物
6及其鑑定的代謝物的總積分MS峰面積的5.7%。所鑑定的每種其他代謝物均為化合物
6及其鑑定的代謝物的總積分MS峰面積的 ＜ 1%。
提出了H10來源於在化合物
6的3H-咪唑[4,5-b]吡啶部分的2C位置處的羥基化。提出了H4a來源於在化合物
6的3H-咪唑[4,5-b]吡啶部分處的水合以及在3H-咪唑[4,5-b]吡啶部分的2C位置處的葡糖苷酸。提出了H11a來源於H10的3H-咪唑[4,5-b]吡啶部分的葡糖苷酸化。提出了H7來源於化合物
6的2-甲氧基
-吡啶部分的
O-去甲基化。提出了H6是化合物
6的葡糖苷酸接合物。所提出的主要代謝途徑（
圖 15）包括AO介導的羥基化，然後是葡糖苷酸化以及水合與葡糖苷酸化的組合。其他觀察到的代謝途徑包括非AO介導的羥基化然後葡糖苷酸化、直接葡糖苷酸化、去甲基化然後是葡糖苷酸化、以及氧化脫胺基化然後氧化的組合。
It is proposed that H10 originates from hydroxylation at the 2C position of the 3H-imidazo[4,5-b]pyridine moiety of
實例example 1616 ： 體外小神經膠質細胞刺激研究 : In Vitro Microglial Stimulation Study
為了確定在CSF1刺激後對人類小神經膠質細胞細胞介素/趨化介素產生的影響，進行以下實驗。To determine the effect on human microglia cytokine/chemokine production following CSF1 stimulation, the following experiments were performed.
測試物品：●
DMSO●
化合物 6RA16100017(Batch: WA.JZO-001139.001) - 將儲備溶液（10 mM）用培養基稀釋以得到100 µM工作溶液並且將小神經膠質細胞在1.5625 nM、3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM或200 nM下處理。
●
化合物 24 RA16100016- 將儲備溶液（10 mM）用培養基稀釋以得到100 µM工作溶液並且將小神經膠質細胞在1.5625 nM、3.125 nM、6.25 nM、12.5 nM、25 nM、50 nM、100 nM或200 nM下處理。
●
重組人 CSF1（R&D Systems，目錄號216-ML/CF，批號MVN1218101） - 通過將50 µg溶解於500 µl PBS中製備100 µg/mL儲備溶液並且將小神經膠質細胞用100 ng/mL處理。
Test items: ● DMSO ● Compound 6RA16100017 (Batch: WA.JZO-001139.001) - stock solution (10 mM) was diluted with culture medium to obtain 100 µM working solution and microglia were incubated at 1.5625 nM, 3.125 nM, Treatment at 12.5 nM, 25 nM, 50 nM, 100 nM or 200 nM.
方法method iCelliCell 小神經膠質細胞的處理和刺激Processing and stimulation of microglia
iCell小神經膠質細胞（人類iPSC來源的小神經膠質細胞）獲自FujiFilm（目錄號C1110，批號105458）。根據製造說明書，將這些細胞解凍並且維持在iCell小神經膠質細胞完全培養基中。對於此測定，將iCell小神經膠質細胞以5 x 10
5個細胞/mL的濃度懸浮並且將100 µL的此細胞懸浮液添加到96孔板的內部60個孔中。允許在37ºC，5% CO
2下，將iCell小神經膠質細胞靜置過夜。在第二天晚上，將培養基去除並且用含有B27添加劑（Gibco，目錄號17504044）的Neurobasal培養基（Gibco，目錄號21103049）替代。再次，允許在37ºC，5% CO
2下，將小神經膠質細胞靜置過夜。第二天晚上，在37ºC，5% CO
2下，將細胞用二甲基亞碸（DMSO）、
化合物 6或
化合物 24處理30分鐘。然後將細胞用100 ng/mL重組人CSF1刺激24小時。刺激後，將培養上清液從每個孔中去除，並且等分至兩種不同的96孔板用於後續ELISA測定。
CellTiter Glo 2.0 生存力測定 iCell Microglia (human iPSC-derived microglia) were obtained from FujiFilm (Cat. No. C1110, Lot No. 105458). These cells were thawed and maintained in iCell microglia complete medium according to the manufacturer's instructions. For this assay, iCell microglia were suspended at a concentration of 5 x 105 cells/mL and 100 µL of this cell suspension was added to the inner 60 wells of a 96-well plate. Allow iCell Microglia to rest overnight at 37ºC, 5% CO 2 . On the second night, the medium was removed and replaced with Neurobasal Medium (Gibco, Cat. No. 21103049) containing B27 Supplement (Gibco, Cat. No. 17504044). Again, allow the microglia to rest overnight at 37ºC, 5% CO 2 . The next evening, cells were treated with dimethylsulfoxide (DMSO),
使用CellTiter Glo 2.0發光細胞生存力測定法（Promega，目錄號G9242）測定細胞生存力。首先允許測定試劑平衡至室溫30分鐘。去除培養上清液後，將100 µL新鮮室溫培養基添加到每個孔中。隨後，將100 µL的測定試劑添加到每個孔中。然後將測定板振搖兩分鐘，並且然後靜置10分鐘。將100 µL從每個孔轉移至白板中，並且在具有SoftMax Pro軟體的FlexStation3多模式微板讀取器（Molecular Devices，目錄號Flex3）上立即讀取發光。 小鼠 MCP-1 ELISA Cell viability was determined using the CellTiter Glo 2.0 Luminescent Cell Viability Assay (Promega, catalog number G9242). The assay reagents were first allowed to equilibrate to room temperature for 30 minutes. After removing the culture supernatant, add 100 µL of fresh room temperature medium to each well. Subsequently, 100 µL of assay reagent was added to each well. The assay plate was then shaken for two minutes and then left to stand for 10 minutes. 100 µL from each well was transferred to a white plate and luminescence was read immediately on a FlexStation3 multimode microplate reader (Molecular Devices, cat# Flex3) with SoftMax Pro software. Mouse MCP-1 ELISA
將培養上清液用Quantikine人類MCP-1 ELISA套組（R&D Systems，目錄號SCP00）測定。將樣品用校準品稀釋液1 : 10稀釋。然後將200微升的標準品和稀釋的樣品添加到孔中。將板通過輕輕敲擊框架混合，並且用膠帶密封。將板在室溫下培育2小時。培育後，將板使用噴射瓶用大約400 µL的洗滌緩衝液洗滌5次。在最後一次洗滌之後，將板在紙巾上輕輕敲擊以去除過量水分。將200微升的人類MCP-1接合物添加到每個孔中，用新的膠帶條覆蓋，並且在室溫下培育2小時。培育後，如上所述將板洗滌。然後將200微升的受質溶液添加到每個孔中，並且在黑暗中在室溫下培育30分鐘。培育後，將50微升的酸終止溶液添加到每個孔中，並且在450 nm下在具有SoftMax Pro軟體的FlexStation3多模式微板讀取器（Molecular Devices，目錄號Flex3）上讀取板。 結果 The culture supernatant was assayed with the Quantikine Human MCP-1 ELISA Kit (R&D Systems, catalog number SCP00). Samples were diluted 1:10 with calibrator diluent. Then 200 microliters of standards and diluted samples were added to the wells. The plate was mixed by tapping the frame lightly and sealed with tape. Plates were incubated at room temperature for 2 hours. After incubation, the plate was washed 5 times with approximately 400 µL of wash buffer using a squirt bottle. After the last wash, the plates were tapped on paper towels to remove excess water. 200 microliters of human MCP-1 conjugate was added to each well, covered with a new strip of tape, and incubated for 2 hours at room temperature. After incubation, plates were washed as described above. 200 microliters of substrate solution was then added to each well and incubated for 30 minutes at room temperature in the dark. After incubation, 50 microliters of acid stop solution was added to each well, and the plate was read at 450 nm on a FlexStation3 multimode microplate reader (Molecular Devices, cat# Flex3) with SoftMax Pro software. result
將iCell小神經膠質細胞（人類iPSC來源的小神經膠質細胞）以50,000個細胞/孔鋪板並且靜置過夜。去除含有生長因子的培養基，並且然後允許細胞再次靜置過夜。接下來，將細胞用DMSO或
化合物 6或
RA16100017 化合物 24預處理30分鐘，並且然後經受CSF1刺激。利用Promega的Cell Titer Glo測定套組評估細胞生存力。將來自此實驗的細胞培養上清液在MCP1 ELISA中加工，以確定刺激/處理是否影響趨化介素產生。
iCell Microglia (human iPSC-derived microglia) were plated at 50,000 cells/well and left overnight. The medium containing growth factors was removed, and the cells were then allowed to rest overnight again. Next, cells were pretreated with DMSO or
在此實驗中 RA16100017在評估的濃度下，
化合物 6不影響人小神經膠質細胞的生存力（
圖 16）。
圖 16描繪了如上所述的CSF1R抑制劑處理和CSF1刺激後的細胞生存力。將iCell人小神經膠質細胞以50,000個細胞/孔鋪板，並且在生長因子饑餓後靜置過夜。將細胞用DMSO或RA16100017預處理30分鐘，並且然後經受CSF1刺激。24小時後，利用Promega的Cell Titer Glo 2.0測定套組評估細胞生存力。CSF1刺激誘導細胞生存力增加，而CSF1R抑制劑對此作用沒有影響。每個數據點代表單個孔，而圖形柱代表六個孔的平均值和標準差。
如
圖 17所示，CSF1刺激誘導MCP-1（CCL2 - 趨化介素）的釋放顯著增加。
圖 17示出了在此實驗中化合物6對CSF1誘導的MCP-1產生的阻斷作用。將iCell人類小神經膠質細胞以50,000個細胞/孔鋪板，並且在生長因子饑餓後靜置過夜。將細胞用DMSO或RA16100017預處理30分鐘，並且然後經受CSF1刺激。利用R&D MCP1 Elisa套組，24小時後評估MCP-1分泌。CSF1R抑制劑處理以濃度依賴性方式顯著降低了MCP1產生（普通單因子變異數分析）。每個數據點代表單個孔，而圖形柱代表六個孔的平均值和標準差。
As shown in Figure 17 , CSF1 stimulation induced a significant increase in the release of MCP-1 (CCL2 - a chemokine). Figure 17 shows the blocking effect of
化合物 6 RA16100017以濃度依賴性方式顯著降低了MCP1產生（普通單因子變異數分析，p ＜ 0.0001）。CSF1刺激誘導MCP1產生的顯著增加，並且
化合物 6的CSF1R抑制以濃度依賴性方式消除了這種作用。
圖 18將
化合物 6與
化合物 24的MCP1產生進行比較，其顯示對MCP1的作用類似。
實例example 1717 ：: MOG-EAEMOG-EAE
實驗性自身免疫性腦脊髓炎（EAE）主要用作CNS的自身免疫性發炎疾病的非臨床模型，並且類似於人類多發性硬化症的許多方面。髓鞘質少突膠質細胞糖蛋白（MOG）誘導的EAE模型對於探索神經發炎和脫髓鞘的這種免疫介導的機制是理想的。在以下實驗中，在小鼠c57BL/6 EAE模型中測試化合物24和化合物6以評價降低疾病得分的可能功效。
物種： 8-9 週 齡雌性 C57BL/6J
每天對小鼠進行臨床評估以瞭解麻痺疾病的體征，並且間歇性地稱重以記錄體重減輕。研究結束時，將1/2腦固定用於組織學檢查，並且收集腦/肝/血漿用於暴露。收集脊髓和全血用於流式細胞術。保存另外的血漿等分試樣用於後續分析。 方法： EAE 誘導和評分 Mice were clinically assessed daily for signs of paralytic disease and weighed intermittently to record weight loss. At the end of the study, 1/2 brain was fixed for histological examination and brain/liver/plasma was collected for exposure. Spinal cord and whole blood were collected for flow cytometry. Additional plasma aliquots were saved for subsequent analysis. Methods: EAE induction and scoring
將雌性C57BL/6J小鼠用在完全弗氏佐劑（CFA）中的MOG
35-55肽（250 µg/小鼠）乳劑免疫。通過兩次皮下注射將乳劑以每個注射部位100 µL的體積遞送至後側腹。在第0天和第2天以在200 µL的PBS中的280 ng/只動物的劑量經由尾靜脈注射投予
百日咳鮑特菌毒素（PTX）。在EAE誘導後，每天監測小鼠的麻痺症狀並且使用進展評分系統對它們的臨床表現進行評分（得分0：無疾病；得分1：尾巴鬆垂；得分2：後肢無力；得分3：後肢麻痺；得分4：前肢無力或部分麻痺；得分5：死亡）。
Female C57BL/6J mice were immunized with an emulsion of MOG 35-55 peptide (250 µg/mouse) in complete Freund's adjuvant (CFA). The emulsion was delivered to the posterior flank by two subcutaneous injections in a volume of 100 µL per injection site. Bordetella pertussis toxin (PTX) was administered via tail vein injection at a dose of 280 ng/animal in 200 µL of PBS on
只要動物達到疾病得分為1，就將其納入研究。每天，將第一次達到得分一的動物平均分佈在處理組中，並且當晚開始處理。將媒劑和化合物用顏色編碼，使得對研究進行評分的人員對處理組不知情。將動物處理七天。在最後一劑（共14劑，處理7天）後一小時，將動物麻醉，並且經由眶後取血將血液收集到EDTA管中。然後將動物用冰冷的PBS灌注，並且收集適當的組織用於研究終點。 結果 Animals were included in the study as soon as they achieved a disease score of 1. Each day, animals that first achieved a score of one were evenly distributed among treatment groups, and treatment began that evening. Vehicle and compound were color coded such that those scoring the study were blinded to treatment group. Animals were handled for seven days. One hour after the last dose (total of 14 doses, 7 days of treatment), animals were anesthetized and blood was collected via retro-orbital bleeding into EDTA tubes. Animals were then perfused with ice-cold PBS and appropriate tissues collected for study endpoints. result
所述方案旨在用更高濃度的MOG
35-55和CFA乳劑誘導EAE模型。在得分為1或更高時，將EAE小鼠隨機分為三個不同的處理組：媒劑、化合物24（15 mg/kg）或化合物6（15 mg/kg）。此研究中的平均疾病得分（
圖 19）證明了MOG35-55誘導的多發性硬化症C57BL/6 EAE模型的標準疾病過程。資料點和誤差條分別代表組平均值和平均值的標準誤差。如
圖 19所示，兩種CSF1R抑制劑均顯著平均疾病得分。然而，與非氘化
化合物 24相比，氘化CSF1R抑制劑
化合物 6以令人驚訝的程度改善了麻痺症狀。
The protocol was aimed at inducing the EAE model with higher concentrations of MOG 35-55 and CFA emulsions. At a score of 1 or higher, EAE mice were randomized into three different treatment groups: vehicle, compound 24 (15 mg/kg), or compound 6 (15 mg/kg). The mean disease score in this study ( Figure 19 ) demonstrated a standard disease course in the MOG35-55-induced multiple sclerosis C57BL/6 EAE model. Data points and error bars represent group mean and standard error of the mean, respectively. As shown in Figure 19 , both CSFlR inhibitors significantly increased mean disease scores. However, the deuterated
圖 1A 和圖 1B示出了在CSF-1刺激後，例示性CSF-1R抑制性化合物（化合物 24）對MCP-1趨化介素產生的影響。示出了平均值（ 圖 1A）和標準差（ 圖 1B）。 Figures 1A and 1B show the effect of an exemplary CSF-1R inhibitory compound (Compound 24 ) on MCP-1 chemokine production following CSF-1 stimulation. The mean ( Figure 1A ) and standard deviation ( Figure 1B ) are shown.
圖 2A 和圖 2B示出了在CSF-1後，例示性氘化CSF-1R抑制性化合物（化合物 6）對MCP-1趨化介素產生的影響。示出了平均值（ 圖 2A）和標準差（ 圖 2B）。 Figures 2A and 2B show the effect of an exemplary deuterated CSF-1R inhibitory compound (Compound 6 ) on MCP-1 chemokine production following CSF-1. The mean ( Figure 2A ) and standard deviation ( Figure 2B ) are shown.
圖 3A 和圖 3B示出了圖1的實驗的IC 50曲線。 Figures 3A and 3B show the IC50 curves for the experiments of Figure 1 .
圖 4A 和圖 4B示出了圖2的實驗的IC 50曲線。 4A and 4B show the IC50 curves for the experiment of FIG . 2 .
圖 5A- 圖 5C示出了CSF-1R抑制劑化合物6（ 圖 5A）和PLX3397對照（ 圖 5C）以濃度依賴性方式顯著降低了MCP-1產生。化合物49的結果示於 圖 5B中。 Figures 5A - 5C show that CSF-1R inhibitor compound 6 ( Figure 5A ) and PLX3397 control ( Figure 5C ) significantly reduced MCP-1 production in a concentration-dependent manner. The results for compound 49 are shown in Figure 5B .
圖 6A- 圖 6C示出了CSF-1刺激顯著增加了Iba1 +面積，並且用CSF-1R抑制劑化合物 6（ 圖 6A）和對照PLX3397（ 圖 6C）的處理以濃度依賴性方式顯著消除了這種作用。化合物49的結果示於 圖 6B中。 Figures 6A - 6C show that CSF-1 stimulation significantly increased the Iba1 + area, and treatment with the CSF-1R inhibitor compound 6 ( Figure 6A ) and the control PLX3397 ( Figure 6C ) significantly abolished this in a concentration-dependent manner . kind of effect. The results for compound 49 are shown in Figure 6B .
圖 7A- 圖 7C示出了CSF-1刺激增加了培養物內的細胞數量（如通過DAPI +細胞證明），以及CSF-1R抑制劑化合物6（ 圖 7A）和對照PLX3397（ 圖 7C）以濃度依賴性方式減少了這一數量。化合物49的結果示於 圖 7B中。 Figures 7A - 7C show that CSF-1 stimulation increases the number of cells in culture (as evidenced by DAPI + cells), and that the CSF-1R inhibitor compound 6 ( Figure 7A ) and the control PLX3397 ( Figure 7C ) increase in concentration The dependent approach reduces this amount. The results for compound 49 are shown in Figure 7B .
圖 8A- 圖 8B示出了在野生型（
圖 8A）或SOD1（
圖 8B）細胞中用DMSO（對照）或化合物
6和CSF-1刺激進行預處理後的細胞生存力。
Figures 8A - 8B show cell viability after pretreatment with DMSO (control) or stimulation with
圖 9A- 圖 9B示出了在野生型（
圖 9A）或SOD1（
圖 9B）細胞中用DMSO（對照）或化合物
6和LPS刺激進行處理後的細胞生存力。
Figures 9A- 9B show cell viability following stimulation with DMSO (control) or
圖 10A- 圖 10B示出了在野生型（
圖 10A）或SOD1（
圖 10B）細胞中用DMSO（對照）或化合物
6和CSF-1刺激處理後的MCP-1產生。
Figures 10A - 10B show MCP-1 production after stimulation with DMSO (control) or
圖 11A- 圖 11B示出了在野生型（
圖 11A）或SOD1（
圖 11B）細胞中用DMSO（對照）或化合物
6和LPS刺激處理後的IL-12p40產生。
11A- 11B show IL-12p40 production in wild-type ( FIG . 11A ) or SOD1 ( FIG. 11B ) cells after treatment with DMSO (control) or
圖 12A示出了在不存在AO抑制劑肼苯噠嗪的情況下在人冷凍保存的肝細胞中培育後化合物
24的體外代謝曲線。
Figure 12A shows the in vitro metabolic profile of
圖 12B示出了在存在AO抑制劑肼苯噠嗪的情況下在人冷凍保存的肝細胞中培育後化合物
24的體外代謝曲線。
Figure 12B shows the in vitro metabolic profile of
圖 13示出了提出的在不存在和存在AO抑制劑肼苯噠嗪的情況下在冷凍保存的人肝細胞中化合物
24的代謝途徑。
Figure 13 shows the proposed metabolic pathway of
圖 14A示出了在不存在AO抑制劑肼苯噠嗪的情況下在人冷凍保存的肝細胞中培育後化合物
6的體外代謝曲線。
Figure 14A shows the in vitro metabolic profile of
圖 14B示出了在存在AO抑制劑肼苯噠嗪的情況下在人冷凍保存的肝細胞中培育後化合物
6的體外代謝曲線。
Figure 14B shows the in vitro metabolic profile of
圖 15示出了提出的在不存在和存在AO抑制劑肼苯噠嗪的情況下在冷凍保存的人肝細胞中化合物
6的代謝途徑。
Figure 15 shows the proposed metabolic pathway of
圖 16示出了在CSF1R抑制劑處理和CSF1刺激後的細胞生存力。 Figure 16 shows cell viability after CSF1R inhibitor treatment and CSF1 stimulation.
圖 17示出了在此實驗中化合物6對CSF1誘導的MCP-1產生的阻斷作用。
Figure 17 shows the blocking effect of
圖 18A- 圖 18B將 化合物 6（ 圖 18A）與 化合物 24（ 圖 18B）的MCP1產生進行比較，顯示了對MCP1的作用類似。 Figures 18A -18B compare the MCP1 production of Compound 6 ( Figure 18A ) with Compound 24 ( Figure 18B ), showing a similar effect on MCP1.
圖 19A- 圖 19B示出了兩種CSF1R抑制劑（化合物6和化合物24）顯著地平均疾病得分。與非氘化
化合物 24相比，氘化CSF1R抑制劑
化合物 6以令人驚訝地更大的程度改善了麻痺症狀。
Figures 19A - 19B show significant mean disease scores for two CSF1R inhibitors (
圖 20示出了化合物
6形式A的XRPD圖。
Figure 20 shows the XRPD pattern of
圖 21示出了化合物6形式A的PLM圖像。
Figure 21 shows the PLM image of
圖 22示出了化合物
6形式A的TGA（上圖）/DSC（下圖）覆蓋圖。
Figure 22 shows the TGA (upper panel)/DSC (lower panel) overlay of
圖 23示出了化合物
6形式A的HPLC。
Figure 23 shows the HPLC of
Claims (22)
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